---------------------------------------------------------------------------
    Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997 
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 Perspective

    * The Economic Impact of a Bioterrorist Attack: Are
      Prevention and Postattack Intervention Programs
      Justifiable?
           A.F. Kaufmann, M.I. Meltzer, G.P. Schmid

 Synopses

    * Hantaviruses: A Global Disease Problem
           C. Schmaljohn and B. Hjelle
    * Japanese Spotted Fever: Report of 31 Cases and Review of
      the Literature
           * F. Mahara
      This article is also available in Japanese at
      http://www.cdc.gov/ncidod/EID/eid.htm
    * Polycystic Kidney Disease: An Unrecognized Emerging
      Infectious Disease?
           M.A. Miller-Hjelle, J.T. Hjelle, M. Jones, W.R.
      Mayberry, M.A. Dombrink-Kurtzman, S.W. Peterson, D.M.
      Nowak, and F.S. Darras
    * Borna Disease
           C.G.Hatalski,A.J.Lewis,and W.I. Lipkin
    * The Rickettsia: an Emerging Group of Pathogens in Fish
           J.L. Fryer and M.J. Mauel
    * Rhodococcus equi and Arcanobacterium haemolyticum: Two
      "Coryneform"Bacteria Increasingly Recognized as Agents of
      Human Infection
           R. Linder
    * Is Creutzfeldt-Jakob Disease Transmitted in Blood?
           M.N. Ricketts, N.R. Cashman, E.E. Stratton, S.
      ElSaadany

 Dispatches

    * A New Tick-borne Encephalitis-like Virus Infecting New
      England Deer Ticks, Ixodes dammini
           S.R. Telford III, P.M. Armstrong, P. Katavolos, I.
      Foppa, A.S.O. Garcia, M.L. Wilson, A. Spielman
    * An Unusual Hantavirus Outbreak in Southern Argentina:
      Person-to-Person Transmission?
           R.M. Wells, S.S. Estani, Z.E. Yadon, D. Enria, P.
      Padula, N. Pini, J.N. Mills, C.J. Peters, E.L. Segura,
      and the Hantavirus Pulmonary Syndrome Study Group for
      Patagonia
    * Pertussis in the Netherlands: an Outbreak Despite High
      Levels of Immunization with Whole-Cell Vaccine
           H.E. de Melker, M.A.E. Conyn-van Spaendonck, H.C.
      Rmke, J.K. van Wijngaarden, F.R. Mooi, and J.F.P.
      Schellekens
    * Invasive Haemophilus influenzae type b Disease in Elderly
      Nursing Home Residents: Two Related Cases
           T.C. Heath, M.C. Hewitt, B. Jalaludin, C. Roberts,
      A.G. Capon, P. Jelfs, and G.L. Gilbert
    * Seroepidemiologic Studies of Hantavirus Infection Among
      Wild Rodents in California
           M. Jay, M.S. Ascher, B.B. Chomel, M. Madon, D.
      Sesline, B.A. Enge, B. Hjelle, T.G. Ksiazek, P. E.
      Rollin, P.H. Kass, and K. Reilly
    * Gestational Psittacosis in a Montana Sheep Rancher
           D.M. Jorgensen
    * Lack of Serologic Evidence for an Association between
      Cache Valley Virus Infection and Anencephaly and Other
      Neural Tube Defects in Texas
           J.F. Edwards and K. Hendricks
    * Rabies Postexposure Prophylaxis Survey Kentucky
           M. Auslander and C. Kaelin

 Commentary

    * Biologic Terrorism Responding to the Threat
           P.K. Russell

 From the 1st International Conference on Emerging Zoonoses
 The articles in this section were originally presented at the
 1st International Conference on Emerging Zoonoses, Jerusalem,
 Israel, November 24-28, 1996. The conference was cosponsored
 by the Centers for Disease Control and Prevention and the
 Israel Center for Disease Control.

    * The Hantaviruses of Europe: from the Bedside to the Bench
           J. Clement, P. Heyman, P. McKenna, P. Colson, and T.
      Avsic-Zupanc
    * Brucellosis: an Overview
           M.J. Corbel
    * Global Aspects of Emerging and Potential Zoonoses: a WHO
      Perspective
           F.-X. Meslin
    * Epidemiology of Emerging Zoonoses in Israel
           A. Shimshony
    * Electronic Media and Emerging Zoonoses
           S.A. Berger

 Letters

    * The Reemergence of Aedes aegypti in Arizona
           D.M. Engelthaler, T.M. Fink, C.E. Levy and M.J.
      Leslie
    * Treatment of Exudative Pharyngitis
           P.D. Ellner
    * Reply to P.D. Ellner
           H.S. Izurieta, P.M. Strebel, T. Youngblood, D.G.
      Hollis, and T. Popovic

 News and Notes

    * Molecular Epidemiology and Evolutionary Genetics of
      Pathogenic Microorganisms
    * Simian Virus 40 (SV40)
    * Foodborne Pathogens: Implications and Control
    * Emerging Infectious Diseases in the Pacific Rim
    * Emerging Zoonotic Infectious Diseases
    * Hantavirus Conference
    * International Conference on Emerging Infectious Diseases
    * Emerging Infectious Diseases Laboratory Fellowship
      Program
    * Emerging Infections: Clinical and Pathologic Update II
    * Rabies Conference
    * Erratum


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                                 About EID 

Emerging Infectious Diseases is indexed in Index Medicus/Medline, Current
Contents, and several other electronic databases.

Emerging Infectious Diseases is part of CDC's plan for combatting emerging
infectious diseases; the plan is outlined in a recently published document,
Addressing Emerging Infectious Disease Threats--A Prevention Strategy for
the United States. One of the main goals of CDC's plan is to enhance
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Editors

Editor
Joseph E. McDade, Ph.D., National Center for Infectious Diseases
Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA

Perspectives Editor
Stephen S. Morse, Ph.D.,The Rockefeller University,
New York, New York, USA

Synopses Editor
Phillip J. Baker, Ph.D., Division of Microbiology and Infectious Diseases,
National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland, USA

Dispatches Editor
Stephen Ostroff, M.D., National Center for Infectious Diseases,
CDC, Atlanta, Georgia, USA

Managing Editor
Polyxeni Potter, M.A., National Center for Infectious Diseases,
Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA
---------------------------------------------------------------------------

Liaison Representatives

Anthony I. Adams, M.D., Chief Medical Adviser,
Commonwealth Department of Human Services and Health,
Canberra, Australia

David Brandling-Bennett, M.D., Deputy Director,
Division of Communicable Diseases, Pan American Health Organization,
World Health Organization
Washington, D.C., USA

Gail Cassell, Ph.D.,
Liaison to American Society for Microbiology
University of Alabama at Birmingham
Birmingham, Alabama, USA

Richard A. Goodman, M.D., M.P.H., Editor, MMWR,
CDC, Atlanta, Georgia, USA

Thomas M. Gomez. D.V.M.,M.S., Staff Epidemiologist
U.S. Department of Agriculture Animal and Plant Health Inspection Service
Riverdale, Maryland, USA

Joseph Losos, M.D., Director General,
Laboratory Center for Disease Control
Ontario, Canada

Gerald L. Mandell, M.D., Liaison to Infectious Diseases Society of America,
University of Virginia Medical Center,
Charlottesville, Virginia, USA

William J. Martone, M.D., Senior Executive Director
National Foundation for Infectious Diseases
Bethesda, Maryland, USA

Phillip P. Mortimer, M.D., Director, Virus Reference Division
Central Public Health Laboratory
London, United Kingdom

Robert Shope, M.D., Director, Yale Arbovirus Research Unit,
Yale University School of Medicine,
New Haven, Connecticut, USA

Natalya B. Sipachova, M.D., Ph.D.
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Russian Republic Information & Analytic Centre
Moscow, Russia

Bonnie Smoak, M.D.
U.S. Army Medical Research UnitKenya
Unit 64109
Box 401
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Robert Swanepoel, B.V.Sc., Ph.D., Head, Special Pathogens Unit,
National Institute for Virology,
Sandrinham 2131, South Africa

Roberto Tapia-Conyer, M.D.
Direccin General de Epidemiologa
Secretara de Salud
Mxico
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Emerging Infectious Diseases (EID) is a peer-reviewed journal established
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EID has an international scope and is intended for professionals in
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Perspectives: Contributions to the Perspectives section should provide
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influence the emergence of infectious diseases, including microbial
adaption and change; human demographics and behavior; technology and
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approximately 3,500 words and should include references, not to exceed 40.
Use of additional subheadings in the main body of the text is recommended.
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Synopses: Submit concise reviews of infectious diseases or closely related
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however, timely updates of other diseases or topics are also welcome.
Synopses should be approximately 3,500 words and should include references,
not to exceed 40. Use of subheadings in the main body of the text is
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experimental procedures should immediately follow the body of the text.
Photographs and illustrations are encouraged. Provide a short abstract (150
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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Perspective

The Economic Impact of a Bioterrorist Attack: Are Prevention and Postattack
Intervention Programs Justifiable?

Arnold F. Kaufmann, Martin I. Meltzer, and George P. Schmid
Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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      Understanding and quantifying the impact of a bioterrorist
      attack are essential in developing public health preparedness
      for such an attack. We constructed a model that compares the
      impact of three classic agents of biologic warfare (Bacillus
      anthracis, Brucella melitensis, and Francisella tularensis)
      when released as aerosols in the suburb of a major city. The
      model shows that the economic impact of a bioterrorist attack
      can range from an estimated $477.7 million per 100,000 persons
      exposed (brucellosis scenario) to $26.2 billion per 100,000
      persons exposed (anthrax scenario). Rapid implementation of a
      postattack prophylaxis program is the single most important
      means of reducing these losses. By using an insurance analogy,
      our model provides economic justification for preparedness
      measures.

Bioterrorism and its potential for mass destruction have been subjects of
increasing international concern. Approximately 17 countries (including
five implicated as sponsors of international terrorism) may have active
research and development programs for biologic weapons (1). Moreover,
groups and individuals with grievances against the government or society
have been known to use or plan to use biologic weapons to further personal
causes.

Only modest microbiologic skills are needed to produce and effectively use
biologic weapons. The greatest, but not insurmountable, hurdle in such an
endeavor may be gaining access to a virulent strain of the desired agent.
Production costs are low, and aerosol dispersal equipment from commercial
sources can be adapted for biologic weapon dissemination. Bioterrorists
operating in a civilian environment have relative freedom of movement,
which could allow them to use freshly grown microbial suspensions (storage
reduces viability and virulence). Moreover, bioterrorists may not be
constrained by the need for precise targeting or predictable results.

The impact of a bioterrorist attack depends on the specific agent or toxin
used, the method and efficiency of dispersal, the population exposed, the
level of immunity in the population, the availability of effective
postexposure and/or therapeutic regimens, and the potential for secondary
transmission. Understanding and quantifying the impact of a bioterrorist
attack are essential to developing an effective response. Therefore, we
have analyzed the comparative impact of three classic biologic warfare
agents (Bacillus anthracis, Brucella melitensis, and Francisella
tularensis) when released as aerosols in the suburbs of a major city and
compared the benefits of systematic intervention with the costs of
increased disease incidence (from the economic point of view used in
society).

Analytic Approach

Scenario Assumptions

We compared the impact of a theoretical bioterrorist attack on a suburb of
a major city, with 100,000 population exposed in the target area. The
attack was made by generating an aerosol of an agent (B. anthracis spores,
B. melitensis, or F. tularensis) along a line across the direction of the
prevailing wind. The meteorologic conditions (thermal stability, relative
humidity, wind direction and speed) were assumed to be optimal (2), and the
aerosol cloud passed over the target area within 2 hours. We projected
impact on the basis of 10% and 100% of the target population being exposed
to the aerosol cloud.

We assumed that, when inhaled, the infectious dose50 (ID50) was 20,000
spores for B. anthracis and 1,000 vegetative cells for B. melitensis and F.
tularensis. The rate of physical decay for airborne particles 5 mm or less
in diameter was estimated to be negligible during the 2-hour transit time.
The rate of biologic decay of the particulate agents was estimated to be
negligible for the B. anthracis spores and 2% per minute for the B.
melitensis and F. tularensis vegetative cells. Viability and virulence did
not dissociate. Persons who were exposed to the B. anthracis cloud at any
point during the 2-hour transit time inhaled one ID50 dose, and persons who
were exposed to either the B. melitensis or F. tularensis cloud inhaled one
to 10 ID50 doses, depending on their proximity to the origination point of
the aerosol cloud.

The epidemic curve for anthrax by days after exposure was assumed to be <1
day, 0% of cases; 1 day, 5%; 2 days, 20%; 3 days, 35%; 4 days, 20%; 5 days,
10%; 6 days, 5%; and 7 or more days, 5% (3-5). Case-fatality rates were
also assumed to vary by the day symptoms were first noted. The
case-fatality rate was estimated as 85% for patients with symptoms on day
1; 80% for patients with symptoms on day 2; 70% for those with symptoms on
day 3; 50% for those with symptoms on days 4, 5, and 6; and 70% for those
with symptoms on and after day 7. The increased death rate in persons with
an incubation period of 7 or more days is calculated on an assumption of
delayed diagnosis, with resultant delayed therapy.

When estimating days in hospital and outpatient visits due to infection, we
assumed that 95% of anthrax patients were hospitalized, with a mean stay of
7 days. Patients not admitted to a hospital had an average of seven
outpatient visits, and surviving hospitalized patients had two outpatient
visits after discharge from the hospital. Persons who received only
outpatient care were treated for 28 days with either oral ciprofloxacin or
doxycycline. No significant long-term sequelae resulted from the primary
infection, and no relapses occurred.

The epidemic curve for brucellosis by days after exposure was assumed to be
0 to 7 days, 4% of cases; 8 to 14 days, 6%; 15 to 28 days, 14%; 29 to 56
days, 40%; 57 to 112 days, 26%, and 113 or more days, 10% (4, 6-9). The
case-fatality rate was estimated to be 0.5%. Fifty percent of patients were
hospitalized, with an average stay of 7 days. Nonhospitalized patients had
an average of 14 outpatient visits, and hospitalized patients had seven
outpatient visits after discharge from the hospital. Outpatients received a
combination of oral doxycycline for 42 days and parenteral gentamicin for
the first 7 days of therapy. Five percent of patients had a relapse or
long-term sequelae, and required 14 outpatient visits within 1 year.

The epidemic curve for tularemia by days after exposure was assumed to be:
<1 day, 0% of cases; 1 day, 1%; 2 days, 15%; 3 days, 45%; 4 days, 25%; 5
days, 10%; 6 days, 3%; and 7 or more days, 1% (4,10-11). The estimated
case-fatality rate was 7.5%; and 95% of patients were hospitalized, with an
average stay of 10 days. Nonhospitalized patients had an average of 12
outpatient visits, and hospitalized patients who survived the acute illness
had two outpatient visits after discharge from the hospital. Outpatients
received oral doxycycline for 14 days and parenteral gentamicin for 7 days.
Five percent of patients had a relapse or long-term sequelae and required
an average of 12 outpatient visits.

The efficacy of intervention strategies is unknown; our projections are our
best estimates based on published clinical and experimental data (4,12-14).
For anthrax, the projected intervention program was either a 28-day course
of oral ciprofloxacin or doxycycline (assumed to be 90% effective), or a
28-day course of oral ciprofloxacin or doxycycline plus three doses of the
human anthrax vaccine (assumed to be 95% effective); for brucellosis, a
42-day course of oral doxycycline and rifampin (assumed to be 80%
effective), or a 42-day course of oral doxycycline, plus 7 days of
parenteral gentamicin (assumed to be 95% effective); for tularemia, the
intervention program was a 14-day course of oral doxycycline (assumed to be
80% effective), or a 14-day course of oral doxycycline plus 7 days of
parenteral gentamicin (assumed to be 95% effective). Only 90% of persons
exposed in the target area were assumed to effectively participate in any
intervention program. Because the target area cannot be precisely defined,
we estimated that for every exposed person participating in the
intervention program, an additional 5, 10, or 15 nonexposed persons would
also participate.

Economic Analyses of Postattack Intervention

To analyze the economic factors involved in establishing an intervention
program, we compared the costs to the potential savings from such an
intervention. Following the recommendation of the Panel of
Cost-Effectiveness in Health and Medicine (PCEHM), we used estimates of
actual costs rather than financial charges or market prices, which usually
incorporate profit (15). We calculated the net savings (cost reductions) by
using the following formula: Net savings = (number of deaths averted x
present value of expected future earnings) + (number of days of
hospitalization averted x cost of hospitalization) + (number of outpatient
visits averted x cost of outpatient visits) - cost of intervention.

When we calculated the costs of hospitalization and outpatient visits, we
assumed that only persons with symptoms (i.e., case-patients) would use
medical facilities. The remainder of the exposed and potentially exposed
populace would receive postexposure prophylaxis.

Present Value of Expected Future Earnings

The cost of a premature human death was nominally valued at the present
value of expected future earnings and housekeeping services, weighted by
the age and sex composition of the work force in the United States (16).
The undiscounted average of future earnings is $1,688,595. As recommended
by PCEHM (17), the stream of future earnings was discounted at 3% and 5%,
to give values of $790,440 and $544,160, respectively. The present value of
expected future earnings was estimated with 1990 dollars, adjusted for a 1%
annual growth in productivity (16). However, in constant terms (1982
dollars), the average hourly earnings in private industry fell from $7.52
in 1990 to $7.40 in 1994 (18); therefore, the estimate of future earnings
was not adjusted upwards.

Cost of Hospitalization

In 1993, the average charge for a single day of hospitalization was $875
(19). To derive true cost, we multiplied the average charge by the
cost-to-charge ratio of 0.635, (the April 1994 statewide average
cost-to-charge ratio for urban hospitals in New York state) (16). On this
basis, we estimated true hospitalization costs at $556/day (Table 1).
Hospital costs included all professional services, drugs, x-rays, and
laboratory tests. Lost productivity during hospital stay was valued at
$65/day (the value of an "unspecified" day's earnings, weighted for age and
sex composition of the U.S. work force) (16).

Table 1. Costs of hospitalization and outpatient visits (OPVs) following a
bioterrorist attack
---------------------------------------------------------------------------
                                Anthrax       Tularemia      Brucellosis
                              Base   Upper   Base   Upper   Base    Upper
---------------------------------------------------------------------------
Hospitalized patient
  Days in hospital                 7      7     10      10      7        7
  Cost per day ($)(a)            556    669    556     669    556      669
  Lost productivity ($/day)       65     65     65      65     65       65
  Follow-up OPVs (no.)             2      2      2       2      7        7
  Cost 1st OPV ($)                28     44     28      44     28       44
  Cost other OPVs, ea. ($)        13     24     13      24     13       24
  OPV laboratory ($)(b,c)         87    174     87     174    131      261
  OPV x-rays costs ($)(d)         66     66      0       0      0        0
  Lost productivity
($/OPV)(e)                        16     16     16      16     16       16
Total costs ($)                4,541  5,380  6,338   7,582  4,584    5,587
Avg. costs/day ($/day)           649    769    634     758    655      798
% increase: Base to upper
estimate                                 18             20              22
Nonhospitalized patient
  Number of OPVs                   7      7     12      12     14       14
  Cost 1st OPV ($)                28     44     28      44     28       44
  Cost other OPVs, ea. ($)        13     24     13      24     13       24
  Lost productivity
($/OPV)(e)                        16     16     16      16     16       16
  Laboratory costs ($)(b,f)      131    174    261     522    261      522
  X-ray costs ($)(d)              66     66     66      66     66       66
  Drugs used(g)                    D      C    D+G     D+G    D+R    D+R+G
  Cost of drugs ($)                6    181     29      29    220      246
Total costs ($)                  422    810    722   1,120    972    1,418
Avg. costs/day ($/day)            60    116     60      93     69      101
% increase: Base to upper
estimate                                 93             55              46
---------------------------------------------------------------------------
Notes: All costs rounded to the nearest whole dollar.
(a)Hospital costs assumed to include all costs such as drugs, laboratory
tests, and x-rays.
(b)Laboratory tests consists of general health panel (CPT code 80050) and
an antigen or antibody test (modeled on the cost of a Streptococcus
screen, CPT code 86588).
(c)Follow-up OPVs for hospitalized patients included two laboratory test
sets for anthrax and tularemia patients and three laboratory test sets for
brucellosis patients.
(d)X-ray costs (CPT code 71021), included two sets taken at different
OPVs.
(e)Productivity lost due to an OPV was assumed to be one-quarter of an
unspecified day's value.
(f)For OPVs of nonhospitalized patients, one set of laboratory tests is
assumed for every two visits.
(g)Drugs used: D = doxycycline; C = ciprofloxacin; R = rifampin.
Sources: See text for explanation of sources of cost estimates.

Cost of Posthospitalization Outpatient Visits

After discharge from the hospital, a patient was assumed to have follow-up
outpatient visits, the number of which varied by disease (Table 1).
Outpatient visit costs were valued by using the Medicare National Average
Allowance (20), which was chosen to represent the equivalent of bulk
purchase discounted costs (i.e., actual costs) (Table 1). The first visit
has a Current Procedural Terminology (CPT) code of 99201, which is
classified as a "level 1" visit, requiring a physician to spend an average
of 10 minutes with a patient (20). Subsequent level 1 visits, with the
physician spending an average of 5 minutes with each patient, have a CPT
code of 99211 (20). During outpatient visits, a general health panel test
incorporating clinical chemistry tests and complete blood counts (CPT code
80050) and a single antigen or antibody detection test (e.g., CPT code
86558) were assumed to be ordered (20). Although data on Medicare
allowances for office visits and many other procedures were available, data
on Medicare allowances for laboratory tests were not. Thus, to establish
the costs of the tests, we arbitrarily divided the lowest allowable charge
for each test in half. X-rays (CPT code 71021) were valued according to the
Medicare National Average Allowance (Table 1). In terms of lost
productivity, we assumed that each outpatient visit cost the equivalent of
2 hours, or one-quarter, of the value of an unspecified day (16).

Cost of Outpatient Visits of Nonhospitalized Patients

For nonhospitalized outpatients, the cost of each visit, laboratory test,
x-ray, and lost productivity was the same as an outpatient visit for
discharged hospital patients and varied by disease (Table 1). We assumed
that one set of laboratory tests would be ordered every other visit and
that two sets of x-rays (CPT code 71021) would be ordered during the
therapeutic course. Drug costs are discussed below.

Cost of an Intervention

The costs of an intervention can be expressed as follows: Cost of
intervention = (cost of drugs used) x ([number of people exposed x
multiplication factor] - number killed - number hospitalized - number of
persons who require outpatient visits).

The intervention costs per person depend directly on the costs of the
antimicrobial agents and vaccines used in a prophylaxis program (Table 2).
We obtained drug prices from the 1996 Drug Topics Red Book and used the
lowest cost available for each drug (21). The cost of doxycycline ($0.22
per 200 mg total daily dose) was the Health Care Financing Administration
cost, whereas the cost of gentamicin ($3.76 per 160 mg total daily dose),
ciprofloxacin ($3.70 per 1,000 mg total daily dose), and rifampin ($5.01
per 900 mg total daily dose) were wholesale costs from pharmaceutical
companies. The cost of anthrax vaccine was $3.70 per dose (Helen
Miller-Scott, pers. comm., 1996). The cost of administering one vaccine
dose or gentamicin injection was estimated at $10.00, on the basis of the
1992 cost of administering a vaccine in a clinical setting (Valerie Kokor,
pers. comm., 1996). In estimating the cost of administering oral
antimicrobial agents, we assumed weekly visits, during which the drug would
be distributed and counseling would be given ($15.00 for the first visit
and $10.00 for each subsequent visit).

Table 2. Costs of prophylaxis following a bioterrorist attack
---------------------------------------------------------------------------
          Level of
        effectiveness           Anthrax      Tularemia      Brucellosis
---------------------------------------------------------------------------
Lower
Effectiveness (%)                       90             80               80
Drugs used(a)                       D or C              D              D+R
Cost of drugs ($)(b)              6 or 181              3              220
No. of visits(c)                         4              2                6
Total cost/
  person ($)                     51 or 226             28              285
Upper
Effectiveness (%)                       95             95               95

Drugs used(a)                       D+V or            D+G              D+G
                                       C+V
Cost of drugs ($)(b )            17 or 193             29               36
No. of visits(c)                         4              7               12
Total cost/
  person ($)                     62 or 238            104              161
Minimum No.
  participants(d)                  451,912        418,094          423,440
Maximum No.
  participants(e)                1,492,750      1,488,037        1,488,037
---------------------------------------------------------------------------
Notes: All costs are rounded to the nearest whole dollar.
(a)Drugs used: D = doxycycline; C = ciprofloxacin; V = anthrax vaccine; G
= gentamicin; R = rifampin.
(b)See text for explanation of drug costs.
(c)Cost of visit to drug-dispensing site: 1st visit = $15/person;
follow-up visits = $10/person/visit.
(d)Estimate assumed that the prophylaxis program was initiated on
postattack day 6 for anthrax and tularemia and postattack day 113 for
brucellosis, that the prophylaxis program had the lower effectiveness
level, and that the multiplication factor for unnecessary prophylaxis
given to unexposed persons was 5.
(e)Estimate assumed that prophylaxis was initiated on postattack day 0
(day of release), that prophylaxis had the upper effectiveness level, and
that the multiplication factor for unnecessary prophylaxis given to
unexposed persons was 15.

We assumed that more people would receive prophylaxis than were actually
exposed because of general anxiety and uncertainty about the boundaries of
the attack, the timing of the attack, and the time it would take
nonresidents to travel through the attack area. Three different
multiplication factors (5, 10, and 15) were used to construct within the
population. Finally, ongoing intelligence gathering would detect possible
bioterrorist threats. The cost of these prerequisite activities can be
calculated if they are seen as a form of insurance, the goal of which is to
"purchase" the maximum net savings through preparedness to manage the
consequences of an attack and reduce the probability of an attack. The
"actuarially fair premium" for the "insurance" can be defined as follows
(22): Actuarially fair premium = reduction of loss probability x value of
avoidable loss.

The term "reduction of loss probability" indicates that, although increased
surveillance and related activities can reduce the odds of an attack, they
cannot guarantee absolute protection. The term "avoidable loss" refers to
the fact that, even if a postexposure prophylaxis program were implemented
on the day of release (day zero), some deaths, hospitalizations, and
outpatient visits would be unavoidable.

Various reductions of attack probability illustrated the impact of these
estimates on the calculation of actuarially fair premiums. Such reductions
included reducing the probability from 1 in 100 years (0.01) to 1 in 1,000
years (0.001), a reduction of 0.009, and reducing a probability from 1 in a
100 years (0.01) to 1 in 10,000 years (0.0001), and from 1 in 100 years
(0.01) to 1 in 100,000 years (0.00001). The attack probability of 0.01 in
the absence of enhanced preventive actions was selected for illustrative
purposes and does not represent an official estimate.

A range of minimum and maximum values of avoidable loss was derived from
the net savings calculations. The values reflect differences in
effectiveness of the various prophylaxis regimens, the reduced impact of
delayed prophylaxis on illness and death, and the two discount rates used
to calculate the present value of earnings lost because of death.

Sensitivity Analyses

In addition to the scenarios discussed above, three sensitivity analyses
were conducted. First, the impact of increasing the cost of hospitalization
and outpatient visits was assessed by using a set of upper estimates (Table
1). The cost of a hospital day was increased to $669 by increasing the
cost-to-charge ratio from 0.634 to 0.764 (the ratio for Maryland) (16). The
costs of outpatient visits (first and follow-up) were increased by assuming
each visit was a "level 2" visit, doubling the average time a physician
spends with each patient. The alternative cost-of-intervention scenarios
that take into account persons who were not at risk but participated in the
prophylaxis program. Thus, if 100,000 people were exposed, we assumed that
the maximum number seeking prophylaxis was 500,000, 1,000,000, or
1,500,000.

Economic Analysis of Preparedness: Insurance

The analyses outlined above consider only the economics of an intervention
after an attack and include several assumptions: First, stockpiles of
drugs, vaccines, and other medical supplies would be available and could be
rapidly moved to points of need. Second, civil, military, and other
organizations would be in place and have the capability to rapidly identify
the agent, dispense drugs, treat patients, and keep order costs of
laboratory tests were increased to the full amount of the allowable charge
(20).

The second sensitivity analysis considered a reduced impact, in which only
10% of the original 100,000 target population were considered exposed. All
other estimates were held constant. The third sensitivity analysis
considered the threshold cost of an intervention, given differences due to
the effectiveness of various drug regimens, and discount rates used to
calculate the present value of expected lifetime earnings lost to a death.
The threshold cost occurs when net savings equal $0. Thus, the threshold
value represents the maximum that could be spent per person on an
intervention without having the intervention cost more than the loss from
no intervention.

Findings

Postattack Illness and Death

In our model, all three biologic agents would cause high rates of illness
and death. In the absence of an intervention program for the 100,000
persons exposed, the B. anthracis cloud would result in 50,000 cases of
inhalation anthrax, with 32,875 deaths; the F. tularensis cloud in 82,500
cases of pneumonic or typhoidal tularemia, with 6,188 deaths; and the B.
melitensis cloud in 82,500 cases of brucellosis requiring extended therapy,
with 413 deaths.

The speed with which a postattack intervention program can be effectively
implemented is critical to its success (Figure 1). For diseases with short
incubation periods such as anthrax and tularemia, a prophylaxis program
must be instituted within 72 hours of exposure to prevent the maximum
number of deaths, hospital days, and outpatient visits (Figure 1). Some
benefit, however, can be obtained even if prophylaxis is begun as late as
day 6 after exposure. The relative clinical efficacy of the intervention
regimen has a lesser but definite impact on observed illness and death
rates (Figure 1).

[Figure 1] [Figures not available in ASCII version]
Figure 1. Total deaths, hospital days, and outpatient visits associated
with aerosol releases of B. anthracis, B. melitensis, and F. tularensis by
the postattack day of prophylaxis initiation and level of prophylaxis
effectiveness.

A disease with a long incubation period such as brucellosis has a similar
pattern (Figure 1); an important difference is the time available to
implement an intervention program. Having more time available to implement
an intervention program can make a marked difference in its effectiveness.
However, the prolonged incubation period creates a greater potential for
panic in potentially exposed persons because of the uncertainty about their
health status.

Economic Analyses of Postattack Intervention: No Program

Without a postexposure prophylaxis program, an attack with B. anthracis is
far costlier than attacks with F. tularensis or B. melitensis (Table 3).
The differences between agents in medical costs as a percentage of total
estimated costs are due to the large differences in death rates attributed
to each agent (Figure 1).

Table 3. Costs(a)($ millions) of a bioterrorist attack with no postexposure
                            prophylaxis program
---------------------------------------------------------------------------
                             Anthrax       Tularemia        Brucellosis
---------------------------------------------------------------------------
Direct costs
  Medical: Base
  estimates(b)
    Hospital                      194.1           445.8              170.3
    OPV(c)                          2.0            10.5               48.9
  Medical: Upper
  estimates(d)
    Hospital                      237.1           543.3              211.7
    OPV(c)                          4.4            18.5               78.3
Lost productivity
  Illness(e)
    Hospital                       21.6            50.9               18.8
    OPV(c)                          0.7             3.9               15.0
Death
  3% discount(f)               25,985.7         4,891.2              326.5
  5% discount(f)               17,889.3         3,367.3              224.7
Total costs
  Base estimates
    3% discount(f)             26,204.1         5,402.4              579.4
    5% discount(f)             18,107.7         3,878.4              477.7
  Upper estimates
    3% discount(f)             26,249.7         5,507.9              650.1
    5% discount(f)             18,153.1         3,983.9              548.4
---------------------------------------------------------------------------
(a)Assuming 100,000 exposed.
(b)Medical costs are the costs of hospitalization (which include follow-up
outpatient visits) and outpatient visits (Table 1).
(c)OPV = outpatient visits.
(d)Upper estimates calculated with data in Table 1.
(e)Lost productivity due to illness is the value of time spent in hospital
and during OPVs (Table 1).
(f)Discount rate applied to calculate the present value of expected future
earnings and housekeeping services, weighted by age and sex composition of
the United States workforce (16), lost due to premature death.

Net Savings Due to a Postexposure Prophylaxis Program

If the postexposure prophylaxis program is initiated early, it reduces the
economic impact of all three diseases, especially anthrax (Figure 2).
Regardless of drug costs, the largest cost reductions are obtained through
a combination of the most effective prophylaxis regimen (i.e., 95%
effective, Table 2), the smallest multiplication factor to adjust for
persons who unnecessarily receive prophylaxis, and a 3% discount rate to
calculate the present value of the expected value of lifetime earnings.

In the case of anthrax, either doxycycline or ciprofloxacin could be used
in the intervention program (Table 2), but the use of doxycycline generated
the largest savings. The largest difference in net savings between the two
drugs was approximately $261.6 million. This difference occurred when it
was assumed that the program began on day zero (day of release), each drug
was used in combination with the anthrax vaccine, a 3% discount rate was
used, and a multiplication factor of 15 for unnecessary prophylaxis was
used. This amount is equal to approximately 1.2% of the maximum total net
savings generated by using a regimen of doxycycline plus the anthrax
vaccine.

Some scenarios, particularly those in which prophylaxis programs were
started late, generated negative net savings (i.e., net losses). In the
case of tularemia, at a 5% discount rate, net losses of $10.7 to $115.1
million occurred when a post-exposure program was delayed until day 6 after
exposure, and a prophylaxis regimen of doxycycline and gentamicin
(estimated 95% efficacy) was used. For the same scenario, but with a 3%
discount, a net savings of $1,513.3 million was observed when a
multiplication factor of five for unnecessary prophylaxis was used.
However, multiplication factors of 10 and 15 generated net losses of $49.8
and $102.0 million, respectively. With the same drug combination, beginning
the program 1 day earlier (day 5 after exposure) resulted in net savings in
all scenarios except when a multiplication factor of 15 and a discount rate
of 5% were used. Under the latter two assumptions, net savings result only
for prophylaxis initiated by day 4 after exposure.

In the case of brucellosis, the use of a doxycycline-rifampin regimen
(estimated 80% efficacy), a multiplication factor of 15 for unnecessary
prophylaxis, and a discount rate of either 3% or 5% generated net losses
regardless of when intervention began (Figure 2). The
doxycycline-gentamicin regimen (estimated 95% efficacy) generated net
losses only when it was assumed that the start of a program was delayed
until 113 or more days after exposure.

             [Figure 2] [Figures not available in ASCII version]
             Figure 2. Ranges(a) of net savings due to
             postattack prophylaxis by disease and day of
             prophylaxis program initiation.

             (a)Maximum savings (l) were calculated by
             assuming a 95% effectiveness prophylaxis regimen
             and a 3% discount rate in determining the present
             value of expected lifetime earnings lost due to
             premature death (16) and a multiplication factor
             of 5 to adjust for unnecessary prophylaxis.
             Minimum savings (n) were calculated by assuming
             an 80% to 90% effectiveness regimen and a 5%
             discount rate and a multiplication factor of 15.
             In tularemia prophylaxis programs initiated on
             days 4-7 postattack, the minimum savings were
             calculated by assuming a 95% prophylaxis regimen
             effectiveness rather than an effectiveness of 80%
             to 90%.

Preparedness: Insurance

The annual actuarially fair premium that can be justifiably spent on
intelligence gathering and other attack prevention measures increases with
the probability that a bioterrorist attack can be decreased by such
measures (Table 4). However, the potential net savings attributed to
reduced probability are minor compared with the potential net savings from
implementing a prophylaxis program. Depending on the level of protection
that can be achieved, the annual actuarially fair premium in an anthrax
scenario would be $3.2 million to $223.5 million (Table 4). The lower
premium would be justifiable for measures that could reduce the risk for an
attack from 0.01 to 0.001 and provide the ability to mount an intervention
program within 6 days of the attack. The higher premium would be
justifiable for measures that could reduce the risk from 0.01 to 0.00001
and allow immediate intervention if an attack occurred.

Table 4. The maximum annual actuarially fair premium(a) by reduction in
probability of event and size of avoided loss: Anthrax
---------------------------------------------------------------------------
                                             Actuarially fair annual
                                              premium ($ millions)
                                      -------------------------------------
       Days           Preventable        0.01      0.01          0.01
      post-               loss            tp        to            to
    attack(b)         ($millions)       0.001     0.0001       0.00001
---------------------------------------------------------------------------
Maximum loss estimate(c)
0                             22,370.5    201.3       221.5          223.5
1                             20,129.4    181.2       199.3          201.1
2                             15,881.5    142.9       157.2          158.7
3                              8,448.0     76.0        83.6           84.4
4                              4,200.1     37.8        41.6           42.0
5                              2,076.1     18.7        20.6           20.7
6                              1,013.8      9.1        10.0           10.1
Minimum loss estimate(d)
0                             14,372.4    128.9       141.8          143.1
1                             12,820.1    115.4       126.9          128.1
2                             10,049.1     90.4        99.5          100.4
3                              5,200.1     46.8        51.5           51.9
4                              2,429.7     21.9        24.1           24.3
5                              1,004.2      9.4        10.3           10.4
6                                351.2      3.2         3.5            3.5
---------------------------------------------------------------------------
(a)See text for definition.
(b)No. of days from attack to effective initiation of prophylaxis.
(c)Maximum loss preventable (potential net savings) occurs with the
doxycycline-anthrax vaccine prophylaxis regimen, a multiplication factor
of 5 for unnecessary prophylaxis, and a discount rate of 3% (Table 2).
(d)Minimum loss preventable (potential net savings) occurs with the
ciprofloxacin prophylaxis regimen, a multiplication factor of 15 for
unnecessary prophylaxis, and a discount rate of 5% (Table 2).

Sensitivity Analyses

The upper estimates of the cost of hospitalization increased average costs
per day by 18% to 22%, and upper estimates of the cost of outpatient visits
increased average costs per day by 46% to 93% (Table 1). However, the upper
estimates only increased medical costs by 1% to 6% of the total medical
costs associated with a bioterrorist attack (Table 3). The largest increase
was for brucellosis, for which upper estimates increased medical costs from
38% to 44% of total costs (Table 3).

When the number of persons infected during an attack was reduced tenfold,
the patient-related costs were reduced proportionately (Table 3). In most
cases, however, the net savings in total costs are less than 10% of the net
savings when 100% of the target population was presumed infected. The
shortfall in savings is caused by an increase in the number of unexposed
persons receiving prophylaxis. In the case of anthrax, when intervention
programs are initiated within 3 days of exposure, savings are 4.1% to 10%
of those in the original scenario (Figure 2). Delaying initiation of
prophylaxis until days 4, 5, or 6 after exposure, however, results in net
losses of $13.4 to $283.1 million. Losses occur regardless of prophylaxis
regimen, discount rate, or multiplication factor used to adjust for
unnecessary prophylaxis by unexposed persons.

In scenarios in which a multiplication factor of 15 was used to adjust for
unnecessary prophylaxis, the threshold value of intervention was always
above the prophylaxis cost for anthrax but not above the prophylaxis costs
for tularemia and brucellosis (Table 5). For tularemia, the threshold
intervention costs exceeded disease costs up to day 5 in the scenario with
95% effectiveness and a 5% discount, and for brucellosis, at all levels in
the scenarios with 80% effectiveness and up to day 56 in the scenarios with
95% effectiveness. This is consistent with the lower range of estimated net
savings (net losses) given in Figure 2. Reducing the number of unexposed
persons receiving prophylaxis increases the cost thresholds, making the
program cost beneficial. For example, changing the multiplication factors
for unnecessary prophylaxis to 5 and 10 increases the cost thresholds to
$659 and $319, respectively, for a brucellosis prophylaxis program
initiated 15 to 28 days after exposure, with a 5% discount rate. If a
discount rate of 3% is used instead of 5%, the cost thresholds increase to
$799 and $387. All these cost thresholds are above the estimated
prophylaxis cost of $285 per person for the doxycycline-rifampin regimen
and $161 per person for the doxycycline-gentamicin regimen (Table 2).

Table 5. Cost thresholds(a) of interventions ($/person) by day of
intervention initiation, prophylaxis effectiveness, and discount rates.
--------------------------------------------------------------------------
         Threshold costs for intervention ($/person, multiplication factor
                                     of 15(b))
         Anthrax               Tularemia               Brucellosis
Post-     Disc. rate(c)   Post-    Disc. rate   Post-       Disc. rate
attack                    attack                attack
day(d)      5%      3%     day     5%     3%     day       5%       3%
--------------------------------------------------------------------------
  90% effectiveness(e)    80% effectiveness(e)    80% effectiveness(e)
0           9,838  14,238       0 1,891  2,633      0-7     233*     282*
1           8,851  12,809       1 1,873  2,609     8-14     224*     272*
2           7,022  10,162       2 1,599  2,227    15-28     211*     255*
3           3,775   5,463       3   756  1,053    29-56     179*     217*
4           1,893   2,739       4   258    366   57-112      86*     104*
5             944   1,366       5    79    110     113+      24*      30*
6             468     677       6   20*     28
Prophylaxis
cost(c)              $226                  $28                       $285

  95% effectiveness(e)    95% effectiveness(e)     95% effectiveness(e)
0          10,370  15,007       0 2,229  3,104      0-7      274      333
1           9,359  13,544       1 2,207  3,074     8-14      264      320
2           7,427  10,948       2 1,898  2,644    15-28      248      301
3           3,995   5,782       3   898  1,251    29-56      211      256
4           2,004   2,900       4   328    457   57-112     102*     124*
5           1,000   1,447       5   93*    131     113+      29*      35*
6             496     718       6   23*    32*
Prophylaxis
cost(e)              $238                 $104                       $161
--------------------------------------------------------------------------
*Threshold value is below estimated cost of prophylaxis.
(a)Cost threshold is the point where cost of intervention and net savings
due to the intervention are equal.
(b)Multiplication factor to adjust for persons who participated in the
prophylaxis program but were unexposed.
(c)Applied to present value of expected future earnings and housekeeping
services (weighted average for age and sex).
(d)Postattack day on which prophylaxis was effecively implemented.
(e)See Table 2 for prophylaxis regimens assumed to give the stated levels
of effectiveness and cost/person of prophylaxis.

Conclusions

The economic impact of a bioterrorist attack can range from $477.7 million
per 100,000 persons exposed in the brucellosis scenario to $26.2 billion
per 100,000 persons exposed in the anthrax scenario (Table 3). These are
minimum estimates. In our analyses, we consistently used low estimates for
all factors directly affecting costs. The ID50 estimates for the three
agents are twofold to 50-fold higher than previously published estimates
(5,6,10,11), resulting in a possible understatement of attack rates. Also,
in our analyses we did not include a number of other factors (e.g.,
long-term human illness or animal illnesses) (Table 6) whose cumulative
effect would likely increase the economic impact of an attack.

Our model shows that early implementation of a prophylaxis program after an
attack is essential. Although the savings achieved by initiating a
prophylaxis program on any given day after exposure has a wide range, a
clear trend of markedly reduced savings is associated with delay in
starting prophylaxis (Figure 2). This trend was found in the analysis of
all three agents studied.

Table 6. Potential factors affecting the economic impact of a bioterrorist
attack
---------------------------------------------------------------------------
Potential                                     Relative
impact on                                    magnitude
Factor                                       net savings      of impact
---------------------------------------------------------------------------
Higher than projected                         Increase          ++++
  case-fatality rate
Long term illness (physical                   Increase           ++
  and psychological)
Decontamination and disposal                  Increase           ++
  of biohazardous waste
Disruptions in commerce                       Increase           ++
  (local, national, and
  international)
Animal illness and death                      Increase            +
Lower than projected                          Decrease          - - -
  effectiveness of prophylaxis
Adverse drug reactions due                    Decrease            -
  to prophylaxis
Postattack prophylaxis                        Decrease            -
  distribution costs, including
  crowd control and security
Training and other skill                      Decrease            -
  maintenance costs
Procurement and storage of                    Decrease            -
  antimicrobial drugs and
  vaccines before attack
Criminal investigations                       Variable           +/-
  and court costs
---------------------------------------------------------------------------

Delay in starting a prophylaxis program is the single most important factor
for increased losses (reduced net savings). This observation was supported
by the actuarially fair premium for preparedness analysis (Table 4).
Reductions in preventable loss due to early intervention had significantly
greater impact on the amount of an actuarially fair premium than reductions
in probability of an attack through intelligence gathering and related
activities.

Although implemented at different times in a threat-attack continuum, both
attack prevention measures and prophylaxis programs are forms of preventive
medicine. Attack prevention measures seek to prevent infection, while
prophylaxis programs prevent disease after infection has occurred.

Using an actuarially fair premium analogy in which cost and benefit are
required to be equal, we find that the incremental rate of increasing
prevention effectiveness (the marginal increase) declines rapidly as
probability reduction targets go from 0.001 to 0.0001 to 0.00001. Because
the loss probability is decreasing on a logarithmic scale, the potential
increment in marginal benefit drops comparably, resulting in ever smaller
increments in the protection above the preceding base level.

Conversely, delaying a prophylaxis program for anthrax, a disease with a
short incubation period and a high death rate, increases the risk for loss
in a manner akin to a semilogarithmic scale. Arithmetic increases in
response time buy disproportionate increases in benefit (prevented losses.)
The potential for reducing loss is great because an attack is assumed, thus
increasing the actuarially fair premium available to prepare for and
implement a rapid response.

Large differences between prophylaxis costs and the threshold costs for
most scenarios, particularly if prophylaxis is early (Table 5), suggest
that the estimates of savings from prophylaxis programs are robust. Even
with large increases in prophylaxis cost, net savings would still be
achieved.

The ability to rapidly identify persons at risk would also have significant
impact on costs. For example, the threshold costs for brucellosis
prophylaxis are often lower than intervention costs when the ratio of
unexposed to exposed persons in the prophylaxis program is 15:1 (Table 5).
This finding provides an economic rationale for preparedness to rapidly and
accurately identify the population at risk and reduce unnecessary
prophylaxis costs.

The maximum amount of the annual actuarially fair premium varies directly
with the level of risk reduction and the rapidity of postattack response
(Table 4). The calculated amount of actuarially fair premiums, however,
should be considered a lower bound estimate. A higher estimate (called the
certainty equivalent) can also be calculated; however, this requires the
determination of a social welfare function (22), and such complexity is
beyond the scope of this study.

Our model provides an economic rationale for preparedness measures to both
reduce the probability of an attack and increase the capability to rapidly
respond in the event of an attack. The larger portion of this preparedness
budget (insurance premium) should be allocated to measures that enhance
rapid response to an attack. These measures would include developing and
maintaining laboratory capabilities for both clinical diagnostic testing
and environmental sampling, developing and maintaining drug stockpiles, and
developing and practicing response plans at the local level. These measures
should be developed with a value-added approach. For example, the
laboratory capability could be used for other public health activities in
addition to preparedness, and drugs nearing their potency expiration date
could be used in government-funded health care programs. However, these
secondary uses should not undermine the preparedness program's
effectiveness.

Arnold Kaufmann is a retired Public Health Service officer, formerly
assigned to the National Center for Infectious Diseases.

Address for correspondence: Martin I. Meltzer, Mail Stop C-12, National
Center for Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, GA 30333; fax: 404-639-3039; e-mail: qzm4@cdc.gov.

References

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  4. Benenson AS, editor. Control of communicable diseases manual. 16th ed.
     Washington (DC): American Public Health Association, 1995.
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  6. Kaufmann AF, Fox MD, Boyce JM, Anderson DC, Potter ME, Martone WJ, et
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  7. Olle-Goig JE, Canela-Soler J. An outbreak of Brucella melitensis
     infection by airborne transmission among laboratory workers. Am J
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  8. Staszkiewicz J, Lewis CM, Colville J, Zervos M, Band J. Outbreak of
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  9. Trever RW, Cluff LE, Peeler RN, Bennett IL. Brucellosis I.
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     al. Doxycycline-rifampin versus doxycycline-streptomycin in treatment
     of human brucellosis due to Brucella melitensis. Antimicrob Agents
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     Siegel JE, Russell LB, Weinstein MC, editors. Cost-effectiveness in
     health and medicine. New York: Oxford University Press, 1966:214-35.
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---------------------------------------------------------------------------


   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Synopses

Hantaviruses: A Global Disease Problem

Connie Schmaljohn* and Brian Hjelle
*United States Army Medical Research Institute of Infectious Diseases, Fort
Detrick, Frederick, Maryland, USA; and University of New Mexico,
Albuquerque, New Mexico, USA
---------------------------------------------------------------------------


      Hantaviruses are carried by numerous rodent species throughout
      the world. In 1993, a previously unknown group of hantaviruses
      emerged in the United States as the cause of an acute
      respiratory disease now termed hantavirus pulmonary syndrome
      (HPS). Before then, hantaviruses were known as the etiologic
      agents of hemorrhagic fever with renal syndrome, a disease that
      occurs almost entirely in the Eastern Hemisphere. Since the
      discovery of the HPS-causing hantaviruses, intense
      investigation of the ecology and epidemiology of hantaviruses
      has led to the discovery of many other novel hantaviruses.
      Their ubiquity and potential for causing severe human illness
      make these viruses an important public health concern; we
      reviewed the distribution, ecology, disease potential, and
      genetic spectrum.

The genus Hantavirus, family Bunyaviridae, comprises at least 14 viruses,
including those that cause hemorrhagic fever with renal syndrome (HFRS) and
hantavirus pulmonary syndrome (HPS) (Table 1). Several tentative members of
the genus are known, and others will surely emerge as their natural ecology
is further explored. Hantaviruses are primarily rodent-borne, although
other animal species harboring hantaviruses have been reported. Unlike all
other viruses in the family, hantaviruses are not transmitted by arthropod
vectors but (most frequently) from inhalation of virus-contaminated
aerosols of rodent excreta (1). Human-to-human transmission of hantaviruses
has not been documented, except as noted below.

 Table 1. Members of the genus Hantavirus, family Bunyaviridae
 --------------------------------------------------------------------------

     Species      Disease    Principal     Distribution   Distribution of
                             Reservoir       of Virus        Reservoir
 --------------------------------------------------------------------------
 Hantaan (HTN)    HFRS(a) Apodemus         China,       C Europe south to
                          agrarius         Russia,      Thrace, Caucasus,
                          (striped field   Korea        & Tien Shan Mtns;
                          mouse)                        Amur River through
                                                        Korea to E Xizang
                                                        & E Yunnan, W
                                                        Sichuan, Fujiau, &
                                                        Taiwan(China)
 Dobrava-Belgrade HFRS    Apodemus         Balkans      England & Wales,
 (DOB)                    flavicollis                   from NW Spain,
                          (yellow-neck                  France, S
                          mouse)                        Scandinavia
                                                        through European
                                                        Russia to Urals, S
                                                        Italy, the
                                                        Balkans, Syria,
                                                        Lebanon, & Israel
 Seoul (SEO)      HFRS    Rattus           Worldwide    Worldwide
                          norvegicus
                          (Norway rat)
 Puumala (PUU)    HFRS    Clethrionomys    Europe,      W Palearctic from
                          glareolus        Russia,      France and
                          (bank vole)      Scandinavia  Scandinavia to
                                                        Lake Baikai, south
                                                        to N Spain, N
                                                        Italy, Balkans,W
                                                        Turkey, N
                                                        Kazakhstan, Altai
                                                        & Sayan Mtns;
                                                        Britain & SW
                                                        Ireland
 Thailand (THAI)  nd(b)   Bandicota        Thailand     Sri Lanka,
                          indica                        peninsular India
                          (bandicoot rat)               to Nepal, Burma,
                                                        NE India, S China,
                                                        Laos, Taiwan,
                                                        Thailand, Vietnam
 Prospect Hill    nd      Microtus         U.S., Canada C Alaska to
 (PH)                     pennsylvanicus                Labrador,
                          (meadow vole)                 including
                                                        Newfoundland &
                                                        Prince Edward
                                                        Island, Canada;
                                                        Rocky Mountains to
                                                        N New Mexico, in
                                                        Great Plains to N
                                                        Kansas, & in
                                                        Appalachians to N
                                                        Georgia, U.S.
 Khabarovsk (KHB) nd      Microtus fortis  Russia       Transbaikalia Amur
                          (reed vole)                   region; E China
 Thottapalayam    nd      Suncus murinus   India        Afghanistan,
 (TPM)                    (musk shrew)                  Pakistan, India,
                                                        Sri Lanka, Nepal,
                                                        Bhutan, Burma,
                                                        China, Taiwan,
                                                        Japan, Indomalayan
                                                        Region
 Tula (TUL)       nd      Microtus         Europe       Throughout Europe
                          arvalis                       to Black Sea & NE
                          (European                     to Kirov region,
                          common                        Russia
                          vole)
 Sin Nombre (SN)  HPS(c)  Peromyscus       U.S.,        Alaska Panhandle
                          maniculatus      Canada,      across N Mexico
                          (deer mouse)                  Canada, south
                                                        through most of
                                                        continental U.S.,
                                                        excluding SE & E
                                                        seaboard, to
                                                        southernmost Baja
                                                        California Sur and
                                                        to NC Oaxaca,
                                                        Mexico
 New York (NY)    HPS     Peromyscus       U.S.         C and E U.S. to S
                          leucopus                      Alberta & S
                          (white-footed                 Ontario, Quebec &
                          mouse)                        Nova Scotia,
                                                        Canada; to N
                                                        Durango & along
                                                        Caribbean coast to
                                                        Isthmus of
                                                        Tehuantepec &
                                                        Yucatan Peninsula,
                                                        Mexico
 Black Creek      HPS     Sigmodon         U.S.         SE U.S., from S
 Canal                    hispidus                      Nebraska to C
 (BCC)                    (cotton rat)                  Virginia south to
                                                        SE Arizona &
                                                        peninsular
                                                        Florida; interior
                                                        & E Mexico through
                                                        Middle America to
                                                        C Panama; in South
                                                        Amer ica to N
                                                        Colombia & N
                                                        Venezuela
 El Moro Canyon   nd      Reithrodontomys  U.S., Mexico British Columbia &
 (ELMC)(d)                                              SE Alberta,
                          megalotis                     Canada; W and NC
                          (Western                      U.S., S to N Baja
                          harvest mouse)                California &
                                                        interior Mexico to
                                                        central Oaxaca
 Bayou (BAY)(d)   HPS     Oryzomys         U.S.         SE Kansas to E
                          palustris                     Texas, eastward to
                          (rice rat)                    S New Jersey &
                                                        peninsular Florida
 --------------------------------------------------------------------------
 Probable species:(e)
 --------------------------------------------------------------------------

 Topografov (TOP) nd      Lemmus           Siberia      Palearctic, from
                          sibiricus                     White Sea, W
                          (Siberian                     Russia, to
                          lemming)                      Chukotski
                                                        Peninsula, NE
                                                        Siberia, &
                                                        Kamchatka;
                                                        Nearctic, from W
                                                        Alaska E to Baffin
                                                        Island & Hudson
                                                        Bay, S Rocky Mtns
                                                        to C B.C., Canada
 Andes (AND)(d)   HPS     Oligoryzomys     Argentina    NC to S Andes,
                          longicaudatus(f)              approximately to
                                                        50 deg S latitude,
                          (long-tailed                  in Chile &
                          pygmy                         Argentina
                          rice rat)

 To be named(d)   HPS     Calomys laucha   Paraguay     N Argentina &
                          vesper mouse                  Uruguay, SE
                                                        Bolivia, W
                                                        Paraguay, and WC
                                                        Brazil
 Isla Vista       nd      Microtus         U.S.         Pacific coast,
 (ISLA)(d)                californicus                  from SW Oregon
                          (California                   through
                          vole)                         California, U.S.,
                                                        to N Baja
                                                        California, Mexico
 Bloodland Lake   nd      Microtus         U.S.         N & C Great
 (BLL)(d)                 ochrogaster                   Plains, EC Alberta
                          (prairie vole)                to S Manitoba,
                                                        Canada, S to N
                                                        Oklahoma &
                                                        Arkansas, E to C
                                                        Tennessee & W West
                                                        Virginia, U.S.;
                                                        relic populations
                                                        elsewhere in U.S.
                                                        & Mexico
 Muleshoe         nd      Sigmodon         U.S.         See Black Creek
 (MUL)(d)                 hipidus                       Canal
                          (cotton rat)
 Rio Segundo      nd      Reithrodontomys  Costa Rica   S Tamaulipas & WC
 (RIOS)(d)                                              Michoacan, Mexico,
                          mexicanus                     S through Middle
                          (Mexican                      American highlands
                          harvest mouse)                to W Panama; Andes
                                                        of W Colombia & N
                                                        Ecuador
 Rio Mamore       nd      Oligoryzomys     Bolivia      C Brazil south of
 (RIOM)(d)                microtis                      Rios Solimoes-
                          (small-eared                  Amazon &
                          pygmy                         contiguous low
                          rice rat)                     lands of Peru,
                                                        Bolivia, Paraguay,
                                                        & Argentina.
 --------------------------------------------------------------------------
 (a)HFRS, hemorrhagic fever with renal syndrome
 (b)nd, none documented
 (c)HPS, hantavirus pulmonary syndrome
 (d) not yet isolated in cell culture
 (e) viruses for which incomplete characterization is available, but for
 which there is clear evidence indicating that they are unique
 (f) suspected host, but not confirmed
 Adapted from (57,72) and from (9,13,23,38,42,43,50-71)

The recognition of a previously unknown group of hantaviruses as the cause
of HPS in 1993 is an example of virus emergence due to environmental
factors favoring of the natural reservoir; a larger reservoir increases
opportunities for human infection. We reviewed the global distribution of
hantaviruses, their potential to cause disease, and their relationships to
each other and to their rodent hosts.

History of HFRS and HPS

"Hemorrhagic fever with renal syndrome" denotes a group of clinically
similar illnesses that occur throughout the Eurasian landmass and adjoining
areas (2,3). HFRS includes diseases previously known as Korean hemorrhagic
fever, epidemic hemorrhagic fever, and nephropathia epidemica (4). Although
these diseases were recognized in Asia perhaps for centuries, HFRS first
came to the attention of western physicians when approximately 3,200 cases
occurred from 1951 to 1954 among United Nations forces in Korea (2,5).
Other outbreaks of what is believed to have been HFRS were reported in
Russia in 1913 and 1932, among Japanese troops in Manchuria in 1932 (2,6),
and in Sweden in 1934 (7,8). In the early 1940s, a viral etiology for HFRS
was suggested by Russian and Japanese investigators who injected persons
with filtered urine or serum from patients with naturally acquired disease
(2). These studies also provided the first clues to the natural reservoir
of hantaviruses: the Japanese investigators claimed to produce disease in
humans by injecting bacteria-free filtrates of tissues from Apodemus
agrarius or mites that fed on the Apodemus mice. Mite transmission was
never conclusively demonstrated by other investigators, and it was not
until 1978 that a rodent reservoir for HFRS-causing viruses was confirmed
by investigators who demonstrated that patient sera reacted with antigen in
lung sections of wild-caught Apodemus agrarius and that the virus could be
passed from rodent to rodent (9). The successful propagation of Hantaan
(HTN) virus in cell culture in 1981 provided the first opportunity to study
this pathogen systematically (10). The history of HFRS has been explored
(2,11,12).

HPS was first described in 1993 when a cluster of cases of adult fatal
respiratory distress of unknown origin occurred in the Four Corners region
of the United States (New Mexico, Arizona, Colorado, and Utah). The
unexpected finding that sera from patients reacted with hantaviral antigens
was quickly followed by the genetic identification of a novel hantavirus in
patients' tissues and in rodents trapped near patients' homes (13-15).

Prevalence and Clinical Course

Approximately 150,000 to 200,000 cases of HFRS involving hospitalization
are reported each year throughout the world, with more than half in China
(16). Russia and Korea also report hundreds to thousands of HFRS cases each
year. Most remaining cases (hundreds per year) are found in Japan, Finland,
Sweden, Bulgaria, Greece, Hungary, France, and the Balkan countries
formerly constituting Yugoslavia (16). Depending in part on which
hantavirus is responsible for the illness, HFRS can appear as a mild,
moderate, or severe disease (Table 2). Death rates range from less than
0.1% for HFRS caused by Puumala (PUU) virus to approximately 5% to 10% for
HFRS caused by HTN virus (16). The clinical course of severe HFRS involves
five overlapping stages: febrile, hypotensive, oliguric, diuretic, and
convalescent; it is not uncommon, however, for one or more of these stages
to be inapparent or absent. The onset of the disease is usually sudden with
intense headache, backache, fever, and chills. Hemorrhage, if it occurs, is
manifested during the febrile phase as a flushing of the face or injection
of the conjunctiva and mucous membranes. A petechial rash may also appear,
commonly on the palate and axillary skin folds. Sudden and extreme
albuminuria, around day 4, is characteristic of severe HFRS. As the febrile
stage ends, hypotension can abruptly develop and last for hours or days,
during which nausea and vomiting are common. One-third of deaths occur
during this phase because of vascular leakage and acute shock. Almost half
of all deaths occur during the subsequent (oliguric) phase because of
hypervolemia. Patients who survive and progress to the diuretic phase show
improved renal function but may still die of shock or pulmonary
complications. The final (convalescent) phase can last weeks to months
before recovery is complete (3,5,12).

 Table 2. Distinguishing clinical characteristics for HFRS and HPS
 --------------------------------------------------------------------------
           Disease             Pathogens  Distinguishing Characteristics*
 --------------------------------------------------------------------------
 HFRS (moderate-severe)      HTN, SEO,    hemorrhage +++
 Death rate                  DOB          azotemia/
 1%-15%                                   proteinuria +++/++++
                                          pulmonary capillary leak +/++
                                          myositis +/+++
                                          conjunctival injection ++/++++
                                          eye pain/myopia ++/++++
 HFRS (mild)                 PUU          hemorrhage +
 Death rate <1%                           azotemia/
                                          proteinuria +/++++
                                          pulmonary capillary leak -/+
                                          myositis +
                                          conjunctival injection +
                                          eye pain/myopia ++/++++
 HPS (prototype)             SN, NY       hemorrhage +
 Death rate >40%                          zotemia/
                                          proteinuria +
                                          pulmonary capillary leak ++++
                                          myositis -
                                          conjunctival injection -/+
                                          eye pain/myopia -
 HPS (renal                  BAY, BCC,    hemorrhage +
 variant)                    Andes        azotemia/
 Death rate>40%                           proteinuria ++/+++
                                          pulmonary capillary leak
                                          +++/++++
                                          myositis ++/++++
                                          conjunctival injection -/++
                                          eye pain/myopia -
 --------------------------------------------------------------------------
 *Minimum/maximum occurrence of the characteristic: - rarely reported;
 + infrequent or mild manifestation; ++, +++, ++++ more frequent and severe
 manifestation.

More than 250 cases of HPS have been reported throughout North and South
America. Although the disease has many features (e.g., a febrile prodrome,
thrombocytopenia, and leukocytosis) in common with HFRS (Table 2), in HPS
capillary leakage is localized exclusively in the lungs, rather than in the
retroperitoneal space, and the kidneys are largely unaffected. Most of the
174 cases of HPS in the United States and Canada have been caused by Sin
Nombre (SN) virus. In HPS, death occurs from shock and cardiac
complications, even with adequate tissue oxygenation. Cases of HPS in the
southeastern United States, as well as many in South America, are caused by
a newly recognized clade (a group that shares a common ancestor) of viruses
that includes Bayou (BAY), Black Creek Canal (BCC), and Andes viruses. As
with HFRS, clinical differences can be observed among patients with HPS
caused by different hantaviruses. For example, although HPS due to SN virus
infection can sometimes be associated with renal insufficiency after
prolonged hypoperfusion, renal impairment is only rarely observed early in
disease, and chemical evidence of skeletal muscle inflammation (increased
serum levels of the muscle enzyme creatine kinase) is rare (17). In
contrast, both renal insufficiency and elevated creatine kinase levels are
observed at much higher frequency, although not universally, with Andes,
BAY, and BCC virus infections (18-20; J. Davis, J. Cortes, and C. Barclay,
pers. comm.). In an outbreak of HPS recently described in Paraguay, a novel
hantavirus, carried by Calomys laucha, was identified as the etiologic
agent (21). The relationship of this virus to other HPS-causing
hantaviruses remains to be established.

Ecology and Epidemiology

Hantavirus infection is apparently not deleterious to its rodent reservoir
host and is associated with a brisk antibody response against the virion
envelope and core proteins and chronic, probably lifelong infection. In
natural populations, most infections occur through age-dependent horizontal
route(s). The highest antibody prevalence is observed in large (mature)
animals. A striking male predilection for hantavirus infection is observed
in some rodent species such as harvest mice and deer mice, but not in urban
rats (Rattus norvegicus) (22-24). Horizontal transmission among cage-mates
was experimentally demonstrated (25), but vertical transmission from dam to
pup is negligible or absent both in wild and experimental settings
(22,24,25).

Outbreaks of hantaviral disease have been associated with changes in rodent
population densities, which may vary greatly across time, both seasonally
and from year to year. Cycles respond to such extrinsic factors as
interspecific competition, climatic changes, and predation. Spring and
summer outbreaks of HFRS in agricultural settings in Asia and Europe are
linked to human contact with field rodents through the planting and
harvesting of crops (16,26). PUU outbreaks in Scandinavia and the HPS
outbreak in the Four Corners region of the United States were associated
with natural rodent population increases, followed by invasion of buildings
by rodents (27,28). The ecologic events that led to 1994 and 1996 outbreaks
of Andes virus-HPS in Patagonia, a region in southern South America, are
being investigated. Human interventions, such as the introduction of Old
World plant species (e.g., rosas mosquetas and Scottish brougham) to
Patagonia, with associated alteration in rodent population dynamics, have
been suggested as possible factors. Recent fires and a mild winter in
Argentina's Rio Negro and Chubut Provinces may also have had a positive
effect on the carrier rodent, the colilargo, Oligoryzomys longicaudatus (M.
Christie and O. Pearson, pers. comm.).

Although the aerosol route of infection is undoubtedly the most common
means of transmission among rodents and to humans, virus transmission by
bite may occur among certain rodents (29) and may also occasionally result
in human infection (30) (often inside a closed space, such as a
rodent-infested grain silo, garage, or outbuilding used for food storage).
Epidemiologic investigations have linked virus exposure to such activities
as heavy farm work, threshing, sleeping on the ground, and military
exercises. Indoor exposure was linked to invasion of homes by field rodents
during cold weather or to nesting of rodents in or near dwellings (16,31).
Genetic sequencing of rodent- and patient-associated viruses has been used
to pinpoint the precise locations of human infections, which has supported
the role of indoor exposure in hantavirus transmission (32,33). Many
hantavirus infections have occurred in persons of lower socioeconomic
status because poorer housing conditions and agricultural activities favor
closer contact between humans and rodents. However, suburbanization,
wilderness camping, and other outdoor recreational activities have spread
infection to persons of middle and upper incomes.

Nosocomial transmission of hantaviruses has not been documented until very
recently (34) and must be regarded as rare. However, viruses have been
isolated from blood and urine of HFRS patients, so exposure to bodily
fluids of infected persons could result in secondary transmission. Only
rarely have multiple North American HPS cases been associated with
particular households or buildings. During recent outbreaks of HPS in South
America, however, clustering of cases in households and among personal
contacts appeared to be more common (M. Christie, pers. comm.). During a
recent outbreak of Andes-virus-associated HPS in Patagonia, a Buenos Aires
physician apparently contracted the infection after minimal exposure to
infected patient blood (34; D.A. Pirola, pers. comm.). An adolescent
patient in Buenos Aires apparently contracted hantavirus infection from her
parents, who were infected in Patagonia. This unprecedented observation of
apparent person-to-person spread of a hantavirus clearly requires
laboratory confirmation, especially by careful comparative analysis of the
viral sequences (32,33).

Hantaviruses have also caused several laboratory-associated outbreaks of
HFRS. Laboratory-acquired infections were traced to persistently infected
rats obtained from breeders (35-37), to wild-caught, naturally infected
rodents (38-40), or to experimentally infected rodents (39). No illnesses
due to laboratory infections have been reported among workers using
cell-culture adapted viruses, although asymptomatic seroconversions have
been documented (40).

Hantavirus Distribution and Disease-causing Potential

The worldwide distribution of rodents known to harbor hantaviruses (Table
1) suggests great disease-causing potential. Each hantavirus appears to
have a single predominant natural reservoir. With rare exception, the
phylogenetic interrelationships among the viruses and those of their
predominant host show remarkable concordance (Figure; 41). These
observations suggest that hantaviruses do not adapt readily to new hosts
and that they are closely adapted for success in their host, possibly
because of thousands of years of coexistence. As many as three hantaviruses
can be found in a particular geographic site, each circulating in its own
rodent reservoir, with no apparent evolutionary influence on one another
(42).

[Figure 1] [Figures not available in ASCII version]
Figure. Phylogeny of hantaviruses and their relationships to natural
reservoirs. The trees were constructed by comparing the complete coding
regions of the S segments of hantaviruses or of 330 nucleotides
corresponding to those of the M segment of Hantaan virus (strain 76118)
from nucleotides 1987 to 2315. Abrreviations for viruses are as in Table
1. For each analysis, a single most parsimonious tree was derived by using
PAUP 3.1.1 software. For the S segment tree, boostrap values resulting
from 100 replications were all greater than 87% except for the branch
leading to BCC (78%) and the branch leading to DOB (52%). The next most
common placing of DOB was on a branch with HTN.

All known hantaviruses, except Thotta-palayam (TPM) virus, have been
isolated or detected in murid rodents. Because only one isolate of TPM
virus was made from a shrew (Order Insectivora), it is not clear if Suncus
is the true primary reservoir or an example of a "spillover" host, i.e., a
secondary host infected through contact with the primary host. Spillover is
common in sympatric murid rodents, including those identified as the
predominant carrier of another hantavirus; thus, the opportunity for
genetic exchange among hantaviruses is present in nature. Spillover hosts
are believed to have little or no impact on hantaviral distribution or
associated disease. However, rodents other than the primary reservoirs can
play an important carrier role. For example, Microtus rossiaemeri-dionalis
may play a role in maintenance of Tula virus in some settings (43), and
Peromyscus leucopus and Peromyscus boylii can be important reservoirs for
SN virus in the western United States (T. Yates and B. Hjelle, unpub.
data). Apparent spillover may also be the result of laboratory errors such
as polymerase chain reaction (PCR) contamination or misidentification of
rodent species. However, spillover is probably under-appreciated in many
studies that rely on reverse transcriptase PCR for identifying specific
viruses because many primer pairs may not detect an unexpected spillover
virus. In either case, because mistaken identities and cell culture
contaminations with other hantaviruses have occasionally been reported,
investigators should verify unusual findings to prevent further confusion.

Antigenic and Genetic Diversity among Hantaviruses

Hantaviruses have been characterized by a combination of antigenic and
genetic methods. For viruses propagated in cell culture, the
plaque-reduction neutralization test is the most sensitive serologic assay
for differentiation (44,45); nine hantaviruses have been defined by this
test: HTN, Seoul (SEO), PUU, Prospect Hill, Dobrava-Belgrade (DOB),
Thailand, TPM, SN, and BCC viruses (44-48). Genetic relationships among
hantaviruses are mirrored in their antigenic properties. A direct
correlation between genetic and antigenic relationships is difficult;
however, it can be assumed that the plaque-reduction neutralization test
measures differences in the M segment gene products, i.e., the G1 and G2
envelope glycoproteins. Comparing the deduced G1 and G2 amino acid
sequences, therefore, may provide clues to the antigenic as well as genetic
diversity among hantaviruses.

Of characterized hantavirus isolates, SEO virus is the most genetically
homogeneous. Isolates of SEO virus, regardless of their geographic origin,
display M segment nucleotide and deduced amino acid sequence homologies of
approximately 95%, and 99%, respectively (41,47). A reported exception, the
R22 isolate from China, had a slightly lower homology; however, the
original data suggest that an error in the nucleotide sequence, resulting
in a frame shift reading error, may account for almost all of the
additional changes. PUU virus isolates vary the most, with M segment
nucleotide and amino acid sequence homologies of 83% and 94% observed
between a Finnish and Russian isolate. HTN virus also appears to be quite
stable in nature. Comparing the M segment sequences of prototype HTN virus
(from Apodemus) and those of two human isolates obtained at a 6-year
interval from HFRS patients in Korea produced nucleotide and deduced amino
acid sequence homologies of 94% and 97%, respectively (48). For SN virus,
comparing the complete M or S segment sequences of three strains from
California or New Mexico produced homologies of 87% to 99%. Partial
nucleotide sequence comparisons of the M or S segments of SN viruses from
adjacent counties in California, detected in deer mice captured 19 years
apart, were 97.5% homologous (49). Similarly, a retrospective analysis of
archived tissue samples collected in Mono County, California, in 1983
showed viruses with partial M and S segment nucleotide sequence homologies
of about 87% with SN from an 1993 HPS patient from New Mexico (50). In all
cases, the amino acid sequences encoded by these genes differed between
cognate proteins by much less than 5%. These values are similar to those
observed among strains of HTN virus. Studies have just begun to appear in
which the nature of quasispecies in natural rodent hosts is defined
(43,51). Such investigations should provide more definitive data concerning
the genetic diversity among hantaviruses in nature.

Evolution of Hantaviruses

Phylogenetic trees derived by comparing complete or partial S (Figure), M,
or L segment nucleotide sequences (41,52,53) show two major lineages of
hantaviruses, one leading to HTN, SEO, Thailand, and DOB viruses, and one
leading to PUU, Prospect Hill, SN, and other New World hantaviruses. TPM
virus, the first hantavirus isolated in cell culture (54), may be the most
antigenically and genetically disparate member of the genus; however,
comparison of the complete nucleotide sequence of the TPM S segment (A.
Toney, B. Meyer, C. Schmaljohn, unpub. data) suggests that TPM virus is
more closely related to HTN, SEO, and DOB viruses than to any of the other
viruses in the genus (Figure). Nucleotide sequence homologies of the M and
S segments of any two hantaviruses have approximately the same degree of
divergence between each of the three segments, which suggests similar
evolutionary rates for these two gene segments. A slightly higher homology
among L segments sequenced to date perhaps indicates a greater need for
conservation of either RNA or protein functions (47). Point mutations
appear to account for most of the genetic drift among hantaviruses.
Recombination has not been reported for hantaviruses, although segment
reassortment within a particular species appears common (52,55). The
exchange of gene segments is suggested to be nonrandom, with a higher
propensity for M segment swapping, than for S or L (55). Whether it
contributes to the pathogenesis of hantaviruses is not known, but
reassortment certainly provides an avenue for more rapid accumulation of
changes than could occur by point mutation. There is no evidence that
reassortment can occur between different species of hantaviruses; however,
this has not been studied systematically.



Murid rodents have probably harbored inapparent hantavirus infections for
thousands, perhaps millions of years. It is likely that the genus
Hantavirus evolved in the Old World and that viruses were carried by
rodents across the Bering land bridge when they migrated during the
Oligocene, and into South America in the Pliocene (71). Humans are
incidental hosts, the victims of spillover infections from the natural host
rodents. One of the two major forms of hantaviral diseases is endemic in
each hemisphere. Both HFRS and HPS can be divided into distinct clinical
subtypes, and the viral strain is a key determinant of the severity and
nature of the clinical abnormalities. Not covered in this review are
clinical studies of HFRS and HPS patients, which suggest that pathogenesis
may be immunologic and may be mediated by cytokine responses (72). New
outbreaks with novel hantavirus strains are still being uncovered,
especially in South America. However, the largest clinical caseload and
largest number of deaths occur in Asia and Europe.

Dr. Schmaljohn is chief, Department of Molecular Virology, USAMRID. Current
research interests include the development of molecular vaccines for 
hantaviruses, filoviruses, and flaviviruses.
Dr. Hjelle has been active in studies of the molecular biology, evolution,
epidemiology, and clinical aspects of hantavirus disease. His laboratory is
a reference diagnostic center for hantavirus infections of humans and
animals and has recently received funding to develop innovative vaccine
strategies against HPS and other emerging viral diseases.

Address for correspondence: Connie Schmaljohn, Virology Division, USAMRID,
Fort Detrick, Frederick, MD 21702-5011; fax: 301-619-2439; e-mail:
cschmaljohn@detrick.army.mil

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]
Synopses

Japanese Spotted Fever: Report of 31 Cases and Review of the Literature

Fumihiko Mahara
Mahara Hospital, Tokushima, Japan
---------------------------------------------------------------------------

This article is also available in Japanese. The Japanese version can be
accessed at http://www.cdc.gov/ncidod/EID/eid.htm

      Spotted fever group (SFG) rickettsioses, which are transmitted
      by ticks, were long thought not to exist in Japan. Three
      clinical cases of Japanese spotted fever (JSF) were first
      reported in 1984. The causative agent was isolated and named
      Rickettsia japonica. Through October 1996, 31 cases were
      diagnosed as JSF in Tokushima Prefecture. Infected patients
      typically had acute high fever, headache, and characteristic
      exanthema; eschar was observed in 90%. After the discovery of
      JSF, more than a hundred cases were reported in southwestern
      and central Japan. Recent surveys show ticks to be the most
      probable vectors. As an emerging infectious disease, JSF is not
      commonly recognized by clinicians; therefore, even though it
      has not caused fatal cases, it merits careful monitoring.

The spotted fever group (SFG) rickettsioses, which are transmitted by
ticks, have a worldwide distribution. Japanese spotted fever (JSF) is one
of the newcomers of this group (1); the first clinical cases were reported
in 1984 (2). The causative agent was isolated and named Rickettsia japonica
(3). Because outbreaks were sporadic and limited, clinical reports
concerning JSF, especially from specialists in dermatology and physiology
and from general practitioners, were scarce. JSF was first found in
Tokushima Prefecture, on the island of Shikoku in southwestern Japan;
Tsutsugamushi disease, an important rickettsiosis in Japan, was found there
soon afterwards (4). Through October 1996, 31 clinical cases of JSF and 11
cases of Tsutsugamushi disease were diagnosed at Mahara Hospital, Tokushima
Prefecture. During the same period, 45 cases of human tick bites were
recorded in this JSF-endemic area in the same hospital. This article
describes JSF's history, clinical characteristics, and differences from
Tsutsugamushi disease and summarizes current information about the
epidemiology, vectors, and causative agent of JSF.

History

In the 1980s, clinicians believed that Tsutsugamushi disease (scrub typhus)
was the only rickettsial disease in Japan except for sporadic outbreaks of
epidemic typhus in the 1950s. In Tokushima Prefecture, neither disease has
been reported in the last two decades. In May 1984, a 63-year-old woman
(the wife of a farmer) was hospitalized at Mahara Hospital with high fever
and erythematous nonpruritic skin eruptions over the entire body.
Antibiotics (-lactam and aminoglycoside) used for common febrile
infections were not effective, but the patient gradually became afebrile in
2 weeks without effective treatment. In May and July 1984, two additional
patients with similar symptoms were treated at the same hospital.
Doxycycline was markedly effective in these cases. Before the onset of
illness, the patients had collected shoots from bamboo plantations on the
same mountain. In two of the patients, an eschar was observed.
Tsutsugamushi disease was suspected. However, results of Weil-Felix tests
showed positive OX2 serum agglutinins, and OXK were negative in all three
cases. These results did not indicate Tsutsugamushi disease, but rather
OX2-positive infections, i.e., SFG rickettsioses (1).

The cases were subsequently confirmed by complement fixation test with
antigens of SFG rickettsiae (5,6). The name Japanese spotted fever was
proposed for these infections (7) and has been commonly used since then
(8-10). Oriental spotted fever (11) is a synonym for JSF. The causative
agent was isolated in 1986 (12) and named R. japonica (3).

Clinical Features

The clinical features of the 31 patients whose illness was diagnosed as JSF
at Mahara Hospital from 1984 to October 1996 were analyzed. The disease
developed abruptly, with the common symptoms of headache (25 [80%] of 31
patients), fever (31 [100%] of patients), and shaking chills (27 [87%]).
Other major objective symptoms of JSF included skin eruptions (31 [100%])
and tick bite eschars (28 [90%]). Most patients (28 [90%]) complained of
malaise; joint and muscle pain or numbness of the extremities was rarely
mentioned. In the acute stage, remittent fever accompanied by shaking
chills was frequently observed. In severe cases, high fever (40C or more)
continued for several days (Figure 1). The maximum body temperature was
38.5C to 40.8C (mean 39.5C), which was higher than that seen in patients
with Tsutsugamushi disease (38.5C ~ 39.1C). With abrupt high fever or, a
few days after onset, fever of unknown origin, the characteristic erythemas
developed on the extremities and spread rapidly (in a few hours) to all
parts of the body including palms and soles, without accompanying pain or
itching. These eruptions were the size of a grain of rice or soybean, and
the margin of each of the spots was unclear (Figure 2). The erythemas
became remarkable during the febrile period and tended to spread more over
the extremities than the trunk. Palmar erythema, a characteristic finding
not seen in Tsutsugamushi disease, disappeared in the early stage of the
disease. The erythemas became petechial after 3 to 4 days, peaked in a week
or 10 days, and disappeared in 2 weeks. However, in severe petechial cases,
the brown pigmentation remained for 2 months or more. Eschar was observed
on the hands, feet, neck, trunk, and shoulders of patients (Figure 3). This
eschar generally remained for 1 to 2 weeks, but in some cases it
disappeared in a few days. Eschars in JSF patients are smaller than those
seen in patients with Tsutsugamushi disease and may be missed without
careful observation. Regional or generalized lymphadenopathy, which is
observed in almost all cases of Tsutsugamushi disease, was not remarkable
in JSF patients. Swelling of the liver and spleen was observed in a few
patients. One patient had cardiomegaly (5), and in another area, a patient
had central nervous system involvement (13).

[Figure 1] [Figures not available in ASCII version]
Figure 1. Fever and clinical course, 62-year-old woman.

   [Figure 2] [Figures not available in ASCII version]
   Figure 2. Skin eruptions, hospital day 3.

[Figure 3] [Figures not available in ASCII version]
Figure 3. Small and shallow eschar on admission, which disappeared in a
few days.

Laboratory Examinations

The results of laboratory examinations of JSF patients are almost the same
as those of patients with common SFG rickettsioses. During clinical
examinations at the initial stage of the disease, urinalyses registered a
slight positive reading for protein and occult blood, which may lead to a
misdiagnosis of urinary infection. In the acute stage, leukocytosis may
also be found with leukopenia (3,600~12,800), and a left shift in leukocyte
count was observed. Thrombocytopenia (6.8~35.3) may also be found. In week
1 to 2, leukocyte counts increased slightly, and lymphocyte counts tended
to increase. Among biochemical examinations, C-reactive proteins were
strongly positive, and liver functions were slightly impaired but returned
to normal in 2 to 3 weeks.

Serologic Results

Serodiagnosis for JSF is usually performed by the indirect immunoperoxidase
(IP) or immunofluorescence (IF) techniques, with antigens prepared from R.
japonica or other SFG rickettsiae. With the IP test, IgG and IgM antibodies
were detected in the sera beginning on day 9 after the onset of fever;
titers of IgG antibodies were higher than those of IgM antibodies (14). The
IF test had similar results (15). In the 31 clinically diagnosed cases of
JSF in Tokushima Prefecture at Mahara Hospital, all patients had
significant changes in serum IP antibody titers to R. japonica, and 27
(87%) had significant changes in OX2 agglutinin titers by the Weil-Felix
technique (10,14; F. Mahara, unpub. data).

Treatment

Antibiotics such as penicillins, -lactams, or aminoglycosides, commonly
used in the empiric treatment of febrile disease, were completely
ineffective, but doxycycline and minocycline were markedly effective in
treating the JSF patients (16).

On the first day of hospitalization in one patient with severe disease
(Figure 1), fever of more than 40C with shaking chills persisted after the
antibiotics cefazolin and fosfomycin were administered. The general
condition of the patient worsened and on the morning of the third day,
generalized edema and confusion developed. On day 3, the patient was given
drip infusion of doxycycline 300 mg per day, which was dramatically
effective; the fever decreased to 38C during the first drip infusion.

In an in vitro study, minocycline was the most effective antibiotic against
R. japonica, followed by other tetracycline antibiotics (17,18). In
contrast, the sensitivity to -lactam and penicillin was lower or
negligible, but quinolones were effective against the JSF agent (18). Three
patients were treated with a new quinolone (tosufloxacin 300 mg per day in
three divided doses per os), which proved effective in two cases (9).
Patients with dehydration received drip infusion of doxycycline or
minocycline (200 mg to 300 mg per day for 3 to 7 days), and after becoming
afebrile, received 200 mg per day 2 divided doses to prevent relapse.
Patients suspected of having JSF should receive empiric therapy with
minocycline or doxycycline without waiting for serologic confirmation of
illness.

Epidemiology

From 1984 to 1995, 144 cases of JSF were reported by the National
Institutes of Health in Japan (19; Figure 4). Case reports included only
the number of cases and prefectures where cases were reported, including
Tokushima Prefecture. According to this information, JSF-endemic
prefectures are located along the coast of southwestern and central Japan
in a warm climate (Figure 5). The landscape is diverse, including bamboo
plantations, crop fields, coastal hills, and forests (20). Of the 31 JSF
patients in Tokushima Prefecture, nine were male, and 22 were female. Ages
were 4 to 78 years, but most patients (68%) were 60 to 70 years old. The
onset of the disease was 2 to 8 days after work in the fields. In this
prefecture, farmers work in the forest to gather bamboo shoots in the
spring and chestnuts in the autumn; cases occurred from April to October
(6,18; Figure 6). In this area, we can distinguish seasonal differences in
the occurrence of JSF and Tsutsugamushi disease; whereas JSF occurs from
spring to autumn, Tsutsugamushi disease occurs in winter
(November-February). Although seasonal differences occur in the western
parts of Japan, the prevalent seasons for Tsutsugamushi disease vary in
other parts of Japan (21).

          [Figure 4] [Figures not available in ASCII version]
          Figure 4. The number of Japanese spotted fever patients
          in Japan (1984-1995).

              [Figure 5] [Figures not available in ASCII version]
              Figure 5. Geographic distributions of JSF and
              Tsutsugamushi disease in Japan.
             [Figure 6] [Figures not available in ASCII version]
             Figure 6. Seasonal prevalence of Japanese spotted
             fever and Tsutsugamushi disease.

Like other SFG rickettsioses, JSF is presumed to be transmitted by a tick
bite. The high proportion of patients with tick bite eschars supports this
hypothesis. Thirteen JSF patients (28.9%) recalled tick bites before the
onset of illness; however, the ticks had been lost, and no specimens from
the patients were available for further study. From 1984 to October 1996,
45 persons with tick bites were recorded at Mahara Hospital (Table 1).
Identified ticks included three genera and eight species.

      Table 1. Cases of human tick bites, Mahara Hospital, 1984-1996
      ---------------------------------------------------------------
                                    Female Male  Nymph Larva Total
      ---------------------------------------------------------------
      Haemaphysalis flava           1            4           5
      Haemaphysalis longicornis     8      1     3     2     14
      Amblyomma testdunarium        4      1     12          17
      Ixodes ovatus                 1                        1
      Ixodes nipponensis            4                        4
      Ixodes persulcatus                         2           2
      Ixodes tanuki                 1                        1
      Haemaphysalis kitaokai        1                        1
      Totals                        20     2     21    2     45
      ---------------------------------------------------------------

Three genera and six species of ticks have been reported as positive for R.
japonica in JSF-endemic areas (Table 2). Hemolymph samples from Dermacentor
taiwanensis, Haemaphysalis flava, Haemaphysalis formosensis, Haemaphysalis
hystricis, Haemaphysalis longicornis, and Ixodes ovatus were positive when
tested by the IP technique using a species-specific monoclonal antibody
against R. japonica (22). R. japonica was also detected by IF in the
hemolymph of H. longicornis (23). A polymerase chain reaction (PCR)
technique using species-specific primers detected R. japonica in H.
hystricis (24), H. flava, and I. ovatus (25). The agent has also been
detected in H. longicornis by restriction fragment length polymorphism of
PCR product (23). Of these, H. flava, H. longicornis, and I. ovatus
commonly feed on humans in Japan (26). Recent tick surveys in Japan have
identified two serotypes or species of SFG rickettsial isolates other than
R. japonica, which are of uncertain clinical significance (27,28).

Table 2. Reported tick species positive for Rickettsia japonica and their
prevalence in endemic disease areas
--------------------------------------------------------------------------
                                     No. positive/no. examined
                           ------------------------------------------------
       Tick species           Tokushima          Kochi        Kanagawa
--------------------------------------------------------------------------
Dermacentor taiwanensis    3/5(a), 1/1(b)   -               -
Haemaphysalis flava        6/36(a)          3/10(a)         1-3/90(c)
Haemaphysalis formosensis  -                10/16(a)        -
Haemaphysalis hystricis    +(d)             5/9(a)          -
Haemaphysalis longicornis  -                1/1(a), 6/9(e), -
                           5-33/33(f)
Ixodes ovatus              1/13(a)          -               1/16(c)
---------------------------------------------------------------------------
(a)Hemolymph test by immunoperoxidase stain using R. japonica-specific
monoclonal antibody (22).
(b)Isolation (28).
(c)Polymerase chain reaction (PCR) using the primers designed for
amplifying the genomic DNA from only R. japonica (25).
(d)PCR method same as the above (24). Number was not shown.
(e)Hemolymph test by immunoflurescein stain using R. japonica-specific
monoclonal antibody (23).
(f)Restriction fragment length polymorphism of PCR product (23).

Pathogen

The etiologic agent was first isolated from a patient (in Kochi Prefecture)
in 1986 (12). In 1987, the causative rickettsia was also isolated from a
JSF patient in Tokushima Prefecture (29-31). The former isolate is the type
strain YH (ATCC VR-1363), later named R. japonica, a new SFG rickettsia;
the latter strain (Katayama) was the first isolate from JSF-endemic areas
outside Kochi (3,32). The Katayama strain type and R. japonica were
demonstrated by serologic analysis using monoclonal antibodies (33).
Serotyping by use of the reciprocal crossreactions of mouse antisera to six
human isolates from Tokushima and the type strain YH or R. japonica also
indicated that these are the same species (34). In 1988, another strain was
isolated from a patient in Awaji Island, Hyogo Prefecture, which is
considered a new area of JSF-endemic disease (35).

Recently, an isolate from a febrile patient in Wakayama Prefecture was also
reported as R. japonica (36). In an electron microscopy study, R. japonica
were generally recognized as short rods or pleomorphic coccobacillary forms
less than 2 mm in length and 0.5 mm in diameter and could be found not only
in the cytoplasms but also in the nuclei of the host cells (37). A
multilayered mesosome-like structure was observed in the rickettsiae
multiplying in a host cell (38). This unique structure has not been
reported in other species except in Rickettsia prowazekii (39). After the
initial isolation, at least 20 rickettsial strains have been isolated from
JSF patients by cell culture techniques or nude mouse passage in Tokushima,
Kochi, Hyogo, Chiba, and Wakayama Prefectures. However, it has not been
determined if their strains differ in virulence.

Ten diseases caused by SFG rickettsiae have been reported in humans (40):
Rocky Mountain spotted fever, Mediterranean spotted fever, Siberian tick
typhus, African tick bite fever, Queensland tick typhus, Japanese spotted
fever, Israeli spotted fever, Astrakhan spotted fever, Flinders Island
spotted fever, and rickettsialpox. The clinical symptoms of JSFa triad of
high fever, skin eruptions, and tick bite escharare similar to those of
typical SFG rickettsioses. With regard to skin eruptions, eschar, and
severity of the disease, JSF is more akin to Mediterranean spotted fever
and Siberian tick typhus than to Rocky Mountain spotted fever. Recent tick
surveys indicated that the most probable vectors of JSF are H. flava, H.
longicornis, and I. ovatus.

In Japan, the clinical features of JSF are similar to those of
Tsutsugamushi disease; however, close clinical observation exposes the
differences between the two diseases. Widespread outbreaks of Tsutsugamushi
disease have been reported repeatedly in recent years (21). No fatal cases
of JSF have been reported. However, death rates from other SFG
rickettsioses (approximately 2.5% for Mediterranean spotted fever [41] and
3% to 7% from Rocky Mountain spotted fever [42]) suggest that unless JSF is
treated appropriately, it can pose the same risk. If JSF is suspected,
empiric treatment should begin without delay during the early stages of
disease.

Acknowledgments

The author thanks Professor Emeritus Suto T., Department of Microbiology,
Akita University, for serologic confirmation; Professor Jan Kazar, Slovak
Academy of Sciences, for his helpful advice; Drs. Fujita H., Ohara Research
Laboratory and Takada N., Fukui Medical School, for epidemiologic studies;
and all colleagues who collaborated in the investigations.

Dr. Mahara is chief director of Mahara Hospital, Tokushima, Japan. He
serves as a counselor of the Japanese Association for Infectious Diseases
and is a member of the International Eurasian Academy of Sciences.

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     based on reactivities to monoclonal antibodies. Jpn J Med Sci Biol
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     serological identification of causative rickettsiae from Japanese
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     patient with spotted fever group rickettsiosis in Japan on Awaji
     Island, Hyogo. J Jpn Assoc Infect Dis 1990;64:413-8.
 36. Fujita H. Isolation of Rickettsia japonica from a febrile
     patient-Wakayama. Infectious Agents Surveillance Report 1995;16:30.
 37. Iwamasa K, Okada T, Tange Y, Kobayashi Y. Ultra-structural study of
     the response of cells infected in vitro with causative agent of
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 38. Amano K, Hatakeyama H, Sasaki Y, Ito R, Tamura A, Suto T. Electron
     microscopic studies on the in vitro proliferation of spotted fever
     group rickettsia isolated in Japan. Microbiol Immunol 1991;35:623-9.
 39. Silverman DJ, Wisseman CL, Jr. Comparative ultra-structural study on
     the cell envelopes of Rickettsia prowazekii, Rickettsia rickettsii,
     and Rickettsia tsutsugamushi. Infect Immun 1978;21:1020-3.
 40. Beati L, Raoult D. Spotted Fever Rickettsiae. Kazar J, Toman R,
     editors. Proceedings of the Vth International Symposium on Rickettsiae
     and Rickettsial Diseases. Publishing House of the Slovak Academy of
     Science, Bratislava, Slovak 1996;134-69.
 41. Raoult D, Weiller PJ, Chagnon A, Chaudet H, Gallais H, Casanova P.
     Mediterranean spotted fever. Am J Trop Med Hyg 1986;35:845-50.
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---------------------------------------------------------------------------

   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Synopses

Polycystic Kidney Disease: An Unrecognized Emerging Infectious Disease?

Marcia A. Miller-Hjelle,* J. Thomas Hjelle,* Monica Jones,* William R.
Mayberry, Mary Ann Dombrink-Kurtzman, Stephen W. Peterson, Deborah M.
Nowak,* and Frank S. Darras*
*University of Illinois College of Medicine at Peoria, Peoria, Illinois,
USA; East Tennessee State University, Johnson City, Tennessee, USA; and
U.S. Department of Agriculture, Agricultural Research Service, Peoria,
Illinois, USA
---------------------------------------------------------------------------


      Polycystic kidney disease (PKD) is one of the most common
      genetic diseases in humans. We contend that it may be an
      emerging infectious disease and/or microbial toxicosis in a
      vulnerable human subpopulation. Use of a differential
      activation protocol for the Limulus amebocyte lysate (LAL)
      assay showed bacterial endotoxin and fungal (1[-]3)--D-glucans
      in cyst fluids from human kidneys with PKD. Fatty acid analysis
      of cyst fluid confirmed the presence of 3-hydroxy fatty acids
      characteristic of endotoxin. Tissue and cyst fluid from three
      PKD patients were examined for fungal components. Serologic
      tests showed Fusarium, Aspergillus, and Candida antigens. IgE,
      but not IgG, reactive with Fusarium and Candida were also
      detected in cyst fluid. Fungal DNA was detected in kidney
      tissue and cyst fluid from these three PKD patients, but not in
      healthy human kidney tissue. We examine the intertwined nature
      of the actions of endotoxin and fungal components, sphingolipid
      biology in PKD, the structure of PKD gene products, infections,
      and integrity of gut function to establish a mechanistic
      hypothesis for microbial provocation of human cystic disease.
      Proof of this hypothesis will require identification of the
      microbes and microbial components involved and multifaceted
      studies of PKD cell biology.

Examining the hypothesis that polycystic kidney disease (PKD) is an
emerging infectious disease and/or microbial toxicosis in a vulnerable
population of humans must begin with a review of the conceptual tools that
relate disease etiology and progression to the identification of microbes,
their cellular components, and shed toxins in affected persons (1). An
emerging infectious disease can be defined, in part, as an existing disease
for which microbes are newly recognized as a causative factor and/or as a
factor contributing to disease progression. The microbe may be 1) present
at the site of a lesion, 2) disseminated throughout the body, or 3)
localized to an anatomic site separate from the primary lesions. In this
third case, one or more toxins released by the microbe into distribution
fluids, usually blood, produce the pathologic effect(s); this process
resembles microbial toxicosis, where the patient is exposed only to the
toxin(s) present in the diet, environment, and gut microflora.
Distinguishing between an infectious disease and microbial toxicosis is
essential if the source of the toxin is to be removed or reduced.
Concurrent infection and microbial toxicosis (e.g., endotoxicosis,
mycotoxicosis) can also occur. Although mycotoxicosis is commonly
understood to refer to the absorption of small organic fungal toxins, such
as aflatoxin, fumonisin, or trichothecene, detection of toxin in body
fluids and tissues is often difficult. Exposure to toxin may be episodic.
For many known and newly discovered microbial toxins, analytical methods
pertinent to the levels of toxins involved in acute and chronic disease,
especially in vulnerable subpopulations, need to be developed and verified
in human tissues and fluid. We have taken the view that the presence of
signature components of microbial genera/species that produce toxins may
indicate that all components of the microbe, including its shed toxins, are
also present. As a corollary, absorption of the detectable microbial
components, which are often larger in molecular size than the classical
mycotoxins and endotoxins, should presume the absorption of other
components and toxins from this organism.

On its face, PKD would not appear to be an emerging infectious disease.
Classically viewed as an inherited disease that follows Mendelian genetics,
PKD in its autosomal dominant form affects 600,000 people in the United
States at a rate of 1 in 400-1,000 persons; the autosomal recessive form
occurs in 1 per 44,000 births. An acquired form of PKD occurs in patients
on renal dialysis. Animal models of inherited and chemically induced PKD
have been described (2-4). The current consensus is that fundamental
anomalies in cell differentiation or maturation explain the array of
anomalies in cystic renal epithelium. Although cysts can sometimes be
detected in utero, loss of kidney function to the point of endstage renal
disease occurs by the sixth decade in 50% of autosomal-dominant PKD
patients. Despite substantial progress in molecular genetics studies of
PKD, the role of defective gene products in its causation and progression
to end-stage renal disease has remained elusive (3,5,6). Indeed, additional
factors have been proposed as being necessary for progression of PKD to
end-stage renal disease. PKD patients have higher rates of renal and
urinary tract infections and higher rates of illness and death from
infection in general than healthy persons (7,8). Since these infections in
otherwise-healthy-persons do not lead to PKD, PKD patients could be
exhibiting heightened vulnerability to infection and sensitivity to
microbial cell components and toxins. How such sensitivity and
vulnerability might occur is unknown. The disease does not appear to
involve an immunocompromised state. Thus, alternative possibilities need to
be considered, such as altered colonization of the uroepithelium and
altered bowel structure and colonization.

Microbes and Microbial Components

Werder et al. (9) performed perhaps the critical experiment in establishing
a pathogenic role for microbes in PKD. Using genetically cystic mice, they
found that mice grown under germ-free conditions survived substantially
longer than cystic littermates grown under ambient conditions.
Gardner et al. (10,11) extended these observations when they reported that
rats fed a chemical cystogen also had lower rates of renal cystogenesis
when raised under germ-free conditions; moreover, coadministering the
ubiquitous cell wall component of gram-negative bacteria,
lipopolysaccharides (LPS), and the chemical cystogen produced higher rates
of cyst formation than using cystogen alone. Thus, LPS was considered a
provocateur of the underlying genetic (or chemically induced)
susceptibility to renal cystogenesis. From this line of research has
emerged the possibility that LPS, either alone or in combination with other
microbial or environmental factors, promote disease progression, and
removal of such provocateurs would slow the progression of PKD to end-state
renal disease.

Endotoxin is the mixture of LPS and other bacterial cell wall proteins and
constituents that is found in bacterial infections (12). LPS is isolated by
solvent extraction of bacterial cell walls; its chemical purity may vary
across preparations. The portion of LPS responsible for most of its known
biological activities is Lipid A; the carbohydrate side chains also elicit
responses in humans (13). The chemical composition of LPS depends on the
genera and species of bacteria from which it is isolated (13,14). As
knowledge of the structure and biological activities of LPS has increased
and merged with the infectious disease vocabulary based on endotoxin, these
terms are sometimes used interchangeably. For example, the highly purified
LPS used as positive reference material in the Limulus amebocyte lysate
(LAL) assay is called Control Standard Endotoxin. Even though interchanging
the terms LPS and endotoxin may promote appreciation of microbial
involvement in disease, we prefer the term endotoxin in discussing disease
because it respects the structural and pharmacologic heterogeneity of LPS
and mixtures of other microbial components present in vivo.

When Miller et al. (15) measured putative endotoxin levels by the classic
LAL assay in culture-negative urine samples of male PKD patients with no
clinically apparent infection, 80% contained detectable LAL-positive
material, while the urine samples of healthy male volunteers did not. In
cyst fluid from PKD patients, Gardner et al. (16) reported LAL-positive
material and cytokines typically induced by LPS. The origins of the renal
and urinary endotoxin were not known; possible sources include the gut
microflora, occult urogenital infections, cryptic colonizations, and
abnormal handling by the liver of the normal daily load of absorbed
endotoxin. Enhanced exposure to endotoxin in the PKD patient is important
since endotoxin plays a fundamental role in human health and disease. In
low doses, endotoxin enhances the immune system, but in larger amounts it
is lethal. In combination with drugs and other substances, it can cause
tissue damage and severe disease; examples of endotoxin synergy with other
substances resulting in disease include alcohol in cirrhosis, aspirin in
Reyes syndrome, and trichothecene T-2 toxin (a mycotoxin from Fusarium) in
gastrointestinal tract damage (17). Endotoxin can also promote the
translocation of bacteria from the gut into the blood (18). In susceptible
persons, chronic exposure to endotoxin has been linked to rheumatoid
arthritis (19); tentative links with atherosclerosis have also been
reported (20). Although suggestive of endotoxin in PKD, the LAL methods
used above were not specific for endotoxin, since they were susceptible to
false positive and/or negative results.

Bacterial Endotoxin in Human PKD Renal Cysts

In this report, we describe our recent findings concerning 1) endotoxin
levels recorded after recent improvements in LAL methods, 2) analysis of
fatty acids specific for bacterial endotoxin, and 3) the presence of other
microbial components in human cyst fluid. The LAL assay (Figure 1) is the
classic method used to detect the low levels of endotoxin in biologic
fluids and pharmaceuticals.

Both endotoxin (in eight of eight patients) and (1[-]3)--D-glucans (in two
of eight patients) were detected when methods based on differential
activation of the two Limulus pathways were applied to cyst fluids from six
autosomal-dominant PKD patients, one autosomal-recessive PKD patient, and a
patient with simple cysts, where 200 ml from a single simple cyst were
recovered (Table 1). None of these patients exhibited clinical evidence of
infection, had been on hemodialysis, or received human immunologic products
before nephrectomy, which could have accounted for -D-glucan reactivity
(26). For six patients (four autosomal dominant, one autosomal recessive,
and one with simple cysts), only endotoxin was detected in cyst fluids; gel
clot end points for both the LAL and LAL+laminarin assays were equal for
each fluid tested, indicating that the -D-glucan stimulated pathway was
not activated. When exposed to polymyxin B, LAL-positive cyst fluids became
negative. This is consistent with the conformational changes that occur
when this antibiotic binds to Lipid A to form a stoichiometric complex that
is inactive in the assay (25). In contrast, kidney tissue from one male
patient (donor 4) and one female patient (donor 5) yielded cyst fluids that
contained separately endotoxin or -D-glucan; however, no single fluid
contained both substances (Table 1). Fluids that were LAL positive in the
presence or absence of polymyxin B, but negative in the presence of
laminarin are consistent with the detection of -D-glucan. Donor 6 had been
on peritoneal dialysis for slightly more than 1 year before nephrectomy.
Although cyst fluids were all initially LAL negative, when the incubation
time of the LAL assay was increased from 1 to 2 hours, 4 of 13 cyst fluids
were endotoxin positive, albeit at much lower concentrations than the
fluids from patients not on dialysis. None of the cyst fluids from donor 6
were positive for -D-glucan after longer incubations. Longer incubation
times might also have produced LAL-positive material in LAL-negative cyst
fluids from the other donors; therefore, we view the percentage of
LAL-positive cyst fluids for each patient as a minimum value.

            [Figure 1] [Figures not available in ASCII version]
            Figure 1. Cascade of biochemical interactions and
            reactions leading to gel clot formation in the
            Limulus amebocyte lysate assay. Endotoxin binding
            to Factor C via the Lipid A moiety results in its
            activation and in turn the sequential activation of
            Factor B, which results in the subsequent
            activation of the proclotting enzyme. Binding of
            polymyxin B to endotoxin blocks Limulus reactivity
            in those samples where endotoxin is the initiating
            molecule. In contrast, (1[-]3)--D-glucans bind to
            a different component, Factor G, in the Limulus
            assay reagent, thereby leading to its activation
            and subsequent cascade of reactions to gel
            formation. The addition of laminarin, an inhibitor
            of glucan binding to Factor G, to Limulus assay
            reagent blocks reactivity of samples containing
            glucan (21).

            Lipid A binds to and activates Factor C resulting
            in a cascade of enzymatic reactions leading to gel
            clot formation, the positive endpoint (22). Also
            present in the classic LAL reagents is Factor G,
            which is responsible for a side cascade (i.e., the
            alternative pathway). Factor G is stimulated by (1
            [-]3)--D-glucans, associated primarily with fungal
            cell walls (23). Addition of laminarin, an
            inhibitor of glucan binding to Factor G (24),
            permits a more specific measurement of
            endotoxin-induced gel clot formation and provides a
            means of screening for (1[-]3)--D-glucans in
            biological samples, when used in a differential
            activation protocol with other inhibitors and
            activators. As two internal controls, polymyxin B
            can block the LAL reactivity of endotoxin (25), and
            known quantities of Control Standard Endotoxin can
            be added to duplicate samples to detect inhibition
            of the assay.

      Table 1. Limulus assay results from human kidney cyst fluids
      ---------------------------------------------------------------
                                   Cysts ET positive
                                          (%):       Cysts -D-glucan
       Donor    Patient  No. Cysts     Endotoxin      positive (%):
        No.      data      Tested     units/ml(a)         pg/ml
      ---------------------------------------------------------------

         1    AD(b) 45 yr5         3 (60%): 0.48     nd(c)
              F                    EU/ml

         2    AD 52 yr F 6         4 (67%): 1.71     nd
                                   EU/ml

         3    AD 48 yr M L(d) 9    5 (56%): 1.45     nd
                                   EU/ml

                         R(e) 11   6 (55%): 2.27     nd
                                   EU/ml

         4    AD 41 yr F 16        3 (19%): 0.88     3 (19%): 120
                                   EU/ml             pg/ml

         5    AD 37 yr M 11        2 (18%): 3.84     6 (55%): 106
                                   EU/ml             pg/ml

         6    PD(f) AD 4313        4 (31%): 0.26     nd
              yr F                 EU/ml

         7    AR(g) 5 wk pool(8)   3.84 EU/ml        nd
              M

         8    SC(h) 48 yr1(200ml)  3.84 EU/ml        nd
              F
      ---------------------------------------------------------------
      (a)Mean EU/ml or pg (1[-]3)D-glucan/ml
      (b)Autosomal-dominantpolycystic kidney disease (PKD)
      (c)Not detected
      (d)Left kidney
      (e)Right kidney
      (f)Peritoneal dialysis
      (g)Autosomal recessive PKD
      (h)Simple kidney cyst (not PKD)
      Kidneys were from seven patients who had received or were
      awaiting renal allografts. Cyst fluid was obtained ex vivo from
      both surface (cortex) and sub-surface (medullary) cysts by
      aseptic aspiration from seven excised kidneys
      (post-nephrectomy) of PKD patients; simple cyst fluid was
      recovered by radiologically guided needle aspiration. All
      kidneys and cyst fluids were collected under an approved
      Institutional Review Board protocol. The mean number of cysts
      aspirated per kidney was 10 (range 1-21); two kidneys were
      obtained from one of the patients with both being used for
      studies. Fluid from individual cysts in each kidney were
      collected separately, except for the autosomalrecessive PKD
      patient in whom cyst volumes were too small (~ 0.1 ml) for
      individual testing. Therefore, fluid from eight cysts in close
      proximity were pooled. All procedures used pyrogen-free
      materials in the collection, handling, and processing of
      kidneys and cyst fluids and preparation of commodities and
      reagents for the Limulus assay. Depyrogenation was carried out
      at 250C for 4 hours, which eliminates both endotoxin and
      (1[-]3)--D glucans (27). Cyst fluids were stored at -70C.
      Assessment parameters of cysts included color and size; for
      fluids, color, turbidity, pH, mg protein/ml, specific gravity,
      and blood. A commercial dipstick was used to detect and
      semiquantitate cyst fluid pH, specific gravity, and blood (Ames
      10 SG Multistix, Miles, Inc., Elkhart, IN, USA). Blood
      sensitivity was 0.015-0.062 mg/dL hemoglobin, equivalent to
      5-20 intact red blood cells per microliter. Results were
      recorded as 1-4+. For specific gravity, determinations
      permitted ranged from 1.000-1.030; for pH, 5.0-9.0. Protein was
      by the method of Lowry et al. (28).

      The LAL gel clot end point assay (22) was performed according
      to the manufacturer's instructions (Charles River ENDOSAFE,
      Charleston, SC). The mechanism of the LAL assay is illustrated
      in Figure 1. To quantify endotoxin, undiluted samples and
      serial twofold dilutions through 1:64 were tested. To detect
      false positives due to activation of the alternate (1[-]3)--DG
      pathway, LAL assay reagent fortified with the glucan inhibitor
      laminarin was used (23). Laminarin was courtesy of Dr. J.
      Cooper, Charles River ENDOSAFE, Charleston, SC. Laminarin had
      been rendered endotoxin free by treatment with 0.2 N NaOH at
      50-60C for 6 hours; treatment did not affect activity of
      laminarin in the LAL assay (23). The standard gel clot end
      point method was used, except laminarin was added to the LAL
      assay reagent (23). Minimum sensitivity of both assays was
      0.03-0.06 Endotoxin Units (EU)/ml (3-6 pg control standard
      endotoxin, CSE; 10 EU/ng of EC-5 (E.coli O133) U.S. Standard
      endotoxin; for -DG, 10 pg/ml (29).

      Known endotoxin-positive (tap water) and -negative
      (pyrogen-free water) samples were run as controls along with a
      CSE curve. The endotoxin concentration was estimated by
      multiplying the maximum dilution producing a positive test
      against the sensitivity of lysate that was verified daily. The
      content of -DG in cyst fluid was estimated by the difference
      in titers between the endotoxin specific assay (LAL+laminarin)
      and the conventional LAL assay (30). Portions of all samples
      were additionally spiked with 0.25 EU CSE and tested for
      inhibitory substances. If inhibition was observed in the LAL
      assays, samples were boiled for 5 minutes, cooled to room
      temperature, and retested. Samples were incubated, in some
      cases after boiling, for 30 min at room temperature with
      polymyxin B (5 mg/assay) before LAL testing. Polymyxin B
      inhibits the LPS-activated pathway via Factor C by its direct
      binding to LPS, which yields a nonreactive complex (25). An
      alternative approach was to use 20% dimethyl sulfoxide (DMSO)
      to inhibit Factor C directly (23); DMSO was not used to permit
      detection of nonendotoxin, but LAL-reactive materials that were
      not inactivated by binding to polymyxin B. However, in our
      system DMSO did completely block the LAL reaction in the
      presence of CSE.

      After centrifugation of the cyst fluid, the LAL-positive
      material was located only in the supernate for
      autosomal-recessive- and simple cyst-derived fluids. For
      autosomal dominant-derived fluids, the reactive material was
      located in the pellet. Thus, centrifugation of fluids to remove
      particulate material before the LAL assay may influence
      results.

      LAL assay inhibition was detected in nearly two-thirds of 73
      cyst fluids tested, including autosomal recessive and simple
      cyst fluids. Dilution of cyst fluid through 1:16 was often
      required to overcome the inhibitory effect in unboiled
      specimens. Boiling of entire fluids or supernates and pellets
      for 5 minutes eliminated LAL assay inhibition in virtually all
      specimens at all dilutions. Exceptions were some undiluted and
      1:2 dilutions of chocolate-colored cyst fluids, where
      inhibition was not eliminated in both test and spiked specimens
      of autosomal dominant fluids.

The concentrations of endotoxin-specific material in autosomal dominant
cyst fluids was 0.12 to 3.84 EU/ml, with substantial levels of endotoxin
(3.84 EU/ml) also found in a pool of eight cysts from one autosomal
recessive kidney and simple cyst fluid from one donor. The mean
concentration of endotoxin across all of the auto-somal dominant fluids
tested (including those that were LAL negative) was 0.65 EU/ml. Using 10
EU/ng as a conversion factor yields 65 pg endotoxin/ml cyst fluid; normal
plasma levels of endotoxin are <4-5 pg/ml (31). Intravenous injection of 5
EU/kg body weight (350 EU/70 kg) can induce shock in the average adult male
(21). We estimate that a single cystic kidney in an adult male (3-5 kg
kidney weight of which 33% is cyst fluid) contains 648 to 1,080 EU/kidney
or about two lethal doses of endotoxin per kidney. Unexplained fever and
flank pain in PKD patients have been attributed to release of IL-1 from
rupture or hemorrhage of cysts (27). On the bases of the high levels of
endotoxin observed in this study and the high sensitivity of humans to
endotoxin, we propose that the release of endotoxin from the cyst into the
peritoneum or blood may be an important initiator of a cascade of biologic
events after leakage or rupture of renal cysts.

For one donor (donor 3) both kidneys were available for cyst fluid analysis
(nine left and 11 right kidney cysts, respectively). When the frequency of
LAL-positive fluids (endotoxin) was compared, no significant difference was
found (Table 1). However, a threefold difference in the frequency of
inhibitor detection was observed in left kidney cyst fluids compared with
the right kidney (90% vs. 31%, respectively). Thus, LAL testing of fluids
for endotoxin requires vigilance for both false negative and positive
materials.

Signature Bacterial Fatty Acids

If endotoxin is present in renal cysts, fatty acids unique to endotoxin can
be used to confirm its presence. The Lipid A region of LPS contains on
average four 3-hydroxy (3-OH) acyl groups of various chain lengths (13,14).
Acid hydrolysis of LPS releases the 3-OH fatty acids and other fatty acids.
Diverse bacterial genera exhibit characteristic ratios of fatty acids of
various chain lengths and patterns of hydroxylation; compilations of such
signature fatty acid profiles are available for many genera (14).
Initially, aliquots of five cyst fluids were analyzed in a single blind
manner by gas chromatography-mass spectrometry as described by Mayberry and
Lane (32). 3-Hydroxy (3-OH) fatty acids of nC:12:0 and nC:14:0 carbon
length were detected but not quantified in the three cyst fluids that were
positive in the endotoxin specific LAL assay; they were not detected in the
two cyst fluids that were LAL negative. Thus, complete concordance of LAL
reactivity and signature fatty acid analysis was observed. 3-OH fatty acids
(C:14, C:16) were also reported in LAL-positive cyst fluids by a separate
reference laboratory (Microbiological Insights, Inc., Knoxville, TN).
Additional chemical analyses of cyst fluid are required before a classic
signature fatty acid profile and linkage to one or more bacterial genera
are possible. Also, we cannot discount the possibility that 3-OH fatty
acids released during degradation of endotoxin were incorporated into
mammalian sphingolipids that were positive in the LAL assay, thereby
accounting for both the presence of such fatty acids and LAL reactivity in
cyst fluids.

(1[-]3)--D-Glucans (-DG)

In addition to endotoxin, (1[-]3)--D-glucans were identified in cyst
fluids from two patients by differential activation of the LAL assay (Table
1). The range of -DG reactive material in cyst fluids was estimated at 40
to 160 pg/ml. In plasma of healthy persons, levels of -DG are lower than
6.9 pg/ml (26). -DG, a ubiquitous constituent of filamentous fungal and
yeast cell walls (33), is exceeded only by endotoxin in its reactivity in
the LAL assay; glucans lacking a 1[-]3 linkage are not LAL reactive (34),
nor are mannans, dextrans, and cellulose (24,26). Although diverse forms of
glucans are components of fungal cell walls, those with (1[-]3)- linkages
are the most potent in causing hypersensitivity pneumonitis and other
severe pulmonary illnesses (35). Although -DGs are indicative of fungal
cell walls, their occurrence alone does not identify the genera and species
of the fungus that produced it. To confirm the presence of fungal
components and determine the source of -DG, serologic tests were performed
on three -DG positive and two -DG negative cyst fluids from three
autosomal-dominant PKD patients (Table 2). Cyst fluid from donor 6 that was
negative for LAL-reactive material was also free of detectable fungal
antigens and anti- from both kidneys containing -DG-positive material
(donors 4 and 5; Table 1) were also positive for fungal antigens. Fluid
from donor 5 also exhibited fungal antibodies; fluid from donor 4 was not
tested for antibodies to fungi.

      Table 2. Serologic results of five cyst fluids from three
      autosomal-dominant PKD patients tested for fungal antigens or
      antibodies to fungal components
      ---------------------------------------------------------------
      Donor;Cyst
      No.           LAL(a) Findings  Serologic Findings
      ---------------------------------------------------------------
      D6;1          Neg. Et(b);Neg.  Antigens not detected
                    -DG(c)
      D6;2          Neg. ET;Neg.     IgE and IgG reactive with fungi
                    -DG             were not detected
      D4;3          Neg. ET;Pos.     Antigens: Fusarium solani at 1:2
                    -DG             dilution; Aspergillus
                                     galactomannan at >50 ng/ml
      D5;4          Neg. ET;Pos.     Antigens: Aspergillus
                    -DG             galactomannan at >50 ng/ml;
                                     Candida albicans serotype A
                                     mannan at 10 ng/ml
      D5;5          Neg. ET;Pos.     IgE reactive with Fusarium
                    -DG             moniliforme; C. albicans
                                     (weak positive)
      ---------------------------------------------------------------
      (a)Limulus amebocyte lysate assay
      (b)Endotoxin
      (c)-D-glucan

      Fungal serology was performed on three cyst fluids from donors
      4 and 5; (Table 1) with -DG positivity and two cyst fluids
      from donor 6, which were negative for both endotoxin and -DG
      after extended incubation in the LAL assay. Fluids were tested
      for antibody to Penicillium notatum (Penicillium chrysogenum)
      and Penicillium frequentans, Aspergillus fumigatus, Candida
      albicans (yeast), and Fusarium moniliforme. The Pharmacia CAP
      systems RAST FEIA (IgE) and IgG FEIA were employed (Pharmacia &
      Upjohn Diagnostics, Kalamazoo, MI, USA). The tests were
      modified by substituting cyst fluid for plasma; interference of
      fluid with the test was not observed. Tests to detect fungal
      antigens were performed by the Centers for Disease Control and
      Prevention, Atlanta, GA. To detect Fusarium sp. antigen, a
      modified aspergillosis microimmunodiffusion test (36) was used
      with experimental rabbit anti-Fusarium solani antibody; kidney
      cyst fluid was substituted for plasma. A double-antibody
      sandwich enzyme immunoassay was used to detect C. albicans
      serotype A mannan concentrations in cyst fluids; specificity of
      this test in sera is reported as 100% (37). An experimental
      ELISA inhibition assay was used to detect Aspergillus
      galactomannan (Steven Hurst, unpub. protocol) Standard serum
      reference curves were used for extrapolating quantities of C.
      albicans mannan and Aspergillus galactomannan in cyst fluids.
      However, these tests were not designed for cyst fluids, but for
      serum antigen detection, and must, therefore, be viewed as
      estimated quantities.

Fungal serologic tests provided insights into potential sources of -DG.
Initial measurements showed Fusarium solani antigen. These findings suggest
the antigen to be from F. solani or a shared antigen present in other
Fusarium species (L. Kaufman, pers. comm.). IgE (but not IgG) antibody to
Fusarium moniliforme was also detected. Aspergillus galactomannan antigen
and Candida albicans serotype A. mannan were present in selected cyst
fluids. Cross-reactivity of Fusarium with Aspergillus and Penicillium is
unlikely as 1) the Fusarium antibody (prepared against the mycelium) was
cross-absorbed with Aspergillus, and 2) the immunodominant group of
Aspergillus and Penicillium species, galactomannan with a galactofuranosyl
moiety (38), is absent in Fusarium species (39). As the serologic tests for
Fusarium and Aspergillus in cyst fluids are experimental and have not been
evaluated clinically, these data are indirect presumptive indication of the
presence of these fungal antigens. The paucity of fungal serologic and
chemical methods for detection and identification of emerging fungal
pathogens in human tissues and body fluids and fungal components in the
human diet has been noted by others.

Also detected were IgE antibodies to Candida. IgE antibodies to both
Fusarium and Candida in cyst fluid suggest recruitment of immunologic
defenses against fungi. The site of production of these IgEs and route of
entry into the cyst are unknown; a hypersensitivity reaction cannot be
discounted. In human and experimental PKD, the severity of cystic disease
correlates with the numbers of circulating neutrophils; neutrophilia is
related to exposure to endotoxin and cytokines, both of which have been
reported in cyst fluids (16). Our finding of fungal antigens and antibodies
raises the additional possibility of leukocyte activation in PKD by -DG
and mannan released from fungi, as -DG and mannan have been reported to
stimulate cytokine production (40).

Serologic testing is frequently used to detect fungal infection. Cyst
fluids collected from PKD patients at sequential times during progressive
stages of the disease were not feasible. Therefore, quantitative and/or
qualitative changes in antigen or antibody titers could not be used to
determine active fungal infections in these patients. Although antigens of
Aspergillus or Fusarium species are generally interpreted as apparent or
covert infection, another possibility is the absorption and distribution of
fungal components independent of infection (i.e., mycotoxicosis versus
infection). While Aspergillus enters primarily by inhalation, this route
does not appear to be the predominant source of Fusarium; fewer than 1% of
air samples contained Fusarium species (41). Fumonisin from Fusarium is
present in grains, rice, and corn consumed by humans (42), with regional
variations in levels and the type and processing of dietary product.
Because of their occurrence in the food supply and their effect on
experimental animals, it has been proposed that fumonisins may contribute
to kidney disease in humans (43).

-D-Glucans and mannans are shed by fungi during growth (26,39) and thus,
like bacterial endotoxin, can potentially be distributed through blood and
lymph. Diverticular disease and other anomalies in the structural and
functional integrity of the gut may occur in up to 80% of PKD patients
(2,44). In addition to diminished barriers to the absorption of gut-derived
endotoxin, the outpouchings (diverticula) become overgrown by minor
subpopulations of gut microflora (45), thereby generating increased
quantities of unusual mixtures of potentially absorbable microbial
components. Diverticular disease (44) and acquired renal cystic disease
(46) occur in patients on hemo- and peritoneal dialysis. To complete the
symmetry of this line of reasoning, outpouchings of the renal tubule in PKD
are described as both cysts and diverticula (2). Direct measurement of
intracyst pressure and elasticity of the basal lamina have led to the
rejection of the obstruction hypothesis (i.e., a physical balloon
inflation) of cystogenesis in favor of a cell-mediated restructuring of the
basal lamina coupled with electrolyte transport into the cyst to expand
intracyst volume (47). Although diverticular disease is thought to be a
pressure-driven lesion, gut diverticula in PKD and dialysis patients may
also involve a cellular lesion further promoted by pressure. Thus,
diverticula in kidney and gut are associated with microbial components. It
is not known if the processes of renal cystogenesis, formation of gut
diverticula, and the walling off of infecting material are all facets of
the same defense response in humans. Endotoxin in relatively high
concentrations was found in fluid from a simple cystic kidney (Table 1);
intestinal or biliary obstruction and urinary tract infections have been
associated with simple kidney cysts (2). Although endotoxin has been
proposed as being associated with "high sodium cyst fluids" (47), we
observed endotoxin in cyst fluids of low and high sodium content (data not
shown).

Relevant to our study is the report of the capacity of -DG to prime cell
systems, which results in sensitization to bacterial endotoxin and
infection (48), an example of -DG-endotoxin enhancement; -DG and
endotoxin also demonstrate a strong synergistic effect on macrophages (49).
In addition, there have been reports of synergy between mycotoxin and
endotoxin (50).

Detection of Fungal DNA

To identify fungal DNA as evidence of past or current fungal infection
and/or the absorption and distribution of fungal components to kidney
cysts, polymerase chain reaction (PCR) methods were used to amplify and
thus detect fungal DNA in PKD cyst fluids and kidney tissue. Six cyst
fluids from three patients and two samples of kidney tissue from two
autosomal-dominant PKD patients were tested; all were positive for fungal
DNA. A sample of normal human kidney was negative for fungal DNA (Figure 2
A-C). Because culture confirmation was not achieved from the cyst fluids,
expanded studies with species-specific probes are warranted.

             [Figure 2] [Figures not available in ASCII version]

             Figure 2. Amplification results of normal and PKD
             kidney tissue and cyst fluids with universal
             fungal primers ITS 1 and NL 4. 2A: DNA from
             healthy human kidney tissue diluted 1:10, 1:100
             and 1:1,000 (lanes 1-3); control fungal DNA, A.
             tamarii (lane 4); negative control (lane 5); 1 kb
             ladder (lane 6); arrow indicates migration front.
             2B (NL 4) and 2C (ITS 1): two cyst fluids, donor
             6, negative for detectable endotoxin and -DG
             (lanes 1 and 5); two cyst fluids, donor 4,
             positive for -DG (lanes 2 and 3) and positive
             for Fusarium solani antigen (lane 3); two cyst
             fluids, donor 5, positive for -DG (lanes 4 and
             6); two PKD kidney tissues, donor 5 (lane 7) and
             donor 4 (lane 8); negative control (lane 9).
             Large arrows in 2B and 2C point to 560 bp
             molecular weight marker; small arrows point to
             product bands.

             Polymerase Chain Reaction

             Specimens for PCR included 1) normal human kidney
             tissue (0.46 g) from a 7-year-old girl who died
             of a head injury; the kidney was not
             transplantable because of the presence of a
             hematoma but was otherwise normal; the section
             used for PCR excluded the area of hematoma. 2)
             0.5 g of cystic tissue, devoid of cyst fluid,
             from each of two autosomal-dominant PKD patients
             (donors 4 and 5, Table 1). 3) multiple cyst
             fluids (0.5-1.0 ml each; donors 4, 5, and 6). The
             positive control consisted of DNA extracted from
             A. tamarii (NRRL 26010) plus primer. The CTAB
             extraction method for fungal genomic DNA was
             employed. The universal oligonucleotide primer
             pair ITS 1 and NL4 for fungi, as previously
             described (51), was used; NL 4 is the reverse
             primer. PCR master mix (37) with 1.5 mM MgCl2 and
             primers in a final reaction volume of 100 ml was
             subjected to the following thermal cycler
             parameters (Perkin Elmer Thermo Cycler 1):
             96C30 sec; 53C30 sec and 72C30 sec for 40
             cycles, followed by a final 7 min extension at
             72C and soak at 4C. The presence of the
             expected molecular weight PCR products (600 bp)
             was confirmed by ethidium bromide staining after
             separation on a 1.0 % agarose gel. The amplified
             products were compared visually for similarity on
             the basis of the presence or absence of bands;
             variations in band intensity were not considered.

             A-C represent the banding patterns obtained by
             agarose gel electrophoresis of healthy kidney
             tissue, cystic kidney tissues, and cyst fluids
             following PCR amplification with the universal
             fungal primer pair, ITS 1 and NL 4. Amplified
             fungal products were not detected in the negative
             PCR controls (2A, lane 5; 2B and C, lanes 9) or
             normal human kidney tissue (2A, lanes 1-3). In
             marked contrast, distinct bands of the predicted
             size of each fungal primer used ( ~ 600 bp; 45)
             were detected in all cystic kidney tissues and
             fluids examined from the three patients (2B-C);
             minor fragments were not detected. The migration
             patterns were consistent with the positive fungal
             DNA control (2A, lane 4).

             Although fungal DNA might have been anticipated
             in tissues and fluids from PKD kidneys showing
             -DG or positive serologic findings, fungal DNA
             was also found in PKD cyst fluid from donor 6 (2B
             and C: Lanes 1 and 5), which lacked these
             components. The -DG positive cyst fluids tested
             from donors 4 and 5 are in Lanes 2 and 3 and 4
             and 6, respectively. The fluid depicted in Lane 3
             also had positive serologic results for F. solani
             antigen; serologic testing was not done on the
             other fluids assayed by PCR. Each cyst fluid must
             be considered an independent sample, requiring
             direct measurement; assumption of cyst content on
             the basis of other fluids within the same kidney
             is not valid (16). Fungal DNA was also detected
             in kidney tissue from donors 4 and 5 (Figure 2B
             and C; Lanes 8 and 7). Differences were noted
             between the two primers used; ITS 1 yielded
             amplified products in all PKD samples while NL 4
             amplified less well, being more concordant with
             the detection of -DG. The amplicons are
             presumptive evidence of fungal DNA.

Despite substantial effort, we have not been able to culture either
bacteria or fungi from PKD cyst fluids by axenic methods, but fungi were
detected in cultured epithelial cells from PKD kidney tissue (see below);
methods appropriate to culture L-forms or other cell-wall-defective
microbes were not performed. Gram stains of cyst fluid supernates and
pellets did not show intact bacteria. Electron microscopy also did not show
intact bacteria, but occasionally, a field consistent with fungal cell
walls was observed (not shown); intact fungi were not observed.
Nonetheless, it was only from PKD kidneys where -DG was identified in cyst
fluid that fungal organisms were frequently isolated from PKD epithelial
cell cultures. Other human and animal cells isolated and propagated in the
laboratory by the same reagents and work spaces were free of fungal growth.
The fungi were identified as belonging to the genera Paecilomyces and a new
species of Penicillium (manuscript in preparation). Fungi have been
reported, albeit infrequently, as etiologic agents in renal infections in
PKD (8). Given all of the above, it is possible that viable fungi were
present in the PKD renal tissue, but only their remnants were in the cyst
fluid. Emerging human awareness of infectious disease may well describe our
experience in this study of PKD.

Infectious Disease, Microbial Toxicosis, and Sphingolipid Biology

Emerging knowledge of sphingolipid biology and PKD's vulnerability to
microbial toxins offer an opportunity for a fresh look at PKD. As Spiegel
and Merrill (52) have noted, sphingolipids fall into two broad categories,
both of which are altered in PKD (53,54). Complex sphingolipids (i.e.,
ceramide with a carbohydrate or phosphocholine head group on carbon-1)
interact with growth factor receptors and the extracellular matrix and
adjacent cells and act as binding sites for gram-positive and -negative
bacteria and microbial toxins. In the second category of sphingolipids,
ceramide sphingoid bases (i.e., sphingosine, sphinganine, phytosphingosine)
and their 1-phosphates modify the activity of protein kinases and
phosphatases, ion transporters, and a growing number of signal transduction
processes. However, the dynamics of human sphingolipid biology occur within
the context of a microbe-dominated environment.

Thus, we propose a third category called "microsphingoids,"
sphingolipid-like molecules of microbial origin that mimic or antagonize
the actions or metabolism of human sphingolipids. Examples include, but are
not limited to, various mycotoxins (42,55-57), bacterial sphingolipids
(58), and endotoxin (59,60). In this report, we have shown the presence of
endotoxin and fungal antigens or antibodies to at least Fusarium,
Aspergillus, and Candida in human PKD tissues and fluids; from human PKD
cells in vitro we have encountered Paecilomyces and Penicillium. These
fungi produce mycotoxins that inhibit multiple enzymes required for
sphingolipid biosynthesis (55; Figure 3) and alter the activity of various
elements of signal transduction cascades, such as protein phosphatases,
calmodulin, and GTP-binding proteins (56,61). Such mycotoxins are also
found in the human diet, which is itself an emerging concern in food safety
(42,43,55). Rietschel et al. (13) noted the similarity of LPS to
glycosphingolipids. Wright and Kolesnick (59) have reviewed the structural
similarity of the Lipid A region of bacterial LPS with ceramide and the
ability of LPS to mimic the ceramide-induced alterations in protein kinase
and phosphatase activities. Endotoxin is also reported to alter the
structure of mammalian sphingolipids (62) and to initiate cytokine-mediated
cascades that generate ceramide, sphingosine-1-phosphate, and
lysosphingolipids, all of which exhibit biologic activity (13,63). Not to
be ignored are bacterial sphingolipids formed by genera such as
Bacteriodes, which represent approximately 30% of the gut microflora (58).
The role of bacterial sphingolipids and their metabolites in human biology
and their bioavailability in disease are poorly understood. The term
"microsphingoid" is intended to highlight the contribution of microbes to
sphingolipid biology in human health and disease.

    [Figure 3] [Figures not available in ASCII version]
    Figure 3. Sites of impact of endotoxin and mycotoxins on
    sphingolipid metabolism. In PKD, renal sphingolipid formation is
    altered. Such compromised sphingolipid pathways would be expected
    to be vulnerable to these highly potent microbial toxins,
    especially during chronic exposure within renal cysts.

Could pertubations in sphingolipid biology caused by genetic anomalies
and/or "microsphingoids" account for both infantile autosomal recessive and
adult onset forms of kidney cystic disease? Hannun (64) has reviewed the
role of sphingolipids as "biostats" that regulate diverse cellular programs
executed in response to various stimuli. Such biostatic regulation could
account for both acute and chronic changes in cellular behavior and
differentiation in the kidney (65). Calvet et al. (2,66) have proposed two
models to explain the immaturity or dedifferentiated state of epithelial
cells found in hereditary and acquired cystic kidneys: In infantile cystic
disease, the epithelial cells never reach terminal differentiation and are
trapped in an immature state, while in adult forms of cystic disease,
toxin-induced injury to an initially mature renal epithelium is followed by
inability of the epithelium to recover to a fully differentiated state. In
genetically cystic cpk/cpk mice (a model for autosomal recessive PKD),
Deshmukh et al. (53) reported altered levels of ceramide and complex
glycosphingolipids in kidney tissue from cystic mice, but not in their
phenotypically normal littermates. Lower levels of ceramide and sulfatide,
but higher levels of glucosyl- and lacto-sylceramide and ganglioside GM3,
were measured. Our research in infantile and adult human PKD kidney tissue
and cyst fluids showed no detectable free sphinganine, the primary
sphingoid base formed during de novo biosynthesis of mammalian
sphingolipids (54). Thus, anomalies in sphingolipid biology exist in cystic
kidney tissue.

It is difficult to separate concepts of sphingolipid biology from
considerations of infectious disease, microbial components, and
"microsphingoids" in PKD. Potential consequences of alterations in
glycosphingolipids on the surface of PKD cells include enhanced microbial
colonization due to the availability of complex sphingolipids as binding
sites. Binding of microbes or their components to such sphingolipids may
even promote cystogenesis, as binding to complex sphingolipid has been
reported to cause changes in differentiation and morphology of cells in
vitro (67). Altered biosynthesis of sphingoid bases affects the ratios of
sphinganine to sphingosine with consequences to signal transduction (61)
and may even alter the antimicrobial environment, as sphingoid bases are
reported to have direct antifungal and antibacterial activity (68). The
levels of sphingosine in human cyst fluid (Hjelle, unpub.) are comparable
to the levels that induce a state of cytoresistance (cellular
dedifferentiation) in kidney cells in vitro (69). In addition to products
of mammalian sphingolipid metabolism, endotoxin in relatively high amounts
was found in cyst fluids from infantile and adult forms of PKD and in
simple cysts (Table 1). Could "microsphingoids" or microbial toxins injure
and then prevent repair of renal epithelial cells? Data from diverse
scientific disciplines support this possibility.

LPS influences nephron formation (70) and renal cystogenesis (11).
Fumonisins are potent nephrotoxins (43,55), alter repair mechanisms in
kidney cells in vitro (71), and induce apoptosis (72,73). Renal cysts are
occasionally reported after chronic exposure of rodents to fumonisins (74).
Rates of programmed cell death are abnormal in PKD kidney tissue (75,76);
ceramide is a pivotal messenger in apoptosis (64). In PKD, altered
processing and sorting of cell membrane proteins and secretory material
occurs (2-4); sphingolipids influence processing, sorting, and movement of
membranes and ion transporters (64,65), as does fumonisin in kidney cells
in vitro (77). In PKD, various electrolyte transport systems are altered
(2). cAMP-mediated electrolyte fluxes were proposed as important in cyst
formation (3). Fumonisins are reported to activate cAMP response elements
(78). Cyst fluid contains uncharacterized substances that promote
cystogenesis in vitro (3). Although LPS isolated from Escherichia coli did
not induce the full array of anticipated cystogenic responses in kidney
cells in vitro, the structure, potency, and array of elicited biologic
responses of endotoxin depend on the genera and species of bacteria from
which it was isolated (13). Coupled with the presence of fungal components,
cyst fluid likely represents a complex mixture of "microsphingoids" and
toxins that may change over time with dynamic
consequences to cyst formation. The heterogeneity of cyst volume and
content of growth factors and cytokines in adult PKD has been noted (3,16).

Concepts of infection and "microsphingoids" appear to converge with the
expanding knowledge of PKD gene products. The structures of the PKD1
(polycystin) and PKD2 gene products share homology with a1E subtype of
voltage-activated, calcium channels (3,6) and a sea urchin protein likely
involved in calcium fluxes during fertilization (79). LPS alters
voltage-activated, calcium channels (80) and osmoregulation (81) in
mammalian cells. The PKD1 gene product also contains regions of homology
with ligand and receptor domains putatively involved in binding to adjacent
cells and extracellular matrix (3); one such domain is similar to C type
lectins that can bind microbes (82). Tissues, including kidney and gut,
expressing polycystin in adult PKD, may exhibit a relatively greater
leakiness to normal molecular and particulate traffic, as seen in kidney
cysts (3) and susceptibility to diverticulosis (3,44). Polycystin also
shows homology with apoprotein from low density lipoprotein. Because LPS
and sphingolipids bind to and are transported by serum lipoproteins, the
low-density lipoprotein binding site in polycystin may also enable
accumulation and/or transport of "microsphingoids."

Secondary mutations in PKD genes appear to be required for clonal cyst
formation (3,5), as is a loss of renal tissue through dysregulation of
apoptosis (76). Alterations in the expression of the bcl-2 gene product in
mice results in polycystic kidneys and dysregulation of apoptosis (83).
Although infection increases translocation of the bcl-2 gene in human
lymphoid tissue (84), it is not known if infection in PKD patients causes
such a dysregulation of bcl-2 in kidney tissue.

Regarding infection and sphingolipids, ceramide is emerging as a pivotal
molecule in the immune system (63) and fumonisins are reported to alter
immune function (85). It is ironic that our findings potentially link
Fusarium to PKD, as fumonisin is used as a molecular tool to study
sphingolipid metabolism and signal trandsduction in PKD. Not to be
overlooked are the classic bacterial sphingomyelinases encountered during
infection and released from gut microflora (e.g., Staphylococcus aureus and
Clostridium perfringens) that can stimulate ceramide formation by
hydrolysis of sphingomyelin. Thus, the vulnerability of PKD patients to
infection may be related, in part, to anomalies in sphingolipid biology
that influence 1) "biostatic" mechanisms of cell regulation, 2) the
structure of the plasma membrane and the function of PKD gene products, 3)
the availability of glycosphingolipids as binding sites for microbes and
their components, 4) the bioavailability of microbial components present in
the gut, and 5) antimicrobial defenses in general. Such vulnerability is
likely influenced by repeated courses of antimicrobial therapy that provide
selection pressure for colonization with a modified microflora. The extent
to which sphingolipid biology in PKD is influenced by genetic defects
rather than microbial factors is yet to be defined.

Anomalies caused by the PKD gene defect(s) alone cannot explain
cystogenesis. As shown by Werder et al. (9), raising genetically cystic
mice in a germ-free environment essentially eliminated cystogenesis and
increased survivorship to nearly 100% over that of littermates raised in
ambient environment for 18 months. Even in cyst fluid from infantile PKD,
relatively high levels of endotoxin were found (Table 1). This suggests
that microbial toxins are available early in this disease. Prenatal
exposures to toxins and "microsphingoids" are unknown. Although our finding
of fungal DNA in eight of eight samples of autosomal-dominant PKD tissue
and cyst fluids examined suggests an intimate association of fungal
exposure to renal cysts, a contributing multifaceted microbial toxicosis
involving diet and gut microflora cannot be excluded. By itself, the
finding of microbial components at the site of lesion does not prove a
causal role for the microbe(s) in disease progression (1). However, a
working hypothesis can be formed on the basis of evidence that microbes
promote progression of the primary disease and components of the microbes
act on mammalian biology to cause effects plausibly related to the known
pathophysiology of the disease.

Although there is an established body of knowledge that PKD is a genetic
disorder, our data indicate that bacterial endotoxin, -DG, and likely
other microbial components are available within the kidney to provoke
cystogenesis. We have provided chemical and advanced LAL assay-based
evidence of bacterial endotoxin and -DG in human PKD cyst fluids. From
cyst fluids and PKD kidney tissue we have provided evidence of fungal DNA
and cell components by serologic testing and PCR with universal fungal
primers. We have integrated these findings into a working hypothesis based
on emerging knowledge of PKD gene products, altered sphingolipid metabolism
in PKD, the effects of LPS on renal cystogenesis, the mimicry of ceramide
by LPS and the effects of mycotoxins on mammalian sphingolipid biology and
signal transduction, the occurrence of infection in PKD, and the impact of
altered gut permeability and microbial colonization on progression of PKD.
Is PKD a genetic disease promoted by microbial influences? Tests of this
multifaceted hypothesis require awareness of a breadth of issues drawn from
numerous scientific disciplines. Identification of the microbes and
microbial components involved will require a concerted analysis using
highly sensitive and specific methods. As awareness of the importance of
sphingolipid biology in health and disease grows, so will the appreciation
that microbial influences will need to be considered in pharmacologic
studies that seek to manipulate ceramide and complex sphingolipid
biochemistry in disease. The ubiquitous and highly potent bacterial
endotoxin is again one of the usual suspects examined as provocateur of
disease; in this case, endotoxin's modus operandi of coopting the signal
transduction machinery of ceramide to cause chronic disease may ultimately
be revealed.

Acknowledgments

This research was supported by grants and gifts from the Methodist Medical
Center Foundation; the Children's Miracle Network Telethon; and the Campus
Research Board, University of Illinois. We thank Dr. Jim Cooper for his
advice, counsel, and support during the endotoxin experiments and
preparation of the manuscript; Dr. Kerry O'Donnell for analysis of
specimens by PCR for fungal DNA; Dr. Christine Morrison (Candida mannan),
Dr. Leo Kaufman (Fusarium antigen), and Mr. Steven Hurst (Aspergillus
galactomannan) for fungal serologic tests for antigens; and Dr. Hun-Chi Lin
for fungal serologic tests for antibodies. We also thank Jonathan R. Lane
and Robert V. Acuff, for the use of, and advice regarding, the
gas-chromatograph-mass spectrometer.

Dr. Miller-Hjelle is professor of Microbiology and Diplomate, American
Board of Medical Microbiology.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Synopses

Borna Disease

Carolyn G. Hatalski, Ann J. Lewis, and W. Ian Lipkin
University of California, Irvine, California, USA
---------------------------------------------------------------------------


      Borna disease virus, a newly classified nonsegmented
      negative-strand RNA virus with international distribution,
      infects a broad range of warm-blooded animals from birds to
      primates. Infection causes movement and behavioral disturbances
      reminiscent of some neuropsychiatric syndromes. The virus has
      not been clearly linked to any human disease; however, an
      association between infection with the virus and selected
      neuropsychiatric disorders has been suggested. We reviewed
      recent advances in Borna disease virus research, focusing on
      evidence of infection in humans.

Although Borna disease was first recognized in the early 1800s as a
neurologic syndrome with an infectious basis, Borna disease virus (BDV) has
only recently been characterized as the causative agent. BDV is the
prototype of a newly recognized virus family, Bornaviridae, within the
nonsegmented negative-strand RNA viruses (order Mononegavirales) (1,2).

The molecular biology of the virus has several unusual aspects, including
nuclear localization for replication and transcription (3), overlap of open
reading frames and transcription units (4,5), posttranscriptional
modification of subgenomic RNAs (6,7), and marked conservation of coding
sequence across various animal species and tissue culture systems (8,9).
BDV replicates at lower levels than most known viruses (10,11), is not
lytic, and persists in the nervous system despite a vigorous immune
response. In the classic syndrome, infected animals exhibit movement and
behavior disorders (10,12,13); however, clinical signs may be dramatic,
subtle, or inapparent depending on the integrity and intensity of the host
immune response to viral gene products (14).

Natural Infection and Transmission

Originally described as a disease of horses, Borna disease has also been
found in sheep, llamas, ostriches, cats, and cattle (15). Because an even
larger variety of species has been infected experimentally, the host range
is likely to include all warm-blooded animals; no data exist concerning
infection of species other than warm-blooded hosts.

The geographic distribution of BDV is unknown. Natural infection has been
reported only in Central Europe, North America, and parts of Asia (Japan
and Israel). However, this apparent geographic restriction may reflect lack
of reliable methods and reagents for diagnosis of infection or failure to
consider the possibility of BDV infection. Recent reports of asymptomatic
naturally infected animals suggest that the virus may be even more
widespread than previously thought (16-18).

Neither the reservoir nor the mode of transmission of natural infection is
known. An olfactory route for transmission has been proposed because
intranasal infection is efficient, and the olfactory bulbs of naturally
infected horses show inflammation and edema early in the course of disease
(19,20). BDV nucleic acid and proteins in peripheral blood mononuclear
cells (PBMC) also indicate a potential for hematogenous transmission
(21-28). Experimental infection of rodents results in virus persistence and
is associated with the presence of viral gene products in saliva, urine,
and feces (28); such secreta/excreta are important in the transmission of
other pathogenic viruses (e.g., lymphocytic choriomeningitis virus and
hantaviruses). Thus the rodent provides the potential for both a natural
reservoir and vector; however, because natural BDV infection has not been
reported in rodents, their role in BDV transmission to other domesticated
animals and humans remains speculative. No data concerning vertical
transmission of BDV in natural or experimental hosts have been published.

Animal Models for BDV Pathogenesis

Borna disease in naturally infected horses and sheep is characterized by
agitated aggressive behavior that progresses over weeks to paralysis and
inanition (29). Because its immune-mediated disease most closely resembles
that in naturally infected horses and sheep, the Lewis rat has been
selected as a rodent model. Rats infected as adults exhibit hyperactivity
and exaggerated startle responses coincident with viral gene products in
limbic system neurons and infiltration of mononuclear cells into the brain
(13,30). The inflammation recedes over several weeks, but the virus
persists and animals show stereotyped motor behavior, dyskinesias, and
dystonias associated with distinct changes in the central nervous system
(CNS) dopamine system (12,31), as well as decreased activity and cachexia
(13). In contrast, rats infected as neonates have a disease characterized
by stunted growth, hyperactivity, subtle learning disturbances, and altered
taste preferences and do not mount a cellular immune response to the virus
(32,33).

Behavioral disturbances have been reported in experimentally infected
primates: tree shrews and rhesus monkeys. Infected tree shrews have altered
social and sexual behavior, manifested as abnormal dominance relationships
and failure to mate (34). Infected rhesus monkeys are initially hyperactive
and subsequently become apathetic and hypokinetic (35).

BDV and Neuropsychiatric Disease

Serology (Table 1)

Its broad host and geographic range suggests that BDV might cause human
neuropsychiatric disease. Because the behavioral disturbances in animals
resembled those of affective disorders, particularly bipolar depression,
initial studies investigated these disorders. The earliest work to suggest
a link between BDV and human mental illness came from a serologic survey in
1985 of 285 patients with affective disorders in the United States, 694
psychiatric patients in Germany, and 200 healthy controls (36). An indirect
immunofluorescence assay (IFA) was used to detect antibodies reactive with
a BDV-infected cell line; sera from 12 (4.3%) patients from the United
States and four (< 1%) patients from Germany were immunoreactive; no sera
from controls were immunoreactive. Sera from many of these patients were
subsequently analyzed by a Western immunoblot assay based on BDV
nucleoprotein (N) and phosphoprotein (P) purified by affinity
chromatography from infected rabbit kidney cells (37). In this study of 138
patients with affective disorders and 117 healthy controls, antibodies to N
were found in 53 (38%) patients versus 19 (16%) controls; antibodies to P
were found in 16 (12%) patients versus five (4%) controls; antibodies to
both proteins were found in nine (6.5%) patients versus one (< 1%) control.

Table 1. Serum immunoreactivity to Borna disease virus in various diseases
---------------------------------------------------------------------------
Disease                                  Prevalence          Assay    Ref.
                          Disease    (%)    Controls  (%)
---------------------------------------------------------------------------
Psychiatric               (4/694)    0.6     (0/200)     0     IFA    (36)
(various)                (13/642)      2    (11/540)     2     IFA    (60)
                        (200-350/    4-7        (10/     1     WB/    (61)
                            5000)              1000)          IFA
                          (18/60)     30     (1/100)     1      WB    (24,
                                                                       25)
                         (18/132)   13.6     (3/203)   1.5      WB    (27)
                          (13/55)   23.6      (4/36)  11.1     IFA    (51)
                           (6/49)   12.2                       IFA    (38)
Affective                (12/265)    4.5     (0/105)     0     IFA    (62)
                         (12/285)    4.2     (0/200)     0     IFA    (36)
                         (53/138)     38    (19/117)    16      WB    (37)
                           (6/52)   11.5     (3/203)   1.4      WB    (27)
                          (10/27)     37                       IFA    (38)
Schizophrenia             (29/90)     32      (4/20)    20      WB    (43)
                         (16/114)     14     (3/203)   1.5      WB    (27)
                            (1/4)     25                       IFA    (38)
CFS                        (6/25)     24                        WB    (26)
MS                       (15/114)     13    (10/483)   2.1     IP/    (63)
                                                              IFA
HIV-                     (36/460)    7.8    (11/540)     2     IFA    (60)
positive
HIV-early                (61/751)    8.1    (10/483)     2     IP/    (61)
                                                              IFA
HIV-LAP                  (34/244)     14    (10/483)     2     IP/    (63)
                                                              IFA
Schisto/                 (19/193)    9.8    (10/483)     2     IP/    (63)
malaria                                                        IFA
---------------------------------------------------------------------------
Abbreviations: IFA, immunofluorescence assay; WB, Western immunoblot; IP,
immunoprecipitation; CFS, chronic fatigue syndrome; MS, multiple
sclerosis; HIV, human immunodeficiency virus; LAP, lymphadenopathy;
Schisto/malaria, schistosomiasis or malaria.

To establish a correlation between immunoreactivity to BDV and the duration
and severity of psychiatric disease, Bode et al. performed IFA on multiple
serum samples taken at different times from 71 patients with various
diagnoses including minor or major depression, paranoid psychosis,
schizophrenia, anxiety disorder, and personality disorder (38). Overall,
the prevalence of immunoreactivity to BDV was greater than 20%, a marked
increase from the 2% to 4% found in an earlier study that assayed specimens
from each patient at one time. Thirty-seven percent of patients with major
depression, 25% with paranoid psychosis, and only 6% or fewer with reactive
depression and other neurotic conditions were seropositive by day 17 of
illness. Because BDV gene products have been identified in PBMC of infected
rats (28), PBMC from patients with neuropsychiatric diseases were examined
for viral antigens by fluorescence activated cell sorting analysis (39). Of
the 70 patients tested, more than 40% were antigen carriers, twice the
number predicted by the previous serologic survey.

Some similarities between particular animal models of BDV infection and
schizophrenia have been reported. The subtle signs of disease caused by
infection in neonatal rats (e.g., learning deficiencies, hyperactivity with
CNS abnormalities including cerebellar disorganization and loss of dentate
gyrus granule cells [32,33,40,41]) are consistent with a longstanding
hypothesis that schizophrenia reflects an early brain insult (e.g.,
infection) resulting in abnormal brain development (42) and prompted
investigation of the role of BDV infection in the pathogenesis of
schizophrenia (43). A Western immunoblot assay based on BDV N, P, and
matrix protein (M) purified from infected human neuroblastoma cells was
used to examine sera from 90 schizophrenic patients and 20 healthy
controls.
Antibodies to one BDV protein (N, P, or M) were detected in 29 (32%)
patients and four (20%) controls. Antibodies to two or more BDV proteins
were detected in 13 (14.4%) patients and zero controls. Antibodies to M
protein were found in 12 (13.3%) patients and zero controls.
Immunoreactivity to two or more BDV proteins or M protein was significantly
associated with abnormal brain morphology in magnetic resonance image
analysis (MRI) and the clinical diagnosis of deficit syndrome (a
schizophrenia subgroup characterized by social withdrawal, neurologic
dysfunction, and neuroanatomic abnormalities). Similar findings indicated
an association between antibodies reactive with BDV proteins and MRI
evidence of cerebral atrophy in schizophrenic patients (44).

Molecular Epidemiology (Table 2)

Interest in the potential role of BDV as a human pathogen and in
BDV-infected animals as models for human neuropsychiatric diseases guided
efforts to identify the infectious agent. Because of low viral productivity
and tight association of BDV with plasma membranes, classic methods for
virus isolation have been unsuccessful; however, BDV nucleic acids have
been independently cloned from horse isolates by using a purely molecular
subtractive cloning approach (45,46). The viral genome was subsequently
cloned from viral particles (4) and nuclear extracts of infected cells
(47). With the advent of viral sequence information, new diagnostic
reagents for BDV infection have been introduced, including recombinant
proteins for serology as well as oligonucleotide primers and probes for
molecular epidemiology.

Table 2. Borna disease virus nucleic acid in patients with various
diseases
---------------------------------------------------------------------------
Disease         Tissue                 Prevalence        Divergence* Ref.
                                Disease (%)  Controls(%)
---------------------------------------------------------------------------
Psychiatric                PBMC   (4/6)66.7    (0/10)  0        0-3.6 (22)
(various)                  PBMC  (5/12)41.7    (0/23)  0          0-4 (27)

                           PBMC (22/60)  37   (8/172)4.7               (24,
                                                                       25)

                 PBMC-coculture  (3/32) 9.4     (0/5)  0    0.07-0.83  (23,
                                                                       53)
Affective                  PBMC   (1/3)33.3    (0/23)  0              (27)
                           PBMC   (1/6)16.7    (0/36)  0              (51)
                           PBMC   (0/9)   0                           (56)
Schizophrenia              PBMC  (7/11)63.6    (0/23)  0              (27)
                           PBMC  (5/49)10.2    (0/36)  0              (51)
                           PBMC                               4.2-9.3 (54)
                          Brain   (0/3)   0     (0/3)  0              (55)
                            CSF  (0/48)   0     (0/9)  0              (55)
                           PBMC   (0/9)   0     (0/9)  0              (55)
                           PBMC  (0/26)   0                           (56)
CFS                        PBMC  (3/25)  12                    6.0-14 (26)
Hippocampal
sclerosis                 Brain   (4/5)  80                           (53)
---------------------------------------------------------------------------
Abbreviations: PBMC, peripheral blood mononuclear cells; CSF,
cerebrospinal fluid; CFS, chronic fatigue syndrome.
*Divergence of P-gene nucleotide sequence from common BDV isolates (strain
V [4] and He/80 [9]).

The earliest experiments in BDV molecular epidemiology examined whether
conserved virus sequences could be identified across several host species.
Reverse transcription polymerase chain reaction (RT-PCR), as well as
extensive experimental passage through rabbit and rat brains and cell lines
from various species, was used to amplify and clone coding and noncoding
sequences from virus strains divergent by over 50 years' growth in nature.
Because of inaccuracies in viral RNA-dependent RNA polymerases, most
single-stranded RNA viruses have sequence divergence of 10 to the 3rd power
to 10 to the 4th power per site per round of replication (48-50). Sequence
analysis of BDV isolates showed a much lower rate of divergence. For N and
P sequences, maximum variability was 4.1% at the nucleotide level and 1.5%
at the predicted amino acid level (8). Similar sequence conservation was
later found for sequences from naturally infected donkeys, sheep, and cats
(9, H. Ludwig, pers. comm.). The extent to which sequence conservation in
BDV represents enhanced polymerase fidelity, or more likely, selective
environmental pressures is unknown.

Extending molecular analysis to human materials is both complex and
controversial. If BDV infects neuropsychiatric disease patients, viral
nucleic acids are present at lower concentrations in human brain and PBMC
than in previously studied naturally or experimentally infected hosts
because investigators who report isolation of BDV nucleic acid from human
PBMC must use the highly sensitive technique of nested RTPCR. Groups in
Germany and Japan have detected viral nucleic acid in PBMC of patients with
neuropsychiatric diseases: Bode and colleagues found BDV nucleic acids in
four (66.7%) of six patients (22); Sauder and colleagues found BDV
sequences in 13 (50%) of 26 neuropsychiatric patients (seven patients with
schizophrenia, one with affective disorder, and five with other psychiatric
disorders) (27); Kishi and co-workers found BDV nucleic acids in PBMC of 22
(37%) out of 60 neuropsychiatric patients (24) and eight (4.7%) out of 172
blood donor controls (25); Igata-Yi et al. detected BDV nucleic acids in
six (10.9%) of 55 neuropsychiatric patients (five patients with
schizophrenia, one with depression) versus zero of 36 blood donor controls
(51); and Nakaya et al. reported BDV nucleic acid in three (12%) of 25
chronic fatigue syndrome patients (26). Additionally, BDV nucleic acids and
immunoreactivity have been detected in the hippocampus of four of five
North American patients with postmortem diagnosis of hippocampal sclerosis
(52).

Genomic analyses of BDV isolates from human patients have yielded differing
estimates of nucleotide sequence conservation (Table 2). Extensive
conservation of the N and P open reading frames, consistent with previous
findings in field and tissue culture isolates (8), has been reported
(22,27). Infectious BDV has been isolated after cocultivation of PBMC from
neuropsychiatric patients with a human oligodendroglial cell line (23).
Sequences of these isolates were highly conserved with previously
identified sequences (53); other groups have described greater sequence
divergence (26,54). However, differences in levels of sequence conservation
may reflect variations in methods for RT-PCR amplification rather than true
strain differences (27).

In two reports RT-PCR did not yield evidence of BDV nucleic acid in
neuropsychiatric disease patients (Table 2): No BDV nucleic acid was found
by RT-PCR analysis of brain tissue, cerebrospinal fluid, or PBMC from
patients with schizophrenia (55); similarly, no BDV nucleic acid was found
in RT-PCR studies of PBMC from 26 patients with schizophrenia and nine with
affective disorders (56).

Special Considerations for BDV Diagnostics

Although a wide variety of assays (IFA, Western immunoblot,
radioimmunoprecipitation, and enzyme-linked immunosorbent assay) and
antigen preparations (infected cells, infected cell extracts, and
recombinant proteins produced in prokaryotic or baculovirus systems) have
been used for BDV serology, generally accepted standards for diagnosis of
human BDV infection have not been established. Because only limited data
concerning interassay comparability for serology within individual
laboratories (and none between different laboratories) have been
established, discrepancies between investigators may reflect differences in
clinical populations, assay sensitivity, or other factors.

Similarly, agreement concerning methods for molecular diagnosis of BDV
infection is limited. Most investigators use RT-PCR; some use a method
sensitive to 100 to 300 copies of RNA template (nested RT-PCR, 80 to 100
cycles), while others use less sensitive methods (no nesting, 30 cycles).
Only investigators using the more sensitive method have reported BDV gene
products in human materials (PBMC, brain).

RT-PCR, particularly nested RT-PCR, is prone to artifacts because of
inadvertent introduction of template from laboratory isolates or
cross-contamination of samples. Because putative human isolates detected by
nested RT-PCR are similar in sequence to known animal and tissue culture
isolates, it has been argued that they represent low-level contaminants.
However, the finding of sequence conservation is consistent with previous
analyses of well-characterized isolates disparate by host species and
geography (8,9) and cannot be used to discount the validity of positive
nested RT-PCR results.

Future Directions

The broad potential host range of BDV suggests that humans are targets for
infection. Rodents with persistent BDV infection and minimal overt signs of
disease and domestic animals and livestock could serve as vectors. Serum
antibodies reactive with BDV have been detected in asymptomatic farmworkers
exposed to ostriches with a Borna disease-like syndrome (57).
Immunoreactivity to BDV and neurologic disturbances have been reported in a
farm worker exposed to seropositive asymptomatic horses and sheep (58).
Finally, although no human cases of disease have been linked to feline
infection, there is evidence of BDV infection in house cats in Europe (59)
and Japan (18). However, even though BDV could infect humans and is likely
to do so, a number of questions remain unanswered: The sources and routes
of potential human infection are not clear, no detailed epidemiology has
been done in animal populations, and transmission from domestic animals to
humans has not been demonstrated.

Viral gene products are readily detected in CNS of natural and experimental
hosts without such sensitive methods as nested RT-PCR. The need to use
sensitive methods to detect BDV nucleic acid in humans indicates that the
virus is present only at low levels. Thus, if BDV can be implicated as a
factor in human neuropsychiatric disease, mechanisms for pathogenesis may
be different from those found in other natural and experimental hosts.
Although a higher prevalence of markers for BDV infection has been reported
in neuropsychiatric patients than in controls, no single neuropsychiatric
disease has been correlated with BDV infection. Efforts to link BDV with
neuropsychiatric disease have not used accepted epidemiology standards.

To rigorously address the issues of BDV epidemiology and pathogenesis,
multicenter groups in Europe and the United States are collaborating to
collect and perform blinded analysis of human clinical materials by
standardized methods and reagents. The objectives of these projects will be
to determine the prevalence of serum antibodies to BDV in patients and
controls and the extent to which various assays for antibodies are in
accord, the prevalence of BDV nucleic acids in brains and PBMC of patients
and controls, and the correlation between antibodies to BDV or viral
nucleic acids and a particular neuropsychiatric disease.

Acknowledgments

The Laboratory for Neurovirology is supported by a grant from the National
Institutes of Health (NS29425), as well as awards from the Wayne and Gladys
Valley Foundation, Cure Autism Now Foundation, and Lucille P. Markey Trust.

Dr. Hatalski is a postdoctoral fellow in the Department of Anatomy &
Neurobiology, University of California, Irvine, California, USA.

Address for correspondence: W. Ian Lipkin, Laboratory for Neurovirology,
Department of Neurology, University of California, Irvine, CA 92697-4290;
fax: 714-824-1229; e-mail: ilipkin@uci.edu.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Synopses

The Rickettsia: an Emerging Group of Pathogens in Fish

John L. Fryer and Michael J. Mauel
Oregon State University, Corvallis, Oregon, USA
---------------------------------------------------------------------------


      Piscirickettsia salmonis is the first of the previously
      unrecognized rickettsial pathogens of fish to be fully
      characterized. Since the recognition of P. salmonis in 1989,
      the impact of rickettsial pathogens in fish has become
      increasingly apparent. Growing awareness of the emergence of
      these fastidious intracellular organisms has led to the
      discovery of rickettsial diseases among diverse species of fish
      from different geographic locations and aquatic environments.
      The source, reservoir, and mode of transmission of these agents
      as well as appropriate methods of disease prevention and
      control remain to be established.

The Emergence of Rickettsial Diseases in Fish

The earliest report of a rickettsialike organism in fish occurred in 1939
during examination of diseased Tetrodon fahaka from the Nile River in
Egypt. The microorganisms were observed within stained tissue cells, but no
attempts were made to culture them (1). In 1975 Ozel and Schwanz-Pfitzner
(2) first cultured rickettsialike organisms from fish while examining
rainbow trout (Oncorhynchus mykiss) for Egtved virus. While cultivating the
virus in RTG-2 cells (3), they observed that cells also contained an
intracellular rickettsialike organism. However, research on the organism
was limited, and its taxonomic position and relevance to disease were not
determined. The microorganism was not maintained and is no longer available
for evaluation. No further reports of rickettsia in fish appeared until
1986 when Davies observed rickettsialike organisms in the tissue of
dragonets (Callionymus lyra L.) collected in waters off the coast of Wales
(4) (Table 1).

The role of rickettsiae as emerging pathogens of fish became apparent in
1989 (5), when a previously unrecognized bacterium (6) was isolated in the
chinook salmon (Oncorhynchus tshawytscha) embryo cell line, CHSE-214 (7)
and was demonstrated to be the cause of epizootics in marine-netpen-reared
coho salmon species and (Oncorhynchus kisutch) in Region X, Chile (8).

Table 1. Unidentified rickettsialike organisms observed in and/or isolated
from fish
---------------------------------------------------------------------------
Fish host                     Geo.         Salt/Fresh      Obs./   Ref.
species                       location     Water           Iso.
---------------------------------------------------------------------------
Fokaka                        Egypt        F(a)            O(c)    1
(Tetrodon fahaka)
Rainbow trout                 Europe       F               I(d)    2
(Oncorhynchus mykiss)
Dragonet                      Wales        S(b)            O       5
(Callionymus lyra)
Masu salmon                   Chile        S               O
(Oncorhynchus masou)(e)
Mozambique tilapia            Taiwan       S & F           I       15
(Oreochromis mossambicus)
Nile tilapia
(O. niloticus)
Blue tilapia
(O. aureus)
Redbelly tilapia
(Tilapia zillii)
Wami tilapia
(T. hornorum)
Blue-eyed plecostomus         Colombia     F               O       16
(Panaque suttoni)
Atlantic salmon               Chile        F               I       19
(Salmo salar)
Sea bass                      France       S               O       17
(Dicentrarchus labrax)
---------------------------------------------------------------------------
(a)F = Host collected from fresh water
(b)S = Host collected from salt water
(c)O = Observed only
(d)I = Isolated in cell culture
(e)Sandra Bravo, Puerto Montt, Chile

Piscirickettsia salmonis, was the first rickettsialike organism isolated
from fish, characterized, and demonstrated to cause disease in
experimentally infected hosts (6). Initially, rickettsial infections of
fish were thought to be confined to coho salmon in southern Chile, but the
agent and the disease it causes have since been observed in other locations
(Table 2). In British Columbia, Brocklebank et al. (9,10) observed that
sea-farmed Atlantic salmon (Salmo salar) also manifested pathologic
features indistinguishable from those of salmonids in Chile. The causation
of the disease was established by experimental infection followed by
isolation in CHSE-214 cells. The organism was morphologically similar to P.
salmonis, but comparatively less virulent for salmonids. These researchers
also provided anecdotal reports that the same condition occurred as early
as 1970 in pink salmon (Oncorhynchus gorbuscha) held in sea water tanks in
British Columbia; beginning in the 1980s, the condition was also seen in
coho and chinook salmon from local sea farms rearing salmonids. Rickettsiae
morphologically similar to those implicated in the diseases in Chile and
Canada also caused low level mortality in Atlantic salmon held in sea water
cages in Norway (11) and Ireland (12). We have identified all of these
agents as P. salmonis by direct fluorescence antibody tests using the
polyclonal antiserum (unpub. data; 13).

Until 1994, all recognized rickettsial diseases of fish had been observed
in species of salmonids cultured in sea water. However, it is now apparent
that these pathogens affect fish over a broad host and geographic range and
in both fresh water and marine environments. Chern and Chao (14) reported
an epizootic rickettsial disease in several species of tilapia in both
marine and fresh water ponds in Taiwan (Table 1). Death rates associated
with this disease reached 95% at certain sites. Khoo et al. (15) reported a
rickettsialike organism in moribund specimens of a fresh water tropical
fish, the blue-eyed plecostomus (Panaque suttoni), shipped to the United
States from Colombia. In France, Comps et al. (16) found rickettsialike
organisms in the brain of moribund juvenile seabass (Dicentrarchu labrax)
exhibiting abnormal swimming behavior and in Chile, Gaggero et al. (17)
isolated P. salmonis from diseased coho salmon and rainbow trout held only
in fresh water. A different, and as yet unidentified, bacterium has been
isolated in fish cell cultures from diseased Atlantic salmon in Chile that
had been reared only in fresh water.

Table 2. Piscirickettsia salmonis LF-89(T) observed in and/or isolated
from fish
---------------------------------------------------------------------------
                Fish                              Salt
                Host                    Geo.      Fresh    Obs./
              Species                 Location    Water    Iso.     Ref.
---------------------------------------------------------------------------
Coho salmon                        Chile        S(a)    I(c)      6
(Oncorhynchus kisutch)
Rainbow trout                      Chile        S       O/I(d)    9
(Oncorhynchus mykiss)
Chinook salmon
(Oncorhynchus tshawytscha)
Atlantic salmon
(Salmo salar)
Chinook salmon                     British      S       O/I       11
                                   Columbia
Atlantic salmon                    Canada
Coho salmon
Pink salmon
(Oncorhynchus gorbuscha)
Atlantic salmon                    Ireland      S       O(e)      13
Atlantic salmon                    Norway       S       O/I       12
Coho salmon                        Chile        F(b)    I         18
Rainbow trout
---------------------------------------------------------------------------
(a) S = Host fish collected from salt water
(b) F = Host fish collected from fresh water
(c) I = Isolated in cell culture

The organism is smaller than P. salmonis, ca. 0.2-0.8 vs. ca. 0.5-1.5 in
diameter, and both are usually coccoidal in shape. The unidentified
bacterium was intracellular in spleen and kidney, did not grow on several
types of bacteriologic media, and reportedly did not react with polyclonal
antibodies against P. salmonis (18). The rickettsialike organisms observed
in and/or isolated from nonsalmonid fish have not been characterized
sufficiently to determine their relationship to P. salmonis. Taxonomic
placement of these agents requires additional study.

Neorickettsia helminthoeca, the cause of the "salmon poisoning" disease of
canids, is associated with fish but is not a fish pathogen. It is carried
by the digenetic trematode, Nanophyetus salmincola, a parasite of salmonid
fish in the Pacific Northwest (19). N. helminthoeca has been cultured in
canine and murine cells but does not grow in cells of salmonid fish (20).
Phylogenetically, N. helminthoeca is more closely related to the ehrlichiae
and the rickettsia than it is to P. salmonis based on an analysis using the
April 3, 1997, 16S rRNA gene sequence database containing 6,000 sequences.
In addition, when P. salmonis was tested with N. helminthoeca antiserum by
indirect immunofluorescence, no reaction was observed (6).

There is no indication that P. salmonis or other rickettsial pathogens of
fish cause disease in humans or other warm blooded animals. We speculate
that the optimum temperature of 15C to 18C with no growth at 25C and
above prevents P. salmonis from becoming established in warm-blooded
animals.

P. salmonis

Taxonomic Position

P. salmonis is the best characterized of the rickettsiae observed in and/or
isolated from fish. The type species LF-89(T) was isolated from a diseased
coho salmon from a sea water netpen site in southern Chile during an
epizootic. The organism has been placed in a new genus in the order
Rickettsiales, family Rickettsiaceae based primarily on their similar
morphology and obligate intracellular nature (6). It has been deposited
with the American Type Culture Collection as ATCC VR 1361.

Many rickettsiae observed in or isolated from fish since 1989 have been
identified serologically as P. salmonis (Table 2). We tested isolates from
coho salmon and rainbow trout from Chile and from Atlantic salmon reared in
Chile; British Columbia, Canada; and Norway. All five isolates are
morphologically similar to LF-89(T) and react with polyclonal antibodies
against the type strain. Certain monoclonal antibodies developed in our
laboratory can differentiate between these isolates (unpub. data). The 16S
ribosomal DNA of these five isolates from three geographic locations were
amplified by PCR to assess the genetic variability in this species or
species-complex. The PCR products were sequenced and compared with other
bacterial small subunit rRNA sequences. The genus Piscirickettsia belongs
within the gamma subdivision of the proteobacteria and is not closely
related to species of Erlichiae, Rickettsia, or Neorickettsia from the
subdivision of alpha proteobacteria (Figure 1). The similarities between
Piscirickettsia, Ehrlichiae, and Rickettsia species are approximately 80%.
Similarities between Piscirickettsia, Coxiella, and Wolbachia are
approximately 86% to 88%. The five P. salmonis isolates form a tight
monophyletic cluster with relatedness of 99.7% to 98.5%. Two of the
isolates from Chile and those from Norwegian and Canadian fish are closely
related (>99.4% similarity). One isolate from Chile, EM-90, differed from
the other four isolates (21). Similarity values for EM-90 were 98.5% to
98.9%.

[Figure 1] [Figures not available in ASCII version]
                               2% divergence
Figure 1. Phylogenetic relationships of Piscirickettsia salmonis, selected
rickettsiae and bacteria. Evolutionary distances were calculated by the
method of Jukes and Cantor (34). After eliminating regions of ambiguity
and uncertain homology 1,313 positions of the 16S rDNA gene were compared.

To further clarify the genetic variability in this genus, the internal
transcribed spacer (ITS) and 23S rDNA of six isolates have been analyzed.
One spacer sequence was identified per isolate, and the region did not
contain a tRNA gene. The ITS sequences were 311-bp in length and varied
among the isolates (95.2% to 99.7% similarity). Only one ITS sequence was
obtained for each of the P. salmonis isolates, suggesting the presence of
one rRNA operon, which agrees with reports for other slow growing organisms
(22).

Approximately 1,900-bp of the 23S rDNA gene have been analyzed for the six
isolates, and similarities ranged from 97.9% to 99.8%. Three Chilean
isolates and the Norwegian and Canadian isolates are closely related (99.1%
to 99.7% ITS and 99.3% to 99.8% 23S rDNA similarities). The sequence of
isolate EM-90 differed from those of the other five isolates (similarities
ranged from 95.2% to 96.9% ITS and 97.6% to 98.5% 23S rDNA).

Phylogenetic trees constructed with the 16S, ITS, and 23S rDNA data showed
similar topography, further reinforcing the relationships indicated between
the isolates. Comparison of the P. salmonis ITS with the 16S rDNA region
shows that it has diverged on average 3.15 times faster than the 16S rDNA
gene, while the 23S rDNA gene has diverged 1.6 times faster than the 16S
rDNA gene. This is similar to findings with other obligate intracellular
bacteria (23).

Characteristics of the Type Strain LF-89(T)

P. salmonis, type strain LF-89(T) is a nonmotile, gram-negative,
obligatedly intracellular bacterium. It is predominately coccoid (ca.
0.5-1.5 mm in diameter) and also occurs as rings or pairs of curved rods
(Figure 2). It replicates within membrane-bound cytoplasmic vacuoles
(Figure 3) in selected fish cells lines and in the cells of tissues
throughout infected fish. Thin sections of the bacterium examined by
electron microscopy display typical gram-negative cell walls and the
protoplasmic structure of a prokaryote. Giemsa stains the rickettsiae cells
dark blue. LF-89(T) does not react with the monoclonal antibody made
against the group-specific chlamydial LPS antigen (5).

             [Image] [Figures not available in ASCII version]

             Figure 2. Piscirickettsia salmonis within a
             cytoplasmic vacuole in CHSE-214 cell line 4 days
             post inoculation. Note organisms dividing within
             vacuole. May Greenwald-Giemsa stain. Bar = 1 m.

                 [Image] [Figures not available in ASCII version]

                 Figure 3. Piscirickettsia salmonis
                 undergoing apparent binary fission within
                 a vacuole in the cytoplasm of infected
                 CHSE-214 cells. Bar = 10 m.

P. salmonis can be cultivated in certain fish cell lines where it produces
a cytopathic effect. It does not replicate on any known cell-free media,
and its in vitro growth characteristics have been described (6).
Replication is optimal at 15C to 18C, retarded above 20C and below 10C,
and does not occur above 25C. It replicates to titers of 10 to the 6th
power to 10 to the 7th power TCID50 ml to the -lth power in fish cell
cultures. Titers are decreased 99% or more by a single cycle of freeze-thaw
at -70C. The addition of 10% DMSO to the freezing medium acts as a
cryopreservative. In vitro, it is sensitive to a broad range of
antibiotics.

Tenfold dilutions of spent medium from an LF-89(T)infected cell culture
injected into groups of 40 juvenile coho salmon and groups of 30 juvenile
Atlantic salmon resulted in death rates of 88% to 100% during a 42-day
experiment (8). Typical signs of the disease were present in the inoculated
experimental coho, whereas in Atlantic salmon, the only gross sign of
disease was death. The LD50 was not obtained, but death in both species
followed a dose-response pattern, and LF-89(T) was reisolated from each
group inoculated.

Extracellular survival of LF-89(T) was tested under selected environmental
conditions (24); infectivity remained for at least 14 days in preparations
of semipurified LF-89(T) suspended in high salinity sea water at 5C, 10C,
and 15C. Infectivity was rapidly reduced in preparations suspended in
fresh water. Titers dropped below the level of detection [10 to the 2th
power TCID(subscript 50)/ml] immediately after suspension in fresh
water, and no infectious material could be recovered from these
preparations.

Piscirickettsiosis

P. salmonis produces an epizootic disease of fish called
piscirickettsiosis. Death rates associated with piscirickettsiosis in
salmonids range from a high of 90% among coho in Chile (25) to a low of
0.06% in Canada and Norway (9,11). All species of salmonids cultured in
Chile are affected by this disease, but the highest death rates occur in
coho salmon cultured in salt water. Fish in sea water netpens begin to die
6 to 12 weeks after their transfer from fresh water (5). Deaths peak in the
fall and rise again the following spring (26).

A variety of clinical signs are associated with P. salmonis infection, but
few are specific to piscirickettsiosis. Moribund fish collect at the water
surface along the edges of the sea cages; they are lethargic, dark, and
show loss of appetite (9); the gills are pale, and hematocrits are
frequently 25% or less. The first signs observed are often hemorrhages and
lesions of the skin. The lesions range from small areas to shallow ulcers
up to 2 cm in diameter (25). Internally, the kidney is swollen and the
spleen enlarged. Petechial hemorrhages are found on the swim bladder and
viscera. Diagnostic ringshaped, cream-colored lesions are present on the
livers of chronically infected fish (Figure 4). In acute cases, death may
be the only gross sign of disease (27).

The histopathologic symptoms associated with P. salmonis infection and the
disease it causes have been described, but a great deal of work remains to
be done (25-27). Piscirickettsiosis produces marked pathologic changes in
most internal organs of infected fish, where severe changes occur in the
intestine, kidney, liver, and spleen. Necrosis and inflammation may occur
throughout the body, especially in cells adjacent to blood vessels.
Epithelial hyperplasia results in lamellar fusion of the gills. The
bacterium is commonly observed within macrophages and in the cytoplasm of
infected host cells.

             [Figure 4] [Figures not available in ASCII version]

             Figure 4. Coho salmon infected with
             Piscirickettsia salmonis. Note cream- colored
             lesions on liver, enlarged spleen, pale gills,
             and hemorrhaged areas within the peritoneal
             cavity.

Mode of Transmission

The source, reservoir, and means of transmission of P. salmonis continue to
be important areas of study. Although piscirickettsiosis has been observed
in salmonid fish over a widespread geographic area (Table 2), with the
exception of the predictable annual epizootics in Chile, the infections
seem sporadic and of limited virulence in other parts of the world. No
association other than the marine environment and the host species is
apparent between locations in Canada, Norway, and Ireland. One or more
naturally occurring aquatic animals, perhaps only transiently present, may
provide the source and reservoir of these rickettsiae in the widely
separated diverse areas. No alternate host has been identified.

P. salmonis has been experimentally passed by injection (8,26), but the
normal mode of transmission for this agent (horizontal, vertical, or
through a vector) has not been demonstrated. Except in Coxiella burnetii,
an intermediate host or vector is required for transmission of rickettsiae
in the terrestrial environment (28). C. burnetii not only has an
intermediate host but appears to develop a sporogenic phase that protects
it from drying when it is host or vector free. No vector or sporogenic
phase has been observed for P. salmonis. P. salmonis may not require an
intermediate host or sporogenic stage for survival and transmission in the
aquatic environment. The extended extracellular survival time of this
organism in salt water (24) may be of sufficient duration to permit
horizontal transmission without a vector. The almost immediate deactivation
of the rickettsia in fresh water makes direct horizontal transmission
unlikely unless P. salmonis was protected by host(s) cell membranes or
tissue exudates.

Limited research designed to demonstrate the role of horizontal
transmission has provided mixed results. Garcs et al. (8) saw no evidence
of horizontal transmission between injected coho salmon dying of
piscirickettsiosis and uninjected salmon held in a cage in the same tank of
flowing fresh water. Cvitanich et al. (26) reported horizontal transmission
between injected and sham-injected coho salmon held in static fresh and sea
water aquaria. Environmental conditions differed between experiments (e.g.,
flowing water at a mean temperature of 10.5C vs. static water at 15C),
therefore, meaningful comparisons could not be made. Nevertheless, this
research suggests that under certain circumstances, horizontal transmission
is possible.

Although rickettsiae are found in the gonads of infected fish, vertical
transmission of P. salmonis has not been demonstrated. The limited number
of piscirickettsiosis cases reported in fresh water indicate that vertical
transmission is, at best, rare. Until definitive studies are conducted, the
questions of source, reservoir, and normal mode of transmission of P.
salmonis remain unanswered.

Detection and Identification

Inoculation of susceptible fish cell lines is the most sensitive method for
detecting P. salmonis (29). However, isolation of an infectious agent
sensitive to low levels of antibiotics routinely used in cell culture
presents a problem. All cultures must be maintained in antibiotic-free
medium.

Diagnostic specimens collected aseptically in the field may become
contaminated by other bacteria. For this reason, preliminary diagnosis of
piscirickettsiosis is normally made by examining Gram, Giemsa, methylene
blue, or acridine orange-stained kidney imprints or smears, and confirming
their identity by serologic methods, e.g., immunofluorescence (13) or
immunohistochemistry (30).

A nested polymerase chain reaction (PCR) using universal 16S rDNA bacterial
outer primers and P. salmonis-specific internal primers was developed to
detect the genomic DNA. The nested PCR assay allowed detection of less than
one P. salmonis tissue culture infectious dose 50 (TCID50). Using the P.
salmonis-specific primers in a single amplification allowed detection of 60
TCID50. The specificity of PCR was assessed with a panel of four salmonid
and 15 bacterial genomic DNA preparations. Products derived from
amplification were observed only from P. salmonis DNA (31).

Restriction fragment length polymorphism analysis of the 16S rDNA products
from six isolates of P. salmonis demonstrated that one isolate, EM-90, was
different. Two additional primers were developed that differentiate EM-90
from the other five P. salmonis isolates (31).

PCR using DNA extracted from spleens of tilapia or paraffin-embedded tissue
of the blue-eyed plecostomus did not produce amplification products. These
tissues were collected and examined from fish during an epizootic caused by
rickettsialike organism (unpub. data). Amplification using 16S rDNA
universal primers was successful, which suggests that the rickettsialike
organism infecting these fish were not P. salmonis.

Control of the Disease

Although P. salmonis is sensitive in vitro to many of the antibiotics
commonly used to control other infectious diseases of fish, e.g.,
tetracycline, erythromycin, and oxolinic acid (6,26), these preparations
have not been useful in treating fish with piscirickettsiosis. Antibiotic
levels may not reach sufficient concentrations within the host cells in
vivo, to terminate replication of the pathogen.

The lack of effective methods for treating piscirickettsiosis has
encouraged the development of disease prevention techniques. Vaccines have
been successfully used for control of certain gram-negative bacterial
diseases of fish; however, at present no efficacious preparations have been
developed to protect fish against P. salmonis.

Questions remain concerning P. salmonis and the disease it causes in fish.
The reservoir(s) of the infectious agent should be determined if the spread
of the disease is to be controlled (32). There is a need to determine the
mode(s) of transmission of P. salmonis to fully understand the pathogenesis
of piscirickettsiosis. The infectivity of P. salmonis for native,
nonsalmonid fish has not been investigated. Difference in virulence between
P. salmonis in Chile and salmonids in the Northern Hemisphere is an
important consideration. It must be determined if intrinsic differences in
the isolates, the host fish, the environment, or some combination of
factors is responsible for these differences. The rickettsiae from salmonid
fish in the fresh water and marine environments should be compared, and the
relationships between the rickettsiae from salmonid fish and those from
other fish species should be determined.

A new group of fish pathogens was recognized in 1989 with the isolation and
identification of P. salmonis LF-89T from cultured salmonids in Chile
(5,6). The rickettsial etiology of an infectious disease of fish in a
variety of locations and host species suggests that they are an important
group of emerging fish pathogens (33). These pathogens have caused severe
mortality among cultured salmonids in Chile and are not present among
stocks of important food fish cultured in British Columbia, Canada, Norway,
and Ireland. Taiwan has experienced serious losses amon talapia, another
important food fish. At least one species imported for aquaria are also
infected with a rickettsialike agent. The lack of antimicrobial drugs and
vaccines for prevention and control emphasize the need for additional
research. The method of transmission of P. salmonis is not understood and
needs to be researched. The movement of fish and spread of the agent make
the develoment of improved diagnostic techniques important.

Acknowledgments

We thank Dr. Sandra Bravo of Puerto Montt, Chile, for the photograph of a
diseased fish in Figure 4. Preparation of this paper was supported in part
by the Oregon Sea Grant with funds from the National Oceanic and
Atmospheric Administration Office of Sea Grant, Department of Commerce,
under grant NA36RG0451, project R/FSD-22. This is Oregon Agricultural
Experiment Station Technical Paper Number 11,090.

Dr. Fryer has been a faculty member of the Department of Microbiology at
Oregon State University (OSU) for more than 35 years, serving as chair for
20 of those years. His research has focused primarily on the infectious
diseases of Pacific salmon and other species of fish. He is professor
emeritus and director of the Center for Salmon Disease Research at OSU.

Dr. Mauel received his degree from the Department of Microbiology at Oregon
State University in 1996 and is a postdoctoral fellow at the Center for
Vector-borne Disease, University of Rhode Island. His scientific interests
concern the molecular biology of fastidious intracellular pathogenic
bacteria.

Address for correspondence: John L. Fryer, 220 Nash Hall, Department of
Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA; fax:
541-737-2166; e-mail: fryerj@bcc.orst.edu.

References

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Synopses

Rhodococcus equi and Arcanobacterium haemolyticum: Two "Coryneform"
Bacteria Increasingly Recognized as Agents of Human Infection

Regina Linder
Hunter College, New York, New York, USA
---------------------------------------------------------------------------


      Rhodococcus equi and Arcanobacterium haemolyticum, formerly
      classified in the genus Corynebacterium, are members of the
      loosely defined taxon "coryneform" bacteria. Although they are
      the etiologic agents of distinct human infections, both
      organisms are frequently overlooked, which results in missed or
      delayed diagnoses. R. equi, long known as an important pathogen
      of immature horses, has become in the past three decades an
      opportunistic pathogen of severely immunosuppressed humans.
      Most cases are secondary to HIV infection. When specifically
      sought in throat swab cultures, A. haemolyticum is found
      responsible for 0.5% to 2.5% of bacterial pharyngitis,
      especially among adolescents. These two microorganisms
      represent a spectrum of disease in humans: from a mild, common
      illness to a rare life-threatening infection. Each organism
      elaborates lipid hydrolyzing enzymes (cholesterol oxidase by R.
      equi and sphingomyelinase D by A. haemolyticum) that are toxic
      to animals and humans and damaging to mammalian cell membranes.
      The participation of the cytotoxins in pathogenicity is
      discussed. Greater awareness of the properties of these two
      bacteria may promote faster, more accurate diagnoses and better
      clinical management.

A variety of factors contribute to the underreporting of human infections
caused by bacteria in the genus Corynebacterium and closely related genera.
The group, often referred to as "coryneform," comprises taxonomically
diverse gram-positive rods resembling Corynebacterium diphtheriae and
displaying pleomorphism and irregular cellular arrangements (1,2). The
group includes human and animal pathogens, as well as commensal bacteria.
The control of diphtheria in industrialized countries and the subsequent
deemphasis of the genus Corynebacterium have contributed to discounting
isolates characteristic of the genus as contaminants. Even reference
laboratories report difficulty in the speciation of gram-positive
pleomorphic rods that resemble corynebacteria (1). Because of the emergence
of a number of coryneform bacteria as important human pathogens, rigorous
biochemical and molecular tools have increasingly been applied to isolates.
The resulting improved epidemiology and taxonomy have led, for example, to
the definition of CDC groups JK and D-2 in the genus Corynebacterium, now
recognized as important opportunistic pathogens (1). Similarly, more
accurate characterization of some species caused them to be removed from
the genus Corynebacterium. Excellent reviews of the pathogenicity and
epidemiology of these diverse genera have been published (1,2). This
article explores two pathogenic coryneform bacteria: Rhodococcus equi, a
rare often fatal human pathogen, in which virtually all human infections
occur among compromised hosts; and Arcanobacterium haemolyticum, which is
responsible for many respiratory infections in healthy people. This article
aims to bring about improved recognition of these two easily overlooked
pathogens and considers mechanisms underlying the diseases, the immune
response of the hosts, and treatment protocols.

Eidemiology and Clinical Presentation

R. equi

Originally isolated by Magnusson in 1923 from granulomatous lung infections
in young horses (3), Corynebacterium (now Rhodococcus equi) remains an
important pathogen of foals. Much of the considerable body of knowledge
about R. equi, including its pathogenicity and immune response to
infection, derives from veterinary studies and has been recently updated
(4).

R. equi is readily found in soil, especially where domesticated livestock
graze (5). The stool of horses and other animals is the source of soil
contamination. Infection in humans derives from environmental exposure
(2,5), and the organism may be ubiquitous in soil (6). While early cases
occurred mostly in persons with a history of contact with horses, only 20%
to 30% of recent cases can be traced to such contact (7). A review of cases
in the three decades since the first reported human infection in 1967 is
presented in Table 1.

 Table 1. Rhodococcus equi case reports in humans: 1967-1996
 -------------------------------------------------------------------------
                                           Primary
                                            site
                                             of
                Predisposing condition    infection
  Years  Cases         (number)           (number)  Deaths  References(a)
 -------------------------------------------------------------------------
 1967-76 7     Corticosteroid (1)        Lung (6)   0      8-10
               Cancer/immunosuppressant  Lymphatic
               (3)                       (1)
               Renal transplant (2)
               None(b)(1) 

 1977-86 15    Corticosteroid (1)        Lung (14)  8      8-10,22
               Cancer/immunosuppressant
               (4)                       Blood (1)
               Renal transplant (2)
               HIV (7)
               Alcoholism (1)

 1987-96 93    Cancer/immunosuppressant  Lung (72)  34     8-13,22
               (8)

               Renal transplant (3)      Lymphatic
                                         (2)
               HIV (67)                  Blood (8)
               Other(c) (8) (1(b))       Wound (6)

               None (7) (4(b))           Other(d)
                                         (5)
 -------------------------------------------------------------------------
 (a)In the interest of space, case compilations are cited in lieu of
 individual case reports.
 (b)Child
 (c)Includes intravenous drug use, lab infection, emphysema, kidney
 disease.
 (d)Central nervous system, gastrointestinal

R. equi is a rare opportunistic pathogen found in severely compromised
patients, and most commonly in recent years, in human immunodeficiency
virus (HIV)-infected persons. Early cases, most in patients receiving
immunosuppressant therapy, were more likely to be successfully treated with
antimicrobial agents than cases in AIDS patients (8). Most often, patients
have a slowly progressive granulomatous pneumonia, with lobar infiltrates,
frequently developing to cavitating lesions visible on chest x-ray. Other
sites of infection include abscesses of the central nervous system, pelvis,
and subcutaneous tissue, and lymphadenitis (7,8-10). Cases of lung
infection caused by inhalation and cutaneous lesions caused by wound
contamination have been documented; the latter are almost the only R. equi
infections reported in healthy persons, frequently children (11). Delays in
accurate diagnosis of R. equi are still common (2,7), despite increased
awareness of this organism as an opportunistic pathogen in humans. Factors
for delayed diagnosis include the insidious onset of disease, clinical
resemblance of the infection to mycobacterial, fungal, and actinomycotic
infections, and the relatively nondescript bacteriologic profile of R.
equi. Morphology, partial acid fastness, and a distinctive histopathologic
profile in bronchial specimens (Figure 1 A and B) contribute to accurate
diagnosis. Numerous polymorphonuclear leukocytes with intracellular
pleomorphic gram-positive bacteria, microabscesses, pseudotumors, and
malakoplakia are noted on tissue (7,11). Malakoplakia is a relatively rare
granulomatous inflammation not typically associated with histology of lung
infection and can be of help in forming a differential diagnosis (11,12).
Firm diagnosis and differentiation from similar pathogens require the
isolation and identification of R. equi from sputum, bronchial washings,
open-lung biopsy, or other specimens reflective of pathology. Blood
cultures from severely immunosuppressed patients with focal R. equi
infection often contain the organism. In sixty-five percent of cases
secondary to HIV infection, the organism is found in patients' blood
cultures (11). Deaths exceed 50% among AIDS patients with documented R.
equi pneumonia and are almost always preceded by multiple relapses, which
are common even when successful treatment is ultimately achieved (Table 1;
13).

                   A[Figure 1A] [Figures not available in ASCII version]

                   B[Figure 1B] [Figures not available in ASCII version]

                   Figure 1. A. Bronchial tissue Gram
                   stain showing intrahistiocytic
                   coccobacillary forms of Rhodococcus
                   equi. Original magnification, x
                   1,000. B.Open lung biopsy showing
                   coalescent microabscesses with
                   numerous histiocytes containing
                   Rhodococcus equi organisms. PAS
                   stain. Original magnification x 250.
                   Figure provided by Dr. Margie Scott,
                   Vanderbilt University Medical
                   Center.

Corynebacterium haemolyticum

A. haemolyticum was first described and named by MacClean et al. (14), who
isolated it from pharyngeal infections in U.S. soldiers and natives in the
South Pacific. Classification of the organism generated controversy until
the definition in 1982 of a new genus, Arcanobacterium (secretive
bacterium), in which it remains the only species (15).

Unlike R. equi infection, where invasive clinical disease underscores the
need to detect and identify the causative agent of infection, A.
haemolyticum infection is often reported from deliberate screening for the
organism of a large number of patients with sore throats. After it was
identified during World War II from patients with pharyngitis (14), it was
occasionally reported from Europe, the United States, and in 1981, Sri
Lanka (16 cases) (16). Most cases involve pharyngitis and/or tonsillitis,
and approximately 50% are exudative. Throat infections are often
accompanied by cervical lymphadenopathy (17,18). Diagnosis of cases
(distinct from screening studies) often occurs only after recurrent
infections, which are thought to be related to incorrect initial diagnosis,
resulting in less-than-optimum treatment (19). Infection is most common in
15- to 25-year-old persons, and is thought to result from droplet transfer
from infected persons (20). Symptoms resemble those of -hemolytic
streptococci or viral infection. The spectrum of disease ranges from sore
throat to, in rare cases, a life-threatening membranous pharyngitis
resembling diphtheria (18,20). An erythematous morbilliform or scarlatinal
rash of the trunk, neck, or extremities is associated with 20% to 25% of
cases (19), enhancing the possibility of misdiagnosis as streptococcal
infection or penicillin allergy, because -lactam therapy is frequently
initiated without accurate diagnosis. A recent report describes in detail
the dermatologic manifestations of A. haemolyticum infection (20).

The demonstration that A. haemolyticum is not a component of the human
commensal flora was essential to establishing its role in human infection.
Studies of more than 2,000 cases each found the organism only in
association with clinical symptoms (17,19). Table 2 summarizes several case
compilations, including the incidence of infection among culture-positive
bacterial sore throats, as well as data on coinfection. Some 0.5% to 3% of
cases of pharyngitis can be traced to A. haemolyticum depending on the
population studied, with the highest numbers among 15- to 30-year-old
patients (19). Clearly, accurate diagnosis depends on differentiating A.
haemolyticum from more common pathogens. A. haemolyticum occurs relatively
often in polymicrobic infections together with typical respiratory
pathogens such as streptococci. The isolation of classical pathogens from
specimens that also contain A. haemolyticum exacerbates the tendency to
overlook the organism.

Table 2. Representative pharyngitis screenings for Arcanobacterium
haemolyticum
---------------------------------------------------------------------------
  Period of                                                    Clinical
    study     Number of                        Incidence(a)    features
 (reference)  isolations Rash   Coinfection        (%)       (population)
---------------------------------------------------------------------------
1978-80 (16) 16         0    13 (C.            NR           Symptomatic
                             diphtheriae,                   throat
                             S. pyogenes, E.               swabs and
                             coli,                          pyoderma (Sri
                             P. aeruginosa)                 Lanka)
1981-85 (19) 81(b)      37   NR                2.0          Symptomatic
                                                            throat
                                                            swabs (Sweden)
1990-92 (17) 42         17   NR                0.36         Symptomatic
                                                            throat
                                                            swabs; 5 cases
                                                            onospot
                                                            positive
                                                            (Ottawa,
                                                            Canada)
1991-92 (42) 19         5    11                0.49         Symptomatic
                             (streptococci,                 throat swabs,
                             groups A, B, G)                3922(c);
                                                            (Finland)
1992-95 (43) 16         12   5 (streptococci,  0.75         Symptomatic
                             groups A, B,                   throat swabs,
                             -hemolytic)                   2121(c);
                                                            (Czech
                                                            Republic)
---------------------------------------------------------------------------
NR-not reported
(a) Incidence refers to the proportion of sore throat cases in each study
cited yielding A. haemolyticum.
(b)1 of 550 asymptomatic specimens yielded A. haemolyticum.
(c)Figures refer to the total specimens screened.

Taxonomy, Bacteriology, and Differential Identification

R. equi

On the basis of the chain length of mycolic acids and other properties of
its lipids, R. equi was reclassified in the suprageneric taxon nocardioform
actinomycetes (1,2,5). R. equi is a strictly aerobic gram-positive
bacterium displaying rod-to-coccus pleomorphism, with fragmenting and
occasionally palisading forms. It is nonfastidious. Colonies on blood agar
from clinical specimens can be mucoid and coalescing. Typical salmon pink
pigmentation develops on blood agar, but often only after 2 to 3 days
incubation. Growth on Lowenstein-Jensen medium allows earlier detection of
pigment (M. Scott, pers. comm.). Positive routine biochemical tests include
catalase and urease, but R. equi is generally nonreactive. Acid-fast
staining of direct smears and fresh isolates is helpful in identification
but is rarely observed on subculture (5,7). Some diagnostic laboratories
use a commercial kit (API Coryne strip (bioMerieux-Vitek, Hazelwood, MO)
for identification.

Also helpful in identifying R. equi is synergistic hemolysis (resembling
the CAMP test), displayed by cross-streaking on sheep blood agar with any
of a number of other bacteria, including A. haemolyticum, Staphylococcus
aureus, and Corynebacterium pseudotuberculosis (21; Figure 2). Synergistic
hemolysis is discussed among mechanisms of pathogenesis (below). In
addition, antagonism between imipenem and other -lactam antibiotics used
against strains of R. equi provides the opportunity of differentiating the
organism from taxonomically related species (22).

[Figure] [Figures not available in ASCII version]

Figure 2. Cooperative (and antagonistic) hemolytic reactions on sheep
blood agar, demonstrating cooperative hemolysis between Rhodococcus equi,
Arcanobacterium haemolyticum, and Staphylococcus aureus. Partial hemolysis
by S. aureus (cross-hatched on diagram) is inhibited in the proximity of
A. haemolyticum.

A. haemolyticum

Organisms are gram-positive rodsslender at first, sometimes clubbed, or in
angular arrangements. Coccal forms predominate as the organism grows. The
organism is facultatively anerobic. Growth is enhanced in blood and in the
presence of CO2. Some sugars are fermented, and the organism is catalase
negative. Hemolysis is best observed on human blood, and Gaston et al. (20)
suggest routine plating of specimens suspected of containing A.
haemolyticum on human blood agar to distinguish Streptococcus pyogenes.
Pitting beneath colonies on human blood agar is helpful in identification.
Synergistic hemolysis with R. equi (or inhibition of the hemolytic zone of
S. aureus, (Figure 2) is useful in identification, especially as it may
rule out group B streptococci. Poor growth on tellurite assists in
differentiation from Corynebacterium diphtheriae.

Pathogenesis and the Immune Response

R. equi

Because R. equi is a rare and recently emergent cause of human infection,
mechanisms of its pathogenicity are not well defined. However, the
much-studied infection in foals and an experimental model in mice provide
data which, together with the available human data, give insight into the
workings of the pathogen. Its nature as a facultatively intracellular
bacterium, able to persist, grow, and ultimately destroy macrophages
(23-25), is the property of R. equi most closely associated with virulence
in each host. Foal alveolar macrophages having ingested R. equi did not
undergo phagosome-lysosome fusion and were irreversibly damaged in electron
micrographs (26). Results of pathology tests (microscopy and
roentgenography) reflect significant inflammation, consistent with that
found in such other intracellular pathogens as Mycobacterium tuberculosis,
which elude pulmonary clearance. Open lung biopsies in humans show numerous
polymorphonuclear leukocytes, foam cells, and cavitating lesions with
intracellular bacteria (7,11; Figure 1). Also as in mycobacteria, cell wall
mycolic acids are present. These acids may contribute to the ability of R.
equi to grow in macrophages; virulence of strains for mice was found
related to the carbon chain length of the molecules (27). Granuloma
formation was observed when killed R. equi strains, regardless of
virulence, were introduced into inbred mice, supporting a role for mycolic
acids or other cell wall glycolipids in pulmonary inflammation (28).

A key contributor to virulence in the foal and the mouse model is a group
of large (85-90 kb) plasmids, encoding 15-17 kDa antigens among strains
isolated from almost all natural infections in foals (29). Strains cured of
the plasmid are cleared in experimental infections, and intracellular
replication in murine macrophages is greatly diminished in its absence
(25,28). In contrast, of 39 strains isolated from humans (29 with AIDS), 31
strains did not express the virulence associated plasmid and were
non-virulent in mice (6). The investigators suggest that intracellular
growth and, therefore, virulence among human strains may not be explained
by the same determinants as foals, while mycolic acids may play a role.

Nordmann (23) found that intracellular growth in mouse and human
macrophages of strains isolated from AIDS patients was related to -lactam
resistance, the production of a bacteriophage, and virulence in inbred
mice. Virulence was not attributable to 17kDa virulence antigens, but
soluble cytotoxic substances were associated with the virulent phenotype.
The cytotoxic activity remains to be characterized, and its relationship to
known cytotoxic activities of R. equi remains to be elucidated. In addition
to numerous hydrolytic enzymes typical of the genus Rhodococcus (5),
strains of R. equi, irrespective of virulence, produce cholesterol oxidase,
which is responsible for the organism's participation in synergistic
hemolytic reactions with other bacteria (30; Figure 2). Experiments using
cultured mouse macrophages with phagocytosed R. equi suggest a role for
cholesterol oxidase in macrophage destruction in infections. Macrophages
undergo oxidation of membrane cholesterol, and the accumulation of oxidized
cholesterol is significantly enhanced by the cophagocytosis of C.
pseudotuberculosis, a related coryneform bacterium producing
sphinomyelinase D (31). Toxicity to vertebrates as a result of enzymatic
oxidation of membrane cholesterol is documented in diverse systems, most
dramatically by lethality to hypercholesterolemic rabbits (31).

Because R. equi is uniquely an opportunistic pathogen in humans, it is of
interest to consider the precise nature of the immune deficiency that
underlies susceptibility to this organism. Recent investigations in
immunodeficient mice are especially instructive. T-cell subsets,
specifically functional CD4+ lymphocytes, are necessary to effect complete
clearance of R. equi challenge (23,32). Specifically, Kanaly et al. showed
that CD4+ Th1 cells (expressing interferon-gamma) are sufficient to achieve
clearance from the lungs of mice (32,33). Consistent with these data,
peripheral mononuclear cells, from patients with AIDS, challenged in vitro
with R. equi failed to secrete high levels of interferon-gamma in
comparison with cells from healthy donors (34). Investigations implicating
a specific defect in the Th1 phenotype in the pathogenicity of AIDS make
these studies especially provocative and suggest a role for immunotherapy
in R. equi infection (33).

While cell-mediated immunity appears to have a primary role in protecting
against R. equi infection, the participation of humoral antibody has been
established in foals. Passive immunization with hyperimmune serum is
efficacious in prophylaxis, and severity of disease is inversely related to
circulating antibody (1,5). Mastroianni et al. (35) demonstrated antibody
to the major antigens of R. equi in four AIDS patients with cavitating
pneumonia. The role of such antibody in the natural history of the
infection remains to be elucidated.

A. haemolyticum

Little is known about the mechanisms by which A. haemolyticum produces
infection or brings about the skin manifestations frequently associated
with it. The organism is known to produce uncharacterized hemolytic
agent(s) (20) and two biochemically defined extracellular products: a
neuraminidase and a phospholipase D (PLD) acting preferentially on
sphingomyelin and generating ceramide phosphate in the target membrane
(36). Of these, PLD is known to bring about tissue damage, as elaborated by
this organism as well as the closely related bacterium, C.
pseudotuberculosis, an important pathogen of sheep. Soucek et al. (36)
found that the enzyme was responsible for the dermonecrotic, as well as the
synergistic hemolytic, activity of the organisms that elaborate it. The PLD
gene from A. haemolyticum has been cloned and shown to have a high degree
of homology with that of C. pseudotuberculosis, where it is thought to
participate in vascular permeability and dissemination of the pathogen
(37). Evidence relates PLD with toxicity of C. pseudotuberculosis. Targeted
mutagenesis of the PLD gene of C. pseudotuberculosis confirmed the role of
the enzyme in virulence and specifically in dissemination in the host (38).
Mutant bacteria had a reduced ability to establish infection in goats and
were unable to disseminate by the lymphatics to secondary sites. A PLD
sharing many properties with corynebacterial PLDs, including biochemical
and biological activities, is responsible for the toxicity of the venom of
the brown recluse spider (39). The role of potentiated cytotoxicity caused
by the combined activity of PLD and cooperative agents such as cholesterol
oxidase in disease is not established, but suggested by in vitro data
involving cophagocytosis as described above (31).

Treatment

R. equi

Increased recognition of R. equi as a cause of life-threatening infection
in severely immunocompromised persons has promoted a number of studies of
in vitro antimicrobial susceptibility of clinical isolates (11,13,40,41).
While variations exist, most strains were susceptible to inhibition by
glycopeptide antibiotics (including vancomycin and teicoplanin) and
rifampin. Macrolide antibiotics, such as erythromycin and clarithromycin,
were also inhibitory to many strains. Resistance to -lactam antibiotics
(with the exception of carbapenems, specifically imipenem) was generally
reported, and is not related to the production of a -lactamase.

Because of relapse in spite of treatment in a majority of cases (11) and
high mortality rate, especially among AIDS patients (Table 1), there is no
standard treatment protocol for pulmonary and/or systemic R. equi
infections. However, several principles reflect the accumulated experience
of investigators. Careful and repeated culture and susceptibility testing
during treatment is required to discover acquired resistance, in a manner
similar to the treatment of mycobacterial infection (11,22). Tolerance to
the cidal effects of some drugs and the need for long-term therapy
(generally 2 months to life-long treatment; 40,41) make bactericidal
testing a useful addition to laboratory studies. In consideration of the
severe immunosuppression of patients and proclivity to relapse,
investigators generally promote a combination of at least two drugs
parenterally (usually including a glycopeptide or rifampin) followed by
oral maintenance therapy (11,41). Recommendation of lipophilic
antimicrobials that penetrate macrophages is controversial (11,41). A
proposed regimen involves parenteral glycopeptide plus imipenem for at
least 3 weeks, followed by an oral combination of rifampin, plus either
macrolides or tetracycline (41). Examples of efficacious protocols for
parenteral treatment are available (Table 1).

Surgical lung resection has been reported occasionally since the emergence
of human cases, especially where large focal lesions develop (9,10), and
has sometimes been efficacious in combination with antimicrobial therapy.
As cases of R. equi continue to be recognized among AIDS patients,
antimicrobial prophylaxis against this opportunistic pathogen may prove a
benefit.

A. haemolyticum

In vitro testing of A. haemolyticum isolated from human infections shows
susceptibility to erythromycin, gentamicin, clindamycin, and cephalosporins
(42). Reports of treatment failure with penicillin in spite of low minimum
inhibitory concentrations have been attributed to tolerance and to failure
to penetrate the intracellular location of the pathogen. Erythromycin has
been proposed as the drug of choice, with parenteral antimicrobial drugs
used for serious infections (20). The general similarity of the
susceptibility pattern of A. haemolyticum to more commonly encountered
pharyngeal pathogens, including S. pyogenes, makes culture and accurate
diagnosis essential if cases are to be recognized for their true etiology.
Its participation in polymicrobic infections (Table 2) requires that A.
haemolyticum be specifically sought in appropriate specimens to obtain
accurate diagnosis and to allow epidemiologic analysis.

R. equi and A. haemolyticum represent distinct poles of infectious disease:
one a ubiquitous soil organism producing life-threatening opportunistic
infections and the other a readily treatable respiratory infection of
healthy young persons. In both instances, a high degree of suspicion is
required to make accurate and timely diagnoses of infections. Diagnostic
failure may result in a graver clinical profile including deaths for R.
equi and, in many undiagnosed or misdiagnosed cases, for A. haemolyticum.
As members of the morphologically defined taxon "coryneform" bacteria,
these organisms exemplify properties of the group that require further
elucidation. Weakly pathogenic and noninvasive, the group includes
environmental bacteria; animal pathogens "crossing-over" to become human
opportunistic pathogens; commensals similarly infecting compromised hosts;
and producers of a wide variety of hydrolytic enzymes bearing a poorly
defined relation to virulence (1). Both R. equi and A. haemolyticum
elaborate a cytotoxic protein (cholesterol oxidase or sphingomyelinase D)
known to be responsible for systemic harm to animals. Coincidentally, these
products potentiate each other's cytotoxic action. The participation of the
agents in harm to a host, alone or in combination with other substances, is
consistent with available data, but yet unproven. Of particular interest is
the role of cholesterol oxidase in destruction of alveolar macrophages in
R. equi pneumonia.

Clarifying the role of synergistic or cooperative cytotoxins in one or more
infectious diseases will surely improve our understanding of others because
of the common occurrence of these agents among bacterial pathogens (21). It
is difficult to envision the potentiated hemolytic combination of R. equi
and A. haemolyticum at the site of an infectious lesion. However,
cooperatively hemolytic combinations have been shown to result from the
partnership of hydrolytic enzymes (e.g., phospholipases, which are
ubiquitous in tissue) with the cytotoxins of pathogenic bacteria (10).
Similarly, the hydrolytic enzymes of commensal bacteria or copathogens that
occur, for example, in A. haemolyticum pharyngitis, can readily be
envisioned to participate in potentiated cytotoxicity in host tissue.
Together with improved recognition of these two pathogens, greater
understanding of their toxic products should prove beneficial.

Acknowledgments

I am grateful to Dr. Margie Scott for providing additional information on
her cases and the micrographs in Figure 1. I thank Dr. Alan Bernheimer for
his critical review of the manuscript and Dr. Patrice Nordmann for helpful
discussions.

Dr. Linder is associate professor of health sciences at Hunter College,
City University of New York, and director of the Medical Laboratory
Sciences Program, which prepares undergraduates for careers in laboratory
medicine. Her research involves the mechanisms of action and role of
bacterial cytotoxins in infection.

Address for correspondence: Regina Linder, School of Health Sciences,
Hunter College, 425 East 25th St., New York, NY 10010,
USA;fax:212-420-9135;e-mail:rlinder@shiva.hunter.cuny.edu.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Synopses

Is Creutzfeldt-Jakob Disease Transmitted in Blood?

Maura N. Ricketts,* Neil R. Cashman, Elizabeth E. Stratton,* Susie
ElSaadany*
*Laboratory Centre for Disease Control, Health Canada, Ottawa, Ontario,
Canada; and Montreal Neurological Institute, Montreal, Canada
---------------------------------------------------------------------------


      Creutzfeldt-Jakob disease (CJD) has been considered infectious
      since the mid-1960s, but its transmissibility through the
      transfusion of blood or blood products is controversial. The
      causative agent's novel undefined nature and resistance to
      standard decontamination, the absence of a screening test, and
      the recognition that even rare cases of transmission may be
      unacceptable have led to the revision of policies and
      procedures worldwide affecting all facets of blood product
      manufacturing from blood collection to transfusion. We reviewed
      current evidence that CJD is transmitted through blood.

Creutzfeldt-Jakob disease (CJD), a rare neurodegenerative disorder, affects
0.5 to 1 persons per million population worldwide each year (1-8). CJD is a
human spongiform encephalopathy; others are kuru, which is associated with
ritualistic cannibalism in the Fore tribe of Papua New Guinea;
Gerstmann-Strussler-Scheinker syndrome, an inherited disorder; and fatal
familial insomnia, inherited as an autosomal-dominant trait. Animal
spongiform encephalopathies include scrapie, bovine spongiform
encephalopathy (BSE), transmissible mink encephalopathy, and wasting
disease of elk most frequently referred to as transmissible spongiform
encephalopathies; other names such as prion dementias, transmissible
degenerative encephalopathies, and infectious cerebral amyloidoses are also
used.

The classic clinical symptoms of CJD are rapidly progressive presenile
dementia, myoclonus, and progressive motor dysfunction. No treatment is
available, and survival averages less than 1 year (most often 2 to 6
months) (9). Diagnosis is based on symptoms, electroencephalograms, and
neuropathologic tests (10,11).

The pathophysiology of CJD is incompletely understood, although it is known
that in persons with the disease, the normal soluble prion protein (PrPC)
is conformationally shifted into a more stable, less soluble -pleated
protein. The term PrPCJD indicates the abnormal isoform found, with a
variety of distribution patterns in the brain of persons with CJD. Limited
proteolysis of PrPCJD by Western blot analysis shows four patterns of
protease-resistant prion protein (12,13).

No screening assay is available to detect PrP in asymptomatic persons.
Cerebrospinal fluid protein markers can distinguish CJD from other
neurodegenerative disorders in certain settings (14,15). However, they are
not CJD-specific and are not markers of PrP. The etiologic agent is
believed to be a prion (proteinaceous infectious particle), although viral
etiologies have been proposed (16-18). Sporadic CJD may also result from
the spontaneous conversion of PrPC into PrPCJD or from somatic mutation in
the prion protein gene.

Three epidemiologic forms of CJD are well recognized: sporadic (the most
common), familial, and infectious/iatrogenic. The familial form (5% to 10%
of cases) results from mutations in the coding sequence of the PrP gene
located on chromosome 20. Polymorphisms at codon 129 have been correlated
with genetic susceptibility to human prion diseases (19). Fewer than 1% of
human cases of CJD are iatrogenic (20). A new variant (nv-CJD), which
occurred temporally in association with BSE in cattle in the United
Kingdom, was recently reported (21); a direct association with food
consumption remains uncertain. Possible transmission of CJD through receipt
of blood or blood products is a concern. Routes of iatrogenic transmission
are summarized below, followed by a discussion of possible bloodborne
transmission.

Iatrogenic CJD

Although first described in the 1920s, CJD was not considered a
transmissible disease until 1966 when kuru was shown to be transmissible
(22). In 1968, CJD was confirmed to be a transmissible spongiform
encephalopathy when it was shown to be transmitted to chimpanzees (23).
Virtually every case of CJD attributed to infection is iatrogenic;
transmission between humans has been clearly demonstrated during
neurosurgical procedures with contaminated instruments and through central
nervous system tissue and extract transfer. Worldwide, more than 100 cases
of transmissible CJD have been detected, and new cases continue to appear
(Table 1; 27).

    Table 1. Transmissible cases of Creutzfeldt-Jakob Disease
    -------------------------------------------------------------------
     Mode of infection     No.       Agent entry     Mean incubation
                       of patients   into brain      period (range)
    -------------------------------------------------------------------
    INSTRUMENTATION
    Neurosurgery       4          Intracerebral    20 months (15-28)
    Stereotactic EEG   2          Intracerebral    18 months (16-20)
    TISSURE TRANSFER
    Corneal transplant 2          Optic nerve      17 months (16-18)
    Dura mater implant 25         Cerebral surface 5.5 years (1.5-12))
    TISSURE EXTRACT
      TRANSFER
    Growth hormone     76         Hematogenous     12 years (5-30)
    Gonadotrophin      4          Hematogenous     13 years (12-16)
    -------------------------------------------------------------------
    EEG = electroencephalogram
    Table taken from (27)

The first report of suspected iatrogenic CJD was published in 1974 (24).
Animal experiments showed that corneas of infected animals could transmit
CJD, and the causative agent spreads along visual pathways (25,26). A
second case of CJD associated with a corneal transplant was reported
without details (27). In 1977, CJD transmission caused by silver electrodes
previously used in the brain of a person with CJD was first reported (28).
Transmission occurred despite decontamination of the electrodes (later
shown to transmit disease to experimental primates [29]) with ethanol and
formaldehyde. Retrospective studies identified four other cases likely of
similar cause (30-32). The rate of transmission from a single contaminated
instrument is unknown, although it is not 100% (31). In some cases the
exposure occurred weeks after the instruments were used on a person with
CJD.

CJD was first reported in a recipient of a dura mater transplant in 1987
(33); a second case was identified in 1989 in a 25-year-old man from New
Zealand, who also received dura mater (34). Because the same company
produced dura mater for both patients, the dura mater was suspected as the
source of iatrogenic CJD. Approximately 15 case reports have been
published, providing information on 20 cases associated with dura mater
transplant; a recent review article indicates that 25 such cases have
occurred throughout the world (Australia, Canada, Germany, Italy, Japan,
New Zealand, Spain, the United Kingdom, and the United States) (27).
However, Japan has very recently reported more than 40 cases of CJD in dura
mater recipients (J. Tateishi, Pers. Comm.). Most dura mater cases have
been associated with a single manufacturer whose manufacturing processes
were inadequate to inactivate the prion agent. This, combined with pooling
of the dura mater has led to the relatively large number of cases. The
earliest reported transmission occurred in a patient who received a dura
mater transplant in 1969 (35). Although recommendations to reduce the risk
were made in 1987, cases have continued to appear because of the long
incubation period. Notably, one of the most important epidemiologic
characteristics of dura mater transmissions is the young age of the first
reported cases.

By 1985, a series of case reports in the United States showed that when
injected, cadaver-extracted pituitary human growth hormone could transmit
CJD to humans (36). Shortly thereafter, it was recognized that human
gonadotropin administered by injection could also transmit CJD from person
to person (37).

Strain typing based on molecular PrP analysis and a polymorphism
(methionine/methionine, valine/valine or methionine/valine) found at codon
129 of the PrP gene has been proposed as a tool for distinguishing between
iatrogenic and sporadic CJD (12,13). Homozygosity at this site may
predispose a person to acquired forms of CJD, may lead to shorter
incubation periods, and has been associated with various phenotypes of the
disease (11,38).The proportion of polymorphism among persons with sporadic
CJD is more similar to the proportion of polymorphism among persons with
iatrogenic CJD than to that in the general population (Table 2), which
suggests that simple stochastic events do not fully explain sporadic CJD;
were that so, the distribution of homozygosity would be the same in both
healthy controls and persons with sporadic cases. The clinical symptoms in
patients with iatrogenic and with sporadic CJD have been compared and are
indistinguishable (18).

Table 2. Amino acid phenotypes for codon 129 in patients with iatrogenic
Creutzfeldt-Jakob Disease
---------------------------------------------------------------------------
                                                       Homo-    Total No.
Tested groups            Met/Met   Met/Val   Val/Val   zygous   tested
                         (%)       (%)       (%)
                                                       (%)      (n)
---------------------------------------------------------------------------
Sporadic CJD (38)        78        12        10        88       73
All Iatrogenic           60        11        29        89       63
Cases (27)
  CNS route
  of infection           80        10        10        98       20
Peripheral route
  of infection           51        12        37        88       43
Healthy controls (27)    37        51        11        49       261
Healthy controls (38)    48        42        10        58       1397
---------------------------------------------------------------------------
Met = methionine; Val = valine


Is CJD Transmitted by Blood Transfusions?

Animal Experimentation Data

Human CJD has been reported to be transmitted to mice by injecting blood
from human patients directly into mouse brain (39,40). However, this
evidence has not been widely duplicated by other laboratories, and a
comprehensive review of three decades of research with non-human primates
by the National Institutes of Health indicated that the blood of humans
with CJD injected either peripherally or centrally into primates did not
transmit CJD (41).

Some evidence indicates that blood of experimentally infected animals
contains an infective agent. PrP infectivity resides predominantly or
exclusively in lymphocytes and monocytes rather than in granulocytes
(42,43); there has been no evidence of infectivity in erythrocytes,
platelets, or plasma, but low infectivity has not been completely excluded.
Animal studies have demonstrated that the agent causing scrapie replicates
first in the spleen and other lymphoid tissues but reaches highest titer in
the brain, where it results in the clinical appearance of disease (44).
Hence, peripheral tissues in contact with blood also harbor PrP
infectivity. Animal transmission data indicate that human spleen, lymph
nodes, serum, and cord blood are irregularly infective for animals,
although few cord blood samples have been tested (27). Brown has recently
reported transmission of mouse scrapie using fractionated blood
administered by intracerebral inoculation (P. Brown, Pers. Comm.). Studies
of experimental CJD in guinea pigs and mice have shown that the infectious
agent is present in the brain, viscera, and blood before clinical disease
develops (43,45).

Several factors must be considered in reviewing animal evidence regarding
transmission of CJD in blood: the level of PrP infectivity of the study
tissue, the species barrier, and the route of administration. Human CJD can
be readily transmitted to nonhuman primates (low species barrier) by
intracranial injection (high efficiency of the infective route) of
contaminated brain (high titer of infectivity). Human CJD is not readily
transmitted to dissimilar species (such as rodents) and is even less
readily transmitted when low-titer tissues (e.g., blood cells) and/or a low
efficiency route of inoculation is used. In animals, peripheral inoculation
of brain tissue transmitted CJD only irregularly, but the incubation
periods were comparable to central administration (41). Barriers to
transmission also include the difficulties in infecting peripheral cells
and crossing the blood brain barrier and possibly the lack of structural
similarity between peripheral PrPCJD and brain PrPC. Strain variation may
also contribute to the difficulties of transmission to animals. Deslys et
al. suggest that the route of inoculation may induce strain
differentiation, which then facilitates subsequent transmission by the same
route (46). Others disagree (47). The evidence from animal studies is
inconclusive regarding transmission of CJD between humans by transfusion,
and study design and laboratory techniques are controversial.

Evidence from Studies of Humans

Human Case Series and Case Reports

Case series and case reports provided information linking CJD to receipt of
dura mater and human growth hormone. However, no human cases have yet been
causatively linked to blood transfusion. A transplant recipient developed
CJD (48) after receiving a liver from a donor who died of a cerebrovascular
accident and had no symptoms of CJD; however, an autopsy was not performed,
and brain tissue was not available for further investigation. The liver
recipient did not have any of the known PrP gene coding sequences
pathognomonic of familial transmission. The donor also provided a heart (to
a recipient who died shortly after surgery) and a kidney (to yet another
recipient; it was removed after 2 months); the latter recipient remains
well. The liver recipient also received transfusions of blood, plasma,
albumin, and anticytomegalovirus immunoglobulin from other donors. None of
the blood donors is known to have had CJD, but one of the albumin donors
died of a rapidly developing undiagnosed dementia.

Four Australians have been reported with CJD following transfusion (49).
The patients had cerebellar signs; however, no other evidence of iatrogenic
cause was described (50). The source of blood transfusions was
undocumented. Genetic testing results were not provided; it is uncertain if
cases were of the familial type, and no other information on alternative
iatrogenic sources was provided.

In Canada, an albumin recipient died of neuropathologically confirmed CJD
after receiving albumin from a pool containing blood from a person who died
of neuropathologically confirmed CJD (D.G. Patry, pers. comm.). Eight
months separated the receipt of albumin and development of symptoms, a much
shorter period by a factor of three than seen in any other putative
iatrogenic case, which makes iatrogenic transmission unlikely. A complete
investigation is under way.

Without an experimental, diagnostic, or epidemiologic tool that can
distinguish sporadic from iatrogenic disease or link the agent from a
source with a recipient, it is difficult to draw any conclusions from the
case reports described above. Statistically, a certain number of persons
exposed to pooled blood would be expected to develop CJD. In addition, bias
is introduced into case reports because of strong suspicion regarding
iatrogenic sources. Verifying the statistical probability of cases by
calculating the expected number of cases is difficult, if not impossible,
because CJD is rare.

Surveillance

Population surveys or surveillance systems of the worldwide epidemiology of
CJD indicate that CJD occurs in the population at a rate of 0.05 and 1.5
cases per million per year. After 1979, the rates are 0.3 to 1.5 cases per
million per year (1-8). The age distribution patterns are consistent and
show that cases in persons under 30 years of age are extremely rare; cases
in persons under 50 years of age are rare; and the peak age of onset is 60
to 70 years. In age-specific data, rates decline in older age groups
(2,6-8,51).

If CJD is transmissible in blood, cases should occur in young persons,
particularly if the incubation period is short. Even if the incubation
period is decades long, one would expect cases in young persons because of
transfusions given to infants and children. However, CJD is rare in young
persons and remains rare over time. Alternatively, the rare diagnosis of
CJD in younger age groups may be due to preferences for neurologic disease
diagnoses in these groups. Recent attention to nv-CJD in 14 persons under
the age of 40 years in the United Kingdom, which has an intensive active
surveillance system, will certainly affect investigations of unexplained
mental deterioration among younger populations (22). The British Paediatric
Surveillance System has initiated investigations into undiagnosed
progressive neurologic disease among children (C. Verity, A. Nicoll and R.
Will, pers. comm.), and Canada will initiate a similar system. In the
United States, the Centers for Disease Control and Prevention has enhanced
surveillance for CJD in persons under 55 years of age (52).

If CJD is transmitted in blood, the last three to four decades might show a
detectable increase in cases reflecting the increasing use of blood
transfusions. In fact, surveillance data demonstrate that CJD rates are
increasing in some countries. Interpretation of this finding is difficult:
most surveillance systems were initiated in the last two decades; in
countries with intensive surveillance, "catch-up" from previous
underreporting led to initial increases in case numbers and rates; few
countries have sufficiently intensive surveillance systems to conclude that
there is no risk from blood; and the death certificates used for
surveillance in some countries are sometimes incomplete, which may
introduce a bias toward easily ascertained cases (8,51).

If CJD is transmitted in pooled blood products, clusters would be detected;
indeed, surveillance systems have expected such clusters. However,
observation of one or two cases often leads to more careful search for
cases. Most such clusters have been attributed to familial disease (1,53).
Cluster investigations in surveillance systems have not systematically
searched for blood-related transmission.

Surveillance systems have found cases of CJD among persons who have
received blood transfusions, but none have been linked to blood
transmission. In the United Kingdom, the well-established surveillance
system identified nv-CJD, despite its rarity, demonstrating the capability
of surveillance to find rare diseases; nv-CJD was recognized quickly
because of the young age of the patients and the novel neuropathologic
findings. However, the absence of evidence is not evidence of the absence
of transmission of CJD through blood, for two reasons: 1) surveillance
systems designed to identify rare diseases need intensive resource
allocation to detect sentinel events and 2) bloodborne transmission may go
unnoticed when examining population data if unaccompanied unique
epidemiologic features (such as extreme youth or novel neuropathologic
features).

If CJD is transmitted in blood, cases could increase in industrialized
countries, where access to blood transfusion is greater. The number of
reported cases is larger; however, cases have been found in every country
in which they have been sought, although some countries have limited
surveillance capacity. While some countries have higher rates of CJD, there
is no evidence that this is due to transmissible forms of the disease.
Rather, the higher rates are most likely due to older age distributions in
industrialized countries' populations, surveillance biases following
intensified surveillance for CJD, and familial clusters.

Finally, in many surveillance systems, the age distribution pattern for CJD
is uniform (2,7,8,51). The pattern is more consistent with early exposure
to an agent with long incubation periods, a common population exposure that
peaked long ago and is now declining, or a heritable disorder that causes
shortened life-span than with a risk which increases with age (e.g.,
spontaneous genetic mutation or stochastic change of the normal prion to
the -pleated form). However, CJD may be uniformly underdiagnosed in older
age groups; because of nv-CJD there will likely be increased attention to
differential diagnoses among elderly persons dying of rapidly progressing
dementing illnesses. We do not suggest that all sporadic cases are due to
external exposure such as blood, but rather we draw attention to an
important epidemiologic characteristic of CJD that is not consistent with
an entirely stochastic or age-related event.

Case Control Studies

In three countries, case-control studies of CJD have included questions
regarding exposure to blood: Japan (54), the United Kingdom (55-58), and
the United States (59). In the Japanese study, only one case-patient and
three controls received blood transfusions. Two of the United Kingdom
studies indicate no difference in exposure to blood between the
case-patients and controls (55,58). A comparison of the frequency of
receipt of blood among persons with CJD and controls matched for age and
sex using data collected in the United Kingdom CJD surveillance system
between 1980 and 1984 (56) showed no difference in the history of blood
receipt between those with and without CJD. Although an odds ratio (OR) is
not calculated for the data, our calculations indicate the OR is 0.78 (95%
confidence interval 0.38, 1.58) with a power of only 30% to detect an OR
different from one (60). Davinipour (mid-Atlantic United States) also found
that blood exposure posed no risk, with an OR for blood transfusion of 0.6
(59). However, with only 26 cases, the confidence intervals or number of
patients exposed was not mentioned, although the OR is described as
significant. Wientjens et al. analyzed pooled data for 178 cases and 333
controls from three case-control studies (Japan, United States, United
Kingdom) and did not find an OR different from one for blood transfusion
(61).

Although not using a formal case-control study, Operskalski et al, compared
rates of human immunodeficiency virus (HIV) dementia among three
HIV-positive groups (persons with hemophilia, "other blood recipients," and
"blood donors") using data from the Transfusion Safety Study (62). They
hypothesized that if CJD was misdiagnosed as HIV dementia but was the true
cause of dementia, there would be an excess rate of HIV dementia in persons
with hemophilia due to their high rates of exposure to blood and blood
products (persons with hemophilia were exposed to the blood or product made
from pools of hundreds of thousands if not millions of donations). In
addition, since the study contained data from as early as the 1950s, long
periods of observation were available. Rates of HIV dementia in the study
populations were compared, and CJD in pooled plasma derivatives did not
pose a risk. However, it cannot be concluded that rare diseases such as CJD
would be detectable in increased rates of misdiagnosed HIV dementia or that
diseases with long incubation periods may have sufficient time to develop
in persons with hemophilia. Any disease with an incubation period longer
than that of HIV would be in competition with AIDS and hepatitis B or C as
a cause of death, and persons with hemophilia and HIV infection die young.
In addition, the data included the cause of death of 1,000 HIV-infected
persons but did not provide information on duration of observation or an
estimate of the rate of disease that would be necessary before even one
additional case would be observed.

The primary weakness found in these published case-control studies is the
use of the categories "exposure to blood" or "blood transfusion" as a
surrogate for exposure to blood containing the agent of CJD. If the agent
of CJD is frequently present in blood used for transfusions, the
case-control study design is sufficiently robust to detect a risk. However,
transmission of CJD through transfusion of blood contaminated with the
agent of CJD appears to be rare. Consequently, a negative finding of risk
attributable to blood in these studies may simply reflect an absence of
exposure to the disease. Study design will have to account not only for a
rare disease, but possibly for a rare exposure as well.

In addition, when hospitalized persons are used as controls, the design
must consider Berkson's bias, a selection bias that occurs when the
selection method for controls affects the rate of exposure to the agent
studied (63). The study design must then show that hospitalized controls do
not have a different rate of potential exposure to blood transfusion from
the general population. The use of population controls may be more
appropriate for studying blood exposure.

Another weakness in the existing studies was the use of a reporter, most
often a relative, to collect information about exposure to blood or blood
products. Oral history of blood exposure tends to underestimate the rate of
exposure to blood. In one Canadian hospital conducting a retrospective
study, approximately 40% of patients did not know if they had received a
blood transfusion (C. Kennealy, Pers. Comm.). In a central Ontario
hospital, 25% of recipients did not know if they had received a blood
transfusion (S. King, Pers. Comm.). Given that CJD and
transfusion-transmitted CJD (if it occurs) are rare, the published
case-control studies lack the power to detect very low ORs.

The 126 hemophilia centers in the United States have been asked to report
deaths of persons with neurologic symptoms for neurologic confirmation of
cause of death; no CJD cases have been reported in persons with hemophilia
(B. Evatt and L. Schonberger, Pers. Comm.). The Canadian Haemophilia
Society has requested assistance from the Health Canada, Laboratory Centre
for Disease Control, to initiate a similar study in Canada (D. Wong-Reiger,
Pers. Comm.). In Canada, a combined active surveillance system and case
control study will begin in 1997 to identify the risk for CJD as a result
of transfusion with blood from a person with CJD. Many of the methodologic
problems in other studies have been addressed in the design of this study.

Cohort Studies

The investigation of a patient with CJD who donated 35 units of blood in 20
years identified 27 persons who definitely received his blood and eight who
probably received blood; for 20 units, the recipients could not be
identified (64). Eighteen (33%) of the identified recipients had died. None
of the recipients had exhibited neurologic disease, although some were
observed only briefly; only eight were observed for longer than 5 years.

In the United States, the American Association of Blood Banks has initiated
a long-term cohort investigation. By linking the names of persons who
received blood from CJD patients with the national death records, the cause
of death for each person will be determined. Of the 147 recipients
identified, 80 have died; for 65, the cause of death is known but no cases
of CJD have been found (M. Sullivan, Pers. Comm.).

Cohort studies are unlikely to accumulate enough cases of exposure to allow
a reasonably precise estimate of risk. However, if pooled product confers
uniform risk to each recipient, data from a large number of exposed
recipients can be collected. The cohort studies under way may be able to
identify higher than expected rates of disease, thus indicating
transmission through blood transfusion without quantifying the rate of
transmission.

Conclusions

Many health professionals are concerned that CJD may be transmissible
through blood. As noted by Brown, "iatrogenic disease from this source
would dwarf in importance all other sources by virtue of the sheer number
of people who theoretically have been or could be at risk" (27).

Animal studies indicate that the infective agent of CJD is present in blood
but in low titer, and sufficient evidence of animal transmission suggests
that the disease has the potential to be transmitted through blood (65).
However, human epidemiologic evidence only indicates that if blood
transmission occurs, it is likely rare. Some researchers suspect that the
agent of CJD is ubiquitous, therefore, we are commonly exposed to it;
perhaps, another, as yet unidentified factor, "turns on" the disease. If
so, is there any part of the human population that is not exposed? Most
people are exposed to some components of blood products through vaccines.
Some people may be protected by strain variation through exposure to
nonvirulent forms, so only subgroups are susceptible to a virulent form
(66). Polymorphism at codon 129 may restrict susceptibility to a small
portion of the population, or most transfusions may not contain a dose
large enough to cause infection.

Can experimental studies answer these questions? Sufficiently large numbers
of study animals could overcome problems such as the species barrier and
low transmission rates due to a low dose or peripheral route of
inoculation. The studies would require strict adherence to proper
laboratory technique and replication in at least one other laboratory.
Unanswered questions include the following: Can the transmissible
spongiform encephalopathies of animals be transmitted, by transfusion,
within the species when spiked blood is used? Within species from
"naturally infected" blood? From humans to animals with spiked blood? From
humans to animals with "naturally infected" blood? Does transmission by
blood become more efficient after passage? Can human blood cells carry the
agent? What are the routes for infection of the brain if infection is
peripheral?

Can epidemiologic methods detect blood transmission of CJD if it is rare?
Epidemiologic tools such as outbreak investigations, case-control studies,
and cohort studies are limited in their ability to detect rare events. In
addition, during the long incubation period, patients often move away from
the location where they received the transfusion; forget their exposure;
and essentially lose their membership in a recognizable cohort. The absence
of a test for exposure, such as an antibody test or gene sequencing of an
agent as used in investigating HIV-related out-breaks, makes the
investigation of iatrogenic CJD extremely difficult, hence the importance
of vigilance, in the form of case reports from alert and informed
clinicians, followed by critical field investigation. Surveillance systems
provide the core resources for identifying and investigating unique or
unexplained events. Followup case-control studies allow the observation and
recording of the actual chain of exposure to blood.

Acknowledgments

The authors thank Drs. Paul Gully, Jamie Hockin, and Martin Tepper for
their thoughtful reviews of earlier drafts of this paper and Drs. Catherine
Bergeron, Michael Coulthart, and Brian Foster for discussion and scientific
assistance.

Dr. Ricketts is a medical specialist in the Bloodborne Pathogens Division,
Laboratory Centre for Diseases Control, Ottawa, Ontario, Canada. Her
research interests include the CJD surveillance system in Canada, a
collaborative international study on the incidence and risk factors for
CJD, and policy and infection control guidelines for CJD and other prion
diseases.

Address for correspondence: Maura N. Ricketts, Bloodborne Pathogens
Division, Bureau of Infectious Diseases, Laboratory Centre for Disease
Control, LCDC Building, PL 060 3E1, Ottawa, Ontario, K1A 0L2 Canada; fax:
613-954-6668; e-mail: mricketts@hpb.hwc.ca.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Dispatches

A New Tick-borne Encephalitis-like Virus Infecting New England Deer Ticks,
Ixodes dammini (See footnote)

Footnote. The taxonomy continues to be controversial (5).

---------------------------------------------------------------------------


      To determine if eastern North American Ixodes dammini, like
      related ticks in Eurasia, maintain tick-borne encephalitis
      group viruses, we analyzed ticks collected from sites where the
      agent of Lyme disease is zoonotic. Two viral isolates were
      obtained by inoculating mice with homogenates from tick
      salivary glands. The virus, which was described by reverse
      transcriptase polymerase chain reaction and direct sequencing
      of the amplification products, was similar to, but distinct
      from, Powassan virus and is provisionally named "deer tick
      virus." Enzootic tick-borne encephalitis group viruses
      accompany the agents of Lyme disease, babesiosis, and
      granulocytic ehrlichiosis in a Holarctic assemblage of emergent
      deer tick pathogens.

American zoonotic foci of the agents of Lyme disease (Borrelia burgdorferi,
a spirochete) and human babesiosis (Babesia microti, a protozoon) were
first recognized in coastal New England and the northern Great Plains
during the 1960s and 1970s (1). Human granulocytic ehrlichiosis (caused by
rickettsia, Ehrlichia microti or Ehrlichia phagocytophila/equi [2]) joined
this guild (3) of deer tick-transmitted pathogens (Ixodes dammini [4,5])
during the 1990s. B. burgdorferi has infected I. dammini and their rodent
hosts at least from the beginning of the 20th century (6). In Eurasia,
closely related and ecologically equivalent ticks (Ixodes ricinus and
Ixodes persulcatus) transmit analogs of each of these agents (7), including
several zoonotic variants of the Lyme disease spirochete, babesiosis
(caused by Babesia divergens), tick-borne fever (caused by E.
phagocytophila), and certain flaviviruses (tick-borne encephalitis [TBE]).
Although analogs of three of the four members of the Eurasian guild of I.
ricinus/persulcatus-transmitted pathogens are well established in North
America, no arbovirus has been reported from I. dammini or other American
members of the I. persulcatus species complex. Accordingly, we determined
whether arboviral infection is present in adult I.dammini from deer or
vegetation in coastal New England, where Lyme disease is frequent.

Partially fed female ticks were removed from carcasses of deer shot by
hunters during November 1995 or were derived from host-seeking ticks swept
from vegetation and permitted to partially engorge on rabbits in the
laboratory. All ticks were held in vials designated by site, at 4C, and in
a humidified chamber until dissection, no more than 1 month after they were
removed from hosts.

Each field-derived tick was dissected into a drop of sterile Hanks Balanced
Salt Solution with 15% fetal bovine serum (HBSS/FBS), and one of its
salivary glands was stained by the Feulgen reaction (8). The corresponding
gland was pooled in 0.5 mL HBSS/FBS with that from four other ticks and was
homogenized; 0.4 mL of each pool was subcutaneously injected into an adult
C3H/HeJ mouse. Blood smears were made 1 week after injection to detect
ehrlichiae or piroplasms. All mice were observed for 1 month.

Neonatal (2 to 5 days after birth; "suckling") or subadult (<18 g) outbred
CD-1 mice (Charles River Laboratories, Wilmington, MA) were used to
propagate putative viral pathogens. Suckling mice were intracerebrally
inoculated with 0.03 mL of a clarified and 0.22 mm filtered 20% suspension
of whole mouse brain in HBSS/FBS. Alternatively, aseptically drawn plasma
from putatively infected mice was directly injected. Siblings of the
suckling or subadult mice received 0.03 mL of sterile HBSS/FBS.

Suspensions of brain tissue from mice that had died were centrifuged, the
pellet was resuspended in Trizol Reagent (BRL Life Technologies, Bethesda,
MD), and RNA was extracted according to the manufacturer's protocol. RNA
was reverse transcribed by using POW-1, POW-2, ENV-A and POW-6 primers.
Primer design was based upon the published cDNA sequences for TBE group
viruses (9). Reverse transcriptase polymerase chain reaction (RT-PCR) was
performed with dedicated pipettors in an area physically separated from
where amplification products were manipulated.

Amplification products were excised from 3% agarose gels, purified by using
the GeneClean kit (Bio 101 Inc.), and cycle sequenced at the University of
Maine Biotechnology Center. Each sequence was verified by sequencing
amplification products from two independent PCR reactions. These sequences
(Ips1, Ips2, CT 390) have been deposited in GenBank (accession numbers
U93287, U93288, U93289).

To determine whether ticks that (as larvae) had fed on putatively infected
mice became infected with a viral pathogen, living nymphs were directly
placed in Trizol Reagent within individual screwcapped microcentrifuge
tubes and allowed to remain overnight. A heat-sealed micropipet tip was
used to crush each tick, and the resulting homogenate was extracted as
described. RNA was analyzed for evidence of TBE group-specific env using
primers POW1/POW2 in the described RT-PCR assay.

Distinctive inclusions were detected by light microscopy in the
Feulgen-stained salivary glands of one tick in each of two pools (Figure
1). The staining properties and shape of these inclusions were distinct
from those of an ehrlichia or a piroplasm. Of the mice that were inoculated
with pooled salivary tissues from these ticks with the unidentified
salivary inclusions, two died approximately 14 days later after a prodrome
that included signs of neurologic involvement. Ehrlichiae or babesiae were
not detectable in their peripheral blood. One had received an inoculum
derived from ticks sampled in a site in Massachusetts and the other from
ticks in Connecticut (Table). Mice that had received salivary gland
material that did not contain similar inclusions remained healthy. A
neurotropic microbial pathogen was suspected.

 [Photo]  [Figure 1] [Figures not available in ASCII version]

          Figure 1. Comparison of morphology and tinctorial properties of
          Feulgen stained, intact tick salivary glands infected by three of
          the deer tick pathogen guild. Spirochetes only transiently
          migrate through salivary tissues, and thus would not be
          visualized by this technique. (A) Ehrlichia microti. Polyhedral
          clusters of rickettsiae (arrows) within hypertrophied salivary
          acinus. (B) Babesia microti, dense stippling of sporoblasts
          (arrows). Each minute dot represents the nucleus of a sporozoite.
          (C) Deer tick virus. Hypotrophied salivary acinus filled with
          amorphous masses of pinkstaining (=Feulgen positive) material
          (arrows). Scale bar = 10 m.

             Table. Prevalence of deer-tick virus infection in
             adult Ixodes dammini sampled from deer or
             vegetation during November 1995 from
             Massachusetts and Connecticut
             --------------------------------------------------

               Sampling area*         No.            No.
                                    examined       infected
             --------------------------------------------------
             W MA                      17             0
             C MA                      15             0
             NE MA                    157             1
             SE MA                    175             0
             NW CT                     7              1
             NE CT                     6              0
             SW CT                     26             0
             SE CT                     23             0
             Total                    465             2
             --------------------------------------------------
             *The deer management zones, or study sites,
             belong to one of four geographic quadrants for
             Connecticut (NE, northeastern; NW, northwestern;
             SE, southeastern; SW, southwestern) or of four
             regions in Massachusetts (W, western; C, central;
             NE, northeastern; SE, southeastern).

To determine whether an arbovirus may have infected the two mice that died,
we injected homogenized samples of brain tissue into suckling mice.
Although we initially anticipated that an atypical rickettsial agent might
have caused the death of these mice, we found that the putative agent could
be passaged by serially inoculating clarified and filtered suspensions of
brain tissue of affected mice into either suckling or adult mice. Blood or
plasma from moribund mice was poorly infectious. We concluded that a
neurotropic infectious agent was present in at least two of the 465 ticks
that were sampled originally.

To identify this microbe, RNA was extracted from an aliquant of each of the
infected salivary gland homogenates and from a suspension of the brain
tissue of each of the two mice that died. This preparation was used as
template for an RT-PCR employing primers designed to amplify a portion of
the TBE group virus nonstructural protein or envelope genes. Amplification
products were sequenced and compared to viral sequences accessioned in
GenBank. Parsimony analysis of the derived sequences for both genes
indicated that the two viral isolates from different sites were identical,
grouped within the TBE complex, but were distinct from known viruses
(Figure 2). Powassan virus was the most closely related organism, with 84%
pairwise similarity in the portion of the envelope gene that was analyzed.
This apparently novel member of the TBE group of flavivirus is
provisionally named deer tick virus (DTV).

             [Figure 2] [Figures not available in ASCII version]
             Figure 2. Maximum parsimony phylogram indicating
             the relationship of deer tick virus (DTV) to
             other tick-borne encephalitis group viruses.
             Sequences in GenBank for representative
             tick-borne flavivirus envelope genes were aligned
             and compared by using yellow fever virus as the
             outgroup. Values above branches indicate
             bootstrapped confidence values. Branch lengths
             are proportional to percent similarity in
             sequence. IPS 001, mouse brain derived DTV
             (Massachusetts isolate); CT390, mouse brain
             derived DTV (Connecticut isolate). POW, Powassan
             virus; TBE, tick borne encephalitis; TSE, Turkish
             sheep encephalitis; GGE, Greek goat encephalitis;
             LI, louping ill virus; SSE, Spanish sheep
             encephalitis; KFD, Kyasanur Forest disease virus;
             TYU, Tyuleniy virus; SRE, Saumarez Reef virus.
             The topology of the phylogram that was
             independently derived from NS-5 does not differ
             from that of env and is not presented.
             Approximately 100 ng of extracted RNA was added
             to a 20 mL reverse transcriptase (RT) reaction
             containing 5mM MgCl2, 50mM KCl, 100mM tris-HCl
             (pH 8.3), 5mM each of the four deoxynucleotides,
             20U RNase Inhibitor, 50U RT, and 25 pmols of the
             appropriate downstream primer (POW-2 [5'
             GCTCTCTAGCTTGAGCTCCCA 3'] or POW-6
             [5'TTGTGTTTCCAGGGCAGCGCCA 3']). To a 50 L PCR
             reaction using the Elongase system (BRL Life
             Technologies) and a final Mg++ concentration of
             1.5mM was added 1/20th of the RT reaction and
             25pmol of primers POW-1 [5'TGGATGACAACAGAAGACATGC
             3'] and POW-2, which amplify a 291 bp region of
             the TBE virus complex nonstructural protein gene
             (NS-5). Alternatively, primers POW-6 and ENV-A
             [5'GTCGACGACGAGGTGCACGCATCTTGA 3'] were added to
             amplify a 689 bp region of the TBE virus envelope
             gene (env).

             Sequences derived from RT-PCR were compared with
             those accessioned in GenBank. Sequence alignments
             were generated with the PILEUP program of the
             Wisconsin Genetics Computer Group, and these
             prealigned sequences were analyzed by parsimony
             using PAUP 3.1 (D. Swofford, Illinois Natural
             History Survey, Champaign, IL) with yellow fever
             virus as the outgroup. Characters were equally
             weighted and treated as unordered. Phylogenies
             were constructed by using the heuristic search
             option with the tree bisection reconnection
             branch swapping method. The robustness of the
             maximum parsimony tree was estimated by
             performing 1,000 bootstrapped replicates.
             Separate comparisons were done for env and NS-5
             because fewer NS-5 sequences were available for
             many of the viruses for which there were env
             sequences.

Suckling mice became paretic and died by 5 days after DTV was
intracranially injected. During the third serial passage, the adult mouse
LD50 for subcutaneous injection of a 20% brain suspension, performed in
adult CD-mice, was estimated at 6.3 x 104/mL. Although adult laboratory
mice died within 14 days of infection, all eight adult P. leucopus survived
without apparent ill effect. White-footed mice (Peromyscus leucopus) were
from a laboratory colony at the Harvard School of Public Health and
represent mice originally collected from Nantucket Island in 1992. Their
ability to survive infection suggests that white-footed mice may serve as
reservoirs of this virus in nature.

A xenodiagnosis (10) was performed to detect cryptic ehrlichial infection.
We permitted noninfected, laboratory-reared larvae of I. dammini to engorge
on one of the original C3H mice that showed signs of disease. After
molting, the derived nymphs were permitted to engorge on outbred Swiss
mice. Both mice died 2 weeks later, suggesting that DTV had been
transmitted by these ticks. Indeed, two of eight nonengorged ticks from the
same feeding contained DTV RNA by an RT-PCR assay. Nymphs from the same
batch of larvae that had been fed on noninfected mice did not contain DTV
RNA. We concluded that subadult I. dammini efficiently transmit DTV.

The presence of this new TBE-like virus in New England deer ticks confirms
that this guild of Ixodes-transmitted pathogens is as diverse in the
Nearctic as in the Palearctic and is consistent with an original ancient
Holarctic distribution of these ticks and their pathogens. We hypothesize
that the guild survived the Pleistocene period in refugia located near the
terminal moraine sites where the deer tick-transmitted zoonoses were first
described (5). The alternative, that the American guild may have evolved in
parallel with that in Eurasia, seems less persuasive.

DTV may derive from Powassan virus, which mainly cycles between North
American woodchucks (Marmota monax) and I. (Pholeoixodes) cookei (11), a
one-host tick that is distantly related to I. dammini and only rarely feeds
on humans or mice. Because deer ticks occasionally feed on woodchucks, they
may carry this virus to the mouse population. Indeed, I. dammini are
competent laboratory vectors of I. cookei-derived Powassan virus (12).
Although the extent of geographic variation in its genes remains unknown,
Powassan is as molecularly distinct from DTV as Central European
encephalitis virus (also known as Western subtype of TBE) is from Russian
spring summer encephalitis (also known as Eastern subtype of TBE) or
Louping Ill viruses (Figure 2). DTV, therefore, may represent a new subtype
of Powassan virus.

Many tick-borne agents may be detected more readily if ticks are allowed to
partially feed, thereby initiating reactivation (13). We regarded as
unlikely the possibility that any infections detected in partially fed
ticks simply represented infections that had been ingested. In that case,
other ticks in the same collections would also tend to be infected. Our
focus on the analysis of the salivary glands also reduces the possibility
that an ingested pathogen rather than one derived from a previous bloodmeal
would be detected. Indeed, the developmental cycle of these agents would
not readily allow rapid invasion and residence in the salivary glands
during the infecting bloodmeal (but see 14). Accordingly, the two
infections detected had been transstadially passaged as opposed to
periprandially acquired.

          [Figure 3] [Figures not available in ASCII version]
          Figure 3. Adult deer tick.

The Feulgen reaction is DNA-specific (15); therefore, it appeared illogical
that inclusions of an RNA virus would be specifically stained in our
microscopy-based assay. Experimental evidence suggests, however, that
glycoproteins may also be stained (16,17) in what is known as the
plasmalogen reaction. The inclusions detected in tick salivary glands may,
therefore, represent envelope proteins from massively replicating virus.

DTV appears to be less infectious than other members of the TBE virus
complex. Suspensions of suckling mouse brain tissue contained 10 to the 4th
power adult mouse LD50 of virus; in contrast, similar preparations of
Central European encephalitis may contain 10 to the 8th power. However,
early passage virus may increase in virulence with subsequent adaptation.
Suckling or adult mice that had received control inocula but lived within
the same cages as virus-infected mice did not become infected, which
suggests that aerosol or contaminative transmission of virus between mice
is rare.

The public health significance of DTV remains undescribed. TBE-like viruses
tend to be virulent, causing an encephalitis with a case-fatality rate that
approaches 40% or with debilitating neurologic sequelae (18,19). Although
the consequences of human DTV infection are unknown, the related Powassan
infection usually causes severe encephalitis (20). On the other hand,
Central European encephalitis infection is frequently mild or silent (21).
Our estimate of the prevalence of viral infection in adult I. dammini
(0.43% of 465 ticks) is consistent with those for enzootic Central European
encephalitis in present-day European sites where the risk for this
infection is well-described. TBE-group viruses, however, exist in microfoci
(22), and local prevalence may be greater. Residents of New England sites
where the other three members of the deer tick pathogen guild are zoonotic
may thus be exposed to DTV, but the infection causes a self-limited febrile
illness of unknown etiology.

Attempts to define the etiology of Lyme disease in the 1970s included an
exhaustive search for evidence of arboviral infection in Connecticut I.
dammini; none was found (23,24). Because deer ticks were less numerous then
than they are now, the pathogens they transmitted might have been
correspondingly infrequent. DTV may constitute an emerging fourth member of
the guild of deer tick-transmitted zoonotic agents in eastern North
America. The incidence of disease associated with the aggressively
human-biting deer tick may continue to rise where deer populations
increase.

Acknowledgments

We are indebted to Charles Seymour for many useful discussions and
suggestions and to John David for reading the manuscript. All animal
experiments were conducted according to the approved IACUC guidelines of
the Harvard Medical Area. This work was supported by NIH grants AI 39002,
AI 37993, AI 34409, and AI 19693; the Gibson Island Corporation; the Chace
Fund; and David Arnold.

Sam R. Telford III,* Philip M. Armstrong,* Paula Katavolos,* Ivo Foppa,* A.
Sonia Olmeda Garcia,* Mark L. Wilson, Andrew Spielman*

*Harvard School of Public Health, Boston, Massachusetts, USA; Universidad
Complutense de Madrid, Spain; and Yale University School of Medicine, New
Haven, Connecticut, USA

References

  1. Spielman A, Wilson ML, Levine JF, Piesman J. Ecology of Ixodes
     dammini-borne zoonoses. Annu Rev Entomol 1985;30:439-60.
  2. Telford SR III, Dawson JE, Katavolos P, Warner CK, Kolbert CP, Persing
     DH. Perpetuation of the agent of human granulocytic ehrlichiosis in a
     deer tick-rodent cycle. Proc Nat Acad Sci USA 1996;93:6209-14.
  3. Telford SR III, Dawson JE, Halupka K. Emergence of tickborne diseases.
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  4. Spielman A, Clifford CM, Piesman J, Corwin MD. Human babesiosis on
     Nantucket Island, USA: description of the vector, Ixodes (Ixodes)
     dammini, n.sp. (Acarina:Ixodidae). J Med Entomol 1979;15:218-34.
  5. Oliver JH, Owsley M, Hutcheson HJ, James AM, Chen C, Irby WS, et al.
     Conspecificity of the ticks Ixodes scapularis and I. dammini
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  6. Marshall WF, Telford SR III, Rhys PN, Rutledge BJ, Mathiesen D,
     Spielman A, et al. Detection of Borrelia burgdorferi DNA in museum
     specimens of Peromyscus leucopus. J Infect Dis 1994;170:1027-32.
  7. Aeschlimann A, Burgdorfer W, Matile W, Peter O, Wyler R. Aspects
     nouveux du role de vecteur joue par Ixodes ricinus L. Acta Trop
     1979;36:181-91.
  8. Piesman J, Mather TN, Donahue JG, Levine JF, Campbell JD, Karakashian
     SJ, et al. Comparative prevalence of Babesia microti and Borrelia
     burgdorferi in four populations of Ixodes dammini in eastern
     Massachusetts. Acta Trop 1984;43:263-70.
  9. Mandl CW, Heinz FX, Stockl E, Kunz C. Genome sequence of tickborne
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     1989;173:291-301.
 10. Donahue JG, Piesman J, Spielman A. Reservoir competence of
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 11. Artsob H. Powassan encephalitis. In: Monath TP, Editor. The
     arboviruses: epidemiology and ecology, Volume IV. Boca Raton (FL): CRC
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 16. Wolman M. On the absence of desoxyribonucleic acid from some
     chemically induced cytoplasmic and intranuclear inclusions, with
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     encephalitis. American Review of Soviet Medicine 1946;5:1-80.
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     Etiologic and epidemiologic observations in New York State. NY State J
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     encephalitis in a high-risk population in Sweden. Eur J Clin Microbiol
     Infect Dis 1992;11:894-900.
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     Raton (FL): CRC Press; 1989;177-202.
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---------------------------------------------------------------------------

   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Dispatches

   An Unusual Hantavirus Outbreak in Southern Argentina: Person-to-Person
                               Transmission?

---------------------------------------------------------------------------


      Hantavirus pulmonary syndrome is a rodent-borne zoonosis first
      recognized in the United States in 1993. Person-to-person
      transmission has not been reported; however, in the outbreak of
      20 cases reported here, epidemiologic evidence strongly
      suggests this route of transmission.

In 1995, a novel hantavirus (Andes virus) was identified in samples from
patients in southern Argentina (1). The patients had hantavirus pulmonary
syndrome (HPS), a disease first described 2 years earlier in the United
States, in association with Sin Nombre virus (a new world hantavirus)
(2,3). Old World hantaviruses (Puumala, Seoul, Hantaan, and Dobrava) have
been recognized causes of hemorrhagic fever with renal syndrome (HFRS) for
at least 40 years and are responsible for more than 100,000 cases each year
in Eurasia (4). Hantaviruses (family Bunyaviridae, genus Hantavirus) are
rodent-borne viral zoonotic agents believed to be transmitted to humans
primarily by inhalation of aerosols of infected rodent excretions (4).
Person-to-person transmission of hantaviruses has not been reported despite
extensive epidemiologic studies. Between September 22 and December 5, 1996,
18 cases of HPS occurred in residents of, or visitors to, the towns of El
Bolsn, Bariloche, and Esquel in southern Argentina. Two additional persons
who had contact with El Bolsn patients but had not visited the area,
contracted HPS during this period (5). Five of the patients were
physicians; three were directly responsible for the clinical care of an HPS
patient. Epidemiologic links between all but four of the cases and evidence
of low rodent population density in the area strongly suggest
person-to-person transmission of HPS during this outbreak.

Sporadic cases of HPS and possible cases of HFRS from as early as 1987,
have been retrospectively identified in Argentina (6). In 1995, genetic
material from Andes virus, a unique hantavirus, was identified in the lungs
of a patient from El Bolsn (population ~15,000) in southern Argentina (1).
The outbreak of HPS reported in this dispatch began on September 22, 1996,
when the 41-year-old index patient (patient I) (also from El Bolsn) became
ill with HPS (Figure 1). El Bolsn, a rural town in Rio Negro Province, is
approximately 350 meters above sea level in the foothills of the Andes.
Patients requiring intensive care are transferred to Bariloche (population
80,000), 150 kilometers to the north (Figure 2).

                 [Figure 1] [Figures not available in ASCII version]
                 Figure 1. Transmission tree for HPS cases
                 in southern Argentina, September-December
                 1996, indicating dates of onset of
                 symptoms, survivor status, and proposed
                 lines of transmission. Lines of
                 transmission are hypothetical since many
                 of the patients had contact with multiple
                 HPS patients. Bold lines denote husband
                 and wife. The two sporadic cases, U and
                 R, are not shown.

                   [Figure 2] [Figures not available in ASCII version]
                   Figure 2. Towns involved in the 1996
                   HPS outbreak in southern Argentina.

Potential cases were identified by local physicians familiar with the
clinical features of HPS (3) and were accepted as cases when enzyme-linked
immunosorbent assay detected IgM antibodies reacting with Sin Nombre virus
and/or when reverse transcriptase and polymerase chain reaction detected
hantaviral RNA (1,2,6,7). Viral sequences indicated a sigmodontine-derived
hantavirus related to, but clearly distinct from, Sin Nombre virus;
additional sequencing and phylogenetic studies are under way.

Twenty-one and 20 days, respectively, after the index patient became
symptomatic, his 70-year-old mother (patient B) and one of his doctors
(patient A) contracted HPS. The doctor's spouse, also a doctor (patient C),
became ill with HPS 27 days after her husband's first symptoms (19 days
after his death). She traveled to Buenos Aires for medical care. In a
Buenos Aires hospital, an admitting doctor (patient D) spent 1 hour taking
a clinical history and examining her. The doctor (patient D) applied
pressure to a venipuncture site on patient C's arm with multiple layers of
gauze; no obvious blood contact occurred. The only other contact between
this doctor and patient C occurred 2 days later, when the doctor briefly
visited the hospital's intensive care unit to attend to another patient.
Twenty-four days after attending patient C, the doctor became ill with HPS.
The doctor, patient D, had not traveled outside Buenos Aires, and she
reported no contact with rodents during the 2 months preceding her illness
(5).

A 40-year-old doctor (patient E) from Buenos Aires contracted HPS 17 days
after patient C was admitted to the hospital. This doctor was a friend of
patients A and C and spent 3 days in El Bolsn after the death of patient
A. She visited patient C often in the hospital but was not directly
involved in the clinical management of any HPS patients. A fifth doctor
with HPS (patient F) reported having close contact with several HPS
patients. He intubated patient B and examined patients I and G. He also had
daily contact with his colleague, (patient A), and spoke briefly with
patient I's sister (patient H), brother-in-law (patient J), and friend
(patient K).

At the funeral of patient B, patient I's housekeeper (patient L) was
already symptomatic. She returned to Buenos Aires by car with patients H,
J, and their daughter (patient M). Symptoms developed in the latter three
occupants of the car 11, 15, and 29 days, respectively, after the drive
back to Buenos Aires. No evidence of rodent infestation was found when the
car was examined 3 weeks later. Although patients H and J had stayed at the
home of patient I for several nights, patient M had not been to El Bolsn
in the preceding months, traveling only as far as Jacobacci (~250 miles
from El Bolsn) for patient B's funeral.

A second group of HPS cases occurred in Bariloche. Four people who had
visited or worked in the hospital to which many of the El Bolsn patients
were transferred (a modern 40-bed facility with a three-bed intensive care
unit) contracted HPS: the hospital's night receptionist (patient N), a
40-year-old woman (patient O) who visited an unrelated patient, a
27-year-old man (patient P), and his wife (patient Q). Patients P and Q
developed a close relationship with patient N during their many visits to
the hospital between September 20 (when their 27-week-gestation baby was
born) and October 27 (when the baby died). The three shared mate (a local
tea drunk through a communal metal straw), and patient P occasionally
rested on patient N's camp bed. The baby got abdominal distention and shock
37 days after birth. The clinical signs suggested necrotizing
enterocolitis, but no serum or tissue specimens from the infant were
available for definitive diagnosis.

The remaining three HPS patients in El Bolsn during this period were men
aged 44 (patient G), 29 (patient R), and 14 (patient T) years. Patients G
and T were friends or acquaintances of one or more of the HPS patients, but
did not recall contact with an HPS patient in the 6 weeks before the onset
of their symptoms.

Both patients R and U likely had coincidental sporadic cases of HPS.
Patient R lived in the Chilean mountains and had prodromal symptoms of HPS
on arrival in El Bolsn. Patient U, a 33-year-old man living and working in
Futalaufqun National Park, Chubut Province (150 km south of El Bolsn),
also contracted HPS during this period but is unlikely to have had contact
with HPS patients in El Bolsn.

Overall, 11 (55%) of the 20 patients were male, the mean age was 38 years
(range 13-70 years), and the case-fatality rate was 50%. The death rate
among cases for which epidemiologic links were identified (Figure 1) was
lower during the latter stage of the outbreak (three of nine patients with
onset of symptoms in November-December died compared with six of seven
patients with onset of symptoms in October). The clinical picture was
similar to that of HPS in the United States, although several atypical
features, such as conjunctival injection and head and neck suffusion, were
noted. Coughing did not occur earlier or more frequently in Argentinean HPS
patients than in U.S. patients, which suggests that coughing was not the
critical factor in person-to-person transmission.

Early in the investigation of this outbreak, attention was focused on the
index patient's brick house, one of several dwellings located on a
semirural property on the outskirts of El Bolsn. Several weeks before
patient I became sick, he and patients B and L moved from a wooden cabin
into the brick house. Two doctors (patients A and F) made visits to this
house, and patients H and J stayed there at least one night during November
1996. The 13 other HPS patients in the outbreak did not visit the house.
One month after the onset of patient I's symptoms, an examination of the
brick house and other nearby structures found the buildings to be well
constructed with no obvious sites for rodent entry and no evidence of
rodent infestation (as confirmed by trapping studies). Trapping in and
around the homesites of the El Bolsn and Bariloche patients yielded few
rodents. Trap success (i.e., the number of rodents captured per 100 trap
nights) in neighboring areas of disturbed or natural vegetation indicated a
low rodent population density. Identification and serologic testing of
captured rodents are under way. The low trap success rate contrasted with
that of the 1993 HPS outbreak in the southwestern United States, when
rodents were plentiful overall and significantly more numerous in and
around homes of patients than in and around homes of controls (8).

The probable transmission of hantavirus from patient C to her doctor
(Patient D) is the best epidemiologic evidence available to support the
hypothesis that person-to-person transmission of hantavirus occurred during
this outbreak. When considered separately, links between other Argentinean
HPS patients are less convincing. However, when the contacts between
patients are viewed collectively, the probability that person-to-person
transmission played a major role in this outbreak strengthens. A comparison
of the relative success of rodent trapping efforts over the last 20 years
indicated that rodent population densities in the general area of the
outbreak during the spring of 1996 were lower than average (Pearson O,
pers. comm. 1996), adding further support for the hypothesis of
person-to-person transmission. It is highly unlikely that the low rodent
population density observed was the result of a precipitous decline in
rodent populations in late spring. Rodent populations in northwest
Patagonia are at minimal levels in the early spring and represent only
animals that have survived the winter. Renewed reproduction increases
population density throughout the spring (Guthmann N, pers. comm. 1997), as
was corroborated by the high percentage of immature animals captured during
trapping. Thus the rodent population density during our trapping in late
spring is likely to have been higher than density when the outbreak began.

The unique pattern of person-to-person transmission in this outbreak has
not been a feature of hantavirus epidemiology; in fact, a study performed
in New Mexico in 1993 found no disease or hantavirus antibodies in serum
specimens taken from 396 health care workers, including 266 who had been
exposed to patients with HPS or their body fluids 2 to 6 weeks earlier (9).
The high case-fatality ratio has precluded a detailed description of the
exact nature of contact between cases. Consequently, the likely mode of
transmissionwhether through direct contact, droplets, infectious aerosols,
or contaminated fomitesis not known. Because hantaviruses are difficult to
isolate and cultivate, few attempts have been made to study virus excretion
from patients or to assess viral stability under different environmental
conditions. Efforts to define the infectious stage of the disease and the
type and duration of contact that may have led to transmission are
continuing. Genetic sequences of viruses from patients and rodents are
being determined to define more clearly the pattern of transmission. In the
absence of any clear-cut evidence for nosocomial transmission of Sin Nombre
or related viruses in the United States, it is premature to suggest
changing the existing guidelines for care of HPS patients. The level of
suspicion should be raised to investigate any suspected person-to-person
spread of this rodent-borne zoonosis, however. Further epidemiologic and
laboratory studies are needed to clarify the requirements for protecting
both healthcare workers and others from hantaviral infections in Patagonia.

Rachel M. Wells,* Sergio Sosa Estani, Zaida E. Yadon, Delia Enria, Paula
Padula, Noemi Pini, James N. Mills,* Clarence J. Peters,* Elsa L.
Segura, and the Hantavirus Pulmonary Syndrome Study Group for Patagonia
*Centers For Disease Control and Prevention, Atlanta, Georgia, USA;
Instituto Nacional de Chagas "Dr. Mario Fatala Chaben," Buenos Aires,
Argentina; Administracin Nacional de Laboratorios e Institutos de Salud,
Buenos Aires, Argentina; Instituto Nacional de Enfermedades Virales
Humanas "Dr. Julio I. Maiztegui," Buenos Aires, Argentina

N. Guthmann, Centro Regional Universitario Bariloche; E. Argelo, F.
Klein, R. Levy, C. Nagel, Fundacin Favorolo; R. Calfin, F. de Rosas, M.
Lazaro, M. Rosales, P. Sandoval, Hospital Bariloche; L.A. Albornoz, R. L.
Antonia, J. C. Becerra, W.H. Benitez, A. Broide, C. Flandes, A. Honik, J.
Meza, L. Paganini, A. Resa, G. Roberts, P. Rodriguez, I.E. Rojo, G. Suppo,
A. Werenicz, S. Wisky, Hospital El Bolson; H. Quiroga, Hospital El Hoyo;
C.A. Cohen Arazi, L. Bogni, R. Lombardelli, B. Salvagno, C. Simeone,
Hospital Esquel; L. Clara, Hospital Italiano de la Ciudad de Buenos Aires;
M. del Castillo, Hospital Nacional de Clnicas "Jos de San Martn"; J.L.
Becker, H. Lopez, M. Palmigiani, Instituto Nacional de Enfermedades Virales
Humanas "Dr. Julio I. Maiztegui"; M. Ameztoy, A. Lawrynowicz, Instituto
Nacional de Epidemiologa "Dr. Juan H. Lara"; A. Edelstein, Instituto
Nacional de Microbiologa "Dr. Carlos Malbran"; A. Aguilera, V.O. Vigil,
Instituto Nacional de Parasitologa "Dr. Mario Fatala Chaben"; R. Chuit, C.
Riva Posse, Ministerio de Salud y Accin Social; C. Barclay, L. Caride, E.
De Orta, M. Furque, A. Picone, L. Samengo, Sanatorio San Carlos; R.
Fernandez, R. Gonzales, Sistema Provincial de Salud, Esquel; O. Arellano,
G. Cantoni, E. Larrieu, J. Vilosio, Sistema Provincial de Salud, PCIA de
Ro Negro; K. Busico, A. Khan, T.G. Ksiazek, W. Terry, J. Young, S. Zaki,
Centers for Disease Control and Prevention

References

  1. Lopez N, Padula P, Rossi C, Lazaro ME, Franze-Fernndez MT. Genetic
     identification of a new hantavirus causing severe pulmonary syndrome
     in Argentina. Virology 1996;220:223-6.
  2. Nichol ST, Spiropoulou CF, Morzunov S, Rollin P, Ksiazek TG, Feldmann
     H, et al. Genetic identification of a hantavirus associated with an
     outbreak of acute respiratory illness. Science 1993;262:914-7
  3. Khan A, Khabbaz RF, Armstrong L, Holman RC, Bauer SP, Graber J, et al.
     Hantavirus pulmonary syndrome: the first 100 cases. J Infect Dis
     1996;173:1297-303
  4. McKee KT, LeDuc JW, Peters CJ. Hantaviruses. In: Belshe R, editor.
     Textbook of human virology, 2nd edition. St. Louis: Mosby Year Book,
     Inc., 1991:615-32
  5. Enria D, Padula P, Segura EL, Pini N, Edelstein A, Riva Posse C, et
     al. Hantavirus pulmonary syndrome in Argentina. Possibility of person
     to person transmission. Medicina (B Aires) 1995;58:709-11.
  6. Parisi M, Enria D, Pini NC, Sabatini MS. Deteccin retrospectiva de
     infecciones clnicas por hantavirus en la Argentina. Medicina (B
     Aires) 1995;56:1-13.
  7. Ksiazek TG, Peters CJ, Rollin PE, Zaki S, Nichol S, Spiropoulou C, et
     al. Identification of a new North American hantavirus that causes
     acute pulmonary insufficiency. Am J Trop Med Hyg 1995;52:1017-23.
  8. Childs JE, Krebs JW, Ksiazek TG, Maupin GO, Gage KL, Rollin PE , et
     al. A household-based, case-control study of environmental factors
     associated with hantavirus pulmonary syndrome in the southwestern
     United States. Am J Trop Med Hyg 1995;52:393-7.
  9. Vitek CR, Breiman RF, Ksiazek TG, Rollin PE, McLaughin JC, Umland ET,
     et al. Evidence against person-to-person transmission of hantavirus to
     health care workers. Clin Infect Dis 1996;22:824-6.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Dispatches

Pertussis in the Netherlands: an Outbreak Despite High Levels of
Immunization with Whole-Cell Vaccine

---------------------------------------------------------------------------


      In 1996, a sudden increase in pertussis incidence was reported
      in the Netherlands (2.1 per 100,000 in 1995, 18 per 100,000 in
      1996). Although not all potential surveillance artifacts could
      be excluded, it is highly probable that the data reflect a true
      outbreak. However, the cause of this increase has not yet been
      determined. Further research is directed to the severity of
      disease and a possible mismatch between the vaccine and the
      circulating Bordetella strains.

In 1996, 2,771 cases of pertussis were reported to the Inspectorate of
Health in the Netherlands (population 15 million), compared with 319 cases
in 1995. With epidemic cycles expected every 3 to 5 years and a recent
outbreak in 1994, this rise was unexpected (1). After the introduction of
pertussis immunization with a whole-cell vaccine in the National
Immunization Program (1952), the incidence of pertussis in the Netherlands
decreased significantly. Children are immunized at ages 3, 4, 5, and 11
months with a diphtheria, tetanus, pertussis, and inactivated polio vaccine
(DTP-IPV), and vaccine coverage for pertussis, for at least three
immunizations, is 96% at the age of 12 months. Until the 1980s, the
incidence of pertussis seemed very low, because only incidental cases were
reported. However, in the last two decades, pertussis has been endemic, and
epidemic peaks have appeared.

The surveillance of pertussis is based mainly on notification, which was
made obligatory by law in 1976. Because the availability and interpretation
of serologic tests changed in the 1980s and the absence of a clear case
definition for notification seemed to have influenced surveillance, a
restrictive case definition that included criteria for laboratory diagnosis
was introduced in 1988. Therefore, we have limited this report to
notification data from 1989 onwards. The annual incidence of pertussis
notification from 1989 to 1995 is shown in Figure 1. In this period, the
case definition includes defined clinical symptoms and laboratory
confirmation. The clinical symptoms are a serious cough lasting more than 2
weeks or cough attacks or cough followed by vomiting in combination with at
least one of the following symptoms/findings: apnea, cyanosis,
characteristic cough with whooping, subconjunctival bleeding, leucocytosis,
or contact with a person with confirmed or suspected pertussis in the
previous 3 weeks. Laboratory confirmation is defined as either positive
culture of Bordetella pertussis or Bordetella parapertussis or positive
two-point serology, i.e., the finding of a significant rise (> 4-fold) of
IgG antibodies against pertussis toxin and/or IgA antibodies against B.
pertussis in paired sera. The case definition is highly specific, but
results in significant underreporting.

             [Figure 1] [Figures not available in ASCII version]
             Figure 1. Annual incidence of pertussis
             estimated from notification by registration
             date, 1989-1995.

We compared the number of cases reported per 4-week-period in 1996 with the
number reported in 1994, the year with the highest incidence in recent
years (Figure 2). It appears that an unexpectedly large number of pertussis
cases was reported (18 per 100,000) in 1996. The reports came from all over
the country. No geographic clustering was observed, even in regions with
pockets of low vaccination coverage or at the borders of the country (close
to Germany, where vaccination coverage for pertussis is low).

             [Figure 2] [Figures not available in ASCII version]
             Figure 2. Number of cases reported by
             registration date, 1994 and 1996 (4-week
             periods).

The age distribution of patients in 1989 to 1996 shows a significant
decrease in the proportion of infants less than 1 year old (from 21% in
1989 to 7% in 1996) and a significant increase in the proportion of 1- to
4-year-old children (from 21% in 1989 to 30% in 1996). A significant
decrease (from 42% in 1989 to 30% in 1995) was also observed in 5- to
9-year-old children; however, in 1996, the proportion increased to 39%. No
changes were observed in the 10- to 14- and 15- to 19-year-old groups,
whereas the proportion of patients 20 years old or older increased from 5%
to 11% (Table).

Table. Age distribution (percentages) pertussis notification in 1989 to
1996
---------------------------------------------------------------------------
      Age       1989   1990   1991   1992    1993   1994   1995     1996
---------------------------------------------------------------------------
0                  21     21     23      21     24     16     13          7
1-4                21     21     26      19     27     34     33         30
5-9                42     37     34      30     26     32     30         39
10-14              10      9     10      17     12      8     10         11
15-19               1      2      2       2      1      1      1          2
[>=]20              5      9      6      11     10      8     12         11

Unknown             0      0      1       1      0      1      1          0
Total (n)         434    471    164     169    294    536    319      2,771
---------------------------------------------------------------------------

Whereas the average vaccine coverage did not change, the proportion of
vaccinated patients increased from 55% in 1989 to 85% in 1996. In all age
groups, except that of 6- to 11-month old infants, the proportion of
vaccinated patients is higher in the years 1994 to 1996 than in 1989 to
1993.

We estimated the annual incidence of pertussis for vaccinated and
unvaccinated children ages 1 to 4 and 5 to 9 years, on the basis of
reporting by registration date (1989 to 1996) (Figure 3). Calculation of
incidence according to vaccination status was deliberately restricted to
ages 1 to 4 years and 5 to 9 years (vaccine coverage estimated at 96%) for
methodologic reasons. The changes in age-specific incidence over the years
for vaccinated and unvaccinated children are identical for 1989 to 1993.
However, from 1994 to 1996, and particularly in 1996, the incidence among
vaccinated 1- to 4-year-olds was considerably higher than in 1989 and 1990,
the years with the highest incidence in 1989 to 1993. In unvaccinated
children, the incidence in 1994 and 1995 was lower than in 1989 and 1990.
The difference in incidence between the period 1989 to 1990 and the year
1996 was much smaller in unvaccinated than in vaccinated persons. A similar
discrepancy can be seen in the 5- to 9-year-old group.

             [Figure 3] [Figures not available in ASCII version]
             Figure 3. Estimated incidence of pertussis for
             unvaccinated and vaccinated children aged 1-4
             years and 5-9 years based on notification
             records and assuming a coverage of 96% for the
             entire population.

In view of the increase in pertussis reporting in 1996, the first issue to
be addressed was whether this reporting reflected a true increase in
pertussis incidence or was a surveillance artifact. If considerable
underreporting is assumed, an increase due to improved compliance to the
notification system or increased alertness should be considered. However,
in the first two quarters of 1996, there is no apparent reason for such an
assumption. Nonetheless, after the first reports of the observed rise, both
publicity and increased awareness of the disease by physicians and patients
might have influenced reporting.

Serodiagnosis for the country as a whole is done by a single reference
laboratory at the National Institute of Public Health and the Environment;
thus, the data provided by the laboratory can be used for nationwide
surveillance. Furthermore, the reference laboratory collects strains of B.
pertussis from regional public health laboratories, and the number of
strains received also reflects the epidemic curve. The epidemiologic curve
derived from these sources over the past years is consistent with the
notification data.

Since laboratory confirmation is part of the case definition, changes in
serodiagnostic practice might have influenced notification. Frequently, no
conclusive results are obtained from serology: a second serum sample may
not be submitted, or the first serum sample may be taken late after onset
of disease, thereby missing the dynamic phase of the immunoresponse.
Recently, cut-off values for antibody titers characteristic for the acute
immunoresponse after natural infection with B. pertussis have been
determined by comparing the titer distribution in the healthy population,
in recently vaccinated children, and in sera longitudinally collected after
natural infection. Since 1993, high titers in acute-phase sera are reported
to be "suggestive for recent infection with Bordetella pertussis" (2). It
can be imagined that such cases are reported, albeit not strictly in
accordance with the case definition.

To exclude the possibility that the rise in notification was the result of
reporting cases diagnosed with positive one-point serology only, we linked
the notification and serodiagnosis databases. (The data were taken from
January 1993 to September 1996.) Special permission to link the databases
was given by the Inspectorate of Health. It appeared that some notification
was based on positive one-point serology as early as 1993, and this
proportion increased from 20% in 1993 to 33% in 1994. It remained at this
level in the subsequent years. Thus, the 1996 increase in notification
cannot be attributed to this change in serodiagnostic practice.

Our surveillance data suggest a decrease in vaccine efficacy, but
estimation of vaccine efficacy from surveillance data should be interpreted
with caution. However, there are no indications of significant bias from
physicians' perceptions of vaccine efficacy that could have caused
selective reporting of vaccinated patients; a higher probability of a
positive serologic test result due to priming in vaccinated persons; or
misclassification of cases with respect to vaccination status.

Although not all potential surveillance artifacts could be excluded, it is
highly probable that the surveillance data reflect a true pertussis
outbreak. The outbreak could have been caused by 1) decrease in vaccination
coverage, 2) decrease in vaccine quality, 3) interference with other
vaccines, 4) changes in circulating strains of B. pertussis that are not
covered by vaccine-induced immunity, or 5) a combination of these factors.
A decrease in vaccination coverage has not been observed in the (accurate)
reporting system. The whole-cell vaccine used was produced in the National
Institute of Public Health and the Environment and meets international
standards. There was no sign of a gradual deterioration of vaccine quality
as determined for product release by the mouse protection test.

In April 1993, vaccination against Haemophilus influenzae type b (Hib) was
added to the immunization program. The polyribosylribitolphosphate tetanus
toxoid conjugated vaccine (PRP-T) was administered simultaneously with the
DTP-IPV, although on different limbs. Interference of Hib vaccination with
the immunoresponse to pertussis vaccine has been described (3). In a
prospective serologic study on the potential interference in the
Netherlands, this effect was not observed (4). Furthermore, the increase of
pertussis in 5- to 9-year-old vaccinated children, who never received Hib
vaccine, was similar to the increase in the 1- to 4-year-old age group
(Figure 3). Decreased vaccine efficacy due to interference of Hib
vaccination is, therefore, very unlikely.

Molecular typing of B. pertussis isolates collected since 1945 indicated a
shift in population structure, possibly due to the introduction of
whole-cell vaccine (5). Further, antigenic variants of B. pertussis
distinct from the strains incorporated in the vaccine appear to have
emerged (6). It might be possible that circulating strains of B. pertussis
have become less sensitive to vaccine-induced immunity. The abrupt increase
in the proportion of vaccinated patients reported in all age groups
suggests such a change.

Data from other countries in Europe indicate that the pertussis epidemic is
restricted to the Netherlands. However, a resurgence of pertussis has been
noted in the United States since the late 1980s and in Canada since 1991.
No single factor has been found to explain this resurgence (7-10).

The decrease in pertussis notification in the Netherlands at the end of
1996 may indicate a seasonal variation in incidence. The 1996 data,
including those on hospital admissions, are still being analyzed. These
data will give insight into the severity of the pertussis epidemic among
infants. Protection of these infants is the main reason for pertussis
vaccination. In January 1997, active (monthly) surveillance by
pediatricians of cases among hospitalized children was added to the routine
surveillance based on notification and laboratory surveillance.

A prospective study is being considered to assess efficacy of the
whole-cell vaccine, including differentiation of severity of illness and
number of immunizations received, if the outbreak continues. Cooperative
studies to determine the distribution of restriction fragment length
polymorphism types and antigenic variants of B. pertussis in various
countries in Europe, Asia, and North America are under way. Also, the
immunogenicity of the Dutch whole-cell vaccine is being assessed: whether
it has changed over time and how it compares to published immunogenicity
data of whole-cell vaccines with known, prospectively determined, vaccine
efficacy (11).

In conclusion, in the Netherlands a sudden increase of pertussis
notification has been observed, which seems to reflect a true increase in
incidence. Nevertheless, the cause of this increase has not been
definitively determined. A possible mismatch between the vaccine and the
circulating Bordetella strains is being investigated.

H.E. de Melker,* M.A.E. Conyn-van Spaendonck,* H.C. Rmke,* J.K. van
Wijngaarden, F.R. Mooi,* and J.F.P. Schellekens*

*National Institute of Public Health and the Environment, Bilthoven, The
Netherlands; and Inspectorate of Health, Rijswijk, The Netherlands

References

  1. Melker HE de, Conyn-van Spaendonck MAE, Rmke HC, Sprenger MJW,
     Schellekens JFP. Kinkhoest in Nederland: 1989-1994. Nederlands
     Tijdschrift Geneeskunde 1995;139:1280-6.
  2. Zee A van der, Agterberg C, Peeters MF, Mooi F, Schellekens JFP. A
     clinical validation of B. (para)pertussis PCR: comparison with culture
     and serology using samples of patients suspected for whooping cough
     from a highly immunized population. J Infect Dis 1996;174:89-96.
  3. Clemens JD, Ferreccio C, Levine MM, Horwitz I, Rao MR, Edwards KM, et
     al. Impact of Haemophilus influenzae type b
     polysaccharide-tetanus-pertussis vaccine. JAMA 1992;267:880-4.
  4. Labadie J, Sundermann LC, Rmke HC, and the DTP-IPV~Hib vaccine study
     group. Multi-center study on the simultaneous administration of
     DTP-IPV and Hib PRP-T vaccines. Part 1. Immunogenicity. National
     Institute of Public Health and the Environment (RIVM) Bilthoven, The
     Netherlands. Report No. 124001003, April 1996.
  5. Zee A van der, Vernooij S, Peeters M, Embden J van, Mooi FR. Dynamics
     of the population structure of Bordetella pertussis as measured by
     IS1002-associated RFLP: comparison of pre- and post-vaccination
     strains and global distribution. Microbiology 1996;142:3479-85.
  6. Mooi FR, Oirschot H van, Peeters J, Willems RJL. Antigenic variation
     of the accellular vaccine component pertactin in the Dutch B.
     pertussis population. National Institute of Public Health and the
     Environment (RIVM) Bilthoven, The Netherlands. Annual Report 1995.
  7. Centers for Disease Control and Prevention. Resurgence of pertussis -
     United States, 1993. MMWR Morb Mortal Wkly Rep 1993;42:952-3,959-60.
  8. Centers for Disease Control and Prevention. Pertussis - United States,
     January 1992-June 1995. MMWR Morb Mortal Wkly Rep 1995;44:525-9.
  9. De Serres G, Bouliane N, Douville Fradet M, Duval B. Pertussis in
     Quebec: ongoing epidemic since the late 1980s. Can Commun Dis Rep
     1995;21:45-8.
 10. Milord F. Resurgence of pertussis in Monteregie, Quebec - 1990-1994.
     Can Commun Dis Rep 1995;21:40-4.
 11. Greco D, Salmaso S, Mastrantonio P, Giuliano M, Tozzi AE, Anemona A,
     et al. A controlled trial of two acellular vaccines and one whole cell
     vaccine against pertussis. N Engl J Med 1996;334:341-8.

---------------------------------------------------------------------------

   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Dispatches

Invasive Haemophilus influenzae Type B Disease in Elderly Nursing Home
Residents: Two Related Cases

---------------------------------------------------------------------------


      We investigated two fatal cases of invasive Haemophilus
      influenzae type b (Hib) infection in a community nursing home
      in western Sydney, Australia. Two elderly women had lived in
      the same room, and the onset of their illness was 5 days apart.
      Hib isolates from blood cultures showed identical profiles by
      pulsed field gel electrophoresis. These findings suggest that
      Hib infection was transmitted within this nursing home. Serious
      Hib disease may be underrecognized in this setting. Continued
      surveillance and serotyping of invasive H. influenzae disease
      is essential for identifying groups at increasing risk that may
      benefit from immunization against Hib.

After the introduction of routine childhood immunization against
Haemophilus influenzae type b (Hib), the incidence of invasive Hib disease
fell dramatically in several countries, including Australia (1-3). This
decline has been most evident among children aged less than 5 years.
Meanwhile, serious Hib infections, such as pneumonia and epiglottitis, have
been increasingly recognized among elderly, debilitated, and
immunosuppressed adults (4-6). However, clusters of Hib infection have
rarely been reported in adults. To examine their relatedness, we
investigated two fatal cases of Hib septicemia in a community nursing home.

Case Reports

Case 1
In June 1996, a 71-year-old female nursing home resident was hospitalized
after 3 days of unproductive cough, fevers, confusion, and increasing
dyspnea. She had a history of cerebrovascular disease, muscular dystrophy,
and congestive heart failure and was confined to a wheelchair. She had
clinical signs of left lower lobe pneumonia and was afebrile. A blood film
was normal, but a chest X-ray showed extensive bilateral pulmonary
infiltrates. Arterial blood gases showed severe hypoxia in room air (PO2 44
mm Hg, saturation 77%). Ceftriaxone 1 g and metronidazole 500 mg were given
intravenously for suspected aspiration pneumonia, but the patient became
severely hypotensive and died 8 hours after admission to the hospital. The
following day, betalactamase-negative Hib was isolated from blood cultures
collected before the patient died.

Case 2
Two days after the first patient was hospitalized, an 80-year-old female
resident of the same nursing home became ill with fever and sore throat.
She had a history of chronic airflow limitation, mild renal impairment, and
dementia. Her sore throat rapidly worsened, and the next day she was
hospitalized with severe dysphagia. On examination, she was febrile
(38.5C) and hypotensive (90/60 mm Hg) and had pretracheal cellulitis. No
clinical evidence of pneumonia was observed, and a chest X-ray was normal.
She was intubated, and extensive epiglottitis, laryngitis, and tracheitis
were noted. Ceftriaxone 2 g, gentamicin 80 mg, flucloxacillin 1 g, and
metronidazole 500 mg were administered intravenously. Inotropes were given
for hypotension and acute renal failure, but the patient's condition
deteriorated rapidly, and she died 9 hours after admission to the hospital.
Beta-lactamase-negative Hib was subsequently isolated from blood cultures
collected before the patient died.

Investigation
For several months, these two women had occupied beds in the same six-bed
room (the largest room) in the 44-bed nursing home. The home employed 37
nursing and ancillary staff. During 3 weeks before the investigation, 20
residents and several staff had been unwell with bronchitis, for which many
had received oral antibiotics. No sputum samples had been collected for
culture from residents or staff. Two other elderly residents had died
during the preceding 10 days, but their case notes did not document any
fever or symptoms suggesting infection. On the first day of our
investigation, two elderly residents were febrile. One woman had bilateral
bronchopneumonia that was not responding to oral cefaclor and was
hospitalized; blood and sputum cultures yielded no pathogens, and urine
samples did not contain Hib capsular antigen. The other woman had been
febrile for 1 week, with a cellulitic leg ulcer and pleuritic chest pain of
uncertain etiology. Because of her other chronic illnesses she was treated
palliatively and died 1 week later. A swab of her leg ulcer yielded group G
streptococcus, blood cultures were negative, and sputum was not obtained.

Throat swabs were collected from all 81 residents and staff at the nursing
home and cultured for Hib on bacitracin chocolate blood agar. We
administered rifampicin chemoprophylaxis to all roommates of the index case
patients and 28 staff who had been in regular close contact with the two
case patients during the week before their illness. No new febrile
illnesses occurred during 3 weeks of follow-up, and Hib was not isolated
from any throat swab cultures. The Hib isolates from the first and second
patients were examined by pulsed field gel electrophoresis and had
identical profiles that were distinguishable from several epidemiologically
unrelated isolates (Figure).

         [Image] [Figures not available in ASCII version]
         Figure. Pulsed field gel electrophoresis of Haemophilus
         influenzae type b (Hib) isolates from blood culture of
         two elderly nursing home residents (lanes 1 and 2)
         compared with epidemiologically unrelated H. influenzae
         isolates sent to our laboratory for typing: lane 3,
         laboratory Hib strain; lane 4, non-H. influenzae; lane 5
         and 12, invasive nontypeable H. influenzae; lane 6-11,
         unrelated Hib isolates; lane 13, H. influenzae type a;
         lane 14, 1 kilobase molecular marker. The enzyme used
         for DNA digestion was Apal.

The relationship of these two fatal cases of Hib disease in time and place
and the clonality of the isolates provide good evidence that transmission
of Hib infection occurred in this nursing home. We were unable to
demonstrate that Hib caused any of the associated reports of bronchitis and
febrile illness among residents and staff, and our prevalence study did not
show any asymptomatic Hib carriage. However, the survey was performed 10
days after the onset of the second case of septicemia following widespread
use of antibiotics, and it may not have accurately reflected the prevalence
of Hib carriage at the time these cases occurred. It is also difficult to
evaluate whether chemoprophylaxis was a useful intervention in this
setting.

To our knowledge, this is the first reported cluster of invasive Hib
infection among adults in a community-based nursing home. Three other
clusters of Hib infection in adults have been described: one occurred in a
nursing home attached to a hospital, and two were nosocomialincluding a
cluster of bronchitic illness (7-9). Our report provides further evidence
that Hib can cause clusters of serious disease among the elderlyat least
in institutions. Given that diagnostic procedures are performed
infrequently in many community nursing homes, Hib infection may be
underrecognized in this setting.

We were especially interested in these two cases in adults as they were the
only instances of invasive Hib disease reported to this Public Health Unit
from its population base of one million persons over 12 monthsa rate of
0.3 per 100,000 population aged 15 years and older. This pattern of
notification differs markedly from that observed in western Sydney at the
time childhood Hib vaccination was first introduced in mid-1992. In 1992,
27 of 30 notifications of invasive Hib disease in this area were of
children aged less than 5 years, or 36.9 per 100,000 population (New South
Wales Health Department, unpub. data). Although invasive Hib infection
remains uncommon in adults in communities where childhood Hib vaccination
is routine, the relative frequency of invasive Hib infection is increasing
in older age groups.

Population-based studies of acute epiglottitis undertaken in northern
California and Rhode Island have also shown that the incidence of childhood
epiglottitis is falling rapidly since the introduction of Hib immunization,
while the incidence of epiglottitis in adults remains steady or is
increasing (10,11). Acute epiglottitis in children is usually caused by
Hib, and the falling incidence of epiglottitis in this age group is
explainable by immunization. In adults, most culture-positive cases of
acute epiglottitis are also caused by Hib (10,12). However, the incidence
of Hib-related epiglottitis in adults does not appear to be changing
appreciablyat least in Rhode Islandand Hib does not account for the
increasing incidence of epiglottitis observed in this age group (10).
Indeed, acute epiglottitis is frequently culture negative in adults, so
explaining its increasing incidence and ascertaining the etiologic agents
in older age groups require further study (10,12).

Invasive Hib disease should continue to be monitored in all age groups
after the introduction of childhood immunization against Hib. Evidence
suggests that Hib immunization reduces the acquisition of Hib carriage in
children, and reduced carriage among children may indirectly prevent Hib
disease in adults (13). However, surveillance data in western Sydney and
metropolitan Atlanta have not supported this hypothesis; rates of adult Hib
disease are remaining approximately unchanged, despite dramatic reductions
in childhood disease (5; New South Wales Health Department, unpub. data).
Only by serotyping isolates of H. influenzae that cause invasive disease
will it be possible to detect changing patterns of invasive Hib disease in
adults during this era of childhood immunization. Serotype-specific
surveillance of invasive H. influenzae disease is also needed to assess
progress towards the elimination of childhood Hib disease. In addition,
periodic cross-sectional serotyping studies of H. influenzae isolates
causing noninvasive disease would help measure the effects of Hib
immunization on the incidence of less serious forms of Hib infection. These
surveillance methods will identify groups at increasing or underrecognized
risk that may benefit from immunization.

Acknowledgments
The authors thank Graham O'Donnell (Department of Microbiology, Westmead
Hospital) and Marianne Kerr and Oanh Nguyen (Western Sector Public Health
Unit). The Master of Applied Epidemiology Program is funded by the
Commonwealth Department of Health and Family Services, Canberra.

Timothy C. Heath,* Moira C. Hewitt, Bin Jalaludin,* Christine Roberts,
Anthony G. Capon,* Peter Jelfs, and Gwendolyn L. Gilbert
*Western Sector Public Health Unit, North Parramatta, New South Wales;
Australian National University, Australian Capital Territory, Australia;
Department of Public Health and Community Medicine, Westmead, New South
Wales; and Institute of Clinical Pathology and Medical Research, Westmead,
New South Wales

References

  1. Adams WG, Deaver KA, Cochi SL, Plikaytis BD, Zell ER, Broome CV,
     Wenger JD. Decline of childhood Haemophilus influenzae type b (Hib)
     disease in the Hib vaccine era. JAMA 1993;269:221-6.
  2. Peltola H, Kilpi T, Antilla M. Rapid disappearance of Haemophilus
     influenzae type b meningitis after routine childhood immunisation with
     conjugate vaccines. Lancet 1992;340:592-4.
  3. Herceg A, Oliver G, Andrews G, Curran M, Crerar S, Andrews R, Evans D.
     Annual report of the National Notifiable Diseases Surveillance System,
     1995. Communicable Diseases Intelligence 1996;20:451.
  4. Alho OP, Jokinen K, Pirila T, Ilo A, Oja H. Acute epiglottitis and
     infant conjugate Haemophilus influenzae type b vaccination in northern
     Finland. Arch Otolaryngol Head Neck Surg 1995;121:898-902.
  5. Farley MM, Stephens DS, Brachman PS, Jr., Harvey RC, Smith JD, Wenger
     JD. Invasive Haemophilus influenzae disease in adults. A prospective,
     population-based surveillance. CDC Meningitis Surveillance Group [see
     comments]. Ann Intern Med 1992;116:806-12.
  6. Casadevall A, Dobroszycki J, Small C, Pirofski LA. Haemophilus
     influenzae type b bacteremia in adults with AIDS and at risk for AIDS
     [see comments]. Am J Med 1992;92:587-90.
  7. Smith PF, Stricof RL, Shayegani M, Morse DL. Cluster of Haemophilus
     influenzae type b infections in adults. JAMA 1988;260:1446-9.
  8. Patterson J, Madden GM, Krisiunas EP, Masecar B, Hierholzer WJ Jr.,
     Zervos NJ, Lyons RW. A nosocomial outbreak of ampicillin-resistant
     Haemophilus influenzae type b in a geriatric unit. J Infect Dis
     1988;1002-7.
  9. Howard AJ, Owens D, Musser JM. Cross-infection due to Haemophilus
     influenzae type b in adults. J Hosp Infect 1991;19:70-2.
 10. Frantz TD, Rasgon BM. Acute epiglottitis: Changing epidemiologic
     patterns. Otolaryngol Head Neck Surg 1993;109:457-60.
 11. Mayo-Smith MF, Spinale JW, Donskey CJ, Yukawa M. Acute epiglottitis -
     an 18 year experience in Rhode Island. Chest 1995;108:1640-7.
 12. Berg S, Nylen O, Hugosson S, Prellner K, Carenfelt C. Incidence,
     aetiology, and prognosis of acute epiglottitis in children and adults
     in Sweden. Scand J Infect Dis 1996;28:261-4.
 13. Barbour ML, Mayon-White RT, Coles C, Crook DWM, Moxon ER. The impact
     of conjugate vacine on carriage of Haemophlilus influenzae type b. J
     Infect Dis 1995;171:93-8.

---------------------------------------------------------------------------

   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Dispatches

Seroepidemiologic Studies of Hantavirus Infection Among Wild Rodents in
California

---------------------------------------------------------------------------


      A total of 4,626 mammals were serologically tested for
      antibodies to Sin Nombre virus. All nonrodent species were
      antibody negative. Among wild rodents, antibody prevalence was
      8.5% in murids, 1.4% in heteromyids, and < 0.1% in sciurids. Of
      1,921 Peromyscus maniculatus (deer mice), 226 (11.8%) were
      antibody positive, including one collected in 1975. The highest
      antibody prevalence (71.4% of 35) was found among P.
      maniculatus on Santa Cruz Island, off the southern California
      coast. Prevalence of antibodies among deer mice trapped near
      sites of human cases (26.8% of 164) was significantly higher
      than that of mice from other sites (odds ratio = 4.5; 95%
      confidence interval = 1.7, 11.6). Antibody prevalence increased
      with rising elevation (>1,200 meters) and correlated with a
      spatial cluster of hantavirus pulmonary syndrome cases in the
      Sierra Nevada.

In spring 1993, a cluster of unexplained severe acute respiratory illnesses
associated with a high death rate was reported in the southwestern United
States (1). The outbreak was linked to a newly recognized hantavirus
strain, Sin Nombre virus (SNV), carried by the deer mouse, Peromyscus
maniculatus (2-5). Sporadic cases of the illness, hantavirus pulmonary
syndrome (HPS), were subsequently identified in other regions of North
America, especially the western United States (6,7). Three additional
pathogenic viruses associated with HPS were later discovered outside the
usual range of P. maniculatus: 1) Black Creek Canal virus, harbored by the
cotton rat, Sigmodon hispidus, in Florida; 2) Bayou virus, identified in
the rice rat, Oryzomys palustris, in Louisiana; and 3) New York virus,
isolated from the white-footed deer mouse, Peromyscus leucopus, in New York
(8-12). These viruses have not been found in California; however, two novel
hantaviruses, El Moro Canyon virus (EMCV) and Isla Vista virus (ISLA), were
recently discovered in that state (13-15). Genetic studies identified the
harvest mouse, Reithrodontomys megalotis, and the California meadow vole,
Microtus californicus, as the reservoirs for EMCV and ISLA, respectively.
Human infection with EMCV and ISLA has not been documented.

Through 31 January 1997, 156 cases of HPS were reported to the Centers for
Disease Control and Prevention (CDC); the case-fatality rate is
approximately 50% (T. Ksiazek, P. Rollin, unpub. data). HPS has been
confirmed in 26 states, with California reporting the third largest number
of cases (14 cases, 8 deaths), after New Mexico (29 cases) and Arizona (22
cases).

Hantaviruses are excreted in the urine, feces, and saliva of asymptomatic
infected rodents (16). Transmission to humans occurs when aerosols
contaminated with the virus are inhaled (16-18). Bites by infected rodents
or exposure to broken skin or mucous membranes may represent alternative
routes. Although specific risk factors are poorly defined, persons engaging
in activities that bring them in contact with rodents and/or their
excretions may be at a higher risk for infection (19).

The present study compiles retrospective and prospective serologic data to
1) confirm P. maniculatus as the primary reservoir of SNV and identify
alternative reservoirs, if any, in California; 2) assess differences in SNV
seroprevalence in vector populations from various geographic regions; and
3) compare seroprevalence rates of reservoirs collected near sites of human
cases with those from other sites.

The Study

Mammal Surveys
The California Department of Health Services (CDHS) contacted 18 agencies
(local vector control districts, local health and environmental health
departments, universities, state and national park and forest services, the
military, and wildlife refuges) involved in cross-sectional surveys of
California mammals to participate in developing a centralized, statewide
database of SNV serologic results and ecologic data.

Historical surveys used archived specimens collected in 1975, 1976, and
1988 and stored at the University of California, Berkeley, Museum of
Vertebrate Zoology, and specimens collected from 1989 through early 1993 by
local vector control districts. Prospective surveys and human case
investigations were conducted from late 1993 through the end of 1995. Wild
rodent trapping and processing methods were as previously described (20).

Reports by participating agencies included ecologic data on individual
mammals (species, age, and sex). Information on survey sites included
county, physical location of trapline, date of survey, elevation, and
habitat. Habitat was categorized by a single dominant vegetation type:
chaparral, conifer trees, grassland, hardwood trees, sage/scrub brush, or
urban environment (21).

Human Cases
Within 2 weeks after a confirmed diagnosis of HPS, the patient or a
surrogate was interviewed to determine potential sites of exposure to
infected rodents. An environmental/ecologic investigation was conducted at
possible sites of exposure (20,22).

Laboratory Studies
Rodent serum samples were tested by one or more of six laboratories (CDC;
CDHS; University of New Mexico; University of California, Davis; and
University of Nevada, Reno). Serum samples were examined for IgG antibodies
to the SNV nucleocapsid protein by Western blot and/or enzyme-linked
immunosorbent assay with CDC reagents (23,24). For archived specimens,
liver tissue from frozen carcasses was used for polymerase chain reaction
(PCR) (22,25).

Data Analysis
Descriptive data were analyzed by Epi Info, Version 6.0 (26). Frequency
distributions were obtained, and chi-square tests of homogeneity for
two-by-two contingency tables were used to examine the statistical
significance of any association. A crude relationship between altitude and
SNV-antibody prevalence was assessed by the Mantel-Cox test for trend.
Adjusted odds ratios and 95% confidence intervals were calculated by
logistic-binomial regression (27). A random effects model was chosen
because of the presence of important clustering effects from the sampling
design. The presence or absence of antibodies to SNV was the primary
dependent variable, and characteristics that were identified in the
descriptive analysis were the independent variables. Data from samples
collected on the Channel Islands were analyzed separately from mainland
data where indicated.

Sin Nombre Virus Antibody Prevalence

California Mammals
A total of 4,626 (4,549 rodent and 77 nonrodent species) mammals
representing 47 species were collected between 1975 and 1995 in California
and serologically tested for IgG antibodies to SNV (Table 1). Wild rodents
(most 3,109, from the family Muridae) made up 98% of the sample, followed
by Sciuridae (1,369), and Heteromyidae (71). Among the Muridae, 78% were
deer mice or related species, 11% were wood rats, 6% were domestic rodents
(house mice, rats), 3% were harvest mice, and fewer than 1% were meadow
voles or cotton rats. Antibodies to SNV were found only among wild rodents.
Prevalence of antibodies reactive with SNV was 8.5% among the Muridae, 1.4%
among the Heteromyidae, and less than 0.1% among the Sciuridae. Nonrodent
species tested included 74 carnivores (five domestic dogs, Canis
familiaris; 26 coyotes, Canis latrans; 25 Island foxes, Urocyon littoralis;
six domestic cats, Felis domesticus; two opossums, Didelphis virginiana;
three striped skunks, Mephitis mephitis; seven raccoons, Procyon lotor),
one black-tailed jackrabbit, Lepus californicus, one Nuttall's cottontail,
Silvilagus nuttallii, and one shrew, Sorex sp.

Table 1. Prevalence of antibodies to Sin Nombre virus among wild rodents
in California, 1975-1995
---------------------------------------------------------------------------
Family/                        Common              No.      No.    %
Species                        Name                Tested   Pos.   Pos.
---------------------------------------------------------------------------
Heteromyidae
    Dipodomys sp.              kangaroo rats       28       1      3.6
    Perognathus sp.            pocket mice         43       0      0.0
SUBTOTAL                                           71       1      1.4
Muridae
Microtus sp.                   meadow voles        29       5      17.2
    M. californicus            California          22       5      22.7
                               meadow vole
    M. montanus                Montane vole        3        0      0.0
    M. townsendii              Townsend's vole     1        0      0.0
Mus musculus                   house mouse         88       0      0.0
Neotoma sp.                    wood rats           330      2      0.6
    N. cinerea                 bushy-tailed        20       0      0.0
                               wood rat
    N. fuscipes                dusky-footed        215      2      0.9
                               wood rat
    N. lepida                  desert wood         95       0      0.0
                               rat
Peromyscus sp.                 deer mice           2430     236    9.7
    P. boylii                  brush mouse         98       0      0.0
    P. californicus            California          159      4      2.5
                               mouse
    P. crinitus                canyon mouse        29       2      6.9
    P. eremicus                cactus mouse        100      1      1.0
    P. maniculatus             deer mouse          1921     226    11.8
    P. truei                   pinyon mouse        123      7      5.7
Rattus norvegicus              Norway rat          11       0      0.0
Rattus rattus                  roof rat            99       0      0.0
Reithrodontomys megalotis      harvest mouse       108      16     14.8
Sigmondon hispidus             cotton rat          14       0      0.0
SUBTOTAL                                           3109     263    8.5
Sciuridae                                          1369     1      <0.1
Ammonospermophilus             antelope            4        0      0.0
leucurus                       ground
                               squirrel
Spermophilus sp.               ground              1205     1      <0.1
                               squirrels
    S. beecheyi                California          856      1      0.1
                               ground
                               squirrel
Tamias sp.                     chipmunks           152      0      0.0
Tamiascurus douglasii          Douglas'            5        0      0.0
                               squirrel
SUBTOTAL                                           1369     1      <0.1
TOTAL                                              4549     245    5.4
---------------------------------------------------------------------------

Deer Mouse Populations
Of 1,921 P. maniculatus, 226 (11.8%) had antibodies to SNV (Table 1). Other
peromyscine-related species (e.g., cactus mice, canyon mice, California
mice, and pinyon mice) also had antibodies to SNV, but prevalence was
lower. In almost all instances, infected P. maniculatus were collected at
the same site as SNV-antibody-positive animals of other species.

Retrospectively, antibodies to SNV were identified in 3 (6.0%) of 50 deer
mice specimens collected in 1975 (1 of 22, 4.5%), 1976 (2 of 17, 11.8%),
1981 (0 of 2), 1992 (0 of 1), and early 1993 (0 of 8). The three
seropositive P. maniculatus collected by the University of California,
Berkeley in 1975 and 1976 were from Alameda, Kern, and Mono Counties.
Frozen liver tissue available from the seropositive Kern County specimen
yielded a PCR sequence of the G1 amplimer of SNV that differed from a P.
maniculatus collected in nearby Mono County in 1994 by only five residues
out of 274. The largest comparative protein dissimilarity was with a P.
maniculatus from the Channel Islands, which differed by seven amino acid
substitutions.

Among the deer mice for which age (1,165 animals, 87% adults) and sex
(1,239 animals, 57% male) were available, SNV antibody prevalence was,
respectively, 13.5% in adults and 6.7% in juveniles and 13.0% in males and
10.1% in females. Differences in age (chi-square = 5.5) and sex (chi-square
= 2.5) were not significant.

Geographic Distribution
Thirty-four of the state's 58 counties were surveyed (Table 2). Antibodies
to SNV were identified in deer mice from 21 (62%) of these counties (Figure
1). Most samples were from coastal counties (56%), followed by
foothill/mountainous (36%) and inland/valley (8%) counties. Among these,
the prevalence was 14.5% of 684 in the foothills/mountains, 11.6% of 112 on
the coast, and 0.7% of 151 in the inland/valley areas.

Table 2. Prevalence of antibodies to Sin Nombre virus among Peromyscus
maniculatus by county, California, 1975-1995
--------------------------------------------------------------------------
                    No. survey   No.           No.        %
County              sites        tested        pos.       pos.
--------------------------------------------------------------------------
COASTAL
Alameda             2            6             1          16.7
Contra Costa        1            36            0          0.0
Del Norte           2            19            0          0.0
Los Angeles         10(8)        112(71)       13(11)     11.6(15.5)
Marin               2            153           3          2.0
Mendocino           1            13            0          0.0
Monterey            2            52            5          9.6
Orange              8            52            3          5.8
San Diego           32           131           7          5.3
San Francisco       1            30            0          0.0
San Luis Obispo     2            4             0          0.0
San Mateo           1            40            8          20.0
Santa Barbara       6(2)         294(81)       86(7)      29.3(8.6)
Sonoma              1            7             0          0.0
Ventura             5(3)         137(9)        0(0)       0.0(0.0)
SUBTOTAL            76(68)       1086(704)     126(45)    11.6(6.4)
INLAND/VALLEY
Imperial            1            2             1          50.0
Riverside           10           57            0          0.0
Sacramento          1            36            0          0.0
San Bernardino      4            49            0          0.0
San Joaquin         1            7             0          0.0
SUBTOTAL            17           151           1          0.7
FOOTHILLS/MOUNTAINS
Butte               12           115           14         12.2
El Dorado           1            25            0          0.0
Glenn               1            4             0          0.0
Inyo                2            3             1          33.3
Kern                5            66            7          10.6
Mariposa            1            46            7          15.2
Mono                8            107           17         15.9
Nevada              1            52            26         50.0
Placer              2            29            2          6.9
Plumas              4            35            1          2.9
Shasta              3            30            4          13.3
Siskiyou            4            117           12         10.3
Tehama              1            35            5          14.3
Tulare              1            20            2          10.0
SUBTOTAL            46           684           99         14.5
TOTAL               139          1921          226        11.8
--------------------------------------------------------------------------
*Numbers in parentheses exclude the Channel Islands in Los Angeles
(Catalina, San Clemente), Santa Barbara (San Miguel, Santa Barbara, Santa
Cruz, Santa Rosa), and Ventura (Anacapa, San Nicolas) counties.

 [Figure 1] [Figures not available in ASCII version]
 Figure 1. Geographical distribution of hantavirus pulmonary syndrome
 cases and occurrence of Sin Nombre virus antibodies among deer mice
 (Peromyscus maniculatus), California, 1975-1995 (n=1,921).

One hundred and thirty-nine individual sites were surveyed within 34
counties with a mean of four sites per county (Table 2). Antibody
prevalence at individual sites was 0.0% to 71.4% (25 of 35 tested on Santa
Cruz Island, Santa Barbara County). In the Sierras, the highest antibody
prevalence (50.0% of 52) was found in deer mice captured in Truckee, Nevada
County, during a human case-patient investigation (Table 3, Case 3). At
least one SNV-antibody-positive mouse was detected at each mainland site
where 38 or more mice were tested.

Table 3. Characteristics of selected hantavirus pulmonary syndrome cases
and prevalence of antibodies to Sin Nombre virus in Peromyscus maniculatus
collected at candidate sites of exposure, California, 1994-1995*
---------------------------------------------------------------------------
        Onset  Outcome    Suspect     Predominant Elevation    No.      %
         Date              County      Vegetation  (meters)  Rodents  Pos.
 Patient                of Exposure                           Tested
---------------------------------------------------------------------------
1       09/94 Nonfatal Mono          conifer,     1801-2100 34        14.7
                                     sage brush
2       03/95 Nonfatal Mono          conifer,     1801-2100 22        13.6
                                     sage brush
3       04/95 Nonfatal Nevada        sage brush   1801-2100 52        50.0
4       06/95 Fatal    Mono          conifer,     1801-2100 11        54.5
                                     sage brush
5       09/95 Fatal    Placer/Nevada conifer      1501-1800 26        7.7
6       10/95 Nonfatal Plumas        conifer      1201-1500 19        5.3
Total                                                       164       26.8
---------------------------------------------------------------------------
*Rodent studies associated with California HPS cases described in greater
detail (20,22,29).

Channel Islands
The Channel Islands, a group of eight islands located south of the Santa
Barbara-Los Angeles coast, are 20 km (Anacapa) to 98 km (San Nicolas) from
the mainland and 5 km to 45 km from each other. Despite the proximity
between them, SNV-antibody prevalence varied significantly between islands.
Antibody prevalence in deer mice trapped on the islands (20.9% of 382) was
significantly higher than that of deer mice from the mainland (chi-square =
40.9, p < 0.001). In addition, Channel Island sequences differed by
approximately 17% to 19% from any mainland California sequences (22,25).
The highest prevalence was found on Santa Cruz and Santa Rosa Islands,
where, respectively, 25 (71.4%) of 35 and 47 (58%) of 81 deer mice were
SNV-antibody positive. Antibody prevalence among deer mice on the other
islands was 7 (17.9%) of 39 on San Miguel, 1 (14.3%) of 7 on Santa
Catalina, and 1 (2.9%) of 34 on San Clemente. However, deer mice sampled on
Anacapa (n=37), San Nicolas (n = 91), and Santa Barbara (n = 58) Islands
were all SNV-antibody negative.

Habitat
A higher SNV-antibody prevalence was observed in the Sierra Nevada, Great
Basin, and southern coastal habitats (Figure 1). Likewise, antibody
prevalence was higher among deer mice trapped in vegetation associated with
these environments: 15.1% of 531 in conifer, 14.8% of 597 in grassland,
13.4% of 86 in hardwood, 11.2% of 165 in sage/scrub brush, and 5.8% of 474
in chaparral. In urban environments, only 2.9% of 68 deer mice were
SNV-antibody positive.

Antibody prevalence among deer mice increased significantly (p < 0.001)
with rising altitude (Figure 2). In the regression model, adjusted odds
ratios steadily increased with elevation, peaking at 4.3 in the 1,800- to
2,100-meter range (95% confidence intervals = 1.3, 16.7) (Table 4). The
increased prevalence of SNV antibodies in deer mice trapped at higher
elevations correlated with an increased incidence of HPS cases at
elevations above 1,200 meters (Table 3).

Table 4. Odds ratios for Sin Nombre virus antibody prevalence among
Peromyscus maniculatus by association with human cases and elevation,
California, 1975-1995(a)
---------------------------------------------------------------------------
                Cases      Controls     Adj.
                (+SNV)     (-SNV)       OR(c)    95% CI(c)     p value
---------------------------------------------------------------------------
Human
case(d)
Absent          101        1274         1.0      NA(c)         NA
Present         44         120          4.5      1.7-11.6      0.002
Elevation
(meters)
0-300           32         580          1.0      NA            NA
301-600         2          101          0.4      0.4-3.5       0.423
601-900         11         83           0.6      0.2-1.8       0.390
901-1200        8          85           2.4      0.7-8.6       0.168
1201-1500       26         234          3.2      1.0-10.0      0.044
1501-1800       23         152          2.6      0.7-9.2       0.151
1801-2100       26         103          4.3      1.3-16.7      0.032
---------------------------------------------------------------------------
(a)Excludes Channel Islands; (b)Adjusted for trapline location and survey
date, vegetation, sex and age of deer mouse; (c)OR, odds ratio; CI,
confidence interval; NA, not applicable; (d)Deer mice collected at
candidate sites of exposure during human case investigations.

             [Figure 2] [Figures not available in ASCII version]
             Figure 2. Prevalence of Sin Nombre virus
             antibodies among deer mice (Peromyscus
             maniculatus) by elevation, California, 1975-1995
             (n=1,539, excluding Channel Islands).

Human Cases
HPS cases were spatially clustered in the state's Sierra Nevada region
(Figure 1). Results from antibody prevalence studies among rodents
collected during the investigation of six HPS cases in California are
presented in Table 3. Antibody prevalence in deer mice trapped at these
sites was significantly higher than at sites not associated with a human
case (odds ratio = 4.5; 95% confidence interval = 1.7, 11.6) (Table 4).

Reservoirs of Hantavirus in California

Extensive serologic testing unequivocally identified P. maniculatus as the
primary reservoir of SNV in California, as did previous studies in the
western United States (2,20,28-30). The overall prevalence of SNV
antibodies in deer mice (11.9%) in California is also comparable with
results published by nearby western states such as Montana (8.0%) and
Nevada (12.5%) (28,30). Other peromyscine rodents (e.g., canyon mice and
pinyon mice) may harbor the virus in California, but at much lower levels.
Because these related wild mice species frequently inhabit the same
environment as P. maniculatus, their infections may represent transmission
from the primary reservoir population rather than from each other, as would
occur in a permanent virus-species relationship. The extremely low
prevalence of SNV antibodies (Table 1) in heteromyids (kangaroo rats,
pocket mice), sciurids (ground squirrels, chipmunks), and Old World murids
(domestic mice and rats), despite a large sample size (> 1,600), suggests
that these animals are not important in the epidemiology of SNV in
California.

Positive serologic test results from California meadow voles and harvest
mice probably represent cross-reaction with SNV antigen by ISLA and EMCV,
respectively (13-15). The high prevalence of antibodies identified in
meadow voles (22.7% of 29) and harvest mice (14.8% of 108) is a cause for
concern, even though human infection by these viruses has not been
documented. Precautionary measures should be taken against exposure to
hantaviruses through all potential wild rodent reservoirs until more is
known about these newly discovered strains.

Temporal and Spatial Trends in Deer Mice Populations
Our data from historical surveys indicate that SNV was already circulating
in deer mice 20 years ago in parts of California. The antibody-positive
deer mouse originally trapped in Kern County in 1975 is the oldest
documented evidence of SNV infection in wild rodents. Notably,
SNV-antibody-positive deer mice identified retrospectively were captured
almost 20 years before the first HPS cases were recognized in California
and in some of the same geographic regions (20,22,29,31). The slight
difference (approximately 2%) between sequences from the Kern County deer
mouse trapped in 1976 and a Mono County deer mouse trapped in 1994
indicates a long-standing stability of the virus, as previously
demonstrated by Nerukar et al. in Mono County (32).

Although SNV infection in deer mice is widespread in California,
represented biotypes vary considerably in seroprevalence levels. For
example, deer mice in foothill/mountainous (14.5% of 684) counties have a
higher seroprevalence than those in inland/valley (0.7% of 151) and coastal
(6.4% of 704, excluding the Channel Islands) counties. The trend of
increasing prevalence with rising elevation found in this analysis has been
observed in other states (Jim Mills, pers. comm.).

The Channel Islands offered a unique opportunity to study SNV infection in
a relatively isolated population of deer mice. Deer mouse populations have
been on the Channel Islands long enough that each island has its own
subspecies and have considerable variation genetically within those
subspecies (33,34). Likewise, Hjelle et al. found significant divergence
between genetic sequences of virus from infected deer mice collected on the
islands and those from the nearby coastal mainland (25). Although travel
from the mainland to the islands and from island to island is common,
evidence suggests that SNV coevolved separately within the deer mouse
populations on each island (San Clemente, San Miguel, Santa Catalina, Santa
Cruz, Santa Rosa). It appears that the virus is not endemic or is present
at very low levels among deer mice on Anacapa, San Nicolas, and Santa
Barbara Islands.

Human Cases
The antibody prevalence of SNV in deer mice collected at potential exposure
sites during the investigation of sporadic HPS cases in California (26.8%)
was similar to the antibody prevalence (30.0%) in deer mice observed during
the 1993 HPS outbreak in the Four Corners region (2). Prevalence was
significantly higher (p = 0.002) in deer mice trapped near human case
exposure sites than in those from survey sites with no cases (Table 4).
Together these findings imply that the percentage of infected deer mice may
be a risk factor for human exposure to SNV. In addition, landscape features
such as high elevation may be another important predictor of hantavirus in
the state. The spatial clustering of HPS cases in the Sierra Nevada range
supports this conclusion.

Characteristics of the mouse population, the environment, or human
lifestyles may explain these geographic differences. The climate and
vegetation in the mountains could be conducive to large populations of deer
mice, a factor which might influence SNV prevalence. In addition, the
occupational and recreational activities of the inhabitants of rural,
mountainous environments bring them into frequent contact with rodents. In
addition, local residences (e.g., old log cabins) are prone to rodent
infestation, a possible risk factor for HPS infection (35).

Otteson et al. documented a higher SNV antibody prevalence among rodents
trapped near buildings, regardless of the presence of a human case; a
case-control study of the Four Corners outbreak had similar findings
(30,36). Information on proximity to human dwellings was not available for
our analysis; however, most mice were collected near buildings during
investigation of human cases. Since other survey sites were probably less
likely to be located near human dwellings, this factor may represent a
source of bias in our study. Other biases may have been introduced because
of nonrandom sampling and small sample size at some survey sites.
Systematic longitudinal studies of SNV infection in deer mice at the key
locations identified in this analysis are needed to further develop a
predictive model for hantavirus infection in California which could
elucidate the natural history of SNV and enhance prevention efforts.

Public Health Implications
Preliminary results indicate a need for health education of residents,
visitors, and workers at high risk, especially in the Sierra Nevada range.
Human dwellings in the mountains may be more vulnerable to deer mice
infestation, especially if the buildings are older and/or intermittently
occupied. In addition, persons working in or cleaning these structures may
be at an even higher risk (22,29,35,36). Local health care providers and
tertiary care centers should be aware of the potential for HPS cases in the
state.

Acknowledgments
The authors thank the many workers who conducted field surveys and
participated in the compilation of a state-wide mammal database; those
providing laboratory support, including David Cottam, Dale Dondero, CDHS;
Mary Lane Martin, George Gallucci, and Brad Hotard, CDC; Jeff Riolo,
University of Nevada, Reno; and Vicki Kramer for her helpful comments and
review of this manuscript. This work was supported in part by Public Health
Service Grant RO1 AI36336.

  Michele Jay,* Michael S. Ascher, Bruno B. Chomel, Minoo Madon, David
 Sesline, Barryett A. Enge, Brian Hjelle, Thomas G. Ksiazek, Pierre E.
                Rollin, Philip H. Kass, and Kevin Reilly*
  *California Department of Health Services, Sacramento, California, USA;
   Viral and Rickettsial Diseases Laboratory, Berkeley, California, USA;
 University of California, Davis, California, USA; California Department
  of Health Services, Ontario, California, USA; University of New Mexico,
 Albuquerque, New Mexico, USA; Centers for Disease Control and Prevention,
                           Atlanta, Georgia, USA

References

  1. Centers for Disease Control and Prevention. Outbreak of acute illness
     - southwestern United States, 1993. MMWR Morb Mortal Wkly Rep
     1993;42:421-4.
  2. Childs JE, Ksiazek TG, Spiropoulou CF, Krebs JW, Morzunov S, Maupin
     GO, et al. Serologic and genetic identification of Peromyscus
     maniculatus as the primary rodent reservoir for a new hantavirus in
     the Southwestern United States. J Infect Dis 1994;169:1271-80.
  3. Hjelle B, Jenison S, Torrez-Martinez N, Yamada T, Nolte K, Zumwalt R,
     et al. A novel hantavirus associated with an outbreak of fatal
     respiratory disease in the southwestern United States: evolutionary
     relationships to known hantaviruses. J Virol 1994;68:592-6.
  4. Elliott LH, Ksiazek TG, Rollin PE, Spiropoulou CF, Morzunov S, Monroe
     M, et al. Isolation of the causative agent of hantavirus pulmonary
     syndrome. Am J Trop Med Hyg 1994;51:102-8.
  5. Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann
     H, et al. Genetic identification of a hantavirus associated with an
     outbreak of acute respiratory illness. Science 1993;262:914-7.
  6. Khan AS, Khabbaz RF, Armstrong LR, Holman RC, Bauer SP, Graber J, et
     al. Hantavirus pulmonary syndrome: the first 100 cases. J Infect Dis
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  7. Centers for Disease Control and Prevention. Hantavirus pulmonary
     syndrome - United States, 1995 and 1996. MMWR Morb Mortal Wkly Rep
     1996;45:291-5.
  8. Morzunov SP, Feldmann H, Spiropoulou CF, Semenova VA, Rollin PE,
     Ksiazek TG, et al. A newly recognized virus associated with a fatal
     case of hantavirus pulmonary syndrome in Louisiana. J Virol
     1995;69:1980-3.
  9. Rollin PE, Ksiazek TG, Elliott LH, Ravkov EV, Martin ML, Morzunov S,
     et al. Isolation of Black Creek Canal virus, a new hantavirus from
     Sigmondon hispidus in Florida. J Med Virol 1995;46:35-9.
 10. Hjelle B, Lee SW, Song W, Torrez-Martinez N, Song JW, Yanagihara R, et
     al. Molecular linkage of hantavirus pulmonary syndrome to the
     white-footed mouse, Peromyscus leucopus: genetic characterization of
     the M genome of New York virus. J Virol 1995;69:8137-41.
 11. Torrez-Martinez N, Hjelle B. Enzootic of Bayou hantavirus in rice rats
     (Oryzomys palustris) in 1983. Lancet 1995;346:780-1.
 12. Song J, Baek LJ, Gajdusek DC, Yanagihara R, Gavrilovskaya I, Luft BJ,
     et al. Isolation of pathogenic hantavirus from the white footed mouse
     (Peromyscus leucopus). Lancet 1994;344:1637.
 13. Hjelle B, Chavez-Giles F, Torrez-Martinez N, Yates T, Sarisky J, Webb
     J, et al. Genetic identification of a novel hantavirus of the harvest
     mouse Reithrodontomys megalotis. J Virol 1994;68:6751-4.
 14. Song W, Torrez-Martinez N, Irwin W, Harrison FJ, Davis R, Ascher M, et
     al. Isla Vista virus: a genetically novel hantavirus of the California
     vole Microtus californicus. J Gen Virol 1995;76:3195-9.
 15. Rowe JE, St. Jeor SC, Riolo J, Otteson EW, Monroe MC, Henderson WW, et
     al. Coexistence of several novel hantaviruses in rodents indigenous to
     North America. Virology 1995;213:122-30.
 16. LeDuc JW. Epidemiology of Hantaan and related viruses. Lab Anim Sci
     1987;37:413-8.
 17. Tsai TF. Hemorrhagic fever with renal syndrome: mode of transmission
     to humans. Lab Anim Sci 1987;37:428-30.
 18. Xu ZY, Guo CS, Wu YL, Zhang XW, Liu K. Epidemiological studies of
     hemorrhagic fever with renal syndrome: analysis of risk factors and
     mode of transmission. J Infect Dis 1985;152:137-44.
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     southwestern United States: interim recommendations for risk
     reduction. MMWR Morb Mortal Wkly Rep 1993;42(RR-11):ii-13.
 20. Turell MJ, Korch GW, Rossi CA, Sesline D, Enge BA, Dondero DV, et al.
     Short report: prevalence of hantavirus infection in rodents associated
     with two fatal human infections in California. Am J Trop Med Hyg
     1995;52:180-2.
 21. Ornduff R. Introduction to California plant life. Berkeley and Los
     Angeles: University of California Press, 1974:152.
 22. Hjelle B, Torrez-Martinez N, Koster FT, Jay M, Ascher MS, Brown T, et
     al. Epidemiologic linkage of rodent and human hantavirus genomic
     sequences in case investigations of hantavirus pulmonary syndrome. J
     Infect Dis 1996;173:781-6.
 23. Yamada T, Hjelle B, Lanzi R, Morris C, Anderson B, Jenison S. Antibody
     responses to Four Corners hantavirus infections in the deer mouse
     (Peromyscus maniculatus): identification of an immunodominant region
     of the viral nucleocapsid protein. J Virol 1995;69:1939-43.
 24. Feldmann H, Sanchez A, Morzunov S, Spiropoulou CF, Rollin PE, Ksiazek
     TG, et al. Utilization of autopsy RNA for the synthesis of the
     nucleocapsid antigen of a newly recognized virus associated with
     hantavirus pulmonary syndrome. Virus Res 1993;30:351-67.
 25. Hjelle B, Chavez-Giles F, Torrez-Martinez N, Yamada T, Sarisky J,
     Ascher M, et al. Dominant glycoprotein epitope of four corners
     hantavirus is conserved across a wide geographical area. J Gen Virol
     1994;75:2881-8.
 26. Dean JA, Coulombier D, Smith DC, Brendel KA, Arner TG, Dean AG. Epi
     Info, version 6: a word processing, database, and statistics program
     for public health on IBM-compatible microcomputers. Atlanta: Centers
     for Disease Control and Prevention, 1994:601.
 27. EGRET. Seattle: Statistics and Epidemiological Research Corporation,
     1995.
 28. Douglass RJ, Van Horn R, Coffin KW, Zanto SN. Hantavirus in Montana
     deer mouse populations: preliminary results. J Wildl Dis
     1996;32:527-30.
 29. Jay M, Hjelle B, Davis R, Ascher M, Baylies HN, Reilly K, et al.
     Occupational exposure leading to hantavirus pulmonary syndrome in a
     utility company employee. Clin Infect Dis 1996;22:841-4.
 30. Otteson EW, Riolo J, Rowe JE, Nichol ST, Ksiazek TG, Rollin PE, et al.
     Occurrence of hantavirus within the rodent population of northeastern
     California and Nevada. Am J Trop Med Hyg 1996;54:127-33.
 31. Shefer AM, Tappero JW, Bresee JS, Peters CJ, Ascher MS, Zaki SR, et
     al. Hantavirus pulmonary syndrome in California: report of two cases
     and investigation. Clin Infect Dis 1994;19:1105-9.
 32. Nerukar VR, Song JW, Song KJ, Nagle JW, Hjelle B, Jenison S, et al.
     Genetic evidence for a hantavirus enzootic in deer mice (Peromyscus
     maniculatus) captured a decade before the recognition of hantavirus
     pulmonary syndrome. Virology 1994;204:563-8.
 33. von Bloeker JC. Land mammals of the southern California islands. Santa
     Barbara (CA): Santa Barbara Botanic Garden, 1967:106.
 34. Gill AE. Evolutionary genetics of California Islands Peromyscus. In:
     Power DM, editor. The California Islands. Santa Barbara (CA): Santa
     Barbara Museum of Natural History, 1980:719-43.
 35. Armstrong LR, Zaki SR, Goldoft MJ, Todd RL, Khan AS, Khabbaz RF, et
     al. Hantavirus pulmonary syndrome associated with entering or cleaning
     rarely used, rodent-infested structures. J Infect Dis 1995;172:1166.
 36. Childs JE, Krebs JW, Ksiazek TG, Maupin GO, Gage KL, Rollin PE, et al.
     A household-based, case-control study of environmental factors
     associated with hantavirus pulmonary syndrome in the southwestern
     United States. Am J Trop Med Hyg 1995;52:393-7.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Dispatches

Gestational Psittacosis in a Montana Sheep Rancher

---------------------------------------------------------------------------


      In humans, psittacosis is primarily a flulike illness following
      exposure to psittacine birds. In rare cases, pregnant women
      exposed to Chlamydia psittaci can contract gestational
      psittacosis: atypical pneumonia, sepsis, and placental
      insufficiency resulting in premature birth or miscarriage. In
      the United States, only two cases of gestational psittacosis
      have been reported, both from exposure to psittacine birds.
      Eleven other cases have been reported worldwide, mostly in the
      United Kingdom, all from exposure to infected birth fluids and
      membranes of farm mammals, notably sheep and goats. In these
      mammals, C. psittaci inhabit the reproductive tract, are
      transmitted sexually or by the fecal-oral route, and cause
      miscarriages. The case of gestational psittacosis in a Montana
      sheep rancher is the first farm animal-related case reported in
      the United States. Pregnant women should avoid close contact
      with C. psittaci-infected animals, particularly sheep and goats
      during the birthing season. Obstetricians should consider this
      diagnosis along with early antibiotic treatment and cesarean
      section delivery in the context of the patient's case history.

Psittacosis is a flulike systemic infection, often with fever, headache,
and atypical pneumonia, caused by Chlamydia psittaci. The number of
reported cases varies from year to year because of periodic outbreaks,
although the true baseline incidence in the United States is thought to be
75 to 100 cases per year with 1 or 2 deaths (1). Montana typically reports
one case per year (Montana Dept. of Health surveillance data). Many cases
are probably not diagnosed. In most reported cases, transmission occurs by
inhaling infectious material from diseased psittacine birds (2).

However, not all cases of psittacosis result from inhaling avian strains of
C. psittaci, nor are they all manifested as a simple flulike illness or
atypical pneumonia. In rare cases, pregnant women contract severe
chlamydial disease after exposure to the infected birth fluids and
membranes of goats and sheep (3). This "gestational psittacosis" can be
defined as a flulike syndrome leading to sepsis, placental infection, and
fetal compromise (4). Only 13 cases have been reported worldwide: Eleven
after exposure to gravid sheep and goats (United Kingdom and France) and
two after exposure to psittacine birds (United States). This is the first
farm animal-related case reported in the United States.

Case Report

In April 1996, a previously healthy 25-year-old Montana sheep rancher,
pregnant for the first time, had cough and congestion for 14 days at weeks
19 to 21 of gestation; the symptoms were followed by 4 days of high fever
(104F), myalgia, headache, fatigue, and backache, and 2 days of abdominal
pain. Upon hospitalization, a chest X-ray was normal, but laboratory tests
showed anemia (Hg 10.7 g/dl), thrombocytopenia (platelets 42,000/ml), and
hepatic dysfunction (SGOT 351 I/L), as well as proteinuria (2+) and
hematuria (1+). Other tests showed a positive lupus anticoagulant and
positive anticardiolipin antibodies. Blood, urine, and sputum cultures
(collected before antibiotics were administered) yielded negative results.
Admitting diagnosis was "early severe and low platelet count) syndrome"
with a possible concurrent viral infection and poor hydration accounting
for the fever and lack of hypertension.

On the second day of hospitalization, the patient underwent emergency
termination of the pregnancy (22-23 weeks gestation). On the following day,
the patient still had high fevers; moderate respiratory distress with
tachypnea developed, and oxygen was required. A chest X-ray showed diffuse
bilateral alveolar infiltrates; antibiotic treatment (IV Ancef) was started
for presumed pneumonia. The fever subsided, the patient's condition
improved remarkably, and she was discharged within 3 days.
The discharge diagnosis was early severe HELLP syndrome (probably related
to lupus anticoagulant and anticardiolipin antibodies) with coincident
pneumonia. On followup, the patient was feeling well and had resumed her
activities around the ranch.

Suspicious of a diagnosis related to environmental exposure and motivated
by a need to explain all of the patient's symptoms, the obstetrician sent
placental and fetal tissue to California and Oklahoma for specialized
pathologic examination, after which patient sera were sent to the Centers
for Disease Control and Prevention (CDC) in Atlanta, Georgia, and animal
sera were sent to the Veterinary Diagnostic Laboratory in Bozeman, Montana.
A concomitant epidemiologic investigation was initiated.

Hematoxylin and eosin stain of the placenta showed placentitis: an intense
acute inflammation of the intervillous space, more on the maternal side,
which suggested infection from hematogenous spread rather than from
ascension from the birth canal. Numerous basophilic intracytoplasmic
inclusions in the cytoplasm of the trophoblast appeared to contain minute
cocci-shaped organisms. The inclusions were identified by genus-specific
fluorescein-tagged monoclonal antibody staining as masses of Chlamydia
(either pneumoniae or psittaci) in various stages of development. Electron
micrographs further defined the round, rather than pear-shaped, morphologic
features of the elementary bodies, which suggested psittaci rather than
pneumoniae as the specific species. The pathologic findings are reviewed in
greater detail, including photographs, in a concurrent publication (4).

Because traditional complement-fixation serologic tests do not adequately
differentiate the chlamydial species in humans, sera were sent to CDC for a
microimmunofluorescence assay, which is more sensitive and specific (5).
The patient's sera had very high (1:512) IgG antibody titer to C. psittaci;
identical titer was seen on sera collected several months later. No IgM to
C.psittaci was detected. Tests for the other chlamydia, as well as for
other placental-acquired infections (e.g., Q fever and brucellosis) were
negative.

The epidemiologic investigation showed that, during the week before
hospitalization, the patient's husband had a similar flulike illness with
mild respiratory symptoms. The spring lambing season began 4 to 6 weeks
before hospitalization, during which the patient and her husband worked
closely with gravid sheep.

Out of a flock of 24 sheep, 19 gave birth to 30 lambs. There were six lamb
deaths but no reported abortions. The patient assisted in most deliveries;
however, she had direct contact on just three occasions: pulling one lamb
during a twin birth, pulling one kid during the twin birth of her pet goat,
and assisting in the premature delivery of a yearling ewe. The latter
resulted in considerable placental exposure. She spent approximately 1 hour
manually extracting the retained placenta, wearing only plastic gloves; she
did not cover the mouth or nose. Serologic testing of five sheep and two
goats was performed by complement fixation for C. psittaci. Only sera from
two sheep were positive, including the yearling ewe from which the patient
manually extracted the retained placenta.

The sheep were purchased from a neighboring flock with reported "pink eye"
and abortions, both of presumed chlamydial etiology. The flock's abortion
problem resolved after tetracycline-fortified feed was administered just
before yearly breeding. Neither antibiotic feed nor the available chlamydia
vaccine was used for the sheep on the patient's ranch. During her
pregnancy, the patient had minimal contact with other ranch animals,
including cattle, horses, and poultry; none of these animals were ill or
had abortions. She had no contact with psittacine birds.

HELLP is a variant of the preeclampsia-eclampsia syndrome, which also
includes epigastric pain and liver tenderness, as well as hypertension,
edema, and proteinuria. An underappreciated early symptom is generalized
malaise and/or flulike illness (6,7). Antiphospholipid antibodies, such as
the lupus anticoagulant and anticardiolipin antibodies, have been
associated with thrombosis, thrombocytopenia, and pregnancy complications,
particularly decidual vasculopathy, placental infarction, fetal growth
retardation, early-onset preeclampsia, recurrent miscarriages, and fetal
death (8). Initially, in this patient, except for the high fever and lack
of hypertension, the symptoms reasonably conformed to those of HELLP
syndrome. However, in this case, epidemiologic, clinical, laboratory (human
and animal), and histopathologic data support the diagnosis of gestational
psittacosis. The most likely mechanism of transmission was inhalation of
C.psittaci while manually extracting the retained placenta from the C.
psittaci-infected sheep that lambed prematurely just 2 weeks before the
patient's hospitalization. The usual incubation time for psittacosis is 1
to 4 weeks (2).

In the United States, pneumonia acquired through contact with birth
membranes or fluids of farm animals is more commonly caused by Coxiella
burnetti (Q-fever) or Brucella sp. (brucellosis) (9). In both diseases, the
flulike syndrome does not lead to fetal compromise in pregnant women.
However, in gestational psittacosis, the flulike illness results in sepsis,
placental infection, and fetal compromise. The disease is relatively new,
first reported in the United Kingdom in 1967, and rare, just 13 cases
reported worldwide (4,10,11). Gestational psittacosis has occurred from
exposure to psittacine birds (the United States: two previous cases; 12,13)
and from exposure to infected birth fluids and membranes of sheep or goats
(the United Kingdom: nine cases, all sheep [10,11,14-19]; France: two
cases, both goats [20,21]). This is, therefore, the third case reported in
the United States, the first related to farm animals (4).

Further review of the clinical details of the 13 other reported cases shows
remarkable similarity between them and the case reported here. For example,
in all 14 reported cases (including this case) the illness is manifested as
a flulike febrile syndrome. Similarly, in the 14 cases, thrombocytopenia
and/or coagulopathy were reported (12 [85%]), as well as pulmonary disease
(12 [85%]) and liver disease (11 [78%]). The outcome of pregnancy is also
similar: 11 (78.5%) of 14 pregnancies ended in fetal death, while the three
surviving neonates were born by cesarean section before 34 weeks gestation
(4). Maternal outcome was much better: 13 of the 14 women recovered fully
after pregnancy termination. The other woman died of overwhelming sepsis.
Erythromycin was administered to six of the 14 pregnant women, including
the mothers of the three surviving infants, early in the illness
(4,10,12,13). Tetracycline, the drug of choice for psittacosis, is usually
contraindicated during pregnancy, although it is still recommended for
cases of gestational psittacosis that appear refractory to erythromycin
(12,13).

C. psittaci is known worldwide as the primary cause of abortion in sheep,
often referred to as "enzootic abortion of ewes", "chlamydial abortion," or
simply "enzootic abortion." Enzootic abortion in sheep was first reported
in Scotland in 1936, where C. psittaci became known as the "ewe abortion
agent" (10,22). Abortion usually occurs within 2 years of contact with
other aborting sheep in the flock. Indeed, pregnant sheep experimentally
infected with an ovine abortion isolate of C.psittaci either abort or give
birth to weak lambs and maintain a persistent systemic antibody response to
the organism for up to 2.5 years postinfection; for sheep that aborted,
detectable amounts of chlamydial antigen are excreted from the reproductive
tract during subsequent estrus cycles (23). Postpartum (or postabortive)
infected sheep are considered protected from subsequent C. psittaci-induced
pregnancy complications, and they remain healthy, fertile, and therefore
able to transmit the organism within the flock, perhaps through sexual
contact (23,24).

Moreover, C. psittaci organisms are found in eye, respiratory, oral, and
fecal material, which suggests other modes of transmission. Indeed,
environmental contamination from diseased placentae or vaginal discharge is
considered the primary source of infection to other sheep, perhaps through
ingestion of infected tissue or contaminated feed during the previous or
current lambing season and subsequent intestinal colonization (23,24). The
carrier state is not evident but is often presumed if a high incidence of
abortions or premature lambing occurs in the flock. Sometimes a high rate
of "pink eye" in the flock indicates high C. psittaci prevalence, although
this is usually caused by a serovar different from the one causing
abortion. Nevertheless, ranchers who suspect C. psittaci in the flock
typically respond by physically isolating infected sheep, giving antibiotic
feed, and/or using the chlamydial vaccine. However, these methods rarely
eliminate the disease from the flock (23).

In Montana, 50 to 100 aborted sheep fetuses are examined each year, more
than 50% of which test positive for C. psittaci by complement fixation on
sera or enzyme-linked immunosorbent assay on placental tissue. For this
reason, masks are always worn by laboratory workers during sheep (and
psittacine bird) autopsies (L. Stackhouse, Montana Veterinary Diagnostic
Laboratory, pers. comm.). It is difficult to estimate the overall rate of
colonization/infection within sheep flocks, although it is presumed to be
high, particularly in the United Kingdom, and in areas of the United States
where sheep abortions have already occurred and feed-antibiotics or
chlamydial vaccine is not routinely used. High rates of sheep infection,
thus far, have not led to high rates of miscarriage in female sheep
ranchers. Nevertheless, given the increasing number of small family farms
or hobby farms in the western United States, a growing number of farmers
could be raising sheep (and other animals) without knowing the potential
hazards to human health and how to prevent them.

Thus, it is strongly recommended that pregnant women avoid contact with
membranes or birth fluids of sheep and goats and close contact with
psittacine birds during pregnancy. Moreover, obstetricians should consider
the diagnosis of gestational psittacosis in pregnant patients initially
presumed to have HELLP syndrome and/or a flulike illness, particularly
during the spring lambing season, so that appropriate early antibiotics and
cesarean section delivery can help reduce illness and death from the
disease.

Acknowledgments

The author would like to thank Drs. Mark Miles (Great Falls, MT), Mary
Ballinger (Belt, MT), Larry Stackhouse (Bozeman, MT), Kurt Benirschke (San
Diego, CA), Scott Hyde (Tulsa, OK), and K.H. Wong (Atlanta, GA) for their
technical contributions to this investigation and their encouragement and
expert advice.

Daniel M. Jorgensen
Montana State Medical Officer

References

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  4. Hyde SR. Gestational psittacosis: report of a case with literature
     review. Mod Path. In press.
  5. Wong KH, Skelton SK, Daugherty H. Utility of complement fixation and
     microimmunofluourescence assays for detecting serologic responses in
     patients with clinically diagnosed psittacosis. J Clin Microbiol
     1994;32:2417-21.
  6. Cunningham F, MacDonald P, Gant N, Levene K, Gilstrap L, editors.
     Hypertensive disorders in pregnancy. In: Williams Textbook of
     Obstetrics. Norwalk (CT): Appleton and Lange, 1994; 763-90.
  7. Tomsen TR. HELLP syndrome (hemolysis, elevated liver enzymes, and low
     platelets) presenting as generalized malaise. Am J Obstet Gynecol
     1995;172:1876-80.
  8. Cunningham F, MacDonald P, Gant N, Levene K, Gilstrap L, editors.
     Connective tissue disorders. In: Williams Textbook of Obstetrics.
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 10. Beer RJS, Bradford WP, Hart RJC. Pregnancy complicated by psittacosis
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 15. Wong SY, Gray ES, Buxton D, Finlayson J, Johnson FW. Acute placentitis
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     1985;38:707-11.
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---------------------------------------------------------------------------

   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Dispatches

Lack of Serologic Evidence for an Association between Cache Valley Virus
Infection and Anencephaly and other Neural Tube Defects in Texas

---------------------------------------------------------------------------


      We tested the hypothesis that Cache Valley Virus (CVV), an
      endemic North American bunyavirus, may be involved in the
      pathogenesis of human neural tube defects. This investigation
      followed a 1990 and 1991 south Texas outbreak of neural tube
      defects with a high prevalence of anencephaly and the
      demonstration in 1987 that in utero infection by CVV was the
      cause of outbreaks of central nervous system and
      musculoskeletal defects in North American ruminants. Sera from
      74 women who gave birth to infants with neural tube defects in
      south Texas from 1993 through early 1995 were tested for CVV
      neutralizing antibody. All tested sera did not neutralize CVV.
      These data suggest that CVV is not involved in the induction of
      human neural tube defects during nonepidemic periods but do not
      preclude CVV involvement during epidemics. Other endemic
      bunyaviruses may still be involved in the pathogenesis of
      neural tube defects or other congenital central nervous system
      or musculoskeletal malformations.

Anencephaly, spina bifida, and encephalocele (the major types of neural
tube defects) are generally due to the failure of the neural tube to close
during early embryonic development (1). Neural tube defects are among the
most common and most severe major birth defects. Anencephaly is caused by
failure of the anterior neuropore to close during embryonic development and
results in total or partial absence of the cranial vault, the covering
skin, and the brain. Infants with anencephaly are stillborn or die shortly
after birth. Spina bifida is caused by a disturbance in the normal closure
of neural walls and results in spinal cord defects. Most infants with spina
bifida survive surgical repair of the defect with residual neurologic
handicaps of varying severity. Encephaloceles are skull defects through
which skin-covered meninges, with or without brain tissue, herniate. Small
to moderately sized encephaloceles are surgically correctable (1-4).

The prevalence of neural tube defects in the United States has been
steadily declining (5) and is currently estimated to be six per 10,000
births. The prevalence of anencephaly in the United States has likewise
declined and is now approximately three per 10,000 live births (4). A vital
record study of anencephaly in Texas showed that from 1981 to 1986 the
prevalence rate was 3.8 to 4.3 cases per 10,000 births (6). This study also
showed that in south Texas during this period the average annual prevalence
of anencephaly was approximately 4.9 per 10,000 births. Women with Hispanic
surnames, three or more previous live births, history of stillbirth, or
residence in east or south Texas were at increased risk for neural tube
defect-affected pregnancies. On the basis of vital record data, the annual
prevalence of anencephaly in south Texas from 1981 through 1986 was
approximately 4.9 per 10,000 live births.

In 1991 three babies with anencephaly were born over a 36-hour period at a
single hospital in Cameron County, the southernmost county in Texas
(Brownsville is the county's largest city). The ensuing study, which used
active multisource case finding rather than vital records, showed that the
neural tube defect prevalence rate increased from 14.7 per 10,000 births in
1986 to 1989 to 27.1 per 10,000 births in 1990 to 1991. The higher rate was
due largely to an increase in anencephaly cases. From 1986-89 to 1990-91,
the average annual anencephaly prevalence rate rose from 9.6 to 19.7 per
10,000 births (7,8).

Despite the high prevalence of neural tube defects and the significant rate
of illness associated with them, much remains to be learned about their
complex multifactorial etiology. Evidence suggests that these defects are
etiologically heterogeneous and may follow fetal insults such as maternal
diabetes, hyperthermia, folic acid deficiency, and anticonvulsant
(valproate) therapy (9-13).

In 1987, before the increase in prevalence of human anencephaly in south
Texas, an outbreak of severe congenital malformations of the central
nervous system and musculoskeletal system of lambs occurred in San Angelo,
Texas (14). At the time of the outbreak, there was no active surveillance
of human birth defects in San Angelo, and no reports were received of human
birth defects in the area. The ovine problem was later found to be caused
by in utero infection by Cache Valley Virus (CVV). Although this
insect-borne bunyavirus had been known to commonly infect North American
ruminants (15), it was not thought to be of clinical significance.

Experimentally, it was determined that CVV infection of the dam in early
gestation and transplacental infection of the ovine fetus could produce
severe brain malformations and arthrogryposis multiplex congenita, an
anomaly characterized by limbs fixed in contracture (14). Central nervous
system malformations associated with experimental and spontaneous CVV
infection include hydrocephalus, hydranencephaly, porencephaly,
micrencephaly, and micromelia. After the syndrome was characterized,
outbreaks of CVV-induced malformations in ruminants were diagnosed
throughout North America, and work by Calisher and Sever (16) also linked
CVV to congenital cases of human macrocephaly in the United States.

Other bunyaviruses can cause identical congenital malformations of the
central nervous system in experimentally infected livestock (14). Human
congenital morbidity has also been correlated with maternal antibody to
bunyaviruses (16). A recent study correlated both human microcephaly and
macrocephaly with antibody to Tenshaw virus in mothers of infants with
these illnesses.

We decided to test the hypothesis that CVV infection was related to human
neural tube defects. Public concern regarding the 1990 to 1991 cluster in
Brownsville, Texas, (7) had resulted in an ongoing project in the 14 Texas
counties that border Mexico. A neural tube defect surveillance and folic
acid intervention project were implemented in 1993, and a case-control
study was begun in mid-1995. Sera from case patients had been banked before
the case-control study began.

Sera from 74 women who lived in the Texas border counties and had neural
tube defect-affected pregnancies (36 with spina bifida, 34 with
anencephaly, and 4 with encephalocele) from 1993 through early 1995 were
examined for a possible link between CVV and neural tube defects. With a
standard microtiter serum dilution neutralization test (17), the sera were
screened at final dilutions of 1:2, 1:4, 1:8, and 1:16. The virus used in
all tests was the prototype CVV (strain 6V-633, provided by the Centers for
Disease Control and Prevention [CDC], Ft. Collins, Colorado), which had
been passaged one time in Vero cells after receipt from CDC. Controls
included sera from women of undetermined CVV status who gave birth to
healthy infants in south Texas [8]; sera collected from sheep before CVV
infection [3]; and normal macaque [4], horse [1], and bovine [1] sera.
Positive controls included CVV-convalescent-phase ovine sera [3] and CVV
antibody-positive sera from a horse and a cow. No serum neutralization
activity for CVV was detected in sera from women who gave birth to healthy
infants or infants with neural tube defects. Had CVV infection been present
in these women during gestation, CVV antibody would have been detectable
postpartum.

Before this study, the relationship between CVV and human neural tube
defects was unknown. Testing an adequate number of controls is critical
when seroepidemiology is used to establish a causal relationship between an
agent and a low frequency event or malformation, particularly when case
patients have evidence of antibodies against the agent of interest. In this
study, there was no evidence that CVV was related to the neural tube defect
cases observed in Texas from 1993 through early 1995. Had CVV antibodies
been detected in serum from women with neural tube defect-affected
pregnancies, it would have been necessary to test control sera from age-
and location-matched women with normal births.

The average annual neural tube defect prevalence rate in Cameron County,
Texas, for 1992 to 1995 has returned to the 1986 to 1989 rate of
approximately 14-15 cases per 10,000 births. These data suggest that CVV is
not involved in the induction of human neural tube defects during
nonepidemic periods but do not preclude CVV involvement during epidemics.
CVV may still be involved in induction of other human malformations. Other
endemic bunyaviruses may be involved in the pathogenesis of neural tube
defects and of other congenital nervous system or musculoskeletal
malformations (18,19).

It would seem valid to continue to investigate the relationship of CVV and
other arboviruses to human developmental illness and death rate. Because of
the wide variety of defects caused by these viruses, laboratory models of
fetal infection by the Bunyaviridae would facilitate the understanding of
viral teratogenesis mechanisms in humans.

J. F. Edwards* and K. Hendricks
*Texas A&M University, College Station, Texas, USA; and Texas Department
of Health, Austin, Texas, USA

References

  1. Copp AJ, Brooks SA, Estibeiro JP, Shum AS, Cockcroft DL. The embryonic
     development of mammalian neural tube defects. Prog Neurobiol
     1990;35:363-403.
  2. World Health Organization. Congenital malformations worldwide: a
     report from the international clearinghouse for birth defects
     monitoring systems 1991. New York: Elseviers Science Publishers,
     1991:41-79.
  3. Campbell LR, Dayton DH, Sohal GS. Neural tube defects: a review of
     human and animal studies on the etiology of neural tube defects.
     Teratology 1986;34:171-87.
  4. Thomas JA, Markovac J, Ganong WF. Anencephaly and other neural tube
     defects. Front Neuroendocrinol 1994;15:197-201.
  5. Yen IH, Khoury MJ, Erickson JD, James LM, Waters GD, Berry RJ. The
     changing epidemiology of neural tube defects: United States. Am J Dis
     Child 1992;146:857-61.
  6. Brender JD, Carmichael L, Preece MJ, Larimer GC, Suarez L.
     Epidemiology of anencephaly in Texas, 1981-1986. Tex Med 1989;85:33-5.
  7. Albrecht LJ. Mystery in Cameron County. Physicians and health
     officials search for cause of high rate of anencephaly in the Rio
     Grande Valley. Tex Med 1994;90:16-18.
  8. Texas Department of Health/Centers for Disease Control. An
     investigation of a cluster of neural tube defects in Cameron County,
     Texas. July 1, 1992.
  9. Ardinger HH, Atkins JF, Blackston RD, Elsas LJ, Clarren SK,
     Livingstone S, et al. Verification of the fetal valproate phenotype.
     Am J Med Genet 1988;29:171-85.
 10. Finnell RH, Taylor LW, Bennett GD. The impact of maternal hyperthermia
     on morphogenesis: clinical and experimental evidence for a fetal
     hyperthermia phenotype. Developmental Brain Dysfunction
     1993;6:199-209.
 11. Mills JL, Baker L, Goldman S. Malformations in infants of diabetic
     mothers occurs before the seventh gestational week. Diabetes
     1979;28:292-3.
 12. Sandford MK, Kissling GE, Joubert PE. Neural tube defect etiology: new
     evidence concerning maternal hyperthermia, health and diet. Dev Med
     Child Neurol 1992;34:661-75.
 13. Willett WC. Folic acid and neural tube defects: can't we come to
     closure. Am J Pub Health 1992;82:666-8.
 14. Edwards JF. Cache Valley virus. Vet Clin North Am: Food Anim Pract
     1994;10:515-24.
 15. Calisher CH, Francy DB, Smith GC, Muth DJ, Lazuick JS, Karabatsos N,
     et al. Distribution of Bunyamwera serogroup viruses in North America,
     1956-1984. Am J Trop Med Hyg 1986;35:429-43.
 16. Calisher CH, Sever JL. Are North American Bunyamwera serogroup viruses
     etiologic agents of human congenital defects of the central nervous
     system? Emerg Inf Dis 1995;1:147-51.
 17. Pantuwatana S, Thompson WH, Watts DM, Hanson RP. Experimental
     infection of chipmunks and squirrels with LaCrosse and trivittatus
     viruses and biological transmission of LaCrosse virus by Aedes
     triseriatus. Am J Trop Med Hyg 1972;21:476-81.
 18. Hall JG. Genetic aspects of arthrogryposis. Clin Orthop
     1985;184:44-53.
 19. Davies-Wynne R, Lloyd-Roberts GC. Arthrogryposis multiplex congenita;
     search for prenatal factors in 66 sporadic cases. Arch Dis Child
     1976;51:618-23.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Dispatches

Rabies Postexposure Prophylaxis SurveyKentucky, 1994

---------------------------------------------------------------------------


      A survey of rabies postexposure prophylaxis administered by
      local health departments for a 1-year period showed that very
      few patients received treatment as a result of exposure to a
      confirmed rabid animal. Most prophylaxis was administered for
      contact with domestic animals in situations where existing
      recommendations for quarantine or laboratory testing of the
      animal were not followed. Because rabies in domestic animals in
      Kentucky is uncommon, these findings suggest that had the
      existing recommendations been followed, the prophylaxis would
      have been unnecessary in most cases.

Rabies postexposure prophylaxis (PEP) is expensive, not totally free of
risk, and overused (1). A national public health objective for the year
2000 is to reduce the number of prophylaxis treatments by 50% (2). In
Kentucky, where PEP is administered in public and private settings, there
are no baseline data on PEP use.

A survey of local health departments was used to determine the nature of
each patient's exposure to rabies. The number of PEP treatments
administered by all providers in Kentucky was estimated from local health
department information on rabies biologics purchases and use.

Survey and Sales Summary

In May 1995, the 1994 invoices of the Kentucky Department for Health
Services, Vaccine Depot, were reviewed to determine which local health
departments received 1.0 ml doses of human diploid cell vaccine for PEP.
(Local health departments used 1.0 ml human diploid cell vaccine for PEP
only, and 0.1 ml human diploid cell vaccine intradermally for all rabies
preexposure prophylaxis). Data from two large health departments that
acquired their vaccine directly from the manufacturer rather than from the
Vaccine Depot were included in the survey. In June 1995, local health
departments that had administered at least one PEP during 1994 were asked
to review the records of patients receiving PEP. Information (patient's age
and sex, the number of doses of human diploid cell vaccine, whether human
rabies immune globulin was administered, exposure information, and method
of payment for the treatment) collected on each patient was recorded on a
standardized form by the same telephone surveyor during a followup
telephone call. All data were entered into an Epi Info Version 5.0 record
file and analyzed in either the Analysis or Statcalc Programs for summary
statistics and/or odds ratios, confidence intervals, Fisher's exact test,
or Chi-square at the .05 significance level (3).

A sales record summary for human diploid cell vaccine sold to all providers
in Kentucky was obtained from the only manufacturer of human rabies vaccine
recording any sales in Kentucky that year (Connaught Laboratories, Inc.,
Swiftwater, PA). The number of PEPs administered in the state by all
providers was estimated by comparing local health department purchases and
use with the total number of human diploid cell vaccine 1.0 ml doses sold
to other providers with Kentucky addresses.

PEP Administration Profile

Vaccine Depot records indicated that 28 health departments treated a total
of 97 patients. The number of PEP regimens administered per health
department ranged from 1 to 23 with a median of 1 PEP for the year.
Fifty-two (53.6%) of the patients were male (Table 1); the median age was
28 years (range 2 to 71); 34 (35.1%) patients were younger than 18 years of
age; 59 (60.8%) were older than 18 years of age; and for 4 (4.1%), age was
unknown. No significant differences were observed in the type of animal
exposure by sex or age. Seven patients (7.2%) had previously received PEP
and were treated with two to three doses of human diploid cell vaccine and
no human rabies immune globulin.

             Table 1. Characteristics of local health
             department patients receiving rabies postexposure
             prophylaxis
             --------------------------------------------------
             Sex
                       Male                               52
                       Female                             43
                       Unspecified                        2
             Age(a)    Youth (2 - 10)                     19
                       Adolescent (11 - 17)               15
                       Adult (18-71)                      59
                       Unspecified                        4
             Health department location(b)
                       Urban                              48
                       Rural                              49
             Previously immunized                         6
             Animal exposure
                       Wild                               15

                       Domestic (50 dogs, 29 cats, 1      80
                       horse)
                       Unspecified                        2
             Type of exposure

                       Bite or contact                    72
                        with saliva
                       No contact with saliva             17
                       Unspecified                        8
             Treatment payer
                       Private insurance                  39
                       Medicaid                           7
                       Medicare                           3
                       Patient                            14

                       Other (employer, worker's          3
                        compensation)
                       Unspecified                        6
                       No reimbursement                   25
             --------------------------------------------------
             (N=97)
             (a) [mean] = 28 yrs.
             (b) Health departments in urban areas, as defined
             by the 1990 census of population for Kentucky.
             Metropolitan statistical areas were more likely
             to administer PEP than rural departments.
             (p=.033)

Urban health departments (in the three metropolitan statistical areas of
the state) were more likely to administer PEP than rural health departments
(odds ratio = 1.54, confidence interval = 1.01, 2.33) (4). Patients did not
significantly differ in age, sex, or type of exposure between urban and
rural health departments.

For 25 (25.8%) of the patients, local health department funds covered the
expense of PEP treatments; no payment was received from private insurance,
Medicaid, Medicare, or the patient. There were no significant differences
in payment characteristics between urban and rural health department
patients.

Bite exposures were responsible for 71 (73.2%) of the 97 PEP treatments, 18
(18.6%) exposures were scratches, licks, or "other," and 8 (8.2%) exposure
types were not recorded. Domestic animals accounted for 80 (82.5%) of the
exposures treated.

Type of Animal Exposure

Sixty-four (77.1%) of 83 animals involved in these incidents were not
available for observation or testing. For wild animals, testing was
performed in 3 (20%) of 15 incidents. Testing or observation occurred in
only 16 (20.0%) of 80 domestic animal exposures.

Stray domestic animals accounted for 26 (26.8%) of all exposures. Another
19 (19.6%) of the incidents involved owned dogs that were unavailable for
testing or observation. Unavailability for testing was due to severe brain
damage caused by clubbing or gunshot by irate owners, death and disposal of
the animal without testing, or the animal's escape. For 36 (37%) incidents,
the reason for not testing or observing the animal was not specified.

Thirteen (13.4%) of the patients were exposed to an animal that was tested
and found to be positive for rabies, and two of these patients had bite
exposures. The remaining exposures to these rabies-positive animals were
either low-risk exposures or not true exposures (Table 2).

Table 2. Patients receiving postexposure prophylaxis for exposure to a
confirmed rabid animal in Kentucky, 1994
---------------------------------------------------------------------------
Species       Type of exposure                       Previous history
                                                      of prophylaxis
---------------------------------------------------------------------------
Bat           Bite                                          No
Cat(a)        Mucus & Saliva                              Yes(b)
Cat(a)        Mucus & Saliva                                No
Cat(a)        Cleaned exam table                            No
Cat(a)        Cleaned exam instruments                      No
Dog(c)        Bite                                         Yes
Dog(c)        Touch                                        Yes
Dog(c)        Touch                                        Yes
Dog(c)        Touch                                        Yes
Dog(c)        Touch                                         No
Dog(c)        Touch                                         No
Horse         Sutured wound                               Yes(b)
Skunk         Touch                                         No
---------------------------------------------------------------------------
(a) Same cat
(b) Veterinarian with history of preexposure prophylaxis
(c) Same dog

Total Estimate of State Rabies Postexposure Prophylaxis

Kentucky sales in 1994 for human diploid cell vaccine 1.0 ml to nonmilitary
providers and distributors totaled 1,603 doses. The health departments
ordered 700 of these doses, of which 445 were used for PEP in that same
year. The other doses remained as inventory. Assuming that other users
administered human diploid cell vaccine 1.0 ml in a similar proportion
(445/700=.64), the private sector administered 578 doses (903 x .64) of
human diploid cell vaccine 1.0 ml. Comparing actual local health department
use of human diploid cell vaccine 1.0 ml and estimated use by others, local
health departments administered 43.5% (445/([445+578]) of the human diploid
cell vaccine 1.0 ml used in the state in 1994. Therefore, the estimated
total number of PEP patients in the state is 223 (97/.435) for 1994.

Exact total costs for PEP administration cannot be calculated since most
treatments were made by private providers. The actual cost of biologics to
local health department patients in 1994 was $68,850. Estimated costs of
biologics used by private providers (based on estimates of hospital
pharmacy costs in Connecticut in 1994) would be $180,180 for a typical
patient (126 patients x $1,430) (5). Estimated total costs of biologics is
$249,030. Unknown costs include medical and hospital care, local health
department investigation of the incident, state health department
consultations, and loss of work income by the patient.

Study Limitations

Because records at the local health departments were not always complete or
as detailed as desired, certain variables could not be analyzed for all 97
cases; information about why the suspect animal was not tested or observed
for rabies was absent from more than 10% of the cases. Since no detailed
information was obtained from the private sector, we assumed that the
number of doses used per patient, inventory, waste, spoilage, and other
factors influencing PEP use in the private sector were similar to those in
the public sector. Kentucky residents receiving PEP in another state and
out-of-state residents receiving PEP in Kentucky would not be specifically
accounted for in our estimate.

The difference in urban versus rural PEP administration could be due to
differences in the number of animals or bite incidents; however, the number
of animals or animal bites statewide is not known. An investigation of
prescribing practices of full-time physicians at large, urban health
departments and part-time or contract physicians at small, rural health
departments might determine if these practices contributed to treatment
disparity.

Guidelines and Noncompliance

Guidelines for determining exposures that warrant PEP exist (6,7). Ideally,
any animal involved in a human exposure should be confined and observed or
tested for rabies, whichever is appropriate. It is understandable that most
of the wild animals might have escaped and not be available for testing.
However, the large proportion of domestic animals unavailable for testing
indicates inappropriate handling of the incident or a breach of existing
laws (5-7).

Six people received PEP due to exposure to a single dog with
laboratory-confirmed rabies. This particular incident illustrates how
"anything that can go wrong will go wrong." First, the dog had been
vaccinated by the owner. It is illegal for individual owners to vaccinate
their own dogs in Kentucky (8). Second, the vaccine may have failed for any
number of reasons, including vaccine failure, improper
handling/administration of the vaccine, or failure to vaccinate. Third,
only one of these patients was bitten; the other five reported only
touching the dog and probably were not exposed. Fourth, none of these
patients had insurance or was able to pay for treatment; thus the local
health departments spent several thousand dollars in unbudgeted expenses.
Furthermore, four of these patients had received PEP before.

Noncompliance with existing public health recommendations and laws
contributes to the number of rabies exposure incidents in Kentucky. PEP
administration in Kentucky could be reduced if existing recommendations and
laws were adhered to by the public and health care providers. Accurate and
complete record keeping is essential for assessing the use of PEP.
Additionally, making PEP a notifiable (reportable) condition would allow
public health agencies to assess PEP administration in the private sector.

Michael Auslander and Colleen Kaelin
Kentucky Department for Public Health, Frankfort, Kentucky, USA

References

  1. Noah D, Smith M, Gotthardt J, Krebs J, Green D, Childs J, et al. Mass
     human exposure to rabies in New Hampshire: exposures, treatment, and
     cost. Am J Public Health 1996;86:1149-51.
  2. Healthy People 2000: National Health Promotion and Disease Prevention
     Objectives. Washington (DC): Public Health Service; 1990:122. DHHS
     publication PHS 91-50213.
  3. Dean A, Dean J, Burton A, Dicker R. Epi Info, Version 5: a word
     processing, database, and statistics program for epidemiology on
     microcomputers. Stone Mountain (GA): USD, Inc., 1990.
  4. Census of Population and Housing, 1990. Summary Tape File 3, Prepared
     by the Data Services Division, Bureau of the Census. Washington (DC):
     U.S. Bureau of the Census; 1990.
  5. Centers for Disease Control. Rabies postexposure prophylaxis -
     Connecticut, 1990-1994. MMWR Morb Mortal Wkly Rep 1996;45:232-34.
  6. Centers for Disease Control. Rabies prevention - United States, 1991.
     MMWR Morb Mortal Wkly Rep 1991;40:1-19.
  7. Centers for Disease Control and Prevention. Compendium of Animal
     Rabies Control, 1996. MMWR Morb Mortal Wkly Rep 1996;45:1-9.
  8. Kentucky Revised Statutes. Rabies Control. KRS 258.005-.090; 1994.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Commentary

Biologic Terrorism  Responding to the Threat

---------------------------------------------------------------------------


The growing awareness of the possibility that a terrorist organization
might use a biologic agent in an attack on a civilian target in the United
States raises important questions about our capability as a nation to
respond effectively to the threat and to deal with the consequences of an
attack. The article by Kaufmann et al. in this issue of Emerging Infectious
Diseases describes three possible biologic attack scenarios and uses an
economic analysis to describe the benefits of a rapid medical response and
early intervention. The authors conclude that major reductions in morbidity
and mortality and consequent cost savings can be achieved by early
intervention. The effectiveness of postattack intervention depends on a
rapid response which requires prior planning, preparation, and training.
Achieving the level of preparedness implied by the assumptions stated in
the article will require a major national effort. This discussion of
possible bioterrorist attack scenarios adds to a growing concern about our
willingness as a nation to commit the effort and resources necessary to
protect our citizens.

Biologic warfare and use of biologic weapons by terrorists have only
recently been discussed openly and realistically. The fall of the Soviet
Union and the defeat of Iraq uncovered extensive biologic weapons programs
of surprising sophistication and diversity. The threat to the nation from
biologic weapons is no longer a debate issue. Now the questions are how
immediate and serious is the threat and how do we respond effectively?

Protecting the armed forces against biologic weapons, although complex and
difficult, is less challenging than protecting the civilian population. The
armed forces are relatively small populations that can be vaccinated
against the major threat agents. Aerosols containing biologic materials can
be detected at a distance, and protective masks and suits are effective.
Military medical personnel are trained to recognize and treat casualties,
and antibiotics, antiviral drugs, and antitoxins can be stockpiled for
military contingencies. The preponderance of scientific expertise for many
of the threat agents is within the military medical research laboratories,
although this capability is now being seriously compromised by budget cuts
and personnel reductions.

The civilian population cannot be protected in the same manner as the armed
forces. We must rely heavily on our intelligence and criminal investigation
agencies and on international efforts to identify specific threats and
deter terrorists. We must also recognize the possibility that a determined
terrorist organization may not be deterred, may evade detection, and may
succeed in releasing an aerosol of a virulent bacterium, virus, or toxin in
a susceptible target area such as an airport or stadium. Our current
capability to effectively respond to such a scenario and minimize the
impact is far less than needed.

The U.S. Armed Forces and the Department of Defense have the greatest
capability in biologic defense, but the responsibility for dealing with the
threat of biologic weapon use by a terrorist falls on multiple federal,
state, and municipal agencies and the civilian health care community. Most
of the organizations are inadequately prepared to deal effectively with the
problem.

The organizational aspects of dealing with an attack on our civilian
population are daunting. Responsibility for recognizing an unusual outbreak
of illness that may be the result of the deliberate release of a biologic
warfare agent will fall on the health care community. Early recognition
will be an important factor in determining the overall outcome and will
depend on the level of suspicion and knowledge of the health care providers
that see the initial cases. Rapid, precise, and reliable diagnosis will be
the responsibility of the federal and state public health laboratory system
with help from their military colleagues. Organizing and managing the care
of patients and mounting the appropriate public health response will
involve local health care and municipal agencies and authorities and state
public health authorities. The effectiveness of coordination, support, and
leadership at the federal level may make huge differences in reducing death
rates and containing the possible secondary spread of a communicable
disease. The Federal Emergency Management Agency has the major
responsibility for planning and coordinating the consequences phase of a
federal response, but the level of preparedness at all levels will
ultimately determine the outcome.

If we take the biologic warfare threat seriously, a major effort will be
needed to develop contingency plans and initiate coordinated and mutually
supportive programs in all involved agencies. Training and education of the
health care community will require a major effort involving several major
professional organizations. Developing and improving diagnostic and
identification capability is essential for medical care, public health,
intelligence, and law enforcement agencies and should be a national
priority.

The science base needed to deal with the broad spectrum of agents on the
threat list, bacteria, viruses, toxins, and parasites, is widely
distributed among several federal laboratories in the Department of Health
and Human Services, the Department of Defense, and the Department of
Energy, as well as in universities and state public health laboratories. In
addition, since many of the biologic agents are not normally large public
health problems or popular subjects of scientific research, critical areas
have inadequate research capability and limited expert personnel.
Deficiencies in our scientific knowledge and a paucity of experts will
ultimately limit our capability to rapidly and precisely identify agents
and respond effectively in a crisis. For example, the global molecular
epidemiology of the agents at the top of the threat list is critically
important for identifying the organisms accurately and differentiating
local from exotic strains. Current databases are inadequate, and no
organized effort is being made to fill in the gaps.

The current public discussion of the threat of biologic terrorism is an
opportunity to evaluate our collective capabilities and to assess
weaknesses and vulnerabilities. Raising the level of national preparedness
will require leadership and action by responsible federal agencies. A
thoughtful analysis of the consequences of unpreparedness provides a
mandate for action.

Philip K. Russell
Johns Hopkins University, Baltimore, Maryland, USA
---------------------------------------------------------------------------


   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

1st International Conference on Emerging Zoonoses

The Hantaviruses of Europe: from the Bedside to the Bench

---------------------------------------------------------------------------


In Europe, hantavirus disease can hardly be called an emerging zoonosis; it
is rather a rediscovered disease. Since 1934 an epidemic condition with
primarily renal involvement has been described in Sweden. Nowadays,
hundreds to thousands of cases per year are registered in Fennoscandia,
fluctuating with the numbers of the specific Arvicoline-rodent reservoir,
the red bank vole, which carries the main European serotype, Puumala (PUU).
In the early 1980s, the rat-transmitted serotype, Seoul (SEO), caused
laboratory outbreaks throughout Europe, and recent reports also suggest
sporadic, wild rat-spread hantavirus disease. In the Balkans, at least four
serotypes are present simultaneously: PUU, SEO, the "Korean" prototype
Hantaan (HTN) or HTN-like types, and Dobrava, the latter causing a
mortality rate of up to 20%. Moreover, recent genotyping studies have
disclosed several PUU-like genotypes spread in Europe and/or Russia by
other genera of the Arvicoline-rodent subfamily: Tula, Tobetsu, Khabarovsk,
and Topografov. Their importance for human pathogenicity is still unclear,
but serologic cross-reactions with PUU antigen might have caused their
misdiagnosis as PUU-infections in the past.

Hantaviruses are often heralded as new or at least as emerging pathogens,
particularly in the New World. However, even on the American continent,
application of the newest genotyping techniques (the bench) comparing RNA
sequences found in both human cases and rodent reservoirs shows a
long-standing coevolution of each newly discovered hantavirus serotype in
its specific rodent host; this coevolution results in remarkable genetic
stability across time and in a certain genetic differentiation in
geographic spread. Hantaviruses inducing hantavirus pulmonary syndrome
emerge not through genetic reassortment or a recent mutation, but through
increased exposure to infected rodents and their excreta. While the same
lines of evidence also apply to the European situation, the problem there
is totally different, from a historical point of view.

History

In most European countries, hantavirus disease has long been known by
various (mostly geographic) names, which suggests a long-standing clinical
presence (the bedside). An epidemic of "trench nephritis" during World War
I may in fact have been hantavirus induced. Thousands of cases of this
illness, considered an entirely new renal disease, were noted on both sides
of the front (Kriegsnephritis or nphrite de guerre). Already in 1934, the
typical mild renal form of hantavirus disease had been described in Sweden
(1); it was then described in all other Scandinavian countries, where the
disease was appropriately called nephropathia epidemica from 1945 on.

During World War II, more than 10,000 cases of a rodent-borne
leptospirosislike disease were noted during the 1942 German campaign in
Finnish Lapland (2). Because the snow melted, great numbers of lemmings and
field mice invaded the German bunkers. Examinations in Munich and Berlin of
these rodents, air-lifted from the war theater, offered no clue. Confronted
with some distinctive clinical symptoms (e.g., acute myopia and localized
edema) and with repeatedly negative findings for leptospirosis in his
patients, a researcher concluded it was a new field-like fever disease (2).

"Old" Hantaviruses in the Old World

The Puumala (PUU) serotype, carried by the red bank vole (Clethrionomys
glareolus), remains the most important Western and Central European
serotype, with at least 1,000 serologically confirmed nephropathia
epidemica cases per year in Finland and hundreds per year in Sweden (3).
The number of documented cases in other European countries was more than
1,000 in the former Yugoslavia (3), 531 in France by the end of 1994 (4),
approximately 250 in Belgium by the end of 1996 (J. Clement, unpub. obs.),
some 200 in Germany by the end of 1995 (5), 138 in Greece by the end of
1993 (6), and 39 in the Netherlands by the end of 1994 (7).

IgG seroprevalence rates reported from some of these countries, measured
mostly by immunofluorescence assay (IFA) and/or enzyme-linked immunosorbent
assay (ELISA) both for PUU and HTN, were 6% for Finland (8), 8% for Sweden
(9), 1.7% for Germany (10), 0.9% for the Netherlands (11), 1.6% for Belgium
(12), <1% for France (4), 0.3% for Spain (13) and 4.0% for Greece (6). Most
of the PUU infections were subclinical. A study comparing nephropathia
epidemica incidence (recorded over 14 years) with IgG IFA PUU-antibody
prevalence in an area of Sweden with high rates of endemic disease found
that the antibody prevalence rate for men and women in the oldest age
groups (>60 years) was 14 to 20 times higher, respectively, than the
accumulated life-risk of being hospitalized with nephropathia epidemica
(14). Thus, hospital admissions for the disease are only the tip of the
iceberg.

In nephropathia epidemica, acute renal failure serum creatinine values peak
above 4.5 mg (>400 mol/L) in only one-third of the cases (15). Early
thrombocytopenia, however, is present in 75% of the cases. Eye symptoms,
and particularly acute myopia, are rare (25%) but distinctive first
symptoms of PUU infections. Mild transient hepatitis is frequent (40%), but
icterus is rare (7%) (15). Noncardiogenic pulmonary edema, the hallmark of
Sin Nombre (or Sin Nombre-like)-induced hantavirus disease in the New
World, has been described in a milder, nonlethal form (acute lung injury)
in some rare European PUU (and Seoul [SEO] virus) cases (16,17).

In the former Soviet Union, hantavirus disease has been recognized since
1934 and officially registered since 1978. Seroprevalence studies carried
out by IFA or direct blocking radioimmunoassay involving 115,765 persons
resulted in an overall seropositivity rate of 3.3%, ranging from 3.5% in
the European part to 0.9% in the Far Eastern part (18). A total of 68,612
cases were registered between 1988 and 1992 (65,906 from the European part
and 2,706 from the Far Eastern part), with morbidity rates of 1.2 (1982) to
8.0 (1985) per 100,000 inhabitants. The peak year was 1985 with 11,413
registered cases (19). In the European part of Russia, most cases were due
to milder infection with PUU-related viruses, with mortality rates of 1% to
2%; whereas in Far East Russia, more severe HTN-like cases also occurred
(19).

The Wild Rat: Omnipresent, but Often Overlooked

In Europe, the parallel spread of PUU and HTN (or HTN-like) viruses has
been noted in such countries as Belgium and the Netherlands (11,20),
Germany (10), and European Russia (21). A partial explanation could be that
the HTN-like infection is due to serologic cross-reaction with SEO. The
wild rat is the only hantavirus reservoir with a worldwide distribution
(22), including Europe, and SEO infections are probably underestimated. The
first documented hantavirus disease in Portugal was an HTN-like infection
with acute renal failure and icterus (23). In Portugal Clethrionomys
glareolus is not prevalent, but hantavirus-seropositive wild rats have been
documented (24). Sixteen cases of acute disease, mostly with acute renal
failure and reacting almost exclusively in IFA against a SEO strain
(R22VP30), have been described in North Ireland (25), another country where
C. glareolus is not prevalent, but the most important hantavirus vector
seems to be the wild rat (26). In France, three SEO-induced cases of acute
renal failure have been reported south of the PUU-endemic region, from
rural areas where the rat is an agricultural pest (4). Moreover, 14
SEO-like cases were detected between December 1991 and February 1992 in the
Tula region (300 km south of Moscow) and confirmed by plaque reduction
neutralization tests and positive virus isolation in three of the cases
(27); these cases are awaiting further confirmation.

An often overlooked fact is that hantavirus has been transmitted from
laboratory rats to animal keepers first in Belgium (1979) and later in
France, in the United Kingdom, and in the Netherlands (28). In the earlier
days, these rat-transmitted infections were described as HTN-like by IFA or
ELISA because of cross-reactions with the proto-type screening antigen HTN
76-118, but they were later confirmed as SEO-like by ELISA and blocking
ELISA (20). Because of the now established close relationship between each
hantavirus serotype and its rodent vector, these earlier laboratory
infections can all be regarded as SEO-induced.

The Balkans: A Complicated Situation

In the Balkans, and particularly the former Yugoslavia, outbreaks of
hantavirus disease have been recorded since the early 1950s, often with a
death rate of 5% to 10% or even higher (29). The elevated rates of illness
and death in early reports suggested the spread of one (or several)
hantaviral strains, in addition to the mild PUU serotype prevalent in the
rest of Europe. These HTN-like viruses were later called Plitvice and
Fojnica. In 1987, an HTN-like virus (Porogia) was isolated from the urine
of a Greek soldier who became ill after a military exercise near the border
in northern Greece and had both acute renal failure and severe pulmonary
edema. Extensive cross-reactivity with HTN 76-118 was demonstrated by IFA
with a panel of Mabs and in plaque reduction neutralization tests (30). No
polymerase chain reaction (PCR) genotyping was available at that time.
However, in Slovenia in 1994, a hantavirus was isolated that was
indistinguishable from the prototype Korean strain HTN 76-118 by PCR
genotyping and other serologic techniques (31). Moreover, a hantavirus
(close but not identical to HTN 76-118) that caused a mortality rate of up
to 20% was first described as a human isolate (Belgrade) in Serbia (32) and
confirmed as a new hantavirus serotype called Dobrava (DOB) after isolation
from its rodent vector, an Apodemus flavicollis (yellow-necked field mouse)
captured in Dobrava, Slovenia (33).

The first genetic evidence for the association between DOB and severe
hantavirus disease was demonstrated by nested reverse transcriptase-PCR on
RNA extracted from whole blood of a Greek and an Albanian patient (34).
During the recent conflict in Bosnia, more than 300 patients, most of them
soldiers exposed in the field, were hospitalized in the Tuzla region
(northeast Bosnia) with acute hantavirus disease due either to PUU or to
DOB, as first documented by IgG and IgM ELISA (35) and later confirmed by
focus reduction neutralization tests (36). These findings suggest that at
least three distinct serotypes (PUU, DOB, and maybe also HTN) are endemic
throughout the Balkans and that easily accessible serologic tests are
needed to permit a differential diagnosis, given the totally different
prognosis for each infection. Moreover, preliminary evidence implicated a
fourth serotype, i.e., SEO, spread by wild rats (Rattus norvegicus or
Rattus rattus). Severe hantavirus disease, apparently due to SEO, was
documented in 1992 in a Canadian soldier (37) and later in 1996 in a
British soldier (17), both stationed in Bosnia. These two patients are
clinically interesting, in that the former had a clear exposure to wild
rats inside an infested building, whereas the latter had acute renal
failure and hemodynamically documented acute lung injury, a complication
hitherto undocumented in SEO cases (38). The exact serotype involved in
both these cases needs to be defined, however, by confirmatory tests such
as plaque reduction neutralization tests or (if technically possible) by
PCR genotyping.

That so many of these Balkan cases are in persons on active military duty
should come as no surprise. Exposure to rodents has been confirmed as the
most important risk factor for developing hantavirus diseases and seems an
unavoidable aspect in the life of the soldier at war. Even exercises
imitating war conditions can put the soldier at risk: the most important
cluster of hantavirus disease in Americans abroad was reported in U.S.
soldiers exercising in January 1990 in southern Germany and camping under
tent in a mice-infested area. Within 2 weeks, 24 acute PUU infections were
documented, and 14 soldiers had to be hospitalized with varying degrees of
acute renal failure (no deaths), whereas no outbreak occurred in the
civilian population of the surrounding area (5).

"New" Hantaviruses: Tula (TUL), Tobetsu (TOB), Khabarovsk (KBR), and
Topografov (TOP)

Apart from the long-standing clinical experience with hantavirus strains in
Europe and Asia, an explosive growth in the number of newly discovered
lineages or genotypes has further complicated matters. All these new
genotypes appear more or less related to PUU (Figure, Table). TUL virus was
first detected by RT-PCR in European common voles (Microtus arvalis and
Microtus rossiaemeridionalis) captured in the Tula region (39). TUL virus
was later also detected in voles from Moravia, the Czech Republic, and
Slovakia (Figure). The three viruses most closely related to TUL have been
detected on the American continent: Prospect Hill, isolated in 1982 from
Microtus pennsylvaticus (meadow vole); Isla Vista virus, recently detected
in Microtus californicus (Californian meadow vole) (40); and Bloodland Lake
virus, detected in Microtus ochrogaster (prairie vole) (Hjelle et al.,
unpub. data). TUL was isolated from the lungs of infected M. arvalis and
showed in cross-focus reduction neutralization tests and
cross-hemagglutination inhibitor tests at least eightfold higher homologous
to heterologous titers when compared with PUU, PH, KBR, and HTN (41). None
of these Microtus-derived hantaviruses is a known pathogen in humans, in
contrast to the PUU viruses, which are spread by another genus of the same
rodent subfamily Arvicolinae (Table, Figure). However, serum of a blood
donor living in Moravia, the Czech Republic, possessed a focus reduction
neutralization test titer to TUL at least 16-fold higher than to PUU or
other hantaviruses, thereby giving the first solid evidence that these
viruses carried by Microtus rodents can infect humans (41). As in other
hantavirus serotypes, antibody response to the TUL N-antigen appeared
highly reactive and cross-reactive. Thus, part of the so-called PUU
infections in European (and particularly in Central European and Russian)
patients may have been due to related TUL viruses. Under that hypothesis,
it would be very remarkable that the even more closely related North
American viruses (Prospect Hill, Isla Vista, and Bloodland Lake) would
appear to be apathogenic to humans. Already in the early 1930s, Tula fever
was one of the many regional synonyms used in Russia for describing
epidemics of a feverish condition, which later appeared to be a hantavirus
infection.

             Table: Classification of old and new PUU-like
             genotypes, according to respective rodent host
             and geographic spread.
             --------------------------------------------------
                 Rodent reservoir      Hantavirus
                   (Arvicolinae       (geographic
                    Subfamily)          spread)    Reference
             --------------------------------------------------
             Clethrionomys
               glareolus             Puumala       (43)
                                     (Europe)
               rufocanus             Tobetsu       (44)
                                     (Japan)
             Lemmus
               sibiricus             Topografov    (42)
                                     (Russia)
             Microtus
               pennsylvaticus        Prospect Hill (45)
                                     (N. America)
               californicus          Isla Vista    (40)
                                     (N. America)
               ochrogaster           Bloodland     Hjelle et
                                     Lake          al., unpub-
                                     (N. America)  lished data
               arvalis               Tula          (39)
                                     (Eurasia)
               rossiaemeridionalis   Tula          (39)
                                     (Eurasia)
               fortis                Khabarovsk    (46)
                                     (Eurasia)
             --------------------------------------------------

    [Figure] [Figures not available in ASCII version]
    Figure: Dendrogram of Old World hantaviruses (upper, left, and
    lower part of the tree) vs. New World hantaviruses (right part of
    the tree). Reproduced with permission (42). Branch lengths are
    proportional to genetic distances. The bootstrap support
    percentages of particular branching points calculated from 500
    replicates are given in ovals. HTN = Hantaan virus, strain 76-118;
    SEO = Seoul virus, strain SR-11; DOB = Dobrava virus, PH = Prospect
    Hill virus; TUL = Tula virus, strains Tula/76Ma/87,
    Moravia/5286Ma/94 and Malacky/Ma32/94; ILV = Isla Vista virus,
    strain MC-SB-1; TOP = Topografov virus; PUU = Puumala virus,
    strains Sotkamo, Vindeln/83-L20 and Udmurtia/458g/88; RIOS = Rio
    Segundo virus, strain RMx-Costa-1; ELMC = El Moro Canyon virus,
    strain RM-97; BAY = Bayou virus, strain Louisiana; BCC = Black
    Creek Canal virus; SN = Sin Nombre virus, strain H10; NY = New York
    virus, strain RI-1; KBR = Khabarovsk virus, another PUU-like virus,
    is not depicted here. PH & ILV (and BLL, not depicted here) are the
    only New World genotypes with PUU-like characteristics, hence their
    position close to the TUL-clade.

TOB was the name preliminarily given to a PUU-like virus detected in
Clethrionomys rufocanus (grey-sided vole) captured in Hokkaido, an island
in the north of Japan (44). Its putative rodent reservoir, Cl. rufocanus,
has a very broad geographic range, extending almost over the whole of
Eurasia: in the north from northern Scandinavia to Kamtchatka, and in the
south from the Urals to Manchuria and down to Korea. Moreover, PUU-like
human infections have been noted in Korea (18) (H.W. Lee, pers. comm.).

KBR (not in the Figure), a hantavirus close to PUU, was recently isolated
from a Microtus fortis (reed vole) captured in Far East Russia (46).
Although no pathogenicity for humans has been documented so far, this new
agent could partially explain PUU-like infections described for many years
in this region and in China, together with the findings of PUU-positive M.
arvalis rodents (19). KBR is more closely related to PUU than to Prospect
Hill, which may reflect that both KBR and PUU are viruses from the Old
World, whereas Prospect Hill has been documented so far only in North
America. The first reports in Russia that describe a hantaviruslike disease
are found in the 1913 archives of a hospital in Vladivostok, Siberia.

TOP is another PUU-like genotype, detected in the lemming (Lemmus
sibiricus) (42). Lemmings, the most important small mammals in the Arctic
tundra regions, are also present in Alaska and Canada. Together with the
wild rat, the lemming is the only rodent reservoir harboring a newly
recognized hantavirus genotype, and living in both the Old and the New
World. Of the newer European viruses, TOP is the most closely related to
PUU (Figure). No human pathogenicity has been recognized. However, lemming
fever has traditionally been reported by Nordic inhabitants, particularly
during lemming years (42); this link was made already in 1942 (another
lemming year) by Stuhlfauth (2) when describing an epidemic in German
troops plagued by lemmings.

With the current explosive growth of knowledge concerning hantaviruses, a
tendency is emerging to globalize at least some strains and symptoms: 1)
The distribution of various new and old strains is giving an ever more
confused picture of infection in Eurasia, with HTN-like strains (HTN and
DOB) in the West, and PUU-like strains (TUL, TOB, KBR, and TOP) in the
East. Moreover, Bloodland Lake and Isla Vista have joined the prototype
North American isolate Prospect Hill as PUU-like strains in the Americas.
2) The wild rat SEO-strain remains probably the most underestimated
(pathogenic) hantavirus strain worldwide, despite recent reports of
SEO-like infections throughout Europe and the Americas. 3) The clinical
symptoms tend also to grow to each other on the global scene: whereas in
the Americas, non-Sin Nombre virus cases of hantavirus pulmonary syndrome,
i.e., induced by Black Creek Canal virus and/or Bayou virus, have renal as
well as lung involvement, and whereas even mild cases have recently been
described, we also find now, albeit rarely, evidence of lung involvement
under the form of acute lung injury in documented PUU and SEO cases in
Europe. 4) Careful reading of the earlier literature, often containing
astute clinical or epidemiologic descriptions of viral hemorrhagic fevers,
can still teach us many lessons, both for the bedside and for the bench.

Acknowledgment

This work was made possible by a grant JSM R&D 96/01 of the Belgian Army.

Jan Clement,* Paul Heyman,* Paula McKenna,* Paul Colson, and Tatjana
Avsic-Zupanc
*Queen Astrid Military Hospital, Brussels, Belgium; Centre de Sant des
Fagnes, Chimay, Belgium; and University of Ljubljana, Ljubljana, Slovenia

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     Manni T, et al. Nephropathia epidemica: detection of antigen in bank
     voles and serologic diagnosis of human infection. J Infect Dis
     1980;141:131-4.
 44. Kariwa H, Yoshizumi S, Arikawa J, Yoshimatsu K, Takahashi K, Takashima
     I, et al. Evidence for the existence of Puumula-related virus among
     Clethrionomys rufocanus in Hokkaido, Japan. Am J Trop Med Hyg
     1995;53:222-7.
 45. Lee PW, Amyz HL, Gajdusek DC, Yanagihara RT, Goldgaber D, Gibbs CJ.
     New haemorrhagic fever with renal syndrome-related virus in indigenous
     wild rodents in United States. Lancet 1982;2:1405.
 46. Hrling J, Chizhikov V, Lundkvist A, Jonsson M, Ivanov L, Dekonenko A,
     et al. Khabarovsk virus: a phylogenetically and serologically distinct
     hantavirus isolated from Microtus fortis trapped in far-east Russia. J
     Gen Virol 1996;77:687-94.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

1st International Conference on Emerging Zoonoses

Brucellosis: an Overview

---------------------------------------------------------------------------


      Brucellosis remains a major zoonosis worldwide. Although many
      countries have eradicated Brucella abortus from cattle, in some
      areas Brucella melitensis has emerged as a cause of infection
      in this species as well as in sheep and goats. Despite
      vaccination campaigns with the Rev 1 strain, B. melitensis
      remains the principal cause of human brucellosis. Brucella suis
      is also emerging as an agent of infection in cattle, thus
      extending its opportunities to infect humans. The recent
      isolation of distinctive strains of Brucella from marine
      mammals has extended its ecologic range. Molecular genetic
      studies have demonstrated the phylogenetic affiliation to
      Agrobacterium, Phyllobacterium, Ochrobactrum, and Rhizobium.
      Polymerase chain reaction and gene probe development may
      provide more effective typing methods. Pathogenicity is related
      to production of lipopolysaccharides containing a poly N-formyl
      perosamine O chain, Cu-Zn superoxide dismutase, erythrulose
      phosphate dehydrogenase, stress-induced proteins related to
      intracellular survival, and adenine and guanine monophosphate
      inhibitors of phagocyte functions. Protective immunity is
      conferred by antibody to lipopolysaccharide and T-cell-mediated
      macrophage activation triggered by protein antigens. Diagnosis
      still centers on isolation of the organism and serologic test
      results, especially enzyme immunoassay, which is replacing
      other methods. Polymerase chain reaction is also under
      evaluation. Therapy is based on tetracyclines with or without
      rifampicin, aminoglycosides, or quinolones. No satisfactory
      vaccines against human brucellosis are available, although
      attenuated purE mutants appear promising.

Brucellosis has been an emerging disease since the discovery of Brucella
melitensis by Bruce in 1887. Subsequently, an increasingly complex pattern
of strains has emerged with the identification of Brucella abortus,
Brucella suis, Brucella neotomae, Brucella ovis, Brucella canis, and, more
recently, types infecting marine mammals. Because each type has distinctive
epidemiologic features, with each new type, the complexity of the
interaction with humans has increased. Because new strains may emerge and
existing types adapt to changing social and agricultural practices, the
picture remains incomplete.

This synopsis reviews major advances in the knowledge of certain
aspectsgenetics, antigenic structure, mechanisms of pathogenicity,
diagnosis, treatment, and prevention of the diseaseof the Brucella genus
and its host interactions.

Epidemiology

Worldwide, brucellosis remains a major source of disease in humans and
domesticated animals. Although reported incidence and prevalence of the
disease vary widely from country to country, bovine brucellosis caused
mainly by B. abortus is still the most widespread form (Tables 1-5). In
humans, ovine/caprine brucellosis caused by B. melitensis is by far the
most important clinically apparent disease. The disease has a limited
geographic distribution, but remains a major problem in the Mediterranean
region, western Asia, and parts of Africa and Latin America. Recent
reemergence in Malta and Oman indicates the difficulty of eradicating this
infection (1). Sheep and goats and their products remain the main source of
infection, but B. melitensis in cattle has emerged as an important problem
in some southern European countries, Israel, Kuwait, and Saudi Arabia. B.
melitensis infection is particularly problematic because B. abortus
vaccines do not protect effectively against B. melitensis infection; the B.
melitensis Rev.1. vaccine has not been fully evaluated for use in cattle.
Thus, bovine B. melitensis infection is emerging as an increasingly serious
public health problem in some countries. A related problem has been noted
in some South American countries, particularly Brazil and Colombia, where
B. suis biovar 1 has become established in cattle (2). In some areas,
cattle are now more important than pigs as a source of human infection.

       Table 1. Brucellosis in animals, Europe, 1994
       --------------------------------------------------------------
                    Bovine       Ovine/          Porcine   Ovine
                                 caprine
       Country      (B. abortus) (B. melitensis) (B. suis) (B. ovis)
       --------------------------------------------------------------
       Albania      -            +               +         +
       Belgium      +            -               -         -
       Bulgaria     -            -               +         +
       Croatia      -            -               +         +
       Czech        -            -               ?         -
       Republic
       France       +            ++              ?         +
       Germany      +            -               ?         +
       Greece       +            ++              ND        ND
       Ireland      +            -               -         -
       Italy        +            +               -         ND
       Latvia       -            -               +         -
       Lithuania    -            -               -         ?
       Macedonia    +            +               -         -
       Malta        +            +               -         -
       Poland       +            +               ?         -
       Portugal     +            +               -         +
       Romania      -            -               +         -
       Russia       ++           ++              +         +
       Slovakia     -            -               ND        -
       Slovenia     -            -               -         +
       Spain        +            +               -         +
       Ukraine      ND           ND              ND        ND
       Yugoslavia   +            +               +         -
       --------------------------------------------------------------
       -   not present
       +   low sporadic incidence
       ++   high incidence
       ?   presence uncertain
       ND   no data
       None of the four types of brucellosis is present in Austria,
       Denmark, Estonia, Finland, Hungary, Iceland, Luxembourg,
       Moldavia, Netherlands, Sweden, Switzerland, and the United
       Kingdom
       Source for Tables 1-8: FAO-WHO-OIE Animal Health Yearbooks,
       1994, 1995.

       Table 2. Brucellosis in animals, Africa, 1994
       --------------------------------------------------------------
                    Bovine       Ovine/          Porcine   Ovine
                                 caprine
       Country      (B. abortus) (B. melitensis) (B. suis) (B. ovis)
       --------------------------------------------------------------
       Algeria      +            ?               ND        +
       Angola       ?            ?               ?         ?
       Botswana     +            ND              -         ND
       Cape Verde   ?            ?               ?         +
       Central      ++           ND              +         ND
       African
       Republic
       Chad         ++           ?               ?         ND
       Congo        +            -               -         -
       Cte d'Ivoire+            -               -         +
       Egypt        +            +               ND        -
       Eritrea      +            ?               ND        +
       Ghana        +            -               -         -
       Guinea       +            ND              -         ND
       Kenya        +            +               ND        ND
       Libya        +            +               -         -
       Mauritius    -            -               -         -
       Morocco      +            ?               -         -
       Mozambique   ++           +               ++        +
       Namibia      +            -               -         ?
       Niger        +            +               ND        +
       Nigeria      ++           +               +         ND
       Seychelles   +            -               -         -
       South Africa ++           +               -         +
       Sudan        ++           +               -         -
       Tanzania     +            ND              ND        ND
       Tunisia      +            ++              -         -
       Zaire        +            ND              +         ND
       Zimbabwe     +            +               -         +
       --------------------------------------------------------------
       -   not present
       +   low sporadic incidence
       ++   high incidence
       ?   presence uncertain
       ND   no data
       No data on any of the four types of brucellosis are available
       for Gambia, Mali, and Mauritania

       Table 3. Brucellosis in animals, Asia, 1994
       --------------------------------------------------------------
                          Bovine     Ovine/         Porcine Ovine
                                     caprine
       Country            (B.        (B.            (B.     (B.
                          abortus)   melitensis)    suis)   ovis)
       --------------------------------------------------------------
       Afghanistan        +          +              ND      ND
       Bangladesh         +          +              ND      ND
       Bhutan             +          -              -       ND
       China              +          +              +       +
       Hong Kong          ND         ND             ?       ND
       India              +          +              ?+      -
       Indonesia          +          ND             +       +
       Iran               +          +              -       -
       Israel             -          +              -       -
       Iraq               +          +              ND      ND
       Jordan             -          ++             -       -
       Korea (S)          ++         -              ?+      -
       Kuwait             ++         ++             -       -
       Malaysia           +          -              ?-      -
       Mongolia           ++         +              -       +
       Myanmar            +          ND             +       ND
       Oman               ++         ND             ND      ND
       Qatar              ND         ND             ND      ND
       Sri Lanka          ++         +              -       +
       Syria              +          ND             ND      ND
       Thailand           +          -              +       -
       Turkey             ++         ++             -       ND
       UAE                -          +              -       +
       Yemen              +          +              -       -
       --------------------------------------------------------------
       -   not present
       +   low sporadic incidence
       ++   high incidence
       ?   presence uncertain
       ND   no data
       None of the four types of brucellosis is present in Bahrain,
       Cyprus, Japan, Malaysia (Sabah), Philippines, or Singapore
       No data for countries of the former Soviet Union or Qatar

       Table 4. Brucellosis in animals, the Americas, 1994
       --------------------------------------------------------------
                    Bovine      Ovine/           Porcine   Ovine
                                caprine
       Country      (B. abortus)(B. melitensis)  (B. suis) (B. ovis)
       --------------------------------------------------------------
       Antigua/     ?           -                -         -
       Barbuda
       Argentina    ++          -                +         ++
       Belize       -           -                -         ND
       Bolivia      ++          +                +         ND
       Brazil       ++          -                +         -
       Canada       -           -                -         +
       Chile        ++          -                -         +
       Colombia     +           -                -         -
       Cuba         ?           -                ++        -
       Dominican    ++          -                +         -
       Republic
       Ecuador      ++          ND               ND        ND
       El Salvador  ++          ND               +         ND
       Guatemala    +           -                +         -
       Haiti        +           -                -         -
       Honduras     ?           -                ++        -
       Jamaica      ?+          -                -         -
       Mexico       +           +                ND        -
       Nicaragua    ++          ND               ND        ND
       Peru         ++          ND               ND        ++
       Paraguay     +           ND               -         +
       Uruguay      +           -                -         +
       United
       States       +           -                (+)       +
       Venezuela    ++          -                ++        ?
       --------------------------------------------------------------
       -   not present
       +   low sporadic incidence
       ++   high incidence
       ?   presence uncertain
       ND   no data
       None of the four types of brucellosis is present in Barbados,
       Falkland Islands, Surinam, or St. Kitts/Nevis

       Table 5. Brucellosis in animals, Oceania, 1994
       --------------------------------------------------------------
                    Bovine       Ovine/          Porcine  Ovine
                                 caprine
       Country      (B. abortus) (B. melitensis) (B. suis)(B. ovis)
       --------------------------------------------------------------
       Australia    -            -               (+)      +
       Cook Island  -            ND              -        ND
       New Caledonia-            -               -        -
       New Zealand  -            -               -        ++
       Samoa        +            ND              ND       ND
       --------------------------------------------------------------
       ++   high prevalence
       +   present
       (+)   limited presence
       -   not present
       ND   no data
       None of the four types of brucellosis is present in Vanuatu

The true incidence of human brucellosis is unknown. Reported incidence in
endemic-disease areas varies widely, from <0.01 to >200 per 100,000
population (3). While some areas, such as Peru, Kuwait, and parts of Saudi
Arabia, have a very high incidence of acute infections, the low incidence
reported in other known brucellosis-endemic areas may reflect low levels of
surveillance and reporting, although other factors such as methods of food
preparation, heat treatment of dairy products, and direct contact with
animals also influence risk to the population.

Consumption of contaminated foods and occupational contact remain the major
sources of infection. Examples of human-to-human transmission by tissue
transplantation or sexual contact are occasionally reported but are
insignificant (4). Prevention of human brucellosis depends on the control
of the disease in animals. The greatest success has been achieved in
eradicating the bovine disease, mainly in industrialized countries (Table
6); however, most countries have control programs. B. melitensis infection
has proved more intractable, and success has been limited (Table 7).

       Table 6. Countries reporting eradication of bovine
       brucellosis, 1994
       --------------------------------------------------------------
       EUROPE
       Bulgaria         Croatia               Czech Republic
       (1958)           (1965)                (1964)
       Denmark          Estonia               Finland
       (1962)           (1961)                (1960)
       Hungary          Iceland               Latvia
       (1985)           (never recorded)      (1963)
       Lithuania        Luxembourg            Netherlands
       (1952)           (1993)                (1993)
       Romania          Slovak Republic       Slovenia
       (1969)           (1964)                (1970)
       Sweden           Switzerland           U.K.
       (1957)           (1963)                (1993)
       AFRICA
       Mauritius
       (1986)
       AMERICAS
       Belize           Canada
       (1980)           (1989)
       ASIA
       Cyprus           Israel                Japan
       (1932)           (1984)                (1992)
       Jordan           N Korea               Papua New Guinea
       (1992)           (1959)                (1974)
       Philippines      U.A.E.
       (1989)           (1992)
       OCEANIA
       Australia        French Polynesia
       (1989)           (1984)
       New Zealand      Vanuatu
       (1989)           (1992)
       --------------------------------------------------------------

       Table 7. Countries reporting eradication of other forms of
       brucellosis, 1994
       --------------------------------------------------------------
                    Ovine/caprine       Porcine       Ovine
       Region       (B. melitensis)     (B. suis)     (B. ovis)
       --------------------------------------------------------------
       Europe
                    Bulgaria            Denmark       Czech Rep.
                    (1941)              (1951)        (1951)
                    Croatia             Estonia       Germany
                    (1991)              (1988)        (1986)
                    Czech Rep.          Lithuania     Latvia
                    (1951)              (1991)        (1989)
                    Germany             Sweden
                    (1986)              (1957)
                    Switzerland
                    (1963)
       Africa
                    Ghana               None          Ghana
                    (1993)                            (1993)
                    Namibia
                    (1990)
       Americas
                    United States       Belize        Falkland Is.
                    (1972)              (1985)        (1991)
                    Chile               Honduras
                    (1987)              (1992)
                                        Colombia      Mexico
                                        (1982)        (1991)
       Asia
                    Cyprus              Singapore     Yemen
                    (1993)              (1989)        (1989)
       Oceania
                    Not present         None          None
       --------------------------------------------------------------

Although few recent outbreaks of disease caused by B. suis biovar 4 have
been reported (5), foci of the infection persist in the Arctic regions of
North America and Russia and constitute a potential hazard for the local
population. B. ovis has not been demonstrated to cause overt disease in
humans, although it is widespread in sheep (Tables 1-5). B. canis can cause
disease in humans, although this is rare even in countries where the
infection is common in dogs (6). Precise information on prevalence is
lacking, but B. canis has been recorded in the United States, Mexico,
Argentina, Spain, China, Japan, Tunisia, and other countries. The recent
isolation of distinctive Brucella strains, tentatively named Brucella
maris, from marine animals in the United Kingdom and the United States
extends the ecologic range of the genus and, potentially, its scope as a
zoonosis (7,8).A hitherto unreported incident of laboratory-acquired
infection suggests that this type is pathogenic for humans. Infection could
result from occupational contact with infected seals or cetaceans.

Molecular Genetics

Characterization of the molecular genetics of Brucella has taken place
almost entirely within the past 10 years. The average molecular complexity
of the genome is 2.37 x 10 to the 9th power daltons and the molar G + C
58-59% (9).The genus itself is highly homogeneous with all members showing
>95% homology in DNA-DNA pairing studies, thus classifying Brucella as a
monospecific genus (10). However, the nomenclature proposed by Verger and
colleagues, in which all types would be regarded as biovars of B.
melitensis, has not been generally adopted on practical grounds. For this
reason, although its shortcomings are well known, the old nomenclature has
been retained with the former species' names B. abortus, B. melitensis, B.
suis, Brucella neotomae, B. ovis, and B. canis being used for the
corresponding nomen species (11,12). Within these, seven biovars are
recognized for B. abortus (1,7-10,12,13), three for B. melitensis (1,7,8),
and five for B. suis (1,7-10,12). The other species have not been
differentiated into biovars, although variants exist (14). The current
biotyping system does not encompass all known variants even of the
principal species. Thus, variants of B. melitensis have been described;
this suggests that the scheme should be extended (11,13,15). The strains
isolated from marine animals clearly form a separate group and have been
unofficially designated B. maris (E. S. Broughton, unpub. data). At least
two subdivisions of this strain can be distinguished, corresponding
approximately to strains isolated from cetaceans and seals, respectively
(7,8).

Restriction fragment patterns produced by infrequently cutting
endonucleases provide support for the current differentiation of the nomen
species (16). Restriction endonuclease analysis has generally been
unsuccessful for typing when applied to the whole genome (17) but
polymerase chain amplification of selected sequences followed by
restriction analysis has provided evidence of polymorphism in a number of
genes including omp 2, dnaK, htr, and ery (the erythrulose-1-phosphate
dehydrogenase gene) (18-20). The omp2 gene is taxonomically important
because it determines dye sensitivity, one of the traditional typing
methods for biovar differentiation (21). Its polymorphism and capacity for
posttranslational modification of its product may explain the tendency for
variation in dye sensitivity patterns and have been used as the basis for a
genetic classification of Brucella (22,23). The dnaK gene of B. melitensis
is cleaved into two fragments by Eco RV endonuclease, whereas the genes of
the other nomen species all produce a single fragment (24). The ery gene is
reported to have undergone a 7.2 kbp deletion in B. abortus strain 19 (20).
This could explain this strain's erythritol sensitivity, a major factor in
its attenuation.

The genome of Brucella contains two chromosomes of 2.1 and 1.5 mbp,
respectively. Both replicons encode essential metabolic and replicative
functions and hence are chromosomes and not plasmids (25,26). Natural
plasmids have not been detected in Brucella, although transformation has
been effected by wide host range plasmids after conjugative transfer or
electroporation (27).

rRNA sequencing has defined the phylogenetic relationship of Brucella. Its
closest known relation, Ochrobactrum anthropi, is an environmental
bacterium associated with opportunistic infections (28); this organism is
also detected by a polymerase chain reaction (PCR) procedure that is
otherwise specific for Brucella (29). Possibly more closely related is the
incompletely characterized Vibrio cyclosites, which displays >90%
similarity of 5S rRNA sequence (30). Less closely related but within the
same subgroup of the -2 Proteobacteria are Agrobacterium, Phyllobacterium,
and Rhizobium, which also possess multiple replicons and a capacity for
intracellular growth. The Bartonella group also shows some affinity to
Brucella on the basis of rRNA, but not DNA, similarity (31). Other
similarities have been noted in cell membrane lipid composition and
intracellular growth.

Antigenic Composition

A substantial number of antigenic components of Brucella have been
characterized. However, the antigen that dominates the antibody response is
the lipopolysaccharide (LPS). In smooth phase strains (S), the S-LPS
comprises a lipid A (containing two types of aminoglycose); distinctive
fatty acids (excluding -hydroxymyristic acid); a core region containing
glucose, mannose, and quinovosamine; and an O chain comprising a
homopolymer of approximately 100 residues of 4-formamido-4,6-dideoxymannose
(linked predominantly [alpha]-1,2 in A epitope-dominant strains with every
fifth residue linked [alpha]-1,3 in M dominant strains) (32).

The difference in linkage influences the shape of the LPS epitopes. The
A-dominant type is rod-shaped and is determined by five
consecutive [alpha]-1,2 linked residues, whereas the M-dominant type is
kinked and determined by four residues, including one linked [alpha]-1,3
(33). Strains that react with antisera to both A and M epitopes produce LPS
of both types in approximately equal proportions (30), consistent with the
original hypothesis of Wilson and Miles (34). The presence of 4-amino, 4,6
dideoxymannose in the LPS is also responsible for the antigenic
cross-reactivity with Escherichia hermanni and Escherichia coli O:157,
Salmonella O:30, Stenotrophomonas maltophilia, Vibrio cholerae O:1, and
Yersinia enterocolitica O:9 LPS (32). The structure of the LPS of nonsmooth
strains (R-LPS) is basically similar to that of the S-LPS except that the
O-chain is either absent or reduced to a few residues. The specificity of
the R-LPS is, therefore, largely determined by the core polysaccharide.

Numerous outer and inner membrane, cytoplasmic, and periplasmic protein
antigens have also been characterized. Some are reognized by the immune
system during infection and are potentially useful in diagnostic tests
(35). Hitherto, tests based on such antigens have suffered from low
sensitivity as infected persons tend to develop a much less consistent
response to individual protein antigens than to LPS. Thus, tests such as
immunoblotting against whole-cell extracts may have some advantages over
more quantitative tests that employ purified individual antigens (36).

Recently, ribosomal proteins have reemerged as immunologically important
components. Interest in these first arose more than 20 years ago when crude
ribosomal preparations were demonstrated to stimulate both antibody and
cell-mediated responses and to confer protection against challenge with
Brucella (37). However, the individual components responsible for such
activity were not identified until recently. It has been established that
the L7/L12 ribosomal proteins are important in stimulating cell-mediated
responses. They elicit delayed hypersensivity responses as components of
brucellins (38),and as fusion proteins, they have been shown to stimulate
protective responses to Brucella (39). They appear to have potential as
candidate vaccine components.

Mechanisms of Pathogenicity

Virulent Brucella organisms can infect both nonphagocytic and phagocytic
cells. The mechanism of invasion of nonphagocytic cells is not clearly
established. Cell components specifically promoting cell adhesion and
invasion have not been characterized, and attempts to detect invasin genes
homologous to those of enterobacteria have failed. Within nonphagocytic
cells, brucellae tend to localize in the rough endoplasmic reticulum. In
polymorphonuclear or mononuclear phagocytic cells, they use a number of
mechanisms for avoiding or suppressing bactericidal responses. The S-LPS
probably plays a substantial role in intracellular survival, as smooth
organisms survive much more effectively than nonsmooth ones. Compared with
enterobacterial LPS, S-LPS has many unusual properties: a relatively low
toxicity for endotoxin-sensitive mice, rabbits, and chick embryos; low
toxicity for macrophages; low pyrogenicity; and low hypoferremia-inducing
activity. It is also a relatively poor inducer of interferon (and tumor
necrosis factor) but, paradoxically, is an effective inducer of interleukin
12 (40,41).

S-LPS is the main antigen responsible for containing protection against
infection in passive transfer experiments with monoclonal and polyclonal
antibodies. The protection is usually short-term and incomplete, however.
The elimination of virulent Brucella depends on activated macrophages and
hence requires development of Th1 type cell-mediated responses to protein
antigens (42).

An important determinant of virulence is the production of adenine and
guanine monophosphate, which inhibit phagolysosome fusion; degranulation
and activation of the myelo-peroxidase-halide system; and production of
tumor necrosis factor (41,43). The production of these inhibitors is
prevented in pur E mutants, which are substantially attenuated in
consequence. Cu-Zn superoxide dismutase is believed to play a significant
role in the early phase of intracellular infection (44). However,
conflicting results have been reported, and this role needs to be
confirmed.

Survival within macrophages is associated with the synthesis of proteins of
molecular weight 17, 24, 28, 60, and 62 kDa. The 62 kDa protein corresponds
to the Gro EL homologue Hsp 62, and the 60 kDa protein is an acid-induced
variant of this. The 24 kDa protein is also acid-induced, and its
production correlates with bacterial survival under acidic conditions
(<pH4). The 17 and 28 kDa proteins are apparently specifically induced by
macrophages and correlated with intracellular survival (45).

Another stress-induced protein, HtrA, is involved in the induction of an
early granulomatous response to B. abortus in mice and is associated with a
reduction in the levels of infection during the early phase. Howevr, it
does not prevent a subsequent increase in bacterial numbers, and
htrA-deficient mutants ultimately produce levels of splenic infection
similar to those given by wild-type B. abortus (46). Similarly,
recA-deleted mutants produce a lower initial spleen count than
recA-positive strains but still establish persistent infection (47). The
role of iron-sequestering proteins or other siderophores in the
pathogenesis of brucellosis is still unknown. In general, the low
availability of iron in vivo restricts microbial growth. However, high iron
concentrations promote the killing of Brucella, probably by favoring
production of hydroxylamine and hydroxyl radical.

The mechanisms of pathogenesis of Brucella infection in its natural host
species and in humans are still not completely understood, and further
studies are needed.

Diagnosis

The clinical picture in human brucellosis can be misleading, and cases in
which gastrointestinal, respiratory, dermal, or neurologic manifestations
predominate are not uncommon (48-52). Because unusual cases with atypical
lesions continue to be reported, diagnosis needs to be supported by
laboratory tests (52). Blood culture is still the standard method and is
often effective during the acute phase; the lysis concentration method
gives the best results (53). Automated incubation-detection methods are
effective, but allowance should be made for the relatively slow growth of
the organism (54). Presumptive identification is made on the basis of
morphologic, cultural, and serologic properties. Confirmation requires
phage-typing, oxidative metabolism, or genotyping procedures. Reliance
should not be placed on gallery type rapid identification systems as these
have misidentified Brucella as Moraxella phenylpyruvica, with serious
consequences for laboratory staff (55).

PCR with random or selected primers gives promising results, but
standardization and further evaluation are needed, especially for chronic
disease (56). Similarly, antigen detection methods are potentially useful
but have not been validated. Combinations of these with PCR, such as
immuno-PCR, have considerable potential but require evaluation. Enzyme
immunoassay is now widely used for serologic diagnosis of the disease in
humans and other species. IgA and IgG antibodies seem the most useful
indicators of active infection (57,58). Western blotting against selected
cytoplasmic proteins may be useful in support of screening tests to
differentiate active from past or subclinical infection (35).

Treatment

Despite extensive studies over the past 15 years, the optimum antibiotic
therapy for brucellosis is still disputed. The treatment recommended by the
World Health Organization for acute brucellosis in adults is rifampicin 600
to 900 mg and doxycycline 200 mg daily for a minimum of 6 weeks (59). Some
still claim that the long-established combination of intramuscular
streptomycin with an oral tetracycline gives fewer relapses (60). There is
some evidence of physiologic antagonism between rifampicin and
tetracyclines, but recent studies suggest that the two regimens have very
similar results given adequate time. Quinolones in combination with
rifampicin seem as effective as either of these regimens (61). Controlled
clinical trials with other antibiotics, including new macrolides and
-lactams, have either give inferior results or involved too few patients
for proper evaluation.

Infections with complications, such as meningoencephalitis or endocarditis,
require combination therapy with rifampicin, a tetracycline, and an
aminoglycoside (62). Rifampicin has been recommended as the treatment of
choice for uncomplicated disease in children, with cotrimoxazole as an
alternative. Both are associated with a high relapse rate if used singly,
and best results are achieved by using them in combination (63).
Co-trimoxazole is an alternative but also has a high relapse rate. A
combination of the two agents gives the best results.

Prevention

Prevention of brucellosis in humans still depends on the eradication or
control of the disease in animal hosts, the exercise of hygienic
precautions to limit exposure to infection through occupational activities,
and the effective heating of dairy products and other potentially
contaminated foods. Vaccination now has only a small role in the prevention
of human disease, although in the past, various preparations have been
used, including the live attenuated B. abortus strains 19-BA and 104M (used
mainly in the former Soviet Union and China), the phenol-insoluble
peptidoglycan vaccine (formerly available in France), and the
polysaccharide-protein vaccine (used in Russia). All had limited efficacy
(64) and in the cases of live vaccines, were associated with potentially
serious reactogenicity. Subunit vaccines against brucellosis are still of
interest. The live vaccines have provoked unacceptable reactions in
individuals sensitized by previous exposure to Brucella or if inadvertently
administered by subcutaneous rather than percutaneous injection. These will
probably require a combination of detoxified lipopolysaccharide-protein
conjugate and protein antigens such as the L7/L12 ribosomal proteins
presented in an adjuvant or delivery system favoring a Th1 type immune
response. pur E mutants of B. melitensis appear safe in animals (65) and
may have potential application as human vaccines if their safety and
efficacy is confirmed in clinical trials. New vaccines have been evaluated
for use in animals, including the B. suis strain 2 live vaccine given
either orally or parenterally (66,67). This vaccine has proved inferior to
the Rev.1. strain for the prevention of B. melitensis infection in sheep
and goats and ineffective against B. ovis infection in sheep. B. abortus
strain 19 still appears to be as effective as any for the prevention of B.
abortus infection in cattle. However, the RB51 strain of B. abortus, an R
mutant used as a live vaccine, has been licensed in the United States. This
does not interfere with diagnostic serologic tests, but in laboratory
trials, its efficacy appeared comparable with that of strain 19 (68).
Similar rfb mutants of B. melitensis and B. suis are under development for
the prevention of ovine/caprine and porcine brucellosis.

Substantial progress has been achieved in understanding the molecular basis
of the genetics of Brucella and the pathogenesis of the infection. However,
further progress is needed, especially in relation to diagnostic procedures
and therapy. An effective and safe vaccine against human brucellosis is
also some way in the future.

Michael J. Corbel
National Institute of Biological Standards and Control, Hertfordshire,
United Kingdom

Address for correspondence: National Institute of Biological Standards and
Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG,
United Kingdom; fax: 44-1-707-646-730; e-mail: mcorbel@nibsc.ac.uk.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]
1st International Conference on Emerging Zoonoses

Global Aspects of Emerging and Potential Zoonoses: a WHO Perspective

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      Many new human pathogens that have emerged or reemerged
      worldwide originated from animals or from products of animal
      origin. Many animal species as well as categories of agents
      have been involved in the emergence of diseases. Wild (e.g.,
      bats, rodents) as well as draught animals (e.g., horses) and
      food animals (e.g., poultry, cattle) were implicated in the
      epidemiologic cycles of these diseases. Many of the agents
      responsible for new infections and diseases in humans were
      viruses (e.g., hantaviruses, lyssaviruses, and
      morbilliviruses), but bacteria, especially enteritic bacteria
      (e.g., Salmonellae and Escherichia coli) and parasites (e.g.,
      Cryptosporidium) of animal origin, were also involved in major
      food and waterborne outbreaks. The public health relevance of
      some of these agents (e.g., new lyssaviruses and
      morbilliviruses) is not yet fully assessed. In addition the
      zoonotic nature of some other human diseases, such as Ebola and
      the new variant form of Creutzfeldt-Jakob disease, is suspected
      but not yet demonstrated. Finally, the possible future use of
      xenografts may lead, if precautions are not taken, to the
      emergence of new diseases called xenozoonoses.

Emerging and Reemerging Zoonotic Diseases

In both the developing and industrialized worlds, a number of zoonoses have
emerged either as new pathologic entities or as already known agents,
appearing in areas or species in which they had not been previously
reported. In addition known zoonotic agents have reemerged sometimes after
many years of absence in areas where they had been reported before. In this
connection, limited and sometimes important outbreaks of otherwise endemic
zoonoses such as rabies, brucellosis, leptospirosis, anthrax, a number of
zoonotic foodborne diseases (caused by Salmonella enteritidis, Salmonella
typhimurium, and Escherichia coli) and arbovirus infections involving
production animals (Venezuelan equine encephalitis, Congo-Crimean
hemorrhagic fever) have continued to appear in many industrialized and
developing countries. Some examples of both emerging and reemerging
zoonotic infections are given below.

Emerging Zoonoses

    S. typhimurium in the United Kingdom

Multidrug-resistant S. typhimurium DT 104 initially emerged in cattle in
1988 in England and Wales. Subsequently, the strain has been isolated from
poultry, sheep, pigs, and horses. Antimicrobial therapy is used extensively
to combat S. typhimurium infection in animals. The evolution of a strain
resistant to the commonly used antibiotics has made infections with
S. typhimurium DT 104 in food animals difficult to control and likely to
remain a zoonotic problem. Furthermore, in S. typhimurium DT 104 of R-type
ACSSuT, multiple-drug resistance has become an integral part of the genetic
material of the organism. Unlike other salmonella serotypes,
multidrug-resistant S. typhimurium DT 104 is, therefore, likely to retain
its resistance genes even when antimicrobial drugs are no longer used.
Unlike S. enteritidis, which is mainly associated with poultry and eggs,
multidrug-resistant S. typhimurium DT 104 can be found in a broad range of
foods. Outbreaks in the United Kingdom have been linked to poultry, various
meats and meat products, and unpasteurized milk. In addition to infections
from contaminated food, cases have also occurred from human contact with
infected cattle. A small proportion of cases may have been caused by pets
such as cats and dogs, which can also be infected with this strain of
Salmonella. Like humans, pets probably acquire the infection through
consumption of contaminated raw meat, poultry, or poultry-derived products.
The emergence of Salmonella strains resistant to antibiotics in common use
is important to clinicians, microbiologists, and those responsible for the
control of communicable disease.

    Equine Morbillivirus in Australia

Equine morbillivirus (EMV) was isolated in September 1994 in Queensland,
Australia. Fourteen horses died as a result of infection with EMV in
southeast Queensland during September 1994. Seven additional horses were
infected by the virus and were humanely killed. The first death (of a mare)
attributed to the infection occurred on 9 September 1994. The virus
produced severe damage to the lungs with the accumulation of massive
amounts of fluid. Two persons who had close contact with the sick mare
became infected with the virus and also had respiratory illness, in one
case with fatal consequences. During October 1995, a third human infection
with EMV was diagnosed. The infected man came from a property in Mackay
where two horses had been diagnosed as dying from avocado poisoning and
snakebite during August 1994.

    Rabies in Australia

On 24 May 1996, a black flying fox with signs of neurologic illness was
found in Ballina, New South Wales, Australia, and was submitted for
autopsy. Histopathologic examination of the brain showed severe
nonsuppurative encephalitis. Tissues were examined for EMV infection at the
Animal Research Institute, Brisbane. Additional fixed tissues were sent for
EMV and rabies testing. Results were negative for EMV. However,
immunoperoxidase testing on fixed brain tissue was positive for Lyssavirus
antigen and was subsequently confirmed by immunofluorescence testing.
Subsequent investigations have indicated that the Lyssavirus present is not
classic rabies, serotype 1. A range of cell cultures and mice inoculations
were carried out to characterize the specific Lyssavirus involved.

    Rabies in the United Kingdom

Since 30 May, four people in the vicinity of Newhaven, on the south coast
of England, were thought to have been in contact with an insectivorous bat
with rabies. After the bat was caught and humanely killed, on 3 June, the
carcass was examined, and the diagnosis of rabies was confirmed. In the
past 30 years, three human deaths have been associated with bat rabies in
Europe, one in Finland and two in the former Soviet Union. Bat rabies is
mainly confined to serotine bats in Europe, particularly in Denmark,
northern Germany, and the Netherlands. Serotine bats are relatively
uncommon in the United Kingdom. In the United States, four cases of human 
rabies, caused by different variants of rabies viruses associated with 
insectivorous bats, were reported in 1995.

Reemerging Zoonoses

    Venezuelan Equine Encephalitis in Colombia and Venezuela

An outbreak of Venezuelan equine encephalitis (VEE), which began in early
September 1995, was reported on the border between Colombia and Venezuela.
As of 21 September 1995, 825 human cases of VEE had been reported, with
five confirmed by laboratory analysis and four resulting in death in
Venezuela, mainly in the state of Zulia. In Colombia, more than 450 cases
have been reported in the Department of La Guajira. VEE is the most severe
of several viruses causing disease in horses and can be transmitted to
humans by mosquito bites. Most infections are relatively mild, and symptoms
include abrupt onset of severe headache, chills, fever, muscular pain,
nausea, and vomiting. Clinical cases occur in 11% to 20% of the exposed
population, and death occurs in fewer than 1% of those cases. Large-scale
epidemics of VEE occurred in Colombia, Peru, Trinidad, and Venezuela in the
1950s and Central America and Mexico in the late 1960s, reaching the United
States (Texas) in 1971. Outbreaks can be prevented by the regular
immunization of horses in the framework of public health programs.

    Leptospirosis in Nicaragua

On 1 November 1995, health authorities in Nicaragua established a task
force to determine the cause of an unidentified illness that affected
hundreds of residents of the Achuapa area, causing some 15 deaths. A task
force investigated the nature and origins of the unknown disease, whose
symptoms ranged from high fever to internal bleeding. On 6 November 1995,
tests conducted by the Centers for Disease Control and Prevention (CDC),
Atlanta, Georgia, USA, indicated that the cause of the disease that killed
at least 16 people in Nicaragua was an unusual form of leptospirosis.
Initial reports that the disease was dengue fever, which has been common in
the region, were ruled out after several autopsies showed severe
respiratory hemorrhaging, and laboratory tests for dengue fever proved
negative. Tests on three of the four specimens were positive for
leptospirosis. Leptospirosis, while not rare in the Americas or Asia,
usually affects the liver or kidneys, rather than the respiratory system.

    Enterohemorrhagic E. coli Infection in Japan

An outbreak of enterohemorrhagic E. coli (EHEC) infection among
schoolchildren was announced in Sakai City (population ca. 800,000), in the
region of Osaka, Japan, in July 1996. Most of the reported cases were in
children 6 to 12 years of age from 62 public elementary schools of the
municipality. EHEC serotype O157 was detected in patients' stool samples.
School lunch, which was prepared in individual schools from the foods
delivered by a central supply station, was the suspected cause of this
outbreak; the responsible food is still unknown. Epidemiologic
investigation has shown that fresh radish sprouts (kaiware-daikon) were
among the foods eaten by the schoolchildren. However, samples of radish
seeds and sprouts, water, and soil from the environment of the farm
concerned and stool samples from the farm workers showed no trace of this
organism. As of 26 August 1996, 9,578 cases of E. coli serotypes O157:H7
and O157:H- infection, in both outbreaks and sporadic infections resulting
in 11 deaths, had been reported in Japan. Although most of the cases are
believed to be foodborne, the responsible foods have not been identified
with certainty except in a few isolated cases. Investigations of outbreaks
in other countries have established that animals, especially cattle, are
major reservoirs of Shiga-like toxin producing E. coli and that foods of
bovine origin, such as ground beef and unpasteurized milk, are the
principal sources of human infection. The possibility that the source of
the responsible agent is of animal origin should be further investigated in
Japan.

Potential Zoonoses

The natural cycle of Ebola is not known, and its reservoir (i.e., the
vertebrate playing an essential role in the maintenance of the agent and
its life cycle, if one exists) remains to be identified. On the other hand,
exposure to the agent of bovine spongiform encephalopathy (BSE), a known
disease of cattle, is suspected to be the origin of a new variant form of
Creutzfeldt-Jakob disease (CJD) in humans in the United Kingdom. These
potential zoonoses have very recently had a considerable impact on public
health, animal production, and laboratory animal sectors worldwide in view
of the need to minimize the risks for humans.

    Bovine Spongiform Encephalopathy

BSE is a fatal disease of cattle that first came to the attention of the
scientific community in November 1986 with the appearance in cattle of a
newly recognized form of neurologic disease in the United Kingdom. Between
November 1986 and 31 May 1996, approximately 160,000 cases of this newly
recognized cattle disease were confirmed on approximately 33,400 farms. The
disease is now on the decline in the United Kingdom. Epidemiologic studies
suggested that the source of disease was cattle feed prepared from
carcasses of dead ruminants. Modifications to the process used for
preparing cattle feed introduced in 1981 to 1982 may have been a risk
factor by not eliminating the agent from the feed. By May 1996, BSE had
been reported from 10 countries and areas outside the United Kingdom. In
one group of countriesFrance, Portugal, the Republic of Ireland, and
Switzerlandthe disease occurred in native cattle and could be linked to
importation of potentially infected cattle feed from the United Kingdom. In
another groupthe Falkland Islands, Oman Sultanate, Germany, Canada, Italy,
and Denmarkcases were only identified in cattle imported from the United
Kingdom.

BSE is associated with a transmissible agent, the nature of which is not
yet fully understood, and is one of several forms of transmissible brain
disease of animals. A number of similar severe and fatal neurologic human
diseases are due to non-conventional agents. These diseases include kuru, a
disease that was transmitted by human ritual handling of bodies and brains
of the dead and was identified in Papua, New Guinea in the 1950s, and the
various forms (i.e., sporadic, familial, and iatrogenic) of CJD, a rare
disease with a worldwide distribution.

After the identification of BSE, because the above human and animal
diseases shared a number of characteristics, including their ability to be
transmitted experimentally to a range of animals, the World Health
Organization (WHO) held, or coorganized as a regular activity, four
consultations during 1991 to 1995 to study these diseases (called
transmissible spongiform encephalopathies). An express purpose of these
consultations was to review the possible human public health implications
of animal spongiform encephalopathies, with a special emphasis on BSE.

The sudden announcement on 20 March 1996 by the United Kingdom of a cluster
of 10 human cases identified with what appears to be a variant of CJD
necessitated a fifth and sixth consultation at WHO because the results of
the investigation indicated that, although there was no direct evidence of
a link, the most likely hypothesis was that these cases might be related to
exposure to BSE in the United Kingdom. The fifth consultation reassessed
current recommendations in light of these new developments and issued
recommendations to further minimize risks for consumers particularly in
relation to foods and food products of animal origin. This was achieved
through the organization of expert consultations first held in April 1996.
The sixth consultation by WHO was organized in May 1996 to propose to its
member states a protocol for global surveillance of the new variant form of
CJD and identify the main areas for research on these diseases. Other
consultations may be held when new findings with a bearing on public health
become available.

    Ebola and Ebola-related Viruses

In 1976, Ebola attracted worldwide attention with the outbreaks of Yambuku,
Zaire, with 318 cases and Nzara, Sudan, with 284 cases, characterized by
very high case-mortality rates (53% in Sudan and 88% in Zaire). In the
first cases at the origin of the outbreaks (index cases) the patients may
have had contacts with infected animals or their products (bats and/or
rodents in Sudan, meat from monkeys or wild antelopes in Zaire), but
contact could not be proven, and investigations of the possible animal
reservoir remained inconclusive.

During 1977 to 1989 a small number of suspect, sporadic cases, or
relatively small outbreaks were reported (e.g., in Nzara, 1979). In 1989, a
new Ebola-related virus was isolated from macaques originally captured in
the wild in the Philippines dying from acute hemorrhagic fever and imported
into the United States. The same virus, called Reston, was isolated again
in different animal colonies in macaques imported from the same country in
1990 (United States), 1992 (Italy), and 1996 (United States).

In 1994, 15 years after the last Ebola outbreak (Nzara, 1979) reported in
Africa, a Swiss ethologist became infected by a new Ebola variant after
doing a postmortem on a chimpanzee from the Tai forest in Cte d'Ivoire.
The infection was demonstrated in this chimpanzee, and a number of deaths
reported in these animals in 1994 in the Tai forest were associated with
this agent. Other outbreaks may have occurred in the chimpanzee population
of the Tai forest before 1994. In January 1995, a new Ebola epidemic was
reported in Kikwit, Zaire, with 315 cases and 244 deaths. The initial
source of the infection of this outbreak may have been a forest worker
involved in charcoal making. A year later, in January 1996, an outbreak
occurred in Makokou, Gabon, with 37 cases and 21 deaths. The investigation
of this outbreak showed that most patients had contacts with dead
chimpanzees that they butchered. In October 1996, a second outbreak was
reported in the Makokou region. The number of cases was 24 with 17 deaths
on 12 November 1996. The index case was a hunter who fell ill in July 1996
and died in August in Booue hospital 200 km from Makokou.

Nothing definite is known on the reservoir of Ebola virus. A number of
hypotheses have been proposed involving rodents or insects and even viruses
of plant origin. It is generally agreed that the species of monkeys and
apes in which the virus stains have been isolated so far are only victims
of the disease, because in view of the high mortality rates usually
observed, these populations could not sustain themselves or the agent for
very long. These animals, however, participate in virus amplification by
facilitating further virus transmission, including to humans, through
handling of monkey carcasses or consumption of their meat. The
identification of a new variant of Ebola in chimpanzees in its natural
environment is a unique opportunity; so far the agent was only isolated
from monkeys captured in the wild but (at time of the disease) located far
from their natural environment. WHO, in collaboration with scientists from
many countries including Switzerland, France, the United Kingdom, Sweden,
Canada, and the United States, has, therefore, initiated a
multidisciplinary study in the Tai forest in Cte d'Ivoire to identify the
natural reservoir of the virus on the basis of the behavior of the
chimpanzees. This identification would be essential to understanding the
mechanisms for transmission in nature and facilitating the prevention of
future Ebola outbreaks.

Xenografts and Xenozoonoses

Successful xenotransplantation (the transfer of animal organs or tissues
into human recipients) may quickly become a biomedical reality. The
development of immunosuppressive drug protocols to prevent both organ
rejection and graft vs. host disease, as well as advances in surgical
techniques is opening the door to a field that can save thousands of human
lives each year.

A risk of disease transmission is present in any transplantation system. In
allotransplantation, disease transmission is a major cause of illness and
death in recipients. This is largely due to the required level of recipient
immunosuppression but is also attributable to infected, but usually
asymptomatic, human donors. Cases of viral, bacterial, fungal, and
parasitic transmission are documented. In some cases, the presence of the
infectious agent was known before transplantation. The decision was made to
proceed because of the lack of alternative donors. The field of
xenotransplantation also brings with it the potential for introducing
unwanted animal-origin infectious agents, both known and as yet unknown,
into the human population. These agents could cause disease in their new
host. Dissemination beyond the original recipient into the general
population could lead to epidemics. Xenotransplantation, therefore, has the
potential for being both of great benefit and of detriment to humans. WHO
is developing guidelines to ensure the development of xenotransplant donors
free of zoonotic and other agents representing a potential risk for the
recipient.

    Reasons for the Increasing Trend-Control Measures

As shown in this paper, in both the developing and industrialized worlds, a
number of zoonoses have emerged either as new pathologic entities or as
already known agents, appearing in areas or species where they had not been
previously reported or where they had seemingly disappeared. The reasons
for this increasing trend are complex, but some have been identified as
follows: 1) alteration of the environment affecting the size and
distribution of certain animal species, vectors, and transmitters of
infectious agents of humans; 2) increasing human populations favoring an
increased level of contact between humans and infected/affected animals; 3)
industrialization of foods of animal origin; changes in food processing and
consumer nutritional habits; and, 4) increasing movements of people as well
as trade of animals and animal products and decreasing activities for the
surveillance and control of major zoonoses.

Although the prevalence of major zoonoses, such as brucellosis,
tuberculosis, and dog rabies, has been greatly reduced in the
industrialized world, these diseases have not been eliminated altogether;
zoonoses prevention and control will remain an area of major concern in
most developing countries. Recent observations show that expenses related
to the prevention of zoonotic diseases in humans are likely to increase
dramatically in these countries in the near future if no programs for their
control and elimination in animal reservoirs are implemented. Diseases such
as brucellosis, rabies, and bovine tuberculosis will certainly be brought
under control during the first decade of the second millennium, but this
will require constant efforts for the next 15 to 20 years. Also, as the
trade of animal products and the movements of people become more intense,
the risks of introduction/reintroduction of certain diseases in a country
increase. It is very likely, in view of the foreseeable global changes over
the next few decades (e.g., population growth, urbanization, and climatic
changes), that this trend will continue and even increase. Human disease
patterns will be affected by high densities and movements of human
populations within and between countries and changes in lifestyles; animal
disease patterns will be affected by changing land-use patterns, new
farming practices, displacement of animals, and environmental
contamination.

Infectious diseases, including zoonoses, will remain the major health
problems in most developing countries, together with opportunistic
infections, including zoonotic diseases as a very important component. In
industrialized countries, where cardiovascular diseases and cancers will
remain the main causes of illness and death in spite of the AIDS pandemic,
special attention will need to be paid to human-animal relationships
especially with groups at higher risk such as the elderly, who already
represent a significant part of the population of industrialized countries,
as well as children and childbearing women, HIV-infected persons, and AIDS
patients.

As zoonoses and animal diseases with the potential to affect human health
likely will continue to emerge and reemerge, zoonoses surveillance will
need to be reinforced and maintained at the national and international
levels. In view of the foreseeable unplanned urbanization phenomenon and
the associated lack or shortage of basic facilities, a strategy to respond
to human and animal health problems in an urban environment will need to be
developed for cities of both industrialized and developing countries.
Comprehensive plans will need to be developed to reduce the rural-urban
migration flow to reverse the current and anticipated trend. Major
components of these plans should include improving rural employment and
food availability by promoting animal production projects and improving
health through zoonoses control and reduction of environmental pollution
related to animal rearing.

Further collaboration is essential between all professions involved in food
technology development, food industry control, and promotion of new
production techniques at the primary production level in order to ensure
food protection and response to the needs of the world population.

F.-X. Meslin
Division of Emerging and Other Communicable Diseases-Surveillance and
Control, World Health Organization, Geneva, Switzerland

References

  1. Food and Agriculture Organization. Zoonotic diseases in the Near East
     Region. Cairo: Food and Agriculture Organization, Regional Office for
     the Near East, 1993.
  2. Sthr K. The impact of zoonotic salmonella on public health and
     economics. Proceedings of the Second Asia-Pacific Symposium on Typhoid
     Fever and other Salmonellosis; 1994 Nov 7-9 1994; Bangkok, Thailand:
     South East Asian Journal of Tropical Medicine and Public Health. In
     press.
  3. Meslin F-X. Surveillance and control of emerging zoonoses. World
     Health Stat Q 1992;45:200-7.
  4. Meslin F-X. Global changes: future directions for public veterinary
     medicine. Paper presented at the occasion of the WHO/Mrieux
     Foundation/OIE Seminar on Management and International Cooperation in
     Veterinary Programmes, Veyrier-du-Lac, Annecy, France, 30 May - 5 June
     1993.
  5. Outbreak of Venezuelan Equine Encephalitis in Colombia and Venezuela.
     Wkly Epidemiol Rec 1995;40:283.
  6. Unidentified illness in Nicaragua/Honduras. Wkly Epidemiol Rec
     1995;44:316.
  7. Lyme disease. Wkly Epidemiol Rec 1995;45:320.
  8. Leptospirosis identified in Nicaragua. Wkly Epidemiol Rec 1995;45:322.
  9. Ebola haemorrhagic fever: Confirmed case in Cte d'Ivoire and suspect
     cases in Liberia. Wkly Epidemiol Rec 1995;50:359.
 10. Ebola Haemorrhagic fever in Gabon. Wkly Epidemiol Rec 1996;9:71.
 11. Ebola Haemorrhagic fever in Gabon. Wkly Epidemiol Rec 1996;17:125.
 12. Ebola Haemorrhagic fever in Gabon. Wkly Epidemiol Rec 1996;42:320.
 13. Equine morbillivirus in Queensland. Wkly Epidemiol Rec 1996;27:208-9.
 14. Enterohaemorrhagic Escherichia coli infection in Japan. Wkly Epidemiol
     Rec 1996;30:229.
 15. Bat rabies in the UK. Wkly Epidemiol Rec 1996;34:256.
 16. Enterohaemorrhagic Escherichia coli infection in Japan. Wkly Epidemiol
     Rec 1996;35:267.
 17. Human health aspects of a possible Lyssavirus in a black flying fox.
     Wkly Epidemiol Rec 1996;45:339.
 18. Report of a WHO consultation on public health issues related to animal
     and human spongiform encephalopathies, Geneva, 12-14 November
     1991(WHO/CDS/VPH/92.104).
 19. Report of the WHO informal consultation on bovine spongiform
     encephalopathy in the United Kingdom, Geneva, 7 May 1993
     (WHO/CDS/VPH/93.119).
 20. Report of a WHO Consultation on Public Health Issues Related to Human
     and Animal Transmissible Spongiform Encephalopathies, Geneva, 17-19
     May 1995 (WHO/CDS/VPH/95.145).
 21. Report of a WHO consultation on public health issues related to human
     and animal transmissible spongiform encephalopathies, Geneva, 2-3
     April 1996 (WHO/EMC/DIS/96.147).
 22. Report of a WHO consultation on clinical and neuropathological
     characteristics of the new variant of CJD and other human and animal
     transmissible spongiform encephalopathies, Geneva, 14-16 May 1996
     (WHO/EMC/ZOO/96.1).
 23. Report of the WHO working group meeting on shiga-like toxin producing
     Escherichia coli (SLTEC) with emphasis on zoonotic aspects, Bergamo,
     Italy, 1 July 1994. WHO/CDS/VPH/94.136.

---------------------------------------------------------------------------

   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

1st International Conference on Emerging Zoonoses

Epidemiology of Emerging Zoonoses in Israel

---------------------------------------------------------------------------


The epizootiologic, climatic, and ecologic conditions in the Middle East,
combined with socioeconomic and agricultural structures in Israel, have
created a unique and hazardous veterinary public health situation in this
country. In this paper, emerging zoonotic diseases are newly introduced
disease agents or endemic zoonoses whose previous epidemiologic patterns
have changed.

As of 1996, 15 zoonotic diseases of animals, anthrax, brucellosis,
cysticercosis (bovine), erysipelas, leishmaniasis (canine), leptospirosis,
listeriosis, malleus (glanders), psittacosis (chlamydiasis), Q fever,
rabies, Rift Valley fever (RVF), salmonellosis, trichinosis, and
tuberculosis (bovine and avian) (Table 1), have been declared notifiable in
Israel. When diagnosed in animals, these diseases are officially reported
to the state veterinary officers, who in turn report them to the medical
officers. Of these notifiable zoonoses, RVF has never been recorded in
Israel, and glanders has not been reported since 1951; ten are notifiable
in humans at the national level.

            Table 1. Selected zoonotic diseases in Israel, 1996
            ----------------------------------------------------
                                   Animal           Human
                                Year    Occur-    Year    Occur-
                             Notifiable rence  Notifiable rence
            ----------------------------------------------------
            Anthrax          1945       1993   1949       1984
            Brucellosis      1952       +++    1949       +
            Cysticercosis    1981       +      NN         +
            (bovine)
            Erysipelas       1945       (+)    NN         +
            Leishmaniasis    1945       ( )    1949       1994
            (canine)
            Leptospirosis    1945       +      1949       (+)
            Listeriosis      1961       +      1993       +
            Malleus          1945       1951   NN         never
            (glanders)
            Psittacosis      1952       +      NN         +
            Q fever          1952       +      1949       (+)
            Rabies           1955       +      1949       1960
            Rift Valley fever1978       never  NN         never
            Salmonellosis    1959       +++    1949       ++
            Trichinosis      1982       ( )    No         +
            Tuberculosis     1945       1993   1949
            (bov., av.)
            Botulism         NN         +      1993
            (types C,D)
            Echinococcosis   NN         ++     1980       (+)
            ----------------------------------------------------
            NN Not notifiable
            0000 Never reported
            - not reported, probably no occurrence
            Year Year of last occurrence
            (+) exceptional occurrence
            + low sporadic occurrence
            ++ enzootic/endemic
            +++ High occurrence

This article examines five of the reportable animal diseases whose
epidemiologic features have changed in Israel during the last decade:
Brucella melitensis in cattle, Salmonella enteritidis in poultry, rabies,
canine leishmaniasis, and trichinosis in wild boars. Two nonreportable
diseases in animalsbotulism in ruminants and echinococcosiswill also be
discussed briefly. (Table 1). Recently, the possibility that bovine
spongiform encephalopathy (BSE) may be a zoonotic disease has been
discussed worldwide. Thus, we also describe steps adopted by the Veterinary
Services and Animal Health (VSAH) to prevent the introduction of BSE into
Israel. The data on zoonotic diseases in animals are from VSAH's annual
reports to the Office International des Epizootics in Paris. If not
otherwise mentioned, the data on the diseases in humans are from the weekly
and monthly reports of the Ministry of Health in Jerusalem.

Brucella melitensis in Dairy Cattle

Bovine brucellosis is usually caused by Brucella abortus, less frequently
by B. melitensis, and rarely by B. suis. The disease is manifested by
abortion and excretion of the organisms in uterine discharges and in milk.
Brucellosis is highly pathogenic for humans, B. melitensis is regarded as
more pathogenic than B. abortus. B. melitensis is the main causative agent
of caprine and ovine brucellosis; it appears to occur mainly in the
Mediterranean regions, but infection occurs worldwide.

As of 1996, the dairy cattle of Israel, approximately 120,000 lactating
cows and 230,000 younger animals on 1,500 farms, are free of infection with
B. abortus. Since 1984, VSAH's ongoing, compulsory surveillance of all
farms has reported no cases of B. abortus. The surveillance includes
periodic bulk milk testing. In the event of a positive reaction, the test
is repeated on bulk milk from groups of cows, and the cows of a suspected
group undergo individual blood testing. Cows with positive test results are
slaughtered with full state compensations, and their organs are
bacteriologically examined in the State Reference Laboratory for
Brucellosis in the Kimron Veterinary Institute. In spite of the absence of
B. abortus, vaccinations of all 2- to 6-month-old dairy heifers with B.
abortus strain 19 vaccine has not been discontinued.

The status of B. melitensis in small ruminants, however, is different.
Brucellosis, which has been present in sheep and goat flocks (mainly in the
Bedouin sector) for decades, has increased considerably since the mid-1980s
A countrywide serologic survey was carried out during 1994-1995, in which
906 (10.1%) out of the 4,738 examined flocks were found infected. The total
number of reacting animals was 4,785 (3.16%) out of 151,409 examined sheep
and goats. Accordingly, a sharp rise in the occurrence of B. melitensis in
humans has been recorded from one case per 100,000 in 1983 to 11 cases per
100,000 (total 498 cases) in 1988 to 11 cases per 100,000 (total 498 cases)
in 1988. The most common sources of human infection are locally prepared
unpasteurized goat and sheep cheeses and milk (1).

An important phenomenon regarding B. melitensis in Israel is its
penetration into cattle herds. Until the late 1980s, these were mainly
small herds of locally bred Baladi beef cattle. In sporadic exceptional
cases, introduction of B. melitensis into dairy cattle farms was also
observed. Such a case was recorded for the first time in 1977 when raw
unpasteurized whey, a by-product of sheep cheese, from a central dairy
plant was fed to the lactating cows in one of the largest and finest dairy
herds in Israel, in the centrally situated Valley of Yezreel (2). The case
came to the attention of VSAH only after three dairy workers were
hospitalized with Malta fever. It took 2 years and the slaughter of 28 cows
for the herd to become disease-free again. According the VSAH "test and
slaughter" policy, all cattle in a farm where the seropositivity rate does
not exceed 50% are subject to monthly repeated serologic examinations.
Animals with positive test results are slaughtered, and compensations are
paid to the owners. The farm may be declared free of brucellosis, and
quarantine measures are discontinued after three consecutive negative
serologic tests of the entire herd. If more than 50% of the cows react
positively to the serologic test, the entire herd is slaughtered. Since
this incident, only pasteurized whey has been permitted as animal feed;
however, a second major outbreak occurred in late 1988, when cattle on a
dairy farm with 765 animals in western Galilee tested positive on the
periodic milk-ring test and was infected with B. melitensis (3).
Circumstantial evidence implicated contaminated whey from sheep or goat
milk as the most likely vehicle of infection. The epidemiologic
investigation was inconclusive, but researchers hypothesized that the
pasteurized whey could have been recontaminated when transported in tankers
not properly disinfected after having transported raw goat's milk. The
dairy herd was put under quarantine, and test and slaughter policies were
applied; implementation of these policies led to the compulsory slaughter
of 175 animals (23% of the herd). The farm was declared free of
brucellosis a year after the initial infection and two consecutive herd
tests with negative results. No human cases were involved. From this
outbreak, it could be concluded that vaccination of cattle with strain 19
vaccine did not confer adequate protection against infection with B.
melitensis, although it may have prevented abortions. These observations
were later supported by additional data from other outbreaks in dairy
cattle.

Since 1988, eight additional outbreaks of B. melitensis in large dairy
cattle farms throughout Israel have been recorded (Figure 1). The largest
outbreak occurred in 1994 when the entire cattle population (761 cattle) of
a large kibbutz dairy farm had to be slaughtered because the infection rate
exceeded 60%. In this outbreak, more than 30 members of the kibbutz
community, as well as the attending veterinarian, were infected, most by
direct contact with the infected cows or placenta and by the consumption of
unpasteurized milk. At present, an additional outbreak with human cases is
being investigated.

             [Figure 1] [Figures not available in ASCII version]
             Figure 1. Brucella mellitensis in dairy cattle,
             Israel 1988-1996.

The last eight outbreaks have been attributed to indirect contact with
infected nomadic sheep and goat flocks, which apparently contaminated the
pasture from which fodder was supplied to the lactating cows. Another route
of infection might have been dogs, most probably infected by consuming
infected placentas. Serologic examinations of dogs were carried out on two
of the farms, and antibodies were found in samples of 9 of the 39 dogs
tested. The infected dogs were then euthanized. It should be noted that
Israel is free of B. canis. The introduction of B. melitensis into dairy
cattle farms, a serious public health hazard, may be almost inevitable in a
highly contaminated environment.

Salmonella enteritidis

Salmonellosis is an infectious disease of humans and animals caused
primarily by intestinal bacteria of which 2,375 serovars are recognized, a
number constantly increasing. Salmonellosis appears to be most prevalent in
areas of intensive animal husbandry, especially of poultry or pigs. S.
enteritidis, especially of the phage-type TP4, emerged as a serious public
health hazard in Europe during the late 80s, mainly as a contaminant of
table eggs because of its transovarian infection in laying hens (4).
National programs have been implemented in some countries to control S.
enteritidis in poultry and protect the consumer.

Since the initial report from Europe, in 1988, of the emerging animal and
public health problem caused by S. enteritidis PT4 in poultry, Israeli
policy and activities have undergone a three-phase evolution:

1) 1988-1991: To prevent introduction of S. enteritidis, Israel implemented
strict quarantine conditions, including testing (serologic and
bacteriologic), on the import of breeding poultry, chicks, and hatching
eggs. From 1988 to 1991, 11 imported breeding flocks (five broiler
chickens, five laying chickens, one turkey), 103,100 birds, and 149,970
hatching (layer) eggs were found infected with S. enteritidis PT4 and
destroyed. The policy of strict border control of imported day-old chicks
and hatching eggs probably contributed to the delay in the appearance of
human infection until 1992.

2) 1992-1993: For the first time local breeding flocks were found infected
with S. enteritidis PT4 and destroyed along with their offspring laying
chick flocks. A surveillance program was implemented in the 200 local
breeding flocks. The program included bacteriologic examinations of dead
birds forwarded to the regional poultry disease laboratory, as well as
monthly visits of state poultry disease officers to each breeding farm for
sanitation control that included drag swab tests, sampling of caecal
contents for bacteriologic examinations, and in several flocks, rapid
slight agglutinations. Hatcheries were included as well. After the
surveillance, infected flocks (31 [10 broilers, 20 layer, one goose] which
comprised 272,188 birds and 2.8 million hatching eggs) were destroyed. The
state had to compensate the owners approximately 1.5 million U.S. dollars;
$300,000 in 1992, $1.17 million in 1993). "Competitive exclusion," a
preventive-curative technique developed in Finland and tried experimentally
on six breeding flocks produced inconclusive results (5). During 1992 and
1993, a significant increase in laboratory-contracted human cases of S.
enteritidis PT4 infection was recorded by the Ministry of Health: nine
cases in 1991, 203 in 1992, and 473 in 1993.

During 1993, to evaluate the S. enteritidis infection rate on commercial
farms, VSAH surveyed 88 broiler and layer breeding farms and found 18
(26.8%) of the 67 surveyed broiler breeder farms and 6 (28.5%) of the 21
layer breeder farms infected by direct cultures. In another investigation,
35 flocks of laying hens were examined in slaughterhouses, of which 12
(30.8%) were infected with S. enteritidis. Out of the said 10 isolates,
five were identified as PT4. In addition, 39 hatcheries throughout the
country were surveyed, of which 12 (30.8%) were infected with S.
enteritidis. These findings, combined with the alarming increase human S.
enteritidis infection rates, led to a reevaluation of the general policy,
which examined experience in other countries and local data. One of the
main decisions was to concentrate the efforts in the layer line and
discontinue the stamping out policy in the broilers line. The first step
was to prohibit the simultaneous incubation of layer- and broiler-hatching
eggs in the same hatchery.

The updated policy to concentrate and intensify the control efforts upon
the layers breeding stock while generally improving sanitary conditions in
all lines and to implement the control upon handling of table-eggs seems to
have contributed to the improvement of the public health situation
regarding S. enteritidis PT4.

3) 1994-
In 1993, after the publication of state regulations forbidding the
simultaneous incubation of layer and broiler hatching eggs, VSAH
discontinued intensive surveillance and, eventually, stamping out of
broiler breeding farms, to concentrate efforts on the layer line.
Consequently, the number of stamped-out broiler poultry flocks decreased
from 24 in 1993 to nine in 1994 to only one in 1995. VSAH implemented four
steps: a) Strict control of the layers breeding stock and hatcheries,
including bacteriologic examinations of meconium, offspring, and laying
birds and monthly sampling of drag swabs from the hatcheries and the
breeding farms; b) improvement of sanitary conditions in poultry farms; c)
introduction of an (inactivated) S. enteritidis vaccine, combined with the
live attenuated Salmonella typhimurium vaccine in parent-stock breeding
flocks; and d) enforcement of new state regulations regarding the handling
of table-eggs, their collection, transportation, packing, marking,
chilling, and marketing.

These measures were possible because of a single, centralized veterinary
system that combines modern diagnostic facilities, field activities, and
legal enforcement tools. The results of the measures described are
reflected in the number of reported human S. enteritidis isolates (Table
2).

       Table 2: Reported Salmonella enteritidis isolates in humans
       --------------------------------------------------------------
              S. enteritidis        S. enteritidis (PT4)
       --------------------------------------------------------------
       1989   357                   -
       1990   878                   -
       1991   987                   9
       1992   926                   203
       1993   1376                  473
       1994   1450                  750
       1995   997                   557
       --------------------------------------------------------------
       Furnished by Dr. Vered Agmon, Ministry of Health, Jerusalem.
       Preliminary figures for 1996 indicate that decline continues.

Rabies

Rabies, a major zoonosis caused by the neurotropic Lyssavirus,
transmissible to all mammals, occurs in all parts of the world. It is
especially prevalent in the temperate zones and where there is a large
canine population. Generally, in countries where the virus reservoir is
predominantly within the stray dogs which are the main vectors, the term
"urbanic rabies" is applied. On the other hand, when the reservoir is
mainly the wildlife population, the term "sylvatic rabies" is used. Urban
rabies predominates in Asia, Africa, and South America.

The evolution of rabies in vector animals, since 1948 is presented in Table
3. Annual vaccination of owned dogs became compulsory in 1957; vaccination
coverage is estimated at 60%. In 1979, rabies became sylvatic (involving
mainly fauna [foxes] rather than dogs) and remained so until 1990. A
transitory change was observed during 1991-92 when a sharp rise in the
number of rabid dogs converted the picture into "urban rabies" (dogs/fauna
ratio:2/1). This change, combined with the penetration of the disease into
the densely populated coastal region where rabies had been practically
absent for more than 30 years, could have been an outcome of the Gulf war
at the beginning of 1991 when many people (nearly 50% of the inhabitants of
Tel Aviv and other coastal cities) left their homes for safer areas during
the Iraqi missile attacks. Many of them abandoned their pets, establishing
a large stray-dog population. Subsequent steps to eliminate stray dogs may
have reversed the situation during 1992. Since 1993 (up to November 1996),
the pattern of predominantly sylvatic rabies has been reestablished.

 Table 3. Mean annual number of rabies cases in animals and humans,
 1948-1995
 --------------------------------------------------------------------------
                       Domestic          Wildlife
             No. of    animals                      Other  Fauna   Human
    Years     years   Dogs   Cats  Foxes  Jackals   fauna   (%)    cases
 --------------------------------------------------------------------------
 1948-1957   10      72.0   3.9   0.1     9.9      0.4     12.0   23
 1958-1966   9       19.4   0.9   0.3     0.8      0.1     5.6    2
 1967-1978   12      8.6    0.3   1.3     0.3      0.16    16.5   2
 1979-1990   12      6.2    0.25  12.6    1.25     1.3     70.1   0
 1991-1992   2       27.0   0     11.0    2        0       32.5   0
 1993-1995   3       14.25  1.25  35.25   4        4       72.2   0
 --------------------------------------------------------------------------

Rabies is practically endemic in most of Israel; clusters of rabies cases
in foxes are observed mainly in areas around the cities of Beer-Sheva,
Arad, Jerusalem, and Nazareth, but sporadic cases are scattered in other
areas (Figure 2).

             [Figure 2] [Figures not available in ASCII version]
             Figure 2. Rabies in fauna in Israel, 1992-1996.

The wildlife potentially involved with rabies comprises mainly foxes
(Vulpes vulpes) but in recent years, a significant increase in the number
of jackals (Canis aureus) has been observed in the central and northern
parts of the country. During 1995, 63 foxes and 14 jackals were tested at
the central rabies diagnostic laboratory at the Kimron Veterinary
Institute; 30 (47.6%) foxes and 2 (14.3%) jackals were infected. The
figures for 1996 are similar. Other wildlife species, involved with rabies
are the mongoose (Herpestes ichneumon ichneumon), badgers (Meles meles
canescens), and the stone marten (Martes foina syriaca). During 1986 to
1996, rabies was diagnosed in 251 foxes, 31 jackals, four mongooses, four
badgers, and three martens, which were sent to the Kimron Veterinary
Institute. The actual incidence of the disease was much higher. In view of
the growth of the jackal population, rabiesif spread within that
speciesmay increase considerably the already serious threat to humans and
farm animals. Recently, seven cattle herds in northern Israel were found
infected, more than twice the mean annual number for decades. This might
reflect the increased rabies incidence among the extremely dense
populations of foxes and jackals in that region.

Oral vaccination of wildlife in Israel has been considered, but vaccine and
baits evaluation is still in its experimental stages. At any rate,
vaccination will only be practical if it is effective for both foxes and
jackals and involves entire ecologically related regions (the West Bank,
the Jordan Valley, and probably the western parts of Jordan and southern
Lebanon).

Canine Leishmaniasis

Canine leishmaniasis is a chronic viscera-cutaneous disease caused by
Leishmania infantum (in the Old World), of which the dog acts as the source
reservoir. The vectors of leishmaniasis are phlebotomine sandflies
belonging to the genera Phlebotomus (Old World) and Lutzomyia (New World).

Canine leishmaniasis was common before 1948 but seemed to disappear after
that. Since 1955, no cases have been reported in dogs. However, four human
cases of visceral leishmaniasis (Kala-azar) (two in 1980 and two in 1982)
were reported by the Ministry of Health in two restricted areas in eastern
and western Galilee. The disease became regarded as endemic in these areas,
although no canine cases were detected there.

From January 1995 through November 1996, 30 clinical cases of canine
leishmaniasis were reported from five communities in the Judean hills
northwest of Jerusalem and from one location in western Galilee (Figure 3).
Diagnosis was carried out by the Koret Veterinary School and the Kuvin
Center of the Hebrew University, which reported that the cases were caused
by L. infantum (G. Benet, pers. comm.).

             [Figure 3.] [Figures not available in ASCII version]
             Figure 3. Canine Leishmaniasis in Israel,
             1995-1996.

Trichinosis

Trichinosis is a parasitic disease. Human infections are established
through the consumption of insufficiently cooked pork infected with the
causative nematode Trichinella spiralis.

No cases of trichinellosis (trichinosis) have been recorded in domestic
pigs in Israel since 1948, though a substantial number of pigs are
slaughtered (more than 100,000 in 1995) and each batch is sampled for
representative microscopy examinations. In light of the accumulated data
about the absence of infection in the housed pigs, the sample rate is
limited. Trichinosis in animals was declared notifiable in 1982 when its
potential hazard was reflected by the admission to Israeli hospitals of 20
clinically infected persons from southern Lebanon, close to the northern
Israeli border.

According to a state regulation published in 1977, the entire carcass of
hunted wild boars should be presented to accredited veterinary inspectors
at specified inspection stations. Until 1991, 100-200 such carcasses,
approximately 10% of the actual number of killed boars, were annually
presented for microscopy examinations without detecting any infections.
However, in 1992, hunters from northern Galilee were hospitalized with
symptoms of acute trichinosis after eating pork derived from game.
Consequently, public awareness of the disease grew, and the number of
samples from wild boars submitted for laboratory examination increased two-
to three-fold from previous years. In May 1992, the first case was found in
a wild boar from the Lebanese frontier, followed by 19 additional cases
between 1993 and 1996, all in wild boars shot in three areas: (upper
Galilee; central and western Galilee, and the Carmel Hills). By October
1996 alone, no fewer than nine cases of trichinosis were diagnosed.(see
footnote) According to the Animal Diseases Regulations, carcasses of
animals found infected are condemned; no alternative measures, such as
cooking or freezing, are officially permitted.

    Footnote. Dr. John Wortabet (1827-1908), an Armenian-Lebanese
    physician who worked in the St. John Hospital in Beirut and
    lectured in the medical college there during the second half of
    the 19th century diagnosed trichinosis in humans in south Lebanon
    during a massive outbreak in the village of El-Chiam in November
    1880more than 115 years ago. His exemplary observations about the
    outbreak, which involved 262 cases, including six deaths, were
    published in Lancet (6). A second outbreak, in the Northern Golan,
    was reported by Dr. Wortabet in Lancet (4 August 1883). In this
    outbreak, 40 inhabitants of the village Ein-Kinya, were infected
    after consuming raw pork from a wild boar. However, in this
    outbreak, no deaths were recorded. His footnote, at the end of the
    report, is still timely: "From personal observation and experience
    I have found the use of pork in Syria decidedly unhealthy. The
    wild boar in winter is a delicacy, but unless previously examined
    with the microscope, according to German law, or cooked more
    thoroughly than is usually done, its use cannot be free from the
    danger of communicating trichinae to man." Data about Dr. Wortabet
    courtesy of Drs. Mertyn Malkinson and Arieh Sheskin.

Botulism

Botulism is a highly fatal toxemia caused by ingestion of the toxin of
Clostridium botulinum. There are eight types and subtypes of Cl. botulinum,
of which types A, B, and E are of most importance in human botulism; types
C and D affect farm animals, mainly cattle, horses, chickens, and sheep.

In 1978, a major outbreak of botulism (type D) caused the deaths of many
dairy cattle and small ruminants from farms in central and northern Israel
(7). Spores of Cl. botulinum type D, as well as the toxin itself, spread to
many farms from two central animal feed plants by recycled poultry manure
that included remains of avian cadavers and was not properly sterilized.
Many farms are suspected to have been contaminated by the spores through
the centrally produced and countrywide distributed infected feed.
Consequently, annual botulism vaccinations of cattle and of other
vulnerable animals with a vaccine, including toxoids of Cl. botulinum types
C and D adsorbed onto aluminum hydroxide gel, are conducted in most parts
of Israel. Sporadic cases involving unvaccinated animals (mainly young
cows) occur every year, along with an exceptionally large number of
outbreaks, (23) recorded in 1995, found by the Kimron Veterinary Institute
to be caused by botulism type D. In all these cases, poultry manure was the
suspected source of intoxication.

Although botulism types C and D are not generally regarded pathogenic to
humans, it has recently been decided to prohibit the release of meat from
ruminants with suspected cases for human consumption.

Echinococcosis

Echinococcosis, also called hydatid disease, is caused in humans by
ingestion of eggs of the cestode Echinococcus, of which the most
predominant and epidemiologically significant is Echinococcus granulosus.
The adult tapeworm is carried by dogs and other carnivores. The
intermediate hosts in which the cystic forming larval stage develops
include various food animals and humans. The infection cycle is maintained
by the availability of infected organs to carnivore hosts. Data from
slaughterhouses show that the countrywide infection rate per 1,000 animals
in 1995 was 0.9 for cattle and 39.9 for sheep (8).

In 1981, human hydatid disease was declared notifiable by the Ministry of
Health. During 1991 to 1995, 38 human cases were officially recorded in the
country, but the actual number is much higher. For example, El-On et al.
(9) conducted epidemiologic studies in 1988 in the town of Yirka, a
semirural Druze community of 8,200 persons in northern Israel, and found a
cumulative percentage of confirmed present or recent past hydatid
infections of 12 (1.6%) of 758, leading to an extrapolated rate of 1,583
per 100,000 persons, which is comparable with that in hydatidosis-endemic
areas worldwide. This remarkable situation could be explained by four
combined factors: 1) High infection rates in sheep and goats (10%); 2)
high infection rates in owned dogs (14.2%); 3) widespread practice of
illegal slaughter of sheep and goats, performed without veterinary
inspection and followed by uncontrolled disposal of the infected slaughter
offal; and 4) a large number of hunting dogs kept at the homes of many of
the Druze people, who traditionally hunt.

The prevalence of hydatid disease in neighboring Moslem villages was lower,
mainly because of the smaller number of dogs kept at homes and the general
aversion in the Moslem community to any contact with dogs.

Israel's Measures to Prevent the Introduction of BSE

BSE, a fatal neurologic disease of adult cattle first discovered in the
United Kingdom in 1986, is one of the transmissible spongiform
encephalopathies caused by unconventional agents extremely resistant to
heat and chemical treatments.

Israel has adopted the following rigorous steps (taken since the very first
announcement of the new disease in the United Kingdom) to prevent the
introduction of BSE and to safeguard early detection of eventual occurrence
of the disease in the local livestock:

  1. State veterinarians, farm animal practitioners, slaughterhouse
     veterinarians, and cattle breeders associations were notified and
     updated by a) articles and updates in the monthly Veterinary Bulletins
     (more than 350 items since December, 1987; b) workshops and lectures,
     central and regional, some of them with participation of British
     experts and demonstration of British videotaped clinical BSE cases;
     circulars, including detailed descriptions of clinical BSE
     epidemiology and symptoms.
  2. The import of meat and bone meals, derived from ruminants from the
     United Kingdom was banned since December 1988, and from all other
     countries since July 1990.
  3. BSE was declared an officially notifiable disease in 1992.
  4. A pathologist from the Kimron Veterinary Institute was trained in
     contemporary diagnostic techniques at the Central Veterinary
     Laboratory, Weybridge, United Kingdom (October 1992).
  5. Since 1993, bovine brains presented to the Kimron Veterinary Institute
     after any CNS-related symptoms, are histopathologically examined for
     BSE changes. During 1993-1996, 583 brains (1993, 72; 1994, 134; 1995,
     167; 1996, 210) were examined, all found negative. Most of the brains
     were of bovines older than 2 years. This sample size exceeds the
     number of brains to be examined annually to detect BSE on a country
     level, as recently recommended by the Office International des
     Epizootics. The recommendation was to examine a minimum of 50 brains
     annually if the national cattle population, 209 months of age or
     older, is up to 500,000 and 91 brains if the cattle population is
     1,000,000. The Israeli cattle population of said age group is less
     than 250,000.
  6. In April 1996, an interministerial BSE expert advisory panel was
     established to recommend new/updated steps for BSE
     prevention/monitoring.Detailed recommendations were presented in July
     1996, most of which are already adopted.
  7. The panel paid special attention to the import conditions for meat and
     offals. This issue is related to the documented presence in Israel, of
     a considerable population-cluster that may be exceptionally vulnerable
     to transmissible spongiform encephalopathies. Accumulated scientific
     evidence indicates that this population, composed mainly of Jewish
     persons of Libyan and Tunisian origin, demonstrates a genetically
     linked susceptibility to CJD, probably related to a codon 200 mutation
     (10). This is expressed by an annual CJD incidence of nearly 100 per
     million, compared to less than 1 per million in the general population
     (E. Kahana, pers. comm.). Probable enhanced vulnerability of these
     persons, if exposed to the BSE agent, cannot be ruled out.
  8. Another recommendation referred to adding immunohistochemistry and
     immunoblotting techniques to the routinely used conservative
     histopathologic examination of BSE and further increasing the number
     of examined bovines.
  9. CJD was declared a notifiable disease in humans by the Ministry of
     Health in June 1996.
 10. Since August 1996, the feeding of farm animals, including poultry and
     fish, with meat and bone meals derived from mammals has been
     officially banned.
 11. Imported meals from poultry or fish origin are systematically examined
     at the port of entry to confirm the obtained written certification
     regarding the absence of mammalian ingredients.

The emergence of zoonotic diseases in Israel may be due to unique
epizootiologic, climatic, and ecologic conditions in the regions as well as
specific conditions within the country.

Nomadism, prevalence of disease carriers and their vectors, close proximity
of ultramodern farms housing susceptible livestock of exotic breeds or
their crosses to traditionally maintained flocks of disease-endemic breeds,
and dense overpopulated protected wildlife are the major contributing
factors to emerging zoonoses. But the cardinal factors predominant in the
Middle East are the need for improved veterinary infrastructure, direct
information exchange, and cooperation between the countries of the region.

Out of the seven diseases we reviewed, at least four, brucellosis, rabies,
leishmaniasis, and echinococcosis, can be effectively controlled only if
cooperation and animal health information systems are established on a
regional level.

The assistance of the international community to the establishment of
regional veterinary cooperation in the Middle East is of great importance.

Acknowledgements

Thanks to Dr. Van Ham of the Department of Epidemiology, Veterinary
Services and Animal Health, Beit Dagan, Israel, for the maps used in this
article.

Arnon Shimshony
Veterinary Services and Animal Health, Beit Dagan, Israel

References

  1. Slater PE, Costin C, Seidenbaum M, Ever-Hadany S. Epidemiology of
     human brucellosis in Israel. Public Health Reviews, 1992;18:159-60.
  2. Shimshony A. Activities of the Israeli Veterinary Services, 1973-1982.
     Refuah Vet 1983;40:143-203.
  3. Davidson M, Shimshony A, Adler H, Banai M, Cohen A. Protection of
     brucellosis-free areas from reinfection. In: Adams LG, editor.
     Advances in brucellosis research. College Station (TX): Texas A&M
     University Press, 1990;400-22.
  4. Hopper SA, Mawer S. Salmonella enteritidis in a commercial layer
     flock. Vet Rec 1988;123:351.
  5. Nurmi E, Nutto L, Shnetz C. The competitive exclusion concept:
     development and future. Int J Food Microbiol 1992;15:237-240.
  6. Wortabet J. An outbreak of trichinosis (?) from eating the flesh of a
     wild boar. Lancet 1881;454-5.
  7. Egyed MD, Shlosberg A, Klopfer U, Nobel TA, Mayer E. Mass outbreaks of
     botulism in ruminants associated with ingestion of feed containing
     poultry waste: I. Clinical and laboratory investigation. Refuah Vet
     1978;35:93-9.
  8. Davidson M, editor. Annual report of VSAH for 1995 (in Hebrew);
     Ministry of Agriculture and Rural Development, Beit-Dagan 50250,
     1996;114-124.
  9. Nahmias J, Goldsmith R, Schantz P, Siman M, El-On J. High prevalence
     of human hydatid disease (echinococcosis) in communities in northern
     Israel: epidemiologic studies in the town of yirka. Acta Tropica
     1991;50:1-10.
 10. Gabizon R, Kahana E, Hsiao K, Prusiner SW, Meiner Z. Inherited prion
     disease in Lybian Jews. In: Prusiner SW, et al., editors. Prion
     diseases of humans and animals. London, England: Ellis Horwood Ltd,
     1992:168-179.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

1st International Conference on Emerging Zoonoses

Electronic Media and Emerging Zoonoses

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As indicated by the term "emerging," the principal challenge in following
emerging diseases is their extremely dynamic epidemiology. Changes in these
diseases or the pathogens causing them cannot be followed through standard
texts and journals. The traditional mode of reporting an outbreak in a
peer-reviewed journal several months after it occurred or in a textbook one
or more years later is not appropriate. Weekly publications of the World
Health Organization and reports from national ministries of health are more
reliable but are still limited by delays in data acquisition, processing,
editing, and production. To avoid such delays, some weekly publications are
now being distributed electronically.

In response to the need for quick dissemination of information, several
electronic sites specifically tailored to information in the fields of
infectious diseases, emerging diseases, and zoonoses have become available.
Some have subspecialized to a single disease, animal, vector, or pathogen.
Most combine zoonotic and non-zoonotic diseases (e.g., mosquito-transmitted
diseases) or emerging and nonemerging diseases. Even though they provide
quality control, better sites urge users to verify the source of individual
reports. Moreover, as the number of electronic sites has grown, data have
become more and more diffuse, and users are unable to access and collate
materials that appear over time.

Computer programs can facilitate data access by storing and indexing
reports according to country, pathogen, vector, and animal reservoir. The
University of Tel Aviv has developed a computer software program for
information, diagnosis, and disease simulation that incorporates the status
of infectious diseases (zoonotic and others) in all countries. This program
includes information published in print and electronically. The program can
generate a ranked differential diagnosis for rash and fever following a
tick bite in Uganda or present the biology, bacteriology, epidemiology,
therapy, clinical features, historical background, and current status of
tularemia in Norway. The program--Global Infectious
Diseases and Epidemiology Network (GIDEON, CY Informatics, Ltd, Ramat
Hasharon, Israel)--also contains modules for the identification and
characterization of pathogens and the pharmacology and usage of
antiinfective drugs and vaccines. The program incorporates 319 infectious
diseases; however, even though the GIDEON program is updated through
quarterly diskettes, its information is not adequate during disease
outbreaks. In this case, therefore, electronic communication is
complementing, rather than replacing, traditional communication media.

Various communication media have advantages and deficiencies. Data
presented in a textbook, on the Internet, or in a software program may have
unlimited scope. In contrast, data presented in journals and government
reports are generally narrow, focused, and targeted to a specific
readership. Major journals and texts benefit from peer review and
documentation of sources. The availability of electronic media and software
is limited by access to hardware, user training and sophistication, and in
some situations, adequate electricity and telephone services.

Even though electronic communication has become an inevitable and
irreplaceable adjunct to the field of emerging diseases, many problems
remain in the areas of data access, credibility, standardization, and ease
of use.

Stephen A. Berger
Tel Aviv Medical Center, Tel Aviv, Israel; and the Sackler Faculty of
Medicine, University of Tel Aviv, Tel Aviv, Israel
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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Letters

The Reemergence of Aedes aegypti in Arizona

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To the Editor: Aedes aegypti, primarily an urban, tropical mosquito, is a
competent vector of dengue and yellow fever viruses. In the early 1900s,
Ae. aegypti was found in every country in the Western Hemisphere except
Canada. In the United States, repeated attempts to eradicate it have
failed, and the mosquito is now well established in the southern states,
from Texas to South Carolina, and more recently in Maryland and New Jersey
(1,2).

Although the arid landscape of southern Arizona is an unlikely habitat for
Ae. aegypti, these mosquitoes were identified in the cities of Tucson
between 1931 and 1946 (3,4) and Yuma in 1951 (5). Elsewhere in the western
United States, Ae. aegypti has been conspicuously absent, except for
periodic reports of populations in New Mexico during this same period
(3,6).

Beginning in 1969, the Arizona Department of Health Services initiated an
arbovirus surveillance program involving state and local officials in
routine monthly mosquito sampling between May and October of each year.
Until 1994, no Ae. aegypti specimens had been identified in Arizona through
routine surveillance, which involved adult collection with CO2 and New
Jersey light traps and larval dipping surveys, or through other mosquito
research (7,8).

In August 1994, a University of Arizona entomology professor reported
finding several Ae. aegypti in his Tucson backyard. Followup surveys in
September and October 1994 by state and county health officials identified
a number of Ae. aegypti in that same neighborhood as well as in central
Tucson. During September 1995, additional specimens were collected in
Nogales and again in Tucson. The adult mosquitoes were collected with CO2
traps in various locations in these two cities. In Tucson, trapping from
four of five sites yielded 85 adult Ae. aegypti (8.5/trapnight). In
Nogales, trapping from two of four sites yielded 122 adults
(12.2/trapnight). Trapping done earlier in 1995 at these sites yielded no
Ae. aegypti adults.

Between 1994 and 1995, Ae. aegypti were trapped exclusively after the
monsoon season (late July to early September), when late summer
precipitation allowed for sufficient breeding conditions in backyards.
However, in late March 1996, the Arizona Department of Health Services
responded to a report of "ankle-biting" mosquitoes in central Tucson.
Subsequently, two adult Ae. aegypti were trapped in the complainant's home.
Since then, adult Ae. aegypti have been found in several new areas in and
around Tucson (0-10/trapnight). Adult specimens have also been found for
the first time in the Arizona-Mexico border towns of Douglas (<1/trapnight)
and Naco (<1/trapnight; an additional 17 adults were collected by
aspiration).

It is not certain whether Ae. aegypti mosquitoes were newly introduced in
southern Arizona, or if they have been present at low, undetectable levels
until favorable weather conditions allowed the population to proliferate.
However, trapping data from the last three decades suggest the former.

Oviposition trapping has been the method of choice for Ae. aegypti
surveillance (9). In Arizona hay infusion-enhanced oviposition traps were
used (10). Because of the climate, initial attempts to use oviposition
traps for Ae. aegypti surveillance were unsuccessful: the containers did
not maintain enough water long enough for the hay infusion to attract
egg-laying. Future oviposition trapping attempts will use variations on
this trap, such as more infusion medium and/or larger containers, and will
focus on careful trap placement. These changes may yield a more appropriate
Ae. aegypti trap for use in the arid deserts of Arizona.

The establishment of populations of Ae. aegypti in Arizona is of particular
concern to the local health services because of the presence of more than
400 laboratory-confirmed cases of dengue fever in the bordering Mexican
state of Sonora in 1996 (R. Navarro Coronado, pers. comm.). While no cases
of endemic dengue have been reported in Arizona, two imported cases were
identified in 1994. Records show that between 1941 and 1946 nine cases of
dengue fever were reported in Arizona's residents, eight of which were from
the Tucson and Nogales areas. No exposure or travel history for those cases
is available. Imported cases of yellow fever were reported southeast of
Tucson in the late nineteenth century. The mere presence of infected
patients allowed for possible endemic disease transmission, because of the
simultaneous presence of Ae. aegypti populations. If, or when, new cases of
dengue are identified in Arizona residents, this same predicament will
again exist. Ae.aegypti surveillance throughout southern Arizona will be
expanded in the coming years, and surveillance will continue for new dengue
cases, imported or otherwise.

David M. Engelthaler, T. Michael Fink, Craig E. Levy, and Mira J. Leslie
Arizona Department of Health Services, Phoenix, Arizona, USA

References

  1. Sweeney K, Cantwell M, Dorothy J. The collection of Aedes aegypti and
     Ae. albopictus from Baltimore, Maryland. J Am Mosq Control Assoc
     1988;4:381-82.
  2. Donnelly J. Aedes aegypti in New Jersey. J Am Mosq Control Assoc
     1993;9:238.
  3. Bequaert J. Aedes aegypti, the yellow fever mosquito, in Arizona.
     Bulletin of the Brooklyn Entomological Society 1946;41:157.
  4. Murphy D. Collection records of some Arizona mosquitoes. Entomological
     News 1953;14:233-8.
  5. Richards CS, Nielsen LT, Rees DM. Mosquito records from the Great
     Basin and the drainage of the lower Colorado River. Mosquito News
     1956;16:10-6.
  6. Ferguson F, McNeel TE. The mosquitoes of New Mexico. Mosquito News
     1954;14:30-1.
  7. Smith HH, Janssen RJ, Mail GA, Wood SA. Arbovirus activity in southern
     Arizona. Am J Trop Med Hyg 1969;18:448-54.
  8. McDonald JL, Sluss TP, Lang JD, Roan CC. Mosquitoes of Arizona. Tucson
     (AZ): University of Arizona Agricultural Experiment Station; 1973
     Technical Bulletin No.: 205.
  9. Service MW. Mosquito ecology: field sampling methods. New York (NY):
     John Wiley & Sons, 1976.
 10. Rieter P, Amador M, Colon N. Enhancement of the CDC ovitrap with hay
     infusions for daily Monitoring of Aedes aegypti populations. J Am Mosq
     Control Assoc 1991;7:52-5.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Letters

Treatment of Exudative Pharyngitis

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To the Editor: The dispatch by Izurieta et al. (Emerg Infect Dis
1997;1:65-8) reporting exudative pharyngitis possibly due to
Corynebacterium pseudodiphtheriticum was very interesting, especially with
the resurgence of diphtheria in the former Soviet Union. However, I was
somewhat surprised at the treatment received by the 4-year-old patient
whose case is reported. Erythromycin is an effective antibiotic in
diphtheria, but it is secondary in importance to diphtheria antitoxin.

The presence of a thick grayish white adherent pseudomembrane, adenopathy
and cervical swelling, and low grade fever should certainly provoke a high
index of suspicion of diphtheria, especially in a child who has not
received pediatric immunization. The diagnosis of diphtheria is primarily
made presumptively on clinical grounds and confirmed by the recovery of
toxigenic Corynebacterium diphtheriae by the laboratory.

Antitoxin treatment cannot wait for laboratory confirmation. Prompt
administration of antitoxin is important because diphtheria toxin binds
rapidly and irreversibly to tissue sites. Delay in initiating antitoxin
treatment is associated with increased incidence of myocarditis, paralysis,
and death. Also, it would have been good practice to have placed this child
in isolation until the diagnosis was established by the laboratory. The
primary care physician in this case is indeed fortunate that the patient
did not have diphtheria; the results could have been tragic.

Paul D. Ellner, Ph.D.
Professor Emeritus of Microbiology and Pathology, Columbia University
College of Physicians and Surgeons


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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

Letters

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Reply to P.D. Ellner: We agree that diphtheria antitoxin should be
administered promptly on the basis of a presumptive clinical diagnosis of
respiratory diphtheria. Because laboratory confirmation may be delayed, the
decision to treat with antitoxin and the dose of antitoxin must be based on
the site and size of the diphtheritic membrane, the degree of toxicity, and
the duration of illness (1,2). The presence of a thick grayish white
adherent pseudomembrane, adenopathy and cervical swelling, and low grade
fever should certainly provoke a high index of suspicion of diptheria,
especially in a child who has not received pediatric immunization. The
diagnosis of diptheria is primarily made presumptively on clinical grounds
and confirmed by the recovery of toxigenic Corynebacterium diphtheriae by
the laboratory.

Respiratory diphtheria is rare in the United States. From 1980 to 1995,
only 41 cases were reported (zero to five cases in any given year) (3).
With this low incidence, the likelihood that a patient with membranous
pharyngitis has respiratory diphtheria is low. In addition, membranous
pharyngitis could be associated with infections by other organisms such as
streptococci, Epstein Barr virus, Candida albicans, Borrelia vincenti,
Herpes simplex virus, Arcanobacterium hemoliticum, nontoxigenic
Corynebacterium diphtheriae, and Corynebacterium pseudodiphtheriticum as in
the case we reported (4-10).

The diagnosis and clinical management of exudative pharyngitis with a
pseudomembrane in a country where diphtheria is extremely rare represent a
dilemma for the practitioner. In weighing the benefits and risks of
diphtheria antitoxin treatment, it is prudent to err on the side of using
antitoxin.

H.S. Izurieta,* P.M. Strebel,* T. Youngblood, D.G. Hollis,* and T.
Popovic*
Centers for Disease Control and Prevention, Atlanta, Georgia, USA; and
Rogers Pediatric Clinic, Rogers, Arkansas

References

  1. Farizo KM, Strebel PM, Chen RT, Kimbler A, Cleary TJ, Cochi SL. Fatal
     respiratory diphtheria due to Corynebacterium diphtheriae: case report
     and review of guidelines for management, investigation and control.
     Clin Infect Dis 1993;16:59-68.
  2. American Academy of Pediatrics. Diphtheria. In: Peter G, editor. 1994
     Red Book: Report of the Committee on Infectious Diseases. 23 ed. Elk
     Grove Village (IL): American Academy of Pediatrics, 1994:177-81.
  3. Bisgard KM, Hardy IRB, Popovic T, Strebel PM, Wharton M, Hadler SC.
     Virtual elimination of respiratory diphtheria in the United States.
     Abstracts of the 36th Interscience Conference on Antimicrobials and
     Chemotherapy; 1996 Sep 15-18; New Orleans, LA; Abstract No. K166:280.
  4. Wehrle PF. Diphtheria. In: Evans AS, Feldman HA, editors. Bacterial
     infections of humans. Epidemiology and control. New York: Plenum
     Medical Book Company, 1982:215.
  5. Diphtheria. In: Krugman S, Katz SL, Gershon AA, Wilfert CM, editors.
     Infectious diseases of children. St. Louis (MO): Mosby Year Book,
     1992:50.
  6. MacGregor RR. Corynebacterium diphtheriae. In: Mandell GL, Bennett JE,
     Dolin R, editors. Principles and practice of infectious diseases, 4th
     ed. New York: Churchill Livingstone, 1995:870.
  7. Kain KC, Noble MA, Barteluck RL, Tubbesing RH. Arcanobacterium
     hemoliticum infection: confused with scarlet fever and diphtheria. J
     Emerg Med 1991;9:33-5.
  8. Robson JMB, Harrison M, Wing LW, Taylor R. Diphtheria: may be not!
     Communicable Disease Intelligence 1996;20:64-6.
  9. Barksdale L, Garmise L, Horibata K. Virulence, toxinogeny, and
     lysogeny in Corynebacterium diphtheriae. Ann NY Acad Sci
     1960;88:1093-108.
 10. Izurieta HS, Youngblood T, Strebel PM, Hollis DG, Popovic T. Exudative
     pharyngitis possibly due to Corynebacterium pseudodiphtheriticum, a
     new challenge in the differential diagnosis of diphtheria: report of a
     case and review. Emerg Infect Dis 1997;3:65-8.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

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Meeting Summaries

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The First International Workshop on Molecular Epidemiology and Evolutionary
Genetics of Pathogenic Microorganisms

Under the auspices of the Centers for Disease Control and Prevention (CDC),
ORSTOM (the national French agency for scientific research in developing
countries), and CNRS (the national French agency for basic research), the
First International Workshop on Molecular Epidemiology and Evolutionary
Genetics of Pathogenic Microorganisms was held in Atlanta, from June 16 to
19, 1996. The workshop was cosponsored by the National Institutes of Health
(NIH), the Burroughs Wellcome Fund, the National Foundation for CDC,
Boehringer Mannheim, the French Ministry of Foreign Affairs, and Emory
University. Five hundred participants (health care providers, public health
professionals, and laboratory scientists) from 25 countries attended the
3-day workshop, whose purpose was to exchange information on the use of
molecular tools and approaches in areas of molecular epidemiology and
evolutionary genetics in studies of emerging, reemerging, and endemic
diseases. The workshop provided an opportunity for CDC, NIH, the World
Health Organization, the Walter Reed Army Institute of Research, the Kenya
Medical Research Institute, and ORSTOM to present jointly their
perspectives on meeting the challenges of emerging infectious disease.

During the workshop, public health and laboratory science-based
presentations on parasitic, fungal, bacterial, and viral diseases
identified information gaps in the areas of disease and pathogen detection;
laboratory-based presentations focused on the use of molecular tools and
approaches in pathogen identification and evolution; and other
presentations focused on specialized themes, such as the definition of a
strain, tools and approaches in molecular epidemiology, emerging
infections, concomitant infections, insect disease vectors, opportunistic
infections, and tropical parasites.

Many of the challenges of dealing with emerging and reemerging pathogens
are common to parasitologists, virologists, bacteriologists, and
mycologists. Many pathogens cannot be maintained or propagated in culture
or in animal models often because the biology and physiology of these
pathogens are not known. This difficulty highlights the advantage of moving
directly to molecular probes, polymerase chain reaction amplification, and
sequence-based identification for substantiating epidemiologic
relationships. Infectious disease clinicians and epidemiologists are faced
with whether the disease under investigation is caused by a recently
acquired infection or a recrudescent infection and whether an infection is
caused by multiple species/strains. In addition, host and pathogen genetic
factors that influence susceptibility and pathogenesis and environmental
factors that influence transmission of pathogens are critical in the
assessment of risk factors for acquiring infections. Molecular approaches
to identifying emerging, reemerging, and endemic pathogens were described
as most likely to yield the tools needed by epidemiologists to assess the
source and risk factors, thus allowing the formulation of needed prevention
and control guidelines.

Various approaches and tools now used in detecting pathogens and in
studying evolution were examined, and molecular biology and evolutionary
genetics applications in the following areas were discussed: 1) diagnosis
of known pathogens and development of rapid means to identify unknown
pathogens; 2) strain characterization for epidemiologic tracking; 3)
ecologic and biologic factors that influence emergence of pathogens; 4)
reassessment of taxonomy using molecular biologic data; 5) evaluation of
the impact of genetic diversity of microorganisms on vaccine, drug, and
insecticide efficacies; 6) gene flow in natural populations of vectors and
pathogens; and 7) the role of vectors in the evolution of pathogens.
Regardless of the organism under study, a unified approach in evolutionary
genetics and population biology was recommended.

Two other topics related to emerging infections were also examined. The
first concerned the risk for infection of human recipients of xenogeneic
agents through xenotransplantation (and the subsequent transmission of
these pathogens to the general population). The second concerned the role
of immune activation caused by chronic infections (parasitic and bacterial)
and immunization (pneumococcal and influenza) in HIV-infected persons in
promoting the replication of HIV and associated progression of disease
manifestations. The former is a concern in areas of sub-Saharan Africa and
Asia where HIV coexists with parasitic (e.g., malaria and schistosomiasis)
and bacterial (e.g., tuberculosis) infections; the latter is a concern in
the United States, where pneumococcal and influenza vaccinations are
recommended for HIV-infected persons. Field-based, prospective, and
longitudinal studies are needed for a complete picture of the extent of
interaction between vaccination and pathogen-induced immune activation, HIV
replication, and associated rapid progression to AIDS.

The need for a global partnership to facilitate a more rapid identification
of infectious agents in a manner that discriminates among closely related
strains and species and uses genetic information to study evolution,
emergence, and dispersal of infectious agents was emphasized. To address
emerging infectious disease threats, CDC has a strategic plan that
emphasizes surveillance and applied research for a strong public
health-based defense against infectious disease. A goal of this plan is the
integration of laboratory science and epidemiology to develop and use tools
to detect and promptly identify emerging and reemerging pathogens and
investigate factors that influence their emergence. To promote
international collaborations and interaction between clinicians,
epidemiologists, and laboratory scientists, CDC, ORSTOM, and CNRS will
cosponsor the 2nd International Workshop on Molecular Epidemiology and
Evolutionary Genetics of Pathogenic Microorganisms at ORSTOM, Montpellier,
France from May 26 to 28, 1997.

Altaf A. Lal* and Michel Tibayrenc
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA; Centre
d'Etudes sur de Polymorphisme des Microrganismes, UMR CNRS/ORSTOM,
Montpellier, France
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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

News and Notes

Meeting Summaries

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Simian Virus 40 (SV40), a Possible Human Polyomavirus (Workshop Held at
NIH)

During the past 4 years, polymerase chain reaction (PCR) assays have
detected DNA sequences related to SV40 (an oncogenic simian polyomavirus)
in a variety of human tissues, especially choroid plexus tumors,
ependymomas, mesotheliomas, and osteosarcomas (1-7). These findings were
supported by the isolation of infectious SV40 from a choroid plexus tumor
(8).

Although another paper reported the failure to detect SV40 DNA in
mesotheliomas (9), these studies have reawakened interest in inadvertent
human exposure to SV40 in the late 1950s and early 1960s when polio and
adenovirus vaccines prepared in rhesus monkey cells containing SV40 were
used (10,11). In response to the implications of detecting SV40 DNA in
human tumors, the Food and Drug Administration, National Institutes of
Health, National Vaccine Program Office, and Centers for Disease Control
and Prevention sponsored a workshop on SV40 on January 27-28, 1997 at the
National Institutes of Health to examine the possibility that SV40 is an
infectious agent in humans.

The workshop first reviewed the biology of SV40 and the human
polyomaviruses JC and BK and the data associating SV40 DNA with human
tumors. In addition to tumors, SV40 DNA sequences have been detected in
human pituitary gland tissue, peripheral blood mononuclear cells, and
seminal fluids from healthy persons (3,5,7). Two laboratories were unable
to detect SV40 DNA by PCR assays in human tissue, including mesothelioma;
researchers noted the ability of the PCR primers used in these assays to
amplify DNA sequences from JC and BK viruses as well as from SV40 and
discussed whether each set of primers in the PCR reaction requires specific
conditions to amplify virus-specific DNA. Furthermore, preliminary data
suggested that primers considered to be SV40-specific could, under certain
conditions, amplify what appeared to be host DNA sequences. Two
laboratories demonstrated that the sensitivity of different PCR primers to
detect SV40 DNA was 1-10 to 10-1,000 SV40 genomes. These discussions
emphasized the need for caution in interpreting PCR data and the need for
standardized, quantitative PCR assay procedures.

National Institute for Biological Standards Control scientists described
the use of PCR assays to search for SV40 DNA in current and early lots of
polio vaccines and concluded that polio vaccines used in the United Kingdom
in 1971 to 1996 did not contain SV40 DNA, while early vaccines prepared in
rhesus monkey cells contained easily detectable amounts of SV40 DNA. To
evaluate the relationship between exposure to SV40 in the early polio
vaccines and the development of tumors (choroid plexus tumors, ependymomas,
mesotheliomas, and osteosarcomas), scientists described epidemiologic
surveys that used tumor registries in two countries (two in the United
States, one in Sweden). The surveys compared tumor incidence data in
persons who could have been exposed to SV40 in polio vaccines with those
who, because of their date of birth, could not have been exposed directly;
no discernible relationship between exposure to SV40 and development of
tumors was found. The surveys also found no association between exposure to
SV40-contaminated polio vaccines and the incidence of tumors of the brain
and ovaries. These results support the findings of most of the earlier
epidemiologic studies (10,11) and help mitigate public health concerns
about the use of SV40-contaminated polio vaccines.

Whether SV40 is a human infectious agent that might play a role in human
neoplastic disease was also discussed. If SV40 DNA sequences are present in
choroid plexus tumors in children born many years after vaccines were SV40
free (8), the possibility that SV40 is present in the population must be
considered. Researchers reviewed data on SV40 antibodies in sera taken
before 1954 (12) and in sera from persons in remote regions (13,14) not
exposed to SV40-contaminated polio vaccines; the data suggest that SV40
might have been present in humans before the polio vaccines were introduced
in 1954 (11). Because of crossreactivity between BK, JC, and SV40
antibodies (15) and the lack of standardized serologic assays to identify
SV40 specificity of antibodies present in single samples of human serum, it
is difficult to determine whether SV40 was present in humans before the
population was exposed to SV40 in the early polio and adenovirus vaccines.
Thus, determining whether SV40 is an infectious agent in humans and whether
humans were exposed before the polio vaccine was introduced requires
further study.

Two preliminary studies showed that SV40 T proteins are expressed in some
cells in mesotheliomas and these proteins can bind to both the p53 and Rb
cell-cycle control proteins. The SV40 T protein-p53 and SV40 T protein-RB
protein interactions are thought to contribute to neo-plastic
transformation; however, the role of these interactions in SV40-induced
neoplastic transformation is unresolved. Further attempts to assess the
link between SV40 DNA sequences and neoplastic processes in humans will
require more conclusive data about whether SV40 DNA sequences are present
in tumors, whether this viral DNA is integrated or extrachromosomal, and
whether it is expressed. Two new experimental approaches were suggested to
assess the ability of SV40 to contribute to neoplastic development in
humans: prospective studies on the presence of SV40 in mothers and other
family members of children with choroid plexus tumors and studies comparing
the function of what may be an SV40-associated defect in the
p53-independent cell-cycle control gene SEN6 in SV40-transformed human cell
with cells from tumors containing SV40 DNA sequences.

The sponsors of the workshop were reassured by the independent
epidemiologic surveys in the United States and Sweden that the incidence of
neoplastic diseases in persons exposed to SV40 in viral vaccines has not
increased. However, the sponsors should take the following steps to resolve
questions raised by human exposure to SV40 in viral vaccines prepared in
rhesus monkey cells in the 1950s and 1960s: 1) Form working groups to a)
analyze the sensitivity and specificity of PCR reactions for detecting SV40
DNA in human tissues and develop standardized conditions to ensure
confidence in the data generated by such reactions; b) develop methods for
assessing the specificity of human polyomavirus neutralizing antibodies in
plaque neutralization assays and consider other assays that can measure
antibodies to virus-specific epitopes on the virions of polyomavirus; and
c) develop ways to search for SV40 in the environment. 2) Encourage
additional attempts to isolate SV40 from human tissues and increase the
number of completely sequenced SV40 chromosomes obtained from SV40 field
isolates. 3) Develop standardized reagents and make them available to
laboratories who wish to assess the sensitivity and reliability of their
PCR assay for detecting SV40 DNA. 4) Identify reagents such as archived
tumor specimens, serum specimens and databases useful for epidemiologic
evaluations, and any other specimens critical for evaluating when SV40 or
SV40-like viruses entered the population.

For information about transcripts and audio and video recordings of the
workshop, contact the Food and Drug Administration Freedom of Information
Staff HFI-35, Rm. 12A-16, 5600 Fishers Lane, Rockville, MD 20857; phone:
310-443-1813. The proceedings of the workshop will be published in
Developments in Biological Standardization.

References

  1. Bersagel DJ, Finegold MJ, Butel J, Kupsky WJ, Garca R. DNA sequences
     similar to those of simian virus 40 in ependymomas and choroid plexus
     tumors of childhood. N Eng J Med 1992;326:988-93.
  2. Carbone M, Pass HI, Rizzo P, Marinetti M, Di Muzio M, Mew DJY, et al.
     Simian virus 40-like DNA sequences in human pleural mesothelioma.
     Oncogene 1994;9:1781-90.
  3. Woloschak M, Aiqin Y, Kalmon DP. Detection of polyomaviral DNA
     sequences in normal and adenomatous human pituitary tissues using the
     polymerase chain reaction. Cancer 1995;76:490-6.
  4. Cristaudo A, Vivaldi A, Sensales G, Guglielmi G, Ciancia E, Elisei R,
     et al. Molecular biology studies on mesotheliomas tumor samples:
     preliminary data on H-Ras, p21 and SV40. J Environ Pathol Toxicol
     Oncol 1995;14:29-34.
  5. Carbone M, Rizzo P, Procopio A, Guiliano M, Pass HI, Gebhardt MC, et
     al. SV40-like sequences in human bone tumors. Oncogene 1996;13:527-35.
  6. Pepper C, Jasani B, Navabi H, Wynfodr-Thomas D, Gibbs A. Simian virus
     40 large T antigen (SV40LTAg) primer specific DNA amplification in
     human pleural mesotheliomas tissue. Thorax 1996;51:1074-6.
  7. Martini F, Iaccheri L, Lazzerin L, Carinci P, Corallini A, Gerosa M,
     et al. SV40 early region and large T antigen in human brain tumors,
     peripheral blood cells and sperm fluids from healthy individuals.
     Cancer Res 1996;56:4820-5.
  8. Lednecky JA, Garcea RL, Bergsagel DJ, Butel J. Natural simian virus 40
     strains are present in human choroid plexus and ependymoma tumors.
     Virology 1995;212:710-7.
  9. Strickler H, Goedert JJ, Flemming M, Travis WD, Williams AE, Rabkin
     CS, et al. Simian virus 40 and pleural mesothelioma in humans. Cancer
     Epidemiology, Biomarkers and Prevention 1996;5:473-5.
 10. Lewis AM Jr. Experience with SV40 and adenovirus-SV40 hybrids. In:
     Hellman A, Oxman MN, Pollock R, editors. Biohazards in Biological
     Research. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory,
     1973:96-113.
 11. Shah K, Nathanson N. Human exposure to SV40: review and comment. Am J
     Epidemiol 1976;103:1-12.
 12. Geissler E, Konzer P, Scherneck S, Zimmerman W. Sera collected before
     introduction of contaminated polio vaccine contain antibodies against
     SV40. Acta Virol 1985;29:420-3.
 13. Brown P, Tsai T, Gajdusek C. Seroepidemiology of human papovaviruses.
     Discovery of virgin populations and some unusual patterns of antibody
     prevalence among remote peoples of the world. Am J Epidemiol
     1975;102:331-40.
 14. Brown P, Morris JA. Serologic response to BK virus following human
     infection with SV40. Proc Soc Exp Biol Med 1976;152:130-1.
 15. Penny JB, Narayan O. Studies of the antigenic relationships of the new
     human papovaviruses by electron microscopy agglutination. Infect Immun
     1973;8:299-300.

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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

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Meeting Summaries

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Conference on Foodborne Pathogens: Implications and Control

More than 400 food protection and public health professionals from 18
countries, including microbiologists, epidemiologists, physicians, and
health policy makers in industry, academia, and government, attended the
Conference on Foodborne Pathogens: Implications and Control. The conference
participants examined the response of the food industry and its related
public health/food safety regulatory agencies to the emergence of new
microbiologic threats and to the reemergence of known pathogens in
previously unimplicated foods. The 3-day conference was held in Alexandria,
Virginia, USA, March 24-26, 1997. It was organized by the International
Life Sciences Institute North American (ILSI N.A.), the Centers for Disease
Control and Prevention, the U.S. Department of Agriculture, and the U.S.
Food and Drug Administration, in cooperation with the Food and Agriculture
Organization and the Pan American Health Organization.

The specific goals of the conference were to identify factors that foster
the emergence/reemergence and dissemination of foodborne microbial hazards,
explore scientific and food safety strategies to identify and address these
hazards, determine future research needs, and review the lessons learned
and knowledge gained concerning the emergence and dissemination of
food-related microbial threats to health.

The rapid emergence and dissemination of microbial foodborne pathogens and
human diseases is affected by factors related to the pathogens themselves,
their hosts, and the food production and consumption environment. The
conference explored the role of the rapid mutation of foodborne pathogens
such as Escherichia and Salmonella; the increasing numbers of susceptible
persons; the effect of current livestock production practices, produce
handling and food processing practices, and aquaculture; and changes in
consumer lifestyles and food preferences.

Identifying and anticipating new foodborne microbial hazards require
concerted efforts. The changing epidemiology of foodborne disease calls for
improved surveillance including rapid sub-typing methods, cluster
identification, and collaborative epidemiologic investigation (including
case-control studies). Also examined was the need for better integrated,
coordinated, and standardized animal disease surveillance and health
monitoring programs. Several speakers stressed the importance of risk
assessment (a component of overall risk analysis that combines science and
policy) as a decision support tool and the need to effectively communicate
risk to consumers. Because microbes do not respect national borders, they
need to be addressed at a global level through strengthened infrastructure
and standardized trade that will ensure the health of consumers.

The new problems of foodborne disease require new control and prevention
strategies (as well as further research) to ensure that food in both
domestic and international trade is safe. The development of the Hazard
Analysis Critical Control Point (HACCP) process was presented as a first
step toward an analytic process for identifying hazards and their points of
control. Other research needs in the area of foodborne pathogen control
were also examined. Topics included a need for multidisciplinary teams that
can provide "just in time" research; for basic research to explain factors
associated with food production and processing that contribute to new
foodborne microbial threats; for prompt evaluation and implementation of
innovative preservation methods (e.g., food irradiation) to meet consumer
demand for fresh foods; for a centralized system accessible electronically,
with information on pathogenic organisms in a standardized format; for the
use of emerging molecular methods (e.g., DNA hybridization and polymerase
chain reaction) to examine emerging viral and parasitic foodborne disease
organisms; and for models to predict the probability of a particular
microbial event (e.g., growth and death), which may be useful in the design
of HACCP programs and in defining processes, formulations, and storage
conditions to yield foods with acceptable shelf life and safety
characteristics.

Lessons learned from outbreaks in the last 15 years contribute to
developing strategies for the mobilization of resources to respond rapidly
to emerging foodborne microbial hazards. Retrospective analyses of data
from cases of E. coli O157 infections identified risk factors, variations
in treatment, and estimates of the incidence of hemolytic uremic syndrome.
Unusual foods have been associated with outbreaks of Clostridium botulinum,
including potatoes baked in aluminum foil, bean dip, cheese sauce, and
mascarpone cheese; nontoxigenic clostridia could emerge as a new pathogen
with the transfer of botulism toxin genes. The multistate outbreak of
Salmonella serotype enteritidis underscored the value of molecular
subtyping and public health action based on epidemiologic data in
identifying outbreak cases when dispersed in a larger group of unrelated
infections. Finally, epidemiologic data were presented from a multistate
outbreak of Cyclospora infection associated with consumption of raspberries
from Guatemala. These examples emphasized that the future of foodborne
disease epidemiology will involve new technology and greater coordination
among local, state, and federal public health and regulatory agencies.

Papers from this conference will be published in the Emerging Infectious
Diseases journal.
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Meeting Summaries

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International Conference on Emerging Infectious Diseases in the Pacific
Rim, Bangkok, Thailand

Approximately 200 participants gathered in Bangkok, Thailand, March 6-8,
1997, to discuss issues related to emerging infectious diseases in the
Pacific Rim. The meeting was organized under the auspices of the U.S.-Japan
Cooperative Medical Science Program. Scientists from the United States,
Japan, the host country, and 15 other nations of the region, as well as
from the World Health Organization (WHO) attended. The meeting focused on
research topics relevant to emerging diseases and discussed surveillance
and disease prevention. Formal presentations focused on themes of special
interest to the region: enterohemorrhagic Escherichia coli (EHEC), dengue
and dengue hemorrhagic fever (DHF), and the growing problem of
antimicrobial resistance. Summaries of the WHO global and regional plans to
address emerging infectious diseases were presented along with summaries
from participating countries of their national plans and problems relevant
to these diseases.

The session on EHEC included presentations on the status of this important
pathogen in the United States, Japan, Australia, and Thailand, as well as a
summary of recent efforts to develop better strategies to detect, treat,
and prevent EHEC illness. Presentations on dengue included a discussion of
atypical infections and brief mention of new results regarding the
stability of dried whole blood samples for serologic examination and the
use of insecticide-impregnated screens to control vector mosquitoes; the
prospective clinical study of DHF under way in Bangkok, carried out as a
multicenter collaborative study involving Thai, United States, and Japanese
scientists, was described. Presentations on nosocomial and
community-acquired resistant infections, acute respiratory infections, and
tuberculosis underlined the growing problem of antimicrobial resistance.

Several themes emerged from country reports: the growing importance of
dengue fever/DHF and Japanese encephalitis in many countries of the region;
increasing problems with diarrheal diseases and other food or waterborne
diseases, including cholera; antimicrobial resistance and the need for
assistance in laboratory culturing and sensitivity testing; the need for
regional surveillance to better define the current patterns of
antimicrobial resistance and for the establishment of regional quality
control and proficiency testing as one aspect of the regional response;
frustration with existing surveillance systems and need for assistance in
developing improved surveillance tools and easier information sharing; the
need for improved laboratory support, especially the regional availability
of high quality diagnostic reagents and development of regional reference
facilities; and the desire for a regional approach to addressing emerging
infectious diseases.

The meeting concluded with recognition of the need for both greater
research in the areas of the epidemiology, diagnosis, treatment, and
prevention of EHEC and other Shiga-toxin producing organisms; further
studies on DHF, including pathogenesis, clinical intervention, viral
genetic variability, and genomic analysis; vaccine development; and
improved vector control and fundamental strengthening of public health
practices to address emerging infectious diseases including improved
laboratory capacity, better surveillance programs, easier and more open
communications and information sharing, and assistance in outbreak
responses. Participants highlighted the need for greater training
opportunities for scientists of the region and for development of regional
reference facilities and centers of excellence. The meeting did not cover
human immunodeficiency virus and AIDS, although there was clear recognition
of its importance within the region.

James W. LeDuc
Centers for Disease Control and Prevention,
Atlanta, Georgia, USA
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International Conference on Emerging Zoonotic Infectious Diseases, Taipei,
Taiwan

The International Conference on Emerging Zoonotic Infectious Diseases,
cosponsored by the Taiwan Departments of Health and Defense and the Centers
for Disease Control and Prevention, was held March 1-4, 1997 in Taipei,
Taiwan. The conference brought together scientists from Australia, France,
the United States, and Taiwan and highlighted local work on dengue,
Japanese encephalitis, plague, and rodentborne hantaviral infections.

The opening session outlined current efforts in the United States and
internationally to improve and coordinate surveillance, laboratory
diagnosis, and research of emerging infectious diseases. An example of a
disease (yellow fever) whose threat has not been realized was described and
reassessed in the context of globalization and other factors favoring and
mitigating against the virus' dissemination. Although the possibility of
epidemic yellow fever in Asia is small, it is important to reduce the
disease at its sources in Africa and South America to further minimize this
possibility. Ongoing efforts to elucidate the pathogenesis of dengue
hemorrhagic fever, a growing problem in Taiwan and a leading cause of
childhood illness and death in Asia and the tropics were summarized. Recent
studies in Thai children have defined early clinical immunologic markers
that differentiate febrile patients who contract dengue hemorrhagic fever
from those with self-limited dengue fever; these findings suggest potential
approaches to early recognition and specific intervention.

A session on viral hemorrhagic fevers reviewed recent Ebola virus outbreaks
and the discovery of a rapidly growing number of arena-and hantaviruses,
their phylogeny and associations, and their specific rodent hosts. The
virtual explosion of viruses identified in rodent reservoirs has left
studies of their biologic, clinical, and epidemiologic correlates lagging;
many of the newly discovered agents are orphan viruses. A report of local
rodent surveys showed the presence of several hantaviruses in numerous
species in Taiwan; human disease has not been recognized but epidemiologic
studies are planned to define the spectrum and incidence of human
infection. Approaches toward producing recombinant hantavirus vaccines and
efforts to produce naked DNA vaccines for related vectorborne infections
were reviewed.

Summaries of the recent emergence of dengue and dengue hemorrhagic fever
globally and on Taiwan led to a series of talks on dengue vaccine
development. Various approaches were discussed, including candidate live
attenuated vaccines, purified inactivated and recombinant subunit antigens,
and infectious clone-derived viruses and their engineered chimeras. A
similar session focused on Japanese encephalitis (JE), its changing ecology
and epidemiology on Taiwan and regionally in Australia, the molecular
taxonomy of JE viruses, and recent developments in producing much needed
rapid diagnostic kits.

The cellular and molecular basis of JE pathogenesis was addressed in a
series of reports on the protective role of bcl-2 in viral-induced
apoptotic death, viral inhibitory activity of cell derived NO2, and viral
genetic determinants of virulence and attenuation. Alternatives to the only
internationally accepted JE vaccine, the relatively reactogenic and
expensive inactivated mouse brain-derived vaccine, were discussed,
including the live-attenuated SA14-14-2 vaccine produced in China, a Vero
cell-derived inactivated vaccine under development in Taiwan, and a
chimeric JE vaccine engineered upon a yellow fever 17D virus infectious
clone.

The final session concerned plague; it described the history and current
status of plague globally and on Taiwan; reviewed new developments in the
molecular taxonomy of Yersinia pestis; compared the performance
characteristics of various serologic and PCR-based diagnostic tests; and
described plague pathogenesis and vaccine development. F1 and V antigens
were defined as important virulence factors in mouse and primate parenteral
and aerosol challenge models. Preliminary studies indicate their promise as
constituents of a recombinant subunit vaccine.

Theodore Tsai
Centers for Disease Control and Prevention
Ft. Collins, Colorado, USA
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News and Notes

The 4th International Conference on Hantaviruses, Atlanta, Georgia
March 5-7, 1998

The Centers for Disease Control and Prevention in Atlanta and cosponsors
will host the 4th International Conference on Hantaviruses to allow
exchange of scientific information on hantaviruses in the areas of
epidemiology, clinical management, ecology, molecular biology, laboratory
diagnostics, pathogenesis, drugs, and vaccine development.

The meeting will host plenary sessions with invited speakers as well as
oral and poster sessions based on accepted abstracts.

Deadline for abstract submission is October 31, 1997. For more information,
call 404-639-1510.
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News and Notes

International Conference on Emerging Infectious Diseases, Atlanta, Georgia,
March 8-12, 1998

Preliminary Information and Call for Abstracts

The Centers for Disease Control and Prevention (and other cosponsors) will
convene a conference to 1) encourage the exchange of scientific and public
health information on global emerging infectious disease issues, 2)
highlight programs and activities that address emerging infectious disease
threats, 3) identify program gaps, 4) increase emerging infectious disease
awareness in the public health and scientific communities, and 5) enhance
partnerships in addressing emerging infectious diseases.

The meeting will host plenary sessions and symposia with invited speakers
as well as oral and poster sessions based on accepted abstracts. Major
topics will include current work on the surveillance, epidemiology,
research, and prevention of emerging infectious diseases as well as on
emergency preparedness and response. Abstracts should address new,
reemerging, or drug resistant infectious diseases that affect human health,
e.g., foodborne, tropical, sexually transmitted, and respiratory diseases;
diseases transmitted by animals and arthropods or acquired in health care
settings; diseases in infants and children, immunodeficient persons, and
minority and other populations at risk; and diseases related to blood
safety and xenotransplantation.

Conference attendance will be limited to 2,500 participants. Deadline for
abstract submission is October 31, 1997. Proceedings of the conference will
be published in the Emerging Infectious Diseases journal.

For additional information on registration and abstract submission go to
http://www.cdc.gov/ncidod/ncid.htm or call 202-942-9248.
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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

News and Notes

Emerging Infectious Diseases Laboratory Fellowship Program: Recruitment
Begins for a Third Class of Fellows

The Emerging Infectious Diseases Advanced Laboratory Training Fellowship, a
1-year program designed for bachelors or master level scientists,
emphasizes the practical application of technologies, methods, and
practices related to emerging infectious diseases. Fellows participate in a
core curriculum session at the Centers for Disease Control and Prevention
(CDC)/Atlanta to gain a general understanding of the public health
laboratory system and how it relates to infectious disease surveillance,
prevention, research, and control. Fellows are placed within federal and
state public health laboratories to receive advanced infectious disease
laboratory-related training tailored to each fellow's areas of interest,
high-priority laboratory personnel needs, and host laboratory capabilities.

The Emerging Infectious Diseases Post-Doctoral Laboratory Research
Fellowship, a 2-year program designed for doctoral level (Ph.D., M.D.,
D.V.M.) scientists, awards fellowships for the conduct of research or
development in infectious diseases areas relevant to public health. This
program's fellows also participate in the core curriculum session at
CDC/Atlanta and are then placed within federal and state public health
laboratories to conduct approved research.

For further information and application materials, contact EID Laboratory
Fellowship Program, ASTPHLD, 1211 Connecticut Avenue, N.W., Suite 608,
Washington, D.C. 20036, phone: 202-822-5227, fax: 202-887-5098. Fellowship
application deadline: June 12, 1997.
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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

News and Notes

Emerging Infections: Clinical and Pathologic Update II

This year, the Armed Forces Institute of Pathology course on infectious
disease (November 8-11, 1997) will be held in collaboration with Emory
University School of Medicine and the Centers for Disease Control and
Prevention. The course, which will be held at the Emory University
Conference Center, will be directed by Drs. Ann Marie Nelson and C. Robert
Horsburgh, Jr. and will focus on newly emerging and reemerging diseases
(including yellow fever, dengue hemorrhagic fever, leptospirosis, AIDS,
bovine spongiform encephalopathy, cholera, diphtheria, tuberculosis,
Mycobacterium avium complex, chancroid, meningitis, Escherichia coli
O157:H7, fungal infections, malaria, babebiosis, filariasis, and emerging
infections in captive wildlife). The course will also cover antibiotic
resistance and the role of zoonotic infections and will feature a
roundtable discussion of emerging infectious disease issues. The
epidemiology and clinical features, as well as the pathology and
pathogenesis of each disease, will be presented by experts in emerging
infectious disease. An optional slide review session of 10 hours is
available the day following the lecture series. (Approximately 30 CME
credits)

For further information, contact the Department of Education Services, 14th
and Alaska Ave., NW, Washington D.C. 20306-6000; telephone (202) 782-5021,
toll free (800) 577-3749 (U.S. only); fax (202) 782-7164 DSN 662-5021.
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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

News and Notes

Rabies Conference

The 8th Annual Rabies Conference in the Americas will take place in
Kingston, Ontario, Canada, November 2-6, 1997. For more information,
contact Dr. Christopher Nunan, Ontario Ministry of Natural Resources, 300
Water Street, P.O. Box 7000, Peterbourough, Ontario, Canada, K9J8M5;
telephone (705) 775-1554; e-mail: nunanc@gov.on.ca or Dr. Rolly Tinline,
Director, GIS Center, Queens University, Kingston, Ontario, Canada, K7L3N6;
telephone (613) 545-6039; e-mail: tinliner@qucdn.queensu.ca.
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   [Emerging Infectious Diseases * Volume 3 * Number 2 * April-June 1997]

News and Notes

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 Erratum: Vol. 3, No. 1 In the article "Exudative Pharyngitis Possibly
 Due to Corynebacterium pseudodiptheriticum, a New Challenge in the
 Differential Diagnosis of Diphtheria," by H.S. Izurieta, P.M. Strebel,
 T. Youngblood, D.G. Hollis, and T. Popovic on page 68, the following
 were omitted from the list of references:

 45. Feery BJ, Forsell P, Gulasekharam J. Streptococcal sore throat in
 general practicea controlled study. Med J Aust 1976;1:898-91.

 46. Clarridge JE. When, why and how far should coryneforms be
 identified? Clin Microbiol Newslett 1986;8:32-4.

 We apologize to our readers for this error.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: ftp://ftp.cdc.gov/pub/EID/vol3no2/vol3no2.txt
Updated: 97/05/27