**Esocid Lymphosarcoma In Lake Champlain**

[Thomas E.Jones]{.underline}

Vermont Fish & Wildlife Department, 103 South Main Street, 10 South,
Waterbury, Vermont 05671-0501

Esocid lymphosarcoma has been observed on northern pike in Lake
Champlain for a number of years by governmental fisheries management
agencies as well as the sport fishing public. Through physical
observation of adult pike caught during electrofishing activities during
the spring months, estimates of prevalence of the disease are as high as
20%. Observations of lesions on pike appear to be seasonally dependant
with the majority of lesions reported during late fall, winter and
spring months. Beyond this account, little is known about the impact of
this disease on northern pike or other Esocids in Lake Champlain.

**The Case Of A Worm In A Bottle**

[Paul R. Bowser]{.underline}

Aquatic Animal Health Program, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
New York 14853-6401

A case submission of 28 June 2005 to the Fish Disease Diagnostic
Laboratory at Cornell consisted of what appeared to be a nematode. The
sample was presented by a Cornell University faculty member who received
it from a colleague in Minnesota. The sample was submitted with the
request that the organism be identified and with the question of
potential risk to humans. The nematode had been found in a fresh Chinook
salmon fillet purchased in a grocery store. The nematode was identified
as *Anisakis simplex,* also known as the "sushi worm." The parasite does
pose a zoonotic risk. Signs in humans may include gastrointestinal pain,
nausea and vomiting. The infection is often misdiagnosed.

**'Bloat' -- The Most Abused Term In Fish Health?**

[John S. Lumsden]{.underline}, Shohreh Hesami, Spencer Russell and
Véronique M. LePage

Fish Pathology Laboratory, Department of Pathobiology, University of
Guelph, Guelph, ON, Canada, N1G 2W1

How many different diseases or pathophysiologic pathways can you think
of that might lead to the clinical presentation of a fish with a
distended abdomen? 'Bloat', a colloquial term used widely for the
clinical symptom of a distended abdomen, is used not just by hobbyists
and on the internet but by aquaculturists and by diagnosticians in the
literature. 'Bloat' is used to describe superficially similar syndromes
in a variety of species of farmed salmonids that are
pathophysiologically distinct. Using gastric dilation and air sacculitis
(GDAS) in salmonids as one pertinent example of 'bloat' and outlining a
novel occurrence of this syndrome in cultured sturgeon in Ontario; the
pitfalls of superficial pathologic descriptions will be illustrated.

**Not Your Average Patient: Whale Shark Medicine 101**

^1^Tonya Clauss**,** ^1^[Alistair D.M. Dove]{.underline}, ^1^Christian
Schreiber, ^1^Timothy Binder, ^1^Christopher Coco, ^1^Ray Davis,
^1^Ashlie Vinson, ^1^Nicole LaBove, ^1^Michele Moses, ^1^Michael
Maslanka, ^1^Michael Walsh, and ^2^Jill Arnold

^1^Georgia Aquarium, 225 Baker Street, Atlanta, Georgia 30313;
^2^National Aquarium in Baltimore, Pier 3, 501 East Pratt Street,
Baltimore, Maryland 21202

The whale shark, *Rhincodon typus*, is the largest fish in the world; at
seven meters, a whale shark may weigh as much as 1500 kilograms. This
sheer size creates numerous logistical challenges when handling these
animals for medical procedures. Principal among these is capture and
restraint in a manner that is safe for the people and the animal
involved. Additional challenges include the collection of morphometric
information and diagnostic samples, diagnostic imaging techniques and
the delivery of medications, supplements or fluids. Perhaps the most
significant of the obstacles, however, is analysis of the biological
specimens and subsequent interpretation and action. There is currently
no database of clinical information on whale sharks and there remains
much to learn about the basic anatomy, physiology and ethology of this
species. Nonetheless, the experiences gained from husbandry and
veterinary practices with the four whale sharks acquired by the Georgia
Aquarium will be invaluable in future efforts to better understand and
appreciate these enigmatic elasmobranchs in both aquariums and their
natural environment. We have overcome many of the challenges involved in
handling and performing medical procedures with these animals in a
captive setting. Exciting collaborations doubtless lie ahead with the
other institutions maintaining whale sharks, in a collaborative effort
to increase the knowledge base information and conservation efforts for
this species.

**Unusual Myocardial Pathology Of Farm Reared Atlantic Halibut
(*Hippoglossus hippoglossus*)**

^1^Peter Sykes, ^2^[David Groman]{.underline}, and ^1^Larry Hammell

^1^ Center for Aquatic Health Sciences and ^2^ Aquatic Diagnostic
Services, 550 University Ave., Atlantic Veterinary College, UPEI,
Charlottetown, P.E.I. C1A4P3 Canada

Production of Atlantic halibut (*Hippoglossus hippoglossus*) is an
ongoing alternative species aquaculture industry in Norway, Iceland,
Scotland and more recently Atlantic Canada. Inflammation within the
epicardium and on the pericardial surfaces of aquaculture reared
fingerling halibut has been documented in Norwegian, Icelandic and
Canadian stocks, with suggestion of a nutritional or bio-mechanical
etiology. To date there has been no clear linkage to pathogens. Recent
observations of "grower" halibut, reared in production systems in
Atlantic Canada, have revealed an unusually high prevalence of gross
pericardial nodules and adhesions. Bacteriology and virology analysis of
tissue from affected fish have proved negative for infectious agents.
Histological examination of heart tissue from a range of fish with gross
lesions has revealed a mix of proliferative, inflammatory and vascular
changes. Gross and histopathology data will be presented with hypotheses
as to etiology.

**A Walk On The Wild Side: Selected Histopathologic Findings From
Endocrine Disruptor Studies**

[Jeffrey C. Wolf]{.underline} and Marilyn J. Wolfe

Experimental Pathology Laboratories, Inc., 456000 Terminal Drive,
Sterling, Virginia, 20166

Although the concept of endocrine disruption (ED) is decades old,
research efforts in this area have intensified during the past fifteen
years primarily due to government mandates and heightened public
awareness. Increasingly, chemical production companies and regulatory
agencies find themselves under pressure to rapidly provide data for use
in ED risk assessment analysis. A downside of this accelerated pace of
investigation is that the biological characteristics of a test species
may not be fully understood prior to the creation and performance of ED
experiments. Potential consequences of this are suboptimal study design
and misinterpretation of study results, which may in turn lead to
misconceptions that persist in the scientific literature. A specific
problem in aquatic animal bioassays is that information concerning the
occurrence of spontaneous histopathologic anomalies in control or
reference site animals is often lacking. The primary focus of this
presentation will be the illustration and brief discussion of various
reproductive tissue abnormalities, some of which could be considered
"background" lesions that have been identified in toxicological studies.

**An Epizootic Of Cyprinid Herpesvirus 2 (Goldfish Herpesvirus) In A
Goldfish - Koi Production Farm**

[^1^Janet V.Warg]{.underline}, ^2^Jennifer Haugland, and ^3^James W.
Provo

^1^ USDA-APHIS-VS, National Veterinary Services Laboratories, Diagnostic
Virology Laboratory, P.O. Box 844, Ames, Iowa 50010; ^2^ North Carolina
Veterinary Diagnostic Laboratory System, Raleigh, NC; ^3^
USDA-APHIS-VS,930 Main Campus Drive Raleigh, North Carolina 27606.

The cause of goldfish mortalities at a goldfish-koi production farm was
investigated. The clinical signs and lesions were consistent with those
of herpesviral hematopoietic necrosis. The goldfish were lethargic,
swimming at the top, and gathering around the diffuser. Three dead and 3
live goldfish were received for diagnostic work-up. Petechial
hemorrhages of the skin were observed. The gills were pale pink to tan
and some had pinpoint white foci within the filaments. No fluid in the
peritoneum was found. Some fish had pinpoint white foci in the spleen
and kidneys. Other kidneys were pale and small. A few fish had mildly
enlarged spleens. One fish had exophthalmia and the corneas were cloudy.
Histopathological examination revealed subacute inflammatory lesions and
intranuclear inclusion bodies in the brain, gills, liver, intestine,
pancreas, and adipose tissue. The caudal kidney and spleen exhibited
lymphohematopoietic necrosis and intranuclear inclusion bodies. A 10%
tissue homogenate made from a pool of kidney and spleen from four
goldfish was used for virus isolation. A sample of cell culture fluids
from koi fin cell cultures demonstrating CPE was processed for electron
microscopy and nucleic acid extraction for use in PCR. Testing performed
in the diagnostic workup including histopathology, bacteriology, virus
isolation, electron microscopy, and molecular characterization of the
viral isolate supported the diagnosis of cyprinid herpesvirus 2. This is
the first report of successful cyprinid herpesvirus 2 isolation on KF-1
cell cultures in the United State.

**Right Treatment; Wrong Results**

^1^Carolyn Gunn, ^1^[Peter Walker]{.underline}, ^1^Mandy Colburn, and
^2^Charlie Smith

^1^Colorado Aquatic Animal Health Laboratory, P.O. Box 128, Brush,
Colorado 80723; ^2^ 212 Story Hill Road, Bozeman, Montana 59715

*Flavobacterium psychrophilum* was isolated from the spleens of
10-centimeter rainbow trout in a lot of 160,000 at Pueblo State Fish
Hatchery in south central Colorado in late December, 2006. The fish
quickly responded to a regime of oxytetracycline administered in the
feed, but mortalities returned to normal for less than two weeks before
a second round of bacteremias of much greater severity broke out. This
time virtually all fish were affected. Clinical signs included
fluid-filled blisters progressing to open sores, exophthalmia, pale
gills and livers, enlarged spleens, and grossly swollen and pale renal
kidneys. Histopathology of the hind kidney demonstrated necrosis of some
kidney tubules characterized by pyknotic nuclei. Many cells within
hematopoietic tissue were enlarged and had large, often pleomorphic
nuclei that often contained 2 and occasionally 3 nucleoli.   Liver cells
were also swollen and often displayed double, swollen nucleoli,
while others were degenerate demonstrating karyorectic nuclei.  
Macrophages were swollen and commonly seen within blood vessels often
attached to vessel walls primarily of of livers and
kidneys. Steiner staining showed that macrophages were engorged with
short rods of two distinct morphologies. Cultures from kidney and spleen
on TYES Agar failed to grow *F. psychrophilum*; however, two species of
gram negative, gelatinase positive, beta-hemolytic short rods were
isolated on Blood Agar. These were identified as strains of *Aeromonas
hydrophila* and *Pseudomonas fluorescens*. Both isolates were totally
resistant to oxytetracycline, Romet-30, and florfenicol. The entire lot
of trout was buried and the raceways disinfected.

**Pigmented Tumors In Gizzard Shad From The Black Warrior River,
Alabama**

^1^[Amelia C. Knight]{.underline}, ^1^Jeffery S. Terhune, ^1^John M.
Grizzle, and ^2^William E. Garret

^1^Department of Fisheries and Allied Aquacultures, Auburn University,
Alabama, 36849; ^2^Alabama Power Co. Environemntal Affairs, P.O. Box
2641, Birmingham, Alabama

Some types of neoplasia in wild fish populations are thought to be
linked to environmental contaminants. However, other types of neoplasia
may be caused by viruses or inherited traits. In our study, gizzard shad
(*Dorosoma cepediaum)* were examined (n=200) from the Black Warrior
River at Plant Gorgas, a fossil fuel electric steam plant operated by
Alabama Power Company, in the summer of 2006. Four gizzard shad had
raised, pigmented tumors, varying in size from 0.2 to 0.4 to 0.7 cm to
1.0 x 1.6 x 1.7 cm, located in the skin of the frontal bone, and less
frequently in the skin of the fins. These tumors were cutaneous
melanomas, and this diagnosis was verified by the Registry of Tumors in
Lower Animals. Since the 2006 summer sample, an additional 400 gizzard
shad have been collected and subsequent pigmented tumors were observed
in six additional fish. Histological examination of these latter samples
is pending. Similar tumors in gizzard shad have been described from
several lakes in Oklahoma. Genetics, viruses, and chemical carcinogens
have not to been linked to the tumors in Oklahoma. Likewise, the cause
of the gizzard shad tumors in Alabama is presently unknown.

**Online Fish Disease Course At Kentucky State University**

[Robert M. Durborow]{.underline} and Boris I. Gomelsky

Aquaculture Research Center, Kentucky State University, Frankfort, KY
40601

Phone (502)597-6581

The Kentucky State University Division of Aquaculture plans to offer
Fish Diseases as a 3-credit graduate (AQU 511) and undergraduate (AQU
411) course in the spring 2008 semester. Course lectures were video
recorded when the course was taught in the fall of 2006. These lectures
will be made available to students taking the class in 2008 in Flash
video format and will be downloadable onto hand-held iPods. Students who
have taken other aquaculture courses in this video format preferred it
over online courses that were presented in text and pictures only. They
were able to re-watch lectures to reinforce and/or review certain
concepts that they may have missed after the initial viewing of the
lectures. For fish diseases, video format has the advantage of being
able to present moribund behavior of sick fish and videos of fish
parasites and bacteria. In addition, course documents including video
clips of fish pathogens and infected fish will be posted for student
access online on Blackboard^®^. Messages to students and grades will be
accessible on Blackboard^®^, and timed exams will be administered
online. Offering Fish Diseases online will make the course available to
a greater number and wider range of students compared to teaching the
course only to on-campus students. Online courses in aquaculture at KSU
have typically had more than 20 students enrolled compared to the
typical class size of about 9 for on-campus only courses. If enrollment
increases by 11 students when teaching a course online and tuition is
\$900.00, university income will increase by almost \$10.000.00 per
course per semester. And students who may not be able to quit their job
and go back to school full time will be able to take this online course
during their free time during evenings and weekends.

**The Current Status Of The U.S. National Aquatic Animal Health Plan**

[Robert Bakal]{.underline}

U.S. Department of Interior, Fish and Wildlife Service, National Aquatic
Animal Health Coordinator, 4700 Hillsborough St., Raleigh, NC 27606

The Joint Subcommittee on Aquaculture's (JSA) National Aquatic Animal
Health Task Force was tasked with developing a national aquatic animal
health plan (NAAHP) for aquaculture in cooperation with industry,
regional organizations, state, local, tribal governments and other
stakeholders. The NAAHP is intended to foster and support efficient
aquaculture; protect the health of our nation's wild and cultured
aquatic resources; and meet both national and international trade
obligations. Development of the NAAHP required input from working groups
composed of individuals who represent certain sectors of the aquaculture
industry. The final draft of the NAAHP was completed and submitted to
the JSA in June of 2007. The plan will not be codified into regulation;
however, implementation of certain elements, such as import
requirements, may require revisions to existing laws, regulations or
policies. In this presentation, a summary of key milestones will be
presented and discussed with regard to future goals.

**Comparison Of The Biology Of *Hematodinium* Infections In Blue Crabs
(*Callinectes sapidus*) And Snow Crabs (*Chionoecetes opilio*)**

^1^[Jeffrey D. Shields]{.underline}, ^2^David M. Taylor, and ^1,3^Hamish
J. Small

^1^Virginia Institute of Marine Science, Gloucester Point, VA 23062;
^2^Department of Fisheries and Oceans, CP5567, White Hills, St. John's,
Newfoundland, CANADA A1C 5X1; ^3^Centre for Environment, Fisheries &
Aquaculture Science, Weymouth Laboratory, Weymouth, Dorset DT4 8UB, UK

Differences in the patterns of infection, life history and
histopathology between disparate hosts may lead to a better
understanding of the biology of *Hematodinium*. We compare
*Hematodinium* infections in the temperate blue crab, *Callinectes
sapidus*, with that in the boreal snow crab, *Chionoecetes opilio*. In
heavy infections, crabs become weak and lethargic and die from handling
stress, but blue crabs do not show a discoloration of the carapace that
is a hallmark of infected snow crabs. Snow crabs infected with
*Hematodinium* sp. develop an unusual bitter flavor that does not occur
in infected blue crabs. We speculate that both the discoloration of the
carapace and the bitter flavor result from the duration of the
infection. In the blue crab, infections result in a relatively short
(\<40 d), acute disease, whereas in the snow crab, infections are a
lengthy (12 mo+), chronic process. Snow crabs live in temperatures as
low as --1.3° C, which decreases metabolic rates, perhaps resulting in
longer infections. Histological findings indicate potential differences
in the life histories of the parasites in their disparate hosts. The
plasmodial filamentous trophont occurs in early infections in the
hemolymph of blue crabs but does not occur in the hemolymph of snow
crabs. However, a different sheet-like plasmodial stage occurs in the
hepatopancreas of snow crabs but not in that of blue crabs. Such life
history differences can be used to further taxonomic and pathological
comparisons between these infections.

**Characterization Of *Panulirus argus* Virus 1 From The Caribbean Spiny
Lobster**

^1^[Robert E. Ratzlaff]{.underline}, ^1^Megan M. Montgomery-Fullerton,
^1^Michelle Todd, Bill ^2^Newcomb, ^2^Jay Brown, ^3^Jeffrey D. Shields,
and ^[1]{.underline}^Roland A. Cooper

^1^Department of Biological Sciences, Old Dominion University, Norfolk,
VA 23529; ^2^Department of Microbiology, University of Virginia School
of Medicine, Box 800734, Charlottesville, VA 22908; ^3^Virginia
Institute of Marine Science, The College of William and Mary, Gloucester
Point, VA 23062

*Panulirus argus* virus 1 (PaV1) is a pathogenic virus that infects the
Caribbean spiny lobster (*Panulirus argus*) in the Florida Keys. PaV1
was isolated from Florida Bay spiny lobsters with signs of disease.
Purified virions were morphologically similar to PaV1 previously
reported in tissue biopsies and were infectious to healthy lobsters.
PaV1 shares similarity with Herpesviridae, Iridoviridae, and Ascoviridae
families based on BLAST characterization of DNA fragments cloned from
the virus.

**Primary Culture Of Hemocytes From The Caribbean Spiny Lobster,
*Panulirus argus* And Their Susceptibility To *Panulirus argus* Virus 1
(PaV1)**

[Caiwen Li]{.underline} and Jeffrey D. Shields

Virginia Institute of Marine Science, The College of William and Mary,
Gloucester Pt, VA 23062

Primary cultures of hemocytes from the Caribbean spiny lobster
*Panulirus argus* were developed for studies on the *in vitro*
propagation of *Panulirus argus* Virus 1 (PaV1). A modified Leibovitz
L-15 medium supported the best survival of hemocytes in *in vitro*
primary cultures. However, degradation of the cultures occurred rapidly
in the presence of granulocytes. A Percoll step gradient was used to
separate hemocytes into three subpopulations enriched in hyalinocytes,
semigranulocytes, and granulocytes, respectively. When cultured
separately, hyalinocytes and semigranulocytes maintained high viability
(\~ 80% survival) after 18 days incubation compared with granulocytes,
which degraded over 2-3 days. Susceptibility of the cell types was
investigated in challenge studies with PaV1. Hyalinocytes and
semigranulocytes were susceptible to PaV1. Cytopathic effects (CPE) were
observed as early as 12 h post-inoculation, and as the infection
progressed, CPE became more apparent, with cell debris and cellular
exudates present in inoculated cultures. Cell lysis was noticeable
within 24 hrs of infection. The presence of virus within cells was
further confirmed by *in situ* hybridization using a specific DNA probe.
The probe gave a unique staining pattern to cells infected with PaV1
24-h post inoculation. Cells in the control treatment were intact and
negative to hybridization. This assay was further applied to the
quantification of infectious virus in hemolymph using a 50% tissue
culture infectious dose assay (TCID~50~) based on CPE. These tools will
now allow the quantification of PaV1 using established culture-based
methods.

**Ailments In Captive Crabs**

[Gretchen Messick]{.underline}

Cooperative Oxford Laboratory, Center for Coastal Environmental Health\
& Biomolecular Research at Charleston, USDOC/NOAA/NOS/NCCOS, 904 S.
Morris Street, Oxford, Maryland 21654-1323

The blue crab *Callinectes sapidus* is an essential component of
Chesapeake Bay's ecology and supports an important commercial fishery.
Research indicates that extended captivity can impart stress on blue
crabs, leading to enhanced prevalence and intensity of disease. Captive
crabs can have a high incidence of shell disease. Although initial
mortalities are often attributed to generalized bacterial infections
associated with inflammation and nodule formation in tissue, mortalities
occurring in crabs held in captivity for prolonged periods may also be
due to viral infections. Viruses that have been encountered in captive
crabs during investigations include reolike (RLV) virus and rhabdolike
(RhVA) virus, a virus sequenced as aquareovirus reoviridae, Chesapeake
Bay virus (CBV) and Bifaces virus (BFV). In addition to bacterial and
viral infections, other pathogens in captive crabs have included
microsporidians, fungus, rickettsia-like infections and ciliates.
Histological abnormalities have also been observed, including scab-like
gill lesions and an unusual strand-like structure in the lumen of the
hepatopancreas. Some or all of these parasites or manifestations of
disease may be latent in the crab until stress causes infections to
become patent. The development of molecular detection methods for
viruses or other parasites would provide diagnostic tools needed to
assess environmental impacts on crab populations in the Chesapeake Bay,
and monitor the health status of captive crabs.

**Parasites And Diseases Of Blue Crab, *Callinectes sapidus* From Tampa
Bay, Florida**

[Yasunari Kiryu]{.underline}, Jan H. Landsberg, Anne L.
McMillen-Jackson, Charles R. Crawford, Susan D. Gerhart, Angie M.
Machniak, Jay Abbott, Catalina E. Brown, and Noretta E. Perry

Florida Fish and Wildlife Conservation Commission, Fish and Wildlife
Research Institute, 100 Eighth Avenue Southeast, Saint Petersburg,
Florida 33701-5095

In spite of recent significant declines in blue crab, *Callinectes*
*sapidus*, commercial fishery landings, we have little knowledge about
the general health status of this species in Florida. A preliminary
study was conducted from June 2004 -- July 2006 to monitor the general
health of blue crabs in Tampa Bay (Florida central west coast). Blue
crabs were collected weekly from traps routinely monitored at five
locations: BH, Bayboro Harbor; AB, Apollo Beach; AR, Alafia River; AP,
St. Petersburg/Clearwater International Airport; and SH, Safety Harbor.
At each site, randomly selected live blue crabs were brought back to the
lab for necropsy. Hemolymph was drawn and observed by light microscopy
(LM) (using Giemsa stain) and bacterial isolations were attempted.
Internal tissues were prepared for wet mount LM, routine H&E histology.
From histology and stained hemolymph slides, the prevalence of the
dinoflagellate parasite *Hematodinium* was 9.5% (BH; N=21), 13.0% (AB;
N=23), 20.0% (AR; N=20), and 7.3% (AP; N=41). Prevalence of the
microsporidian parasite, *Thelohania* sp. was 8.7% (AB), 10.0% (AR), and
9.8% (AP). Neither *Hematodinium* nor microsporidia were detected from
SH (N=32). *Microphallus basodactylophallus* metacercarial cysts were
detected at all sites (10.0% -- 30.4%). Tetraphyllidean cestodes were
ubiquitous, with a high prevalence at AR (45.0%). *Vibrio* was commonly
isolated from the hemolymph. Results will be reviewed for potential
management applications.

**Aspects Of The Molecular Biology Of *Hematodinium* sp.**

^1^[Eric Schott]{.underline}, ^2^Gretchen Messick, and ^1^Allen Place

^1^Center of Marine Biotechnology, 701 East Pratt Street, Baltimore, MD
21202; ^2^Cooperative Oxford Laboratory, (CCEHBR) USDOC/NOAA/NOS/NCCOS,
904 S. Morris St., Oxford, Maryland 21654

*Hematodinium* sp. is a parasitic dinoflagellate that infects the blue
crab (*Callinectes sapidus*) and other crustaceans. Blue crab
populations in the MD and VA coastal bays, and the lower Chesapeake Bay,
have experienced occasional losses as a result of *Hematodinium* sp.
infections. While *Hematodinium* sp. is most prevalent in high salinity,
it may be carried by crabs as they migrate into and out of water of
lower salinity. We describe the use of a highly sensitive, quantitative
PCR assay (Q-PCR) that can detect parasite DNA corresponding to less
than a single cell. We are using the Q-PCR assay in parallel with
histology to assess *Hematodinium* sp. prevalence and intensity in blue
crabs from Chesapeake Bay and MD coastal bays. In a retrospective study,
we have also applied the Q-PCR assay to archived DNA samples of crabs
from mesohaline areas of the Chesapeake Bay, and have detected a 10%
prevalence of light infections. The Q-PCR assay is directed towards the
multicopy rRNA locus. By comparing Q-PCR standard curves of parasite
cell dilutions versus Q-PCR standard curves on dilutions of the cloned
rRNA gene target (gift of R. Lee, SKIO) we find evidence for nearly
40,000 copies of the rRNA gene cluster per cell. Investigations are
underway as to whether there is variation in the DNA sequence among
these copies, and whether structural genes of *Hematodinium* sp. also
exist in multicopy.

**Shrimp Hemocyte Immunity: Peroxinectin Expression In Response To
*Vibrio* Infection**

^1^[Erin J. Burge]{.underline}, ^2^Louis E. Burnett, and ^2^Karen G.
Burnett

^1^ Department of Marine Science, Coastal Carolina University, PO Box
261954, Conway, SC 29528; ^2^ Grice Marine Laboratory, College of
Charleston and Hollings Marine Laboratory NOAA/NOS, 331 Fort Johnson
Rd., Charleston, SC 29412

The purpose of this study was to examine changes in the tissue-level
hemocyte distributions of the Pacific white shrimp, *Litopenaeus
vannamei*, in response to challenge with a bacterial pathogen*.* We
quantified by real-time PCR the gene expression of peroxinectin, a
multi-functional immune protein expressed by shrimp hemocytes.
Biological functions of peroxinectin include roles in cell adhesion,
degranulation, opsonization, encapsulation and peroxidase activity.
Peroxinectin gene expression (as mRNA) was quantified in 5 tissues and
in circulating hemocytes for 48 h following intramuscular injection of
shrimp with *Vibrio campbellii*. Peroxinectin signal increased
significantly in the muscle at the site of injection between 1 and 4 h
and returned to control levels within 24 h of injection. Surprisingly,
changes in peroxinectin expression in the circulating pool of hemocytes
were not correlated with *Vibrio* injection, suggesting that recruitment
of hemocytes to the site of injection is from existing tissue localized
hemocyte subpopulations or that recruited hemocytes substantially
upregulate peroxinectin within 4 h of infection. *Vibrio*-injected
shrimp experience an increase in peroxinectin expression within the gill
by 48 h post-injection, supporting the hypothesis that localization and
encapsulation of bacteria in the gill is an important aspect of
crustacean immunity to bacteria. Supported by NSF Grant No. IBN-021292
to KGB and LEB.

**The Role Of Microorganisms In Development Of Epizootic Shell Disease
In The American Lobster**

[Andrei Chistoserdov]{.underline}^1^, Sai Laxmi Guballa^1^, Roxanna
Smolowitz^2^

^1^University of Louisiana at Lafayette, P.O. Box 42451, Lafayette, LA
70504-2451; ^2^ Marine Biological Laboratory, 7 MBL Street, Woods Hole,
MA 02542-1015

Epizootic shell disease found at high levels in wild populations of the
American lobster appears to have bacterial etiology. The microbial
community associated with epizootic shell disease lesions consists of
two to five dominant strains of bacteria. DGGE of PCR amplified 16S rRNA
genes (PCR-DGGE) from DNA isolated from lobster lesions, indicated that
lesions of lobsters always harbor at least two microorganisms: a strain
of *Aquimarina* sp. and either an unidentified a α-proteobacterium or a
*Pseudoalteromonas* sp. strain. Although our attempt to verify the Koch
postulate for *Aquimarina* sp. was not successful, the importance of
*Aquimarina* in pathogenesis should not be underestimated. It is
conceivable that other precursory pathogens weaken the immune system of
lobsters, preventing effective defence against epizootic shell disease.
If such pathogens existed, they must cause a latent, otherwise
unnoticeable infection. Necropsies of over 60 lobsters carried out over
the last two years, demonstrated that lobsters with shell disease have
cubicula lesions but are otherwise healthy. To investigate the
possibility that unknown eukaryotic microbe may be involved, we carried
out PCR-DGGE analysis of 18S rDNA amplified from DNA isolated from
lesions and hemolymph of diseased lobsters. No other eukaryotic DNA is
amplifiable from hemolymph except lobster's one. 18S rDNA of a
bacteriovorus nematode (*Geomonhysteria disjuncta*) was detected in
lesion of all tested lobsters, and that of barnacles, bryozoans and
protozoans in lesions of some.

**Effects Of Lipopolysaccharide On Total Hemocyte Counts Of The Blue
Crab, *Callinectes sapidus***

[Natasha J Sharp]{.underline}, Rachel A Ryan and Marius Brouwer

Gulf Coast Research Laboratory, University of Southern Mississippi, 703
East Beach Drive, Ocean Springs, MS 39564

In contrast to vertebrates, the crustacean immune response does not
include an adaptive component based on immunological memory, yet their
immune systems are extremely complex, efficient and highly developed. In
crustaceans, circulating hemocytes perform essential roles in immunity;
phagocytosis, encapsulation, and lysis of foreign cells.
Lipopolysaccharides (LPS), or endotoxins, are an integral component of
the outer membrane of gram-negative bacteria. LPS exposure induces rapid
degranulation of hemocytes resulting in haemocytopenia (hemocyte
depletion). We investigated the effects of injected LPS on circulating
hemocytes of the blue crab, *Callinectes sapidus*, to determine: 1) the
concentration which induced the most severe haemocytopenia, 2) the time
to reach maximum haemocytopenia, and 3) the time course of recovery from
sub-lethal doses. Severe haemocytopenia was induced at 0.5µg/ul per gram
of wet weight; LPS concentrations of 1, 2 and 5µg/ul resulted in
mortality. At 0.1µg/ul LPS maximum haemocytopenia was reached within 2
hours post-injection, with recovery to pre-injection levels by 12 hours.
Our data showed that dramatic decreases in the number of circulating
hemocytes can be induced after exposure to non-self molecules such as
LPS. Haemocytopenia induced during infection constitutes a serious
threat to the health of an animal by compromising its ability to fight
off invading microorganisms therefore, the rapid recovery of hemocytes
is essential to combat infections. The ability of the blue crab to
rapidly replenish hemocytes may be a necessity given the environment in
which it lives.

**An Overview Of Clam Health In The Chesapeake Bay**

^1^[Shawn M. McLaughlin]{.underline}, ^2^Lewis M. Collier, ^3^Robert M.
Bingaman, and ^1^Dorothy W. Howard

^1^NOAA/NOS/CCEHBR; ^2^Maryland Department of Natural Resources; ^3^JHT
Services, Inc.; Cooperative Oxford Laboratory, 904 S. Morris Street,
Oxford, Maryland 21654

Mollusks are infaunal benthic organisms important to the ecology of
estuaries. Clams and other members of the benthic community are reliable
and sensitive indicators of habitat quality because they reside in
bottom sediments where exposure to contaminants and oxygen stress
frequently occur. Benthic organisms are known to respond to multiple
types of environmental stress and to reflect environmental conditions
that vary over time. In 2006-2007, native and exotic clams were
collected monthly from relatively clean and polluted sites in the
Chesapeake Bay and their tissues examined for histopathology. An
overview of the health status of several clam species will be presented.
For example, infection prevalence and intensity of *Perkinsus* sp.
parasites increased in the late summer in Baltic clams at two sites in
the Tred Avon River. High mortalities of Baltic clams were associated
with increasing parasite burdens coupled with heat stress. Disseminated
sarcomas were also observed in clam tissues. Insights into historical
and current impacts of potential pathogens on *Macoma balthica*,
*Rangia* spp., and *Corbicula fluminea* will enhance the development of
diagnostic and predictive tools needed to more adequately assess
ecosystem health.

**Antimicrobial Peptides From The Suppression Subtractive Hybridization
cDNA Library Of Zebra Mussel (*Dreissena polymorpha*) Byssus Glands**

[Wei Xu]{.underline} and Mohamed Faisal

Pathobiology & Diagnostic Investigation, College of Veterinary Medicine,
Michigan State University, East Lansing, MI 38824

As a special apparatus located at the root of the zebra mussel
(*Dreissena polymorpha*) foot, byssus maintains the adhesion through its
entire life span. However, the current studies on the attachment
mechanisms employed by zebra mussels are extremely limited. In this
study, 2418 Expressed Sequence Tags (ESTs) were obtained from the
Suppression Subtractive Hybridization (SSH) cDNA library constructed
with zebra mussel foot. 750 new aligned ESTs remained after the analysis
of sequences alignment program, Contig Assembly Program 3 (CAP3). The
homologues search with BLASTx reveals that 470 ESTs had putative
functions, while 280 of them were unpredictable. All the ESTs were
divided into 5 groups according to the putative functions of their
homologues, which included attachment related genes, exocrine gland
secreted genes, defense related genes, normal cell functioning genes,
and unknown byssal genes. The full length antimicrobial peptides genes
were cloned and sequenced by Rapid Amplified CDNA Ends (RACE). The
expression analysis of the molecules induced by different stimulation
was analyzed by Real-time PCR and the recombinant expression of the
antimicrobial peptides was applied in the biological activity assay to
detect the inhibition of the growth of Gram-positive and Gram-negative
bacteria. As a result of this study, a number of previously undiscovered
zebra mussel byssal gland ESTs were added to the ESTs database providing
a powerful tool for the further study of zebra mussel attachment
mechanism.

**Identification Of Microbial Communities Associated With The Zebra
Mussel (*Dreissena polymorpha)***

^1^[Andrew D. Winters]{.underline}, ^2^Terence L. Marsh, ^1,3^Mohamed M.
Faisal

^1^Department of Fisheries and Wildlife, Natural Resources Building,
^2^Department of Microbiology and Molecular Genetics, ^3^Department of
Pathobiology and Diagnostic Investigation, Michigan State University
Plant, East Lansing, Michigan 48824

Although much research has been conducted concerning the ecology and
physiology of zebra mussels since they were first introduced into the
Great Lakes region, little is known concerning microbial communities
associated with zebra mussels in Great Lakes ecosystems. In these
studies, microbial community diversity was investigated in zebra mussel
gill, gut, and hemolymph samples collected from inland lakes and
tributaries in Michigan's Lower Peninsula. To determine microbial
diversity we applied two molecular techniques -- sequencing of 16S rRNA
genes through the construction of clone libraries and community analysis
using T-RFLP (Terminal-Restriction Fragment Length Polymorphism)
genotyping. Clone library analysis identified ecologically diverse
bacteria from 11 phyla and revealed distinct microbial assemblages
associated with each sample type. Cyanobacteria accounted for 24.1% of
bacteria isolated from gut samples. Members of the Microbacteriaceae
family were only found to be prevalent in gill samples. T-RFLP may
represent a powerful tool to be used to identify and track disease in
zebra mussel populations. These findings have implications for the
fields of biological control, limnology, and water quality management
and warrant further investigation.

**Health Evaluations Of Impinged Fishes At Steam Generating Power
Plants**

^1^[Amelia C. Knight]{.underline}, ^1^Jeffery S. Terhune, and ^2^William
E. Garret

^1^Dept. of Fisheries and Allied Aquacultures, Auburn University,
Alabama, 36849; ^2^Alabama Power Co. Environmental Affairs, P.O. Box
2641, Birmingham, Alabama.

Section 316(b) of The Clean Water Act of 1972 Section 316(b) requires
that the best available technology (BTA) be implemented in location,
design, construction, and capacity of cooling water system intake (CWSI)
structures in efforts to reduce adverse environmental impacts (AEI).
Since its enactment the interpretation of how to define and measure
BTA's and AEI's has been highly debated. A major component of AEI is
impingement. Impingement occurs when larger organisms, unable to pass
through the screens or trash racks of the CWSI, are trapped against the
intake screens by the water velocity of the intake. Impinged organisms
trapped against the screen and may incur injury and possibly death.
Previous studies have examined factors that may affect impingement rates
of fishes including: CWSI velocity, temperature, turbidity, pH, and
dissolved oxygen. The purpose of this study is to (1) to evaluate the
prevalence of disease in impinged fish versus designated treatment and
open water river reference fish from the steam generating plants of
interest, (2) to evaluate disease prevalence in impinged fish versus
open water river reference fish populations over multiple seasons and
geographic regions. The overall goal of this study is to obtain a better
understand how and why fish are impinged, and to ultimately lower the
impingement rates of fish at steam generating power plants. Study sites
include four plants in Alabama and four plants on the Ohio River.
Impinged fishes were collected from all plants, and reference fish
collected at each plant by electroshocking and gill netting. The
complexity of the CWSI structure at Plant Gorgas (Alabama Power Co.)
called for two additional control treatments, the intake canal, and the
intake pit. Preliminary results suggest there are significant
differences between impinged and reference fish, as well as additional
Plant Gorgas treatments in pathogen prevalence (p\<0.001). Further
interactions between treatments and power plants, species, and disease
factors will also be examined and discussed.

**Strategies For Field Sampling When Large Sample Sizes Are Required**

^1^[Julie A. Bebak]{.underline}, ^1^Craig A. Shoemaker, ^2^Covadonga R.
Arias, and ^1^Phillip H. Klesius

^1^Aquatic Animal Health Research Laboratory, Agricultural Research
Service, United States Department of Agriculture, 990 Wire Rd., Auburn,
AL 36832; ^2^Department of Fisheries and Allied Aquaculture, Auburn
University, Auburn, AL 36849

Estimates of prevalence or incidence of infection with a pathogen
endemic in a fish population can be valuable information for development
and evaluation of aquatic animal health management strategies. However,
hundreds of unbiased samples may be required in order to accurately
estimate these parameters on a catfish farm, which is an especially
inhospitable environment. The high ambient summer air temperatures, lack
of shelter and electrical power, and the large number and size of the
ponds contribute to the challenging conditions. One strategy that can be
employed to reduce the number of samples needed is the use of the
"herd", or epidemiological unit, concept. Even with the use of the
epidemiological unit, however, hundreds of samples will most likely be
needed, so it will be impractical to process each fish at the pond side.
Quick freezing fish on dry ice and storage at -70°C for assay at a later
time may be a viable option, but should be validated before use in the
field. The "herd" concept and the validation of the use of frozen fish
as samples for pathogen assay will be discussed during this
presentation.

**Genomics And Proteomics: What Do These Add To Fish Health Research?**

[Laura L. Brown]{.underline}

National Research Council of Canada (NRC) Institute for Marine
Biosciences, 1411 Oxford Street, Halifax, NS, B3H 3Z1, Canada

Genomics and proteomics technologies have resulted in an explosion of
data in the biological sciences. Examples of tools and methods are
Expressed Sequence Tags (ESTs), whole genome sequencing, Serial Analysis
of Gene Expression (SAGE), microarray-based transcript profiling,
mass-spectrometry analysis of protein expression. These can all provide
important data to understand the biochemical and molecular mechanisms of
infection. However, caution must be exercised when contemplating using
these methods. Capital equipment can be costly, operating expenses are
high and advanced training is usually required. Moreover, some of the
data that are generated must be analysed by specialized methods. This
session will examine some of those technologies and will illustrate the
advantages and disadvantages of each, using presenters' research
programs as examples. The objective of this session is to show when,
why, and how genomics and proteomics techniques can be used to benefit
fish health research programs.

**Genomic Approaches For Studies Of Fish Responses To Pathogens And
Pollutants**

[Matthew L. Rise]{.underline}

Ocean Sciences Centre, Memorial University of Newfoundland, St. John's,
NL, Canada A1C 5S7

Pathogens and pollutants are serious threats to fish populations.
Genomic techniques, such as high-complexity cDNA library construction
and microarray hybridizations, have been used to identify fish genes
responsive to pathogens (bacterial, viral, fungal, protozoal) or
environmental toxicants (e.g. heavy metals, pesticides, dioxins). There
are excellent genomic resources available for several fish species. For
example, there are more than one million zebrafish (*Danio rerio*)
expressed sequence tags (ESTs) and over 436,000 Atlantic salmon (*Salmo
salar*) ESTs in the National Center for Biotechnology Information (NCBI)
EST database. Available fish DNA microarrays include a \~16,000 gene
(16K) salmonid cDNA microarray from the consortium for Genomic Research
on All Salmon Project (cGRASP) and commercially available resources such
as a 21K zebrafish oligonucleotide microarray (Agilent). While currently
available fish DNA microarrays can provide valuable information on the
molecular pathways altered by exposure to pathogens or toxicants, these
resources are missing some important defense-relevant genes. A complete
understanding of the molecular mechanisms by which infections and
pollutants alter fish physiology will require the development of new
genomic resources such as suppression subtractive hybridization (SSH)
cDNA libraries generated from immune- and toxicant-challenged fish
tissues. SSH libraries have been shown to be effective means of
identifying novel fish genes responsive to immunogenic stimuli.
Functional genomic research may lead to the development of new tools for
detecting and combating emerging diseases of fish.

**Application Of Genomics To Study Host-Pathogen Interactions In
Oysters**

[Marta Gómez-Chiarri.]{.underline}

University of Rhode Island, Department of Fisheries, Animal and
Veterinary Science, 20A Woodward Hall, Kingston, RI 02881

Oysters are one of the oldest and most productive segments of US
aquaculture. Unfortunately, habitat degradation, over-fishing, disease,
and lack of successful sets have severely impacted oyster populations in
United States coastal waters. Parasites like *Perkinsus marinus* and
*Haplosporidium nelsoni* place a large economic burden on the oyster
industry. Basic knowledge of innate immunity and the mechanisms used by
pathogens to evade host responses is crucial to develop strategies for
promoting disease resistance and managing epizootics. The recent
development of genomic resources for oysters has provided the means for
an explosion in oyster disease research. These resources include several
collections of expressed sequence tags (ESTs) from a variety of
laboratories, subtractive libraries of genes differentially expressed in
response to infection, oyster microarrays, and BAC libraries. More
importantly, the Joint Genome Initiative has agreed to sequence 150,000
cDNA clones from the Pacific oyster and 133 BAC clones. We are using
these resources to isolate and characterize genes involved in
host-pathogen interactions in oysters. Although genomics resources,
combined with other more traditional approaches, have already led to the
identification and characterization of several molecules with immune
function in oysters, the large majority of the genes in oyster databases
do not match sequences in other databases and have unknown functions.
Unraveling the biological function of the large amount of genes derived
from the oyster genome project will be a major challenge.

**Real-Time Immunology: Analysis Of Gene Expression To Determine Fish
Health**

[Mark D. Fast]{.underline}

Stony Brook University -- Marine Sciences and Research Center (MSRC),
Stony Brook, New York, USA 11794-5000

The bane of the fish health field has long been the paucity of
biomarkers and reagents to accurately assess immunological status for
diverse fish species. As we slowly address the issue of antibody and
ELISA approaches to quantify immune effector molecule regulation,
expansion in fish genomics have opened a door to study host-pathogen
interactions in another way. The use of suppression subtractive
hybridization and microarray analysis are powerful tools for isolating
and identifying, 10s to 100s; to possibly 1000s of differentially
expressed transcripts, over a short period of time. Real-time PCR is a
technique, which can build upon SSH and microarray analysis to
accurately quantify these expression differences. It has an accurate
dynamic range of 7 to 8 log orders of magnitude without
post-amplification manipulation, can discriminate between mRNAs with
nearly identical sequences and is extremely sensitive (less RNA
required). Here we will discuss the considerations and pitfalls of this
technology, highlighting the different chemistries, many types of
normalization, selection of housekeeping and effector genes and most
importantly how these have and will be applied to fish health.

**DNA Microarrays To Study Bacterial Pathogenesis In Fish Health
Research**

[John H.E. Nash]{.underline}

National Research Council of Canada (NRC) Institute for Biological
Sciences, 100 Sussex Drive, Ottawa, ON, K1A 0R6, Canada

Using a draft sequence of the genome of *Aeromonas* *salmonicida* subsp.
*salmonicida*, we designed and constructed a DNA microarray of 2068
genes selected on the basis of "involvement in some aspect of
virulence". This microarray was used to study the comparative genomics
of several wild and clinical isolates of *A. salmonicida* as well as to
study gene expression of the pathogen grown under *in vitro* low iron
conditions and within *in vivo* implants. The latter experiments were
performed to test the hypothesis that that genes associated with iron
regulation would be differentially expressed in *A. salmonicida* grown
under low-iron *in vitro* culture, and inside *in vivo* implants, as
compared to under iron-replete *in vitro* conditions. Many of the genes
observed to be up-regulated by *in vitro* growth in low iron are
involved in iron acquisition or transport, heme synthesis, contain
iron-sulphur clusters, or are iron-regulated. Complementary proteomics
studies of the same *A. salmonicida* samples indicated that the levels
of \>60 proteins increased or decreased by at least 50% in low iron
medium. Many of the up-regulated proteins are involved in iron
homeostasis, and several match genes up-regulated in our low iron
microarray experiments. We have also identified additional genes and
proteins, not associated with iron regulation, that are differentially
expressed under *in vivo* implant growth conditions. We are correlating
these results with data from other studies from our laboratories to
determine molecular mechanisms of *A. salmonicida* pathogenicity.

**Metabolomics; Continuing Post-Genomics Refinement**

^1^[Andrew Dacanay]{.underline}, ^1^Ian Burton, Tobias Karakach,
^1,2^Kirty Solanky, ^1^John Walter

^1^National Research Council of Canada Institute for Marine Biosciences,
1411 Oxford Street, Halifax, NS, Canada; ^2^Current Address: Bruker
Biospin, 15 Fortune Drive, Billerica, Massachusetts 01821

Despite the relative novelty of genomics it is often said that we are
already in the post-genomics era, defined by gene function rather than
gene presence. Transcriptomics, proteomics and metabolomics respectively
attempt "global" surveys of the successive molecular events occurring in
a biological system under given conditions. Metabolomics aims to measure
the amounts and fluxes of low molecular weight metabolites, which are
products of complex interactions among cellular components that
originate from the protein products of RNA transcription and subsequent
post-translational modifications. Metabolites may be measured
principally by Mass Spectrometry (MS) or Nuclear Magnetic resonance
(NMR) spectroscopy. This talk will concentrate on proton (^1^H)
NMR-based metabolomics, which has advantages that complement other
"--omics" technologies. Usually applied to biofluids or tissue extracts,
it measures signals from all hydrogen-containing small molecules in
proportion to their concentrations, provided that the molecules are in
free solution and are not adsorbed to macromolecular or solid surfaces
or otherwise restricted in their motional freedom. In some
circumstances, ^1^H NMR can be applied to whole organisms, organ
preparations or cell cultures. It permits metabolites to be surveyed
with little or no bias, relative to other spectroscopic or spectrometric
methods. Although the proportionality of signal area to concentration is
close to constant for each hydrogen atom in a molecular species, the
detailed shapes of peaks in ^1^H NMR spectra are complicated by coupling
and exchange phenomena, which are in turn indicative of molecular
structure. Unless combined with separation techniques or specialized
pulse sequences that selectively detect particular molecular classes,
the method produces spectra, which are sums of the spectra of individual
metabolites, and concentration changes in minor components may be
obscured by overlapping signals from major components. Comparisons can
be made between individual samples, or between two or more groups each
consisting of multiple individuals, by means of principal components
analysis or other statistical techniques. Metabolites contributing
significantly to inter-group differences can be chemically identified.
Metabolomics is a valuable complement to existing nucleic acid and
protein-based technologies, with numerous uses in human medicine.
Increasing applications to aquatic systems include studies of aquatic
animal health from which illustrations will be drawn.

**Expression of Antimicrobial Peptide Genes (Hepcidin 1 And 2) In
Largemouth And Smallmouth Bass**

[Laura S. Robertson]{.underline} and Luke R. Iwanowicz

USGS-BRD, Leetown Science Center, Aquatic Ecology Branch, 11649 Leetown
Road, Kearneysville, WV 25430

The coincidence of intersex and fish lesions in largemouth (*Micropterus
salmoides*) and smallmouth (*M. dolomieu*) bass in the Potomac and
Shenandoah Rivers suggests a putative relationship between the presence
of estrogenic endocrine-disrupting compounds (EEDCs), endocrine
disruption, and altered immune function. Estrogen or EEDCs, which may
cause intersex in smallmouth bass, may also interfere with the
expression of genes involved in innate immunity, *e.g.* hepcidin, thus
increasing susceptibility of fish to bacterial infection. We have
identified two distinct hepcidin sequences in both largemouth and
smallmouth bass (four hepcidin genes). Hepcidin is a small, cationic,
disulfide-bonded antimicrobial peptide that exhibits antibacterial and
antifungal activity *in vitro*. In mammals, hepcidin is an important
iron regulatory hormone that blocks uptake of iron from the intestine
and release of stored iron from macrophages, thus decreasing available
iron. Expression of mammalian hepcidin is stimulated by interleukin-6
and expression of interleukin-6 is inhibited by estrogen. Hepcidins have
been identified in several species of fish and are predominantly
expressed in the liver. Expression of these antimicrobial peptides is
induced by bacterial infection (or structural components of bacteria,
including lipopolysaccharides). We will discuss the constitutive and
inducible tissue-specific expression of two hepcidin genes in largemouth
and smallmouth bass and the effect of estrogen on these expression
patterns.

**Immunohistochemical Localization Of Two Putative Defense Lectins,
Rainbow Trout Ladderlectin And Intelectin In Diseased Rainbow Trout
(*Oncorhynchus mykiss*) Tissue**

[Spencer Russell]{.underline}, M. Anthony Hayes, Karrie M. Young, John
S. Lumsden

Fish Pathology Laboratory, Department of Pathobiology, University of
Guelph, Guelph, Ontario, Canada N1G 2W1

To investigate the role of lectins in susceptibility and resistance to
disease, we isolated and characterized ladder lectin (RTLL) and
intelectin (RTIntl) from rainbow trout (*Oncorhynchus mykiss*) plasma
based upon their carbohydrate-dependent affinity. Both RTLL and RTIntl
bound to *Aeromonas salmonicida* subsp. *salmonicida*, *Aeromonas*
*hydrophila*, *Yersinia ruckeri*, *Pseudomonas* spp, chitin and artemia
cyts (chitinous walls). As both lectins appear to be associated with
peripheral blood leukocytes, their role in recognition and/or processing
of foreign material is suggested. We used immunohistochemistry for the
localization of rainbow trout ladderlectin and intelectin in tissue from
rainbow trout submitted for examination from clinical cases of
infectious disease. Based on these results, we discuss the possible
biological and defensive function of these lectins.

**Analysis Of A Novel And Phylogenetically Conserved Pattern Recognition
Receptor (PRR)**

[L. Jaso-Friedmann]{.underline}, M. A. Connor, J. H. Leary III, L. D.
Moss and D. L. Evans

Department of Infectious Diseases, College of Veterinary Medicine,
University of Georgia, USA

Pattern recognition receptors (PRR) are important components of the
vertebrate immune system for recognition of microbial structures and the
subsequent inflammatory responses to danger signals. We have previously
described a member of the PRR family (scavenger receptor) which is
present on nonspecific cytotoxic cells (NCC) of catfish. NCC are teleost
NK-like cells important in innate immune mechanisms against parasites,
viruses and bacteria. In the present study we describe the expression of
another PRR in NCC which is a novel and phylogenetically conserved
molecule. This new PRR is expressed on the membrane, in the cytosol and
in granule extracts from NCC and it is referred to as nonspecific
cytotoxic cell cationic antimicrobial protein-1/ncamp-1. Its name
derives from its bifunctional activities: on the membrane ncamp-1 binds
ligands (dinucleotide, polyguanosine, etc.) and in recombinant form
ncamp-1 lyses bacteria. The relevance of ncamp-1 in innate immune
responses was recently supported by finding a mammalian orthologue on
mouse leukocytes. Using an anti-ncamp-1 monoclonal antibody we
identified its expression in subsets of mouse leukocytes. These studies
demonstrated that teleosts, similar to mammals express heterogeneous
families of PRR that may be relevant in the design of future vaccine
formulations against infectious agents. Research supported by BARD and
USDA.

**A Novel Pattern Recognition Receptor On Nonspecific Cytotoxic Cells
Has Bimodal Functions Of Membrane Expression And Bactericidal Activity**

[M. A. Connor]{.underline}, J. H. Leary III, L. Jaso-Friedmann and D. L.
Evans

Department of Infectious Diseases, College of Veterinary Medicine,
University of Georgia, Athens, GA.

A novel cationic antimicrobial protein (nonspecific cytotoxic cell
cationic antimicrobial protein-1/NCAMP-1) was identified from catfish
(cf) NCC. Ncamp-1 is believed to have a bimodal action: as a membrane
protein it may be a pattern recognition receptor (PRR) and in soluble
recombinant form is a potent antimicrobial. When expressed in
recombinant form, ncamp-1 has bactericidal activity. Expression of
ncamp-1 as full length or truncated peptides and testing in killing
assays determined that the bactericidal activity against Gram^-^ and
Gram^+^ pathogens was associated with expression of lysine based motifs
of the synthetic protein. To assess the biological significance of
ncamp-1 in cf innate immunity, clinically relevant bacteriological
isolates from 8 clinical cases and a vaccine strain of *Edwardsiella
ictaluri* were tested in *in vitro* antimicrobial assays. The
bactericidal activity of recombinant ncamp-1 was also determined against
*E. coli* from chickens and from bovine mastitis. In order to evaluate
the relationship between ncamp-1 membrane expression and immune
functions, an anti-ncamp-1 monoclonal antibody (mab 9C9) was derived.
This mab was utilized to investigate the potential signaling activity of
ncamp-1. Flow cytometric analysis, using mab 9C9 binding, indicated that
PMA activation up-regulated the membrane expression of ncamp-1 on NCC.
The *in vivo* role of ncamp-1 in *Edwardsiella* infection models is
currently being investigated. Research supported by USDA and Veterinary
Medical Experiment Station.

**Immune Response Of Hybrid Striped Bass (*Morone saxatilis ***

***x M. chrysops)* To A Commercial *Vibrio* Vaccine**

[John W. Machen]{.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,
Virginia 24061-0442

Striped bass (*Morone saxatilis*) and hybrid striped bass (*Morone
saxatilis* X *M. chrysops*) are both a sport fish and food fish, with
current commercial aquaculture significance. Striped bass and their
hybrids are susceptible to a diverse variety of pathogens, including
viral, bacterial, fungal and parasitic agents. Of these pathogens,
bacteria of the genus *Vibrio* are a major pathogen of cultured marine
species. This study was undertaken to evaluate the immune response of
hybrid striped bass to vaccination with a commercial *Vibrio* vaccine
(Vibrogen 2, Aqua Health Ltd.). Two test groups (IP injected and bath
immersion) and a PBS control group of fish (n=80 fish each) were
evaluated for antibody levels to the *Vibrio* vaccine for 56 days
post-vaccination. An ELISA was developed to assess specific
immunoglobulin levels using a previously developed affinity-purified
rabbit anti-striped bass antibody as one of the ELISA reagents. Fish
(n=8) were non-lethally bled on days 0 (pre-), 4, 7, 14, 21, 28, 35, 42,
49, and 56. Blood was allowed to clot, the serum separated by
centrifugation, and frozen until ELISA analysis. ELISA results of the
three groups were compared to each other over the course of the
experimental period.

**Girlie Boyz: Low-Level Endocrine Disruption In Fatheads**

[Andrew S. Kane]{.underline} and [James D. Salierno]{.underline}

Aquatic Pathobiology Center, Department of Epidemiology and Preventive
Medicine, University of Maryland, 8075 Greenmead Drive, College Park,
Maryland 20742

The ability of male fathead minnows, *Pimephales promelas*, to compete
for, maintain, and defend a spawning substrate is paramount to
reproductive success. This study quantified alterations in male fathead
minnow reproductive behaviors after exposure to environmentally-relevant
concentrations (0, 10, 20 or 40 ng/L) of 17α-ethinylestradiol (EE~2~)
for 21 days. A video-based behavioral quantification system was used to
examine changes in male-male competitive behaviors (i.e., chasing and
head butting), and the ability of males to maintain spawning substrates
(i.e., nibbling and scrubbing). Male behaviors analyzed included time
under the spawning substrate, frequency of substrate cleaning, and
conspecific aggression. Plasma hormone levels (11-KT, T, and E~2~),
vitellogenin (VTG), secondary male characteristics (tubercle count and
dorsal nape pad rank), gonadal somatic index (GSI), and gonad histology
were also evaluated. Exposure to 40 ng/L EE~2~ decreased the ability of
exposed males to compete with control males for spawning substrates (p =
0.09). Further, exposed males displayed reduced frequency of substrate
cleaning activities as well as chasing male competitors (p ≤ 0.05).
11-KT, T, and E~2~ were lower, and VTG was notably higher, in
EE~2~-exposed males compared with control males (p ≤ 0.03). EE~2~
exposure in males was also associated with reduced number and size of
tubercles, lower GSI and gonadal maturity ranks, and presence of an
ovipositor (p \< 0.001 for each endpoint). These data reveal clear
alterations in male reproductive behavior that coincide with decreased
hormone levels and secondary sex characteristics. Behavioral endpoints
to discern potential ecological consequences in fish exposed to low
concentrations of endocrine disrupting compounds may provide sensitive
and functional indices of exposure.

**Assessment Of Environmental Nitrate Stress Effects In Fishes**

[Samuel J. Lupica]{.underline} and John W. Turner

Department of Physiology, Pharmacology, Metabolism and Cardiovascular
Sciences, University of Toledo, College of Medicine

Aquatic environments are in decline worldwide due to both natural and
anthropogenic stressors including coastal development, agricultural
runoff and sewage. Chronically elevated cortisol in response to stress
can lead to impairment of immune response, reproduction and growth.
Cortisol is frequently determined in plasma samples as a measure of
stress. Since some free cortisol is excreted in feces, measurement of
fecal cortisol can serve as a stress response indicator. Fecal samples
offer ease of collection, minimal associated stress and elimination of
potentially misleading acute hormone spikes seen in blood. The aim of
this study was to determine the effects of nitrate, a common aquatic
pollutant, on fecal cortisol levels in fishes. Butterfly koi (*Cyprinus
carpio*) were chosen as a conservative model species, since they are
hardy and tolerate poor water quality. Fishes were maintained group-wise
(n=7) in 96 L aquaria. Nitrate (as sodium nitrate) was added to the
water in 0.4 g/L increments approximately weekly beginning at 0.4 g/L
and ending at 1.6 g/L. Fecal samples were collected daily and
alternative-day samples were extracted and assayed for cortisol by EIA.
Nitrate effects were compared in both baseline (12 days) vs. treatment
(12 days) and control group (n=7) vs. treatment group (n=7) conditions.
Nitrate treatment significantly (t-test, p\<0.0001) increased cortisol
excretion (9-fold, from a pre-treatment mean of 49.5 pg cortisol/g dry
feces to a treatment mean of 329.1 pg cortisol/g dry feces). Additional
nitrate increments produced no further increases in cortisol. This study
demonstrates that fecal cortisol measurement in fishes can be useful as
an indicator of nitrate stress in the aquatic environment.

**Advances In Our Understanding Of Phaeohyphomycosis**

^1^[Akinyi Nyaoke]{.underline}, ^2^E. Scott Weber, ^2^Charles Innis,
^3^Donald Stremme, ^3^Cynthia Dowd, ^4^Christian Keller, ^1^Lynn
Hinckley, ^5^Timothy Gorton, ^6^Brian Wickes, ^7^Elizabeth Thompson,
^7^Deanna Sutton, ^8^Sybren de Hoog, and ^1^Salvatore Frasca Jr.

^1^Department of Pathobiology and Veterinary Science and ^5^Center for
Excellence in Vaccine Research, University of Connecticut, Storrs, CT
06269-3089; ^2^New England Aquarium, Boston, MA 02110-3399; ^3^Adventure
Aquarium, Camden, NJ 08103-1060; ^4^Tennessee Aquarium, Chattanooga, TN
37401-2048; ^6^Department of Microbiology and ^7^Fungus Testing
Laboratory, Department of Pathology, The University of Texas Health
Science Center at San Antonio, San Antonio, TX 78229-3900;
^8^Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht,
The Netherlands

Melanized, also known as phaeoid, fungi have been identified as agents
of cutaneous lesions or systemic infections with greater frequency among
submissions from aquaria to the Connecticut Veterinary Medical
Diagnostic Laboratory (UConn, Storrs, CT) during the last 3 years. In
conjunction with several commercial aquaria, a concerted effort to
culture tissue samples from specimens identified as having fungal
lesions has led to identification of several *Exophiala* species,
including a novel species isolated from seadragons (*Phyllopteryx
taeniolatus* and *Phycodurus eques*). Recent efforts have resulted in
isolation of *Exophiala angulospora* from lumpfish (*Cyclopterus
lumpus)* with cutaneous and systemic lesions. Association of *E.
angulospora* with lesions in a vertebrate animal represents a novel
finding regarding this species, which is a well-known environmental
isolate. Phylogenetic analyses of a broad assortment of melanized fungi,
including *Exophiala* species from aquarium sources, have provided
insights into relationships between these fungi, their hosts and growth
conditions.

**Histopathologic Interpretation Of Lesions In Cephalopods**

^1^[Salvatore Frasca Jr]{.underline}., ^2^Richard J. Montali, and
^3^Roxanna Smolowitz

^1^Department of Pathobiology and Veterinary Science, University of
Connecticut, Storrs, CT 06269-3089; ^2^Department of Molecular and
Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore,
MD 21205; ^3^Marine Biological laboratory, Woods Hole, MA 02543

Cephalopod mollusks, which include octopus, squid, cuttlefish and
nautilus, have unique features regarding their neurobiology, immunology,
anatomy and behavior that impact management of these invertebrates and
the occurrence of disease in aquaria. Reports describing lesions and
mortalities associated with captive cephalopod populations have
highlighted integumentary and ophthalmic lesions as among those
frequently encountered by biologists, clinicians, researchers and
pathologists. Consistent with previous reports, mantle ulcers, erosive
and ulcerative dermatitis involving bacteria and protozoa alone or in
combination, and ophthalmic lesions involving both globe and lens, were
among the most common findings from aquarium submissions to the
Connecticut Veterinary Medical Diagnostic Laboratory (UConn, Storrs, CT)
during the period from 2002 to 2007. At the MBL (Woods Hole, MA), the
most common dermal problems resulted from trauma-induced erosions and
ulcerations. Recognition of salient histologic features of the skin and
eyes of several cephalopod species contributes to an improved
histopathologic interpretation of lesions and is essential to meaningful
comparative assessments of histopathologic changes. This may in turn
provide useful insight into the management of disease conditions in tank
and other aquarium environments.

# **Microbiology Testing For Aquatic Systems: Findings And Potential Implications**

[Scott Weber]{.underline}, Susan Goodridge, and Kathryn Charron

Animal Health Department, New England Aquarium, Central Wharf, Boston,
Massachusetts 02110.

Analyzing environmental quality for aquatic systems is an essential
component for preventive health. Most aquarists measure water for a
standard set of physical and chemical parameters that typically
includes: temperature, pH, salinity, DO (Dissolved Oxygen), ammonia
(Total Ammonia Nitrogen), nitrite, nitrate, phosphate, and alkalinity.
Depending on resources and laboratory capabilities additional chemical
and physical testing can also be done to check more comprehensive water
quality for more complex systems (i.e. coral reef exhibits), monitor
chemical/treatment additions, and identify toxic metal/chemical
contamination. In public aquaria, microbiology is also performed to
assess total coliform levels for systems designed to maintain marine
mammals for display purposes. At the New England aquarium, microbiology
testing has been expanded to test for Enterococcus and total coliforms
in aquatic systems, which house only pokilotherms. The following
presentation will discuss some preliminary findings for these systems,
problems associated with high levels of bacterial detection, and
potential implications for animals and staff.

**Phagocytosis In Nurse Shark (*Ginglymostoma cirratum*) Peripheral
Blood Leukocytes**

[Christina A. Gargan]{.underline}

National Aquarium in Baltimore, 501 East Pratt Street, Baltimore, MD
21202; University of Maryland, Baltimore, Department of Medical and
Research Technology, 100 Penn Street, Baltimore, MD 21201

Chondrichthyans (elasmobranchs and holocephalans) are the oldest extant
class of vertebrates to have a fully functional adaptive immune system.
Molecular studies examining the immune system elements are ongoing.
However, studies on phagocytosis and evaluation of the function of
peripheral blood leukocytes (PBL) have been confusing due to
nomenclature discrepancies as well as variations in species of
elasmobranch used, targets, and treatment of cells. This study attempts
to begin standardization for the identification of cell types based on
function, using a cell atlas derived from sandbar shark (*Carcharhinus*
*plumbeus)* PBL. Whole PBL were exposed to fluorescein isothiocyanate
(FITC) conjugated bacterial targets and phagocytosis was evaluated by
flow cytometry, microscopic examination, and differentiation of cell
types. Bacterial species examined included *Escherichia coli*,
*Staphylococcus aureus*, and *Vibrio alginolyticus*. Opsonization of the
bacteria with shark plasma and IgM blocking studies were also performed.
Results showed the most actively phagocytic cell to be the coarse
eosinophilic granulocyte (CEG). Interestingly, some lymphocytes were
also shown to be phagocytic. Opsonized *E. coli* and *Vibrio* sp. were
taken up by cells, but nonspecifically. Opsonized *S. aureus* were taken
up in significant amounts by washed PBL when compared to unwashed PBL,
and blocking studies to determine the involvement of shark antibodies in
the uptake suggested that other serum factors were involved.

**Head and Lateral Line Erosion Syndrome In Ocean Surgeonfish
(*Acanthurus bahianus),* Current Efforts To Determine Etiologies**

[Allison L. Corwin]{.underline}, Anne J. McCoy, Michele M. Kittell, and
M. Andrew Stamper

Disney's Animal Programs, The Seas, Epcot^®^, Walt Disney World^®^
Resort, EC Trl. W-251, 2016 North Avenue of the Stars, Lake Buena Vista,
FL 32830-1000

# 

Head and lateral line erosion syndrome (HLLES) or chronic erosive
dermatopathy (CED) in fish has long been a frustration of the aquarium
and pet industry. The anatomy of the head and body regions along the
lateral line has been clearly described as well as the pathology of a
disease. The etiology of this syndrome, however, is still unknown and
has been a focus of much debate. Implicated causative agents include
(not all-encompassing) activated carbon, parasites, carbon dust, heavy
metals such as copper, electrical currents, endotoxins, ozone exposure
products, ultraviolet radiation exposure products, as well as nutrient
deficiencies including vitamins A and C. Of these, the authors could
only determine that heavy metals have been formally studied in a
controlled situation. Over the last several years, staff from The Seas
at Epcot and the University of Florida has been systematically examining
possible etiologic agents for HLLES in ocean surgeonfish (*Acanthurus
bahianus)*. To date, in-line ultraviolet radiation, activated carbon,
foam fractionation, ozone, and vitamin A have been preliminarily
examined with controls. Even though these applications need to be
examined in more detail, only carbon and ozone have produced dermatitis
in distinct and consistent areas of the skin, including the lateral line
region. Future studies plan to investigate causative agents in further
detail by varying applications rates and concentrations of these agents.

**Gas Supersaturation In A Large Artificial Reef Exhibit: An Unusual
Presentation And Resolution**

[Catherine A. Hadfield]{.underline}, Leigh A. Clayton, Stacey R. Gore,
Leah M. Neal

National Aquarium in Baltimore, 501 E. Pratt St., Baltimore, MD 21202

A 335,000-gallon artificial seawater exhibit containing 380 teleost fish
(Perciformes, Tetraodontiformes, and Beryciformes) and three stingrays
has been in operation at the National Aquarium in Baltimore for 26
years. In early September 2006, acute mortalities were seen in two blue
runners (*Caranx crysos*). The animals were in good body condition, and
showed recent food intake and no evidence of significant pathogens. The
gills showed gas emboli within the blood vessels and telangectasia.
Other clinical signs in the exhibit included decreased food intake and
increased gilling rate and effort in the medium-to-large sized fish.
Total gas pressure was recorded at 102-103% with a peak on September 27
of 105-106%. Two blue runner mortalities occurred on September 27 and
October 1. Mortalities were not seen in any other species. Acute gas
supersaturation in an aquarium is usually due to air injection into a
pump that allows gas to become trapped under high pressure. The five
pumps on this system were working normally. The source of the gas
supersaturation was an elevated water level in the exhibit that created
flow within a 40-ft vertical overflow pipe into the biotower; there was
insufficient off-gassing before the pumps brought the water to the
exhibit. The water level was corrected on October 7. Total gas pressure
has subsequently been 99-100% and no further clinical signs have been
seen.

**Retrospective Summary Of Diseases Affecting Captive Seahorses At The
Toronto Zoo**

^1^[Véronique M. LePage]{.underline}, ^2^Jeff Young, ^2^Cynthia Lee,
^1^Spencer Russell, ^1^Karrie Young, ^1^Lowia Al-Hussinee, and ^1^John
S. Lumsden

^1^Fish Pathology Laboratory, Department of Pathobiology, University of
Guelph, Guelph, ON, Canada, N1G 2W1; ^2^Metro Toronto Zoo, 361A Old
Finch Avenue, Scarborough, ON, Canada, M1B 5K7

Wild seahorse populations are in sharp decline from over-fishing for
aquaria, curios and traditional medicine. Captive rearing of seahorses
has been practiced for decades and husbandry has improved yet seahorses
still provide a challenge for aquarists. Infectious disease and
management deficiencies are common causes of morbidity and mortality but
are often poorly documented. We are conducting a five-year retrospective
review of seahorse cases submitted by the Toronto Zoo. To date,
approximately half of cases (of 100 total) have been reviewed. The major
reoccurring problems are; a bilaterally symmetrical multifocal chronic
active myopathy, enteritis with bacterial overgrowth, bacterial
dermatitis ('redtail'), a suspect viral enteritis, protozoan dermatitis
and enteritis, and a variety of systemic bacterial lesions, among other
less common diagnoses. The myopathy was found to be the most prominent
problem, occurring to varying degrees in approximately 80% of cases
reviewed thus far. Because the myopathy was found to be bilaterally
symmetrical, it is presumed that it is nutritional in origin.
Supplementation of the diet (brine shrimp primarily) did seem to
resolve, but not eliminate the myopathy. Lesions caused by bacteria and
protozoa were observed in approximately 20 and five percent of cases,
respectively.

**Surgical Treatment Of Lymphoma In A California Moray Eel, *Gymnothorax
mordax***

# [Caryn P. Poll]{.underline}

Medicine Section Chief, Department of Animal Health, John G. Shedd
Aquarium, 1200 South Lake Shore Drive, Chicago, IL 60605

An adult California moray eel, *Gymnothorax mordax*, was presented to
the Veterinary Service at the John G. Shedd Aquarium for evaluation of
an ovoid, intracoelomic mass. Diagnostics including plain and contrast
radiography, ultrasound, and fine needle aspiration with cytology and
bacterial culture suggested an undifferentiated, malignant neoplasm of
mesenchymal origin. The eel was anesthetized and surgery was performed
to resect the mass, which was associated with the gastrointestinal tract
and spleen. A section of stomach and intestine was resected and
anastomosed in a 3.5 hour surgery. Medical and nutritional
post-operative care along with the eel's natural wound healing ability
resulted in an uneventful recovery. Histopathology confirmed the mass as
a high-grade lymphoma.

**Fish Sutures: Tying Things Together**

[Gregory A. Lewbart]{.underline} and Craig A. Harms

Department of Clinical Sciences, North Carolina State University College
of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606

Many suture types have been used successfully in fish. Monofilament
absorbable skin sutures are generally recommended to decrease the risk
of wicking bacteria into the wound (synthetic absorbable sutures may not
be readily absorbed in fish). Other factors in suture selection are
tissue reactivity and healing time. Needles with a cutting tip
facilitate skin penetration. Simple continuous, simple interrupted and
continuous Ford interlocking patterns have all been used for skin
closure with satisfactory results. Continuous patterns provide less
surface area for bacterial and epibiont colonization, and subjectively
have been associated with less suture reaction than interrupted
patterns, but if not placed with adequate tension or without excellent
knot security at either end, dehiscence is more likely. Single or
two-layer closure can be employed depending on the thickness of the body
wall. Studies have shown that surgical adhesives (e.g. cyanoacrylate),
either alone or in combination with sutures, can contribute to
dehiscence, delay healing, and cause local irritation. Results from a
study in koi (*Cyprinus carpio*) indicate that monofilament materials
like nylon and polyglyconate (Maxon®) are slightly superior to braided
suture. Once sutures have been placed, a topical antimicrobial, like
povidone iodine (Betadine®) ointment or triple antibiotic ointment, can
be applied with care to the wound. In many cases suture materials are
removed in 10-14 days; fish wounds heal quickly when optimal
environmental parameters are present.

**To Cut Is To Cure: Surgical Cases In Fish Medicine**

[Helen E. Roberts]{.underline}

Aquatic Veterinary Services of WNY/5 Corners Animal Hospital, 2799
Southwestern Blvd, Suite 100, Orchard Park, NY 14127

Surgical treatments have been historically used commonly in many
companion animal species, including dogs and pets. With the increasing
awareness of the availability of companion animal/exotic animal
veterinarians that are willing to treat pet fish, a few procedures have
become relatively common in pet fish surgery. Epidermal mass removals,
enucleations, repair of traumatic injuries and exploratory celiotomies
are examples of the typical surgeries that may be seen in companion
animal practice. Fortunately, fish make excellent surgical candidates. A
few select case reports of surgical cases seen in private aquatic
veterinary practice will be presented.

**Endoscopic Sex Determination And Sterilization Of Gulf Sturgeon
(*Acipenser oxyrinchus* Desotoi)**

^1^[Robert S. Bakal]{.underline}, ^2^Stephen J. Hernandez-Divers,
^3^Brian H. Hickson, ^2^Clarence A. Rawlings, ^2^Heather G. Wilson,
^2^MaryAnn Radlinsky, ^2^Sonia M. Hernandez-Divers, and ^4^Samuel R.
Dover

^1^U.S. Fish and Wildlife Service, 4700 Hillsborough St. Raleigh, NC
27606; ^2^ Department of Small Animal Medicine & Surgery, College of
Veterinary Medicine, University of Georgia, Athens, GA 30602; ^3^U.S.
Fish and Wildlife Service, Warm Springs Regional Fisheries Center, 5308
Spring St., Warm Springs, GA 31830; ^4^Channel Islands Marine and
Wildlife Institute, Santa Barbara, CA 93117

Seventeen Gulf sturgeon underwent endoscopic sex determination and
various procedures as part of a study to determine whether sterilized
animals could serve as sentinels. The fish were anesthetized with
tricaine methanesulphonate (MS-222) and maintained on a re-circulating
anesthesia machine. A 6 mm cannula was placed just off ventral midline,
midway between pectoral and pelvic fins, which allowed insertion of a 5
mm telescope. Emptying the swim bladder coupled with CO~2~ insufflation
provided excellent visualization of the celoemic cavity. Two additional
cannulae were placed laterally, cranial and caudal to the first cannula
for introduction of surgical instruments. Sex determination was
successfully performed in all fish; with 5 of 17 fish requiring gonadal
biopsy. Bilateral ovariectomy or orchidectomy was successfully performed
in 3 males and 4 females. Unilateral ovariectomy and bilateral ligation
of the müllerian ducts was accomplished in an additional three females.
Endoscopy has provided tremendous advantages over classical surgical
techniques allowing us to minimize trauma to the animals and improve our
success rates.

**Ophthalmic Surgical Procedures In Public Aquarium Fishes**

[Charles J. Innis]{.underline}

New England Aquarium, Central Wharf, Boston, MA 02110-3399

Ophthalmic disorders, including corneal abrasions, ulcers, exophthalmia,
buphthalmia, uveitis, hypopyon, hyphema, cataracts, lens luxation, gas
bubble disease, proptosis, and various retrobulbar disorders are very
common in aquarium fish. Deep corneal ulcers may require surgical
debridement and grid keratectomy. Protection of the ulcer with
cyanoacrylate surgical glue has been effective in some cases, but
appears to have caused tissue reaction in other cases. Lens removal may
improve vision, decrease pain, and eliminate uveitis in cases of lens
luxation or advanced cataracts. While advanced techniques such as
phacoemulsification could be used, good results are often seen via
simple keratectomy and lens extraction. The cornea is closed with 6-0 to
8-0 suture. Unilateral or bilateral pseudobranch ablation via bipolar
cautery has shown approximately 50% success in cases of recurrent gas
accumulation in the anterior chamber or retrobulbar space. Enucleation
is a reasonable alternative for incurable ocular disease, but closure is
challenging in a fish with a very large orbit. Several reports of
successful ocular prostheses in fish have been described.

**Anesthesia Of Fish: I Don\'t Want To Make The Wrong Mistake (Yogi
Berra)**

[Alan M. Klide]{.underline}

Section of Critical Care (Anesthesia), School of Veterinary Medicine,
University of Pennsylvania, Philadelphia, PA 19104

Many substances have been used to anesthetize fish. Tricaine is probably
the most commonly used agent and is the only one approved by the Food
and Drug Administration for use in food fish in the United States,
however, it has a withdrawal time of 21 days. Depending on the specific
drug and the circumstances they may be administered by intramuscular
injection intravenous injection, orally, the drug may be put in water
and fish placed in water or the drug may be put in the water and the
water pumped into the fish's mouth and over the gills. It needs to be
pumped fast enough to deliver enough anesthetic and to ventilate the
fish i.e. maintain normal arterial oxygen and carbon dioxide
concentrations. The system should have an aerator stone in it which
bubbles air into the water. The basic scheme of stages of anesthesia in
the fish can be described as: awake, excited, turn to lateral or dorsal
recumbency, sink, anesthetized. Heart rate may be monitored by use of an
ECG, a Doppler flow probe, or a pulse oximeter. These different monitors
worked inconsistently between fish and sites in each fish, but when they
work they are very useful. For recovery the fish should be ventilated
with water free of anesthetic until they make strong opercular movements
and then be put in the recovery tank. The water should have an aerator
stone in it, which bubbles air into the water. There is much discussion
and disagreement about the question of pain in fish. Until the issue is
resolved with assurance, analgesics should be administered.

**Comprehensive Wound Care Management In A Cownose Ray (*Rhinoptera
bonasus)* And A Blue Catfish (*Ictalurus furcatus*)**

^1^[Tonya M. Clauss]{.underline}, ^2^Anthony C. Capomacchia, ^3^Richard
E. Wooley, and ^3^Branson W. Ritchie

^1^Georgia Aquarium, Veterinary Services and Conservation Medicine, 225
Baker Street, Atlanta, GA 30313; ^2^University of Georgia, College of
Pharmacy, Athens, GA 30602; ^3^University of Georgia, College of
Veterinary Medicine, Emerging Diseases Research Group, Athens, GA 30602

The primary function of the integumentary system is to provide a
protective barrier between the internal organs and the environment. Fish
have integumentary adaptations that permit them to live in an aqueous
habitat. Anatomical or physiological insult to the skin may compromise
homeostasis, immune competency and sensory mechanisms leading to
increased morbidity and mortality. Wounds of varying severity and origin
are one of the most common problems in captive aquatic species. The
unique and sophisticated characteristics of fish integument and exposure
to a constantly moist substrate enable fish to heal rapidly. However,
factors such as wound severity, medical condition of the animal,
nutrition and water quality can influence healing time. Comprehensive
wound management in fish may entail mechanical procedures such as
debridement or closure, local and systemic chemotherapeutics, stress
reduction, proper nutrition and provision of optimal water quality. At
the Georgia Aquarium, the use of Tricide-Neo, a potentiated
antimicrobial preparation, and Regranex™, a recombinant platelet-derived
growth factor, were used to treat a cownose ray and a blue catfish with
injuries sustained from tank mate aggression. The complicated nature of
their wounds required a multifaceted approach to control infection and
facilitate healing.

**Comparison Of Lethal And Non-Lethal Sampling Techniques For The
Detection Of *Aeromonas salmonicida* And Other Fish Pathogens In
Populations Of Ontario Salmonids**

# [Robert A. Quinn]{.underline} and Roselynn Stevenson

Fish Health Laboratory, Department of Molecular and Cellular Biology,
University of Guelph, Guelph, Ontario, Canada N1G 2W1

To monitor bacterial fish pathogens for the Ontario Ministry of Natural
Resources (MNR), the Fish Health Laboratory routinely uses conventional
bacteriological culture of fish organs collected by lethal sampling. As
a non-lethal approach, we used culture, PCR and PCR-Denaturing Gradient
Gel Electrophoresis (DGGE) to detect *Aeromonas salmonicida* and other
fish pathogens in fish mucus. Wild chinook and coho salmon (n=60 and 52)
from the Credit River and lake trout (n=27) from a MNR culture station
were sampled lethally and non-lethally. *A. salmonicida* was isolated on
Coomassie Brilliant Blue agar from mucus of 43 chinook and 31 coho,
while it was cultured from kidneys of 28 chinook and 15 coho. PCR for
the *vap*A gene was positive in the mucus of 30 chinook and 21 coho. *A.
salmonicida* was not detected in mucus or kidney tissue of the lake
trout by either culture or PCR. When mucus samples were used for a
nested PCR amplification of a 193bp segment of eubacterial 16S rDNA, 5
lake trout had bands coincident to the migratory distance of the bands
amplified from control cultures of *A. salmonicida.* Bands were also
found suggesting *Y. ruckeri* (4 fish) and *A. hydrophila* (9 fish). As
DGGE can separate species-specific sequences from a mixed microbial
community, PCR-DGGE may be useful in indicating the presence of multiple
pathogens in fish mucus simultaneously. However, analysis of results may
be complex, in terms of matching band migration patterns and discerning
the presence of bacteria with multiple band patterns, including *A.
salmonicida*.

**Efficacy Of Common Disinfectants Against *Aeromonas* spp. And
*Edwardsiella* spp.**

[Stephen A. Smith]{.underline} and Mary E. Mainous

Aquatic Medicine Laboratory, Department of Biomedical Sciences and
Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine,
Virginia Polytechnic Institute and State University, Blacksburg,
Virginia 24061-0442

Bacterial pathogens are one of the main disease-causing agents of the
aquaculture industry. As many potentially pathogenic bacterial species
are water-borne, disinfection is essential for both disease control and
for enhanced biosecurity. This study examined a variety of commercially
available compounds for their efficacy to reduce or eliminate specific
bacteria from the water, and included Clorox (50,000 ppm, 200 ppm, 100
ppm, 50 ppm), ethanol (70%, 50%, 30%), Roccal (1:256), Lysol (1%),
iodine (100 ppm, 50 ppm), formalin (250ppm), Chloramine-T (15mg/L),
glutaraldehyde (2%) and Virkon (1%). Each compound was tested for
various time intervals against *Edwardsiella ictaluri, E. tarda*,
*Aeromonas hydrophila*, *Aeromonas salmonicida*, and *Aeromonas
salmonicida subsp achromogenes*. In addition, the three *Aeromonas*
species were tested at various temperatures (22C, 15C, 10C, 5C). Both
species of *Edwardsiella* were killed within one minute of contact time
by all compounds except Chloramine-T and formalin, which were both
ineffective, even after 60 minutes. Similarly, all compounds, except
Chloramine-T and formalin, killed the three *Aeromonas* species within
one minute of exposure time regardless of temperature or organism. The
effects of Chloramine-T were temperature-dependent, and also varied with
the organism. *A. hydrophila* was killed within five minutes exposure
regardless of temperature. *A. salmonicida* was also killed within five
minutes, but only at 22C, 15C and 10C, while an exposure of 10 minutes
was required at 5c. In contrast, *A. salmonicida* *subsp achromogenes*,
survived up to 30 minutes exposure at all temperatures, while no growth
was detected at 60 minutes. Formalin was the least effective
disinfectant tested, producing less than a one log reduction in
bacterial growth even after one hour of exposure regardless of
temperature or organism.

**Florfenicol Uptake And Depletion In Tilapia (*Oreochromis niloticus*)
Of Various Sizes**

^1^Paul R. Bowser, ^1^Rachel E. Kosoff, ^1^Chun-Yao Chen, ^1^[Gregory A.
Wooster]{.underline}, ^1^Rodman G. Getchell, ^2^Amel Clifford, ^2^Scott
E. Wetzlich and ^2^Arthur L. Craigmill

^1^Aquatic Animal Health Program, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
New York 14853-6401; ^2^Department of Environmental Toxicology,
University of California -- Davis, Davis, California 95616

Nile tilapia *Oreochromis niloticus*, of three different size groups
were medicated with florfenicol (Aquaflor, Schering-Plough) via a
medicated ration at 15 mg florfenicol/Kg fish body weight/day for 10
days to compare elimination kinetics of the test article. This study was
a part of a larger effort to develop a species-grouping concept for the
labeling of therapeutic compounds for cultured fish. Tests were
conducted on tilapia weighing 100 gm, 250 gm and 500 gm at a temperature
of 25º C to determine if depletion of florfenicol was impacted by fish
size. Animals were acclimated for a minimum of 7 days before
administration of the medicated feed and held for up to 28 days after
the 10-day medication period. Fifteen fish were sampled at the following
time points: 5 days after the initiation of the medicated feeding period
(mid-point of medication period) and at 1 hr, 1 d, 2 d, 4 d, 8 d, 14 d
and 28 d post-medication period. During acclimation and after the
medicated feed was administered a control diet was given. Results
indicated that for tilapia at 25º C there is a trend for more rapid
elimination in smaller fish. For both 100 and 250 g fish, concentrations
of florfenicol declined below the action level within 8 days and for 500
g fish, within 14 days.

**Minimum Inhibitory Concentration Testing of *Flavobacterium
columnare***

^1^[Ahmed M. Darwish]{.underline}, ^1^Bradley D. Farmer, and ^2^John P.
Hawke

^1^Harry K. Dupree-Stuttgart National Aquaculture Research Center,
Agricultural Research Service, United States Department of Agriculture,
P. O. Box 1050, 2955 Hwy 130 East, Stuttgart, Arkansas 72160;
^2^Department of Pathobiological Sciences, Louisiana State University,
School of Veterinary Medicine, Baton Rouge, Louisiana 70803

A simple, accurate and reliable microdilution method has been developed
to test the susceptibility of *Flavobacterium columnare* to antibiotics.
The method has been used to determine the minimum inhibitory
concentration (MIC) of 23 *F. columnare* isolates. The developed method
conducted at 28 °C for 48 h used standardized inoculum, Mueller Hinton
broth at 1/5 the full strength (4g/l), reference isolate, a positive and
a negative control wells and a standardized custom made microtitre
plates, Sensititre® Susceptibility Plates for Aquaculture (Trek
Diagnostic Systems, Inc.). *Escherichia coli*, ATCC25922 was used as the
reference isolate and produced MIC values within the range published by
the Clinical and Laboratory Standards Institute (CLSI), formally the
National Committee for Clinical Laboratory Standards (NCCLS). Mueller
Hinton broth at 1/5 the full strength (4 g/l) was found to support
significantly better growth for *Escherichia coli*, ATCC25922 and *F.
columnare* type strain, ATCC23463, than Mueller Hinton broth at 1/7 the
full strength.

**Sequence Analysis Of The *Yersinia ruckeri* Multidrug Resistance
Plasmid pYR1**

[Timothy J. Welch]{.underline}

National Center for Cool and cold Water Aquaculture, USDA-ARS, 11876
Leetown Road, Kearneysville, WV, 11871

Antimicrobial resistance in *Yersinia pestis* constitutes a significant
threat considering that antimicrobials are used for both plague
treatment and for the prevention of human-to-human transmission. For
this reason, the discovery of a multiple antimicrobial resistant (MDR)
isolate of *Y. pestis* (strain IP275) in 1997 caused considerable alarm.
This isolate contains a large self-transmissible plasmid (pIP1202) that
confers resistance to at least eight antimicrobials including several of
the drugs recommended for plague treatment and prophylaxis. Comparative
analysis of the DNA sequence of pIP1202 revealed an IncA/C plasmid
backbone that is shared by MDR plasmids isolated from *Salmonella
enterica* serotype Newport SL254 and the fish pathogen *Yersinia
ruckeri* YR71. The high degree of sequence identity and gene synteny of
this shared plasmid backbone suggests recent acquisition of these
plasmids from a common ancestor. In addition, PCR-based plasmid typing
and rapid detection methods were used to screen a collection of MDR
isolates of *Salmonella*, *Escherichia coli* and *Klebsiella* for the
pIP1202 backbone. Strains tested were isolated from retail meat samples
collected between 2002 and 2005. The *Y. pestis* pIP1202-like plasmid
backbone was detected in 65 isolates, including several serotypes of
*Salmonella* as well as several *Escherichia coli* and *Klebsiella*
strains. The plasmid-positive strains were found in bacteria associated
with beef, chicken, turkey and pork, and were found in samples from the
following states: CA, CO, CT, GA, MD, MN, NM, NY and OR. These studies
reveal a common plasmid backbone that is broadly disseminated among
zoonotic pathogens associated with agriculture. These plasmids represent
a reservoir of mobile resistance determinants and have the potential to
disseminate to *Y. pestis* and other human and zoonotic bacterial
pathogens of public health importance.

Pharmacokinetics Of Oxytetracycline In The Horseshoe Crab

[Michael W. Nolan]{.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
24061-0442

The American horseshoe crab, *Limulus polyphemus*, is commonly
maintained in research laboratories and public aquaria. Concerns over
the health of these captive animals make the clinical diagnosis and
treatment of pathological conditions in *L. polyphemus* essential. This
study investigated the pharmacokinetics of oxytetracycline in male and
female adult horseshoe crabs following either intravenous or oral
dosing. A non-compartmental model was developed to describe the
pharmacokinetics of oxytetracycline in the horseshoe crab. The following
parameters were determined for a single intravenous bolus of 25 mg/kg
oxytetracycline: AUC = 9524.60 µg\*h/mL, MRT = 443.65 h, Cl~b~ = 0.044
mL/min/kg, V~d(ss)~ = 1.164 L/kg, t~1/2~ = 128.3 h, C~max~ = 55.90
µg/mL, C~ave~ = 27.39 µg/mL. After a single oral bolus of 20 mg/kg, the
following parameters were determined: AUC = 5861.81 µg\*h/mL, MRT =
395.89 h, Cl~b~ = 0.071 mL/min/kg, V~d(ss)~ = 1.688 L/kg, t~1/2~ = 210.0
h, C~max~ = 7.83 µg/mL, C~ave~ = 2.89 µg/mL, F = 61.56%. As compared
with the intravenous route (i.e. 100% bioavailability), oral absorption
of oxytetracycline was observed at a rate of 61.56% in the horseshoe
crab. Because absorption is significantly reduced in the oral route as
evidenced by the relatively low bioavailability, it is recommended that
intravenous injection is a more appropriate route for therapeutic dosing
of oxytetracycline in the horseshoe crab. This preference in route of
administration is further supported by the ease with which an
intravenous bolus can be delivered into the cardiac sinus of horseshoe
crabs.

**Use Of 17-Alpha-Methyltestosterone For Masculinization Of Ornamental
Fish**

^1^[Roy P. Yanong]{.underline}, Chris J. Daniels, Jeff E. Hill, and
Craig A. Watson

Tropical Aquaculture Laboratory, Department of Fisheries and Aquatic
Sciences, Institute of Food and Agricultural Sciences, University of
Florida, 1408 24^th^ St. SE, Ruskin, FL 33570

The compound 17-alpha-methyltestosterone (MT) has been used in
aquaculture for many years. In the U.S., MT is available through an FDA
INAD for use as a masculinizing feed additive for more efficient
production of tilapia. MT has additional potential applications in
ornamental species. Many ornamentals, including swordtails, African
cichlids, dwarf gouramis, rosy barbs, and rainbowfish are sexually
dimorphic, and phenotypic males often having greater economic value than
phenotypic females. MT may also provide a mechanism to reduce population
growth in aquarium exhibits, by allowing stocking of all males/male
phenotypes and/or by sterilization of fish, which may also be desirable
in other situations. Under a current FDA INAD for ornamental swordtails,
ongoing studies in Florida through the Tropical Aquaculture Laboratory
have demonstrated 100% masculinization in fish fed 3% or 5% body weight
of a 60 mg MT/kg feed, as defined by development of a caudal fin
extension ("sword") of seller size phenotypic female fish during in-tank
trials. Commercial field trials for swordtails are currently planned.
Other laboratory trials using limited combinations of different life
stages (\~2-week-old, \~6-week-old, and broodstock) and treatment
regimens (10% or 5% BW feeding of 60 mg MT/kg feed; for 28, 56, or 84
days)in zebra danios have also been completed. Histological analysis of
representative fish as well as breeding experiments demonstrated
differences in effectiveness depending upon the size/age of fish
exposed, with 2-week-old fish appearing to be masculinized most
successfully. Additional studies are planned for other species.

**Preliminary Investigations Into The Use Of Metomidate Hydrochloride
(Aquacalm^®^) In Ornamental Cichlids**

^1^[Kathy H. Kilgore]{.underline}, ^1^Jeffrey E. Hill, ^1^Roy P. E.
Yanong, and ^2^James F. F. Powell

^1^Tropical Aquaculture Laboratory, Department of Fisheries and Aquatic
Sciences, Institute of Food and Agricultural Sciences, University of
Florida, 1408 24^th^ Street SE, Ruskin, FL 33570; ^2^Syndel
International, Inc., 9211 Shaughnessy Street, Vancouver, British
Columbia, V6P 6R5, Canada

Metomidate hydrochloride (Aquacalm^™^) is used for anesthesia or
sedation of fish for procedures such as tagging, weight sampling,
spawning, and transportation. However, its usefulness as a sedative
during transport of ornamental fish has yet to be fully determined.
Based on the results of several pilot studies, convict cichlids (\~4.5
cm TL) *Archocentrus nigrofasciatus* (Cichlidae) were chosen as a test
species to evaluate sedation with metomidate during transport. Three
metomidate concentrations (0.2 ppm, 0.5 ppm, and 1.0 ppm) and a control
were evaluated. Fish were stocked at a density of twenty-five per bag,
and eight replicate bags were run for each metomidate concentration.
After approximately 24 hours of exposure to metomidate (including 4.5
hours of air transport), the fish in each shipping bag were transferred
to a 5-gallon "recovery" tank for data collection over a 7-day
observation period. Water quality data (including total ammonia
nitrogen, dissolved oxygen, temperature, and pH), mortality data, and
appearance and behavior scores were collected at various times
throughout the observation period. Statistical analyses of cumulative
mortality data as well as mean ranks of appearance and behavior scores
revealed that a high dose (1.0 ppm) of metomidate resulted in fewer
mortalities and improved appearance during shipping.

**Gene Discovery In *Ichthyophthirius multifiliis*: A Complementary DNA
Resource**

[Donna M. Cassidy-Hanley]{.underline} and Ted G. Clark

Dept. of Microbiology and Immunology, Veterinary Medical Center, Cornell
University, Ithaca, NY 14853

*Ichthyophthirius multifiliis* (Ich) is a major pathogen of freshwater
fish with substantial impact on commercial aquaculture. No vaccines are
currently available for this agent, and treatment options are restricted
to the use of chemicals that are both costly and toxic to the
environment. We have assembled and analyzed a comprehensive collection
of 24,741 ESTs representing genes expressed at 2 different stages of the
Ich life cycle, the mature trophozoite (trophont) and the free swimming
infectious theront. Analysis of these sequences has resulted in the
identification of 5311 unique transcripts (UniScripts), 3666 from the
trophont stage and 2083 from the theront stage. We have documented
significant differences in gene expression between the stages examined,
which strongly reflect the predominant physiological activities
occurring at each developmental stage. This annotated EST collection
should aid in the discovery of potential drug and vaccine candidates for
the treatment and prevention of "white spot" disease, and in the
elucidation of metabolic pathways that control virulence and parasitism
in *I. multifiliis* and more distantly related parasitic protozoa within
the alveolate clade. Expression profiling of the genes that are active
at different stages of the life cycle, along with comparative genomic
analyses with free-living ciliates and apicomplexans, should also lend
fundamental insight into the genes involved in ciliate pathogenesis.

**Cormorants As Mechanical Vectors Of *Heterosporis* sp.**

^1^[Mark A.J. Quinnell]{.underline}, ^1^Julia Parkman, ^1^Dale Smith,
^1^Spencer Russell, ^2^Dan Sutherland, and ^1^John S. Lumsden.

^1^Fish Pathology Laboratory, Department of Pathobiology, University of
Guelph, Guelph, ON, Canada, N1G 2W1; ^2^Department of Biology,
University of Wisconsin-La Crosse, La Crosse, Wisconsin 54601

*Heterosporis* sp., an emerging microsporidial fish pathogen in North
America, has a wide host range and infects skeletal muscle rendering it
unpalatable for human consumption. The potential for cormorants
(*Phalacrocorax auritus) to* spread this parasite was investigated.
Eight cormorants were used: three control birds; and five experimental
birds each fed 20g of *Heterosporis* sp.-infected yellow perch (*Perca
flavescens*) muscle daily for three days. The affected yellow perch
muscle was confirmed to contain *Heterosporis* sp. by nPCR and
sequencing of the product obtained. Feces were collected daily from each
group of birds for one week after the first bolus of infected muscle was
given. Fathead minnows (*Pimephales promelas*) were used to detect the
presence of viable parasites. Two tanks housed control fish that were
fed daily for one week; brine shrimp mixed with 2 ml of fresh feces from
the control birds. Two tanks of fish were exposed to feces from
cormorants fed infected muscle following a similar procedure. One tank
of fish was given on two successive days, 0.1 ml of feces *per os*, from
birds fed infected muscle. Another tank of fish was fed macerated
infected yellow perch muscle for three days, and served as positive
controls. At approximately monthly intervals over four-and-a-half
months, groups of fish were euthanized and examined for evidence of
*Heterosporis* sp. by histology and nPCR. *Heterosporis* sp. was
detected only in the fish fed infected muscle tissue. At least under the
experimental conditions employed here, cormorants do not appear to be
mechanical vectors of *Heterosporis* sp.

**Renal Trematodiasis Of Largemouth Bass And Development Of PCR
Methods**

^1^[Roxanna M. Smolowitz]{.underline}, ^2^R.S.Shaff, ^1^J.E.Defaveri,
and ^3^S.A.Roberts

^1^Marine Biological Laboratory, Woods Hole, MA 02543; ^2^College of
Veterinary Medicine, U. of Illinois Urbana-Champaign, Urbana, IL 61801;
^3^School of Aquatic and Fisheries Science, U. of Washington, Seattle,
WA 98195

*Acolpenteron ureteroecetes* (suborder Monopisthocotylea, family
Caleostomatidae), is a monogenean trematode that lives in the renal
tubules of large mouth bass, *Micropterus salmoides*. In July, 2005, a
new fresh water MA aquaculture venture purchased fingerlings from a
commercial hatchery. The fish were placed in recirculating raceways upon
arrival in MA. In order to get the best growth of the large mouth bass
in this system, and to make the venture economically profitable, the
temperature of the water was held at 27ºC. By November, 2005, when the
fish averaged 9\" in length, mortality counts of 75 animals/day were
routine. Moribund fish from the facility showed lesions consistent with
renal trematodiasis. The water temperature was lowered to 24ºC and
mortality decreased significantly. But, the growth rate also decreased
resulting in a poor, long term economic outlook. The grower requested a
method for screening fingerlings. A PCR based diagnostic test provides a
quick and sensitive method. DNA was initially amplified using family
specific primers. Species specific primers were then developed and used
on samples of infected kidney tissue, trematodes only and eggs only.
Results showed that the primers selected for our PCR test method were
successful at identifying parasites and eggs. This test could provide
uninfected fish that can be maintained at higher, growth promoting
temperatures which will decrease time to market and will increase
economic viability of bass aquaculture.

**Biology And Management Of A Monogenean Trematode (*Acolpenteron
ureterocetes*) Infecting Largemouth Bass (*Micropterus salmoides*)**

^1^[Jennifer Dill]{.underline}, ^1^Stephen K. O'Shea, ^2^Roxanna
Smolowitz, and ^3^Dale Leavitt

^1^Department of Chemistry, Roger Williams University, One Old Ferry
Road, Bristol, RI 02809, ^2^Marine Biological Laboratories, MBL Street
Woods Hole, MA 02543, ^3^Department of Marine Biology, RWU

Recently a fish farm in Massachusetts experienced mortalities in
largemouth bass (*Micropterus salmoides*) starting three months after
delivery as fingerlings from a commercial hatchery. Upon examination,
Dr. Roxanna Smolowitz, DVM found numerous trematodes in the kidney and
ureters. The diagnosis indicated a unique and understudied monogenetic
trematode, *Acolpenteron ureterocetes*, as the primary cause of fish
mortality. Bass fingerlings from the same cohort, housed at Roger
Williams University, were also examined and also found to host the
trematode but with less severity of infection. To better understand the
etiology of this parasite, the major focus of this project was to
develop a rapid and accurate diagnostic protocol to evaluate the degree
of parasite load in fish. A fresh tissue smear technique was developed
and compared with a histopathological inspection/evaluation protocol.
The quicker, easier and less expensive fresh smear protocol proved to be
reliable and a quantifiable indicator of parasite prevalence. The
effects of the commercial anti-trematode treatment, Praziquantel, were
also studied. This research focused on the tissue uptake of the drug,
administered under controlled conditions (1.5mg/L with 24 hour exposure)
to largemouth bass fingerlings (8-10cm) and compared to an ethanol
control treatment (112 ppm with 24 hour exposure). The body burden of
praziquantel in the bass kidney, liver, gills, and muscle was measured
by HPLC, UV/Vis spectroscopy (λ =220nm) and fluorescence spectroscopy
(λex=260nm and λem=285nm, 100ng detection limit). The degree of uptake
by the four bass tissue compartments was quantified along with the
elapsed time post-treatment where detection of residual praziquantel and
metabolites in the fish tissue were measurable.

**Prevalence And Impact Of Leeches Parasitizing Fish Of Lake St. Clair,
Michigan**

^1^[Carolyn A. Schulz]{.underline}, ^2^Michael V. Thomas, ^3^Scott
Fitzgerald, ^1,3^ Mohamed Faisal

^1^Department of Fisheries and Wildlife, Michigan State University, East
Lansing, MI 48824; ^2^Michigan Department of Natural Resources (MDNR) --
Lake St. Clair Fisheries Research Station, Mt. Clemens, MI 48045;
^3^Department of Pathobiology and Diagnostic Investigation, Michigan
State University, East Lansing, MI 48824

Lake St. Clair (LSC) plays a critical role for recreational and
conservation fisheries. The purpose of this study was to identify the
most prevalent leech species parasitizing LSC fish and to assess damage
caused by the leeches to their host. Fish and leeches were collected via
trapnets by the MDNR during May 2006. Leeches were identified
morphologically according to several keys. Three species of leech were
identified: *Actinobdella pediculata*, *Myzobdella lugubris*, and
*Placobdella montifera*. Of the three, *M. lugubris* (83.29%) was the
most widespread. Freshwater drum, *Aplodinotus grunniens,* (18.21%) and
channel catfish, *Ictalurus punctatus*, (18.13%) were the most prevalent
leech hosts. The pectoral fins (29.21%) were the most common attachment
site. Leech attachment sites were associated with epidermal erosion,
hemorrhages, edema, massive expansion of the connective tissue, and
congestion. These findings show that leeches do cause damage to their
hosts and could potentially be assisting other pathogens to cause harm.

**Toxin Production And Virulence Factors In A Polymicrobial Disease Of
Corals**

[Laurie L. Richardson]{.underline}, Miroslav Gantar, Longin Kaczmarsky,
Jamie L. Myers, and Raju Sekar

Department of Biological Sciences, Florida International University,
Miami, Florida 33199

Black band disease (BBD) of corals is a complex, polymicrobial infection
that migrates as a distinct mat across the surface of coral hosts,
lysing coral tissue. We have been investigating the etiology of this
disease using microbiological, molecular, and analytical techniques. It
appears that multiple toxins and virulence factors are involved in
disease pathogenesis. First, aerobic respiration by BBD community
members results in permanently anoxic zones in the middle and base of
the 1 mm thick band, extending up into the diffusive boundary layer
above the mat at night. The anoxic conditions select for
sulfate-reducing bacteria, which generate toxic sulfide that accumulates
to concentrations in the band that are lethal to coral. Experiments
using the inhibitor Na molybdate revealed that while sulfide production
is not required for coral tissue lysis and disease progression in an
actively migrating band, it is required for disease initiation. We have
found a second BBD toxin, the cyanotoxin microcystin. Using HPLC/MS we
detected microcystin in 22 field samples of BBD from regions of the
northern Caribbean, but not from the eastern Caribbean or the
Philippines. Microcystin was also produced in cyanobacterial cultures
isolated from BBD. Toxic activity was confirmed by ELISA and the protein
phosphatase inhibition assay. In addition to the above, we have
consistently detected numerous 16S rRNA gene sequences in clone
libraries constructed from BBD samples that are most closely matched to
bacteria associated with Paralytic Shellfish Toxin (PST) producing
dinoflagellates suggesting additional toxins.

**Antibacterial Activity Of Cyanobacteria Associated With Black Band
Disease**

[Miroslav Gantar]{.underline}, Longin Kaczmarsky, and Laurie L.
Richardson

Florida International University, Dept. of Biological Sciences,
University Park, Miami, FL. 33199

One of the most common coral diseases is black band disease (BBD). This
complex microbial community contains a great number of taxonomically and
metabolically diverse microorganisms among which cyanobacteria are
dominant. It is currently believed that the disease is caused by
synergistic action of the members of this consortium. Cyanobacteria are
known to produce a vast number of different biologically active
compounds. In this work, we are testing the "Coral Probiotic Hypothesis"
according to which the "right" microbial population associated with
coral mucus and tissue can provide resistance to pathogens. To assess
the potential of coral reef cyanobacteria to affect bacterial growth, we
tested 19 cyanobacterial strains that were isolated from BBD and non-BBD
associated cyanobacterial mats on the reef. Their effect was tested on
10 strains of heterotrophic bacteria isolated from BBD and 10 strains of
heterotrophic bacteria from the mucus of apparently healthy corals.
Taxonomic identification of all microorganisms was performed by 16S rRNA
gene sequencing followed by a BLAST search. Of 19 cyanobacterial strains
tested, 10 showed antibacterial activity against at least one target
organism. The most active cyanobacterial strain was a *Leptolyngbya*
isolated from a non-BBD source, and the second most active was a BBD
*Geitlerinema*. The most susceptible bacterial strain was *Vibrio
harveyi* that was isolated from BBD and that is known to be a common
pathogen for many marine organisms. These preliminary data indicate that
cyanobacteria indeed produce antibacterial compounds effective against
potential BBD pathogens.

**Comparison of Collection Methodology, Location, Coral Species, and
Health Status on Acroporid Coral Microbial Community Composition**

^1,\ 2^ [Shawn W. Polson]{.underline} and ^2^Cheryl M. Woodley

^1^Marine Biomedicine and Environmental Sciences Center, Medical
University of South Carolina, 221 Fort Johnson Rd., Charleston, SC
29412; ^2^Center for Coastal Environmental Health and Biomolecular
Research, NOAA/National Ocean Service, Hollings Marine Laboratory, 331
Fort Johnson Rd., Charleston, SC 29412

*Acropora* spp., once dominant corals on Caribbean reefs, have suffered
region-wide declines over the past two decades. *A. palmata* and *A.
cervicornis* are the first corals to be listed as threatened species
under the U.S. Endangered Species Act. These corals harbor diverse
microbiota, likely participants in coral health and disease processes.
Several investigators have previously attempted to ascertain the
structure of microbial communities associated with these corals by
various methods, often with conflicting results. This study used a
larger-scale genomic approach to analyze the microbiota. Replicate
biopsies from apparently healthy and diseased acroporid colonies were
collected at six sites in the Florida Keys and Dry Tortugas. Three
different processing methods were used in parallel to process the
samples. Individual 16S rRNA gene libraries were generated from each of
56 samples with over 17,000 clones sequenced to date. Results indicate
that the methodology employed to process a given sample introduces
significant variability into the microbial community composition
detected. Of the three methods used, coral mucus samples reveal the most
consistent microbial composition between similar samples, with minor
variation introduced by differing geographic location and coral species.
Several groups of bacteria that were differentially detected in healthy
versus diseased samples are currently under investigation.

**Defense Mechanisms In Gorgonians Against Infections By *Aspergillus
sydowii***

^1^[Garriet W Smith]{.underline},^2^Chad L Leverette, ^2^Melissa Warren,
and ^1^Marie-Ange Smith

^1^Dept. of Biology and Geology, Univ. of South Carolina Aiken Aiken,
SC, 29801; ^2^Dept. of Chemistry and Physics, USCA

Gorgonian defense mechanisms against infections include accumulation of
amebocytes, increased production of antifungal compounds and elevated
production of gorgonin, melanin and chitinase. The most apparent
response, however, is the production of pigmented sclerites. Until now
the chemical nature of the pigment remained unknown. We report here,
using confocal Raman microscopy, that the pigment is a carotenoid with a
polyene chain containing between 14 to 15 carbon double bonds.
Antifungal activity of carotenoids has been reported and we are
currently testing this against the gorgonian pathogen *Aspergillus
sydowii.*

**Histopathology Of White Plague-Diseased Corals**

^1,2^[Esther C. Peters]{.underline}, ^2^Geoffrey M. Cook, and ^2^Robert
B. Jonas

^1^Tetra Tech, Inc., 10306 Eaton Place, Suite 340, Fairfax, Virginia
22030; ^2^George Mason University, 4400 University Drive, Fairfax,
Virginia 22030-4444

Knowledge of the microscopic structure of cells and tissues provides
clues to their function and is necessary in the study of diseases in all
organisms. One concern about the health of the scleractinian or stony
corals since the early 1970s has been the rapid loss of polyps and
coenenchymal tissue resulting in completely denuded exoskeleton within a
matter of hours. Using standard histological techniques and examining
the stained tissue sections by light microscopy revealed bacterial
aggregates in some affected coral species and not in others, leading to
the separation of the white band and white plague diseases. Coral
tissues have been sampled during disease outbreaks on reefs in the
Caribbean and Indo-Pacific during the last 10 years. Histopathological
examinations have shown that similar gross signs of tissue loss can have
apparently diverse etiologies, including bacteria,
non-bacterial-associated apoptosis, and macropredators. Refined
protocols that trap the receding tissue margin of diseased corals and
stain the tissue for bacteria and mucus have resulted in a clearer
visual image of white plague, at least as it affects Caribbean corals of
the genus *Montastraea*, but have obfuscated the etiology. Corals having
signs matching the current field diagnosis of "white syndrome" require
the application of histopathological and microbiological methods to
distinguish the etiologic agents responsible for rapid tissue loss and
to help explain mechanisms of action.

**Large-Scale Geographical Comparisons Of Bacterial Communities
Associated With Corals Exhibiting Signs Consistent With White Plague
Type II**

^1^[Geoffrey M. Cook]{.underline}, ^1^Karen Santora, ^1^Masoumeh
Sikaroodi, ^1,2^ Esther C. Peters, ^1^Patrick M. Gillevet, and ^1^Robert
B. Jonas

^1^Department of Environmental Science and Policy, George Mason
University MSN 5F2, 4400 University Drive, Fairfax, Virginia 22030-4444;
^2^Tetra Tech, Inc., 10306 Eaton Place, Suite 340, Fairfax, Virginia
22030

The objectives of this project focus on fundamental questions pertaining
to a possible white-disease syndrome affecting hard corals on reefs
throughout the greater Caribbean region. Ultimately, this investigation
aims to establish the cause of this disease by characterizing the
microflora associated with diseased and apparently healthy coral tissue
spanning the reported geographic range of white plague type II: the
North Atlantic Ocean (Bermuda), the Tropical Western Atlantic (Lee
Stocking Island, The Bahamas, the Dry Tortugas, and the Florida Keys),
the Caribbean Sea (Little Cayman and St. Croix, USVI), and the Gulf of
Mexico (the Flower Garden Banks National Marine Sanctuary). Samples of
coral tissue, skeleton, and mucus were collected from colonies of the
*Montastraea annularis* species complex exhibiting signs consistent with
white plague type II. Using culture dependent and culture independent
techniques, the bacterial communities associated with diseased and
apparently healthy coral tissue and skeleton were characterized and
quantified. Preliminary results suggest that microflora associated with
diseased and apparently healthy tissues are not dominated by a single
organism and differ on both small and large spatial scales.

**Molecular Genetic Screening For Putative Antimicrobial Genes In
Anthozoans: A Possible First-Line Defense Against Coral Disease
Pathogen**

[Katherine A. Beauchamp]{.underline}

National Fish Health Research Laboratory, Leetown Science Center, USGS,
11649 Leetown Road, Kearneysville, West Virginia, 25430

Coral reefs worldwide are undergoing a significant decline in species
abundance and changes in community structure. Coral disease is now
thought to be a major cause of reef decline and epizootics have been
reported for several coral species. To date, 18 coral diseases affecting
at least 150 anthozoan and hydrozoan species have been described from
the Caribbean and the Indo-Pacific. Antimicrobial peptides and
pore-forming toxins are widespread in nature and are synthesized by
microorganisms as well as by multi-cellular organisms. These naturally
occurring agents form a first-line of host defense against pathogens and
are involved in innate immunity. Preliminary results of genetic
screening by PCR and sequencing of putative genes in antimicrobial
agents will be presented for corals and sea anemones. Conserved sequence
motifs in these genes in anthozoans will be compared to those same genes
that have been characterized in other organisms. Target genes to be
screened in this study include antimicrobial peptides
(metalloproteinases, cystine and serine proteases) and pore-forming
toxins (equinatoxin, hydralysin, neuroysin). This study will better our
understanding of disease mechanisms and to assess the status coral
health.

**Correlation Between API-ZYM And 16S rRNA-RFLP Profiles Of Ontario
*Flavobacterium psychrophilum* Isolates**

^1^[Shohreh Hesami]{.underline}, ^2^Laura L. Brown, ^1^John S. Lumsden
and ^3^Janet I. MacInnes

^1^Fish Pathology laboratory, ^3^Department of Pathobiology, University
of Guelph, Guelph, Ontario Canada N1G 2W1; ^2^NRC Institute for Marine
Biosciences, 1411 Oxford St., Halifax, NS B3H 3Z1

####### Two genetic lineages (I and II) of *F. psychrophilum* have been described. The goal of the present study was to identify these lineages in 75 isolates *of F. psychrophilum* that were collected from farmed salmonids with coldwater disease in Ontario, Canada. The Ontario isolates were morphologically and serologically homogeneous but two distinct biovars were identified by API-ZYM. A 194 bp PCR product was generated from all Ontario isolates and was digested individually with MaeIII or with MnlI. PCR-RFLP analysis demonstrated four restriction patterns with a correlation between biovar I and digestion with MaeIII (lineage II), and between biovar II and digestion with MnlI (lineage I). MnlI digestion produced two fragments of 105 and 62 bp in 16 of 33 isolates belonging to biovar II, but digested only two of the PCR products from biovar I isolates. Digestion with MaeIII gave rise to fragments of 128 and 64 bp in 40 of 45 isolates belonging to biovar I whereas only one biovar II isolate had a PCR product that was digested with MaeIII. One isolate of biovar I was cut by both, and two isolates of biovar I and 16 isolates of biovar II was cut by neither, of the restriction enzymes. Sequence analysis of the 194 bp ribosomal RNA fragment led to the conclusion that *F. psychrophilum* isolates from Ontario are composed of at least six different PCR-RFLP clusters indicating the existence of polymorphism among the isolates. 

**Cold-Inducible Proteins Of *Flavobacterium psychrophilum,* The Cause
Of Coldwater Disease**

^1^[Arman Yazdanpanah]{.underline}, ^1^Paul Huber, ^2^Janet I. MacInnes
and ^1^John S. Lumsden.

^1^Fish Pathology Laboratory, ^2^Department of Pathobiology, University
of Guelph, Guelph, ON, Canada, N1G 2W1

Coldwater disease is a worldwide problem in temperate freshwater
aquaculture and is caused by the Gram-negative bacterium *Flavobacterium
psychrophilum.* There are numerous manifestations of this organism
including tail rot/peduncle disease, necrotic myositis,
osteochondritis/scleritis and rainbow trout fry syndrome. As the name
'coldwater disease' suggests, low water temperatures are characteristic
of all of the presentations. Our hypothesis is that there will be
differential expression of proteins from a given strain of *F.
psychrophilum* grown at 8^o^C vs. 18^o^C and that these proteins may be
associated with virulence. 2D-PAGE is used to examine spot-pattern
differences of two strains (ATCC and Ontario) of *F. psychrophilum*. The
spots of greatest interest are those that are only present at 8^o^C or
those that are more prominent at 8^o^C vs. 18^o^C. So far the conditions
for 2D-PAGE of *F. psychrophilum* have been established and the
identification of spot differences is being evaluated using SYPRO- and
Coomassie-stained gels and densitometric quantification. Spots will be
manually excised from gels for analysis by mass spectroscopy (MALDI-tof)
and MS/MS (tandem mass spec.) for internal amino acid sequence.

**A Prospective Case-Control Study Of Bacterial Gill Disease Outbreaks
In Ontario, Canada Government Salmonid Hatcheries**

^1^[Christopher M. Good]{.underline}, ^2^Margaret A. Thorburn, ^2^S.
Wayne Martin, ^3^Roselynn M. Stevenson, and ^4^Carl S. Ribble

^1^The Conservation Fund Freshwater Institute, Shepherdstown, WV;
^2^Dept. of Population Medicine, University of Guelph, Ontario, Canada;
^3^Dept. of Molecular and Cellular Biology, University of Guelph,
Ontario, Canada; ^4^ Faculty of Veterinary Medicine, University of
Calgary, Alberta, Canada

Bacterial gill disease (BGD) is an important concern in freshwater
aquaculture. It has been a persistent problem at Ontario Ministry of
Natural Resources (OMNR) salmonid hatcheries and the Alma Aquaculture
Research Station (AARS), where outbreaks have been associated with rapid
and high mortality levels. The causative agent of BGD, *Flavobacterium
branchiophilum*, is considered ubiquitous in fresh water; therefore,
outbreaks are thought to be precipitated by environmental conditions
favoring opportunistic infections. Despite the importance of BGD, little
epidemiological research has been conducted to examine risk factors for
this disease. This paper presents a 14-month (July, 2002 -- September,
2003) prospective case-control investigation at five OMNR hatcheries and
the AARS, to identify and quantify important risk factors for BGD
outbreaks. Daily data for all early-rearing (\<9 months of age) units
were collected, and all outbreaks of BGD were confirmed at the Fish
Health Laboratory (University of Guelph). Control tanks were matched to
individual case tanks based on time, hatchery, and species, and the
case-control data were analyzed using multivariable logistic regression
modeling. Results of the final model indicated that tanks with confirmed
BGD outbreaks were significantly more likely to have lower fish numbers,
lower individual fish weights, higher mortality levels and higher
feeding rates during the week preceding observed BGD outbreaks than were
asymptomatic control tanks. Refinements in the observation and
manipulation of these factors can therefore aid in the prevention of
fish losses associated with BGD outbreak mortality spikes.

Unique Mycobacterial Resistance And Clearance In Channel Catfish
(*Ictalurus punctatus)*

Toby L. Lowry and [Stephen A. Smith]{.underline}

Aquatic Medicine Laboratory, Department of Biomedical Sciences and
Pathobiology , Virginia-Maryland Regional College of Veterinary
Medicine, Virginia Polytechnic Institute and State University,
Blacksburg, VA 24061-0442

A novel model for studying *Mycobacteruim marinum* was compared with an
existing fish model to help expand the knowledge base of the fish immune
function to this bacterial pathogen. Juvenile channel catfish
(*Ictalurus punctatus)* and hybrid striped bass *(Morone saxatilis x M.
chrysops)* were injected I.M. with *M. marinum* at 3.84 X 10^3^ colony
forming units per gram of fish weight. Splenic bacterial counts,
macrophage assays (i.e. respiratory burst and phagocytosis),
histopathology, clinical signs and mortality were observed over an 84
day period. Significant differences were seen between the two species
with hybrid striped bass exhibiting progressive classical signs of
pathology and disease. In contrast, channel catfish developed minimal
pathology at the injection site and no internal organ lesions, based
gross observations and on histopathology. Splenic bacterial counts in
the channel catfish remained low through Day 21 and were completely
absent of *M. marinum* growth after Day 35. There are no reports of
mycobacteriosis in channel catfish, and no other fish species has been
reported to spontaneously clear a mycobacterium infection. Due to the
unique nature of the complete clearance of bacteria from splenic counts
in channel catfish, in conjunction with the occurrence of minimal
histopathology, the channel catfish offers a new model to study *M.
marinum*.

**DNA Fingerprinting of *Edwardsiella ictaluri* Using Pulsed Field Gel
Electrophoresis And Repetitive Sequence-PCR**

^1^[Michele L. Williams]{.underline}, ^1^Mark L. Lawrence, and
^2^Michael J. Mauel

^1^College of Veterinary Medicine, Mississippi State University,
Mississippi State, MS 39762; ^2^Thad Cochran National Warmwater
Aquaculture Center, Stoneville, MS 38776

As a result of the current *Edwardsiella ictaluri* genome sequencing
project, we have discovered the presence of numerous mobile elements.
The sheer number of these mobile elements has led us to investigate how
these strains have changed molecularly, if at all. An epidemiological
analysis of archived *E. ictaluri* isolates was performed. A total of 88
isolates of *E. ictaluri* were analyzed by repetitive sequence PCR using
BOX and GTG-5 primers, and 20 of these strains were subsequently
analyzed by restriction enzyme digest with *Pme*I and *Spe*I followed by
separation by pulsed field gel electrophoresis. These strains were
isolated between 1978 through 2006 from Mississippi, Louisiana, Alabama,
Arkansas, Georgia, and South Carolina.

**Pseudokidney Disease In Captive Salmonine Broodstocks**

[Thomas P. Loch]{.underline} and Mohamed Faisal

Department of Pathobiology and Diagnostic Investigation, College of
Veterinary Medicine, Michigan State University, S-112 Plant Biology
Building, East Lansing, MI, 48824

*Carnobacterium maltaromaticum*, the etiological agent of pseudokidney
disease, has recently been isolated from captive lake trout (*Salvelinus
namaycush*), rainbow trout (*Oncorhynchus mykiss*), and brown trout
(*Salmo trutta*) brood-stock experiencing elevated mortality rates in
Michigan. All isolates were Gram-positive, nonmotile, facultatively
anaerobic, asporogenous rods arranged in palisades. They did not produce
catalase, cytochrome oxidase, or H~2~S, and did not reduce nitrate to
nitrite. Isolates hydrolyzed esculin, produced arginine dihydrolase,
produced acid from inulin and mannitol, and grew on both Tryptic Soy
Agar and Cresol Red Thallium Acetate Sucrose Inulin Agar, a selective
and differential medium for *Carnobacterium* spp. External clinical
signs observed in infected individuals include ulceration, melanosis,
varying degrees of exophthalmia, and ocular hemorrhage. Internal signs
included pseudo-membrane formation, hepatic and renal hyperemia,
splenomegaly, nodule formation within the kidney, and the presence of a
transparent mucoid exudate within the lumen of the swim-bladder. Sites
from which this bacterium was recovered include the liver, spleen,
kidney, swim-bladder exudate, and external ulcerations. Molecular
characterization and phylogenetic analyses of the isolates using 16S and
23S rRNA regions specific to *C. maltaromaticum* will be presented. The
impact of this infection on broodstock will be discussed.

**Molecular Cloning, Expression And Genome Organization Of Channel
Catfish (*Ictalurus punctatus*) Matrix Metalloproteinase-9**

[Hung-Yueh Yeh]{.underline} and Phillip H. Klesius

U.S. Department of Agriculture, Agricultural Research Service, Aquatic
Animal Health Research Unit, 990 Wire Road, Auburn, AL 36832

In the course of studying pathogenesis of enteric septicemia of catfish,
we noted that channel catfish matrix metalloproteinase-9 (MMP-9) gene
was up-regulated after *Edwardsiella ictaluri* infection. In this study,
we cloned, sequenced and characterized MMP-9 gene. The sequence of the
CC MMP-9 cDNA gene consisted of 2562 nucleotides that potentially encode
a 686 amino acid peptide with a calculated molecular mass (without
glycosylation) of approximate 77.4 kDa. In addition, analyzed by the
SiganlIP 3.0 program, the CC MMP-9 included a signal peptide with a
cleavage site at positions 20 and 21 (AWS-HP). CC MMP-9 analyzed by the
NetOGlyc 3.1 program had potentially heavy *O*-glycosylation sites
between positions 447 and 490. CC MMP-9 consisted of many structural
domains, including a signal sequence, matrix metalloprotease domain,
matrixin, fibronectin type II domain 1, fibronectin type II domain 2,
fibronectin type II domain 3 and hemopexin-like repeats. In addition,
the pair-wise comparison of amino acid sequences showed that the MMP-9
was highly conserved among fish species (70% - 74%), but not in
mammalian MMP-9 (e.g. 55% with human MMP-9). The complete sequence of
the MMP-9 genomic DNA consisted of 5663 nucleotides. Analysis of the
sequence revealed that channel catfish MMP-9 contains 13 exons (#1 -
#13). The numbers of base pairs of each exon are as follows (exon 1 --
exon 13): 160, 236, 151, 130, 177, 174, 177, 159, 236, 142, 155, 107 and
108, respectively. These results will provide information for further
exploration of the role of MMP-9 in early stage of infection.

**Viral Hemorrhagic Septicemia Virus (VHSV) Type IV′b′ Experimental
Infection In Ontario Rainbow Trout**

^1^[Lowia Al-Hussinee]{.underline}, ^2^Eva Nagy, ^3^Roselynn M.W.
Stevenson, ^1^Spencer Russell, ^1^Karrie M. Young, ^1^Paul Huber,
^1^Véronique M. LePage and ^1^John S. Lumsden

^1^Fish Pathology Laboratory, ^2^Department of Pathobiology, University
of Guelph, Guelph, ON, Canada, N1G 2W1; ^3^Fish Health Laboratory, Dept.
of Molecular and Cellular Biology, University of Guelph

The purpose of this study was to determine the effect of VHSV IV 'b'
(freshwater drum isolate) on an outbred strain of Ontario rainbow trout
used by commercial aquaculture operations to stock freshwater netpens in
Georgian Bay. Fish were held for five months before the experiment and
were determined to be healthy (VHSV-free) by virus isolation, tissue
RT-PCR, bacteriology and histopathology. Treatment groups used
(triplicate tanks of 40, \~10-12g fish) were intraperitoneal (i.p.)
injection and three graded waterborne dosages that received TCID~50~
titres of 10^7.5^/0.1ml, 10~8.5~/ml, 10^6.5^/ml, and 10^4.5^/ml,
respectively; with three tanks of control fish (media). Fish exposed in
the water were held in 2L of aerated water for two hours before being
returned to the tanks. Fish were housed in 60L tanks with flow through
water at 12^o^C and three fish per tank were sampled at weekly
intervals. We evaluated morbidity (behavior indices), mortality,
histopathology and immunohistochemistry for VHSV, RT-PCR on tissues, and
virus isolation on EPC cells. As early as seven days post infection, the
virus was isolated from trout in the i.p. and the highest waterborne
dosage treatment groups. The infection trial is presently still in
progress.

**Viral Hemorrhagic Septicemia In New York State**

^1^[Geoffrey H. Groocock]{.underline}, ^1^Rodman G. Getchell, ^1^Stephen
A. Frattini, ^1^Sarah H. McConnachie, ^1^Katherine H. Kohler, ^1^Gregory
A. Wooster, ^1^Kelly L. Britt, ^1^Young-Sook Kim, ^2^James R. Winton,
^1^Rufina N. Casey, ^1^James W. Casey JW, and ^1^Paul R. Bowser

^1^Aquatic Animal Health Program, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
NY 14853; ^2^United States Geological Survey, Western Fisheries Research
Center,6505 NE 65^th^ Street, Seattle, WA 98115-5016

In May 2006 mortality events of Round Gobies (*Neogobius melanostomus*)
occurred in the St. Lawrence River and Lake Ontario. Investigation
revealed the isolation of Viral Hemorrhagic Septicemia virus of the IVb
genotype which has previously been described in Muskellunge in Lake St.
Clair. A survey of multiple bodies of water across New York State was
performed using inoculations on cell culture and a newly developed
quantitative PCR technique. The results indicate the presence of Viral
Hemorrhagic Septicemia in a wide range of species in all the Great Lakes
waters of New York State and one inland location, Lake Conesus.

**Emergence And Spread Of VHSV In Michigan**

[Mohamed Faisal]{.underline}

Department of Pathobiology and Diagnostic Investigation, College of
Veterinary Medicine, Michigan State University, S-112 Plant Biology
Building, East Lansing, MI, 48824

Viral hemorrhagic septicemia virus (VHSV) was isolated from muskellunge
(*Esox masquinongy)* caught from the northwest portion of Lake St.
Clair, Michigan from 2002-2005. Affected fish exhibited congestion of
internal organs associated with hemorrhages in the inner wall of the
swim bladder. From mortality episodes affecting Lake St. Clair gizzard
shad and muskellunge in the spring of 2006, VHSV was retrieved in high
titers from these two fish species. Histopathology and electron
microscopy provided evidence that VHSV has caused such mortalities. VHSV
was also isolated from other fish species (northern pike, freshwater
drum, rock bass, shortnose redhorse and silver redhorse suckers) not
experiencing mortalities, yet exhibiting clinical signs. The identity of
all isolates was confirmed to be the North American of VHSV by RT-PCR
and gene sequencing. VHSV seem to have spread to Lake Huron and caused
mortalities in walleyes and lake whitefish. Molecular studies performed
on isolates determined they are members of the North American genotype
of VHSV; however, they are sufficiently distinct to be considered a
separate sub-lineage. The impact of VHSV on Lake St. Clair and the Great
Lakes fish stocks remains to be urgently determined.

**Federal Regulatory Issues Regarding Viral Hemorrhagic Septicemia
(VHS)**

[Peter Merrill]{.underline}

U.S. Department of Agriculture (USDA), Animal and Plant Health
Inspection Service (APHIS), National Center for Import Export, 4700
River Road, Riverdale, MD 20737

A Federal Order (originally effective October 24, 2006 and amended Nov
14, 2006) issued by USDA APHIS stipulated the conditions under which
specified VHS-susceptible live fish may be imported into the United
States from two Canadian provinces (Ontario and Quebec) or be moved
interstate from the eight states bordering the Great Lakes. The Federal
Order was not intended to remain in effect on a long-term basis,
especially as the epizootiology of VHS in Canada and the United States
is still only partially understood. New regulations in an interim rule
would establish requirements for importation and interstate movement of
live VHS-susceptible fish from affected areas. This presentation is
intended to provide an update of the current status of Federal
regulatory issues regarding VHS.

**Molecular Correlates Of Infectious Salmon Anemia Virus Virulence**

^1^[Frederick S. Kibenge]{.underline}, ^1^Molly J. Kibenge, ^2^Yingwei
Wang, ^1,4^Biao Qian, ^1,5^Shebel Hariharan and ^3^Sandi McGeachy

^1^Department of Pathology and Microbiology, Atlantic Veterinary College
and ^2^Department of Computer Science and Information Technology,
University of Prince Edward Island, 550 University Ave., Charlottetown,
P.E.I., C1A 4P3., Canada; ^3^New Brunswick Department of Agriculture,
Fisheries and Aquaculture, Fredericton, N.B., E3B 5H1., Canada; ^4^17
Rosedale Drive, Charlottetown, P.E.I., C1A 6S5., Canada; ^5^Rolling
Hills Pet Hospital, 2457 Fenton St. Chula Vista, California, 91914.

Infectious salmon anemia virus (ISAV) is classified in the family
*Orthomyxoviridae*, genus *Isavirus*. Although virulence variation of
ISAV can be demonstrated experimentally in fish, virus strain
identification is ambiguous because the correlates of pathogenicity
and/or antigenicity of ISAV are not well defined. Thirteen ISAV strains
isolated from different locations and characterized for ability to kill
fish were used to search for markers of virulence. Genetic analyses
revealed two genotypes of ISAV, North American and European. The
European genotype was further separated into two genogroups,
real-European and European-in-North America. Deletion/insertion of ≤ 13
amino acids and the presence of two specific motifs in the HE protein,
in combination with a specific motif very close to the trypsin-cleavage
site of the F protein were correlated with reduced cytopathogenicity and
reduced virulence for Atlantic salmon. A novel phylogenetic software
program, BACKTRACK, we wrote estimated that the North American and
European genotypes diverged between 1879 and 1891, whereas the
European-in-North America genogroup diverged from the real-European
genogroup between 1976 and 1988.

**Diagnostic Methodologies For Detection of Beta-Nodavirus Infections Of
Atlantic Cod (*Gadus morhua*)**

^1^Tokinori Iwamoto, ^1^[David Groman]{.underline}, ^1^Carmencita Yason,
^2^Nellié Gagne, and ^3^Stewart Johnson

^1^ Aquatic Diagnostic Services, 550 University Ave., Atlantic
Veterinary College, UPEI, Charlottetown, P.E.I. C1A4P3 Canada; ^2^
Fisheries and Oceans Canada, Molecular Biology, Aquaculture and
Environmental Sciences, 343 Université, Moncton N.B. E1C 9B6 Canada; ^3^
Aquatic Animal Health, Institute for Marine Biosciences, National
Research Council, 1411 Oxford St., Halifax, N.S., B3H 3Z1 Canada

Current methodologies for detection of Betanodavirus in marine fin-fish
included virus culture in SSN-1 or E-11 cells and confirmation by RT-PCR
using a single primer set designed to amplify the T4 variable region
(427 bases) of *Striped Jack Nervous Necrosis Virus* (SJNNV) coat
protein gene. *Atlantic Cod Nervous Necrosis Virus* (ACNNV) has
routinely been isolated and identified using similar techniques (SSN-1
cells and primers specific to the same region in ACNNV). We undertook a
study to compare the efficiency of virus isolation between SSN-1 and
E-11 cell lines. In addition, we compared the use of Real-time PCR with
RT-PCR technologies using a new primer set constructed from a conserved
region of the coat protein gene of all 4 existing Betanodavirus clads.
Our results indicated that the use of E-11 cells provided a minimum of
24 hr. time advantage over the SSN-1 cells for virus isolation.
Similarly, we confirmed that the Real-time PCR increased sensitivity of
detection by 1 log in titration assays with these new primers. We
propose an improved protocol for isolation and detection of ACNNV which
utilizes E-11 cells and Real-time PCR which targets a conserved coat
protein primer set. Supporting data is presented.

**Tadpole Edema Virus: Comparative Susceptibility Among Early Life
Stages of the Fowler's Toad (*Bufo fowleri*)**

[Christine L. Densmore]{.underline} and Philip E. McAllister

National Fish Health Research Laboratory, U.S. Geological Survey, 11649
Leetown Road, Kearneysville, WV 25430

Tadpole edema virus (TEV) is one of a group of ranaviruses pathogenic to
amphibians. Ranaviruses have been isolated from both non-diseased, and
diseased or deceased amphibians. Factors that influence virulence, host
susceptibility and pathogenesis are not well described. Here, we
investigate the comparative virulence of TEV among aquatic life stages
of the Fowler's toad, a common anuran species, from hatch to
metamorphosis. A single mass of fertilized Fowler's toad eggs was field
collected, and the hatched tadpoles reared under laboratory conditions.
Replicate groups of untreated control eggs/tadpoles or groups challenged
with TEV (aqueous exposures of 10^5^ TCID~50~/ml for 24h) were
maintained for 14 day trials for the following approximated life stages:
Gosner stage 20 (hatchlings), Gosner 25 (mouth parts developed), Gosner
36 (rear limbs developing), and Gosner 42 (metamorphosis). Mortality was
recorded daily, and dead tadpoles and representative survivors were
evaluated for presence and titer of virus using a quantal viral
infectivity assay to determine 50% endpoint in cell culture. Mortality
levels were negligible for all control groups, and no virus was detected
among any control groups or the pre-screened eggs and embryos. Among the
TEV-challenged groups, there was variability in mortality and associated
detection of virus among the 4 life stages. High mortality associated
with TEV was observed among the earliest and latest life stages (Gosner
20 and Gosner 42) while lower mortality levels were observed from the
intermediate aquatic life stages examined. Viral titers among exposed
survivors and tadpoles dying with TEV ranged from 10^3^ to 10^6^
TCID~50~/g. Susceptibility appeared greatest during the periods of
heightened physiological stress near hatch and metamorphosis.

**Pathogens Associated With Native And Exotic Trout Populations**

**In Shenandoah National Park And The Relationships To Fish Stocking
Practices**

^1^[Frank M. Panek]{.underline}, ^2^James Atkinson, and ^3^John Coll

^1^ U.S. Geological Survey, National Fish Health Research Laboratory,
11649 Leetown Road, Kearneysville, West Virginia 25430; ^2^ U.S.
National Park Service, Shenandoah National Park, 3655 U.S. Highway 211
East, Luray, VA 22835; ^3^ U.S. Fish and Wildlife Service, Lamar Fish
Health Center, P.O. Box 155, Lamar, PA 16848

Threats to native fish populations from practices such as fish stocking
and from invasive and non-native species and their associated disease
concerns are important natural resource management issues in National
Parks. Prohibitive or restrictive fish stocking policies in National
Parks were developed as early as 1936 in order to preserve native fish
assemblages and genetic diversity. Despite recent efforts to understand
the effects of non-native or exotic fish introductions, park managers
have limited information regarding the effects of these introductions on
native fish communities. Shenandoah National Park (SHEN) was established
in 1936 and brook trout restoration within selected streams in the park
began in 1937 in collaboration with the Virginia Department of Game and
Inland Fisheries (VDGIF). The only known records associated with park
streams stocked during the 1930s were in the survey reports completed by
the US Fish and Wildlife Service in Leetown, West Virginia. These
reports suggest that most accessible streams were annually stocked by
the Commonwealth of Virginia with hatchery reared fingerlings and
catchable size trout through 1949. This practice was continued by the US
Fish and Wildlife Service through 1955. We know that all fish stocked
within Shenandoah National Park originated from a limited number of
hatcheries including the former Fish Culture Station at what is now the
Leetown Science Center, the Erwin National Fish Hatchery in Erwin,
Tennessee and the Montebello Fish Cultural Station in Nelson, Virginia
operated by the VDGIF. A preliminary sampling of brook, brown and
rainbow trout from 28 streams within SHEN from 1998-2002 revealed the
presence of several pathogens. These pathogens included *Renibacterium
salmoninarum*, *Yersinia ruckeri*, and infectious pancreatic necrosis
virus (IPNv)*.* In order to investigate the relationships of the
occurrence of fish pathogens with stocking histories we classified the
streams in three categories: 1) streams within the park with no known
stocking records, 2) streams within the park that were historically
stocked, and 3) streams within the park that were historically stocked
and presently stocked downstream of the park boundary by the VDGIF. The
occurrences of the three pathogens above were summarized relative to
this stocking history. In general, we found an increase in the
occurrence of pathogens in brook, brown and rainbow trout associated
with stocked streams.

**Perspectives For Trout Management And Fish Health In The Batten Kill**

^1^Thomas A. Wiggins, ^2^Kenneth M. Cox, and ^1^[Thomas E.
Jones]{.underline}

^1^Vermont Fish & Wildlife Department, 103 South Main Street, 10 South,
Waterbury, Vermont 05671-0501; ^2^Vermont Fish & Wildlife Department,
100 Mineral Street, Suite 302, Springfield, Vermont 05156

The Batten Kill stands out as one of Vermont's most famous trout
streams. Between 1994 and 1996, wild brown trout abundance (≥ 6 inches)
declined precipitously despite continued evidence of abundant
reproduction. During this same period, wild brook trout populations
remained relatively stable. In 2002, both brook and brown trout were
found positive for the causative agent of Whirling Disease (*Myxobolus
cerebralis*). This was the first documented occurrence of the pathogen
in Vermont waters. In 2000, the Vermont Fish & Wildlife Department
(VTFWD) established an inter-agency Batten Kill Study Team charged with
investigating a list of likely causes for the brown trout decline. Of
all the possible causes for the brown trout population decline that were
investigated over the next six years, the most compelling finding was
that fish cover in the river is inadequate. Nonetheless, the existence
of WD in the Batten Kill has potential implications for its resident
trout populations but more broadly is a risk to other trout waters in
the region. Therefore, the VTFWD is committed to monitoring for fish
pathogens, including WD in the Batten Kill as well as other state
waters. Additionally, the VTFWD continues efforts to increase public
awareness of the issue, and inform user groups of their role in curbing
the potential spread of pathogens to other waters.

**CSI Shenandoah: The Importance Of Understanding Fish Health In A
Riverine Fish Kill Investigation**

[Stephen J. Reeser]{.underline}

Virginia Department of Game and Inland Fisheries, P.O. Box 996 Verona,
Virginia 24482

Between 2004 and 2006 unexplained fish kills occurred throughout the
Shenandoah River in Virginia. These mortality events involved only the
adults of mainly three species *Micropterus dolomieu*, *Lepomis auritus*
and *Hypentelium nigricans*. Small numbers of other species were also
affected. In some cases an estimated 80% of the adult *Micropterus
dolomieu* and *Lepomis auritus* perished in long (\>50 km) reaches of
the river. The kills involving *Micropterus dolomieu* and *Lepomis
auritus* occurred in the early spring and *Hypentelium nigricans*
mortality took place in late May and again in early December. Some acute
mortality was associated with the beginnings of these kill events, and
these fish showed no outward signs of stress. However, the bulk of the
mortality was chronic with fish exhibiting bacterial lesions, eroded fin
rays, excessive surface mucus production, and fungal infections. Fish
kill episodes lasted from several days to three months. Diagnosis by
fish pathologists indicated that the observed symptoms were "secondary
effects" and not the origin of stress. To date the cause of these fish
kills is unknown. Suspect causes being investigated include:
environmental stressors, pathogens, contaminants, and immune system
suppression. Much of the data collected thus far points to multiple
stressors leading to mortality. Researchers with the Shenandoah River
Fish Kill Task Force and the Environmental Protection Agency's Casual
Analysis/Diagnosis Decision Information (CADDIS) team have placed a high
priority on fish health information in determining the cause(s) of fish
mortality. Histopathology, virology, bacteriology, and parasitology of
fish have been conducted and analysis is pending. Investigators hope to
use fish health diagnostics to solve the mystery of fish stress and
mortality in the Shenandoah River.

**Bio-Indicators Of Estuarine Health: A Multivariate Approach Linking
Source To Resource**

[John M. Jacobs]{.underline}, R. Heath Kelsey, A.K. Leight, Earl. J.
Lewis, Shawn M. McLaughlin, Gretchen Messick, and Bob Wood

NOAA/NOS/CCEHBR/Cooperative Oxford Laboratory, 904 S. Morris Street,
Oxford, Maryland, 21654

There is a recognized need among management entities for descriptive
indices that simply and concisely convey ecosystem health. However,
individual indicators are often inadequate for estuarine systems because
of the inherent variability in these environments and the inability to
infer causality from observed responses. These issues may become more
apparent as assessments are performed over broader spatial scales. The
NOAA/NOS Oxford Lab, in cooperation with federal, state, and academic
partners, is implementing an integrated biotic ecosystem assessment on a
sub-watershed (14-digit Hydrologic Unit Code) scale in Chesapeake Bay.
Sub-watersheds were chosen based on statistical analysis of land use
patterns to represent a gradient from developed to agricultural. A
random stratified design was developed and sampling approaches
coordinated within this structure to allow for robust system
comparisons. The approach is hierarchal, with metrics chosen to
represent a range from community to cellular level responses across
multiple organisms. Particular attention is focused on the use of
pathobiology as a tool for assessing environmental condition. By
integrating the biotic component with water quality, sediment indices,
and land-use information, a holistic evaluation of ecosystem health will
provide management entities with information needed to inform local
decision-making processes and establish benchmarks for future
restoration efforts.

**Evaluating Population Health Of Endangered Fishes By Non-lethal
Analysis Of Microbial Diversity**

[Rocco C. Cipriano]{.underline}, Christopher Ottinger, and Cynthia Baker

US Geological Survey/Leetown Science Center, National Fish Health
Research Laboratory, 11649 Leetown Road, Kearneysville, WV 25430

When a bacterium initiates infection and disease, and is eventually
cleared from an affected population, it influences the normal bacterial
flora on external surfaces of individual fish. Understanding how
microbial diversity is affected by the presence, replication and
clearance of a pathogen can provide an index of population health. Such
analysis is non-lethal, which is necessary when working with an
endangered species. To further understand pathogen and normal flora
interactions, Lost River (*Deltistes luxatus*) and Shortnose
(*Chasmistes brevirostris*) suckers were studied in the Upper Klamath
Lake (OR). Widespread habitat destruction and water quality degradation
have caused drastic declines in both species, which were listed as
endangered in 1988 (53 FR 27130 27134). Occurrence of diseases within
the lake, especially Columnaris Disease, threatens population
recoveries. Analyses of the microbial flora affecting these fishes
become important metrics to monitor population health and environmental
quality, which are vital to species\' recovery plans. In this study,
gills and skin of Lost River and Shortnose suckers were evaluated from
spawning adults (4/06), juveniles (9/06), and in adults prior to winter
(10/06). The microbial flora from blue chubs (*Gila* coerulea) was also
compared to adult suckers (8/06) to determine if the former species
could serve as a surrogate model. Results were based upon 49,421 total
bacterial identifications. Neither sucker species was significantly
impacted by bacterial infections in 2006; - no bacterial epizootics were
reported to this laboratory nor were any obligate pathogens isolated.
*Flavobacterium columnare,* cause of Columnaris Disease, was not
isolated from any sucker and was isolated only from the gills of one
chub. Although there were instances where the incidences of facultative
pathogens (mostly motile aeromonads) increased among adult suckers, they
rarely supplanted the normal hierarchical order of the normal flora and
were not deemed to severely impact population health. There were strong
hierarchical shifts among juvenile fish (9/06) and adults obtained
during August in which motile aeromonads actually displaced the normal
flora of the suckers. When combined with certain environmental
stressors, such shifts could potentially impact survival. Finally, the
microbial flora associated with blue chubs differed from those of the
suckers; therefore, these fish were not appropriate surrogates for the
suckers.