**Appraisal of Recommendations For Disinfecting Eggs Of Atlantic Salmon
(*Salmo salar*) In Iodophor**

Rocco C. Cipriano,^1\*^ Bernard M. Novak, ^2^ Daniel E. Flint^2^, and
Darleen C. Cutting^3^

^1^USGS, National Fish Health Research Laboratory, 11700 Leetown Road,
Kearneysville, WV; ^2^ USFWS, Richard Cronin National Salmon Station, 51
East Plumtree Road, Sunderland, MA; ^3^USFWS, Connecticut River
Coordinator's Office, 103 East Plumtree Road, Sunderland, MA

The federal protocol, which requires disinfection of fish eggs in
50-mg/L of iodine for 30 min followed by a secondary disinfection in
100-mg/L iodine for 10 min, was investigated through six spawns of
Atlantic salmon (*Salmo salar*) at the Richard Cronin National Salmon
Station (Sunderland, MA). These salmon had undergone an epizootic of
furunculosis and the surviving fish maintained a persistent infection of
*A. salmonicida* throughout the course of study. Eggs from twenty
paired-matings of salmon were obtained annually from 1995 through 2000,
except for 1999 when eggs from 35 pairs of salmon were examined.
*Aeromonas salmonicida* was isolated from 19 of 135 total groups of
fertilized eggs that were investigated throughout this study. In these
cases, all isolations of the pathogen were made only from fertilized
eggs before any disinfection in iodophor had occurred. Results also
provided further evidence that A*. salmonicida* is not transmitted
vertically via intra-ovum infection. In contrast to our field results,
*in vitro* assays actually showed that *A. salmonicida* was incompletely
killed when concentrations of the bacterium ranged between 1.0x10^7^ to
1.2x10^8^ cfu/mL. However, even when bacterial concentrations exceeded
1.0x10^7^ cfu/mL no *A. salmonicida* remained viable if treated first
with 50-mg/L iodine for 30 minutes and then with 100-mg/L iodine for 10
minutes, as prescribed by federal policy. Incomplete disinfection of *A.
salmonicida* at cell densities greater than or equal to 1.0x107 cfu/mL
in either 50 mg/L iodophor for 30 minutes or in 100 mg/L iodophor for 10
minutes was observed only in A-layer positive phenotypes of cloned
isolates. Isogenic A-layer negative clones, as well as representative
ten strains of *Yersinia ruckeri* and *Aeromonas hydrophila* were
completely disinfected using a single treatment with either 50 mg/L
iodophor for 30 min or 100 mg/L iodophor for 10 min. At high bacterial
concentrations, more bacterial clumps are produced by A-layer positive
phenotypes of *A. salmonicida.* We speculate that the outer cells of
these clumps may "shield" and protect a small percentage of cells at the
core of the clump, which produces an incomplete disinfection.

**Epidemiological Study Of *Aeromonas salmonicida*, Causative Agent Of
Furunculosis, In Salmonid Fish In Japan**

[Tetsuichi]{.underline} [Nomura^1^]{.underline} Hiromi Honma^2^, Hisae
Kasai and Mamoru Yoshimizu^1^

^1^National Salmon Resources Center, Japan; ^2^Graduate School of
Fisheries Sciences, Hokkaido University, Japan

Furunculosis, caused by the bacterium *Aeromonas salmonicida*, is
serious disease in salmonid fish and this study was carried out for the
purpose of establishing　control methods for the disease. In a recent
epidemiological study, we determined the distribution and prevalence of
*A. salmonicida* in asymptomatic mature chum (*Oncorhynchus keta*), pink
(*O. gorbuscha*) and masu salmon (*O. masou*) in Hokkaido and Honshu.
*A. salmonicida* was distributed widely in the populations of
mature　salmonids in Hokkaido and Honshu. From 1979 to 1999, a total of
21,389 chum,　pink and masu salmon were collected from the rivers in
Hokkaido and Honshu. We　isolated *A. salmonicida* from the chum salmon
in 21 of the 34 rivers examined and the overall prevalence was 12.2 % in
the infected stocks. The prevalence of the agent in pink salmon and masu
salmon were 4.6 % and 1.4 % respectively. The prevalence of *A.
salmonicida* on gill surface, kidney and intestine of chum salmon was
studied using CBB medium, made by dissolving 0.1mg/ml Coomasie Brilliant
Blue R250 in Nutrient agar. Using CBB medium, we also examined the chum
salmon caught at the Chitose River catching site. The prevalence of the
agent on gill surface was high (50 %) at catching site on the Chitose
River, and decreased in fish in the holding pond in the river. The agent
was isolated from the gill surfaces, but could not be found in the
kidneys and intestines. *A. salmonicida* was also isolated from the gill
surfaces of masu salmon that were kept directly behind the chum salmon
holding pond. The bacteria are spread during fish migration within the
river and during transportation of the fish from capture site to holding
pond. The agent was isolated from the gill surface of chum salmon caught
off the coast of Shibetsu, Rausu, but the prevalence was low (1.7%). To
control furunculosis in salmonid fish, fish should mature in ponds under
conditions of low density and should be disinfected to prevent spread of
furunculosis before keeping in the pond for maturation.

**Duration Of Fasting And Its Effect On Stress-Induced *Aeromonas
hydrophila* Bacteremia In Channel Catfish**

[Kenneth E Nusbaum]{.underline}^1^, Edward E Morrison^2^, and John M
Grizzle^3^

^1^Department of Pathobiology, ^2^Department of Anatomy, Physiology, and
Pharmacology, College of Veterinary Medicine, and ^3^Department of
Fisheries and Allied Aquacultures, College of Agriculture, Auburn
University, AL

During a water-borne challenge of channel catfish with *Edwardsiella
ictaluri*, a bacteremia of primarily *Aeromonas hydrophila* was detected
as early as 3 hours post challenge. *Aeromonas hydrophila* bacteremia
could also be induced simply stressing fish by drawing several blood
samples over an 8 hour period. Although *A. hydrophila* were readily
isolated from the head kidney, the suggestion was made that the actual
source of the *A. hdrophila* may have been from the gut. This work
attempts to define the contribution of gut borne bacteria to
stress-induced bacteremias of channel catfish. Channel catfish from two
egg masses were hatched and reared indoors on de- chlorinated city
water. Four groups of ten month old fish (n=10) were held at 20 C in
static aerated water in 38 l tanks. Food was withheld from fish for 1, 2
or 3 days. Fish were then subject to handling stress (netting and blood
sampling) 3 times over 5 hours. Half the fish were then tranquilized at
8 hours of stress and blood samples drawn for quantitative culture on
Rimler-Shotts agar. Fish were then anesthetized and dissected.
Distribution of gut contents was noted and cultured quantitatively on
Rimler-Shotts. Samples of gut were harvested for microscopy to determine
if bacteria were traversing the gut wall. Progress of this investigation
will be reported

**An Examination Of Diversity Of *Flavobacterium psychrophilum* Isolates
From Different Origins And A Note On A Medium Used To Recover Isolates
From Long-Term Frozen Storage**

[Clifford E. Starliper]{.underline}^1^ and Richard K. Cooper^2^

^1^USGS, National Fish Health Research Laboratory, 11700 Leetown Road,
Leetown WV; ^2^Department of Veterinary Science, Louisiana State
University, Baton Rouge, LA.

About 150 isolates of *Flavobacterium psychrophilum* were collected in
1996 from five geographic origins and examined for
similarities/diversity by comparing plasmids and restriction enzyme
profiles on 16s rRna PCR products. Three origins (QC, QN, HT) were from
Washington State and two were from the Eastern U.S., West Virginia and
Pennsylvania. Western hosts were Coho salmon and steelhead trout;
eastern hosts were rainbow and golden trout. Within and between site
diversity, as well as comparisons of Eastern vs. Western and host vs.
host were examined. Clinical signs in the populations from the three
locations in Washington State and the one from WV included external body
surface lesions, internal tissues involvement with chronic to acute
mortality. Clinical signs in rainbows from PA differed; at first
observation the signs could be confused with those of Whirling Disease.
*Flavobacterium psychrophilum* was isolated from external lesions and
from internal tissues of fish from all five origins. Primary isolation
was on *Cytophaga* agar and isolates were frozen in fresh *Cytophaga*
broth. Upon recovery after 6 years in storage, none (0%) re-grew in
*Cytophaga* broth, however, 93.3% did using the following medium
(formulated from four literature citations): 0.5% tryptone, 0.05% yeast
extract, 0.028% sodium acetate-3H~2~O, 0.05% beef extract, 0.02% calcium
chloride, 0.074% magnesium sulfate-7H~2~O, and 5% fetal bovine serum.

**Comparative Challenge Model Of *Flavobacterium columnare* Using
Scarified And Unscarified Channel Catfish, *Ictalurus punctatus*
(Rafinesque**)

[Joel A. Bader]{.underline}1, Kenneth E. Nusbaum2, and Craig
A.Shoemaker1

1United States Department of Agriculture, Agricultural Research Service,
Aquatic Animal Health Research Laboratory, PO Box 0952, Auburn, AL;
2Department of Pathobiology, College of Veterinary Medicine, Auburn
University, Auburn, AL

The early entry of the fish pathogen *Flavobacterium columnare* and
enhancement by scarification was studied in channel catfish,
*[Ictalurus]{.underline} [punctatus ]{.underline}*(Rafinesque), using
the polymerase chain reaction (PCR) and a species specific primer set
for a bacterial 16-S rRNA gene product. Evaluations were conducted
following a typical abrasion bacterial bath immersion challenge with
*[F]{.underline}. [columnare]{.underline}*. The newly developed
Fvp2-Fvp3 primer set was useful in detecting the early entry of
*[F]{.underline}. [columnare]{.underline}*. The pathogen could be
detected in various tissues as early as 5 min after immersion challenge
at 29+/-2 C. The earliest detection of the pathogen was made in mucus,
skin, and gill tissues, occurring at 5 min post challenge. Liver and
kidney tissues required as little as 60 min prior to detection.
Detection times for *[F]{.underline}. [columnare]{.underline}* in blood
varied between 11.6 and 60 min. Scarification or abrasion, a practice
which has been historically used prior to bacterial challenge, had
little or no effect on the timing of early entry of pathogen at 292 C,
which calls into question the efficacy of the *F. columnare* challenge
model for channel catfish. However, survival data following abrasion
challenge with *F. columnare* at 29+/-2C showed that scarification does
significantly affect the survival of the fish.

**Treatment Of *Flavobacterium columnare* Infection In Channel Catfish
(*Ictarulus punctatus*)**

Praparsiri Srisopaporn^1^, [Thomas E. Schwedler^1^]{.underline}, Charles
D. Rice^2^, Ian R. Dorociak^1^

^1^Department of Aquaculture, Fisheries, and Wildlife Biology, Clemson
University, Clemson; ^2^Department of Environmental Toxicology, Clemson
University, Clemson, SC

Columnaris Disease *Flavobacterium columnare* often occurs after
handling, causing significant losses. This study investigates the effect
of two therapeutants reported to control columnaris disease. Subsequent
to severe losses of a group of channel catfish (*Ictarulus punctatus*)to
columnaris post handling, a second group was handled similarly and moved
into 40 aquaria. The fish were assigned randomly to 5 groups of eight
replicates.The groups were 1) a single potassium permanganate
treatment,2) a potassium permanganate every other day treatment,3) an
Oxy-tetracycline prophylactic treatment,4) an Oxy-tetracycline onset
treatment, and 5) a control group. The *F.columnare* infection was
severe with onset of mortality at 2 days and 92% of the control fish
dead by day 4 post handling. The two potassium permanganate treatment
groups appeared to have no therapeutic effect with mortality at day 4
being 89 to 93 %. The prophylactic Oxy-tetracycline group had no
mortalities by day 4 nor had any died by day 14 of the study. The onset
Oxy-tetracycline group began medicated feed on day 2 when 17.5 % of the
fish died. The mortality peaked on day 4 at 26.5 % and remained at that
level until day 14. The two Oxy-tetracycline treatment regiments were
very effective in preventing and curing naturally induced *F.
columnaris* infections.

**Aquatic Animal Health: The International Perspective**

[Gilles_Olivier]{.underline}

Department of Fisheries and Oceans, Gulf Fisheries Centre, Gulf Region,
Moncton, New Brunswick, Canada E1C 9B6

In 1995, several events have confirmed that Aquatic Animal Health was
now recognized at the international level. First, the World Trade
Organization was created January 1, 1995, succeeding the General
Agreement on Tariffs and Trade (GATT). On the same date WTO member
countries also adopted the Agreement on the Application of Sanitary and
Phytosanitary Measures (The SPS Agreement). The SPS Agreement sets out
the basic rules for food safety and animal and plant health standards.
Furthermore, the SPS Agreement states that for animal health and
zoonoses, the standards, guidelines and recommendations are those
developed under the auspices of the International Office of Epizooties
(OIE). It is therefore not surprising that, in 1995, the OIE published
the first Edition of their International Aquatic Animal Health Code
(AAHC) and the Diagnostic Manual for Aquatic Animal Diseases. These
publications were further updated in 1997 and in 2000-2001. The
Department of Fisheries and Oceans is now developing a National Aquatic
Animal Health Program (NAAHP). It is hoped that this presentation will
clearly illustrate that aquatic animal health is being
internationalized.

**Development Of An Aquatic Animal Health Plan For Salmonids In The
Exclusive Economic Zone (EEZ)**

[Kevin H. Amos]{.underline}^1^ and Spencer Garrett^2^

National Seafood Inspection Laboratory, National Marine Fisheries
Service, ^1^Olympia, WA; ^2^Pascagoula, MS

The Department of Commerce, National Marine Fisheries Service (NMFS),
has a directive to encourage and support the development of aquaculture
in the exclusive economic zone (EEZ -- the offshore waters from 3-200
miles under the jurisdiction of NMFS). One of the obstacles to
aquaculture in the EEZ is the absence of rules and policies to manage
fish health issues. The purpose of our project is to develop and pilot
test model fish health regulations that will provide for safe and
effective interstate and international commerce of salmonids cultured in
the EEZ. The model plan will be developed via input received at
workshops held in three regions in the United States (Northwest, Great
Lakes, and Northeast). A steering committee, composed of representatives
from relevant Federal agencies (Agriculture, Commerce, Interior, and
State) will provide oversight and review work products and draft plans
generated by the investigators.

**Clinical Presentations Of *Mycobacterium* sp. In Summer Flounder,
*Paralichthys dentatus*, Held In Recirculating Systems**

[Kathleen P. Hughes]{.underline} and Stephen A. Smith

Aquatic Medicine Laboratory, Department of Biomedical Sciences and
Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute and State University, Blacksburg, VA

A population of 1000 commercially-reared juvenile summer flounder was
housed in recirculating systems at the Aquatic Medicine Laboratory at
the Virginia-Maryland Regional College of Veterinary Medicine for
approximately one year. After approximately 6 months, fish began to
develop oral masses on the lower mandible. These discrete masses were
generally white-yellow in color and encompassed the rostral portion of
the mandible. In addition, other fish developed head swelling,
exophthalmia, coelomic distention and opercular masses. It was estimated
that at least 40% of the entire population displayed one or more of
these clinical signs. Some of the affected fish became lethargic and
emaciated. Impression smears and histopathology of these lesions
(stained with Ziehl Neelson acid-fast stain) revealed a dense population
of acid-fast bacilli. All affected tissues had marked effacing and
coalescing nodular granulomatous inflammation primarily composed of
epithelioid macrophages. There were rare mineralized granulomas
delimited by fibrous capsules with central calcification. This tissue
reaction was not the typical granuloma response seen in fish to
*Mycobacterium sp*. Bacterial cultures of the affected tissues grew on
Lowenstein-Jensen and Middlebrook media and were confirmed to be
acid-fast positive with Ziehl Neelson staining. Further identification
of the bacteria is being conducted.

# 

**Immunomodulatory Effects And Efficacy Of A DNA Vaccine**

For Aquatic Mycobacteriosis

[David J. Pasnik]{.underline} and Stephen A. Smith

Aquatic Medicine Laboratory, Department of Biomedical Sciences and
Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine,
Virginia Polytechnic Institute and State University, Blacksburg, VA

DNA vaccine research has demonstrated that plasmid-based vaccines have
multiple advantages over killed, attenuated, or subunit vaccines. DNA
vaccines have also been shown to stimulate a strong Th1/cell-mediated
immune response. Because of this, immunogenic vaccines have been made
for intracellular pathogens, such as infectious hematopoietic necrosis
virus and bacterial kidney disease. Another intracellular pathogen of
fish is *Mycobacterium marinum*, which causes a chronic progressive
disease that leads to systemic infections and ultimately death. It is an
economically important bacterial disease of virtually all species of
freshwater and marine fish. However, an effective vaccine against
mycobacteriosis has not been developed. Because of its advantages and
potential to induce a protective Th1 response, a DNA vaccine was chosen
as an appropriate construct for aquatic mycobacteriosis. A vaccine was
assembled by cloning the *M. marinum* 85A gene in the pcDNA 3.1
eukaryotic expression vector (Invitrogen, San Diego, CA). In the
resulting pCMV-85A construct, the 85A gene is expressed under a
cytomegalovirus (CMV) promoter after injection into fish tissue. Hybrid
striped bass (*Morone saxatilis* x *M.* *chrysops*), a proven
susceptible species, were used as a model to test the vaccine.
Intramuscular or intraperitoneal vaccinations of the construct were
administered on days 0 and 14 at doses of 0.01, 0.25 or 0.50 µg, along
with two control groups injected with an empty plasmid, pCMV-0, or
saline alone. Every two weeks, blood was drawn from the caudal vessels
of the fish to evaluate the humoral and cell-mediated responses by
ELISA, lymphoproliferative assay, and cytokine assay. Then on day 70,
the fish were challenged with *M. marinum*. Routine histological
examination was performed and splenic bacterial counts evaluated to
determine the relative degree of susceptibility among the different
vaccination groups.

**Identification Of Factors Involved In The Pathogenesis Of
*Edwardsiella ictaluri***

[Ronald L. Thune]{.underline}^1,\ 2^, Denise H. Fernandez^1^, Jennifer
L. Benoit^1^, and Maria Kelly-Smith^1^

^1^Dept. of Veterinary Science, LSU Agricultural Center, ^2^Dept.
Pathobiological Sciences, LSU School of Vet. Med., Baton Rouge, LA

Enteric septicemia of catfish (ESC) is recognized as the most serious
disease facing the commercial channel catfish industry. While much is
known about the pathology of *Edwardsiella ictaluri,* the causative
agent of ESC, little is known about the virulence factors associated
with infection. In an attempt to identify virulence genes of *E.
ictaluri,* mutants were created by signature tagged mutagenesis (STM),
an *in vivo* system that allows for the identification of bacteria
unable to grow in the host due to defects in genes affecting invasion
and survival. To date, 133 different mutant pools containing a total of
1197 individual mutants have been screened and 37 mutants were
identified that did not survive in the catfish host. Cloning and
sequencing thus far has identified several nutritional genes, including
a gene encoding N-acetylglucosamine-6-phosphate deacetylase, a gene that
may be involved in the synthesis of folate, the *miaA* gene, encoding
tRNA delta~2~-isopentylpyrophosphate transferase, the *guaB* gene,
involved in the biosynthesis of guanine, and the glucose inhibited
division protein. Possible virulence genes based on BLASTX searches
include a putative 0-antigen transporter gene, *insA*, which is involved
in Shiga toxin regulation, *pilL*, an essential gene in Type IV pilin
formation in *Salmonella enterica*, several genes involved in type III
secretory systems, and several genes having no matches to the National
Center for Biotechnology Information databases.

**Development Of Oxytetracycline Resistance In Rainbow Trout
(*Oncorhynchus* m*ykiss*) Gut Flora And Sediment In A Recirculating
Culture System**

# W. Bane Schill^1^ and [Graham L. Bullock^2^]{.underline}

^1^USGS, National Fish Health Research Laboratory, 11700 Leetown Road,
Kearneysville, WV; ^2^Conservation fund, Freshwater Institute, P.O. Box
1889, Shepherdstown, WV

Last year we reported that rainbow trout cultured in a recycle system
and fed oxytetracycline (OTC) at 2.5 gm/100 lbs of fish for 14 d
developed resistant gut flora, but similar resistance did not develop
among heterotrophic bacteria found in fluidized sand biofilters. Studies
were conducted this year with feces and sediment (feces and uneaten
food). Heterotrophic counts were performed on R2A and Mueller Hinton
media with and without 100 ug/ml OTC on individual trout feces (50-55
trout each sample period), on 12 fecal pools (5 trout/pool), and on
sediment. Feces and sediment were sampled before OTC feeding; on days 3,
7, 10 and 14 OTC feeding; and 3, 7, 14, 21, 28, 35, and 42 d post OTC
feeding. During the first cycle of OTC feeding, resistance (average % of
total heterotrophic counts on both media) in feces of single and pooled
trout increased from 11% before OTC feeding to 73% by day 14. Upon
completion of OTC feeding resistance dropped to 35.9% by day three,
increased slightly on day 14 post OTC feeding and dropped to 1.26% by
day 21. The second cycle of OTC feeding was begun immediately after
completion of the first cycle. The same general pattern of resistance
was seen using fecal samples of individual fish as had been observed
with pooled samples, and sediment resistance generally reflected that
found in feces. Surprisingly, sediment resistance was no higher than
that found in feces despite the fact that OTC levels in sediment were
determined to be as high as 1900 ppm. A definite bimodal pattern of
resistance was seen with the first peak on day 14 of the second OTC
feeding cycle and the second about 14 days after OTC was withdrawn. A
second population of rainbow trout was obtained to determine if these
patterns of OTC resistance in feces were universal or
population-specific. Two cycles of OTC feeding were given and the same
sampling days of pooled feces and sediment were used. Results from two
trials showed that resistance patterns during the first feeding cycle
were similar to those observed during the two trials of the first fish
population. We did not, however, observe the bimodal response during the
second OTC feedings that we had detected with the first fish population.
Additionally, sediment resistance levels did not reflect those obtained
with feces in these trials.

**Immunomodulation And Disease Resistance In *Myxobolus cerebralis*
Infected Rainbow Trout**

[Christine L. Densmore^1^]{.underline}, Christopher A. Ottinger^1^,
Vicki S. Blazer^1^, and Luke R. Iwanowicz^2^

> ^1^USGS, Leetown Science Center, National Fish Health Research
> Laboratory, 11700 Leetown Road, Kearneysville, WV; ^2^Department of
> Environmental Sciences, Virginia Institute of Marine Science, College
> of William and Mary, Gloucester Point, VA

Populations of rainbow trout (*Oncorhynchus mykiss*) infected with
*Myxobolus cerebralis*, the myxosporean that causes whirling disease,
may experience increased mortality levels when exposed to various
environmental stressors. In this study, we examined rainbow trout for
indications of altered immune function and disease resistance ten to
twelve months after *M. cerebralis* infection. The function of anterior
kidney leucocytes was evaluated in sham-exposed and *M. cerebralis*
exposed fish. In addition, both uninfected and *M. cerebralis* infected
trout, were bath-exposed to a bacterial pathogen, *Yersinia ruckeri.*
Macrophage function as measured by bactericidal activity was
significantly greater in the *M. cerebralis* infected fish. In contrast,
lymphocyte mitogen responses of the *M. cerebralis* infected fish were
lower relative to control fish. Slightly higher mortality levels
attributed to *Y. ruckeri* were observed among the *M. cerebralis*
infected fish compared to controls. Two weeks after *Y. ruckeri*
exposure, survivors were cultured for the organism, and the percentage
of fish testing positive was again just slightly higher for the *M.
cerebralis* infected fish compared to controls. Our findings indicate
some degree of immunomodulation among rainbow trout infected with *M.
cerebralis*.

***Ichthyophthirius:* The Historical Context**

# [Margaret S. Ewing]{.underline}

Zoology Department, Oklahoma State University, Stillwater, OK

Although *Ichthyophthirius multifiliis,* or at least the white spot it
causes, has been recognized as a problem in fish culture since the Ming
dynasty, our knowledge of the parasite's biology, interaction with its
fish host, and its ability to stimulate immune response is of course
much more recent. This presentation will provide some historical context
of life history/biology, pathology, treatment, and immune function
studies that are the foundation for current research on
*Ichthyophthirius*. This discussion of work from 1876 to 2000 is
intended to provide a backdrop for the papers that follow, which will
report current work concerning molecular studies of specific antigens,
antibody response to the parasite, and the problems posed by field
infections.

**A Twenty Year Historical Perspective Of *Ichthyophthirius multifilis*
Epizootics In New York State Fish Hatcheries**

[John H. Schachte]{.underline}

New York State Department of Environmental Conservation

## Fish Disease Control Unit, 8314 Fish Hatchery Road, Rome, NY 

The protozoan parasite, *Ichthyophthirius multifilis* (Ich),has caused
considerable fish mortalities in New York State (NYS) fish hatcheries in
the last twenty years. While generally not considered to be a serious
problem in cold water fish culture, three of our nine trout and salmon
hatcheries, Caledonia, Salmon River and Rome, continue to experience
seasonal epizootics of the parasite. Our three cool water facilities,
which respectively rear muskellunge, tiger muskellunge, and walleye, are
generally unaffected by the parasite. Seasonal water temperature
conditions that favor development of Ich at the cold-water facilities
are believed to be the primary factor. The use of surface waters subject
to mid-summer temperature spikes, at least in part, seems to precipitate
predictable epizootics. The incidence of infection from 1981 to 2001,
seasonality, species most affected and the treatments and success in
controlling the parasite will be discussed.

***Ichthyophthirius* and Aquaculture in the People's Republic of China**

[Lin Tianlong]{.underline}, Yang Jinxian, Liu Xiaudong, Gonghui
Chenqiang, and Xu Binfu

Fish Disease Control Center, Animal Husbandry and Veterinary Medicine
Institute, Fujian Academy of Agricultural Sciences, Fuzhou, China

Chinese aquaculture has developed rapidly since 1978 with an average
annual growth rate of 7%. In the year 2000 aquaculture production
reached about 20 million tons per year, and China now has one of the
largest aquaculture industries in the world. *Ichthyophthirius* is a
major threat to cultured freshwater fishes. More than 28 cultured fish
species in China can become infected with *I. multifiliis*, and heavy
losses have been reported over the past decade. Based on our research
with cultured eel (*Anguilla anguilla*), *Ichthyophthirius* is the most
common and virulent pathogen of this species. Parasites found on
infected eels have different tolerances to heat with some isolates
surviving at temperatures as high as 28^o^C. Based on variation of
surface immobilization antigens, four different serotypes of *I.
multifiliis* were found in eight parasite strains isolated from cultured
eels. Our findings suggest that differences in serotype correlate to
variations in temperature tolerance. A recipe containing ginger,
capsicum, pepper and borneol has been used effectively to treat
*Ichthyophthirius*-infected eels in densely stocked concrete ponds.

**Serum And Cutaneous Antibody Responses Of Channel Catfish (*Ictalurus
punctatus*) Against *Ichthyophthirius multifiliis* Following
Immunization By Infection Or Injection Of Immobilization Antigen
(I-Ag)**

[Joanne L. Maki]{.underline} and Harry W. Dickerson

Department of Medical Microbiology and Parasitology, College of
Veterinary Medicine, University of Georgia, Athens, GA

We are using channel catfish infected with *Ichthyophthirius
multifiliis* (*Ich*) to investigate mechanisms of mucosal immunity. In
this study, serum and cutaneous antibody (Ab) responses of catfish
fingerlings (n=20/group) were evaluated following immunization by either
Ich infection or inoculation with purified i-Ag (5g/fish). Sera and
cutaneous mucus were collected bi-weekly for 14 weeks and Ab levels
measured by ELISA. Abs in sera (1:80) and mucus (undiluted) were
detected with a biotinylated-goat anti-catfish Ig Ab. Both groups
(Ich-exposed and i-Ag inoculated) had mean sera absorbance (A~405~)
values that were significantly higher than pre-treatment
(0.241[+]{.underline}0.047) at 5 weeks and increased through the
sampling period to 0.754[+]{.underline}.264. In mucus samples,
Ich-exposed fish had A~405~ values above pre-treatment levels
(0.210[+]{.underline}0.041) at 3-9 weeks with a peak value
(0.301[+]{.underline}0.066) at 7 weeks. Mucus Ab in inoculated fish
varied by tank and week. In one tank (n=10 fish) A~405~ values were
\>0.251 at 2,3 and 11 weeks. Three fish had A~405~ values \>0.300 at 9
weeks (n=1) and 14 weeks (n=2). In a replicate tank (n=10), mucus Abs
were not detected above background even though fish in both tanks had
similar serum Ab levels. In summary, fish immunized by Ich-exposure or
i-Ag inoculation had relatively high serum Ab levels that increased over
time. Mucus Ab levels were relatively low in both groups, were detected
transiently in the Ich-exposed fish and occurred sporadically in the
inoculated fish. Production of cutaneous and serum Ab against Ich does
not appear to occur synchronously.

**Effect Of Cutaneous Antibodies From *Ichthyophthirius* Immune Fish On
*Ichthyophthirius* Development In Catfish**

[De-Hai Xu]{.underline}, Phillip H. Klesius, and Richard A. Shelby

USDA, Agricultural Research Service, Aquatic Animal Health Research Lab,
P.O. Box 952, Auburn, AL

This study determined the effect of cutaneous antibodies to
*Ichthyophthirius multifiliis* (Ich) development in channel catfish.
Culture conditions were established to observe the attachment of
theronts and development of trophonts in the excised catfish tissues.
Many important events involved for the same population of theronts and
trophonts can be observed continuously with this *in vitro* model, such
as theront invasion, shape transformation, trophonts rotation,
relocation, development and survival. Using tissue culture technique,
cutaneous antibodies were collected from the culture fluid of excised
skin from fish immune to Ich. Culture fluids from excised skin of immune
fish immobilized theronts and immobility of theronts was not observed
after immunoadsorption. Immunofluorescent staining of theronts treated
with immune culture fluid revealed strong and uniform fluorescence on
the cilia and cell surface of theronts. Western blot analysis of the
immune culture fluid revealed a 70 kDa band which corresponded to the
molecular weight of catfish immunoglobulin heavy chain. The treatment of
theronts with the immune culture fluid greatly reduced the attachment of
theronts, trophont size and survival in excised tissues compared to
those treated with the culture fluid from naive fish. *In vivo*, fewer
fish were infected and the infection density was less for fish exposed
to theronts treated with immune culture fluid. Results of this study
show that cutaneous antibodies against Ich were present in and released
from the excised skin from Ich immune fish. The antibodies significantly
reduced theront infectivity by immobilizing and weakening theronts.

**Cloning And Expression Of Surface I-Antigen Genes Of *Ichthyophthirius
multifiliis***

Yuankai Lin, Chia-Cheng Wang, Qiang Chen, and [Theodore
Clark]{.underline}

Department of Microbiology and Immunology, Cornell University, Ithaca,
NY

The i-antigens of *Ichthyophthirius multifiliis* comprise a family of
structurally related GPI-anchored membrane proteins that coat the
parasite surface. Antibodies against these proteins protect against
infection, and the antigens themselves represent important vaccine
candidates for the prevention of 'white-spot' disease in freshwater
fish. I-antigens vary among parasite isolates, and at least 5 serotypes
have been distinguished based on immobilization of swimming cells with
type-specific mono- and polyclonal antibodies. Immunity elicited by the
purified antigens is serotype-specific, and since multivalent vaccines
may be required for protection against field isolates, we are attempting
to isolate and characterize i-antigen genes from a wide range of
parasite strains. These efforts have led to the identification of two
new genes from the most common i-antigen serotype, namely, D. These
genes encode \~ 52/55 kDa antigens that share roughly 40% sequence
identify at the deduced amino acid sequence level. Comparisons of the
products of these genes with the deduced sequence of a previously
characterized 48 kDa antigen from serotype A, have revealed both
conserved and unique elements among the i-antigens of different stains.
We are using this information to 1) map protective epitopes associated
with these proteins, and 2) design PCR primers for the amplification of
i-antigen genes from diverse serotypes. Finally, we have developed
methods that circumvent the non-standard genetic code used by
*Ichthyophthirius* (where UAA and UAG "stop" codons specify glutamine).
Such methods will permit large-scale expression of recombinant proteins
for use in structural studies and vaccine trials.

**Elicitation Of Protective Immunity Against *Ichthyophthirius
multifiliis* In Channel Catfish (*Ictalurus punctatus*) By Immunization
With Immobilization Antigens**

[Harry W. Dickerson]{.underline}^1^, Xuting Wang^1^, Yan Gao^1^, Jane
Noe^1^, Jacek Gaertig^1^, and Theodore G. Clark^2^

^1^University of Georgia, Athens, GA and ^2^Cornell University, Ithaca,
NY

The ciliate *Ichthyophthirius multifiliis* infects all species of
freshwater fish and causes the common disease known as "white spot".
Experiments have revealed a novel form of protective immunity in which
parasites exit the host in response to binding of specific antibody. The
target antigens are a class of abundant surface membrane proteins
referred to as immobilization antigens (i-ags), which are a family of
antigenically related polypeptides in the 40 to 60 kDa size range bound
to the cell membrane by a glycolipid anchor. Affinity-purified parasite
i-ags elicit serotype-specific protective immunity when injected
intraperitoneally into naïve channel catfish. I-ag genes have been
expressed in the free-living ciliate *Tetrahymena thermophila* for the
production of recombinant vaccines. Gene constructs encoding the 48- and
52/55-kDa i-ags of *I. multifiliis* serotypes A and D, respectively,
were targeted by homologous recombination into the β-tubulin-1 gene
locus of *T. thermophila*, and expressed using a cadmium inducible
promoter from a *T. thermophila* metallothionein gene. Up to 10 ng of
recombinant i-ag protein/10 μg total cell protein was produced in this
system. Furthermore, immunogenic recombinant i-ags were targeted and
displayed on the surface of the transformed *Tetrahymena* cells as
determined by indirect immunofluorescence and immobilization of live
cells by specific antibodies. To test for the ability of transformed *T.
thermophila* to induce immunity, channel catfish were injected twice
intraperitoneally at a 2-week interval with 1 x 10^6^ live cells per
fish. Immunized fish produced specific serum antibodies and had an
80-100% survival rate when exposed to lethal parasite challenge.

**Blood Chemistries Of Healthy, Nephrocalcinosis-Affected, And
Ozone-Treated Tilapia In Recirculation Systems**

Chun-Yao Chen^1^, Gregory A. Wooster^1^, Rodman G. Getchell^1^, [Paul R.
Bowser^1^]{.underline} and Michael B. Timmons^2^

^1^Aquatic Animal Health Program, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
NY; ^2^Department of Biological and Environmental Engineering, College
of Agriculture and Life Sciences, Cornell University, Ithaca, NY

Blood chemistries of Nile tilapia (pure strain *Oreochromis niloticus*)
raised in recirculation systems were studied. Plasma samples from 120
healthy tilapia, 30 per season, were collected and twenty-nine blood
chemistry parameters were measured. Besides healthy fish, another 30
nephrocalcinosis-affected tilapia, 30 ozone-treated fish and 30 fish
with liver pathology were also examined. Nephrocalcinosis-affected
tilapia differed significantly from healthy fish in most electrolyte and
metabolite concentrations, and also in ALT, AST and AP activities.
Ozone-treated fish showed significant differences in electrolyte
concentrations and ALT, AST, AP activities. Fish with hepatic lesions
differ from healthy controls only in calcium, phosphate, iron
concentrations, saturation of iron binding capacity and plasma protein
concentration. Discriminant analysis reveals good separation for each
group of fish. The number of blood chemistry parameters can be reduced
from 28 to 12 without losing the ability to separate the groups (both
with 91% percent correct prediction). Most of the incorrect prediction
comes from less satisfactory separation of ozone-treated from healthy
fish, due to the lack of apparent adverse impact on health after this
treatment.

**Studies Of Immunoglobulin Structure And Diversity In Non-Salmonid
Marine Fish**

[Andrew Dacanay]{.underline}^1^, E. B. Bentley^1,\ 2^, L. L. Brown^1^,
S. C. Johnson^1^

^1^National Research Council Canada Institute for Marine Biosciences,
Halifax, Nova Scotia; ^2^ Department of Biology, Dalhousie University,
Halifax, Nova Scotia

Our laboratory is investigating the immune responses of cold-water
marine finfish to vaccination and disease exposure. We have investigated
the vaccination of haddock (*Melanogrammus aeglefinus* L) in terms of
the antibody response, immunoglobulin (Ig) quaternary structure and Ig
diversity. Specific antibody levels and immunoglobulin diversity was
determined for groups of haddock that were immunized at 12^o^C with
either a commercial vaccine against *Vibrio anguillarum* and *Aeromonas
salmonicida*, TNP-KLH conjugate and adjuvant, or saline and adjuvant. A
non-injected group served as a control. We were unable to detect
antibody responses to any of the antigens by ELISA. Two-dimensional gel
electrophoresis was used to investigate immunoglobulin diversity. No
detectable differences were observed in the isoelectrophoretograms
between groups. We found that the overall structure of haddock Ig is
similar to that of salmonids and is comprised of 85 kDa heavy chains and
25 kDa light chains. Data from non-reducing, denaturing
agarose/acrylamide gel electrophoresis and analytical FPLC suggest that
haddock Ig has a similar quaternary structure to salmon Ig. However,
with respect to levels of disulphide cross-linking, haddock Ig appears
to exist in its highest cross-linked form as a trimer. We have recently
expanded upon this study to investigate the physical structure of plasma
immunoglobulin from seven orders and 13 species of marine and anadromous
fish. In their highest cross-linked forms elasmobranch Ig is a pentamer
and, with exception of the Gadiformes species and the rainbow smelt,
teleost Ig is a tetramer. All species examined within the Gadiformes
(Atlantic cod, haddock, pollock, and tusk) have immunoglobulin composed
of trimers and lower forms. From these results it is evident that
phylogenetic position influences the extent of Ig disulphide
cross-linking.

**A Fluorescence Based Cytotoxic Assay For Fish Leucocytes Using Calcein
AM Labeled Adherent Target Cells**

 

 

[Luke R. Iwanowicz]{.underline}^1^, Christine L. Densmore^2^ and Chris
A.Ottinger^2^

 

^1^Department of Environmental Sciences, Virginia Institute of Marines
Sciences, College of William and Mary, Gloucester Point, VA; ^2^USGS,
National Fish Health Research Laboratory, 11700 Leetown Road,
Kearneysville, WV

 

 

Rainbow trout and numerous of teleost fishes posses a population of
natural killer-like cells referred to as nonspecific cytotoxic cells
(NCC). These cells are involved with tumor cell lysis, protozoan
parasite immunity and other innate immune functions. The gold-standard
for evaluating cytotoxicity is the ^51^Cr -release assay, which
unfortunately possesses many of the drawbacks inherent in all
radioisotopic assays. In mammalian research, alternative
non-radioisotopic methods have been developed to address this issue. We
have modified a method using the acetoxymethyl ester, calcein AM, as a
replacement for the conventional ^51^Cr label. Calcein AM is colorless,
nonfluorescent, uncharged molecule that is cleaved into a highly
fluorescent product by non-specific esterases inside a target cell. We
selected the carp epithelioma papulosum cyprini (EPC) cell line as a
biologically relevant target. Cytotoxic activity of purified rainbow
trout anterior kidney leucocytes was evaluated at three leucocyte to
target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8,
and 10 h) with labeled EPC cells at 15^o^C. Spontaneous release values
were consistent with those reported with rainbow trout using the ^51^Cr
method on the same cell line. Peak percent cytotoxic activity value
(mean ± SE n = 11 and 12 respectively) of 4.9 ± 1.11 and 11.53 ± 2.43
following 4 h and 17.2 ± 3.38 and 18.9 ± 3.47 following 8h of incubation
at the 2:1 leucocyte to target cell ratio. The bimodality of the
response was associated with sampling stress.

**Nonspecific Cytotoxic Cells Express Cytosolic And Membrane
Histone-Like Proteins**

[Donald L. Evans]{.underline}, Harjeet Kaur, Mustapha Oumouna and
Liliana Jaso-Friedmann.

University of Georgia, Athens, GA

Many laboratories have previously described the antimicrobial activities
of cellular lysates and tissue extracts obtained from many different
teleost species. Among these "antibiotics", one class of endogenous type
protein represents an especially novel approach to explaining mechanisms
of innate anti-bacterial immunity. In the present study, teleost
nonspecific cytotoxic cells (NCC) were treated *in vitro* for 24h with
synthetic oligodeoxynucleotides (ODNs) and whole cell lysates were
examined for the presence of upregulated proteins by ligand blots using
biotinylated ODNs. An 18 kDa molecule was identified and subjected to
tryptic digestion and analysis. Amino acid sequencing of a 13-mer
peptide revealed it was identical to human histone (H1). In order to
determine whether NCC expressed membrane receptors for ODNs, cells were
activated with ODNs, labeled with NHS-sulfo-Biotin and cell lysates were
prepared. Surface membrane biotinylated lysates were next mixed with
ODN-digoxigenin and these complexes precipitated with immobilized
anti-digoxigenin. Immunoprecipitates in the Ligand Blots were visualized
with Avidin-Peroxidase. Depending on the nucleotide sequence of the
Dig-labeled ODN, 2-3 different molecular weight species of DNA binding
receptors were identified. Additional preliminary studies were done to
determine (by flow cytometry) whether histone-like proteins (HLP) were
expressed on NCC membranes. Purified NCC were stained with commercially
available polyclonal antibodies against a 23 kDa histone (H1) protein
and a 15 kDa histone core protein. Both polyclonals bound to NCC in
relatively high proportions. These data demonstrated that NCC express
both cytosolic and membrane HLP.

**Expression Of Scavenger Receptor-A On Catfish Cytotoxic Cells**

[Harjeet Kaur]{.underline} , Liliana Jaso-Friedmann, Donald L. Evans.

College of Veterinary Medicine, University of Georgia, Athens, GA

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of
mammalian NK cells. In the present study the possible role of NCC in
mediating innate anti-bacterial responses was examined by determining if
these cells expressed membrane receptors for bacterial DNA. Among the
oligodeoxynucleotide (ODN) sequences examined for binding and cellular
activation was a unique 20-mer poly-guanosine nucleotide (dG20). dG20
specifically bound to NCC. Although dG20 binding to NCC was saturable at
nanogram concentrations, "cold" ligand competition experiments using
homologous as well as CpG and GpC containing ODNs demonstrated possible
multiple receptor specificities for bDNA. To examine the repertoire of
receptors on NCC for ODNs, experiments were done to determine whether
these cells expressed Scavenger Receptors (SR). In mammals, these
receptors bind poly-guanosine containing ODNs in the form of G-tetrads.
A subset (23%) of purified NCC from catfish expressed SR-A determined by
staining with an anti-human SR-A (Type I) monoclonal (mab). Cold
competition binding with two other types of ligands known to bind
(mammalian) SR-A (i.e. polyvinyl sulfate and dextran sulfate) decreased
anti-SR-A binding to NCC by 40%. Thus, similar to mammalian cells,
teleost SR-A also has promiscuous ligand specificities. Other receptors
than those recognized by anti-SR-A mab bound dG20 because binding
competition experiments with this mab only produced approximately 40%
inhibition of dG20 binding. These data demonstrated that NCC express a
heterogenous population of DNA receptors. Recognition of bDNA by NCC may
comprise one pathway of innate (anti-bacterial) immunity in teleosts.

**Binding Of Bacterial Genomic DNA And Synthetic Oligodeoxynucleotides
Leads To Activation Of Tilapia Nonspecific Cytotoxic Cells (NCC)**

[Mustapha Oumouna]{.underline}, Liliana Jaso-Friedmann and Donald L.
Evans

College of Veterinary Medicine, University of Georgia, Athens, GA

Previous studies in mammals have shown that nonmethylated bacterial DNA
(bDNA) in the form of oligodeoxynucleotides (ODNs) activates many
different types of cells including lymphocytes. In the present study
genomic bDNA from *Streptococcus iniae* and synthetic ODNs containing
CpG dinucleotide motifs were tested for activation of tilapia
nonspecific cytotoxic cells (NCC). Bacterial DNA treatment of NCC (*in
vitro*) activated the killing of tumor cells. Calf thymus and tilapia
genomic DNA treatments had no effects on NCC activity.
Oligodeoxynucleotides containing CpG motifs and oligodeoxyguanosine
20-mers increased the cytotoxicity more than the ODN composed of GpC
motifs. *In vitro* binding experiments demonstrated that bacterial DNA
as well as synthetic ODNs bound to splenic NCC. Specific binding of bDNA
to NCC was demonstrated by reciprocal cold target inhibition
experiments. To determine if binding occurred *in vivo*, tilapia were
injected (IV) with Rhodamine labelled ODNs and purified NCC from
different tissues were examined by flow cytometry. Percentage positive
NCC for ODN binding was: peripheral blood (38%), anterior kidney (12%),
spleen (17%) and liver (50%). One mechanism of regulation of NCC
activity by bDNA was protection from apoptosis detected by finding a
reduction in the production of DNA hypoploidy in bDNA treated NCC. NCC
treated with calf thymus DNA had a 73% reduction in 2N DNA compared to
31% in cells treated with bDNA. These data demonstrated that both *in
vitro* and *in vivo*, bDNA may participate in the activation of NCC and
as such stimulate anti-bacterial innate immunity.

**Construction And Characterization Of An Activated Nonspecific
Cytotoxic Cells (NCC) cDNA Expression Library From Tilapia Peripheral
Blood**

[Kesavannair Praveen]{.underline}, John Leary III, and Liliana
Jaso-Friedmann

Department of Medical Microbiology, College of Veterinary Medicine,
University of Georgia, Athens, GA

The phylogenetic characteristics of the teleost immune system have made
fish an important model for the study of vertebrate immunity. We have
been analyzing components of innate immunity in fish and have previously
reported that NCC are stimulated as measured by increased cytotoxicity
and resistance to apoptosis as a consequence of exposure of tilapia to
cold temperatures. cDNA expression libraries are powerful tools to
compare the required molecules necessary for the quiescent and activated
states in a specific cell population. An NCC cDNA library was
constructed with transcripts from cold-activated tilapia NCC. Total RNA
was extracted using Trizol reagent, and mRNA was purified using
biotinylated oligo-dT followed by capture with streptavidin immobilized
on paramagnetic particles. Purified mRNA was subjected to reverse
transcription with StrataScript reverse transcriptase and primed with an
oligo(dT) linker containing a *Xho* I restriction site and 5-methyl
dCTP. Double stranded, blunt-end cDNA was ligated with *Eco*R I adapter
to form cohesive ends. Size fractionated cDNA fragments were
directionally cloned to the arms of Uni-ZAP XR vector. After *in vitro*
packaging with Gigapack III Gold packaging extract, the XL-Blue MRF'
strain of *E. coli* was transfected with the lambda phage and
recombinants were verified by color selection. The average insert size
for the library (as shown by PCR amplification) was 1.1 kb. Ongoing
studies are aimed at characterizing the expression of various proteins
involved in the activation of innate immune functions mediated by NCC.
(Support provided by BARD US 3159-99C).

**Assessing The Health Of Corals In The Florida Keys Using A Cellular
Diagnostic System**

#### [Cheryl M. Woodley]{.underline} ^1^, John E. Fauth^2^, Craig A. Downs^3^, John C. Halas^4^, and Richard Curry^5^

^1^Marine Biotechnology Program, NOAA NOS CCEHBR, 219 Ft. Johnson Rd.,
Charleston, SC; ^2^University of Charleston, 66 George St., Charleston,
SC; ^3^EnVirtue Biotechnologies, Inc., 1866-C E. Market Street,
Harrisonburg, VA ; ^4^Florida Keys National Marine Sanctuary, NOAA, P.O.
Box 1083, Key Largo, FL ; ^5^Biscayne National Park, 9700 Southwest,
328^th^ St., Homestead, FL

We have developed a novel environmental biotechnology designed to
diagnose the health condition of key-indicator species within specific
trophic communities of a coral reef. Current methods of assessing
ecosystem health focus on responses at population or community levels
which reflect indirect effects of environmental insults. We are using a
comprehensive array of biomedical-like technologies to rapidly diagnose
the direct effects of such insults by evaluating the cellular
physiological condition of an organism and its responses to specific
classes of stressors. We are examining *Montastraea sp, Coralliophilia
caribaea*, *Halimeda opuntia*, *Haemulon plumieri,* and *Stegastes
partitus,* as representatives of 'key' trophic levels within Florida
Keys reef ecosystems. Variation in their exposure and responses to
environmental stressors at four reef sites in Biscayne National Park and
the Upper Florida Keys National Marine Sanctuary is being used to
evaluate ecosystem health condition. Preliminary results suggest that:
(1) this technology can be used to distinguish between global-level
stressors (e.g., El Nino/La Nina effects) and local-level stressors
(e.g., agricultural runoff) and (2) can help predict the condition of
corals several month before more obvious symptoms appear (e.g, coral
bleaching or coral death).

**A Caribbean Echinoderm Pathogen: Identification And Host Range**

[Kimberly B. Ritchie]{.underline}

MicroGenomics, Inc., Carlsbad CA.

In January of 1997, a massive die-off of the heart urchin *Meoma
ventricosa* was observed in Curacao, Netherlands Antilles. Necropsies of
affected individuals were performed and compared with unaffected
individuals. Comparative microbiological analysis revealed a unique
bacterium associated with catch connective tissue in affected
individuals. It was hypothesized that this may be the cause of spine
loss in affected urchins. The bacterial isolate was used to inoculate a
test urchin, *Lytechinus variegates.* Of ten urchins inoculated during
two experiments, all died within five days of inoculation. None of the
ten control inoculations produced any sign of disease. 16S rDNA analysis
showed the bacterium to be closely related to *Pseudoalteromonas
haloplanktis* subsp. *tetraodonis*. Monoclonal antibody testing revealed
that the isolate produced tetrodotoxin. Host range studies performed in
Bocas del Toro, Panama showed a number of urchins, as well as sea stars
were susceptible to infection by the bacterium, and that a range of
susceptibly patterns exist among echinoderms.

**First Steps In The Identification Of The Causal Agent Of The Dark
Spots Disease In Stony Corals**

[Diego L. Gil-Agudelo]{.underline}^1^, Garriet W. Smith^2^ , Diana P.
Fonseca^3^ and Jaime Garzón-Ferreira^3^

^1^University of South Carolina, Marine Science Program. Earth and Water
Science Building, Columbia, SC; ^2^University of South Carolina at
Aiken, Department of Biology and Geology. Aiken, SC; ^3^Instituto de
Investigaciones Marinas y Costeras INVEMAR, P.O. Box 1016, Santa Marta,
Colombia

Coral reefs are one of the most important and beautiful ecosystems on
our planet. But in the last decades, important decreases of coral reefs
have been seen throughout the world, particularly in the Caribbean.
Although some clues about the causes of such dramatic declines have been
discovered, the general question of what the primary causes are, are
still unanswered. Dark Spots Disease is a new syndrome that has
contributed to the general decline of coral reefs in the Caribbean. In
order to start identifying the potential pathogenic organisms causing
this condition, carbon source utilization patterns of more than 200
bacteria isolated from the surface mucopolysaccharide layers of healthy
*Montastraea annularis* and *Siderastrea siderea* and those showing
signs of Dark Spots Disease were compared to each other and the water
column. Using cluster analysis, we found that the bacterial communities
from each source were different. These differences were greatest for
*Montastraea annularis*. A comparison of individual metabolic taxons,
independent of coral species, showed the presence of 17 metabolic
groups. Metabolic group No. VII showed the most notable community change
among the groups. This group is similar to *Vibrio carchariae*, and it
was found to be unique to diseased samples. Work is continuing on the
possible pathogenic relationship between these isolates and corals.

**Timbuktu Meets Coral Reefs: Chemical Contaminants In African Dust**

[Virginia Garrison^1^]{.underline}, W. Foreman^2^, M. Majewski^3^, C.
Holmes^1^, E. Shinn^1^, R. Smith^4^ and M. Ranneberger^4^

^1^US Geological Survey, St. Petersburg, FL; ^2^US Geological Survey,
Denver, CO; ^3^US Geological Survey, Sacramento, CA; ^4^American
International School, Bamako, Mali

Every year, hundreds of millions of tons of African dust are transported
from the Sahara and Sahel across the Atlantic to the Caribbean, and
southeastern U.S. In the Caribbean, the sky becomes hazy, visibility
decreases to a few kilometers, a fine red dust settles on surfaces, and
residents complain of respiratory ailments. The fine soil particles have
been carried to the Caribbean for thousands of years, however the
quantities of dust reaching the Caribbean have increased dramatically
since the early 1970s. The quantity of dust transported is the result of
global climate systems, geomorphology of source areas, and regional land
use practices. Primarily made up of soil particles so small that human
lungs cannot expel them (\<2.5 μm), the dust may transport a variety of
microorganisms and chemicals that hitch-hike on or within the small
particles. We have recently begun a pilot project to test our hypothesis
that Sahelian dust air masses transport chemical contaminants (from the
burning of biomass and waste, and use of antibiotics, pharmaceuticals,
and pesticides in dust source regions) and radioisotopes (from the
source regions and produced in stratospheric transport) thousands of
kilometers to the Americas, and that those contaminants may be working
synergistically to adversely affect coral reef and human health.

**An Update On The Aspergillosis-Gorgonian Story: Increased Host Range
And Strain Origins**

### Garriet W. Smith

Biology Department, University of South Carolina, Aiken, SC 29801

*Aspergillus sydowii* was show to be pathogenic on the sea-fans
*Gorgonia ventilina* and *G. flabellum*, but recently, *A. sydowii* was
isolated from diseased *Pseudopterogorgia americana*, *P. acerosa* and
other gorgonians. Cross-species inoculations showed that *A. sydowii*
may be a nonspecific pathogen of gorgonians. Secondary chemicals may
also play a role in pathogenesis. Fungal isolates obtained from diseased
coral tissue and reference strains were grown in pure culture and
secondary metabolites extracted and analysed using HPLC. Specific peaks
were observed only with pathogenic strains. Results were confirmed by
inoculation on healthy sea-fan tissue. Other isolates obtained from
sediment samples from the mouth of the Orinoco and African dust showed
positive HPLC peaks and were pathogenic using bioassays. Results show
that reference strains are very different than pathogenic strains and
that a possible origin of the pathogenic strains may be African dust.

**United States East And Gulf Coast Shellfish Study**

> [Earl J. Lewis^1^]{.underline}, Ronald Fayer^2^, James M. Trout^2^,
> Lihua Xiao^3^, Dorothy Howard^1^, Rob Palmer^2^, Kristie Ludwig^2^,
> and Sue Tyler^1^
>
> ^1^NOAA, NOS, Coastal Center for Environmental Health and Biomolecular
> Research, Oxford, MD 21654; 2USDA, Agriculture Research Station, ANRI,
> Animal Waste Pathogen Laboratory, Beltsville, MD 20705; ^3^Centers for
> Disease Control and Prevention, Chamblee, GA 30341

In 1997, scientists from USDA, NOAA, Johns Hopkins University School of
Public Health (JHU), and CDC began a project to determine if: 1)
shellfish might be used to detect *Cryptosporidium* spp. and *Giardia
duodenalis* in estuarine environments, and 2) whether there was evidence
of environmental degradation in Maryland Chesapeake Bay from these
fecal-borne pathogens. Results showed *Cryptosporidium* oocysts survived
salinities up to 30 ppt for 8-12 weeks depending on water temperature
and a high prevalence of *C. parvum* was found within tissues, on gill
surfaces, and in hemocytes of oysters at 13 sites (9 of which were open
to shellfish harvest). Oocysts recovered from oysters on several
occasions were infectious in laboratory mice. Oocysts *of C. parvum*
were also found in hard clams and bent mussels; *G. duodenalis* was
found in *Macoma*, and freshwater *Corbicula* clams. Despite these
findings, there have been no reports of cryptosporidiosis from eating
raw shellfish. These findings however were viewed as a potential
environmental and public health issue that required further
investigation. Recently, USDA, NOAA, and CDC began to investigate
*Cryptosporidium* spp. and *G. duodenalis* in oysters and hard clams
from retail markets along the East and Gulf coasts of the U.S.
Traditional and molecular tools are being used to differentiate species
and to determine their prevalence in shellfish. This study will help to
better understand the distribution of the organisms on a multi-state
basis. Taking advantage of such an extensive study, shellfish were also
processed and fixed for histological examination.

**Update Of The Issues Surrounding The Introduction Of The Asian Oyster,
*Crassostrea ariakensis*, Into Chesapeake Bay**

[Frederick G. Kern]{.underline}

NOAA Ocean Service, Center for Coastal Environmental Health and
Biomolecular Research, Oxford, MD

Attempts to introduce exotic oysters into Chesapeake Bay are not new.
The pioneer oyster biologist Paul S. Galtsoff reported in 1932 that
several unsuccessful attempts had been tried along the East Coast of the
United States. There is good evidence that one of those attempts
resulted in the introduction of the parasite *Haplosporidium nelsoni,*
known commonly as MSX. This parasite has devastated oyster populations
along the East Coast of the United States for the last fifty years.
There are a number of examples where the Japanese oyster species,
*Crassostrea gigas,* has become established and has replaced more
desirable native oysters. With the almost complete collapse of the
oyster industry in Virginia, researchers have begun water testing of
non-native species of oysters under controlled experimental conditions.
The experimental tests were reviewed and approved by an ad hoc panel of
the Chesapeake Bay Program. To prevent the accidental introduction of an
exotic species, triploid oysters (presumed to be sterile) were used in
each experiment. There have been three different tests approved over the
last five years. The first involved the use of *C. gigas*, and the
second and third also tested *Crassostrea ariakensis,* the Chinese
Suminoe oyster. These tests revealed evidence of substantial growth of
*C. ariakensis* under varying environmental conditions, and also
indicated possible resistance to the oyster diseases common to
Chesapeake Bay: MSX and Dermo (*Perkinsus marinus*). Little is known
about the biology of the Suminoe oyster, so it is impossible to predict
the impact on native species in Chesapeake Bay and the East Coast of the
United States. Future testing is being discussed that would greatly
expand the number of oysters being placed overboard. This multifold
increase from a few hundred experimental oysters to millions for testing
in aquacultural settings has many public and private organizations
convinced that a viable population of this new species, *C. ariakensis,*
will become established in Chesapeake Bay and elsewhere along the
Atlantic Coast. Concerns expressed in public hearings will be discussed,
as well as the procedures to be used by the ad hoc panel, to evaluate
the risk of establishing a new species of oyster in Chesapeake Bay.

# THE EFFECT OF pH ON THE KILLING ACTIVITY OF HEMOCYTES IN THE PACIFIC OYSTER, *CRASSOSTREA GIGAS*

[Steven M. Allen]{.underline} and Louis Burnett

Grice Marine Laboratory, University of Charleston South Carolina, 205
Fort Johnson Rd., Charleston, SC

In recent years there has been an increase in the occurrence of summer
mortalities of the commercially important Pacific oyster, *Crassostrea
gigas*. These mortalities occur during the late summer when water and
air temperatures are at their highest. *C. gigas* are grown intertidally
and are, therefore, air exposed for hours at a time. An oyster closed
during air exposure depletes the oxygen stores within the shell and
builds up CO~2~ acidifying the tissues. The average pH of hemolymph from
an oyster which is submerged in well aerated water (18°C) and
ventilating is 7.52 (0.04 SEM; N=35). The average pH of of hemolymph
from an oyster which is aerially exposed for 4h in 30°C air is 6.83
(0.02 SEM; N=26). We hypothesize that stresses associated with air
exposure inhibit the immune system of the oyster and contribute to the
summer mortalities. The focus of the present study was to determine if
the innate immunity provided by hemocytes was decreased by low pH. The
ability of hemocytes to kill the bacterium *Vibrio parahaemolyticus* was
assessed using an *in vitro* killing assay. Hemocytes were treated with
low pH and challenged *in vitro* with *V. parahaemolyticus*. A
tetrazolium dye reduction assay was used to quantify the number of
viable bacteria, from which a killing index was calculated. No
significant difference was found between the two treatments pH 7.6 and
pH 6.6 (p \< 0.01; N=14). ODRP Grant No. NA96RG0488

**Impacts Of Short-Term Exposure To Hypercapnic Hypoxia On The
Susceptibility Of The Shrimp, *Litopenaeus vannamei,* To *Vibrio
parahaemolyticus***

[Karen G. Burnett]{.underline}, Stanley W. Iliff and Louis E. Burnett

Grice Marine Laboratory, University of Charleston, South Carolina

Under conditions of commercial production, aquatic organisms may be
exposed to diurnal and seasonal changes in dissolved oxygen, pH and
temperature. In previous studies, our laboratory demonstrated that
mortality rates of aquacultured shrimp *Litopenaeus vannamei* (1.0 --
1.5 g) injected with an LD~50~ dose of *Vibrio parahaemolyticus*
(10^5^/shrimp) significantly increased when the bacterial challenge was
accompanied by an immediate shift from well-aerated water to sub-lethal
hypercapnic hypoxia (4% O~2~, 2% CO~2~ and pH of 6.8-7.0). A more
realistic scenario is that shrimp raised in intensive culture might be
exposed to sub-lethal hypoxia for several hours each day during peak
production periods. The present study addressed the possibility that
animals might adapt to chronic hypoxia, returning to a more
disease-resistant state. Using the same infection model employed in
prior studies, *L. vannamei* were exposed to hypercapnic hypoxia for
four hours prior to challenge with an LD~50~ dose of *V.
parahaemolyticus*. Mortality rates in animals allowed to adapt to
sublethal hypercapnic hypoxia for four hours prior to challenge were not
significantly different from mortality rates in control animals that
were maintained in normoxia, challenged with the LD~50~ dose of bacteria
and returned to normoxic conditions. These results are consistent with
the possibility that shrimp may adapt to hypercapnic hypoxia within
several hours of exposure, restoring normal levels of disease
resistance. The impacts of diurnal cycling in O~2~, CO~2~ and pH on
disease resistance are being evaluated in the same infection challenge
model. (USDA CSREES 99-35204-8555).

**Effects Of Hypercapnic Hypoxia On The Bactericidal Activity Of
Hemocytes From The Penaeid Shrimp *Litopenaeus vannamei***

[Austin S. Dantzler]{.underline}, Karen G. Burnett, and Louis E. Burnett

Graduate Program in Marine Biology and The Grice Marine Laboratory,
University of Charleston, South Carolina

The high densities of shrimp found in aquaculture ponds often cause
dissolved oxygen levels to fall below 30% air saturation. Low oxygen is
usually accompanied by increased carbon dioxide and low pH. Previous
studies from our lab demonstrated that these conditions of hypercapnic
hypoxia decreased the resistance of aquacultured shrimp *Litopenaeus
vannamei* to infection with the opportunistic bacterial pathogen *Vibrio
parahaemolyticus*. To understand more about the mechanisms that might
underlie this effect of dissolved gasses and pH on the susceptibility to
infectious disease, the current study focused on the changes in *in
vitro* bactericidal activity of hemocytes from the white shrimp *L.
vannamei* under conditions of hypercapnic hypoxia. Antibacterial
activity was measured using a tetrazolium dye-based bacterial killing
assay. Hemocyte preparations exhibited two patterns of killing activity.
The first pattern was characterized by the ability of lysed hemocytes to
support bacterial growth. Hypercapnic hypoxia suppressed the
antibacterial activity of these hemocyte preparations. The second
pattern was characterized by the ability of lysed hemocytes to inhibit
bacterial growth. Hypercapnic hypoxia did not impact the antibacterial
killing activity of these hemocyte preparations. (USDA CSREES
99-35204-8555).

**Characterization And Expression Of C-Type Lysozyme cDNA From Kuruma
Shrimp (*Penaeus japonicus*)**

[Sonomi Minagawa]{.underline}^1^, Jiraporn Rojtinnakorn^1^, Jun-ichi
Hikima^2^, Ikuo Hirono^1^, and Takashi Aoki^1^

^1^ Laboratory of Genetics and Biochemistry, Department of Aquatic
Biosciences, Tokyo University of Fisheries, Konan 4-5-7, Minato-ku,
Tokyo 108-8477, Japan

^2^ Present address: Department of Biochemistry of Molecular Biology,
Medical University of South Carolina, 173 Ashley Avenue, POB 250509,
Charleston SC

Lysozyme is widely distributed in the serum, skin mucus and organs of
fish and plays an important role in bio-defence systems. A c-type of
lysozyme cDNA was cloned from a hemocyte cDNA library of the kuruma
shrimp (*Penaeus japonicus*). The cDNA of the kuruma shrimp c-type
lysozyme consisted of 1,055 bp, which coded for 156 amino acid residues.
The deduced amino acid sequence of kuruma shrimp lysozyme possessed?46%
homology with mouse and 43% homologies with Japanese flounder, rainbow
trout, chicken, and human c-type lysozymes. Comparison of the c-type
lysozymes showed that the catalytic residues and the cycteine residues
were completely conserved. Furthermore, we expressed the kuruma shrimp
c-type lysozyme cDNA in insect cells using baculovirus expression system
and evaluated the activity of the recombinant lysozyme. The activity of
the recombinant kuruma shrimp lysozyme was detected using a lysoplate
assay. We are currently investigating the optimum pH and temperature of
this recombinant lysozyme and the lytic activity against several
bacterial pathogens.

**Virulence Factors Expressed By *Vibrio tubiashii,* A Pathogen Of
Invertebrates And Vertebrates?**

[Ben D. Tall]{.underline}, Mahendra H. Kothary, Sherill K. Curtis,
Rachel Delston, Marion Pereira, Nancy Flores, Steve R. Monday, and
Matthew Jeletic

U.S. FDA, 200 C Street, S.W.,Washington, D.C.

Recent studies in this laboratory have revealed that *Vibrio tubiashii*,
a known cause of bacillary necrosis in mollusks, expresses a number of
virulence factors found in other pathogenic *Vibrio* species. Two of
these factors are an extracellular cytolysin and a metalloprotease. The
cytolysin has a molecular weight of 59 kDa and is inhibited by
cholesterol. It lyses various types of vertebrate erythrocytes, and is
cytotoxic to cells in culture, including Caco-2 cells, Chinese hamster
ovarian cells, and Atlantic menhaden liver cells. The N-terminal
sequence of the cytolysin displays significant homology to that of a
cytolysin produced by *Vibrio vulnificus*. Preliminary results obtained
from degenerative PCR analysis using primers based on the N-terminal
sequence yielded a theoretical 60 bp amplicon. In addition, a
theoretical 1456 bp amplicon was obtained using primers based on the
cytolysin gene of *V. vulnificus* that included the conserved sequence
of the ribosome-binding site. The metalloprotease of *V. tubiashii*
displays biological properties that are indistinguishable from those of
the metalloproteases expressed by *Vibrio cholerae, V. vulnificus,* and
*Vibrio anguillarum*. Its molecular weight is 34 kDa, and the first 20
amino acids of its N-terminal sequence are identical to those of the *V.
cholerae* metalloprotease. Oral feeding of *V. tubiashii* to suckling
mice produced significant intestinal fluid accumulation, equal to that
produced by *V. cholerae*. Collectively these results suggest that this
molluscan pathogen produces at least two virulence factors that are
related to those of other vibrios, and thus has the potential for
causing diarrhea in vertebrates, including humans.

> **Crustacean Hormones As Indicators Of Normal Development,
> Reproduction, And Stress**
>
> [Hans Laufer]{.underline}
>
> Department of Molecular and Cell Biology, University
>
> of Connecticut, Storrs, CT

Healthy lobsters require a properly functional endocrine system in order
to survive. A compromised endocrine system is a sensitive indicator of
impending problems affecting the survival, the development through
critical stages such as metamorphosis, growth to legal size), and
ultimately through maturation to reproduction. A healthy endocrine
system is essential for propagation and maintenance of the species.
Adverse environmental factors (both of human origin and of nature), such
as were present in western Long Island Sound (WLIS) in late 1999, are
known to interfere with appropriate functioning of the major endocrine
factors affecting the health and survival of lobsters. Of particular
relevance are stresses, such as unusually high temperatures, and the use
of the insect growth regulator (IGR) methoprene, a compound being used
to control West Nile Virus by targeting larval mosquitoes. Methoprene is
known to interfere with the proper metamorphosis of mosquito larvae into
pupae, preventing them from becoming adults because methoprene is an
analog of their own juvenile hormone (JHIII). We are investigating the
tolerance and resistance of lobsters to important physiological
stresses. The effects of prolonged elevated temperature and the
endocrine disruptor, methoprene, are being used to examine their effects
on the ability of lobsters to sustain specific protein synthesis and on
their ability to maintain a functionally integrated endocrine system, at
critical developmental stages. Of major importance of our research are
the effects of stresses on methyl farnesoate (MF), a growth hormone
which is involved in differentiation and reproduction, ecdysone, the
molting hormone, and hormones from the central nervous system,
crustacean hyperglycemic hormones (CHHs), molt inhibiting hormones
(MIH), and gonad inhibiting hormone (GIH).

**Acute Effects Of Methoprene On Survival, Cuticular Morphogenesis And
Shell Biosynthesis In The American Lobster, *Homarus americanus*.**

[Michael N. Horst]{.underline}^1^, Anna N. Walker^2^, Robert Vogel^3^,
Thomas G. Wilson^4^, Parshall B. Bush^5^, Ernest Chang^6^, Timothy
Miller^7^

Departments of ^1^Basic Medical Sciences, ^2^Pathology and ^3^Family
Medicine, Mercer University School of Medicine, Macon GA; ^4^Department
of Biology, Colorado State University, Fort Collins, CO; ^5^University
of Georgia, Athens, GA; ^6^Bodega Marine Laboratory, UC Davis, Bodega
Bay, CA; and ^7^Darling Marine Center, University of Maine, Walpole, ME

The1999 lobster mortality in WLIS coincided with the application of
pesticides including methoprene. Lethal effects were also observed on
several species of crabs. In addition to pesticides, stress in
crustaceans may be due to altered temperature and salinity of WLIS after
Hurricane Floyd. Reports of gravid female lobsters that died while
attempting to molt suggest endocrine disruption had occurred, since
molting is normally inhibited in gravid females. Three factors may have
contributed to the WLIS mortality: acute dose of methoprene, lower than
normal salinity and above normal water temperatures. Our objectives
were: (1) to determine both lethal and sublethal doses for methoprene in
all life stages of the American lobster at various salinities and
temperatures; (2) to investigate the cytopathology of methoprene treated
tissues; (3) to conduct biochemical studies on the effects of methoprene
on shell formation in lobsters; (4) to evaluate stress protein induction
in lobsters by methoprene; and (5) to examine bioaccumulation and
metabolism of methoprene in lobster tissues. Methoprene is toxic to
crustaceans from 1.5-125 ppb and causes cellular abnormalities and
decreased shell synthesis in blue crabs. It penetrates the egg case of
blue crabs and disrupts embryonic development before emergence of the
first larval stage. If methoprene acts similarly in lobsters, then
significant decreases in numbers of new lobsters could be observed in
future years. Preliminary results indicated that methoprene localizes in
certain tissues within postmolt and intermolt lobsters, specifically
epithelial cells underlying the shell, the digestive gland or
hepatopancreas and nerve tissue; little methoprene accumulates in
muscle. Methoprene treatment of adult lobsters causes increased
formation of certain proteins in the hepatopancreas and decreased levels
of others. NOAA/Sea Grant as part of the NY/CT Lobster Mortality
Initiative sponsored support.

**Bacterial Assemblages And Histopathology Of Shell Disease**

[Andrei Chistoserdov]{.underline}^1^, [Roxanna
Smolowitz]{.underline}^2^, Feliza Mirasol^1^, Andrea Hsu^2^.

^1^MSRC, SUNY at Stony Brook, Stony Brook, NY; ^2^Marine Biological
Laboratory, Woods Hole, MA

Shell disease in free-living lobster populations along the coast from
Eastern Long Island Sound (ELIS), NY, to the Vineyard Sound, MA, is
characterized by erosions of variable depth on the dorsal carapace,
often resulting in thin, easily compressed shells. Histopathologically,
diffuse, deeply extensive erosions are common. Carapace matrix in the
erosions is usually missing. But in some instances, unlike lesions noted
in impoundment disease, pillars of carapace matrix remain in the pitted
shell. The predominate organism found at the interface of necrotic and
live shell are bacteria. A combination of culture-based and molecular
techniques was used to study these bacterial consortia. Preliminary
denaturing gradient gel electrophoresis (DGGE) data indicate that up to
five independent phylotypes of bacteria are present in lobster lesions.
At least two of them were found in all lobsters from ELIS. This is
consistent with the five to six morphotypes of bacteria, which were
cultured on a marine agar. All isolated bacteria were either aerobic or
facultatively anaerobic. Two different types of fastidious chitinolytic
bacteria were isolated on a medium containing chitin. No correlation has
been found between the presence or severity of shell disease and the
presence of bacteria in hemolymph.

**Improved Analysis Of Shell-Disease Severity In Offshore Populations Of
American Lobster**

[Richard A. Robohm]{.underline}, Diane Kapareiko, John J. Ziskowski, and
George R. Sennefelder

NOAA, NMFS, Northeast Fisheries Science Center, Milford, CT

In previous work we established the prevalence of shell-disease and
measured the lesion areas as well as carapace lengths in populations of
American lobster inhabiting offshore canyons of the northeast coast of
the United States. Subsequently, we found a mathematical relationship (y
= 1.1034 + 1.9677 \* log CL) between carapace length (CL) and surface
area (y) for lobsters within the size-range of our database (manuscript
submitted). The formula allowed us to determine the percentage of total
surface area affected by shell disease on each lobster. This percentage,
multiplied by 10^^, constitutes a "disease severity index"(DSI) that
will be useful in evaluating intensity of disease for various lobster
size-classes in continental-shelf locations. Regression-tree analysis
(binary recursive partitioning) of the DSIs indicated that, unlike
earlier prevalence data showing significant sex differences, the most
important variable was carapace length. Kruskal-Wallis analyses showed
that DSIs for smaller females (\< 8.5-cm CL) were significantly higher
than for larger females (p = 0.05). Smaller males (\< 9.5-cm CL) had
significantly higher DSIs than larger males (p = 0.025). No differences
were found between small males and small females (p = 0.094) or between
large males and large females (p = 0.798). Additional regression-tree
analyses suggested both seasonal differences and site differences.
Complete non-parametric analyses of these variables should clarify any
relationships between shell-disease in lobsters and proximity to
anthropogenic pollution such as the 106-mile sewage-sludge dumpsite.

**Histopathologic And Molecular Aspects Of Paramoebiasis In Lobster
(*Homarus americanus*) With Emphasis On The Long Island Sound Die-Off**

[Salvatore Frasca Jr.^1^]{.underline}, Thomas Mullen Jr.^1^, Thomas
Burrage^2^ and Richard A. French^1^

^1^Department of Pathobiology and Veterinary Science, University of
Connecticut, Storrs, CT; ^2^United States Department of Agriculture,
Agriculture Research Service, Plum Island Animal Disease Center,
Greenport, Long Island, NY

Epizootic mortality of American lobster (*Homarus americanus*) occurred
in western Long Island Sound during autumn of 1999. Necropsies were
conducted on dead or dying, i.e. "limp", lobsters to elucidate a pattern
of lesions accountable for clinical signs of paresis, flaccid paralysis
and death. Histologic evaluation of hemocoelomic viscera, nerve ring
ganglia, ventral nerve cord and peripheral nerves revealed hemocytic
neuritis and ganglioneuritis, with polysystemic interstitial hemocytic
infiltrates and microgranulomata. An amoeba was identified in foci of
neuritis, in hemocytic infiltrates, within neurons and between nerve
fibers. This amoeba possessed a small, round, secondary nucleus
differentially stained using the Feulgen technique. Transmission
electron microscopic examination of amoebae in ganglia confirmed the
presence of this nucleus-like organelle, or Nebenkörper, considered a
feature of members of genus *Paramoeba* Schaudinn, 1896. Molecular data
for the family Paramoebidae is absent. However, by utilizing universal
primers complementary to highly conserved regions of eukaryotic 18S
small subunit (SSU) ribosomal RNA genes, we have PCR amplified, cloned
and sequenced ribosomal DNA (rDNA) from twelve morphologically related
species of lobose amoebae. Consensus SSU rDNA sequences from these
amoebae provide the essential data with which to construct phylogenetic
trees using bioinformatic computer methods and to identify target
sequences for PCR primer and *in situ* hybridization probe design for
detection of this paramoeba in lobster tissue. DNA-based diagnostic
techniques such as these are directly applicable to future management
programs designed to assess and maintain the health of the LIS lobster
population.

**CHO Cell Elongation Factor Found in Limp Lobster Disease *Vibrio
fluvialis* Isolates**

[Ben D. Tall^1^]{.underline}, Mahendra H. Kothary^1^, Marion Periera^1^,
Moraima Ramos-Valle^1^, Nancy Flores^1^, Aminat Johnson^1^, Sherill K.
Curtis^1^, Venugopal Sathyamoorthy^1^, Robert H. Hall^1^, Sukadev
Lavu^1^, Barbara McCardell^1^, and Robert C. Bayer*^2^*

^1^U.S. FDA, Washington, D.C. and ^2^University of Maine, Orono, ME

Recent studies in this laboratory have shown that *Vibrio fluvialis*
isolates causing limp lobster disease possess a number of virulence
factors found in other pathogenic *Vibrio* species. For example, they
express a hemagglutinin whose N-terminal sequence shows significant
homology to that of the mannose-sensitive hemagglutinin expressed by
*Vibrio cholerae*. Further analysis of the *V. fluvialis* isolates
revealed that they produced a factor that elongates(Chinese hamster
ovary(CHO) cells. PCR analysis using primers specific for the *cef*
gene, recently shown to encode the CHO cell elongation factor in *V.
cholerae*, revealed that 4 of 19 isolates gave a strong PCR reaction
product, while the remaining isolates produced a weak reaction product.
Characterization and testing of the *V. fluvialis* isolates using a
sealed suckling mouse assays revealed that the organisms caused fluid
accumulation comparable to that produced by *V. cholerae* or *Vibrio
parahaemolyticus*. The fluid accumulation was dose dependent, but no
microorganisms were recovered from the intestinal contents. Thus, the
elongation of CHO cells and the positive fluid accumulation response in
sealed suckling mouse assays observed in the present study might be
attributable at least in part to the expression of the *cef* gene in *V.
fluvialis*. However, other yet-to-be characterized enterotoxins may also
be produced.

**Status Of Attempts To Isolate And Characterize A Rickettsia-like
Organism (RLO) From Arctic Char (*Salvelinus alpinus)***

[Julie Bebak-Williams^1^]{.underline}, Alicia N. Noble^1^, Ana Baya^2^,
Peter Cooke^3^, Frank Hetrick^2^, Philip E. McAllister^4^, Douglas
McGrogan^5^, Caird Rexroad^6^, W. Bane Schill^4^

^1^Freshwater Institute, Shepherdstown, WV; ^2^MD Dept. of Agriculture,
College Park, MD; ^3^ ERRC, USDA-ARS, Wyndmoor, PA; ^4^USGS NFHRL,
Leetown, WV; ^5^Fish Health Management, Hamilton, Ontario; ^6^NCCCWA,
USDA-ARS, Leetown, WV

Since 1998, Arctic char (Nauyuk and Labrador strains) cultured at the
Freshwater Institute (FI) have experienced outbreaks of respiratory
disease associated with infection by a putative rickettsia-like
organism. Cumulative percent mortality in 5 to 1500 gm fish has ranged
from 1% to 60%. Outbreaks occur in flow-through and recirculating
culture systems. Water quality parameters have always been within normal
limits. Except for abnormalities on gill wet mounts and severe mucus
production on skin, gross necropsy results are unremarkable. Gross
microbiological examinations of fish show no evidence of significant
external bacteria or parasites, nor have systemic bacteria or viruses
been detected by culture. Histopathology of organs, except gills and
skin, is unremarkable. The only consistent histopathologic finding has
been the presence of an intracellular, coccoid organism in gill cells.
Attempts to isolate and characterize this organism include culture on
bacterial media and on fish cell lines, electron microscopy,
immunohistochemistry and polymerase chain reaction assay, and have been
unsuccessful. While horizontal transmissiom has occurred in rearing
units at the FI, source of the organism and mechanisms of transmission
have not been determined. A similar, possibly identical, problem occurs
in cultured Arctic char in Canada.

**Dose-Titration Assay Of Walleye Dermal Sarcoma Tumor Homogenate**

Rodman G. Getchell, Greg A. Wooster, Claudia A. Sutton, James W. Casey,
and [Paul R. Bowser]{.underline}

Aquatic Animal Health Program Department of Microbiology and Immunology,
College of Veterinary Medicine Cornell University, Ithaca, NY

Walleye dermal sarcoma (WDS) is a seasonally limited neoplasm caused by
a retrovirus. Due to the inability to culture this virus, our
pathogenesis, regression, and immunity studies have depended upon the
use of a crude tumor homogenate containing an unknown number of walleye
dermal sarcoma virus (WDSV) particles. To determine the quantity of
dermal sarcoma virus in these preparations, real-time reverse
transcriptase-polymerase chain reaction (qRT-PCR) was performed on
dilutions of the homogenate and compared to RNA standards transcribed
from a plasmid containing a portion of the WDSV gag gene. To approximate
the dose of virus needed to induce walleye dermal sarcoma tumors,
walleyes were challenged with a topical application of cell-free dermal
sarcoma tumor filtrates. Ten-fold dilutions of the filtrate were applied
to the side of the fish, which were allowed to develop grossly visible
tumors at 15°C for 20 weeks. We have determined that there are
approximately 10^10^ viral particles in 100 μl of walleye dermal sarcoma
inoculum. There is an estimated 0.075 g of WDS tumor in the 100 μl of
inoculum that we normally prepare, which converts to 13.3 X 10^10^ viral
particles in 1 g. The minimum dose of walleye dermal sarcoma virus that
could induce tumors by the topical challenge method was the 1,000-fold
dilution of this 10^10^ inoculum or 10^7^ viral particles. The ability
to enumerate the WDS virus may allow renewed attention to conducting
waterborne challenges of walleyes.

**Rescue Of Rainbow Trout (*Oncorhynchus Mykiss*) Gametes Compromised By
Infectious Pancreatic Necrosis Virus (IPNV)**

# [Philip E. McAllister]{.underline}

USGS/LSC, National Fish Health Research Laboratory, 11700 Leetown Road,
Kearneysville, WV 25430

Pair mating is a management tool used to reduce the risk of
gamete-associated generational transfer of infectious pancreatic
necrosis virus (IPNV). Fertilized eggs are segregated until the
virus-carrier status of each parent is determined, and if either parent
is found to carry IPNV, the egg lot is terminated. This practice has
resulted in the destruction of large numbers of potential progeny,
diminishes genetic diversity, and can cause bio-political tension if
threatened or endangered species or programmatic concerns are involved.
We envisioned that gametes could be rescued based on the premise that
gamete-associated transmission of IPNV is a variant form of horizontal
transmission. Using rainbow trout (*Oncorhynchus mykiss*) as a test
species, we devised a simple processing procedure to reduce the initial
virus burden of gametes, which are further treated with IPNV-specific
antibody to neutralize residual virus. In laboratory and operational
trials, IPNV titers in gamete preparations were reduced from ≤10^6^
PFU/ml to undetectable supporting the contention that IPNV may be
associated with, but is not integral to, gamete composition. Egg
viability and sperm motility were preserved. Hatch levels were
comparable between treated and untreated lots of eggs. IPNV infection
has not been detected in progeny.

**A Global Survey Of Transcript Expression In Rainbow Trout Leukocytes
During The Early Response To Viral Hemorrhagic Septicemia Virus
Infection**

[Caroline L. O'Farrell]{.underline},^1,2^ Nikta Vaghefi,^2^ Pierre
Boudinot,^2^ Abdenour Benmansour^2^

^1^Molecular Biology, American Type Culture Collection, 10801 University
Boulevard, Manassas, VA; ^2^Institut National de la Recherche, Unité de
Virologie et Immunologie Moléculaires, Jouy-en-Josas Cedex, France

Viral infections induce changes in the expression of host cell genes. A
global knowledge of these modifications should help to better understand
virus/host cell interactions. We used the suppressive subtractive
hybridization (SSH) methodology in the viral hemorrhagic septicemia
virus (VHSV) model of infection with rainbow trout *Oncorhynchus mykiss*
to examine viral-induced changes in host gene expression. We infected
rainbow trout leukocytes with VHSV, and total RNA from infected and
mock-infected cells was compared at 40 hr post-infection. Twenty-four
viral-induced genes were ultimately retrieved from the subtracted cDNA
library, and their differential expression was further confirmed by
semi-quantitative RT-PCR and northern blot hybridizations. Among these
sequences, three have already been described as VHSV-induced genes. Nine
sequences that have not been previously attributed to viral infection in
trout and had known homologues were chosen for further study and
extended to full-length cDNA using 5'and 3'RACE. They were subsequently
classified into three functional subsets. Four genes were homologous to
mammalian interferon responsive genes, three were similar to
chemo-attractant molecules, and two had nucleic acid binding domains.
The identification of these new viral-induced transcripts provide a
framework for future functional studies of rainbow trout immunity.

**Identification Of A Novel Piscine Retrovirus Associated With An
Outbreak Of Swimbladder Sarcoma In Atlantic Salmon, *Salmo salar***

[Claudia A Sutton^1^]{.underline}, Sandra L Quackenbush^2^, Paul R
Bowser^1^,Joel Rovnak^2^, Rufina Casey^1^, James W Casey^1^

^1^Cornell University, Department of Microbiology and Immunology, Ithaca
NY; ^2^University of Kansas, Department of Molecular Biosciences,
Lawrence KS

An outbreak of salmon swim bladder sarcoma in Atlantic salmon at the
North Attleboro National Fish Hatchery occurred in 1997-8. Affected fish
were juveniles caught in the Pleasant River, ME as part of the New
England salmon restoration project. Besides mortality due to swim
bladder sarcoma, involved fish performed poorly, and did not attain
sexual maturity. Our laboratory identified retroviral *pol* sequences in
the tumors by degenerate PCR; the *pol* amplicon was also detected in
blood DNA from this fish population. The same amplicon was also detected
in blood from other Maine river-specific salmon broodstock, as well as
in broodstock from a commercial facility. We have now deduced the entire
proviral sequence of the retrovirus implicated in the swim bladder
sarcoma outbreak, which we have named SSSV-1. We have recently succeeded
in transmission of the virus to salmon in the lab using swim bladder
tumor filtrate. In the course of the transmission studies, we discovered
that the recipient salmon, from the New York Adirondack hatchery, which
were negative for the SSSV-1 *pol* amplicon, were infected with a
second, highly related virus we call SSSV-2. More recently, a third
highly related virus has been detected in a wild Maine salmon. We
hypothesize that there are several SSSVs existing in the wild, and that
infection of fish with these viruses have negative consequences on fish
viability.

**Infectious Salmon Anemia In The United States: A Synopsis Of Maine's
Experiences With A Devastating Disease**

# [Peter L. Merrill]{.underline}

Micro Technologies, Inc., Richmond, ME

Infectious Salmon Anemia (ISA), an important disease of Atlantic salmon
(*Salmo salar*) caused by the contagious virus ISAV, has caused
large-scale economic losses as well as regulatory and political problems
internationally since the 1980's. As the pathogen and attendant disease
have advanced to new environments, most recently Cobscook Bay in Maine,
private industry, regulators and conservationists in the US have worked
together (and sometimes apart) to deal with the consequences of this
syndrome. A brief overview of the known epidemiological factors involved
in the transmission and spread of ISA will be discussed, along with a
review of the laboratory tests used for detection of the virus. A
detailed history of the progression of ISA from Canada to the US will be
included, and the attempts at dealing with both pathogen and disease
from the US and international regulatory perspectives will be examined.
Vaccine research and development will also be discussed. An up-to-date
review of the types and successes of ISAV monitoring programs on the
private, state and federal levels for both cultured and wild populations
will complete the presentation.

**APHIS Veterinary Services Implements An Infectious Salmon Anemia (ISA)
Program**

##### Otis Miller

USDA, APHIS, 4700 River Road Unit 46, Riverdale, MD

A Notice in the Federal Register of December 20, 2001, from the Office
of the Secretary announced that an emergency threatens the livestock
industry of this country, and USDA funds are available to establish an
ISA program to address the threat to the U.S. salmonid industry.
Approximately \$8.3 million has been authorized for APHIS VS to
implement an ISA control and indemnity program for farm-raised salmon in
the United States, effective as of December 13, 2001. In addition to the
payment of indemnity, these funds are to be used to assist the State of
Maine with program activities such as: depopulation and disposal,
clean-up and disinfection, establishment of surveillance programs,
epidemiology and diagnostic support, and training for producers and
veterinarians. Maine has taken steps to prevent the spread of ISA;
however, Federal assistance is deemed necessary to effectively control
this disease, which poses a threat to animal health and the U.S.
economy. ISA is a foreign animal disease of Atlantic salmon caused by an
orthomyxovirus. While this virus appears to cause disease only in
Atlantic salmon (both wild and farmed), sea runbrown trout, rainbow
trout, and other wild fish such as herring may act as carriers or
reservoirs of the virus. Clinical signs of ISA generally appear within 2
to 4 weeks after infection, and can include lethargy, swelling and
hemorrhaging of the kidneys and other organs, protruding eyes, pale
gills, and swelling of the spleen. Mortality is highly variable, ranging
from 3 percent to more than 50 percent over one production cycle. The
ISA virus was first isolated in Noway in 1984, and has since been found
in Scotland and Canada. The first case of ISA in the United States was
confirmed in Maine on February 15, 2001; in December 2001, 14 marine net
pen sites were confirmed to have been infected. Our goal is to control
and contain the ISA virus through rapid detection and depopulation of
salmon that have been infected with or exposed to ISA. It is believed
that the virus can be controlled within high-risk zones through
surveillance, vaccination, and best management practices. ISA control
requires requires depopulation of all pens holding infected fish.
Indemnification is necessary to provide an incentive for salmon farmers
to report diseased fish and to continue testing.

**Infectious Salmon Anaemia Is Not Vertically Transmitted; Evidence From
Recent Experiments In New Brunswick, Canada**

[Gilles Olivier^1^]{.underline}, Anne-Margaret MacKinnon^1^, Steve G.
Griffiths^2^

^1^Department of Fisheries and Oceans, Science Branch, Gulf Fisheries
Centre, Gulf Region, Moncton, New Brunswick, Canada; ^2^Research and
Productivity Council, 921 College Hill Road, Fredericton, New Brunswick,
Canada

Melville and Griffiths (1999) have provided evidence that Infectious
Salmon Anaemia (ISA) virus is not vertically transferred from
individually infected Atlantic salmon. In the fall of 1999, 15 wild
adult Atlantic salmon held in a land-based facility experienced an ISA
outbreak. The ISA virus was recovered from blood, tissues and ovarian
fluids. Eggs from infected Salmon were separated into two groups, a
disinfected lot and a non-disinfected lot. The progeny of both groups
were kept in quarantine and tested at regular intervals for the presence
of ISAv by IFAT, cell culture and RT-PCR. To this date, fish are alive
and held in quarantine, ISA was never observed in these fish and no ISA
virus was isolated from any fish tested by cell culture. The only
positive results were those obtained by RT-PCR several times during
their regular testing regime. The interpretation and significance of
these results will be discussed.

**An Overview Of Diseases Of Freshwater Ornamental Finfish Aquaculture
In Florida**

# [Roy P. E. Yanong]{.underline}

Tropical Aquaculture Laboratory, Department of Fisheries and Aquatic
Sciences, Institute of Food and Agricultural Sciences, University of
Florida, Ruskin, FL

The ornamental fish industry in Florida comprises approximately 50% of
all aquaculture in the state, and was valued at 43 million dollars farm
gate in 1999. Over 800 varieties of fish are raised in Florida by
approximately 200 producers(Florida Agricultural Statistics Service,
June 2000). Health issues in ornamental finfish production are
compounded by several factors: 1) the number of species and varieties
raised, 2) different production techniques used, and 3) different levels
of sophistication among producers. Fish health problems can be broadly
categorized as management: 1) water quality (including weather); 2)
nutrition; 3)predator/animal control; 4)grading/transport/handling;
5)culture system technology; and pathogens: 1) bacteria; 2)parasites;
3)viruses; 4) fungi. These categorizations are of course, artificial
divisions and in the real world, there is much overlap during disease
events. Management issues of interest include: 1) weather (excess heat
and cold); 2) complete nutrition (growout, in recirculating systems and
deformities {swordtails, barbs, koi}; hepatic lipidosis {African
cichlids, rainbowfish}); 3)predator/animal control (aquatic reptiles,
wading birds, otters). Pathogens of interest: 1) bacteria (*Aeromonas*
spp., *Vibrio* spp., *Flavobacterium columnare*, *Streptococcus* spp.
and related spp., *Mycobacterium* spp.); 2) parasites (in recirculating
systems, *Trichodina* sp.; in ponds, digenean trematodes and
pentastomids; flagellates, especially *Spironucleus vortens* and
*Cryptobia* *iubilans*, and others); 3)viruses (iridovirus, herpesvirus
{?}). "New" chemotherapeutics, vaccines, cell-line development,
histopathological analysis of normal fish and those with viral disease,
nutritional studies, and methods of harvesting, grading, and transport
are a few of the projects currently being investigated through the
University of Florida.

**Fish Health Management At A Wholesale Facility**

[Barbara D. Petty]{.underline}

Segrest Farms, Gibsonton, FL

Segrest Farms is one of the largest ornamental fish wholesalers in the
U.S. Some of the typical problems which affect fishes received by
Segrest Farms include improper handling (net damage, lack of temperature
control), poor water quality management, and inadequate control of
ectoparasites and endoparasites. The wholesale facility generates its
own problems. Quarantine is not economically feasible, and fishes from
different suppliers are mixed in different recirculating systems. The
fishes from suppliers' facilities, which have chronic health problems,
are treated during acclimation before introduction to a recirculating
system. Some employees handle fish carelessly, which can result in
mortalities.Strict attention is given to performance of biofiltration
and sterilization equipment, and water quality. Protocols for
efficacious chemotherapeutant application were developed after
necropsies were performed. Morts are removed three times a day. Fish
that do not appear normal are "held" and diagnostics are performed. New
recirculating systems have been built which should reduce the potential
for spread of pathogens. Mortality is currently \< 3%, and with improved
systems and acclimation, it is hoped that the mortality will be further
decreased.

**Parasitological Findings In Freshwater Ornamental Fish Imported In
Italy: A Five Years Survey**

[Monica Caffara]{.underline}^1^, Federica Marcer^1^, Daniela Florio^1^,
Amedeo Manfrin^2^, Maria L. Fioravanti^1^

^1^Dipartimento di Sanità Pubblica Veterinaria e Patologia Animale, Via
Tolara di Sopra 50 - 40064 Ozzano Emilia (BO) Italy. ^2^Istituto
Zooprofilattico Sperimentale delle Venezie, Legnaro (PD) Italy

From 1997 to 2001 a parasitological survey of 747 imported freshwater
ornamental fish was carried out. The fish were from Singapore (494
subjects), Malaysia (82), Sri Lanka (52), Thailand (45), China (30),
Israel (15), Hong Kong (13), Colombia (10) and the Amazon (6). The
parasitological examinations were carried out using the traditional
techniques. Parasites were detected in 406 (54.3%) out of 747 examined
fish and the highest prevalence was found in fish from Malaysia (90.2%),
Israel (60%) and Singapore (56.1%).

Parasitological findings

+-----------------------+--------------+--------+---------------------+
| **Sarcomastigophora** |              | **     |                     |
|                       |              | Ciliop |                     |
| *Cryptobia* sp.(8.3%) |              | hora** |                     |
|                       |              |        |                     |
| *Ichthyobodo*         |              | *C     |                     |
| sp.(3.3%)             |              | hilodo |                     |
|                       |              | nella* |                     |
| *Piscinoodinium*      |              | sp*.*  |                     |
| sp.(0.5%)             |              | (4.7%) |                     |
|                       |              |        |                     |
| *Hexamita* sp.(0.1%)  |              | *Trich |                     |
|                       |              | odina* |                     |
|                       |              | sp.    |                     |
|                       |              | (5.3%) |                     |
|                       |              |        |                     |
|                       |              | *      |                     |
|                       |              | Tetrah |                     |
|                       |              | ymena* |                     |
|                       |              | sp.    |                     |
|                       |              | (1.7%) |                     |
|                       |              |        |                     |
|                       |              | *Ichth |                     |
|                       |              | yophth |                     |
|                       |              | irius* |                     |
|                       |              | s      |                     |
|                       |              | p.(2%) |                     |
+-----------------------+--------------+--------+---------------------+
| **Myxozoa**           |              | **     |                     |
|                       |              | Micros |                     |
| *Myxobolus*           |              | pora** |                     |
| sp.(25.3%),           |              |        |                     |
| *Sphaerospora*        |              | *P     |                     |
| sp.(0.4%),            |              | leisto |                     |
| *Thelohanellus*       |              | phora* |                     |
| sp.(0.1%)             |              | sp.    |                     |
| *Zschokkella*         |              | (0.1%) |                     |
| sp.(0.1%), *Myxidium* |              |        |                     |
| sp. (0.1%), *Kudoa*   |              |        |                     |
| sp.(0.1%)             |              |        |                     |
+-----------------------+--------------+--------+---------------------+
|                       |              | **A    |                     |
|                       |              | picomp |                     |
|                       |              | lexa** |                     |
|                       |              |        |                     |
|                       |              | *Go    |                     |
|                       |              | ussia* |                     |
|                       |              | sp     |                     |
|                       |              | .(20%) |                     |
+-----------------------+--------------+--------+---------------------+
| **Platyhelminthes**   |              |        |                     |
|                       |              |        |                     |
| Monogenea:            |              |        |                     |
| *Dactylogyrus*        |              |        |                     |
| sp.(21.9%),           |              |        |                     |
| *Gyrodactylus*        |              |        |                     |
| sp.(10.1%),           |              |        |                     |
|                       |              |        |                     |
| Digenea:              |              |        |                     |
| *Centrocestus*        |              |        |                     |
| sp.(17.5%),           |              |        |                     |
| *Transversotrema*     |              |        |                     |
| sp.(0.2%)             |              |        |                     |
+-----------------------+--------------+--------+---------------------+
| **Nematoda**          | **           |        | **Fungi**           |
|                       | Arthropoda** |        |                     |
| *Camallanus* sp.      |              |        | *Branchiomyces*     |
| (0.8%)                | *Livoneca*   |        | sp.(0.4%)           |
|                       | sp.(0.1%)    |        |                     |
+-----------------------+--------------+--------+---------------------+

The risk of introducing non-indigenous parasites by means of the
imported exotic freshwater fish in Italy must be taken into
consideration and the importance of carrying out further surveys in this
field must be stressed. This work was supported by a grant from the
Ministry of Health (IZSVE 06/99).

**Selected Diseases Of Pet And Ornamental Fish: Their Detection,
Identification, And Management**

 

### Emmett B. Shotts

Department of Medical Microbiology and Parasitology ; College of
Veterinary Medicine, University of Georgia; Athens, GA

 

 

The variety of fish health problems (infectious and non infectous)
present an ever elusive array of clinical puzzles to solve. The
investigative approach and most importantlyan open and objective
assessment of the clinical stiuation is essential. The wide number of
species and varieties of ornamental fish and their varying response to
disease agents,water qualities, and management make each investigation a
challenge. Diseases caused by members of the bacterial genera
*Flavobacterium, Aeromonas, Edwardsiella, Vibrio, Streptococcus,* and
*Mycobacterium* often compounded by cryptic viruses add to the myriad of
signs seen in diseased fish. Correction of these conditions may vary
greatly and include nutritional supplements, temperature changes to the
use of antimicrobics.This presentation will consider some of these
problems with the aim of enhancing the knowledge of the fish health
specialist in their quest to understand and correct disease conditions
of pet fish.

**Husbandry And Management Of Aquatics At The Retail Level**

[Stephen A. Smith]{.underline} and Kathleen P. Hughes

Aquatic Medicine Laboratory, Virginia-Maryland Regional College of
Veterinary Medicine, Virginia Polytechnic Institute and State
University, Blacksburg, VA

What happens to fish and other aquatic species once they leave the
commercial import/export or aquaculture facility? How do retail stores
deal with the drastic changes in water quality parameters (temperature,
pH, DO, CO2, and ammonia) that often occur during transport? What are
the major infectious and non-infectious problems seen at the retail
level, and what corrective measures or treatments are used? This
presentation will explore some of these issues and provide basic
recommendations for the retail industry.

**Challenges Of Establishing And Maintaining A Healthy Collection During
The Start Up Of A Public Aquarium**

[Jason T. Crichton]{.underline}, Christopher R. Andrews, and Thomas J.
Sheridan

South Carolina Aquarium, 100 Aquarium Wharf, Charleston, SC

After nearly two decades of planning, development and construction, the
South Carolina Aquarium opened on May 19th 2000. The Aquarium has a
regional theme, showcasing the rich biodiversity of the southeast
Appalachian watershed, including a significant number of freshwater and
marine fishes in most of its 60 exhibits. Three years before the
Aquarium opened, an 8,000 square foot warehouse space was converted into
a fish holding facility in downtown Charleston, incorporating closed
freshwater and seawater life support systems ranging from 50 to 30,000
gallons. It was estimated that approximately 3,000 specimens comprised
of over 200 species would be required by opening day. These specimens
would need to be collected from a range of habitats include freshwater
lakes and streams to salt marshes and offshore pelagic and benthic
zones. The Aquarium staff was able to acquire many of the specimens
using a range of standard fishing techniques (seine nets, long-line,
traps, scuba diving and hook and line). The fish were then accumulated
at the holding facility and whenever possible quarantined using
appropriate freshwater or seawater protocols before being introduced to
the exhibits. Challenges included lack of space to adequately quarantine
all of the fish prior to opening, seasonal availability of some species
and possible deleterious species interactions during holding. In the
months that followed the opening of the Aquarium, disease outbreaks in
the exhibits were minimal and limited to *Benedenia* and *Cryptocaryon*.
With proper planning and thorough quarantining the Aquarium opened with
a diverse and healthy animal collection.

# Fish Health Management In Public Aquaria

# [Martin G. Greenwell]{.underline} and Allen C. Feldman

John G. Shedd Aquarium

1200 S. Lake Shore Dr., Chicago, IL

> The specific challenges of the health management of a large, primarily
> wild-caught, multitaxa fish collection in centrally filtered,
> semi-closed and closed systems are highlighted. Tropical, temperate,
> and coldwater fish from marine, euryhaline, and freshwater
> environments are all represented in this collection comprising some
> 350 families of fish dispersed among eighty-eight life support
> systems. Fish system volumes range from 10 gallons to 200,000+ gallons
> with a total water volume for the fish collection of approximately
> 3,000,000 gallons. Topics to be discussed include system/tank design
> issues, species composition issues, strategies to obtaining healthy
> stock, optimizing quarantine procedures (cost vs. benefit), water
> quality/life support management challenges, disease diagnosis
> challenges, logistics of treating sick/injured specimens, and
> nutritional issues. Tank designs are based upon several sources of
> input from several parties in a team-based approach. Competing
> interests of animal needs, aquarist needs, aesthetic needs, and
> pedagogic needs often take considerable time and money to resolve. The
> fish health manager in a public aquarium must navigate the fine line
> between a cost- and time-effective focus of population health
> management versus focusing on individual specimens of high economic or
> conservation value. Mixed taxa exhibits bring unique and often
> unpredictable challenges in areas such as water quality management,
> disease prevention, differential drug sensitivities, predation and
> territorial behavior (intra- and interspecies aggression). Accurate
> ante- and post-mortem diagnostics depend upon the training and/or
> experience of the diagnostician. Nutritional issues can present many
> challenges. Oftentimes, the natural diets of the myriad species
> exhibited are either unknown or incompletely known. Specialized
> feeders can be particularly challenging to feed. In the case of some
> dietary specialists such as obligate corallivores, captive maintenance
> is all but impossible.

**Summary Of Bacterial Isolates From Fish At The National Aquarium In
Baltimore**

# [Jill E. Arnold]{.underline}^1^ and Christina A. Gargan^2^

^1^ Animal Health Dept., National Aquarium in Baltimore, MD; ^2^The
Henson School of Science and Technology, Salisbury State University,
Salisbury, MD

The National Aquarium in Baltimore maintains over 150 aquaria, comprised
primarily of marine tropical teleosts, as well as two elasmobranch
exhibits and approximately 20 freshwater systems. Bacterial isolates
collected from clinical cases since 1994 parallel animal distribution:
of the total 476 isolates (398 Gram-negative, 78 Gram-positive), there
were 232(31%)from marine teleosts, 133(17.8%) from marine elasmobranchs
and 111 (23%)from freshwater fishes. Isolates were characterized by
standard biochemical tests. The most prevalent bacteria in all three
fish groups were Oxidase-positive, Gram-negative rods (346 isolates,
72.7%) of which 205 were susceptible to the vibriostatic compound 0-129.
The most frequently cultured sites were the visceral organs (219),
external/skin (99), blood (86), and the coelomic cavity (32). During
quarantine for a new seahorse exhibit in 2000, skin and viscera from
sygnathid species were screened for *Mycobacterium sp*., and 33 acid
fast positive isolates were found; 17 were identified by gas
chromatography as *Mycobacterium chelonae* (16) and *Tsukamurella
paurometabolum* (1). Kirby Bauer antibiotic susceptibility for 14 drugs
(n=354--411 isolates) showed the following results, in order of least
resistance (%): Gentamicin (5.1), Ceftazidime (10.7), Chloramphenicol
(11.2), Enrofloxacin (11.3), Amikacin (12.2), Ciprofloxacin (12.6),
Doxycycline (14.2), Ceftriaxone (15.5), Trimeth/Sulfa (15.8),
Tetracycline (21.7), Clavamox (27.7), Piperacillin (36.3), Cephalothin
(46) and Ampicillin (72.3).

# Common Syngnathid Health Problems

# [Ilze K. Berzins^1^]{.underline}, Martin Greenwell^2^ and Brent Whitaker^3^

^1^The Florida Aquarium, Tampa, FL; ^2^John G. Shedd Aquarium, Chicago,
IL; ^3^National Aquarium in Baltimore, Baltimore, MD

Seahorses and related species in the family Syngnathidae are
increasingly popular in the public aquarium industry. Common clinical
problems encountered include bacterial diseases, gas/air entrapment and
parasitic infections. Vibriosis is the most commonly diagnosed bacterial
species. Common clinical signs include erosive and ulcerative lesions on
the skin, sudden death with no visible problems, and edema of the tissue
around the eye and snout. Infections with *Mycobacterium* *sp.* are also
being encountered more frequently. Gas/air entrapment can affect the
skin, brood pouch, and swim bladder. The etiology of these conditions is
still in debate. Low level, chronic supersaturation has been proposed
but remains unproven. Two protozoal ectoparasites, *Amyloodinium
ocellatum* and *Uronema marinum*, have been the most commonly diagnosed.
*Uronema* infestations, however, are usually difficult to eradicate.
Endoparasites reported include intestinal coccidiosis, trematodes
(flukes), nematodes (roundworms), cestodes (tapeworms) and the
microsporidian *Glugea sp.* Preventative medicine is encouraged at all
steps of the acquisition process (collecting, wholesaler,
client/recipient, breeder). Proper handling and shipping procedures,
appropriate water parameters, the establishment of a quarantine
protocol, and adequate nutrition are essential in preventing the common
diseases of Syngnathids.

**Spinal Deformities In Captive Sandtiger Sharks (*Carcharius taurus)***

[Ilze Berzins^1^]{.underline}, Michael Walsh^2^ and Michael Richards^3^

^1^The Florida Aquarium, ^2^SeaWorld Adventure Park Orlando, and
^3^Albuquerque Biological Park, Albuquerque, NM

Abnormal spinal deformities have been noted in captive sandtiger shark
(*Carcharius taurus*) populations but until recently, the incidence and
etiology of the problem received minimal investigation. A preliminary
survey, conducted in 1998, and subsequent surveys have identified at
least 33 cases worldwide. The clinical presentation includes a marked
spinal curvature, usually in the region between the pectoral girdle and
cranial dorsal fin. Gingival hyperplasia, permanent protrusion of the
jaw, and curled pectoral fins have also been noted but it is unknown if
the conditions are related. Radiographic studies reveal single or
multiple subluxations and compressed vertebral bodies. Numerous
etiologies have been proposed including nutritional imbalances, trauma,
exhibit constraints, musculoskeletal disease and atypical biomechanical
stresses. Although studies have been initiated in many of these areas,
obtaining detailed information is vital to delineate contributing
factors. Specific sample guidelines have been developed to assist
facilities with the collection of needed information.

**Amphibian Health In A Public Aquarium**

[Marisa A. Giardina]{.underline}, Brent R. Whitaker

National Aquarium in Baltimore, Pier 3/501 E. Pratt Street, Baltimore,
MD

Although leopard frogs and axolotls have been studied in the laboratory
for more than a century, it is only within the past 20 years that
amphibians have gained significant popularity in zoos and aquariums.
This has led to the need for veterinarians who are knowledgeable about
amphibian husbandry, health assessment and medical care. As with fish,
proper husbandry, which includes housing, environmental parameters,
social environment and nutrition, is essential to maintaining amphibian
health. When an amphibian presents with signs of disease, these factors
must be considered as part of a complete health assessment, along with
evaluating the complete history and the physical exam with appropriate
sample collection. Identification of problems with husbandry or
pathogens, including bacterial, fungal, parasitic and viral agents,
helps the veterinarian to form differential diagnoses and develop a
therapeutic plan for successful medical care.

**Chromomycosis In Amphibians**

# [Brent R. Whitaker]{.underline}

National Aquarium in Baltimore, Pier 3/501 E. Pratt Street, Baltimore,
MD

Chromomycosis, or chromoblastomycosis, in amphibians is a systemic or
integumentary infection caused by ubiquitous pigmented fungi normally
found in soil, wood, and decayed vegetation. It is one of the most
common and often lethal fungal diseases affecting amphibians. Infections
originate in skin wounds or the intestinal tract. Pigmented fungi have
also been observed in crickets used as food for captive amphibians,
although a direct correlation with disease has not been shown. Clinical
signs in affected animals are often non-specific and typically include
anorexia, weight loss, ulcerative or granulomatous skin lesions, and
distended coelom. A diagnosis is easily made by finding brown or black
hyphae, sclerotic bodies or chlamydospores in granulomata, which are
often disseminated throughout the animal. Special stains, such as
periodic acid shift (PAS), can be used to detect the presence of these
fungi in biopsies, impression smears or histological specimens, but this
is rarely needed as the pigmented fungi are readily detected in
unstained tissues. To date, treatment of chromomycosis in amphibians has
not been successful. However, the use of antifungal agents such as
itraconazole, which is effective against many fungi including
dermatophytes and yeast, should be investigated.

**Marine Mammal And Sea Turtle Stranding Efforts In Maryland**

[Cindy Driscoll]{.underline}^1^, Susan Knowles^1^, Tricia Litwiler^1^,
David Schofield^2^

^1^Maryland Department of Natural Resources 904 South Morris Street
Oxford, MD; ^2^National Aquarium In Baltimore 501 Pratt Street
Baltimore, MD

In 1990 the Maryland Department of Natural Resources (MD DNR) and the
National Aquarium In Baltimore (NAIB) joined forces to form a marine
mammal and sea turtle stranding network under the authorization of the
National Marine Fisheries Service. Stranding responsibilities in
Maryland were divided along lines of expertise with live animal rescue
conducted by NAIB and recovery of dead stranded animals performed by MD
DNR. Agencies assist each other on an as needed basis. This cooperative
endeavor has been successfully implemented for over ten years and has
allowed for the examination of over 500 animals many of which are
threatened and endangered species in MD waters. A conservation plan has
been developed by MD DNR to address threats to marine mammals and sea
turtles and to recommend actions for recovery of known species in
Maryland waters. Investigations into the cause(s) of death are conducted
through necropsy examinations. Clinical examination and treatment of
rescued animals by NAIB enhance the knowledge of marine mammal and sea
turtle medicine through thorough health assessment tools and
diagnostics. Satellite tracking of released animals has documented the
successful reintroduction of rescued animals back to the wild. Efforts
of both agencies have led to significant advancements in conservation of
marine mammals and sea turtles in the Mid-Atlantic region.

**Overview Of Research, Health And Recovery Efforts For The Florida
Manatee**

# [Robert K. Bonde]{.underline}

U.S. Geological Survey, Florida Caribbean Science Center, 412 NE 16^th^
Avenue, Room 250, Gainesville, FL

Over three decades of research conducted by federal, state and
institutional biologists has been amassed on the Florida manatee
(*Trichechus manatus latirostris*). This research has focused on
abundance and distribution surveys, survival estimation, life history,
mortality, reproduction, behavior, anatomy, functional morphology,
physiology, feeding ecology, nutrition and captive husbandry studies.
Long-term studies have incorporated techniques for monitoring manatees
using radio-tagging, aerial survey, photoidentification, and genetic
analyses.  Individual health assessment and monitoring has also been an
important component in understanding manatee biology. A well-established
salvage program, coupled with observational data collected from all
regions of the state to estimate survival, have contributed to a better
understanding of vital rates and population dynamics and forms the
foundation for directing recovery efforts. Currently, there are
estimated to be about 3500 manatees in the southeast United States. 
Measures to protect this species appear to be working in some areas, but
information from other areas is lacking.  Threats to manatees are higher
in some regions of their habitat and deaths due to human-related causes
have been on the increase on Florida's east and southwest coasts. 
Epizootic die-offs due to harmful algal blooms (brevetoxin) have
impacted certain manatee populations.  The looming threat of
deregulation of the power industry (interrupted usage patterns) will
certainly affect survival of manatees that have become dependent on
these artificial warm-water refugia during winter. Management recovery
efforts follow guidelines established in the Florida Manatee Recovery
Plan.  Concerted efforts to protect this endangered species include
habitat protection, establishment of sanctuaries and water vessel speed
regulations.

**A Brief Overview Of Morbilliviral Epizootics In Aquatic Mammals**

[Thomas P. Lipscomb]{.underline}

Armed Forces Institute of Pathology, Washington, DC

Prior to 1987, morbilliviral infection was unknown in aquatic mammals.
Since 1987, tens of thousands of seals and dolphins have died during
seven well-documented morbilliviral epizootics that occurred in coastal
or inland waters of three continents. Bottlenose dolphins (*Tursiops
truncatus*), Baikal seals (*Phoca sibirica*), harbor seals (*Phoca
vitulina*), gray seals (*Halichoerus grypus*), striped dolphins
(*Stenella coeruleoalba*), common dolphins (*Delphinus delphis
ponticus*), and Caspian seals (*Phoca caspica*) were affected by these
outbreaks. Fatal morbilliviral disease has also been diagnosed in harbor
porpoises (*Phocoena phocoena*) and pilot whales (*Globicephala melas*).
The Baikal and Caspian seal epizootics were caused by canine distemper
virus, the harbor and gray seal epizootics by the newly recognized
phocine distemper virus, and the dolphin, porpoise and pilot whale cases
by the newly recognized cetacean morbilliviruses (porpoise
morbillivirus, dolphin morbillivirus and pilot whale morbillivirus).
Serologic studies have shown that morbilliviral infection is widespread
in many pinniped and cetacean species. Harp seals (*Phoca groenlandica*)
and pilot whales have high incidences of seropositivity consistent with
enzootic infection and may cause epizootics through contact with
susceptible species. Factors that cause changes in distribution of
aquatic mammal species, such as climate change, may be an underlying
cause of the recent series of epizootics.

**In-Water Sea Turtle Abundance And Health Assessment Study Off The
Coast Of The Southeastern United States**

J. David Whitaker^1^, Bruce W. Stender^2^, Phillip P. Maier^1^, [Al L.
Segars^1^]{.underline}

> ^1^South Carolina Department of Natural Resources, Marine Resources
> Division, PO Box 12559, Charleston, SC; ^2^Christschool, 500
> Christschool Road, Arden, NC

One of the obstacles facing policy makers regarding endangered and
threatened sea turtles is the lack of "real- time" data available on
these populations: numbers, sex ratios, age of animals and beach of
origin. This presentation will review techniques (trawling) and results
of years one and two in NMFS-funded study to develop an in-water index
of abundance for sea turtles off the southeastern United States.
Information gained through this project will supplement data gained
through the ongoing beach surveys of nesting females with information on
subadults and males. The study will develop and provide baseline
information for comparison in future population-monitoring efforts.
Health assessments to include CBC and blood chemistry blood profiles,
biotoxin/anthropogenic toxin assays and correlated immunological studies
will also be discussed.

**Recent Developments In The Spread And Study Of Marine Turtle
Fibropapillomatosis And The Fibropapilloma Associated Turtle
Herpesvirus**

Rebecca J. Murcek^1^, [Claudia A. Sutton^1^]{.underline}, Sandra L.
Quackenbush^2^, Thierry M. Work^3^, Rufina N. Casey^1^, Joel Rovnak^2^,
George H. Balazs^4^, James W. Casey^1^

^1^Cornell University, Dept of Microbiology and Immunology, Ithaca NY;
^2^University of Kansas, Dept of Molecular Biosciences, Lawrence, KS;
^3^USGS, National Wildlife Health Center Honolulu Field Station, HI;
^4^National Marine Fisheries Service, Honolulu Laboratory, HI

Fibropapillomatosis (FP) of marine turtles is a recently emerging
disease that affects turtles worldwide. Polymerase Chain Reaction (PCR)
studies identified the polymerase gene of a herpesvirus, named the
Fibropapilloma Associated Turtle Herpesvirus (FPTHV), in tumor samples
taken from FP-affected turtles. Quantitative PCR (qPCR) has demonstrated
that FPTHV is present at high copy numbers (1 to 50 copies per cell) in
FP tumor tissue. We examined the tissues of five different types of
parasites (marine leeches, amphipods, three species of blood flukes,
*Pyelosomum longicaecum* bladder parasites, and barnacles) of
FP-affected Hawaiian green turtles (*Chelonia mydas)* to determine
whether any of them carried enough virus to be possible vector organisms
for FPTHV. Leeches were found to frequently carry high levels of viral
DNA (10\^6 to 10\^7 copies per parasite organism). We have also found
detectable levels of FPTHV in seawater from Kaneohe Bay. Most recently,
we have cloned and sequenced approximately 30 kb of the FPTHV genome,
confirming its classification as an alphaherpesvirus.

# Acute Causes Of Sea Turtle Death: Identifying Turtle In Your Soup

[M. Katherine Moore]{.underline}^1^, John A. Bemiss^1^, Susan M.
Rice^2^, Joseph M. Quattro^3^, and Cheryl M. Woodley^1^

^1^National Oceanic and Atmospheric Administration, National Ocean
Service, National Centers for Coastal Ocean Science, Center for Coastal
Environmental Health and Biomolecular Research at Charleston,
Charleston, SC; ^2^U. S. Fish and Wildlife Service, Eastern Shore of
Virginia National Wildlife Refuge, Cape Charles, VA; ^3^Department of
Biological Sciences, Baruch Institute, School of the Environment,
Program in Marine Science, University of South Carolina, Columbia, SC

One of the many threats to sea turtle populations is poaching of turtles
and their eggs for consumption and sale. Here, we will address the scope
of the illegal trade and introduce a new method to identify sea turtle
eggs and cooked meats to the species level. Currently, fatty acid
profiles that are used to identify eggs to species cannot resolve
differences between loggerheads and the two ridley species.
Protein-based methods can resolve all species, but can only be used for
fresh meat, not eggs or cooked (or otherwise degraded) meat. In this
study, we present methods to produce species-specific diagnostic
restriction fragment length polymorphism patterns in the cytochrome B
region of the mitochondrial DNA. This method will work on DNA of
sufficient quality, regardless of the tissue of origin, and provides
wildlife law enforcement another tool to combat illegal take of
endangered species.