−層■王

第25巻第2号平成9年6月
内総説
麻疹根絶に向けての戦略

容一一英語圏カリブ海諸国における進展一（英文）
・大山

原

卓昭

49−57

著
妊娠マウスを用いた先天性トキソプラズマ症モデルにおけるRT−PCRによるサイトカイン産生の解析（英文）一何
関谷

インドネシアのスマトラで採集されたブユ（双翅目梛，青才 文江，文宗英，矢野 明彦

ブユ科）3新種の記載（英文）・高岡 宏行，Singgih

恵聖，
59−67

H．Sigit69−80

ネパール国カトマンズ市内の附属大学病院における鉤虫感染状況
一10年間の記録一（英文）・・Shiba

久保

Kumar

Rai，Hari隆，小野Govinda一男，宇賀Shrestha，中西

昭二，松村

守，

武男

81−84

最近ラオスで分離したコレラ菌の性状（英文）一ClaudiaToma，Lay

Sisavath，比嘉

直美，岩永

正明

85−87

ラオスにおける下痢症の原因菌分布およぴ健康者保菌率（英文）一Sithat

Lay

Insisiengmay，Khampheuy

Sisavath，Boumang

比嘉

直美，山城

会報・記録
1997年度（平成9年）第1回理事会記録・・
日本医学会だより………一………・………
1997年度役員名簿…一…………・・………
日本医学会への加盟申請についての公示

Munnalath，Noikaseumsy

Sithivong，

Pathouamas，Khampheng

Chomlasak，

哲，仲宗根

正明・

昇，岩永

89−93

…

一g5

−96−97−98−101

49

Jpn. J. Tro p. Med. Hyg., Vol. 25, No. 2, 1997, pp. 49 57

Review

STRATEGY TOWARDS MEASLESELIMINATIONPROGRESS IN THE ENGLISH‑SPEAKINGCARIBBEAN COUNTRIESTAKAAKI OHYAMA
Received August 2, 1996/Accepted May 6, 1997

Abstract: The strategy currently used to control and eliminate measles was reviewed on the basis of theachievement in Americas, especially in the English‑speaking Caribbean countries. The author participatedin one phase of the strategy; measles follow‑up immunization campaign in Belize, and reviewed theachievement of the carnpaign. Several factors were identified as important factors for the success of mass
irnmunization campaign. According to the review and experience of the strategy and its practice for measles
elirnination in the Americas, the author discussed the importance of political commitment, enthusiastic
community's participation and establishment of effective surveillance system for the success of measles
elirnination program. Although there are still several obstacles for the development of the measles elimina‑tion program to other regions, the most important components are the strong political commitment in eachcountry and international cooperation financially and technically. On this point of view, Japan is able toplay an important roll towards measles elimination in Asia.

Key words: measles, irnmunization, elimination, Pan American Health Organization, English‑speaking
Caribbean countries

INTRODUCTION

rently in use are attenuated live measles virus, and many

countries prefer the combined MMR (measles‑mumps‑

There is a new, Iarge scale measles control program

rubella) vaccine to ensure that immunity is obtained forof Pan American Health Organization (PAHO) /World

all three viruses (Cutts and Markowitz, 1994).
Despite the availability of measles vaccine for more

Health Organization (WHO) , which is intended to elimi‑
nate the circulation of measles virus in the Americas.
Although the program has been functioning successfully

since 1991, there is much to learn about the program
through evaluation and assessment. The author par‑ticipated in one of the ongoing programs, and had theopportunity to review and evaluate one phase of it; themeasles follow‑up immunization campaign in Belize. In
this paper, the author reviewed the strategy and its

practice, and considered its development in otherregions .

1. Public health importance of measles
Although measles is a ubiquitous and highly infec‑
tious compared with other infectious diseases such asvaricella and mumps, there is a strong preventive
measure against measles; vaccine. The vaccines cur‑than thirty years, WHO estirnates that more than one
million children worldwide die each year due to measles

and its complications (EPI/WHO, 1994). Moreover,sequelae from measles, possibly related to nutritional
effects and altered immune status, may be expressed formany months after infection. Thus, the true number
dying annually as a result of measles may be twice theestimated one million if the delayed effects of the dis‑ease are taken into account (Gellin and Katz, 1994).
Therefore, measles ranks as a leading cause of child‑hood mortality in the world, and the impact qf measles
on the lives of millions of young children every year,
particularly in developing countries, indicates the urgent

need for further reductions in measles incidence (Clem‑
ents et al.. 1992). In Septernber 1994, the Pan American

Sanitary Conference decided that measles virus should

Department of Parasitology, School of Medicine Kanazawa University, 13‑1 Takara‑machi, Kanagawa 920, Japan50

no longer be a threat to the health of infants and chil‑dren of the Western Hemisphere and voted unanimously
to establish the goal of measles elimination from thechildren from one to five years of age (follow‑up imluu‑
nization campaigns) are conducted in order to decrease

the accumulated susceptibles to measles. They are

Americas by the year 2000 (PAHO/WHO, 1994).

conducted whenever estimates of the number of suscep‑tibles frorn one to five years of age approaches the

2. Strategies towards measles elimination in Americas

number of an average birth cohort. This arbitrary

The program for measles elimination relied oninterval between campaigns is empirically best and will
be evaluated as the prograrn progresses. A surveillanceexperiences gained during two successful eradication
programs which were conducted worldwide during thepast thirty years; smallpox and poliomyelitis. These
two target diseases as well as measles have common
characteristics which imply that eradication may befeasible; 1) man is the only host of the virus, 2) inexpen‑

sive vaccine provides long‑lasting immunity among
vaccinees. In the smallpox eradication campaign, themajor strategies were surveillance and containment,

system for measles (rash and fever illness surveil‑
lance) is being established and investigation and control

measures for each reported outbreak are being conduct‑ed (outbreak response and prevention) (de Quadros etal.. 1996). These strategies have been implemented
successfully in the English‑speaking Caribbean coun‑tries since 1991, and, so far, they have achieved awhich included surveillance for the detection of small‑measles‑free status, which means there is no indigenous
measles in this area, for the first time in this region's

pox cases and tracing of the source of infection andhistory.

chains of transmission, with vaccination targeted to allcontacts in the infected areas. These strategies wereeffective and led to the ultimate worldwide eradicationof smallpox, in part because smallpox virus spreads
more slowly than measles virus. In the poliomyelitis

3. Achievement in the English‑speaking Caribbean

eradication program, country‑wide immunization wasachieved by national immunization days in order toquickly increase population immunity levels and towas "Measles Immunization Month" during which themeasles catch‑up immunization campaign was conduct‑countries
The English‑speaking Caribbean countries initiatedthe first stage of measles elimination in May 1991. May

interrupt chains of poliovirus transmission. This strat‑

ed in the eighteen countries in order to vaccinate all
children between nine months and fourteen years of age,

egy was supplemented by house‑to‑house vaccination inregardless of their previous history of disease and immu‑

areas with low immunization coverage (mop‑up immu‑

nization. The target population for this campaign was
about 1,775,000 children. The achievement was remark‑ably successful with 91.4% overall coverage. Mop‑up
measles immunization activities were continued throughnization activity) . Since more than 95% of polio virus

infections are inapparent or induce nonspecific symp‑

toms, a containment of outbreaks was unable to be
conducted as was possible with smallpox. Nevertheless,a surveillance system for the detection of suspected
polio cases and a laboratory network were created toconfirm the presence of wild virus. These were impor‑tant in order to monitor progress of the program (deQuadros et al., 1996).
The strategy for measles elimination which is cur‑rently being implemented in the Americas has several
stages. First, the transmission of measles virus is inter‑

rupted by vaccinating all children between nine months
and fourteen years of age in a short period (catch‑up

June and July of 1991, and considerable numbers ofchildren who were missed during May were immunized.
The measles catch‑up immunization campaign tointerrupt measles virus transmission was an importantfirst step in the strategy of measles elimination. Veryhigh levels of immunization coverage have been
maintained by scheduling periodically an "immunization

month", during which mop‑up immunization activities
are conducted in low immunization coverage areas byimproving access to vaccination services, including thetion activity (mop‑up immunization) is then carried out

use of mobile immunization teams for hard‑to‑reachareas. As a result, an overall immunization coveragelevel of over 80% has been maintained in almost all

in low coverage areas. Third, efforts are made tocountries since the measles catch‑up immunization cam‑sustain high immunization coverage by routine immuni‑
zation activities, which are incorporated in the publicpaign in 1991.

immunization campaign). Second, a special vaccina‑

health services, through irnproved access to vaccination

services including mobile house‑to‑house vaccination
services. Fourth, periodic immunization campaigns for

After the campaign, surveillance systems were irn‑
plemented which called for a weekly report of suspected
measles cases (rash and fever illness) . This system wasintended to detect all suspected measles cases, which

51

were defined as either 1) any case which a trained
health care worker suspects to be measles, or 2) any
case with macropapular rash, fever, and one of thefollowing: cough, coryza, or conjunctivitis. Each

unique characteristics of Belize is its variety of ethnic

groups: Mestizo, Creole, Maya, Carifuna, Ketchi, Men‑nonite, and so on (UNICEF, 1995) . As a result, there areseveral difficulties in conducting public health activities,

suspected measles case is supposed to be investigatedbecause some of them use their own language and areusing serological diagnosis, and either classified as "con‑geographically isolated from others.

firmed" or discarded if another diaguosrs rs made. If
confirmed, appropriate control measures are carriedout. Over five hundred reporting sites are incorporated
in this surveillance system. The sites include persons
and institutions which are likely to see cases of measles.

2. Measles control in Belize
Belize had been trying to intensify its immunization

programme in order to achieve an immunization cover‑age of at least 80% among children under one year of

These comprise health centers, hospitals, private physi‑

age against the WHO's Expanded Programme on Immu‑

cians, etc. About 85% of them report every week.
Between January and May of 1991, prior to the cam‑paign, 301 cases of measles were reported from theEnglish‑speaking Caribbean countries. From June

nization (EPD target diseases (diphtheria, pertussis,
tetanus, tuberculosis, poliomyelitis, and measles).

onwards, reports of measles cases rapidly declined.
During September and October of 1991, 105 suspected
measles cases were reported, but only two cases wereconfirmed as measles; one was an imported case, theother was lost to follow‑up. Many of the suspected

the coverage with diphtheria‑tetanus‑pertussis vaccine(DTP) , oral polio vaccine (OPV) and measles vaccine

Although immunization coverage with BacilleCalmette‑Guerin vaccine (BCG) had reached over 80%,

continued to be unsatisfactory (40‑60%). In 1986,
Belize, in collaboration with PAHO, United Nations
Children's Fund (UNICEF) , and Rotary International,

measles cases were diagnosed as dengue fever or rubella(Hospedales, 1992). However, from 1992 until the end ofcoverage with BCG. DTP, OPV and measles vaccine.

1995, there were no confirmed measles cases (PAHO/
WHO, 1995). Thus, measles transmission‑appears to

As a result, Belize achieved a coverage of over 80%
immunization for all the EPI target diseases among

have been interrupted in these countries.

children under one year of age (UNICEF, 1995) . In May

MEASLES ELIMlNATION IN BELIZE
The author had a chance to participate in themeasles follow‑up immunization campaign in Belize,

decided to initiate a national EPI campaign to increase

1991, the measles catch‑up immunization campaign,
which covered all children from nine months throughfourteen years of age, was implemented in the effort toand evaluated it using questionnaires and interviews toeliminate measles from the Americas. During the cam‑paign, 67,200 children (84% of the target group) were
vaccinated with measles vaccine. Since then, the immu‑

public health nurses in each district. Through thisnization coverage with BCG, DTP, OPV and measles

process, several factors were identified as critical points

vaccine have been sustained at a high level (over 80%) ,for the success of mass immunization campaign.

and there have been no reported measles cases in Belize
since 1992 (Ministry of Health, Belize, 1991‑1995).

1. Background of Belize
Belize, one of the member countries of the English‑
speaking Caribbean nations, is a small country, Iocatedon the eastern seaboard of the Central American isth‑mus. It is bordered in the north by Mexico and in thesouth and west by Guatemala. It has a sparse but
extremely diverse population. The estimated total
population in 1993 was 205,000; alrnost 45% of the
population is under the age of fifteen years (Central
Statistic Office, Ministry of Finance, Belize, 1994).
There are six administrative districts: Corozal. Orange
Walk, Belize. Cayo, Stann Creek, and Toledo. In recent
years, Belize has experienced high levels of irnmigration

with approximately 30,000 people each year from neigh‑boring Central American countries. One of the most

3. Measles follow‑up immunization campaign in Belize
The measles catch‑up immunization campaign dra‑
matically decreased the number of susceptibles. How‑ever, during routine immunization activities after the
1991 campaign, the proportion of susceptibles began torise. Yearly immunization coverage remained at 80 to

90% overall and some who were vaccinated were notprotected because the vaccine is only 90 to 95% effective

under field conditions. Additionally, some vaccine may
have been impotent due to cold chain failures or inappro‑

priate vaccine administration. As a result, the numberof susceptibles to measles steadily accumulated.
Since measles immunization is given in Belize at
twelve months of age, there are in ahy year, approxi‑

52mately 7,500 infants (all infants under one year of age)

provided vehicles by UNHCR, and they performed

unimmunzed in Belize. If it is assumed that 30% of

immunization activities much better than would havebeen possible with the one vehicle which they had tothese infants are unprotected either by lack or loss of
ma.ternal antibody during their first year of life, thiswould provide about 2.000 susceptibles in the under one
year age group. In addition, if it is assumed that measles

vaccination coverage was 80% each year and that vac‑cine efficacy was 90%, approximately 2,000 susceptibles

would accumulate each year. Thus, it was estimatedthat, as of 1995, approximately 10,000 susceptibles had
accumulated in Belize since the measles catch‑up immu‑
nization campaign in 1991. It is believed that such anumber of susceptibles would be sufficient to support an

epidemic if measles virus were imported from endemic
countries. In order to sustain measles‑free status in
Belize and to prevent measles outbreaks by irnported

share with other public health activities in the district.

The campaign was also supported by a great deal ofcommunity participation. A number of school teachersand community leaders participated in health education
activities distributing promotional materials provided
by the Ministry of Health, providing parents informa‑tion about specific times and places for immunization
activities, and encouraging them to bring their children
to the immunization units. Some teachers helped publichealth nurse and community health workers in estimat‑
ing target populations, and in recording names, addres‑ses, and ages of vaccinated children.

According to the reports from EPI manager andmeasles cases from endemic countries, it was decided to

public health nurses in each district, overall immuniza‑

reduce the number of accumulated susceptibles by im‑
plementing a special measles follow‑up immunization
campaign.
The measles follow‑up immunization campaign intion coverage of this campaign was 74%, the highest
being 98% in Corozal District, and the lowest 46% in
Orange Walk District.

Belize was conducted in October 1995. The target
population for this campaign was all children from

4. Lessons from measles follow‑up immunization

twelve through fifty nine months of age (approximately

According to reports and questionnaires frorn public

34,000 children) . All were to be given one dose ofhealth nurses who were engaged in the campaign, themeasles vaccine, regardless of the past history of immu‑

unexpectedly low coverage, especially in sorne districts,

nization or disease. Unfortunately, a severe hurricane
attacked the northern area of Belize, especially Orange
Walk District, at the end of September. Following thehurricane, a flood occurred in Orange Walk District, anda number of villages had to be evacuated because of thedamage from the hurricane and flood. Thus, in Orange

could be attributed to three factors. First, the hurricane

campaign in Belize

and flood following it harnpered the implernentation of

the immunization campaign, especially in Orange WalkDistrict. Second, many public health nurses committed
to the campaign complained of the shortage of transpor‑

tation, especially vehicles for mobile immunization

Walk District, the immunization activities were notfully performed because transportation and communica‑tion system had been devastated, and immunization
teams had difficulty in tracing the evacuated people.

teams. In commemoration of the fiftieth anniversary of
the United Nations, UN agencies provided vehicles andother resources, but only for the third week of October.

Since other districts had been damaged very little by the

immunization activities for this campaign on the othe.rweeks because of the lack of transportation. Third, thepromotional activities and materials such as posters and,hurricane and flood, the campaigu was conducted asscheduled in Corozal, Belize, Cayo, Stann Creek, andToledo Districts.
One of the most unique characteristics in the cam‑

Immunization teams were unable to implement enoughleaflets were too late to support the immunization

Nations (UN) agencies such as UNICEF, United

activities, because the promotional activities began andmaterials reached health centers in the middle of Octo‑ber, while the campaign was initiated in the beginning of

Nations High Commissioner for Refugees (UNHCR) ,and United Nations Development Programme (UNDP)

October.
Several points have been identified to be of special

in celebration of the fiftieth anniversary of the UN;
October 24, 1995. Their participation in the campaign
generated much publicity among people in Belize. In

relevance to future immunization campaigns.
1 ) The season of the campaign

addition, they provided vehicles and other resources forwere the hurricane and flood, which destroyed transpor‑

the campaign during the third week of October. As atation routes and community health systems in thenorthern areas, especially in Orange Walk District.

paign was the close collaboration with other Unitedresult, in Corozal District, the irnmunization teams were

The major obstacles for the campaign in Belize

53

Although the occurrence of hurricanes is unpredictable,

it would be better to implement immunization cam‑paigns in months when few or no hurricanes are expect‑ed.

2 ) The degree of collaboration with other organiza‑
tions

The principal UN agencies (UNICEF, UNHCR, andUNDP) participated in the campaign in commemora‑

nization activities during the campaign. These teams
were given great flexibility in scheduling campaign
activities according to each unique situation such as
weather conditions and other routine community activ‑
ities. As a result, very few health centers complainedthat the campaign had hampered their routine publichealth activities at the health center level. Such apartially decentralized system and a flexible schedule intion of the fiftieth anniversary of the United Nations,and provided several vehicles and other resources. This
collaboration greatly enhanced the publicity and effi‑
ciency of the campaign, and facilitated the work of thea mass immunization campaign can be very effective infacilitating immunization activities, although strict

mobile immunization teams. Such collaborations withencourage the voluntary support of parents because
each team had close and friendly communication withother agencies are clearly beneficial and supportive infacilitating immunization activities. In addition, involv‑

ing NGOs, which have close relations with communities,
can enhance community participation and contribute to
the sustainability of mass immunization campaigns.
3 ) The extent of target‑specific and timely prornotion

overall supervision is still necessary.

In addition, the District Health Teams were able totheir communities. The irnportance of close relation‑
ships with the community and the success of community
participation have been reported in mass immunization

campaign in Cameroun (McBean et al.. 1976), Chile(Borgono et al.. 1978) , and Ghana (Belcher et al.. 1978) .

In this campaign, Belize had a special promotion
targeting hard‑to‑reach communities, especially the5 ) The importance of transportation

Mennonites, using their language and photographs of thewere shortages of transportation for mobile immuniza‑tion teams. Witlp enough transportation, they could
conduct satisfac/tbry immunization activities including

Mennonite community. This group used to refuse
modern scientific medicine including immunization, andsome still now reject immunization. This effort was
intended to enhance their community's interest in immu‑
nization and to decrease difficulty in implementing the

campaign. In general, it is important to target such

The principal complaints of the public health nurses

mobile immunization activities as seen in Corozal Dis‑
trict, which chieved the highest immunization coverage(98%) due to vehicles provided by UNHCR. A shortage

hard‑to‑reach communities because of the risk ofof vehicles is not unique to the campaign in Belize. In
Senegal, for example, the mobile vaccination activities

measles outbreaks among them in both developing andwere one of the main strategies of their immunization

developed countries. (Novotny et al.. 1988)

campaign, but these activities were limited by the short‑Many public health nurses complained of the delay
in providing promotional materials and, in some cases,

age of vehicles, and both the provision of vehicles andthe effective performance of mobile teams were recom‑of their unavailability. Obviously the promotional activ‑

mended for successful achievement of immunization

ities must be timely and should be made before thecampaign begins in order to enhance public knowledge

campaigns (Desgrees and Pison, 1994). Thus, in plan‑of the campaign and to facilitate immunization activ‑

issue into consideration, and try to provide effective
transportation as much as possible.
6 ) Missed opportunities to provide vaccination

ities for the campaign. If there are high level of accessto the media, and sufficient expertise and funds avail‑able to develop and produce radio and television adver‑
tisements, promotion for immunization campaig!ns using

ning immunization campaigns, we have to take thisTwo unique factors were identified as importantreasons for failures to irnmunize during the campaign inmass communications can significantly improve immu‑

Belize.

nization coverage (Zimicki et al.. 1994).

Migration: Almost all the public health nurses
complained that the migrating population was a major

4 ) The capability of District Health Teams
During visits in Belize, the author was impressed by

the well‑organized District Health Teams and the closeand friendly communication between central level anddistrict levels. At each health center, public healthnurses were responsible for the campaign activities andseveral cornmunity health workers, who were familiar
with the unique situations of the area, supported immu‑

reason for failures to irnmunize. They explained that it

is very difficult even for cornmunity health workers,
who are quite familiar with their health center areas, toestimate the size of the migrating population and toimmunize them during the campaign.
Private physicians: Some public health nurses com‑plained about interference with campaign activities by54

private physicians who had provided parents inconsist‑

WHO, 1994).

ent information with the plan of the campaign. Such

7 ) The influence of behavioral issues on immunization
In the campaign in Belize, a number of public healthconfusion and misunderstanding of parents werecounter‑productive for the immunization activities.
These two factors were not "missed opportunities",
according to the usual definition, which defines missed

opportunities as "circumstances when a child, who iseligible for immunization and who has no contraindica‑tion to immunization, visits a health service and does
not receive the needed vaccines" (Hutchins et al.. 1993;

nurses complained that some parents exhibited little
interest in measles immunization because of the absenceof measles over the past four years, fear of side effectsof vaccine, and negligence of parents. A study in Togo
reported that the major factor in the acceptability ofchildhood immunization was interest in immunizationon the part of parents rather than logistic and manage‑

EPI/WHO, 1993; EPI/WHO, 1994). Although many

ment problems of the program (Eng et al., 1991).

public health nurses understood "missed opportunities"
to mean failures of parents to bring their children to
immunization units, these factors were very important

Pillsbury (1991) reviewed qualitative researches about

the behavioral perspective of immunization and sum‑

as well as true missed opportunities in order to attain a

as follows:

high performance for the campaign.
In other countries, reasons for poor vaccine cover‑age are diverse. In Mozambique and Conakry in Guinea,

management and logistics problems were largely at

marized the reasons why children did not get immunizedReasons related to characteristics of the motherand other caretakers.
‑Time constraints and competing priorities‑Other socioeconomic constraints

part of health workers and mothers (Cutts et al.. 1991) .

‑Lack of knowledge about immunization‑LOW motivation for immunization

In Puerto Rico, the major problems were non‑availabil‑‑Fearsfault along with poor knowledge and attitudes on the

ity of vaccines, Iack of integration of services, provider‑Cornmunity opinion

misconceptions about contraindications, and failure to

Reasons related to characteristics of the vaccines.

administer vaccines simultaneously (Ginder et al..

‑Side‑effects of the vaccines‑Belief that vaccination is not effective

1993). Missed opportunities to vaccinate children are
major barriers for immunization program not only indeveloping countries, but also in developed countries as

was revealed in United States of America during themeasles epidemic of 1989 and 1990. The major reason
for that epidemic was failure to provide vaccine tovulnerable children on schedule, primarily due to two
types of missed opportunities: 1) observing inappropri‑ate contraindications and 2) failure to vaccinate chil‑dren at each visit for health care (The National Vaccine
Advisory Committee, 1991; Peter, 1992; Cutts et al., 1992;

Wood and Brunell, 1995) .

Reasons related to characteristics of the delivery of

immunization services.
‑Accessibility‑Availability‑Acce ptability‑Af f ordability

Reasons related to communication to the public
about immunization.
‑Inadequate communication
Based on the factors mentioned above, it is essential

for persons who plan and implement immunization

Major barriers to adequate vaccination coverage

programs to understand local perceptions and behavior

are the failures to vaccinate children when they are seen

which influence parental willingness to have their chil‑by health care practioners either during an immuniza‑tion campaign or routine immunization services. Two
major strategies can help to decrease missed opportu‑
nities: 1) encouraging health care providers such as
public health nurses and private physicians to involve

dren vaccinated. A better understanding of local percep‑
tions and behavior related to immunization is especiallybeneficial for extending coverage to hard‑to‑reach com‑munities, enhancing community participation, and build‑ing stistainable programs.

themselves in immunization activities and to takeadvantage of every chance to vaccinate children in needof immunization, 2) providing appropriate health infor‑
mation to parents in order to stimulate their interest in
immunization and to get them to participate. These two
strategies have proved effective under different circum‑

stances (Hutchins et al., 1993; EPI/WHO, 1993; EPI/DISCUSSION
Based on the progress of measles elimination pro‑gram in the Americas and the evaluation of the measles
follow‑up irnmunization campaign in Belize, the author
depicted the important components for the strategy, and55

considered the development of measles elimination pro‑gram to other regions.

1. Important components for measles elimination
strategy

gram, which is called "rash and fever surveillance". In

this system, all cases which have rash and fever as
explicit symptoms should be reported and investigatedboth serologically and epidemiologically. Then, such

Since 1991, the strategy for measles elimination has

information should be returned to the primary reportinglevel as feedback of information. For example, in orderbeen functioning effectively in the Americas, andto provide information to public health workers

achieved great success in the English‑speaking Carib‑
bean countries. This success can be attributed to thethroughout the Americas, PAHO has published a weekly

strong government commitment of the member coun‑tries in the English‑speaking Caribbean countries. The
government of each country has supported the interna‑
tional goal on measles elimination of this region, andcollaborated technically and administratively throughthe international organization (PAHO/WHO, UNICEF,
World Bank, etc. ) and other non‑governmental organi‑zations (Rotary International, etc.).

As discussed previously in the measles follow‑up
irnmunization campaign in Belize, strong political com‑

measles surveillance bulletin since 1994. This bulletinsummarizes the number of current outbreaks by coun‑try, total cases under investigation, and cumulative
annual number of confirmed measles cases by country(de Quadros et al.. 1996). For the technical support
system, it is necessary to establish the international
laboratory network, on which there are several refer‑ence laboratories that can perform advanced laboratory
investigation in order to confirm or rule out measles
virus circulation in one region. Moreover, Iaboratory
sequencing of the measles virus genome from isolates
can help to determine geographic sources of outbreaks
and to identify pathways of transmission of measles

mitment and community's participation are the major
factors not only for the mass immunization campaign
but also the ongoing measles elimination program.

virus.

Without those factors, the program cannot obtainadministrative and logistic support such as money,

2. Development of measles elimination program topersonnel, and equipment. Moreover, if there were nocommunity's support on the basis of strong politicaladvocacy, the program could not be carried out effec‑

tively due to low compliance of mass immunization

other regions
Although the new program and strategy have beenproceeding successfully in the Americas and measles
elimination has been achieved in the English‑speaking

program.
In addition to the commitment by government andCaribbean countries, there may be still several obstacles
for global measles elimination to be developed to othercornmunity, the international collaboration is indispens‑able for the success of the measles elimination program.
Since measles is ubiquitous all over the world, there is

regi ons.

always a risk of importation of measles virus, even if
one country or region has achieved measles elimination.
In order to reduce the possibility of measles virus impor‑

First, the predecessor of measles elimination pro‑gram; polio eradication program, has not been achievedin the rest of the world, while there have been nopoliomyelitis since 1991 in the Americas, and since thenthe program had entered the observation stage. Eventu‑

tation or exportation, each country in some region has
to implement the measles elimination program simulta‑
neously. Under such condition, international organiza‑

cases of polio has decreased drastically in the rest of thetion (WHO, UNICEF, etc.) and non‑governmental orga‑

world, the polio eradication prograrn is now at its very

ally, the eradication of polio in Western Hemisphere

was announced in 1994. Although the number of thenization (Rotary International etc. ) are able to play animportant and critical stage; mass immunization cam‑

important roll in order to organize and integrate eachnational program into international program, as seen inpaign and strengthening surveillance. Under such condi‑
tion, all countries except the Americas are now engaged
in the polio eradication program enthusiastically as thethe measles catch‑up immunization campaign in the

English‑speaking Caribbean countries in 1991
(Hospedales, 1992) .

In general, the health information system about thetarget diseases and the technical support system topriority program of public health. There may be very
little resources for the new measles elirnination pro‑gram.

confirm the information are indispensable for success ofSecond, in developing countries, the health informa‑tion system such as disease notification or surveillance

the disease control program. The former is one of themost critical components for measles elirnination pro‑system has not been developed enough to support publichealth program in general. Therefore, it is quite diffi‑

56

to support global measles elimination program finan‑system, it may be much more easier for such developing
countries in Asia to prornote political commitrnent andcommunity's participation enough to implement measles
elimination program enthusiastically.
In addition to financial and administrative point ofview, basic researches for measles and its epidemiology
are indispensable for the measles elimination program.
On this field, Japan can contribute its potential compe‑
tence to applied technologies for measles elimination.

cially and technically. Moreover, basic researches about

For example, development of high‑titer measles vac‑

measles, such as vaccines, are not conducted enough tocine, which enable vaccine administration in earlier ages

provide new scientific information for the measles elimi‑

such as six months or earlier, or the innovative vaccine

nation program, because of low interest in measles indeveloped countries.
Although they are several obstacles for implement‑delivery system.

ing the measles elimination in other regions, as

speaking Caribbean countries. Although there are stillseveral obstacles for development of the measles elimi‑

cult financially and administratively to introduce a new

disease surveillance system such as "rash and fever
illness surveillance" for measles elimination program indeveloping countries.

Third, in developed countries including Japan,
measles is no more a major concern as priority of publichealth importance, because the mortality from measles
is very low. Therefore, they are not enthusiastic enoughmentioned previously, measles has been a leading killer
for children. Moreover, the American region is able toinitiate the program almost five years ago, and in
English‑speaking Caribbean countries, they attained amarvelous success story through this strategy. There‑
fore, these experiences indicated feasibility of themeasles elimination program. Towards the global elimi‑

In summary, measles elimination program is fea‑
sible, and has already achieved its goal in the English‑nation program to other regions, the most importantcomponents are the strong political commitment in eachcountry and international cooperation financially andtechnically. On this point of view, Japan is able to play

an important roll towards the measles elimination inAsia.

nation of measles, it is indispensable for all countries not

only developing countries, but also developed countries
to participate in this program simultaneously. If eachcountry has its strong political commitment and enthusi‑
astic community's participation, this measles elimina‑tion program will be able to function effectively and we

The author is grateful to Dr. D.A. Henderson in
Department of International Health, School of Hygieneand Public Health, The Johns Hopkins University, forcan expect the glorious achievement which we had

academic guidance throughout this research. The

experienced in the smallpox eradication and poliomyeli‑
not wait until the polio eradication program will achieveauthor also gratefully acknowledges the professional
and administrative support of Dr. Ciro de Quadros, Dr.
B. Hersh, and Mr. P. Carrasco, of the Special Program

the worldwide success.

for Vaccines and Immunization (SVD, Pan American

tis eradication. The measles elimination prograrn can‑ACKNOWLEDGMENTS

On the view of international cooperation and

Health Organization (PAHO), and of Mr. Smith of theregional political commitment in Asia, Japan is able toplay an important roll towards the measles elimination
not only in this region but also in the world. Actually,

Caribbean Epidemiology Center (CAREO , PAHO.

in polio eradication program, Japan has supported thisprogram financially, dispatched expert personnel, anddonated vaccines and other equipment for immunization
program to several countries in Asia in collaboration

dedicated work on the measles follow‑up immunization
campaign in 1995. Financial support for this research
was provided by the Foundation for Advanced Studies
on International Development (FASID) , Japan.

Finally, the author extends his respect and thanks to
EPI manager and public health nurses in Belize for theirwith WHO and UNICEF. For the future perspective of
health development in Asia, Japan has to take the initia‑

tive in the new measles elimination program in order tosupport health development in Asian region.
As mentioned previously, there are several obsta‑
cles for irnplementation of measles elimination program.
When Japan will support this program financially andadministratively, these obstacles such as priority con‑flict of health problems and under‑developed health

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de Guzman, E., Dayrit. M., Fausto, A., Lee, M.B. andAbad, M. (1994): Improving vaccination coverage inurban areas through a health communication campaign‑the 1990 Philippine experience. Bull. World Health

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R., Swint, E.B., Hadler, S.C. and Rullan, J.V. (1993):
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59

Jpn. J. Trop. Med. Hyg., Vol. 25, No. 2, 1997, pp. 59‑67

CYTOKlNE PRODUCTION ASSAYEDBY RT‑PCR IN PREGNANT MICE ･INFECTEDBY TOXOPLASMA GONDII AS A MODEL

OF CONGENITAL TOXOPLASMOSISNA HEl'2, FUMIE AOSA12, MUN HYE‑SEONG2, SOUEI SEKIYA3 AND AKIHIKO YAN02*
Received January 31, 1997/Accepted April 9, 1997

Abstract: To explore the mechanisms of immune responses of host to Toxoplasma gondii ( T. gondii)
infection in pregnant mice, we evaluated roles of cytokines [interferon gamma (IFN‑y) , tumor necrosis
factor a (TNF‑a) , interleukin 6 (IL‑6) and interleukin 4 (IL‑4) J by measuring mRNAS of these cytokines
in placentas, Iungs and spleens. The pathogenic effects of time and duration of the Fukaya infection on
cytokine mRNA Ievels in pregnant mice were analyzed. The abundance of mRNAS encoding these cytokines
was measured by reverse transcriptase (RT) ‑PCR at early and late stages of pregnancy in various organsof both susceptible C57BL/6 and resistant BALB/C pregnant mice infected with T. gondii. IFN‑y and TNF‑
a but not IL‑6 or IL‑4, were predominant in the immune responses of placentas, Iungs and spleens of BALB/

c and C57BL/6 mice during T. gondii infection. Levels of IFN‑y and TNF‑a mRNA in placentas of earlystage pregnant BALB/C mice (infected at one‑week pregnancy and examined on day 4 after infection;
IW4D) were higher than those in corresponding C57BL/6 pregnant mice, which might correlate with the factthat higher parasite numbers in placentas and lungs of C57BL/6 mice (infected at one‑week pregnancy andexamined on day 11 after the Fukaya infection; IW11D) were observed than those in placentas and lungs ofcorresponding BALB/C mice, but not correlate with the result of parasite numbers ( T. gondii No./mg tissue)in spleens of C57BL/6 (O) and BALB/C (120i56) pregnant mice. In the late stage of pregnancy, Ievels of
IFN‑y and TNF‑a did not show definite correlations with T. gondii loads in placentas, Iungs and spleens.
These results indicate that endogenous IFN‑y and TNF‑a of early stage pregnancy may be essential for
inhibition of T. gondii growth in some organs (placentas and lungs) , but not in spleens, and the mechanismsof genetic influence involved in the susceptibility and resistance to acute T. gondii infection may include

several immune responses acting together.
Key words: Toxoplasma gondii, cytokine mRNA, congenital toxoplasmosis

INTRODUCTION
Human congenital toxoplasmosis is caused bymaternal transplacental transmission of T. gondii para‑sites to the fetus, mainly by the acute initial maternal

depressed immune response during pregnancy would be
expected to have a bearing on the severity of T. gondii
infection and hence provides a greater opportunity fortransplacental spread to occur (MCLeod et al., 1989).

infection during pregnancy (Yokota, 1995). Any route

Pathological changes are much more cornmon andmore severe in the placenta than in the fetus, andof T. gondii infection leading to a maternal parasitemia

placental damage is probaly the primary cause of fetal

during preguancy may result in toxoplasrnosis of theplacenta and transmission of the protozoa to the off‑
spring before birth (Cowen and Wolf, 1950). In humancongenital infection of T. gondii, severity of disease
appears to be strongly correlated with trimester ofmaternal acquisition (Luft and Remington, 1982). The

death (Loke, 1982). These facts clearly demonstratethat immune responsiveness of the placenta is criticallyimportant for resistance to the parasite entering the
fetus and study of the pathology of the placenta is ofgreat practical irnportan6e.

There have been several reports on maintenance of1 Department of Medical Zoology, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
2 Department of Parasitology, Chiba University School of Medicine, 1‑8‑1 Inohana Chuo‑ku, Chiba 260, Japan
3 Department of Obstetrics and Gynecology, Chiba University School of Medicine, 1‑8‑1 Inohana Chuo‑ku, Chiba 260, Japan*Correspondence: Akihiko Yano, Department of Parasitology, Chiba University School of Medicine, 1‑8‑1 Inohana Chuo‑ku, Chiba
260, Japan. Phone: (81) 43‑226‑2071. Fax: (8D 43‑226‑2076. Electronic mail address: yano@med. m. chiba‑u. ac. jp

60

pregnancy, and recording the placental expression and/organs of resistant (H‑2d) or susceptible (H‑2b)

or synthesiz'es of both harmful and protective cytokines,
including interleukin 3 (IL‑3) . IL‑4, IL‑6, IL‑10, IFN‑y,strains of pregnant mice. The cytokine (IFN‑y, TNF‑
a, IL‑6 and IL‑4) mRNA Ievels in placentas, Iungs andspleens were analyzed by RT‑PCR.

TNF, colony stimulating factor‑1 (CSF‑1) , granulocyte

macrophage‑CSF (GM‑CSF) , and transforming growth
factor fi (TGF‑p) (Chen et al., 1993; Clark et al., 1995;

De et al., 1992; Hunter et al., 1993; Yelavarthi et al.,

199D. These two patterns of harmful and protective
cytokines overlap substantially with the Thl and Th2
cytokine patterns that were originally identified amonga panel of mouse Th clones (Mosmann et al., 1986) , andmutually inhibit the differentiation and/or activation of
the other (Fiorentino et al., 1989). Thus, the inflamma‑

MATERIALS AND METHODS
Mice and Inatings. Inbred female and male BALB/C(H‑2d) and C57BL/6 (H‑2b) mice (Charles River,
Yokohama, Japan) were housed at the Laboratory
Animal Center for Biomedical Research in Nagasaki
University School of Medicine under conventional condi‑
tions. Eight‑ to ten‑wk‑old mice were used for mating.

tory/anti‑inflammatory balance of cytokines in theplacenta can alternatively be viewed as a Thl/Th2

night for mating and the presence of the vaginal plugs inbalance. The protective Th2 cytokine pattern (together
with TGF‑p) may be functionally dominant over poten‑
tially harmful Thl cytokines during normal, successful

the females was checked on the following morning. The
day on which the plug was observed was considered to
be day O of pregnancy. Pregnant females thus identified

pregnancy (Krishnan et al., 1996). Among Th2 cyto‑were removed from the mating cages and housed ran‑
domly with other female mice.
T. goudii strains and infections of mice. A virulent

kines, IL‑4 has the ability to drive precursor T cells into

a subpopulation of T helper effectors known as Th2

One male and two females were housed per cage over‑cells, which are important regulations of humoral immu‑
nity, eosinophilia, and mastocytosis. In some infectious
disease models, administration of anti‑IL‑4 at the tirneof infection will divert the ensuing response away frorn

strain RH and an avirulent cyst‑forming strain Fukaya(Asai and Suzuki, 1990; Watanabe et al., 1993) of T.
gondii were used for infection experiments and preparedTh2 cells toward the Thl subpopulation of T helper

For the experiment with the RH strain, tachyzoites
were adjusted to 107 viable organisms/0.25 ml in phos‑
phate‑buffered saline (PBS) for inoculation. One‑ or

effectors (Sadick et al., 1990). We reported that theThl‑like human CD4+ cytotoxic T Iymphocytes specificfor T. gondii‑infected melanoma cells were existent inperipheral blood lymphocytes of patients with toxoplas‑
mosis and were secreting IFN‑y and IL‑6 (Yang et al.,as previously described (LUO et al., 1995).

two‑week pregnant mice were infected intravenously
via their retro‑ocular venular plexus with 0.25 ml RH

suspension. A11 pregnant mice died on day 6 after

1995). Both IFN‑y and IL‑6 enhanced HLA‑DR mole‑

infection, therfore the infected mice were sacrificed on

cules of T. gondii‑infected melanoma cells, and thesecytokines may play a role in pathogenicity of toxoplas‑
mosis. IL‑6 enhances intracellular replication of T.

day 4 after infection. The organ samples, placentas,
lungs and spleens, were collected to analyze the abun‑gondii and reverses IFN‑y‑mediated activation of

For the experiment with the Fukaya strain, thebrains from mice infected 2 months earlier with the
Fukaya strain of T. gondii were removed and homogen‑murine peritoneal Mc, and that certain of the interac‑tions between these two cytokines may be at the level of

TNF‑a triggering (Beaman et al., 1994). Conversely,an acute T. gondii infection during pregnancy may
disrupt the maternal‑fetal immunological balance infavor of antiparasitic proinflammatory abortogenic
cytokines such as IFN‑y and TNF‑a (Roberts et al.,1995). Since IFN‑y and TNF‑a may be necessary forresistance to T. gondii infection, it is perhaps more

dances of certain cytokines.

ized in 5 ml of PBS with a mortar and pestle. Then 50
pl of brain suspension was placed on a glass microscopeslide and mounted with a cover‑slip and the number ofcysts determined microscopically by scanning the entire

ballance between the two types of cytokines, which can

preparation under 100 X magnification. One‑ or two‑
week pregnant mice were infected by gavage with 0.5
ml of a brain tissue homogenate in PBS suspension,containing 20 cysts. The mice which were infectedduring the first week of pregnancy were sacrificed on

shift in either direction for a variety of reasons (Clark

day 4 after infection. Mice which were infected at two

et al., 1995).

weeks of pregnancy were sacrificed and examined onrealistic to view the situation in pregnancy as an altered

Our present work studied the immune responses of

day 4 after infection, just before delivery. Each experi‑

the placenta to T. gondii infection as compared to othermental group consisted of three animals. The nurnber of

61

Table

l

Summary

Mousestrainofexperimental

placentas

experimentwasresults

were

shown

to

used

this

present

two

be

or

when

each
three

essentially

and

the

identica1．The

mice

post

time

Abbreviation

infection

week

day4
day11

lW4D

two−week

day4

2W4D

total

group．Every
times

AnalysisStage

infectedone

RH，Fukaya

were9to20in

performed

Pregnant

T．go％4露strain

BALB／c，C57BL／6

examinedgroups

RNA

was

1WIID

reversedtranscribed

in

a

final

volumeof

20μl using the RNA PCR kit（RO19A，Takara，Shiga，Japan）according to the manufacturer s instructions．

anaesthetic

The quantity of RNA used for RT and RT−PCR was
decided as manufacturer7s recommendations．The reac・

Experimental groups． Experimental groups of preg−
nant BALB／c and C57BL／6mice infect d with the RHtion mixture contained10mM Tris・HCl（pH8．3），50
mM KC1，5mM MgC12，1mM of each deoxynucleotide
triphosphate（dATP，dUTP，dCTP and dTTP），20U of
RNase inhibitor，5U of avian myeloblastosis virus
reverse transcriptase XL，and2．5mM random nine−

in

study

were

sacrificedbyoverdose．

ortheFukayaor

two−week

go％撚andonarepregnant

theday40rgroupsstrain

ll

after

in

tried

group

the

which

Table1．One−

were

infected

with

mice

examined

as

to

in

these

infection．

abbreviatedrespectively．We
in

mice

infectivities

arepregnancysummarized

were

These

nucleotide

experimental

IW4D，1Wl1D

establishTanexperimental

mice

were

infectedonhowever，these

mice

did

becomenotdayoligomers

of

lOOμl

with

preg−

dilutedsolution

O

and

was

incubated

at30。C

for10

min，50。C for30min，99。C for5min，and4。C for5min．
The RT product mixture（cDNA）was then diluted toand2W4D

was

used

PCR

the

PCR

1ガーinfectedor
to

tions

of

the

mice

accordingmanufacture．Aftertheinstruc・RNAconcentration was determined spectrophotometricallyusing GeneQuant（Pharmacia LKB Biochrom Ltd，Sci−ence Park，Cambridge CB44FJ，Englan（1）．Oneμg of

Table2

PCR

extraction，theconditions（sequencesmRNA，number

of

PCR

GAPDH
IFN一γ

TNF一α

IL−6
IL−4
Primerphoresedoftheoligonucleotide

andproduct

Primerwere

obtainedfromprimers

sequences

CLONTECH

each

set

but

uninfected

microliter

in

of

each

a1．2％agarose

used

for

PCR

control

PCR

of

gel

mice．

productwithamplification

of

was

electro−ethidiumbromidecytokinepredicated）

3〆 TCCACCACCCJGTTGCTGTA
51 ACCACAGTCCATGCCATCAC
3！ CGACTCCTTTTCCGCTTCCTGAG
5TGAACGCTACACACTGCATCTTGG
3〆 CCAAAGTAGACCTGCCCGGACTC5〆 ATGAGCACAGAAAGCATGATCCGC
31 CACTAGGTTTGCCGAGTAGATCTC
5！ ATGAAGTTCCTCTCTGCAAGAGACT
3GCTCTTTAGGCTTTCCAGGAAGTC5ATGGGTCTCAACCCCCAGCTAGT

sequencespregnantTencyclesCytokineforcytokine genes from total RNA by conventional PCR
and no cytokine genomic DNA was detected in1μg of
total RNA．Each of the above−mentioned groups ofpregnant mice were accompanied by a group of age−matchedsize

optimizedofreaction．The

primers（Table2）．Contaminations of total RNA withg弓nomic DNA were examined by attempting to amplify

pregnant

werewater，and5μl
each

Detection of cytokine mRNA by reverse transcriptase（RT）一PCR．Total cellular RNA was extracted with
Trizo1（Gibco BRL，Grand Island，NY，U．S．A．）from
non−infectedconditions

for

nant．

placentas，1ungsandspleensofTgo％

reaction

double−distilled

Annealing
temperature（。C）

Cycles

Product

55

37

452

60

37

460

60

37

691

56

37

638

54

37

399

Laboratories，Inc．，4030Fabian

Way，Palo

Alto

size

（bp）

CA94303USA、

62

staining for 25 min at 100 V and quantification of RNA

was performed with an IPLab Gel Densitometer (Sigallungs than IW4D BALB/C pregnant mice (P < 0.05) .
In the RH‑infected BALB/C and C57BL/6 pregnant

Analytical Corp., Vienna, VA) . The relative intensity of

mice, no detectable level of IL‑4 was observed inbands for each cytokine mRNA was related to theplacentas and lungs of the early and the late stages ofpregnancy. Significant levels of IL‑4 were only obser‑

intensity of the autoradiogram band used as the internal

control, glyceraldehyde 3‑phosphate dehydrogenase(GAPDH). The results are expressed as the ratio of the
optical density (OD) value of the PCR product of eachcytokine to the OD value of products of GAPDH accord‑

ved in spleens from IW4D BALB/C and C57BL/6 preg‑
nant mice and 2W4D BALB/C pregnant mice.
At the late stage infection with the virulent RH
strain, IFN‑y and TNF‑a mRNA but not either IL‑6 oring to the following formula: (OD of cytokine/OD ofGAPDH) >< 100. The OD value for the blank control

IL‑4 mRNA .were dominant cytokines detected in(distilled water) was set at zero.

The abundances of cytokine mRNA transcripts in

Statistical analysis. Student's t‑test was used to ana‑lyse differences in parasite loads or levels of cytokines

between the various groups of mice. P < 0.05 was

BALB/C and C57BL/6 pregliant mice infected with anavirulent Fukaya T. gondii strain. A marked differ‑ence in susceptibility to the avirulent strain Fukaya

accepted as siguificant.

infection between IW11D BALB/C and IW11D C57BL/RESULTS
The abundances of cytokine mRNA transcripts in
BALB/C and C57BL/6 pregnant mice infected with avirulent RH T. gondii strain. High levels of T. gondii
10ads were detected in placentas, Iungs and spleens from

BALB/C and C57BL/6 pregnant mice in the early(1W4D) and late (2W4D) pregnant stages (Fig. IA) .
To examine the correlation between parasite loads andcytokine responsiveness, mRNA transcripts encoding
IFN‑y. TNF‑a, IL‑6 and IL‑4 were tested in IW4D and
2W4D BALB/C and C57BL/6 preguant mice (Fig. 1).
High levels of IFN‑y mRNA were observed after
infection in placentas, Iungs and spleens of IW4D
BALB/C and C57BL/6 pregnant mice. Also high levelsof IFN‑y were observed in lungs of 2W4D BALB/C miceplacentas of both BALB/C and C57BL/6 mice (Fig. IB) .

6 pregnant mice was observed (Fig. 2). To examine theinvolvement of cytokines in the genetic basis of T.

gondii infectivity in BALB/C and C57BL/6 pregnant
mice, mRNA Ievels of IFN‑y, TNF‑a, IL‑6 and IL‑4 inplacentas, Iungs and spleens were assayed (Fig. 2).

Levels of IFN‑y mRNA in placentas, Iungs andspleens were higher in IW4D BALB/C pregnant micethan in IW4D C57BL/6 pregnant mice (P<0.05). This
correlates with susceptibility to infection in IW11D

pregnant BALB/C and C57BL/6 mice (Fig. 2). The
infection rate in placentas and lungs of IW11D C57BL/
6 pregnant mice was higher than that in IW11D BALB/c pregnant mice (P <0.05) (Fig. 2). However, parasite
10ads in the spleen did not differ significantly between

IW11D BALB/C and C57BL/6 mice. Significant or low
level of IFN‑y was not observed in placentas of IW11D

and 2W4D BALB/C and C57BL/6 pregnant mice, in‑and in spleens of 2W4D BALB/C and C57BL/6 mice

dicating that cytokines produced in the placenta ofafter infection. In placentas of the RH‑infected BALB/C

IW4D BALB/C and C57BL/6 pregnant mice are tran‑

and C57BL/6 pregnant mice, IFN‑y mRNA was detect‑sient and are not sufficient to protect placentas of
C57BL/6 mice from Fukaya infection at a late stage of

ed at a higher level in the early stage (1W4D) than in

the late stage (2W4D) of pregnancy (P <0.05). How‑ever, there was no significant difference in the in‑fectivity of placentas between IW4D and 2W4D preg‑
nant BALB/C and C57BL/6 mice (Fig. IA) .
Significant levels of TNF‑a mRNA were detected
in placentas, Iungs and spleens from IW4D and 2W4D

pregnancy. Levels of IFN‑y in lungs and spleens did not

differ significantly between IW11D BALB/C and
C57BL/6 pregnant mice, and levels of IFN‑y werehigher in lungs than in spleens in the two strains of mice.

Thus the markedly lower T. gondii loads in IW11D
BALB/C pregnant mice than in IW11D C57BL/6 preg‑

BALB/C and C57BL/6 pregnant mice after infection.
IL‑6 mRNA was induced at low levels in placentas,
lungs and spleens of the RH‑infected BALB/C and
C57BL/6 pregnant mice. In the late stage (2W4D) of

nant mice can be attributed to other genetic differences
but not to protection by IFN‑y. The fact that the IFN‑y level in the placenta of the Fukaya‑infected BALB/C
and C57BL/6 mice was higher in the early stage (1W4D)

pregnancy, Ievels of IL‑6 mRNA in placentas, Iungs andthan in the late stage (1W11D or 2W4D) of pregnancy
aggrees with the observations of the RH infection as

spleens of BALB/C and C57BL/6 mice were much lotverthan those in the early stage (1W4D) . IW4D C57BL/6
pregnant mice had higher IL‑6 Ievels in placentas andshown in Fig. l.

Levels of TNF‑a in placentas, Iungs and spleens

63

A
Lymphokine mRNA

T.gondii No.

OrganStages/mg tis ue

1600

2W4D

1 820

Infection

IFN*

IL‑4

1

(+)
(.)

Placenta
1 600

IW4D

IL*6

TNF･ a

1796

(+)
(.)

IS25
1996

2W4D

(+)

H ‑H

H
4

{ ‑‑l

{

(.)

Lung

IW4D

1795
1850

( )

1536

1756 (+)
S pleen

2W4D

rl

H

H

rl

rl

(.)

1 520 (+)

IW4D

iH

(.)

1720

(*)

‑H
i

H

H

ON ff
Oe $ CLie 'C S e e e $ ;‑cec
g e,e e ･ee eoY eo;ceeee eec> e*e S,
N eee(OD of Cytokine/OD of GAPDH) x 100

Figure l(A) Cytokine mRNA Ievels in virulent T. gon;dii‑infected pregnant mice. Time courses ofchange in the abundance of mRNAS encoding IFN‑y, TNFc , IL‑6 and IL‑4 in the placenta,

lung and spleen of BALB/C (D and C57BL/6 (1) pregnant mice after infection with RH
strain. At each time point, experimentally‑infected group of pregnant mice ( + ) was ac=
companied by a group of age"matched uninfected pregnant mice ( ) . Lymphokine mRNAS
were assayed by RTPCR and the data is expressed as described in Materials and Methods. (B).
Cytokine mRNA Ievels in placentas of virulent T. gondii‑infected pregnant mice. Abundanceof IFN‑y, TNF‑(2i, IL6 and IL 4 mRNA in the placenta of BALB/C (abbreviated as B/c) and
C57BL/6 (abbreviated as B6) pregnant mice infected at two‑week after pregnancy with RH
strain and tested an day 4 (2W4D) after infection. MW markers are Hae 111 digest of cxl74.

64

Lymphokine mRNA

T. gondii

OrganStagesNo. / mg Infection IFN‑ Y
19Placenta

Lung

Spleen

TNF‑ atissue

20

IWllD

186
850

( )

1 W4D

o
o

(+)

2W4D

75
82

( )

IW11D

267
1556

( )

1 W4D

70
o

(+)

2W4D

78
95

(+)

1 20

( )

IW11D

O

1 W4D

30
50

H* l

(+)

2W4D

IL‑6

IL･4

1

(.)

1

1

(.)

(.)

H

1

H

1.

H

H

(.)

H

(.)

H

H

(.)

(.)

d

(.)

1

(+)
(.)

e̲

e

¥eGee
'

'8

e*e

*

61

5

e

* ld) u)(OD of Cytokine/OD of GAPDH) x 100
Figure 2 Cytokine mRNA Ievels in avirulent T. gondii‑infected pregnant mice. Time courses of change in
the abundance of mRNAS encoding IFN‑y, TNF‑a, IL‑6 and IL‑4 in the placenta, Iung and spleenof BALB/C ([]) and C57BL/6 (1) pregnant mice after infection with Fukaya strain. At each time
point, the experirnentally‑infected group of pregnant mice ( + ) was accompanied by a group ofage‑matched uninfected pregnant mice ( ‑ ) . Lymphokine mRNAs were assayed by RT‑PCR andthe data is expressed as described in Materials and Methods.

were higher in IW4D (BALB/C pregnant mice than inIW4D) C57BL/6 preguant mice (P <0.05). TNF‑a!

DISCUSSION

production paralleled the production of IFN‑y. How‑ever, in placentas, Iungs and spleens of 2W4D and
IW11D pregnant mice, no significant difference in the

An acute T. gondii infection during pregnancy may
disrupt the delicate hormone‑influenced maternal‑fetal
immunological balance in favor of antiparasitic proin‑

TNF‑a level was observed between infected BALB/C
and C57BL/6 pregnant mice.
No significant or very low levels of IL‑6 mRNA

flammatory cytokines such as IFN‑y and TNF‑awere detected in placentas, Iungs and spleens of infected(Roberts et al., 1995). As shown in Figs. I and 2, at themRNA Ievel, using lungs and spleens as references,
IFN‑y and TNF‑a, but not IL‑6 or IL‑4, were predomi‑

or uninfected BALB/C and C57BL/6 pregnant mice. No
significant IL‑4 mRNA Ievel was detected in any ofnant in the immune response of the placenta, Iung andplacentas, Iungs or spleens from either strain of preg‑
nant mice (Fig. 2).

T. gondii infection. On the other hand, IFN‑y and TNF‑
a have deleterious abortogenic effects, Ieading to fetal
demise in otherwise normal pregnancy (Krishnan et al.,spleen of BALB/C and C57BL/6 pregnant mice during

1996). We observed lower pregnancy ratio and higher

65

intracellular replication of T. gondii and reverses IFN‑

abortion ratio of fetuses in the Fukaya‑infected preg‑
nant mice than in non‑infected pregnant mice (data notshown) , suggesting that a beneficial Thl antiparasite

phages, and that certain interactions between these two

response can prove deleterious to successful gestation as

cytokines may be at the level of TNF‑a triggering

it can influence both placental maintenance and preim‑
plantation events (Krishnan et al., 1996). It has been

the presence of high circulating levels of IL‑6 in a lethal

shown that the Thl response to Leishmania major

T. gondii infection in mice (Beaman et al., 1991) as welly mediated activation of murine peritoneal macro‑(Beaman et al., 1994). Previous studies have detected

increases the risk of pre‑ and post‑implantation preg‑as induction of IL‑6 mRNA in the brains of mice infect‑

nancy loss (Krishnan et al., 1996). Positive correlations

ed with T. gondii (Hunter et al., 1993). We have

have also been reported between abortions and theexpression of IFN‑y, IL‑2 and TNF (Tangri and Ragh‑reported that Thl cytotoxic T Iymphocytes specific for

upathy, 1993). One of the mechanisms of fetal damage

patient with toxoplasmic chorioretinitis secreted highlevels of IL‑6 (Yang et al., 1995). However, we obser‑
ved no significant (2W4D) or very low levels (1W4D) of

in response to cytokine production may be the inductionof natural killer activity by IL‑2 and IFN‑y (Wegmann
et al., 1993). Placenta cells from mice undergoing fetal

abortion were less able to suppress natural killer
reactivity as compared to normal placental cells(Chaouat et al., 1985).

We previously analyzed the mRNA Ievels of cyto‑kines (IFN‑y, TNF‑a, IL‑6 and IL‑4) of brains from
BALB/C and C57BL/6 non‑pregnant mice infected by

T. gondii‑infected melanoma cells derived from a

productions of IL‑6 in placentas, Iungs and spleens of T.

gondii‑infected BALB/C and C57BL/6 pregnant mice(Figs. I and 2). Like IL‑6, IL‑4 has been suggested to
be involved in disease exacerbation. IL‑4 impedes theinduction and effector function of the subpopulation ofThl cells which regulate cell‑mediated immunity (Le etal., 1990). In our experiment, Thl dominancy in the T.

the avirulent strain Fukaya, and the results indicate that

gondii‑infected pregnant mice was more impressive

endogenous IFN‑y and TNF‑a are not sufficient forwhen IL‑4 mRNA Ievels were examined (Figs. I and 2) .

inhibition of T. gondii growth in vivo (Luo and Yano,
1996) . As with IFN‑y, many studies have demonstrated
a protective role for TNF‑a in toxoplasmosis (Gerard

The differences in the levels of IL‑6 and IL‑4 production

et al., 1993; Marchant et al., 1994), although there also

exists some evidence that overproduction of TNF‑a
may be detrimental by increasing mortality (Black etal., 1989). Frorn our data shown in Figs. I and 2(1W4D), TNF‑a production occurs in parallel with
IFN‑y production, which may be consistent with theprevious report that TNF‑a functions as an endogenous
modulator of macrophage activation by triggering IFN‑

y‑primed cells to express antitoxoplasmal activity

between infections with the RH and the Fukaya strains
may be explained by the differences between T. gondii
strain properties, size of inoculum and/or infection
route.
Further study, including pathological investigations

into the transplacental spread of T. gondii and study ofthe immunological status of infected and control
placentas of animals and the characteristics of the Tlymphocytes involved in the process of placental
destruction may provide further insight into the path‑ogenesis of congenital toxoplasmosis.

(Sibley et al., 1991) . In Fig. 2, IFN‑y and TNF‑a levels

in placentas and lungs of IW4D BALB/C and C57BL/6
inversely correlated well with the infectivity of Fukayastrain in IW11D pregnant mice, however, no differencein the level of IFN‑y or TNF‑a in the IW11D mice wasobserved between BALB/C and C57BL/6 pregnant mice.
These results suggest strongly that IFN‑y and TNF‑aproduction in the early stage of pregnancy (1W4D) may
play a very important role and have protective immu‑
nity against parasites in the placenta and other organs in

T. gondii‑infected BALB/C pregnant mice. These spec‑
ulations seem reasonable when parasite loads and cyto‑kine production in IW4D pregnant mice were comparedto those in 2W4D pregnant mice infected with the
Fukaya T. gondii stain.

It has been demonstrated that IL‑6 enhances

ACKNOWLEDGEMEN T
This work was supported by a Grant‑in‑aid for
Scientific Research from the Ministry of Health andWelfare, a grant from the ministry of Education, Sci‑ence, Sports and Culture, J.apan and Ohyama HealthFoundation, Tokyo, Japan.

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69

THREE NEW BLACK FLY SPECIES OFSIMULIUM (DIPTERA= SIMULIIDAE)

FROM SUMATRA, INDONESIAHIROYUKI TAKAOKAI AND SlNGGIH H. SIGIT2
Received February 6, 1997/Accepted March 5, 1997

Abstract: Three new black fly species, i.e., Simulium (Gomphostilbia) padangense sp. nov., S. (Simulium)
sumatraense sp. nov. and S. (S.) minangkabaum sp. nov. are described based on females, males, pupae andmature larvae collected from West Sumatra, Indonesia. S. (G.) padangense is unique within the subgenus
Gomphostilbia in having the pupal gill with one much inflated and seven slender filaments. S. (S.) sumatraen‑
se and S. (S.) minangkabaum are most closely related to S. (S.) celsum from Java and S. (S.) argentipes
from Peninsular Malaysia, respectively.
Key words: black fly, Simuliidae, Simulium. Gomphostilbia. Sumatra

The simuliid fauna of Sumatra has been little inves‑scale‑like pubescence interspersed with several dark

tigated since Edwards (1934) recorded eight simuliid
species from Sumatra. Later, only one new species wasadded (Takaoka and Davies, 1995).
Recently we made surveys on black flies at various
localities of Sumatra, and collected about 20 species
including several new taxa. In this paper we describe

hairs. Antenna composed of 2 + 9 segments, brown

three new simuliid species.
The classification follows that of Crosskey (1969) .
Collecting and rearing methods, as well as d,issection of

anatomical parts for description, were mentioned in
Takaoka (1983). Type specimens will be deposited at

the Natural History Museum, London, U.K. (BMNH),
the Department of Parasitology and Pathology, Facultyof Veterinary Medicine, Bogor Agricultural University,

Bogor, Indonesia (BAU) , and Bogor ZoologicalMu eum, Bogor, Indonesia (BZM).

Simulium ( Gomphostilbia) padangense sp. nov.

DESCRIPTION. Female. Body length 2.3 mm. Head.
Slightly narrower than thorax. Frons dark brown,
whitish gray pruinose, shiny, densely covered withwhitish, recumbent, scale‑1ike pubescence interspersedwith a few, whitish, simple hairs; frontal ratio 1.6: 1.0:

3.3. Frons‑head ratio 1.0: 6.4. Fronto‑ocular area (Fig.

1) well developed. Clypeus dark brown, whitish graypruinose, densely covered with whitish, recumbent,
1

2

except scape, pedicel and base of Ist flagellar segment

yellowish when viewed from above; Ist flagellomere
somewhat elongate, ca. 1.6x as long as 2nd one (rightantenna abnormal, with flagellar segments 1‑3 partiallyconnected on dorsal surface) . Maxillary palp with 5
segments; proportional lengths of 3rd, 4th and 5th seg‑ments 1.0:1.0:2.1; 3rd segment a little enlarged; sensory
vesicle (Fig. 2) of moderate size, ca. 0.33x as long as

3rd segment, with medium opening distally. Maxillary
lacinia with 12 inner and 13 or 14 outer teeth. Mandiblewith ca. 24 sinall inner teeth and lacking outer teeth(though apical part of outer margin very weakly
indented) . Cibarium (Fig. 4) with pair of submedialround projections on posterior margin, and with median
10ngitudinal dark band on floor. Thorax. Scutum darkbrown in ground color, whitish gray pruinose (shining inlight) , with 3 dark, rather broad, Iongitudinal vittae

distinct when viewed anterodorsally; scutum densely
covered with bright, whitish, recumbent pubescence.
Scutellum light brown with whitish pubescence and long,
upright, dark hairs along posterior margin. Postscutel‑

lum dark brown, whitish gray pruinose (shining inlight) , bare. Pleural membrane bare. Katepisternum
dark brown, whitish gray pruinose (shining in light) ,with numerous pale and dark hairs, Ionger than deep;

sulcus distinct. Legs. Foreleg: coxa and trochanter

Department of Infectious Disease Control, Oita Medical University, Hasama, Oita 879‑55, Japan
Department of Parasitology and Pathology, Faculty of Veterinary' Medicine, Bogor Agricultural University, Taman Kencana 3,
Bogor 16151, Indonesia

70yellow; femur dark yellow or light brown with apical

Clypeus brownish black, thickly whitish pruinose (shin‑cap brown on outer surface; tibia largely whitish yellow

ing in light) , covered densely with whitish yellow pubes‑

on outer surface, somewhat dark along inner margin

cence interspersed with several dark hairs. Antennacomposed of 2 + 9 segments, pale yellow with a few
apical segn)ents somewhat darkened; Ist flagellomere

and brown on distal 1/4; tarsi dark brown; basitarsus
slender, ca. 7.1x as long as its greatest width. Midleg:coxa light brown; trochanter yellow; femur dark yellow
or light brown with base yellow and apical cap brown;
tibia yellow on basal 1/3 with somewhat dark subbasal

somewhat elongate, ca. 1.6x as long as 2nd one. Maxil‑
lary palp with 5 segments; proportional lengths of 3rd,

ring, Iight to dark brown on apical 2/3; tarsi brown withsmall, 0.21 x as long as 3rd segment, with medium open‑

basal 1/2 of basitarsus yellow. Hind leg: coxa some‑what dark yellow; trochanter yellow; femur dark yellow
or light brown with apical cap brown; tibia yellow onbasal 1/2 with somewhat dark subbasal ring, graduallydarkened toward apex, brown on apical 1/4; tarsi brownwith basal 3/5 of basitarsus and basal 1/2 of 2nd tar‑somere white; basitarsus slender, parallel‑sided, ca.

ing distally. Thorax. Scutum brown, faintly with 3

7.6 x as long as wide. Calcipala well developed, ca. 1.4 x

4th and 5th segments 1.0:1.1:3.0; sensory vesicle (Fig. 3)

narrow, dark longitudinal vittae, densely covered withbright, yellow, recumbent pubescence; scutum entirely

whitish pruinose on anterodorsal surface when il‑lurninated dorsally and viewed anteriorly; also largely
white pruinose on prescutellar area in certain angle oflight. Scutellum brown, with bright, yellow pubescenceand several dark, Iong hairs along posterior margin.

as long as wide. Pedisulcus well developed. Hind femur

Postscutellum brown, whitish pruinose (shining inand tibia ca. 2.3 x and 2.1 x as wide as hind basitarsus,

light) , bare. Pleural membrane and katepisternum as inrespectively. All tibiae densely covered with white,. Legs. Coloration nearly as inexcept following

shiny, scale‑1ike hairs on outer or posterior surface offeatures: hind tibia whitish yellow on basal 2/5, Iight andbasal 2/3 or 3/4; fore and mid femora, on outer surface,dark brown on rest; hind basitarsus whitish on basal 1/2,

each clustered with numerous, dark, scale‑like hairs

dark brown on distal 1/2. Fore basitarsus slender, ca.

from middle to just before apical dark tip, appearing as

8.4x as long as its greatest width. Hind basitarsus (Fig.

a dark broad band. Claws each with large basal tooth0.5x as long as claw. Wing. Length 2.1 mm. Costa with

7) somewhat inflated, almost parallel‑sided, ca. 4.7x aslong as wide. Hind femur and tibia ca. 1.4>< and 1.5x as

spinules as well as hairs. Subcosta haired. Hair tuft ofwide as hind basitarsus, respectively. Calcipala welldeveloped, ca. 1.1x as long as wide. Pedisulcus welldeveloped. Wing. Length 2.0 mm. Other features as instem vein yellow. Basal portion of radius fully haired.

Basal cell absent. Abdomen. Basal scale dull yellow
with fringe of whitish hairs. Dorsal surface of abdomen

except subcosta bare. Abdomen. Basal scale lightdark yellowish brown or grayish brown on segment 2,
somewhat darker on segments 3 and 4, and dark brown

brown, with fringe of yellow hairs. Dorsal surface ofon segments 5‑9, with dark hairs; tergite of 2nd segment
dark yellowish and whitish pruinose, tergites of othersegment 2 pale, and those of segments 3 and 4 Iight
brown, and covered with yellow and dark hairs; seg‑

segments brown to dark brown, those of segments 6‑9

ments 2, 5‑7 each with dorsolateral pair of shiny, whitish

shiny. Genitalia (Figs. 5 & 6) . Sternite 8 bare medially,pruinose patches; those on segment 2 widely connected

abdominal segments brownish black except that ofwith 30‑35 dark macrosetae on each side. Anterior

in middle. Genitalia (Figs. 8‑14). Coxite nearly rectan‑

gonapophysis thin, membraneous, rounded posterointer‑
nally, covered densely with microsetae with a few shortsetae near anterior border; inner margin well sclerot‑
ized. Genital fork of usual inverted‑Y form, with arms
folded medially, Iacking any projection directed for‑gular in ventral view, ca. 1.7x as long as wide. Style ca.

ward. Paraproct moderately produced ventrally, ca.
0.7x as long as wide in lateral view. Cercus ca. 0.46 x

convex medially in ventral view; ventral plate widely
produced ventrally along posterior margin, with mi‑

as long as wide, with posterior border rounded whencrosetae almost entirely on ventral, posterior and dorsal

viewed laterally. Spermatheca ellipsoidal, well sclerot‑
ized except small area around tubal juncture; internal

thorax. Upper eye consisting of large facets in 11 or '12

surface; basal arms slightly converging. Paramere with
3 Iong parameral hooks and several srnall, indistinct
ones. Median sclerite plate‑like, widened toward tip,with round and transparent tip.
Pupa. Body length (excluding gill filaments) ca. 3.0 mm.

vertical columns and in 12 or 13 horizontal rows.

Head and thorax. Integument pale yellow, moderately

setae absent.

Male. Body length 2.6 mm. Head. Slightly wider than

0.85 >< Iength of coxite, tapered toward apical tip, gently

curved inward, with apical spine (though I of 3 (71examined lacked apical spine) . Ventral plate trans‑verse, with posterior margin nearly straight or slightly

71

磯

o

％

1鷹＼
・雌

ヤ
も閣・禍

4

5

輝、

纏
、

ノ

ず

i

綴譲，、，、ξ盤

り

窮

M
翻

9

7

1、＼纐繍

漁
蕊

讐

8

6

13

11

ピロ

14

ロエずハら

譲齢
10

瀦
17

，灘

轡漫

18

（

密
15

19

16

20

Figs． 1−20S魏銘1勉窺（Oonψhos云歪1伽）勿枷ng召ns6sp．nov．1，♀fronto−ocular
﹃p

alpal

segmentwithsensory

anterior

gonapophyses，genital

paraproctandcercus（1ateralarea；2＆3，3rd

vesicle（2，一♀；3，♂）；4，♀cibarium；5＆6，♀genitalia

fork，paraproct，cercusview）；7，♂hind

tibia

and

and

5，8th

spermatheca初s枷（ventral

tarsi；8−14，♂genitalia

maxillarystemite，

view）；6，

8，coxite，styleand ventral plate初s尭％（left coxite and style omitted，ventral view）；9，style with apical spine（ventromedial view）；10，ventral plate with posterior border somewhat rounded（ventral view）111＆
12，ventral plate（11，1ateral view；12，end vieW）；13，paramere（ventral view）；14，median sclerite；15，
1eft pupal gill（dorsal view）；16，basa11／40f pupal gill showing arrangement of7slender filaments（dorsomedial view）；17，terminal hooks of pupal abdomen（central view）118，1arval head capsuleshowingbranched

hypostomium and postgenal cleft（ventral view）；19，apex of larval mandible；20，dark，
spinules on dorsal surface of larval abdominal segments5−9（scale bar O．02mm）．

72

covered with tubercles. Head trichomes 4 pairs, all longand simple. Thoracic trichomes 6 pairs (3 anterodorsal‑
ly, 2 anterolaterally, I posterolaterally) , all long andsimple. Antennal sheath with smooth surface. Gill
(Figs. 15 & 16) composed of 1 Iong, inflated, tubular
filament (ca. 1.8 mm long by 0.07‑0.11 mm wide) and 7

slender filaments (ca. 1.Imm long by 0.01‑0.016mm
wide) ; inflated filament pale brown, gently curved
inwards, with round distal tip, densely covered withminute tubercles on its surface; 7 slender filaments
arranged in 3 groups, i.e., 2 triplets with short common
stalk arising dorsomedially near base of inflated fila‑
ment, and I isolated filament arising a little far frombase of inflated filament; all slender filaments pale
brown, directing forward, subequal in thickness to one

another, with numerous transverse ridges, moderately
covered with minute tubercles. Abdomen. Terga I and 2
almost transparent, without tubercles; tergum I with 2

abdominal segments 1‑4 moderately covered with mi‑nute, dark spinules with 4‑8 branches dorsally; remain‑ing posterior segments of abdomen densely covered withminute, dark spinules with 2‑11 (mostly 7‑10) branches
dorsally (Fig. 20); Iast segmel)t densely covered withnurnerous uncolored setae on each side of anal sclerite.

Rectal papilla compound, each of 3 Iobes with 7‑9finger‑1ike secondary lobules. Anal sclerite of usual X‑
form, with anterior arms 0.8 x as long as posterior ones,
broadly sclerotized at base. Ventral papillae well devel‑.oped. Posterior circlet with ca. 86 rows of up to 14
hooklets per row.

TYPE SPECIMENS. WEST SUMATRA: Holotype(BMNH), slide‑mounted with pupal exuvia, collectedfrom a stream, crossing a road, nearly midpoint from
Padang to Lubukslasih, near a restaurant just before themountain pass, 1.060 m in altitude, 9. VIII. 1994, by H.

long setae on each side, tergum 2 with 6 simple setae oneach side, I seta much longer than others. Terga 3 and4 each with 4 hooked spines directed forward on each

Takaoka. Paratypes: I c l and 2 mature larvae

side. Terga 6‑9 each with transverse row of spine‑
combs directed caudad on each side. Tergum 9 withalcohol, same data as holotype.

(BMNH), slide‑mounted, 2 c71 together with pupal
exuvia and cocoon, and 6 mature larvae (BAU) , inpair of flat, widened terminal hooks with outer margin

ECOLOGICAL NOTES. Pupae and larvae of thisweakly serrate (Fig. 17). Sternum 4 with I distinct

species were collected from fallen pine leaves in a small,

simple hook and a few minute setae on each side; ster‑

shaded, forest stream with a bedrock and a width of 0.3‑

num 5 with pair of bifid hooks on each side; sterna 6 and

0.5 m. Water temperature was 19'C.

7 each with pair of inner bifid (or trifid) and outer

REMARKS. This new species is characterized by its

simple hooks on each side; Iast segment with a few
grapnel‑like hooklets ventrolaterally on each side.

pupal gills consisting of one inflated tubular and seven

Cocoon. Simple, wall‑pocket‑shaped, moderately

slender filaments. There is no report on such uniquewoven.

pupal gills among species of Gomphostilbia.

Mature larva. Body length 4.6‑5.4mm. Body color

The female and male of this species are similar inlight yellowish gray. Cephalic apotome yellowish with
faint or moderate positive head spots, moderately cov‑
ered with minute, uncolored setae; ventral surface ofhead capsule (Fig. 18) markedly darkened along lateralborder of postgenal cleft. Antenna with 3 segments andleg coloration to S. (G.) sundaicum from Java andapical sensillum, Ionger than stem of labral fan; propor‑

tional lengths of 3 segments from base to tip 1.00:0.89:
0.78. Labral fan with ca. 39 main rays. Mandible (Fig.
19) with comb‑teeth decreasing in size from Ist to 3rdteeth; mandibular serration composed of I Iarge and 1
small tooth, without supernumerary serration. Hypo‑
stomium (Fig. 18) with row of 9 apical teeth; median
tooth somewhat longer than each corner tooth which isslightly longer than 3 intermediate teeth on each side;

lateral margin smooth; hypostomal setae 4 or 5 innumber lying subparallel to lateral margins. Postgenal
cleft (Fig. 18) Ianceolate, deep, 6.0‑7.5x as long as

postgenal bridge, constricted basally. Thorax and

Sumatra and S. (G.) whartoni and S. (G.) cheongi from
Peninsular Malaysia (Takaoka and Davies, 1995; 1996)
but are distinguished by the shape of the cibarium andthe mandible in the female and somewhat inflated hindbasitarsus in the male.
The mature larva of this species is easily separated
by its unique shape of gill histoblast developing on eachside of the thorax.

Simul ium ( Simul ium) sumatraense sp. nov.

DESCRIPTION. Female. Body length 2.5 mm. Head.
Narrower than width of thorax. Frons black, shiny,
with several dark stout hairs along lateral margins;
frontal ratio 1.3:1.0:1.2; frons‑head ratio 1.0:4.0. Fronto‑

ocular area (Fig. 21) well developed. Clypeus black,shiny, thickly whitish gray pruinose, with scattered,73

2nd segment dark brown with large, dorsolateral, whit‑

dark, stout hairs; when illuminated, silvery iridescent.
Antenna composed of 2 + 9 segrnents, dark brown, scapeish iridescent spots broadly connected in middle; tergites

and pedicel yellow. Maxillary palp composed of 5

3‑5 small, dark.brown; tergites 6‑8 Iarge, black, shining,segments, proportional lengths of 3rd, 4th and 5th seg‑ments 1.0:1.0:2.3; 3rd segment of moderate size; sensory
vesicle (Figs. 22 & 23) of mediurn size, elliptical, withrugged surface, 0.37‑0.40 X Iength of 3rd‑ segment, withlarge, round opening a little distad to center. Maxillary
lacinia with 13 inner and 16 outer teeth. Mandible withca. 32 inner and 13‑15 outer teeth. Cibarium (Fig. 25)

with dark hairs. Genitalia (Figs. 33 & 34). Ventral
surface of abdominal segment 7 with submedian pair of
weak sternal plates of irregular form. Sternite 8 wellsclerotized, bare medially, with ca. 24 Iong stout hairs
laterally on each side; anterior gonapophysis inverted

trapezoidal, membraneous, covered with 17‑19 shorthairs and numerous microsetae except narrow portion

with numerous minute tubercles. Thorax. Scutum

along posterior margin bare; inner border nearlybrownish black, shiny, moderately covered with recum‑
bent, dark brown pubescence, interspersed with long,straight, narrowly sclerotized. Genital fork of inverted‑of lights. Scutellum brownish black, with long, darkhairs. Postscutellum brownish black, shiny, silvery

Y form, with well sclerotized slender stem; arms slen‑der, each with strongly sclerotized distal ridge anddistinct projection directed anteriorly. Paraproct inventral view a little wider than long, with ca. 8 minutesensilla on anterointernal surface, covered with ca. 36
short and long hairs ventrally and laterally; in lateral

iridescent when viewed in lights, without hairs. Pleuralview paraproct not produced ventroposteriorly below

membrane bare. Katepisternum longer than deep, bare.
Legs. Foreleg: coxa and trochanter yellow; femur yel‑cercus. Cercus in lateral view rounded posteriorly, ca.

upstanding, dark hairs on prescutellar area; scutum withbright iridescent spot on each shoulder extending poster‑
iorly along lateral border to wing base in certain angles

10w with distal 1/4 brownish black; tibia (Fig. 26) white

0.5x as long as wide, covered with ca. 20 short hairs.
Spermatheca nearly globular, well sclerotized except

with distal 1/4 and basal 1/3 (except outer surface
white) brownish black, outer median portion largely

tube and small area of tubal base, with no definite

iridescent in lights; basitarsus entirely black, moderate‑

setae.

reticulate pattern except near base, with minute internally dilated (W: L=1.0:5.1) , with thick dorsal hair crest;

Male. Body length 2.6 mm. Head. As wide as thorax.

rest of tarsal segments black. Midleg: coxa and tro‑
chanter brownish black except basal 1/2 of trochanter

Upper eye consisting of large facets in 16 horizontal
rows and in 14 vertical columns. Clypeus black, whitish
gray pruinose, strongly iridescent when illurninated,sparsely covered with dark brown hairs. Antenna com‑yellow; femur entirely brownish black; tibia in outside
view (Fig. 28) yellowish white on basal 1/3 and brown
to brownish black on distal 2/3 (except a little morethan basal 1/2 of posterior surface and basal 2/3 ofanterior surface yellowish white) , in certain lights withlarge area of whitish sheen on posterior surface; basitar‑sus whitish except distal 1/5 brownish black; rest of
tarsal segments brownish black except basal 1/3 of 2nd
segment white. Hind leg (Fig. 30) : coxa brownish black;trochanter yellow; femur brownish black except basal
1/5 pale yellow; tibia yellowish white on basal 1/2,

brown to brownish black on distal 1/2, with brown
subbasal patch, in certain lights with large area ofwhitish sheen on posterior surface; tibia 0.83 >< as wide

as femur; tarsal segments brownish black except a little
more than basal 2/3 of basitarsus and basal 1/2 of 2nd

posed of 2+9 segments, dark brown except base of
pedicel and base of Ist flagellar segment pale yellow; Ist

flagellomere elongated, 1.8x as long as 2nd one. Maxil‑
lary palp composed of 5 segments, proportional lengths
of 3rd, 4th and 5th segments 1.0:1.3:2.3; 3rd segment (Fig.

24) of normal size, with small sensory vesicle 0.27><
length of 3rd segment; opening of sensory vesicle small.
Thorax. Scutum brownish black, with silvery iridescentpattern composed of anterior pair of spots on shoulders,large transverse spot on prescutellar area contiguous toanterior spots by band along lateral margins; spot on‑each shoulder curved posteromedially, somewhat point‑
ing posteriorly appearing like a crescent band; scutum

uniformly covered with copper‑colored, recumbent

tarsomere whitish; basitarsus W:L=1.0:5.3, calcipala
short, W:L ratio 1.1:1.0; pedisulcus distinct. Tarsal
claws (Fig. 32) each with small, subbasal tooth. Wing.
Length 2.6 mm; costa with spinules and hairs; subcosta

pubescence, interspersed with long, upright, dark hairs
on prescutellar area. Scutellum, postscutellum, pleuralwith several hairs; basal section of vein R bare; hair tuft

basal 3/4 (though somewhat darkened distally) , brown‑

at base of stem vein dark brown; basal cell absent.
Abdomen. Basal scale black with fringe of dark hairs;

ish black on distal 1/4; tibia (Fig. 27) white except basal

membrane and katepisternum as in . Legs. Foreleg:coxa yellow; trochanter dark yellow; femur yellow on

1/3 and distal 1/3 on inner and lateral surfaces brownish

74

black, with large area of whitish sheen on outer surface;tarsi black; basitarsus sornewhat dilated (W:L= 1.0:5.7) .

Midleg: coxa, trochanter and femur brownish black
except basal 1/3 of trochanter yellow; tibia (Fig. 29)

brown to brownish black with base yellow, and anterior
surface somewhat paler submedially; basal 1/3 on poste‑rior surface with whitish sheen in certain angle of light;
basitarsus whitish except distal 1/5 brownish black (itsborder not well defined) ; rest of tarsal segments brown‑

ish black except base of 2nd and 3rd segments pale.
Hind leg (Fig. 31): coxa brownish black; trochanterdark yellow; femur brownish black except basal 1/5
yellow; tibia brownish black except base pale yellow;
tarsal segments brownish black except basal 1/2 ofpupa, decreasing in length and thickness from dorsal toventral; dorsal filamenf' of upper pair longest 2.6‑2.7mm) , veritral one of lower pair shortest (1.3‑1.4 mm),other filaments intermediate in length (1.5‑2.Imm) ;cuticle of filaments with well‑defined annular furrows
and ridges forming reticulate pattern, uniformly covered

with minute tubercles. Abdomen. Tergum I pale yel‑
10wish brown with I Iong seta on each side. Tergum 2
on each side with I Iong and 5 short simple setae intransverse row along posterior margin, and on eachanterolateral portion with numerous comb‑like groupsof minute spines. Terga 3 and 4 each with 4 hooked
spines along posterior margin on each side. Terga 7 and
8 each with transverse row of spine‑combs on each side.

basitarsus and a little less than basal 1/2 of 2nd tarsal

Tergum 9 with pair of small, cone‑shaped, terminal

segment whitish; hind basitarsus enlarged, wedge‑
shaped (W:L=1.0:3.9), subequal in greatest width tohooks, and comb‑like groups of minute spines. Sternum
4 with a few, minute, simple setae submedially on eachside. Sternum 5 with pair of bifid hooks submedially oneach side. Sterna 6 and 7 each with pair of bifid innerhind femur, but a little narrower than hind tibia; cal‑
cipala small, W: L ratio 1.0:1.0; pedisulcus distinct.

Wing. Length 2.4 mm; other features as in except
subcosta bare. Abdomen. Basal scale blackish withlong, dark hairs. Terga brown to brownish black withdark hairs; segments 2, 4‑7 each with pair of silvery
iridescent areas dorsolaterally, those on segment 2
broadly connected in middle. Genitalia (Figs. 35‑39).

and simple outer hooks widely spaced on each side.
Grapnel‑1ike hooklets absent. Cocoon. Wall‑pocket‑

Coxite in ventral view nearly quadrate, 0.57 x as long asrior 1/2, with 3 distinct darkened areas on posterior 1/2

style; style length 3.7x width at base, nearly parallel‑(1 medially just before posterior border, the other 2subrnedially between head spots) ; all head spots pale,sided throughout its length in ventral view; stylespatulate ventrodorsally, with subterminal spine; noprominent subbasal protuberance present. Ventral plateand basal arms in ventral view forming a broad U‑
shape, having low ventral projection with toothed poste‑rior margins (7 or 8 teeth on each side) ; plate withmidventral ridge anterior to ventral projection, slightly

shaped, thickly woven, with thick anterior rim; there isno ventrolateral extension.

Mature larva. Body length 5.4‑6.0 mm. Body color darkgray. Cephalic apotome pale yellowish white on ante‑

except mediolateral spots sometimes somewhat dark=
ened, then appearing negative type; Iateral surface ofhead capsule widely darkened behind eye spot area, withnegative lateral spots near posterior border; ventral
surface of head capsule (Fig. 41) dark, specially nearborder of postgenal cleft, with negative spots. Antenna

wider than plate, covered with fine appressed setae

composed of 3 segments and apical sensillum, Ionger

anteriorly; basal arms long, curved outwardly and for‑wardly. Median sclerite of shoehorn shape, thin, gradu‑

than stem of labral fan; Iength ratio of segments (frombase to tip) 1.00:1.06:0.65. Labral fan with ca. 36 main

ally widened toward apex. Paramere with numerous
medium‑sized hooks.
Pupa. Body length (excluding gill filaments) 3.0 mm.
Head. Integument dark yellow, very sparsely covered

rays. Mandible (Fig. 42) with mandibular serration

with tubercles, with I facial and 2 frontal pairs of short,

slender, simple trichomes, facial trichomes much longer

than frontal ones. Thorax. Integument yellowish

composed of I Iarge and I small tooth, without supernu‑

merary serration; Ist comb‑tooth much longer thanothers. Hypostomium with 9 small anterior teeth, ofwhich median and each corner tooth are longer thanothers; 5 or 6 hypostomal bristles diverging posteriorly
from lateral border on each side. Postgenal cleft (Fig.

brown, sparsely covered with tubercles, with 2 dorsal
and 2 Iateral pairs of rather long, simple trichomes on41) deep, widest at base, anterior 2/3 gradually nar‑anterior 1/2 plus I Iateral pair of trichomes on posterior

bridge. Thoracic cuticle bare. Abdominal cuticle bare
except dorsolateral areas on last segment moderately
covered with short, uncolored setae. Rectal papilla

1/2, those trichomes are subequal to each other, butmuch longer than facial ones. Gill (Fig. 40) with 6 darkbrown, slender filaments in pairs, very short‑stalked,
gradually tapering toward apex, somewhat shorter thanrowed toward anterior tip, ca. 4.0 X Iength of postgenal

compound, each of 3 Iobes with 10 or 11 slender, finger‑

like, secondary lobules. Anal sclerite X‑shaped, with

75C ) )Q0'ooo,
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31

Figs. 21‑42 Simulium (Simulium) sumatraense sp. nov. 21,
fronto‑ocular area; 22‑24. 3rd maxillary palpal
segment with sensory vesicle (22 & 23, ; 24, c ) ; 25, cibariurn; 26 & 27, fore tibia (26, ; 27, c71);
28 & 29, mid tibia (28, ; 29, c ); 30 & 31, hind leg (30, ; 31, c l); 32,
claw; 33 & 34,
genitalia

33, 8th sternite, anterior gonapophyses, genital fork, paraprocts, cerci and spermatheca in situ
(ventral view) ; 34, paraproct and cercus (1ateral view) ; 35‑39, c71 genitalia
35, coxite, style,ventral plate and pararneres in situ (1eft coxite and style omitted, ventral view) ; 36 & 37, ventral plate(36, Iateral view; 37, end view) ; 38, paramere (1eft side only, end view) ; 39, median sclerite; 40, pupal

gill filaments (outside view, scale bar 0.2 mm) ; 41, Iarval head capsule showing hypostomiurn andpostgenal cleft (ventral view) ; 42, apex of larval mandible.

76

broadened anterior arms ca. 0.5x length of posterior
ones. Posterior circlet with ca. 88 rows of hooklets with

margin and above antennal base; frontal ratio 1.20:1.00:
1.13; frons‑head ratio 1.0:4.0. Fronto‑ocular area (Fig.

up to 13 hooklets per row. Ventral papillae absent.

43) narrowly developed, with triangular lateral tip.
Clypeus brownish black, shiny, thickly white pruinose,

TYPE SPECIMENS. WEST SUMATRA: Holotype

covered moderately with dark hairs except median(BMNH), slide‑mounted, together with pupal exuvia,

portion on upper 1/2 Iargely bare. Antenna composed of
2 + 9 segments, brown except scape, pedicel and base ofIst flagellar segment yellow in dorsal view. Maxillaryjust south of Lake Dibawah, ca. 1,580 m in alt., 21. VIII.

1994, by H. Takaoka. Paratypes: I c l, slide‑mounted,
together with pupal exuvia, I pupa, 3 mature larvae(BMNH), same data as holotype; I , I mature larva(BAU) , just southeast of Lake Diatas, along the roadfrom Alahanpanjang to Surian, ca. 1,470 m in alt., 12.
VIII. 1994, by H. Takaoka.

palp brownish black, composed of 5 segments withproportional length of 3rd, 4th and 5th segrnents 1.0:1.2:

2.4; 3rd segment (Fig. 44) of moderate size; sensory
vesicle medium in size, elliptical, with rugged surface,0.24 x length of 3rd segment, with medium round open‑
ing near distal end. Maxillary lacinia with 11 inner and

ECOLOGICAL NOTES. Pupae and larvae were found13 outer teeth. Mandible with ca. 28 inner and 12 or 13

on twigs caught in the water of two small streams (one,outer teeth. Cibarium (Fig. 45) with ca. 12 minute

0.4‑0.6 m wide, shaded, running through the natural

tubercles. Thorax. Scutum brownish black, shiny, thin‑

forest beyond the cultivated field for tea plantation, andly white pruinose, covered moderately with yellow

the other, 0.3‑0.5 m wide, partially shaded, running frompubescence, interspersed with long, upstanding, darkhairs on prescutellar area. Scutellum brownish black,the forest into the rice field) .

with long, dark hairs and yellow pubescence. Postscutel‑

REMARKS. This species is closely related to S. (S.)
celsum and S. (S.) nebulicola from Java (Takaoka andDavies, 1996). The genitalia of both sexes are almost
the same in these three species. However this species
differs from these two known species in the adults by
the leg coloration: the femora of both female and malemidlegs of this species are entirely dark while those of
the other two species are pale at least basally, and also

the female hind tibia has a subbasal dark spot in thisspecies but lacks such a spot in the other two species.
The pupa of this species is almost the same as that of S.

lum brownish black, shiny, white pruinose, without
hairs. Pleural membrane bare. Katepisternum longer
than deep, bare. Legs. Foreleg: coxa and trochanter
yellow; femur yellow on basal 1/2, dark yellow to yel‑lowish brown on distal 1/2; tibia white with distal 1/6

brown, and with median anterior portion largely white
sheeny in lights; basitarsus entirely brownish black,dilated (W:L=1.0:5.4); rest tarsal segments brownish

black. Midleg: coxa brownish black; trochanter andfemur brown with basal l/3 or 1/2 of trochanter yellow;
tibia white except distal 1/6 brown, posterior surface(S. ) celsum, but differs from that of S. (S. ) nebulicola bylargely with a white sheen when illuminated; tarsi

the longer gill filaments and the presence of the ,terminal

brownish with basal 3/4 of basitarsus, basal 2/5 of 2nd

hooks. This species appears to be distinguished in thelarva from the other two species by the unicolored body.
The female of this species is somewhat similar tothat of S. (S.) fuscopilosum Edwards from Peninsular
Malaysia (Takaoka and Davies, 1995) in several charac‑

segment and base of 3rd segment white. Hind leg: coxa
brownish black; trochanter yellow; femur dark brown tobrownish black except base yellow; tibia white with aters including the unpatterned scutum, claws with ailluminated; tarsi brown with a little less than basal 314

little less than distal 1/2 brown, posterior surface along

basal 1/2 or a little more with a white sheen whensubbasal tooth, genital fork with a strong projection onof basitarsus and basal 1/2 of 2nd segrnent white;each arm, paraproct not produced ventrally, hind tibia
with a subbasal dark spot. However, there are differ‑basitarsus (Fig. 46) parallel‑sided, W:L=1.0:6.1, its

ences in the cibarium, sensory vesicle, Ieg coloration andcalcipala short, L:W ratio 1.0:1.1, and ca. 0.6 >< as wide as

shape of the anterior gonapophyses.

distal tip of basitarsus; pedisulcus distinct. All tarsal

Simulium (Simulium) minangkabaum sp. nov.

width ca. 0.7x that of tibia and ca. 0.6 x that of femur;

claws (Fig. 48) with small subbasal tooth. A11 femora
and tibiae covered with scale‑like setae on outer sur‑face. Wing. Length 2.3 mm; costa with spinules and

DESCRIPTION. FEMALE. Body length ca. 2.5 mm.

hairs; subcosta haired except distal 1/5 or 1/6 bare;Head. Narrower than width of thorax. Frons brownish

basal section of vein R bare; hair tuft of stem vein dark

black, shiny, with several dark hairs along each lateralbrown; basal cell absent. Abdomen. Basal scale brown‑

77

ish black with fringe of pale hairs; all segments brown‑
silvery iridescent spots broadly connected to each othersomewhat dilated, W:L=1.0:5.5. Midleg: coxa brownish
black; trochanter and femur dark brown to brownish
black except base of trochanter yellow; tibia dark

in middle; tergites 3‑5 small and dull; tergites 6‑8 Iarge,

brown with posterior surface of basal 1/3 white, with ashiny, with dark hairs. Genitalia (Figs. 49‑51).

white sheen when illuminated; tarsi brown with basal

Ventral surface of abdominal segment 7 without devel‑

1/2 or a little less of basitarsus pale yellow. Hind leg:

oped sternite. Sternite 8 well sclerotized, bare medially
but with 8‑14 dark, Iong, stout hairs laterally on eachside; anterior gonapophysis tongue‑1ike in shape, mem‑coxa brownish black; trochanter yellow; femur brownish
black except base yellow; tibia brownish black except
extreme base white; tarsi brownish black except basal

braneous, bent ventrally, covered with 6‑8 somewhat

1/2 of basitarsus and of 2nd segment whitish; basitarsus

10ng hairs as well as numerous microsetae; inner borders(Fig. 47) enlarged, W:L=1.0:4.0, and only slightly nar‑concave, widely separated in middle from each other.
Genital fork of inverted‑Y form, with well sclerotizedrower than femur at greatest width, which is slightly

stem; arms narrow, each with strongly sclerotized distalbulge and with distinct projection directed anterodorsal‑
ly. Paraproct enlarged, nearly as long as wide in ventral

Wing. Length 2.2 mm; other features as inexceptsubcosta bare. Abdomen. Basal scale brownish blackview, somewhat produced ventrally and ventroanterior‑with dark hairs; segments 2, 5‑7 each with pair of silvery

ish black; tergite 2 with pair of large, dorsolateralnarrower than tibia; calcipala small, pedisulcus distinct.

with long dark hairs; tergites brown to brownish blackly, covered with 9‑14 short, stout hairs in lateral view;and blueish iridescent spots dorsolaterally; those on

surface of anteroventral corner densely covered withsegment 2 broadly connected in middle. Gen'italia

pale setae; inside surface largely concave and well scler‑with definite reticulate pattern, and with minute internal(Figs. 52‑55). Coxite nearly quadrate. Style elongate,ca. 2.0X as long as coxite, narrowed from basal 1/3 toapical tip, and with apical spine; style flattened dor‑soventrally, having no basal protuberance and the poste‑rior third curved medially. Ventral plate broadly Y‑

setae; tube and small area of tubal base unsclerotized.

shaped, with narrow body in ventral view, having

Male. Body length 2.5‑2.8 mm. Head. Slightly wider

median process directed ventrally, and with a few finesetae on each lateral side; posterior surface of medianotized anteriorly and with several minute setae. Cercus
semilunar in lateral view, covered with numerous shorthairs. Spermatheca nearly globular, well sclerotizedthan thorax. Upper eye consisting of large facets in 18

horizontal rows and in 16 or 17 vertical columns.
Clypeus black, silvery pruinose, strongly iridescent whenprocess with vertical row of 4 small spines on eachlateral margin; basal arms stout, diverged from each

illuminated, moderately covered with dark hairs except
median portion largely bare. Antenna composed of 2 +9 segrnents, dark brown; Ist flagellomere 1.5x as longmeres wide, each with several small hooks. Medianas 2nd flagellomere. Maxillary palp composed of 5

Pupa. Body length (excluding gill filaments) ca. 2.8 mm.

other, with apical tip rernarkably curved inward. Para‑
sclerite wide, thin, and with small medial notch apically.

segments, proportional lengths of 3rd, 4th and 5th seg‑

Head. Integument yellow, covered moderately or

ments 1.0: 1.4: 3.1; 3rd segment of normal size, with small

sparsely with small tubercles except frontal surface
almost bare, and with I facial and 2 frontal pairs of
simple trichomes (facial trichomes much longer thanfrontal ones) . Thorax. Integument yellow, without any

sensory vesicle. Thorax. Scutum black, with silvery
and blueish iridescent pattern composed of anterior pairof large spots on shoulders, Iarge transverse spot onprescutellar area contiguous to anterior spots by broadband along lateral margins; anterior pair of spots widely

protruding ridges, covered moderately with srnall tuber‑cles laterally and posterodorsally but almost bare anter‑

separated from each other in middle, curved postero‑odorsally; thorax anteriorly with 3 dorsal and 2 dor‑

medially with posteromedial end somewhat pointed;

solateral pairs of long, sirnple and somewhat stout tri‑chomes (occasionally I of those bifid) and also posteri‑orly with I Iateral pair of simple trichornes. Gill (Fig.

scutum uniformly covered with brown pubescence, inter‑
spersed with long, upright, dark hairs on prescutellar
area. Scutellurn dark brown with long, upright, darkhairs. Postscutellum dark brown, white pruinose, shiny.

Katepisternum and pleural membrane as in56) usually with 8 short, slender filaments basicallyarranged in 2 groups (i.e., inner and outer groups) , inner. Legs.

group mostly consisting of I middle and 3 dorsal fila‑

Foreleg: coxa and trochanter pale yellow; femur brownwith basal 1/5 or 1/4 yellow or dark yellow; tibia darkments (i.e., 2 paired and I individual filaments) or rarely

brown with outer surface mostly white, with a whiteouter group consisting of 2 middle and 2 ventral paired
filaments with stalk of variable lengths; in about 1/3 ofsheen when illuminated; tarsi brownish black; basitarsus

consisting of 2 dorsal and 2 middle paired filaments, and

78

欝醗
驚．

麹

轡

竃．

嚇・

8。

43

45

44

イ庵 ︐r
壷
薩
塾
ミ
賢︑︑
︸
f
ンy4
〜〆
孤7 ︑

窺
七

戸〆

49

＼

52

47

46

54

㌔

■

、鷹

48

53

55
57

58
65

59
〆

60

1〆4

v磁

oグ

63

64

‡）壬！≡

懸笠

56

霧

61

〜

懸
62

Figs．43−65S勉％」伽郷（S加％1伽窺）郷初αng肋肱％窺sp．nov．43，♀fronto−oculararea；44，♀3rd

maxillarypalpal segment with sensory vesicle；45，♀cibarium；46＆47，hind basitarsus参nd2nd tarsal segment（46，♀；47，♂）；48，♀claw；49−51，♀genitalia
49，8th stemite，anterior gonapophyses，genital
fork，paraproct，cercus and spermatheca初s枷（ventral view）；50＆51，paraproct and cercus（50，ventral view；51，lateral view）；52−55，♂genitalia
52，coxite，style，ventral plate and parameresづn s伽（1eft coxite and style omitted，ventral view）；53＆54，ventral plate（53，1ateral view；54，endview）；55，median sclerite；56，right pupal gill（outside view，scale bar O．2mm）；57−60，basa11／20f
pupal

group
genal

gill

showing various arrangements of imer group filaments（outside view，4filaments of outer
omitted）；61，cocoon（1ateral view）；62，1arval head capsule showing hypostomium and post−

cleft（ventralview）；63，apexoflarvalmandible；64，uncolored

thorax and abdomen（scale bar O．01mm）；65，wholeances on abdominal segments1−5（lateral view）．

body

of

mature

setae

on

larva

dorsalsurfaceofshowing

dorsalprotuber−larval79

Table I Variation in the number of pupal gill filamentsof Simulium (Simulium) minangkabaum sp. nov.
Numbers of pupae collected at two localities
with different numbers of gill filaments are

shown(Fig. 62) uniformly darkened to varying extent except

near posterior margin of hypostomium widely paleyellow. Antenna composed of 3 segments and apicalsensillum, Ionger than stem of labral fan; Iength ratio ofsegments (from base to tip) 1.0: 1.3: 0.6. Labral fan with

Talang

9

o

1

1

2

54‑58 main rays. Mandible (Fig. 63) with 2nd and 3rd
comb‑teeth of equal size, shorter than Ist one; man‑
dibular serration composed of I medium and I small

Lake Diatas

28

o

1

2

10

tooth but without supernumerary serration. Hypos‑

No. filaments (1eft, right or vice versa)
Localities(8, 8) (8, 9) (9, 9) (9, 10) (10, 10)

the pupae examined (Table 1), gill with 9 or 10 fila‑
ments, caused by variations in filarnent number of innergroup (Figs. 57‑60), e.g., individual filament of dorsal
triplet is further divided into 2 filaments, with stalk ofvariable lengths, or additional filament arising from thebase of dorsal triplet, and/or middle filament is bran‑ched into 2 filaments with stalk of variable lengths;
middle filament is occasionally branched into 4 fila‑
ments in pairs while individual filament of dorsal triplet

disappears; gill filaments yellow to brown, as dark as or

darker than thoracic integument, increasing in lengthfrom dorsal to vehtral (ca. 0.8‑1.6 mm long) , tapering
toward tip, with moderate annular ridges and furrows
throughout their length, forming reticulate pattern,covered with minute tubercles. Abdomen. Tergum 1

tomal teeth 9 in number, small, with median and eachcorner tooth longer than others; Iateral margins marked‑
ly serrate apically; 5 or 6 hypostomal bristles diverging

posteriorly from lateral border on each side. Postgenal
cleft (Fig. 62) very deep, rounded, ca. 4.0 >< as long as

postgenal bridge. Thoracic and abdominal cuticle
moderately covered with very minute, flat, uncolored
setae (Fig. 64) dorsally and dorsolaterally. Abdominal
segments 1‑5 each with dorsolateral pair of conicalprotuberances (Fig.,65) . Rectal papilla compound, eachof 3 Iobes with 8‑11 finger‑1ike secondary lobules. Analsclerite X‑shaped, with broadened anterior arms ca.
0.7x as long as posterior ones. Last abdominal segment
bulged laterally but lacking ventral papillae. Posterior
circlet with 90‑96 rows of hooklets with up to 16 hook‑lets per row.

pale yellowish brown, with I simple, slender hair on eachside. Tergurn 2 on each side with 1 Iong, simple seta and

TYPE SPECIMENS. WEST SUMATRA: Holotype

5 short, simple, spinous setae, 3 or 4 of which are some‑(BMNH) , slide‑mounted with pupal exuvia, just south‑
east of Lake Diatas, along the road from Alahanpanjang
to Surian, ca. 1,460 m in alt., 12. VIII. 1994, by H.

what stout. Terga 3 and 4 each with 4 hooked spines
along posterior margin on each side. Tergum 8 withtransverse row of distinct spine‑combs on each side.
Tergum 9 with pair of small but distinct, cone‑shaped,
terminal hooks. Sternum 4 with a few, minute, simple
setae submedially on each side. Sternum 5 with pair ofbifid hooks submedially on each side. Sterna 6 and 7
each with pair of bifid inner and simple outer hooks

widely spaced on̲ each side. Grapnel‑like hooklets
absent. Cocoon (Fig. 61). Shoe‑shaped, with rather low
front wall, thickly woven, but anteriorly with several
open interspaces or windows in webs.
Mature larva. Body length 5.5‑6.1 mm. Body color palereddish brown or yellowish green to greenish gray.
Cephalic apotome usually pale yellow to yellow onanterior 1/2, but darkened to varying extent on poste‑rior 1/2, with median narrow portion darkest just before
the posterior margin; anterior median longitudinal spot

Takaoka. Paratypes: I(BAU) , I c 1 (BMNH), bothslide‑mounted, 4, 4 c l , 4 pupal exuviae, 2 maturelarvae(BAU) , 3 , 3 c71 , 6 Pupal exuviae, 4 maturelarvae (BMNH) , all in alcohol, same dasa as holotype.
Other paratypes: 4 , 4 c71 , 2 Pupal exuviae, I maturelarva (BZM) , just south of Talang, along the road from
Solok to Padang, ca. 970 m in alt., 9. VIII. 1994, by H.

Takaoka.

ECOLOGICAL NOTES. Pupae and larvae of this new
species were found attached to trailing grasses, andleaves and stems of aquatic plant in water in two
medium, moderately flowing streams (3‑5 m wide, 0.2‑
0.4 m deep), exposed to the sun, running through hilly
cultivated fields. Water temperature was 20'C.

usually appearing positive, while other head spots

REMARKS. This new species most closely resembles,

appearing positive or negative depending on the extent
of darkness of the ground color, or merged in groundcolor with pale submedial space on each side of poste‑rior longitudinal spot; ventral surface of head capsulein the female, S. (S.) argentipes Edwards, which was
originally described from female specimens collectedfrorn Peninsular Malaysia (Edwards, 1928), in many
characters including the genitalia with subtle differences

80(Takaoka and Davies, 1995). There are only slightdifferences in the leg coloration between the two

REFERENCES

species: in the latter species, the fore femur was almost

1 ) Crosskey, R.W. (1969): A re‑classification of thebrown, the fore tibia was largely white but brown on the
distal tip and along the inner margin, and the hind tibia

Sirnuliidae (Diptera) of Africa and its islands. Bull. Br.

white with the distal 1/3 brown. There may be somedifferences in the other characters, such as the cibarium,

mandible, maxillary palp and sensory vesicle, of which
no detailed descriptions are, however, available for thelatter species.

The other related known species, i.e., S. (S.)
beludense from Sabah (Takaoka, 1996), S. (S.) can‑laonense from the Philippines (Takaoka, 1983), S. (S.)

tanae from south China (Xue, 1992), and S. (S.)
bidentatum from Japan and China (Takaoka, 1976) are
different from this new species by the coloration of thefemale fore femora, which are almost brownish in theformer two known species, and almost yellowish in theMus. Natur. Hist. (Entomol.) suppl. 14, 195p.

2 ) Edwards, F.W. (1928): Diptera Nematocera from theFederated Malay States Museums. J. Fed. Malay StatesMus., 14, 1‑1393 ) Edwards, F.W. (1934): The Simuliidae (Diptera) of Javaand Sumatra. Arch. Hydrobiol. 13 Suppl. "Tropische
Binnengewasser" . 5, 92‑138
4 ) Takaoka, H. (1977) : Studies on black flies of the Nansei
Islands, Japan (Simuliidae, Diptera) . 111. On six speciesof the subgenus Simulium Latreille. Jpn. J. Sanit. Zool.,
28, 193‑217

5 ) Takaoka, H. (1983): The blackflies (Diptera:
Simuliidae) of the Philippines. Japan Society for the
Promotion of Science, Tokyo, xi + 199p.
6 ) Takaoka, H. (1996): Description of a new species ofSimulium (Simulium) from Sabah, Malaysia (Diptera:
Simuliidae) . Jpn. J. Trop. Med. Hyg., 24(3) , 157‑161

latter two.

ACKNOWLEDGEMENTS
We are grateful to Dr. D.M. Davies, Professor
Emeritus, McMaster University, Ontario, Canada for his

valuable suggestions. This study was supported byIndonesian Institute of Sciences (LIPD and by a grant‑in‑aid (no. 030410653) from the Ministry of Education,
Science and Culture, Japan for International Scientific
Research Program entitled "Medicozoological survey onbiogeography and coevolution of vectors, parasites andhosts in Indonesia" headed by Prof. I. Miyagi, Universityof the Ryukyus.

7 ) Takaoka, H. and Davies, D.M. (1995): A new black fly
species of Simulium (Nevermannia) frorn Sumatra, In‑
donesia. Jpn. J. Trop. Med. Hyg., 23(2), 127‑131
8 ) Takaoka, H. and Davies, D.M. (1995): The black flies(Diptera: Sirnuliidae) of West Malaysia. Kyushu Uni‑v rsity Press, Fukuoka, viii + 175p.

9 ) Takaoka, H. and Davies, D.M. (1996): The black flies(Diptera: Simuliidae) of Java, Indonesia. Bishop Mus.
Bul. Entomol. 6, Bishop Museum Press, Hawaii, viii +8lp.

10) Xue, H.T. (1992): A new species of black fly Simulium
from Yunnan Province, China (Diptera: Simuliidae) .
Acta Zootaxonomica Sinica, 17(1) , 93‑96

J pn. J. Trop. Med. H yg

Vol 25 No 2 1997, pp. 81‑84

81

HOOKWORM INFECTION RECORDED AT
AN UNIVERSITY TEACHING HOSPITALIN KATHMANDU, NEPAL OVERONE DECADE PERIODSHIBA KU.MAR RA11.*, HARI GOVlNDA SHRESTHA2, MAMORU NAKANISH13
TAKASHI KUB03, KAZUO ON04, SHOJI UGA5 AND TAKEO MATSUMURAl
Received March 21, 1997/Accepted May 22, 1997

Abstract: We report an annual prevalence of hookworm infection among Nepalese as studied at Par‑
asitology Laboratory of Department of Pathology, Tribhuvan University Teaching Hospital, Kathmandu,
Nepal over one decade period (1985‑1994). A total of 4,164 to 9,440 (average 7,857) fecal samples were
examined each year for the presence of various intestinal parasites. The annual positivity rate forhookworm infection ranged from 3.8 to 10.7 percent. Throughout the study period, a higher incidence of
hookworm infection was observed among adults compared with children. Hookworm infection was morecommon among females compared with their male counterparts except for the year 1989 and 1990. Of thetwo species of hurnan hookworms, 67.0% were Ancylostoma duodenale while remaining 33.0% were Necatoramericanus.
Key words: hookworm, An(ylostoma duodenale. Necator americanus. Nepal

INTRODUCTION
Hookworms are among the most common humaninfection in the causation of severe anemia has also beenreported (Williams and Naraqi, 1979; Fleming, 1989).
Thus, the hookworm infection has a significant impact

parasite and are transmitted by fecal contamination of
the soil. Approximately one billion people, mainly indeveloping countries, are affected by hookworm infec‑

both on health and economic productivity of an individ‑

tion (Gilles, 1985; Capello et l., 1995). The hallmark ofreportedly vary from less than 5.0% to as high as 90.0%

hookworm infection is gastro‑intestinal blood loss andiron deficiency anemia. Blood loss is attributed to theMani et al. (1993) from southern part of neighboringual.

Hookworm infection rate elsewhere in the world(Maldonado, 1993) and 93.0% (Adams et al., 1994),

direct ingestion by worms and bleeding from trauma
produced by worm attachment and feeding. The loss ofblood into the gut is proportional to the worm load
(Gilles, 1985) but becomes disproportionately great
when worms are crowded (Beaver et al.. 1964). Hook‑worm infection in children cause both physical andintellectual growth retardation. Heavy infection may

country India reported an incidence rate of 45.0%

cause death due to severe hemorrhage and shock.

infantile hookworm disease in China. Chaudhary andJayswal (1984) have also reported infantile hookworm

Zimmermann (1946) reported 60.0% fatality rate due toancylostomiasis in children of less than 16 months in

Guam. An association between malaria and hookworm

among primary school children. An unusual outbreak of
hookworm disease after a Kabaddi game (a local game
which results in much body contact with the ground) has

also been reported from North India (Koshy et al.,1978). Infantile hookworm disease has also been report‑ed. Yu et al. (1995) have reviewed hundreds of cases of

disease from India.

Anemia is a common clinical condition among

2

Department of Medical Zoology, Kobe University School of Medicine, Kobe, Japan
Dapartment of Pathology, Tribhuvan University Teaching Hospital (TUTH) , Kathmandu, Nepal

3

JICA Medical Education Project, TUTH, Kathmundu, Nepal

4

Division of Microbiology, Hyogo Prefectural Institute of Public Health, Kobe, Japan
Department of Medical Technology, Faculty of Health Science, Kobe University School of Medicine, Kobe, Japan

1

5

Address for correspondence: Shiba Kumar Rai, PhD (DMSC), Department of Medical Zoology, Kobe University School of
Medicine, 7‑5‑1 Kusunoki‑cho, Chuo‑ku, Kobe 650, Japan

82

Nepalese to which hookworm infection contribute to a

12

great extent. A good correlation between hookworm

10

infection and hemoglobin depletion has been observed in
Nepalese children (Curtale et al.. 1993). In Nepal, the

a,

O
C
O
CP
>
O
L
aL

hookworm infection rate is variable between less than
5.0% to as high as 83.8% in different specified popula‑tion (Gianotti, 1990; Rai and Gurung, 1986; Estevez et

e

8
6

4

al., 1983). Thus, it is necessary to consider an effective

control measures for the control of hookworm and othersoil‑transmitted helminths while formulating any publichealth intervention programme aimed at reducing ane‑mia prevalence in Nepal. Keeping in view of publichealth importance of hookworm infection, here, weo'85 '86 '87 '88 '89 '90 '91 '92 '93 '94 years

Figure I Annual overall rate of hookworm infection in
Nepal.

report an annual incidence rate of hookworm infectionin Nepal as recorded at Tribhuvan University Teaching
Hospital in Kathmandu, Nepal.

12a,MATERIALS AND METHODS
A total of 4,164 to 9,440 (average 7,857) fecal sam‑ples received from patients visiting Tribhuvan Univer‑sity Teaching Hospital for their some kind of ailments
were examined for the presence of intestinal parasites
each year over a period of one decade (1985 to 1994).

D[ Ad u Its

10
oE:
o
>
oL
,l
cll

ll Ohildren

8
6

4

o

'85 '86 '87 '88 '89 '90 '91 '92 '93 '94 years

Patients were provided a clean, dry and screw capped
plastic container for sample collection. An average of
30 samples/day were received. Routine saline prepara‑

Figure 2 Annual rate of hookworm infection in adults andtion (direct smear) was prepared from each sample

during recent years mainly due to the influx of people
from different parts of the country and construction ofhouses. This, in turn, reduced the chances of exposure

received and examined microscopically. Concentration
technique (formal‑ether sedimentation) was applied
only to few doubtful cases (anemia and melena) . In thelater part of the study, 200 hookworm ova positive
samples were subjected to culture by Harada and Mori
(1955) technique. The hookworm larva thus recovered
were identified either as Ancylostoma duodenale or
Necator americanus on the basis of their morphologicalcharacters. The findings were stratified against age(children: 15 and less than 15 years and adults: morethan 15 years) and sex.

RESULT AND DISCUSSION

children.

to the infective hookworm larva among the farming
community (Jyapu) of Kathmandu valley. On the other
hand, the increasing trend observed after 1991 may
reflect the consequence of unplanned urbanization in
Nepal particularly in Kathmandu valley. Most area in
Kathmandu valley represent the urban slum in develop‑
ing countries where sewerage system is virtually non‑existent or insufficient even if it is existed. Drinking

wafer is highly contaminated with fecal matter (Adhi‑
kari et al.. 1986) . Even now, some people in Kathmandu
valley do not have latrine at their house and go for open

defecation that leads to soil contamination. Thus, a

The annual rate of hookworm infection variedstreet flood up to knee level and with bad smell in somebetween 3.8‑10.7% (Fig. 1) with a mean of 7.0 percent.
A declining trend of incidence was observed till the year

parts of city area, is a common phenomena during rainyseason in Kathmandu valley recently. This might have

1991. This appears to be due to the behavioral change ofcontributed hookworm infection in spite of wearing

Nepalese during recent years. Now a days, most of the
Nepalese wear some kind of shoes or slipper even in the
remote areas which reduce the chances of skin penetra‑

shoes/slipper resulting into a high incidence during thetion by infective larva. The farming land area in
Kathmandu valley has also been reduced significantly

later part of this study. Further, two rivers‑ Bagmati
and Bishnumati in the valley are heavily contaminatedwith sewage during recent years, but some people still
utilize the river water for washing and bathing. Udonsi

83

not available from Nepal till date and rernains to be the12

D Malel Fem ale

10
O
O
>
O
L

duodenale in Nepal was in agreernent with the finding of

Koshy et al. (1978) from North India where they8

recovered A. duodenale from all patients of hookworm
outbreak except four who had received antihelminthic

C l

ae

subject of future study. Higher incidence of A.

4treatment and with that reported frorn China (Yu et al..

O

85 86 87 88 89 90 91 '92 '93 '94 years
Figure 3 Annual rate of hookworm infection in males andf emales.

1995). About one‑fourth of hookworm infection in thisstudy were found to be associated with other parasites
most common being Ascaris lumbricoides and Trichuris
trichiura (data not shown) .

The incidence rate could be higher had the concen‑
tration technique been applied to each of the sample
included in this study or had three consecutive samples

and Amabibi (1992) from Nigeria have also reported
water‑borne transmission of hookworm among fisher‑from each patient been examined. However, we con‑men. Further, an association between urbanization andsider that our present finding sufficiently demonstrateparasitic infection has also been reported by other inves‑

the status of human hookworm infection in Nepal.
Though, there are reports showing less than 5.0%

tigators (Cromton and Savioli, 1993).
Throughout the study period, adults (more than 15

years of age) showed higher incidence rate comparedwith children (15 or less than 15 years of age) (Fig. 2).

(Gianotti, 1990) to as high as 83.8% (Entevez et al..
1983) incidence of hookworm infection in Nepal, such ahigh difference appears to be due to the difference inThis finding was in agreement with those reported byother investigators (Gilles, 1985; Xu et al., 1995).

study population and sample size (less than 40 samples) .

Further, Bakta et al. (1993) from Indonesia have report‑

no protective role against hookworm infection, it isnecessary to take appropriate preventive measures in‑cluding planned urbanization with good sanitary dis‑posal and sewerage facility to prevent the hookworm
associated morbidity, mortality and economic losses in

ed an increase in the prevalence and intensity of hook‑worm infection with age. In this study, females were

found more commonly infected compared with theirmale counterparts except in the year 1989 and 1990 (Fig.
3) . However, the differences were not significant statis‑

tically. These findings showed that both male andfemale have almost an equal chance of exposure tohookworm infection in Nepal.

Since the good nutrition (Roche and Layrisse, 1966) has

Nepal in future.

REFEREN CES

Of the two species of human hookworms, A.

1 ) Adams, E.J., Stephenson, L.S., Latham, M.C. and Kinoti,

duodenale is considered to be more pathogenic as it

S.N. (1994) : Physical activity and growth of Kenyan
school children with hookworm, Trichuris trichiura andsucks 8 to 9 times more blood (260 pl/worm/day)compared with N. americanus (30 pl/worm/day) .
Keeping in view of this fact, in the latter part of thestudy period, we cultured 200 hookworm ova positive

fecal samples to see the type of human hookworm
prevalent in Nepal. Of the total 200 hookworm ova

Ascaris lumbricoides infections are improved after treat‑ment with albendazole. J. Nutr. 124, 1199‑1206
2 ) Adhikari, R.K., Rai, S.K., Pokhrel, B.M. and Khadka, J.

B. (1986): Bacterial study of drinking water of Kath‑mandu valley. J. Inst. Med. (Nepal) , 8, 313‑316

positive samples cultured, 67.0% were A. duodenale
while the remaining 33.0% were N. americanus. Thisfinding revealed that hookworm associated anemia in

3 ) Beaver, P.C., Yoshida, Y. and Ash, L.R. (1964): Mating

Nepal is caused mainly by A. duodenale and correlated
well with a significantly lower hemoglobin level among
hookworm infected subjects (Curtale et al.. 1993). A
higher prevalence of A. duodenale appears to be due to4 ) Bakta, I.M., Widjana, I.D. and Sutisna, P. (1993): Sorne

its ability to undergo arrested development and to infect5 ) Capello, M., Vlasuk, G.P., Bergum, P.W., Huang, S. andAncylostoma caninum in relation to blood loss in their
host. J. Parasitol., 50, 286‑293

epidemiological aspects of hookworm infection among
the rural population of Bali, Indonesia. Southeast Asian

J. Trop. Med. Public Health., 24, 87‑93

also through oral and transmammary/transplacental

Hotez. P.J. (1995) : Ancylostoma caninum anticoagulant(Yu et al.. 1995) routes other than skin penetration.
However, report on infantile hookworm infection was

peptide: a hookworm‑derived inhibitor. of human coagu‑
lation factor Xa. Proc. Natl. Acad. Sci. U.S.A., 92, 6152‑

84

6156

6) Chaudhary, A.K. and Jayswal. S.N. (1984): Hookworm
anemia in an infant of two and half months. J. Indian

Rico's secret killer. P. R. Health Sci. J., 12, 191‑196
16) Mani, G.G.. Rao. S.T. and Madhavi. R. (1993): Estima‑tion of hookworm intensity by anthelmintic expulsion in

Med. Assoc., 82, 25

primary school children in south India. Trans. R. Soc.

7) Cromton, D.W.T. and Savioli, L. (1993) : Intestinal
parasitic infection and urbanization. Bull. World HealthTrop. Med. Hyg., 87, 634‑635
17) Rai, S.K. and Gurung, C.K. (1986): Intestinal parasitic

Organ., 71, 1‑7

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Harada, Y. and Mori, O. (1955) : A new method for

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85

Jpn. J. Tro p. Med. Hyg., Vol. 25, No. 2, 1997, pp. 85‑87

CHARACTERIZATION OF VIBRIO CHOLERAE O 1
ISOLATED IN LAO PEOPLE'S DEMOCRATIC REPUBLIC
CLAUDIA TOMA*1, LAY SISAVATH2, NAOMI HIGA1
AND MASAAKI IWANAGA1'3
Received March 21, 1997/Accepted Apnl 28 1997

Abstract: Thirty‑four strains of Vibrio cholerae O1 serotype Ogawa isolated in Lao People's Democratic
Republic in 1995 were examined for their biological behaviour and cholera toxin productivity. Biotyping
indicated that the 34 strains belonged to the El Tor biotype. All strains were hemolysin positive and resistant

to polymyxin B and cholera phage IV. However, 4 strains were negative for the hemolysin activity as
determined by the test tube method, although they were positive for the antigenic activity. Prophage typing
identified 31 strains of the Celebes type, including 3 cured Celebes type, and 3 Ubol type. All the strains
produced detectable levels of cholera toxin; 15 isolates produced more than 100 ng/ml, 10 strains between
10 and 100 ng/ml and 9 strains less than 10 ng/ml, determined by using AKI medium in a stationary test

tube. The maximum production was 200 ng/ml.
Keywords: Vibrio cholerae. Lao PDR, biotyping, prophage typing

INTRODUCTION

In 1995, V. cholerae biotype El Tor was reported
from all regions of the world. A total of 208,755 cases

The seventh cholera pandemic which began in 1961
shows no signs of declining. El Tor vibrios, the agent

were officially reported to WHO, an almost 50%

responsible for the present pandemic, appears to be
more resistant to adverse environmental factors thanthe classical biotype responsible for earlier cholera
pandemics. However, various changes in the epidemic

globally to 2 4%･ According to the "Weekly
Epidemiological Record" from WHO, Lao People'scholera vibrios have been seen during the past 36 years.

6.3% and 12.7%, respectively. The important efforts
made by the government and WHO in the region, as well

A11 epidemic strains of E1 Tor vibrios were lysogenic
until 1966 when "cured" strains suddenly appeared in

Cambodia and Thailand (Kuyyakanond et al.. 1990).
Moreover, strains of V. cholerae O1 biotype El Tor thatwere susceptible to Mukerjee's cholera bacteriophagedecrease over 1994. The case‑fatality rate was reduced

Democratic Republic (Lao PDR) reported 9,640 cases in1994 and 1,365 cases in 1995 with a case fatality rate ofas the positive response from the donor community, may
have contributed to the decrease in the incidence ofcholera cases. However, the case‑fatality rate in the
Lao PDR still remains high. This study examined the

IV were reported (Iwanaga et al.. 1989) , and changes inhemolytic property of El Tor vibrios were also observed(Nakasone et al.. 1987) . In 1992, a new serogroup V.
cholerae 0139 emerged as a causative agent of cholera in
India and Bangladesh, which rapidly spread to neighbor‑characteristics of V. cholerae serogroup O1 strains iso‑ing countries (Ramamurthy et al.. 1993). The changes
in epidemic cholera vibrios must be considered for
ecology of the organisms and for epidemiology of theBacterial strains: Thirty‑four strains of V. cholerae O1

disease. Therefore, characterization of cholera vibrios
at different places and at different times must be don .

lated in Lao PDR in 1995.

MATERIAL AND METHODS
isolated in 1995 from cholera patients and submitted to
the National Institute of Hygiene and Epiderniology of
Lao PDR were used. V. cholerae H218 (biotype classic,

serotype Ogawa) was used for the study of bacterio‑

1 Department of Bacteriology, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa 903‑01, Japan
2 National Institute of Hygiene and Epidemiology, Vientianne, People's Democratic Republic of Laos
3 Research Institute of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa 903‑01,

Japan* Corresponding author: Nishihara, Okinawa 903‑01, Japan. Tel. 81(98)895‑3331; Fax. 81(98)895‑2951

86phages. The isolates were stocked in nutrient agar
prepared with a double amount of water until use.
Characterization of isolates: The characteristics ofeach isolate were determined on the basis of serotype,biotype, prophage type and cholera toxin production.

Although biotype is decided essentially bykappa phage but was susceptible; and Ubol type did not
produce kappa phage and was not susceptible.

Cholera toxin production: The organisms were cul‑

tured in AKI medium (Bacto‑Peptone 1.5%; yeast
extract 0.4%; NaC1 0.5%; sodium bicarbonate 0.3%) at

hemolytic properties of the organisms, Voges‑Pros‑

37'C for 20 hr in a stationary test tube, after which thecholera toxin in the culture supernatant was titrated bykauer reaction and susceptibility to cholera phage groupusing the reversed passive latex agglutination method

IV, El Tor phage 5 and polymyxin B were also

(Iwanaga and Yamamoto, 1985).

examined. Serotype was determined by the slide agglu‑
tination method using commercial anti‑Ogawa and anti‑
Inaba sera (Denkaseiken Co., Tokyo, Japan) .
Hemolysin activity was titrated by a slight modifi‑
cation (using heart infusion broth with 1% glyceroD of
Feely and Pittman's method (Feely and Pittman, 1963) .
The strains with no hemolytic activity were examined
for the antigenic activity of El Tor hemolysin by the

Table 1

Biotype characterization
positiveb)

Hemolysin*)

100oVoges‑Proskauer

74

26

O

100

Susceptibility to:reversed passive latex agglutination (RPLA) method(Chinen et al.. 1996). The Voges‑Proskauer reaction

cholera phage IV
E1 Tor phage 5

was examined by using Voges‑Proskauer soft agar

polymyxin B(Eiken Co., Japan) .

Susceptibility to polymyxin B was examined with anegativeb)

100

O

O

100

a) Determined by biological or antigenic activity.
b) Percentage of the strains.

plate dilution technitlue to determine the minimum
inhibitory concentration (MIO of the drug. Agar platecontaining the drug at concentrations ranging from 100
to 6.25 pg/ml were prepared, and organisms which grewwith a drug concentration greater than 12.5 pg/ml wereregarded as resistant to polymyxin B.
Susceptibility to cholera phage IV and E1 Tor phage
5 was examined as follows. First, 10 ml of peptone agar
(1% peptone, 0.5% NaCl, 1.5% agar) was poured into a

Table 2 Prophage typing

Prophage type(%) ')

broth were killed with chloroform. The killed culture

kappa phage+

cured Celebes (9%)+

Ubol (9%)
a ) Percentage of the strains.

16 [the organisms (0.2 ml) and 4 ml of soft agar (heart

kappa phage was examined with the method mentioned
above. Production of kappa phage by the organisms
was examined as follows. The organisms cultured in

production of

kappa phage

Celebes (82%)

petri dish and solidified. An overnight broth culture ofinfusion broth with 0.5% agar) kept at 43'C were mixed
and overlaid on the peptone agar plate. Phage solution(15 pl) at the routine test dilution was spotted on thesoft agar plate containing the organisrns.
Prophage typing was done basically as described by
Takeya and Shimodori (1963) using kappa phage and its
host strain H218. Susceptibility of the organisms tosusceptibility to

14
12
(1)

c:
a5

Lv)

10

4J

Y‑

8

Z

6

O
d

4
2

fluid (0.1 ml), a broth culture of strain H218 (0.2 ml),and soft agar (4 ml) were mixed and overlaid on a
peptone agar plate. After overnight incubation at 37'C,

turbid plaque formation was regarded as kappa phage
production. The organisms were classified as Celebes
type if they produced kappa phage and were not suscep‑tible to the phage; cured Celebes type did not produceO‑ I O‑1 OO ‑
10

1 OO

ng/ml

Figure 1 Cholera toxin production in AKI medium in sta‑tionary test tube as determined by the reversed
passive latex agglutination (RPLA) method.

87

RESULTSAll the isolates belonged to serotype Ogawa. The

biotype‑specific reactions are shown in Table 1.

demic strains are regarded as Celebes type El Tor
vibrios. In the present study, 31 of 34 isolates (91%)

belonged to the Celebes type, including 3 cured type.
The remaining 3 isolates were Ubol type.

Hemolytic activity was negative for 4 strains examinedlwanaga and Yamamoto (1985) previously notedby the method of Feeley and Pittman. However, thesethat cholera toxin production by E1 Tor vibrios in AKI
medium usually ranged frorn 20 to 100 ng/ml. However,an increase in productivity was observed in more recent
isolates (Kuyyakanond et al.. 1990) . We also found thatstrains were positive for the antigenic activity of El Tor

hemolysin when tested by the RPLA method. The
Voges‑Proskauer reaction was positive in 25 strains(74%). The cholera phage IV susceptibility test was

44% of the isolates produced more than 100 ng/ml ofnegative for all the strains, while 100% of the strainscholera toxin. Whether this increased productivity is awere susceptible to El Tor phage 5. The MIC ofgeneral tendency of E1 Tor vibrios worldwide or justindicates a dispersion of the parameter will be deter‑polymyxin B against these cholera vibrios was morethan 25 pg/ml, and they were regarded as resistant.
Prophage typing (Table 2) classified the isolates
into 28 Celebes type (82%) , 3 cured Celebes type (9%) ,mined only when epidemic strains are examined at
different places and at different times.

and 3 Ubol type (9%).
All the strains produced detectable levels of cholera
toxin (Fig. 1) ; 15 isolates (44%) produced,more than 100

ng/ml, 10 strains ( 9%) between 10 and 100 ng/ml, and9 strains (27%) Iess than 10 ng/ml. The maximum
production was 200 ng/ml.

DISCUSSION
A11 V. cholerae serogroup O1 strains examined inthis study were positive for, the production of E1 Tor
hemolysin, as determined by biological activity or by
antigenic activity. Historically, the E1 Tor biotype was
identified by its ability to hemolyze sheep erythrocytes,

while classical strains were non‑hemolytic (Feeley,1966). However, by 1972, nearly all E1 Tor isolates
worldwide had become non‑hemolytic, and thereafter,changes in hemolytic property of E1 Tor vibrio wereobserved (Iwanaga et al., 1982). Chinen et al. (1996)
have suggested the presence of some hemolysin inactiva‑tion factor (s) in El Tor vibrios. Although the propor‑

tion of hemolytic strains was increased by using
glycerol‑enriched broth, our findings showed that somestrains produced E1 Tor hemolysin, though biologicalactivity was not detected in the culture supernatants.
Therefore, E1 Tor hemolysin must be antigenically or
genetically detected to avoid false negative results.

Susceptibility to cholera phage IV, E1 Tor phage 5
and polymyxin B were typical findings of E1 Tor vibrios.

Therefore, phage and polymyxin B susceptibility seemto be the most reliable in deciding the biotype of V.
cholerae O1. Kuyyakanond et al. (1990) reported El Tor
vibrios susceptibie to cholera phage IV, but such a strain

was not found in this study.
In the current seventh cholera pandemic, the epi‑

REFERENCES
1 ) Chinen, I., Toma, C., Honma, Y., Higa, N. and lwanaga,
M. (1996): Hemolysin production by Vibrio cholerae as
examined by hemolytic and antigenic activities. Jpn. J.
Trop. Med. Hyg., 24, 151‑155

2 ) Feeley, J.C. and Pittman, M. (1963): Studies onhemolytic activity of E1 Tor vibrios. Bull. W.H.O., 28,
347‑356
3 ) Feeley, J.C. (1966): Minutes of International Subcom‑
mittee on Taxonomy of Vibrio. Intern. J. Syst. Bacter‑iol., 16, 135‑142

4 ) Iwanaga, M.,'Mori, K. and Kaviti, J.N. (1982): Vibrio
cholerae O1 isolated in Kenya. J. Clin. Microbiol., 16,
742‑7435 ) Iwanaga. M. and Yamamoto, K. (1985): New medium
for the production of cholera toxin by Vibrio cholerae O1
biotype E1 Tor. J. Clin. Microbiol., 22, 405‑408

6 ) Iwanaga, M., Nakamura, S. and Kuyyakanond, T.
(1989): Hemolytic Vibrio cholerae O1 that is sensitive to

Mukerjee's cholera phage IV and the phage produced by
the hemolytic vibrio lysogenized with the infection of
Mukerjee's cholera phage IV. Microbiol. Immunol., 33,
609‑6187 ) Kuyyakanond, T., Nakamura. S., Wipawadee, M. andlwanaga, M. (1990): Vibrio cholerae serogroup O1 innortheast Thailand. J. Clin. Microbiol., 28, 872‑875

8 ) Nakasdne, N., Iwanaga, M. and Roger. E. (1987):
Characterization of Vibrio cholerae O1 recently isolatedin Bangladesh. Trans. R. Soc. Trop. Med. Hyg., 81, 876‑

8789 ) Ramamurthy, T., Garg, S., Sharma, R., Bhattacharya, S.
K., Nair, G.B., Shimada, T., Takeda, T.. Karasawa, T.,

Kurazono. H.. Pal, A. and Takeda, Y. (1993): Emer‑
gence of novel strain of Vibrio cholerae with epidemic
potential in southern and eastern India. Lancet, 341,
703‑704

10) Takeya, K. and Shimodori, S. (1963): "Prophage‑
typing" of El Tor vibrios. J. Bacteriol., 85, 957‑958

J pn. J. Tro p. Med.

89

Hyg., Vol. 25, No. 2, 1997, pp. 89‑93

ETIOLOGY OF DIARRHEAL ･DISEASESAND THE HEALTHY CARRIER OFENTEROPATHOGENS IN VIENTIANE,PEOPLE'S DEMOCRATIC REPUBLICOF LAOSITHAT INSISIENGMAY1, KHAMPHEUY MUNNALATH1, NoIKASEUMSY SITHIVONGl,
LAY SISAVATH1, BOUNNANG PANTHOUAMAS1, KHAMPHENG CHOMLASAKl,
NAOMI HIGA2, TETSU YAMASHIR03, NoBORU NAKASONE2
AND MASAAKI IWANAGA2
Received March 19, 1997/Accepted April 30, 1997

Abstract: Etiologic bacteria of 135 cases with diarrheal diseases and the healthy carrier rate in 211
individuals were studied in People's Democratic Republic of Lao. Diarrheagenic Escherichia coli. Shigelia
and Campylobacter were the predominant pathogens isolated. This country does not face the sea, but Vibrio
parahaemolyticus was isolated from 2 cases. Healthy carrier rate of enteropathogenic E. coli as diagnosed byserogroup Class I was 3 times higher than the detection rate in diarrheal patients (10.9% and 3.7%) . Healthycarrier rate of Shigella was 4.3%, whereas the detection rate of Shigella in diarrheal patients was 15.4%. The
isolation frequencies of enterotoxigenic E. coli and Campylobacter jejuni in the patients and healthy individ‑

uals were almost the same. Verotoxin producing E. coli was found in one healthy individual (Toxin type
VT1, O‑antigen O111) . Rotavirus was not detected from the 29 infant diarrhea examined, but detected from
2 adult cases out of 32 examined.
Keywords: diarrhea, enteropathogen, epidemiology, Laos

INTRODUCTION
Although the numbers of death due to diarrheal
disease has markedly decreased during the past 15 years

because of intensive world wide promotion of primary
health care (PHO activities including the enhancementof oral rehydration therapy, diarrheal disease is still a

People's Democratic Republic of Lao (Lao PDR) has
been published. In this communication, the etiologic
agents of diarrhea recently studied in Lao PDR were
described.

PATIENTS, MATERIALS AND METHODS

leading cause of death among young children in develop‑
ing countries. Diarrheal diseases are preventable, cur‑able, and controllable with a simple technique. There isPatients: A total of 135 patients with diarrhea at 3
public hospitals and a private clinic in Vientiane wereno need to die of diarrhea but many children do so.

period of 70 days from October 17 to December 27, 1996.

Epidemiological study is the first step to control thediseases in the society. The isolation frequency and the
distribution of diarrheagenic bacteria in many countries
have been reported, and a different isolation pattern
between regions was shown (Adkins et al..1987; Aihara

Age distribution ranged from 3 months to 73 years

et al., 1991; Iwanaga et al., 1993; Kain et al., 1991; Kim

rectal swab from 70 patients and stool in the plastic
containers from 65 patients.

et al., 1989; Yam et al., 1978), but no report from1

2
3

examined. The patients were examined during theincluding 44 patients older than 15 years, 86 patients
younger than 5, 2 patients of 12 years. There were 3
patients whose ages were not recorded.

Specimens: The contents of 135 specimens were

National Institute of Hygien and Epidemiology, Vientiane, Lao People's Democratic Republic
Department of Bacteriology, Faculty of Medicine, University of the Ryukyus, Okinawa 903‑01, Japan
Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus, Okinawa 903‑01, Japan

90

Microbiological examinations: The target bacteria
for isolation were 4 kinds of diarrheagenic Escherichia
coli except enteroaggregative E. coli (EAggEO , Shigel‑la. Salmonella. Vibrio. Aeromonas. Plesiomonas andCampylobacter for all specimens. Rotavirus wasfrom 0.025 /1 g/ml to 100 /1 g/ml were prepared. The
organisms were cultured in hear.t infusion broth over‑night, and the culture fluids were diluted 1:10 withnormal saline solution for the inoculum (Ca. 107/mD.
They were inoculated by using a microplanter (Sakuma

examined for the stools collected in the plastic con‑tainers by using latex agglutination kit, Rotalex (Den‑

Co. MIT#00257), and determination of MIC was madekaseiken Co. Tokyo, Japan) . Serial 10‑fold dilutions of

medium, SS agar medium, and TCBS agar medium,

Healthy carriers: Carrier of enteropathogens was
examined for a total of 211 individuals including 185
healthy children under 6 years of age in "Children s
School" and 26 adult staff taking care of the children.
All but 15 specimens (stools) were collected in therespectively. The inoculated sample was spread overplastic containers. The methods for isolation and identi‑

one‑third space of the agar plate by a glass bar spreader

fication of the pathogens were the same with those used

and then streaked onto the remaining two‑thirds spaceof the plate with a wire inoculation loop. The colonies
grown on agar plate were identified by routine labora‑

for diarrheal stools.

stool samples were made in normal saline solution, and25 /t I of the dilution at l0‑3, 10‑2, and 10‑1 were

inoculated onto agar plates of modified Drigalsky

after incubation at 37'C for 24 hr.

RESULTS

tory tests. For the diagnosis of diarrheagenic E. coli, 4
to 5 possible E. coli colonies were subcultured to prolif‑

Enteropathogens isolated from the patients: As

erate on a nutrient agar plate. The proliferated organ‑
isms were examined for E. coli O group agglutination byusing diarrheagenic E. coli diagnostic antisera (Den‑kaseiken Co.) . The strains with agglutination were re‑shown in Table 1. Shigella was isolated from 21 patients

examined after heating at 121"C for 15 min. Heat labile

enterotoxin (LT) and verotoxin production wereout of 135 (15.4%). Subgroup distribution revealed S.
flexneri 14, S. sonnei 6 and S. boydii 2. One case, a 3
year‑old girl, excreted 2 subgroups of Shigella (S. flex‑
neri 2a, S. sonnei 1) and LT producing enterotoxigenic
E. coli (ETEO . Salmonella was isolated frorn 3 cases,

examined by using reversed passive latex agglutination

and all 3 strain belonged to O antigen group 4. EPEC

kit (VET‑RPLA, Denkaseiken Co.). Verotoxin produc‑tion was examined for the E. coli strains which agg‑

serogroup was classified into three groups, Class I, Classlutinated with the antisera of enteropathogenic E. coli

Table 1 Enteropathogens isolated from hospital‑visiting(EPEO and enteroinvasive E. coli (EIEO serogroupindicated in the commercially purchased preparation.
Heat stable enterotoxin (ST) was examined by using
immunoenzyme assay kit (COLIST, Denkaseiken Co.) .

diarrheal patients in Vientiane

Isolated pathogens

Case frequency (%)

Shigella spp.

21 (15. 4)

The species of the strains with positive agglutination or

Salmonella spp.

3

(2 .2)

positive enterotoxin production were biochemically

EPEC (serogroup Class D
EPEC (serogroup Class ID
EPEC (serogroup others)
EIEC (serogroup)
ETEC (LT 4, ST 7)

5(3 . 7)

4(3 . O)

identif ied.

Campylobacters were isolated onCampylobacter blood‑free selective medium (Oxoid)
incubated at 42'C for 48 hr and was identified by confir‑

ming the morphology with Gram‑staining and degrada‑tion activity for hippuric acid. Rectal swab samples

7(5 . 2)3(2 . 2)11(8 . 1)EHEC

O

Vibrio parahemolyticus

2(1. 5)(O . 74)

were directly inoculated onto the agar plate by routine
techniques. Rotavirus was examined for the stool sam‑ ‑Vibrio hollizae

1

ples collected in the containers.

Aeromonas spp.

O

Campylobacter jejuni

7(5 . 2)(1 . 5)

Drug sensitivity test: Twenty‑two Shigella isolates

from 21 patients and 9 strains frdm healthy carriers
were examined for susceptibility against ampicillin(ABPO , tetracycline (TO , cefdinir (CFDN) , ofloxacin

Campylobacter coli2

pathogen positive

57 (42%)(OFLX) and erythromycin (EM). Minimum inhibitory
concentration (MIO of the drugs were examined by apathogen negative

78 (58%)

Total

135

plate dilution technique. Heart infusion agar plates
containing the drug at the serial 2‑fold concentration

More than 2 pathogens were isolated from I case.
Of 65 examined, 2 cases were positive for rotavirus.

91

Table 2 Healthy carrier of enteropathogens

Number examined‑age

Isolated pathogens
Total: 211<6y: 185

Shigella spp.

98

Salmonella spp.

O

O

EPEC (serogroup Class D
EPEC (serogroup Class ID
EPEC (serogroup others)
EIEC (serogroup)
ETEC (LT 2, ST 16, LT/ST 2)

23

19>20y: 26
o

5

5

o

15

15

o

6

5

20

20

EHEC (VTI producing)l1Vibrio spp.

1

o
o

1

o

Campylobacter jejuni

11

11

o

Campylobacter coli

6

6

o

RotavirusOOo83 (39%)77 (42%)6 (23%)

128 (61%)108 (58%)

20 (77%)

Aeromonas caviae

pathogen positive
cases ( % )pathdgen negative
cases ( % )More than 2 pathogens were isolated from 16 cases. Serogroup of EHEC was O111.

II, and others. Class I EPEC serogroup includes 026,
055, 086, O111, O119, 0125, 0126, 0127. 0128ac, and0142. Class 11 EPEC includes 018, 044, O112, and O114.

Table 3 Drug sensitivities of Shigella isolates
Number of strains
MIC ( p g/ml)

ABPC

TC

EM

CFDN

OFLX

These antisera were prepared in the batch of diagnostic

o.025 >

o

o

o

o

1

antisera (Denkaseiken Co.). The case frequency with

0.025

o

o

o

o

1

Class I, Class 11 and others were 5 (3.7%), 4 (3.0%) and

0.05

o

o

o

o

1

7 (5.2%), respectively. Three cases excreted EIEC

0.1

o

o

o

3

28

0.2

o

o

o

17

1

0.39

o

o

o

11

o

0.78

o

o

o

o

o

1.56

1

3

o

o

o

3.13

6

1

1

o

o

7 and 2 cases, respectively. In total, bacterial pathogen

6.25

6

o

4

o

o

positive cases were 57 (42%) . Rotavirus was examined

12.5

3

o

7

o

o

for 65 stool samples collected from 29 infants, 32 adults

25

o

5

8

o

o

50

o

3

11

1

o

100

o

17

o

o

o

3

1

o

o

Others EPEC includes O1, 0146. 0151, 0157, 0158.

serogroup. ETEC were isolated from 11 cases in total,
including 4 LT producing strains and 7 ST producing
strains. V. parahemolyticus was isolated from 2 adult ,cases with watery diarrhea containing many leucocytes.
V. hollizae was isolated from a 2 year old boy withbloody diarrhea. Aeromonas and Plesiomonas were notisolated at all. C. jejuni and C. coli were isolated fromand 4 adolescent. The infatile specimens were all nega‑tive but 2 adult cases were positive.

Healthy carrier rate: The isolation frequency ofClass I EPEC was higher than the patients with diar‑rhea, and that of C. jejuni was the same with diarrheal
patients. As shown in Table 2, 9 healthy carrier of
Shigella out of 211 examined (4.3%) were found. The
subgroup of Shigella consists of 4 S. boydii, 3 S. sonnei,

100 <

16

ABPC: ampicillin, TC: tetracycline, EM: erythromycin, CFDN:
cefdinir, OFLX: ofloxacin

92

and 2 S. flexneri. Class I, Class II, and others EPEC were

known about the pathogenesis, epidemiology, and ser‑isolated from 23 (10.9%), 5 (2.4%), and 15 (7.1%),
respectively. Six cases were positive for EIEC sero‑group. A total of 20 ETEC (9.5%) producing LT, ST,

otypes of EAggEC. Exactly speaking, EPEC can be

and LT/ST were isolated from 2, 16, and 2 cases, respec‑tively. Enterohemorrhagic E. coli (EHEO produojng
VT‑1 was isolated from only one case, a 2 year‑old girl.

The strain belongs to the serotype O111.
Drz g sensitivities of Shigella: The isolates weregenerally resistant to ABPC and TC, and sensitive to

identified by detecting bundle forming pili (BFP) and 94

kDa outer membrane protein (intimin) or their genes(bfp, eae) (Donnenberg and Kaper, 1992; Law, 1994;
Levine, 1987). Sereny test or examination of a path‑ogenic gene, such as the gene for invasive plasmid
antigen (Ipa) is required for identification of EIEC
(Levine, 1987). However, in the routine laboratory
diagnosis, so called corresponding O‑antigens are

CFDN and OFLX. The sensitivity to ABPC showed 2

examined by slide agglutination test using traditionallytypes; one was highly resistant (MIC> 100 /1 g/ml) andanother was moderately resistant (MIC= 1.56‑12.5 /t gl

prepared antisera. Although the presence of certain O
antigens (serogroup Class D is considered to correspondml) . Almost 90% of the isolates were highly resistant towell to the presence of bfo and/or eae (Levine, 1987) , theTC. On the contrary, MIC of OFLX was 0.1 /1 glml

diagnosis with serogroups agglutination test may not be
reliable. Anti‑E. coli O antigen sera are cornmerciallyavailable for the screening of diarrheagenic E. coli. In
Japan, the commercially available antiser include someagainst almost all isolates (Table 3) .

DISCUSSION
Surveillance of the disease based on detailepidemiological data is essential for making the strat‑
egy to control diseases in the society. Diarrheal disease,one of the leading cause of death in developing coun‑batches of sera for the screening of EPEC, ETEC, and
EIEC. However, it is unclear what percentage of E. coli
with these serogroups are true pathogens.
C. jejuni is one of the major diarrheagenic organism
in worldwide. The isolation frequency in diarrheal stool

tries, can be controlled by implementing a proper strat‑

was 5.2% (7/135) which may be a universal average

egy. Analysis of the epidemiological data obtained in
the present study would play an important role to con‑ratio including the industrialized countries (Adkins etal., 1987; Aihara et al., 1991; Iwanaga et al., 1993; Kain

trol diarrheal diseases in the related societies.

et al.,1991; Kim et al., 1989; Yam et al., 1978). However,Attention to high frequency of shigellosis and highhealthy carrier rate of Shigella should be paid. Numberof cases with shigellosis was 21 out of 135 diarrheal
patients (15.6%) , and the healthy carrier rate was 4.3%
(9/211). Fortunately, the 9 healthy carriers of Shigellaattention should be paid in the present study to the samein the present study did not manifest the illness, and thepathogens spontaneously disappeared in the re‑exami‑
nation at I month after the first detection of Shl ella. In

other Asian countries, the reported case frequency wasusually more or less 5% of diarrheal patients and thehealthy carrier rate was usually less than 2% (Adkins etal., 1987; Aihara et al., 1991; Iwanaga et al., 1993; Kainet al., 1991; Kim et al., 1989; Yam et al., 1978). Shigel‑losrs rs a typrcal "fecal oral mfectron". These highfrequencies suggest that sanitary condition is not prefer‑able in the society. The enhancement of primary healthcare activities is required.

isolation frequency, in the healthy population (11/211,5.2%) and diarrheal patients (7/135, 5.2%).

High healthy carrier rate of enteropathogen wasseen not only for Campylobacter but also for Shl ella anddiarrheagenic E. coli. The carrier rate of EPEC withClass I serogroup was 11.4% (24/211) which is higher
than the isolation rate of diarrheal patients (3.7%,
5/135). Some studies reported the isolation rate of
ETEC in diarrheal patients and healthy individuals is
almost the same, but EPEC was definitely associatedwith diarrhea (Albert et al., 1995; Kain et al., 1991). In

the present study, however, the carrier rate of EPEC
was 3 times that of diarrheal patients. The reason forthis is unknown, but many healthy individuals may beready to develop diarrhea at any time.
Drug sensitivity was examined for Shl ella isolates.

Generally, diarrheagenic E. coli is most frequently
isolated from diarrheal stool in the tropical countries,
but identification of the pathogen, especially EPEC, is

The results showed that ABPC and TC were not helpful,but oral cephem and new quinolone were very effective.
Almost all isolates were sensitive to nalidixic acid as

not so easy. The identification of ETEC and EHEC isroutinely made by detection of the toxins. EAggEC isexamined by the disc method (data not shown) .

usually not handled in the clinical laboratories, because

shigellosis, because oral cephem and new quinolones areof lacking the proper diagnostic method. Little is

not readily available in developing countries. Anti‑

Nalidixic acid must be the first drug of choice for93

Shigella activity of erythromycin seems poor, but in
1960s, there were many reports in Japan that eryth‑romycin was as effective as tetracycline and chloram‑
phenicol which were excellent antibiotics for the treat‑ment of shigellosis at that time (Saito et al., 1964). In
general, rotavirus is frequently detected in infant diar‑rhea without seasonal variation in the tropical countries(Adkins et al., 1987: Aihara et al., 1991; Iwanaga et al.,

1993; Kain et al., 1991; Kirn et al., 1989; Yam et al.,1978) , whereas the epidemic in Japan is concentrated in
the first 3 months of the year (Fujita, 1990). In thepresent study, rotavirus was detected from only 2 cases

out of the 65 examined (3%). It is unknown whether
this low frequency is the characteristic of Lao PDR ornot. The continuous study throughout the year isrequired.

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JUNE1997

VOL、25NO．2

CONTENTS
Review
Ohyama，T．
Strategy Towards Measles Elimination−Progress in the English−speaking Caribbean

Original

Countries一

ArticleHe，N．，Aosai，F．，Hye−Seong，M．，Sekiya，S．and

Cytokineby・49−57

Production

AssayedToκoμ召s耀go％朔asaby

RT−PCR

Modelof

Takaoka，H．and Sigit，S．H．
Three New Black Fly Speciesofin

Yano，A．

Pregnant

Congenital

Mice

Infected

Toxoplasmosis…・…・

・・59−67

S枷％IJ％吻（Diptera：Simuliidae）fromSumatra，Indonesia…69−80

Rai，S．K．，Shrestha，H．G．，Nakanishi，M．，Kubo，T．，Ono，K，Uga，S．

and Matsumura，T．
Hookworm Infection
Nepal

Over

One

Recorded

DecadeatanPeriod一…

University

Teaching

HospitalinKathmandu，
・81−84

Toma，C．，Sisavath，L．，Higa，N．and Iwanaga，M．
Characterization of四治πo oho伽鷹011solated in Lao People s Democratic Republic・ ・85−87

Insisiengmay，S．，Mumalath，K．，Sithivong，N．，Sisavath，L，Panthouamas，B、，

Chomlasak，K．，Higa，N．，Yamashiro，T．，Nakasone，N．and
Etiology

Peopleofs

Diarrheal

Diseasesand

Democratic

Republic

of

the

Lao

Healthy

CarrierofIwanaga，MEnteropathogens

inVientiane，

・89−93