**Evaluation Of Single Contact Applications Of Iodophor Against The Fish
Pathogen *Aeromonas salmonicida*, Cause Of Furunculosis**

[Rocco C. Cipriano]{.underline}

United States Geological Survey, National Fish Health Research
Laboratory, 11649 Leetown Road, Kearneysville, WV 25430

Optimalization of survival among fertilized gametes is critical for the
success of fish restoration programs that are limited by reduced
population numbers. Iodophors are routinely used to disinfect fertilized
fish eggs in order to ensure that surface pathogens do not kill the egg
and to make sure that egg transfers between facilities do not transmit
diseases. United States Fish and Wildlife Service (USFWS) Fish Health
policy requires that eggs be disinfected in 50 mg/L of iodophor for 30
minutes at the facility where those eggs are fertilized. If the same
eggs are shipped to a second facility, policy further states that those
eggs must undergo a second disinfection in 100 mg/L of iodophor for 10
minutes at the receiving station. It was previously observed that when
either treatment was applied individually, neither treatment could
completely kill concentrations of A-layer positive *Aeromonas
salmonicida* that exceeded 1.0x10^6^ colony forming units (cfu) of the
pathogen. It was further noted that this prevalence occurred on Atlantic
salmon (*Salmo salar*) eggs *in vivo*. At such prevalence, it was
necessary to conduct both treatments in tandem in order to affect
complete decontamination. Experiments conducted by the USFWS Northeast
Fisheries Technology Center (Lamar, PA) also indicated that survival
among Atlantic salmon eggs treated with concentrations of iodophor from
50 to 150 mg/L for up to 90 minutes was more adversely impacted by time
of exposure. Relatively little mortality was attributed to the
concentration of the iodophor, itself. This study was conducted to
examine short-term, high concentration effects of iodophor against *A.
salmonicida*. Times of exposure were conducted for 5, 10, 15 and 30
minutes with iodophor concentrations that ranged from 50 to 500 mg/L,
respectively. This effort was designed to identify a single treatment
regimen that was completely efficacious against high concentrations of
*A. salmonicida* and could be further tested for safety among Atlantic
salmon eggs *in vivo*.

**A Polymerase Chain Reaction Assay To Monitor Channel Catfish
(*Ictalurus punctatus)* Eggs For *Flavobacterium columnare***

## [Joel A. Bader]{.underline}, Richard Whittington, Craig A. Shoemaker, and Philip H. Klesius

# Aquatic Animal Health Research Laboratory, United States Department of Agriculture-Agricultural Research Service, P.O. Box 952, Auburn, AL, 36830

A newly developed species-specific polymerase chain reaction (PCR) assay
was used to determine if *Flavobacterium columnare*, the causative agent
of columnaris disease, was present in channel catfish (*Ictalurus
punctatus*) eggs following spawning. This assay was compared to the
standard culture method and was found to be as effective in detecting
*F. columnare* in channel catfish eggs as the more traditional culture
method. *Flavobacterium columnare* was successfully detected, in eggs
that were artificially infected and in apparently healthy eggs,
collected following spawning. The PCR assay was better than culture for
detecting the bacterium in the naturally exposed samples. Further, PCR
was used to show that povidone-iodine was effective in disinfecting egg
masses of *F. columnare*.

**Isolation And Characterization Of A Bacteriophage Which Infects
*Yersinia ruckeri***

[Timothy J. Welch]{.underline} and Christin K. Campbell

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

Bacteriophages are viruses that infect and replicate in bacteria. While
in theory these bacteria-specific infectious agents have great potential
as potent prophylactic and therapeutic agents, little has been done to
realize this potential. Here we present the isolation and partial
characterization of a lytic bacteriophage (Phage YRNC10) with
pathogenicity to *Y. ruckeri*, the causative agent of Enteric Redmouth
disease (ERM). YRNC10 was isolated using an enrichment technique from
sediments collected at a trout farm where outbreaks of ERM occur
regularly. Nucleic acid analysis revealed that phage YRNC10 has a
double-stranded DNA genome of approximately 60 kb. Spontaneous
phage-resistant variants of *Y. ruckeri* have been identified which are
sensitive to rainbow trout serum and are attenuated for virulence in
rainbow trout. Several of these variants are defective in 0-antigen
production and fail to bind phage, suggesting a role for the
0-polysaccharide in phage binding. Current investigations focused on
assessing the effectiveness of phage YRNC10 for the treatment of *Y.
ruckeri* infections and for the reduction of horizontal transmission of
this pathogen.

**Mycobacteriosis, Is It Truly A Chesapeake Bay Disease Or East Coast
Problem**

[Laurence H. Pieper^1^]{.underline}, and Mark Matsche^2^

^1^Maryland Department of Natural Resources, Fisheries Service, 301
Marine Academy dr., Stevensville, MD.21666; ^2^Maryland Department of
Natural Resources, Fisheries Service, Sarbanes Cooperative Oxford
Laboratory, 904 South Morris St., Oxford, MD 21654

The Maryland Department of Natural Resources, Fisheries Service, Fish
and Wildlife Health Program continues to research and monitor
mycobacteriosis in Maryland's portion of the Chesapeake Bay. Fish were
collected in 2002 from the upper, middle, and lower areas of Maryland's
portion of the bay. Additionally, fish were also collected from the
Choptank River. One hundred and forty fish were collected and examined
histologically for granuloma formation as an indication of
mycobacteriosis. Fifty-one of these fish (36%) were infected. This
percentage has remained in a range of 33-44% for five to six years. The
disease is more prevalent in three to four year old fish. Personnel from
the Virginia Institute of Marine Science (Gloucester Point, VA) and the
National Fish Health Research Laboratory (NFHRL; Kearneysville, WV) have
been doing similar work on the Chesapeake Bay, and there has been a
great deal of good work accomplished; however, much more research and
investigation remains to be done. Investigators at the NFHL started to
adopt standard operating procedures in spring 2002; however, the results
from the microbiology labs have been unclear. Therefore the resources
from various agencies in this research need to be pooled in order to
continue to carry out and improve the work toward a common goal. The
NFHL has taken a lead position in getting all the parties working
together in order to address the real issue, which is management of the
striped bass stock. Stock assessment for the states is regulated by
federal agencies. The National Marine Fisheries Service (NMFS) and
Atlantic States Marine Fisheries Commission (ASMFC) regulate the states
through fishery management plans (FMP). These FMPs incorporate a value
for mortality, which is essential in regulating a stock. The fish health
community needs to form an advisory group for the federal agencies to
develop a figure for disease mortality of striped bass. This figure
could then be incorporated into NMFS and ASMFC's FMP.

**Detection Of Mycobacteria In Paraffin-Embedded, Formalin-Fixed Tissues
Of Striped Bass (*Morone saxatilis*) With Peptide Nucleic Acid (PNA)
Probes**

[David T. Gauthier^1^]{.underline}, Wolfgang K. Vogelbein^1^, and
Kimberly S. Reece^1^

Virginia Institute of Marine Science, College of William and Mary, 1208
Greate Rd., Gloucester Point, VA 23062

An epizootic of mycobacteriosis is currently occurring in striped bass
(*Morone saxatilis*) of the Chesapeake Bay. Visceral and dermal
granulomatous lesions characterize the disease, and infected fish are
often significantly emaciated and/or disfigured. A large number of
different mycobacterial species have been cultured from Chesapeake Bay
striped bass, and it is likely that more than one contributes to
production of disease. Reliable methods for detection and speciation of
mycobacteria in fish tissues are therefore crucial to understanding the
pathobiology of mycobacteriosis in the wild. Acid-fast staining is the
standard method for detection of mycobacteria in histologic sections,
but this method may lack sensitivity and does not allow for
species-specific identification. Culture of mycobacteria does allow for
limited species identification, but is very time consuming and
labor-intensive, and is often logistically unfeasible in a field
setting. As a first step toward developing rapid, convenient
species-specific molecular detection techniques for mycobacteria in
fish, we have designed and are validating genus-specific peptide nucleic
acid (PNA) probes that detect mycobacteria in standard histologic
sections. The PNAs are synthetic oligonucleotides in which standard
bases (A, T, C, G) are attached to an uncharged glycine-derived
backbone. The uncharged nature of the PNA molecule allows for improved
penetration of hydrophobic membranes, such as the cell wall of
mycobacteria. In addition, binding to target DNA is stronger and more
specific than with standard oligonucleotide probes. Genus-specific
probes were designed to target conserved regions of 16S *Mycobacterium*
rRNA. These probes demonstrated strong, specific hybridization with *M.
marinum* in tissue sections from experimentally infected striped bass
and, in some cases, they appeared to have greater sensitivity than
acid-fast staining. Optimization of these probes for other
*Mycobacterium* spp. is underway.

**Evaluating The Effects Of Oxytetracycline On The Microflora**

**Of A Recirculating Aquaculture System**

[S. Serfling]{.underline}, C. Gieseker, R. Miller, E. Squibb, T. Brady,
and R. Reimschuessel

United States Food and Drug Administration, Center for Veterinary
Medicine, Office of Research, Laurel, MD 20708

According to the United Nation's Food and Agriculture Organization,
aquaculture is the fastest growing food producing industry in the world.
Because of this rapid development, and the shift towards more intensive
farming methods, the aquaculture industry has encountered many viral and
bacterial diseases, and the need for treatments to control and prevent
disease. As a result, public health concerns have arisen about chemical
residues and the development of antibiotic resistance in fish tissue. In
the United States there are two antimicrobials approved by the Food and
Drug Administration for aquaculture, oxytetracycline - Terramycin ®, and
sulfamerazine - Romet-30 ®. These antibiotics are added to feed during
the manufacturing process. Any food that is not eaten by fish, and the
drug residues excreted in fish feces, will eventually reach pond
sediments, or be captured in bio-filters used by recirculating
aquaculture systems. Antibiotic residues are adsorbed in these
sediments, which can create selective pressure on the microorganisms
found in this environment. The purpose of this study is to survey
bacteria found in recirculating aquaculture systems and to find a
potential indicator organism. In addition to defining the bacterial
isolation and identification methods, the isolated organisms will be
tested for susceptibility to oxytetracycline following antibiotic
susceptibility methods developed at CVM for aquatic organisms. Data from
this study will help identify potential microorganisms to monitor for
changes in antimicrobial susceptibility patterns following antibiotic
use in recirculating aquaculture systems.

**Mystery Of Epizootic Oral Tumors In Spring Chinook Salmon**

[Jeffrey C. Wolf]{.underline}^1^, Mary Edwards^2^, John Kaufman^3^, Sam
Onjukka^4^, and Marilyn J. Wolfe^1^

^1^The Registry of Tumors in Lower Animals, 22900 Shaw Road, Suite 107,
Sterling, VA 20166; ^2^Nez Perce Tribe Dept. of Fisheries, Enterprise
Field Office, 612 SW 2^nd^ St, Enterprise, OR 97846; ^3^Oregon Dept. of
Fish and Wildlife, 220 Nash Hall, Department of Microbiology, Oregon
State University, Corvallis, OR 97331; ^4^Oregon Dept of Fish and
Wildlife, Eastern Oregon University, Badgley Hall, One University
Boulevard, La Grande, OR 97850

In December 2002, abnormal growths were observed among two-year-old
Chinook salmon (*Oncorhynchus tshawytscha*) in a freshwater and a
saltwater Pacific Northwest hatchery. Fish were collected in 2001 for a
captive breeding program and they initially had red irregular formations
along the jaw, tongue, and/or the roof of the mouth, which eventually
progressed to large tumors that disfigured the lips and oral cavity.
Microscopically, tumors were consistent with those of ameloblastic
(tooth enamel-forming) origin: 1) multiple irregular clefts lined by
palisading epithelial cells; 2) cords of epithelial cells contiguous
with invaginations of the oral mucosa; and 3) areas of stroma that
resembled stellate mesenchyme. By December 2003, tumor prevalence was
94% (n=131) and 100% (n=73) among salmon at the freshwater and saltwater
hatcheries, respectively, but cohorts at these facilities were not
affected. Tumors were also observed in the mouths of several Chinook
salmon that had returned to natal streams during the summer of 2003.
These fish were captured at weirs from three rivers in two watersheds,
and at least one of these fish was of feral origin. Diagnostic
procedures performed at Oregon Department of Fish and Wildlife to
identify potential viral (and other) pathogens included: cultures from
tumors and internal organs; transmission electron microscopy; polymerase
chain reaction (PCR) assay for *Oncorhynchus* masou Virus and
*Herpesvirus salmonis*; modified PCR for reverse transcriptase activity;
injection of homogenates into Spring Chinook fingerlings; and
cohabitation of fingerlings with tumor-laden adults. Investigations have
not supported a viral etiology. A genetic cause was ruled out because
relatedness between affected and unaffected fish was comparable to the
relatedness among affected fish. Exposure to environmental carcinogens
has not been dismissed; however, two site rivers are considered pristine
environments, and a link among collection site contaminants was not
established.

**Coral Mortalities Along The Kenyan Coast Associated With Extensive
Tissue Lysis: Possible Mycotic Etiology**

[Shawn M. McLaughlin]{.underline}^1^, Timothy R. McClanahan^2^

William H. Wilson^3^, Marina Carreiro-Silva^4^, Esther C. Peters^5^,
Kathy L. Price^6^

^1^NOAA NOS Center for Coastal Environmental Health and Biomolecular
Research/Cooperative Oxford Laboratory, 904 South Morris St., Oxford, MD
21654; ^2^The Wildlife Conservation Society, Bronx, NY10460; ^3^Marine
Biological Association, Citadel Hill, Plymouth, PL1 2PB, England;
^4^University of Maryland, Marine and Estuarine Environmental Sciences,
College Park, MD 20742; ^5^Tetra Tech, Inc., 10306 Eaton Place, Suite
340, Fairfax, VA 22030; ^6^Jardon and Howard Technologies, 12424
Research Parkway, Orlando, FL 32826

An apparent new source of coral mortality occurred along 600 km of
coastline from Tanzania to Kenya in early 2002. *Astreopora,*
*Echinopora*, and *Montipora* species were severely affected, with
*Montipora* being nearly eliminated from Kenyan reefs. *Acropora*,
*Platygyra*, *Goniopora,* and massive *Porites* were also affected;
however, *Porites* and *Goniopora* rarely died and often recovered,
whereas death for most other species occurred within two weeks. Scanning
electron micrographs of affected corals revealed the presence of fungal
filaments in affected samples. Examination of histopathology showed
extensive necrosis and lyses of tissues in the three genera most
severely affected by the syndrome. Special staining of tissue sections
also revealed more fungal filaments in these genera than unaffected
genera; however, the role of fungi in the Kenya coral mortalities is
uncertain.

# Catch And Release: The Importance Of TLC

[Susan V. Marcquenski^1^]{.underline} and Hui-Min Hsu^2^

Wisconsin Department of Natural Resources. Box 7921. Madison, Wisconsin
53707^1^; Wisconsin Veterinary Diagnostic Lab. 6101 Mineral Point Road.
Madison, Wisconsin 53705^2^

In May 2003, a moribund muskellunge with external skin lesions was
observed in Lake Wissota, an inland lake in west central Wisconsin. The
fish was netted, euthanized and frozen prior to necropsy. Fish length
was 930 mm. The legal size limit for muskellunge is 850 mm in Lake
Wissota. The fish had several well defined oval to circular lesions on
the caudal peduncle and midbody of the fish. Skin was missing from some
lesions, exposing inflamed and hemorrhagic muscle. Although the fish had
been frozen, cultures were made from the lesions (at the margin of skin
and muscle), kidney, spleen and liver on trypticase soy agar (TSA); the
margin of the lesions, and pieces of kidney, liver, and spleen were
fixed in 10% neutral buffered formalin for histology. Clear red ascites
was present in the body cavity, but this could have been an artifact
from freezing. There were no other significant signs in the viscera and
no evidence of previous hooking injury. Bacterial cultures were
submitted to the Wisconsin Veterinary Diagnostic Lab.
*Streptococcus/Entercoccus* sp. were cultured from the kidney, liver and
spleen. Sections for histopathology were stained with hematoxylin and
eosin (H&E) and Giemsa. Giemsa stained sections showed tremendous
concentrations of long, rod-shaped bacteria in areas of the muscle and
the muscle appeared edematous. Melanomacrophage centers were plentiful
in the kidney, and there were few intact tubules, which may have been an
artifact of freezing or a consequence of bacterial infection. We would
like some feedback on interpreting histopathology when tissues are first
frozen. The locations of the lesions approximate the area where an
angler would have held the fish while being photographed. Muskellunge
fishing is considered trophy fishing in Wisconsin and most fish are
caught and returned back to the water (after photos are taken, of
course!). Anglers should take care when handling all fish to prevent
serious trauma and subsequent bacterial infections.

**First Record Of *Renibacterium salmoninarum* Isolation**

**From The Sea Lamprey *(Petromyzon marinus)***

[Alaa Eissa]{.underline}^1^, Ehab Elsayed^1^, and Mohamed
Faisal^1,\ 2,\ 3^

Colleges of Veterinary Medicine^1^ and Agriculture and Natural
Resources^2^, Michigan State University, and Michigan Department of
Natural Resources^3^, Lansing, Michigan

Bacterial kidney disease, caused by *Renibacterium salmoninarum* is a
serious widespread problem of salmonid fish species. It is currently
unknown how high levels of this bacterium are maintained in the
Laurentian Great Lakes' basin leading to infection prevalence of
unprecedented magnitude. Research involving reservoirs and mechanisms of
*R. salmoninarum* transmission in fish is lacking due to ecological
complexity of heterogeneous habitats. Surprisingly, we isolated *R.
salmoninarum* from the kidneys of the widespread fish parasite, the sea
lamprey. The organism was cultured from 12% of Lake Ontario's lampreys
examined and colonies were verified by *p*57 gene sequencing. It is
highly probable that nonlethal lamprey encounters play a role in the
temporal and spatial spread of *R. salmoninarum* in the Great Lakes
basin.

**A Case Of Severe Helminth Parasitism In Fish In A Small Impoundment**

[P.R.Bowser]{.underline}^1^, S.L. Bartlett^1^, S.G. Grimmett^1^, G.A.
Wooster^1^, R.G. Getchell^1^, T. Baudanza^2^ and R. Pierce^3^

^1^Aquatic Animal Health Program, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
NY 14853; ^2^New York City Dept. of Environmental Protection, P.O. Box
370, Shokan, NY 12481; ^3^New York State Dept. of Environmental
Conservation, 21 South Putt Corners, New Paltz, NY 12561

A case was received that consisted of centrarchids from a 22 acre
impoundment in the lower Hudson River Valley of New York State. The fish
kill was acute in nature and included almost 100% of the fish in the
impoundment. Water quality parameters associated with the fish kill did
not reveal any problems. A wooded area surrounded the impoundment, with
a few private residences set approximately 200 feet from the water's
edge. There were no immediately evident sources from which pollutants or
toxic substances could have entered the water. Fish submitted in October
2003 for examination consisted of bluegill *Lepomis* *macrochirus* (n =
3), redbreast sunfish *Lepomis* *auritus* (n = 1) and largemouth bass
*Micropterus* *salmoides* (n = 2). A subsequent submission of 1
additional bluegill was made three days after the initial submission.
Upon examination, the most significant findings were severe infestations
of the internal organs by encysted digenetic trematodes (*Neascus*),
encysted nematodes (*Eustrongylides*) and larval cestodes
(*Proteocephalus*). Parasites made up approximately 50% of the combined
organ mass of some fish. A most remarkable finding was that when the
mesenteries were removed from the fish and examined with a
stereomicroscope, the warming of the specimen by the illumination light
appeared to activate the encysted nematodes, which perforated their
cysts and began to migrate over the organ mass. Meteorological records
indicated that a warm spell had occurred immediately prior to this
event. Although we could not definitively attribute the mortalities to
the parasites, we hypothesized that the rapid warming of the water in
this shallow impoundment (mean depth = 5 feet) might have caused the
nematodes and cestodes to become active and migrate within the fish,
which caused their deaths.

**An Unusual Case: Walleye Lesions In The Visceral Cavity**

[Andrew J. Mitchell]{.underline}

HKDSNARC/ARS/USDA, P. O. Box 1050, Stuttgart, AR 72160

An unusual disease condition was found in a 1.8 kg walleye from Merritt
Reservoir in northwestern Nebraska. The fish was processed at the
Stuttgart diagnostic laboratory in May of 1990, and when the body cavity
was opened hard cysts or nodules in the swimbladder and visceral fat
were observed. The nodules appeared to be formed around a worm-like
mass. The nodules (10±5 mm in diameter) were composed of five to seven
layers of tightly packed granules, that were at least partially
calcified and the layers were held together by a sticky matrix. The
outer coat of the nodule was convoluted with many protuberances. This is
a first report of such cysts from fish and they appear to be similar to
nodules induced by worms (encysted nematodes) in the intestines of swine
and ruminants. Histopathological results were not conclusive but
supported results found from the original diagnostic workup.

# A Serendipitous Finding While Screening Spotted Musky for *Piscirickettsia* sp.

[Susan V. Marcquenski^1^]{.underline} , Ehab Elsayed^2^, and Mohamed
Faisal^2,\ 3,\ 4^

^1^Wisconsin Department of Natural Resources, Box 7921, Madison, WI
53707; ^2^Colleges of Veterinary Medicine and ^3^Agriculture and Natural
Resources, Michigan State University, and Michigan Department of Natural
Resources, Lansing, MI

In 2002, *Piscirickettsia* sp. was diagnosed in wild spotted muskies
from Lake St. Clair. Prior to this diagnosis, fertile eggs from this
population were transferred to the Wild Rose Hatchery in Wisconsin where
they were hatched and reared for stocking as fall fingerling. A
subsample, (about 200 fish), were held over winter with the intent to
stock the fish in fall 2003. In July 2003, kidney and spleen from ten
spotted muskies were aseptically removed and prepared for tissue
culture. Severe cytopathic effects were observed in FHM and CHSE cell
lines inoculated with samples from three fish, however *Piscirickettsia*
sp. was not detected using the polymerase chain reaction (PCR) method.
Electron microscopy revealed the presence of an icosahedral virus in two
of the three samples and unusual viral particles in the third sample.
Current investigations aim at characterizing the isolated viruses and
determining potential sources of infection.

**Abnormal Growth Associated With An Intracellular Bacterium In The
Yellow Perch (*Perca flavescens*)**

[Mohamed Faisal]{.underline}^1,\ 2,\ 3^, Ehab Elsayed^1^, and Scott
Hanshue^3^

^1^Colleges of Veterinary Medicine; ^2^Agriculture and Natural
Resources; ^3^Michigan State University; Michigan Department of Natural
Resources, Lansing, Michigan

A relatively large, lobulated tissue mass was noticed in the abdomen of
a yellow perch caught from the Big Cedar Lake, Barry County, Michigan.
This unusual mass occupied most of the abdominal cavity, pushing the
visceral organs anteriorly and was loosely attached to the abdominal
wall. In stained tissue sections, the mass consisted primarily of
macrophage-like cells arranged in cords. Darkly stained rounded, 1-2 μm
intracellular bodies were abundant. Transmission electron microscopy
revealed that these intracellular bodies were rickettsia-like bacteria,
which were enclosed within double membranes. The nature of this
bacterium and its relationship to the unusual outgrowth are currently
unknown.

# Introduction To Largemouth Bass Virus

[John M. Grizzle]{.underline}^1^ and Cindy J. Brunner^2^

^1^Southeastern Cooperative Fish Disease Project, Department of
Fisheries and Allied Aquacultures, Auburn University, AL 36849;
^2^Department of Pathobiology, College of Veterinary Medicine, Auburn
University, AL 36849

Largemouth Bass Virus (LMBV) is an iridovirus that causes disease
principally in largemouth bass. The disease caused by LMBV occurs during
the summer and typically affects adult fish, which lose equilibrium and
are found floating at the water surface. Largemouth Bass Virus was first
isolated during 1991 in Florida and has now been found in several bodies
of water in the eastern United States. Fish kills have occurred in some
infected populations of largemouth bass, but in other locations this
virus is found in largemouth bass and other fish species without overt
disease. This virus appears to be the most likely cause of changes in
largemouth bass populations in some reservoirs. Few problems with LMBV
have been documented in hatchery-reared fish, but wild fish moved to
hatcheries sometime experience rapid increases in prevalence of
infection and viral concentration. This virus can replicate in a wide
range of cultured cells and is easily isolated from infected fish.
However, diagnosis of LMBV disease can be difficult because
pathognomonic lesions are not present, concurrent bacterial infections
are common, and isolation of LMBV is not sufficient to indicate that
LMBV disease is the cause of morbidity.

Development Of Non-Lethal Testing Methods For Largemouth Bass Virus

[Robert S. Bakal]{.underline}, Thomas Vaughn, and H. Edward Hohlt

## 

## U.S. Fish and Wildlife Service, Warm Springs Regional Fisheries Center, Warm Springs Fish Health Center, 5308 Spring Street, Warm Springs, GA 31830

Although Large Mouth Bass Virus (LMBV) was first isolated from moribund
largemouth bass (*Micropterus salmoides*) from Santee-Cooper Reservoir
in South Carolina in 1995, archived samples showed that LMBV was
isolated from bass in four Florida lakes as early as 1989. Since these
reports, LMBV has caused many fish kills throughout the southeast and
midwest. It has been suggested the virus has a predilection for larger
"trophy" sized animals and that it can affect population size structure
because trophy fish are impacted. Because of these impacts, it is
important to establish strategies that prevent the spread of LMBV.
Managers must have information on the prevalence and distribution of the
virus, as well as it's overall impact on the population dynamics. The
majority of health assessment tools require sacrifice of animals.
Gathering such data will require collection of hundreds of samples of
various sized animals from hundreds of locations that could have
long-term effects on the population. Lethal sampling also makes it
impossible to follow individuals or potentially, the population over
time. While most individuals recognize it is necessary to sacrifice some
animals for the greater good of a species, destructive sampling creates
public relations problems if either large numbers of animals are
sacrificed or if larger animals are killed. Such techniques also serve
as a focal point for animal rights groups, which are becoming
increasingly aware of fishery issues. By contrast, non-lethal techniques
would allow for collection of statistically relevant samples and
minimize the number of fish that would be lost. Finally, these
techniques may potentially save time, cost less, and require less
expertise, making them more cost effective than traditional techniques.
This study evaluated the suitability of non-lethal sampling for the
detection of LMBV. Non-lethal samples collected included blood, skin
scrape, fin clip, and gill biopsy, which were compared to the standard
kidney/spleen samples. All samples were assayed by viral isolation. In
addition, total white blood cell counts were completed and the presence
of anti-LMBV antibodies in serum was determined by agar gel
immuno-diffusion.

**Host, Pathogen And Environmental Factors That Affect The
Susceptibility Of Largemouth Bass To Largemouth Bass Virus**

[Tony L. Goldberg]{.underline}

University of Illinois Department of Veterinary Pathobiology 2001 South
Lincoln Avenue Urbana, IL 61802

Populations of largemouth bass vary widely in their responses to
infection with Largemouth Bass Virus (LMBV). Some populations experience
epidemic mortality events (fish kills) while others remain apparently
healthy. The reasons for this variation are poorly understood, but may
hinge on factors operating (and interacting) at the level of the host,
pathogen and environment. Preliminary epidemiological analyses indicated
that fish kills tended to occur in warm seasons and within intensively
managed water bodies. Mortality patterns were similar for water bodies
in the same watershed. In a series of experiments, we explored these
risk factors in greater detail using juvenile largemouth bass that were
held in tanks and exposed experimentally to LMBV via intraperitoneal
injection. Analysis of different isolates of LMBV indicated that
multiple viral strains exist, differing both genetically and in their
virulence. Outbreeding depression had a strong effect and was evident by
the fact that hybrid bass suffered higher mortality from LMBV than
non-outbred parental stocks. Ambient temperature was a significant risk
factor: fish held at 30^o^C died more quickly than fish held at 25^o^C,
and had higher viral loads in tissues. Water quality affected host
survival in complex ways. Dissolved nitrate levels were paradoxically
protective (higher nitrate levels in tanks were associated with reduced
mortality and viral loads). Social stress from crowding was associated
with increased mortality and viral loads. Stress from a single,
maximally stressful experimental angling event, however, did not affect
mortality rates or viral loads of infected bass. These results indicated
that host responses to LMBV infection were modulated in complex ways by
multiple, factors operating at the level of the host, pathogen, and
environment. Understanding the effects of these factors will be critical
in order to predict the impact of LMBV on wild populations.

**Application And Implication Of Serology For Largemouth Bass Virus
Infection Of Largemouth Bass (*Micropterus salmoides*)**

[Jack M. Gaskin]{.underline}^1^, Wes Porak^2^, Bill Johnson^2^

^1^Department of Pathobiology, College of Veterinary Medicine,
University of Florida, Gainesville, FL 32610; ^2^Florida Fish and
Wildlife Conservation Commission (FFWCC), Eustis Fisheries Research
Laboratory, 601 West Woodward Avenue, Eustis, FL 32726

We investigated the pathogenicity of Largemouth Bass Virus (LMBV) in
captive juvenile largemouth bass and the dynamics of the humoral immune
response as measured by the agar gel immunnodiffusion (AGID) method. In
experimental studies, 96 juvenile bass, held at either 23^o^ or 29^o^C,
were inoculated intramuscularly with 2x10^6^ infectious units of LMBV.
Uninoculated "sentinel" bass were co-habited with them. Forty-eight
control largemouth bass, inoculated with virus-free cell culture fluid,
were also held in two separate tanks at the two temperatures. At 29^o^C,
bass inoculated with LMBV developed severe necrotic lesions at the site
of inoculation, whereas those held at 23^o^C developed comparatively
mild lesions. Although none of the sentinel fish showed visual evidence
of disease, lateral transmission of infection, as evidenced by AGID
seroconversion, was much more efficient at 29^o^C (8/13; 62%) than at
23^o^C (1/11; 10%). Lateral transmission at 29^o^C occurred over a
3-month interval, suggesting that there was long-term shedding of virus.
Precipitating antibody was detected in experimentally inoculated bass
within three weeks of inoculation and was detected for over nine months
in some bass held at 29^o^C. No controls developed pathologic or
antibody responses. Re-challenge of seven recovered bass with the same
dose of the original virus demonstrated that these previously exposed
fish were resistant to the pathogenic effects of the virus, even at
29^o^C. None of these recovered fish developed lesions, whereas five
similarly challenged naïve bass developed necrotic inoculation site
lesions and two died. Persistence of precipitating antibody along with
seroconversion of sentinel bass after several months of exposure to
inoculated fish, especially at 29^o^C, suggested that some bass might
not clear the viral infection, might become latently infected, and might
shed virus under conditions of stress, such as high ambient water
temperature. Our studies help validate that detection of precipitating
antibody to LMBV is a simple non-lethal means to screen for evidence of
exposure to the virus. Bass with precipitating antibody to LMBV may
harbor the virus as a latent infection, subject to reactivation and
viral shedding during times of environmental or other stress.

# Expansion Of Largemouth Bass Virus Range In Michigan's Lower Peninsula Lakes

[Mohamed Faisal]{.underline}^1-3^, E. Elsayed^2^, K. Ambrose^2^, and G.
Whelan^1^

^1^Fisheries Division, Michigan Department of Natural Resources;
^2^Department of Pathobiology and Diagnostic Investigation, College of
Veterinary Medicine, and ^3^Department of Fisheries and Wildlife,
College of Agriculture and Natural Resources, Michigan State University

Largemouth Bass Virus (LMBV) is a ranavirus (Family *Iridoviridae*),
originally isolated from largemouth bass (*Micropterus salmoides*) in
the Santee-Cooper Reservoir, SC, in the mid-1990's. In less than a
decade, LMBV spread into at least 17 states in the southeastern and
midwestern United States. In 2000, LMBV infection was detected from Lake
George, a border water between Michigan and Indiana. By 2002, LMBV had
been confirmed in 23 Michigan lakes with suspected LMBV related
mortalities in 10-15 other lakes. In the summers of 2002 and 2003, the
Michigan Department of Natural Resources (MDNR) initiated an LMBV
sampling program. Adult largemouth bass were collected from 32 inland
lakes by MDNR and the Indiana Department of Natural Resources collected
adult largemouth bass again from Lake George for LMBV testing. These
lakes represented the western, eastern, and central zones of Michigan's
Lower Peninsula radiating out from the initial confirmed site. Results
indicated that there was a progressive northward, westward and eastward
expansion of LMBV's range in the southern region of Michigan's Lower
Peninsula since it was initially reported in Lake George. Sequence
analyses of the major capsid protein and methyltransferase genes of the
five isolates demonstrated their homology to each other and to the LMBV
strain isolated from the Santee-Cooper Reservoir. While the impact of
this virus on largemouth bass populations has been limited in most
cases, there is a large amount of uncertainty about the pathogen and
there is cause for concern for the health of North America's most
popular sportfish species.

**Cryptobiosis In Cichlidae: Fun For The Whole Family**

[Roy P. E. Yanong^1^]{.underline}^,\ 2^, Eric Curtis^1,\ 2^, Riccardo
Russo^1,\ 2^, Ruth Francis-Floyd^2^, ^3^,RuthEllen Klinger ^3^, Ilze
Berzins ^2,\ 4^, Karen Kelley ^5^, and Sarah L. Poynton^6^

^1^Tropical Aquaculture Laboratory, IFAS, University of Florida, Ruskin,
FL;

^2^Dept. of Fisheries and Aquatic Sciences, University of Florida,
Gainesville, FL;

^3^Dept. of LACS, College of Vet. Med., Univ. of Florida, Gainesville,
FL; ^4^Florida Aquarium, 701 Channelside Dr, Tampa, FL; ^5^Electron
Microscopy Core Lab., ICBR, University of Florida, Gainesville, FL;
^6^Div. of Comp. Med, Johns Hopkins Univ. School of Medicine, Baltimore,
MD

Granulomatous gastritis is common in numerous species of Old and New
World ornamental cichlids. Nutritional disease, clostridiosis,
mycobacteriosis, and flagellated protozoal infections are a few proposed
or identified etiologies. Fungi, rickettsia, amoeba, nematodes, and
foreign bodies can also incite similar responses. Specific etiologies
have been difficult to prove. Based on diagnostics and observations from
clinical cases, and on the scientific literature, infection with
*Cryptobia iubilans* appears a major cause of granulomatous
gastritis/gastroenteritis in cichlids. *C. iubilans* and *Spironucleus
vortens,* both flagellated protozoans found in the gastrointestinal
tract in cichlids, differ in morphology, typical pathology, primary
target organ, and therapy. *S. vortens* does not cause granulomatous
disease, targets primarily the intestinal tract, and can be treated
effectively in ornamental tropical fish with metronidazole. *C.
iubilans* causes primarily granulomatous gastritis; severe infestation
can result in more widespread, systemic granulomatous disease, and
metronidazole is not an effective treatment. Morbidity and mortality in
a population seem affected by many factors: water quality, presence of
other pathogens, diet, fish species, size, and age. Good husbandry
appears important for prevention and severity reduction. Rigorous
diagnostics (including TEM) are important for detection and
identification of the parasite, which has been found alive with in cell
vacuoles and dead within granulomas. Overall epidemiology is poorly
understood; in more advanced disease, mobile, luminal parasites may no
longer be present. Bath treatments of dimetridazole (at 80 mg/L for 24
hours x 3 days) and 2-amino-5-nitrothiazol (at 10 mg/L for a minimum of
3 days), in experimental studies, appear to be promising treatments
against this parasite, reducing prevalence of infection, although
further studies are necessary.

**Fusariomycosis In Captive Parrotfish (Family *Scaridae*) And
Bonnethead Sharks (*Sphyrna tiburo*)**

# 

# [Donald L. Neiffer, V. M. D.]{.underline} and Scott. Terrell, D. V. M., A. C. V. P.

# Disney's Animal Programs, 1200 North Savannah Circle East, Lake Buena Vista, FL, 32830

# Fusariomycosis, caused primarily by *Fusarium solani*, has been identified as suppression of immune function in these species and/or the provision of favorable growing temperatures for the fungus. Parrotfish developed fusariomycosis because they were maintained at lower water temperatures than those in their native range. The bonnethead sharks developed disease following multiple acute low temperature events whereby in-line heater failure resulted in water temperatures dropping below 24 °C (19.9-22 °C) for 1-2 days at a time. To date, treatment of parrotfish and bonnethead sharks has been unsuccessful due, in part, to lack of early recognition of the primary pathogen and failure to attain therapeutic anti-fungal drug levels in plasma. Recent successful treatment of a balloonfish (*Diodon holocanthus*) with aggressive surgical debridement procedures and an extended course of fluconazole provided encouragement for the future treatment of individual cases of fusariomycosis. a significant cause of disease in captive marine fish. At Disney's Typhoon Lagoon and Living Seas Pavilion, bonnethead sharks (*Sphyrna tiburo*) and parrotfish (Family Scaridae) have historically been highly susceptible to infection. Fusariomycosis has accounted for at least 30% of all parrotfish mortalities. Initially, skin ulcerations and loss of scales and/or epidermal tissue on the body and/or jaws were noted. Progression into skeletal muscle and, subsequently, the viscera occurred in most cases. Skeletal destruction also occurred in some cases. Histological evaluation (H & E stain) demonstrated fungal hyphae in most affected tissues. In bonnethead sharks, fusariomycosis developed in 4/6 animals that entered quarantine. Mortality occurred over approximately four months. All animals presented with cutaneous pustules and/or ulcerations associated with the cephalic and the lateral line canal system. Although histopathology revealed lesions in multiple internal organs, fungal invasion and associated damage was limited to the skin, subcutaneous space, and adjacent skeletal muscle. Involvement of the gills was also observed in one case. While trauma and dietary deficiencies may have contributed to development of fusariomycosis, thermal stress due to low temperatures was felt to have precipitated development of the disease. 

**Surgical Cases In Ornamental Fish: A Chance To Heal With Cold Hard
Steel**

[Gregory A. Lewbart^1^]{.underline} and Craig A. Harms^2^

^1^North Carolina State University, College Vet. Med., Dept. of Clinical
Sciences, 4700 Hillsborough Street, Raleigh, NC 27606; ^2^North Carolina
State University, College Vet. Med., Department of Clinical Sciences,
Center for Marine Sciences and Technology, 303 College Circle, Morehead
City, NC 28557

Ornamental fish can be excellent surgical patients. A large percentage
of these animals are nishikigoi, or simply koi (*Cyprinus carpio*),
which are taxonomically an ornamental carp. Most small and exotic animal
hospitals will already possess the necessary surgical instruments and
other supplies necessary to perform surgery on fish. A delicate or
ophthalmologic pack is helpful when working with small patients. Some of
the reasons fish may require surgery include: Traumatic injury from a
predator or fixed object in a pond or aquarium; dystocia (egg-binding);
neoplasia (either external or internal); enucleation of a diseased eye;
foreign body removal; swimbladder repair or removal; surgical biopsy of
an internal or external tissue; and repair of a torn fin. Listed are
some general considerations when performing surgery on a fish that may
differ from surgery on a terrestrial animal: 1) The skin should be kept
moist throughout the surgical procedure. During prolonged procedures, a
red rubber catheter and large syringe can be used to carefully moisten
the fish patient without splashing water into the incision site. 2)
Patient monitoring can be performed with a pulse Doppler or properly
placed ECG leads. 3) A clear plastic avian-style surgical drape is
advantageous. The plastic helps retain moisture around the fish, does
not allow moisture to leak through and compromise the surgical field,
and provides a sterile working surface. A rim of petroleum jelly can be
used to adhere the drape to the fish if desired. 4) Surgical preparation
should minimize disruption of the skin and mucus, because these are
major barriers to infection. A simple swipe along the intended incision
site with a cotton swab soaked in sterile saline, or at most dilute
povidone iodine or chlorhexidine solution, to reduce gross contamination
suffices in place of a traditional surgical scrub. 5) Removing scales
along the incision line facilitates a smooth entry. 6) Retractors are a
valuable tool for visualizing and accessing various internal organs. 7)
Bipolar cautery works well for hemostasis. 8) Needles with a cutting tip
facilitate skin penetration (skin is the holding layer in fish). 9) A
variety of suture types are used with fish but synthetic monofilament
types work best.

**Piscirickettsiosis-Like Syndrome In Tilapia**

Michael J. Mauel

Mississippi State University, Delta Research and Extension Center,
Aquatic Diagnostic Laboratory, P.O. Box 197/ 127 Experiment Station
Road, Stoneville, MS 38776

An epizootic disease affecting tilapia in both marine and fresh water
ponds was reported in 1992 in Taiwan, Hawaii in 1994, sporadically
throughout the world during the 1990's and the United States in the last
few years. All species of tilapia were affected by the disease.
Mortality was greater than 75% in severe outbreaks and averaged around
30%. Diseased tilapia often swam erratically and had trouble staying at
depth. Upon examination, the fish exhibited lethargy, occasional
exophthalmia and petechial skin lesions. The gills were often extremely
necrotic with a patchy white and red appearance. Grossly the spleen and
kidneys were extremely granular in appearance with whitish irregular
nodules throughout. Microscopically, granulomatous infiltrates were
observed throughout the viscera with the exception of the liver. The
granulomas contained organisms that were Geimsa positive, acid-fast
negative and weakly Gram negative. Transmission of the syndrome was
produced when naïve tilapia were co-habitated with diseased tilapia. The
syndrome has been associated with cold stress and poor water conditions.

**Whole Blood and Plasma Cholinesterase Levels In Normal Koi (*Cyprinus
carpio*)**

[Lisa A. Murphy^1^]{.underline}, Gregory A. Lewbart^2^, Gavin L.
Meerdink^3^, Gerald L. Bargren^3^

^1^ASPCA Animal Poison Control Center, 1717 S. Philo Rd., Suite 36,
Urbana, IL 61802 (\*Current address: USDA, APHIS, VS, 160
Worcester-Providence Road., Sutton Square Plaza, Suite 20, Sutton, MA
01590); ^2^Department of Clinical Sciences, North Carolina State
University, College of Veterinary Medicine, 4700 Hillsborough St.,
Raleigh, NC 27606; ^3^University of Illinois, College of Veterinary
Medicine, Veterinary Diagnostic Laboratory, 2001 S. Lincoln Ave., Urbana
IL 61802

Organophosphate and carbamate insecticides produce toxic effects by
binding with cholinesterase enzymes, causing inhibition of acetylcholine
catabolism. Excessive acetylcholine levels cause excessive synaptic
neurotransmitter activity. In most animal species, whole blood is the
preferred sample for evaluation of cholinesterase activity. Previous
reports of cholinesterase activity in carp have involved sampling of
serum, plasma, brain, liver, muscle, and heart. Whole blood samples were
obtained from 12 healthy koi (*Cyprinus carpio*). Whole blood
cholinesterase activity was measured in all 12 samples. Because one
sample was clotted, it was centrifuged and the analysis was performed
primarily on plasma. To evaluate if this may have significantly affected
the result, plasma cholinesterase activity was also measured in four
additional samples. The average of the whole blood cholinesterases was
0.08 mcmol/ml/min (SD=0.024). With the value from the clotted sample
removed, the average became 0.075 mcmol/ml/min (SD=0.019). The results
for the plasma cholinesterases were not very different, with an average
of 0.08 mcmol/ml/min (SD=0.012). Since these reference values are close
to the minimum detection limit for the method of analysis used,
evaluation of blood samples from koi suspected to be suffering from
anticholinesterase toxicity may be difficult.

**Ichthyophthiriasis: Atypical Outbreak InTwo Susceptible Ornamental
Fish Species In Egypt Under The Same Environmental Conditions**

[Ehab Elsayed]{.underline}^1^, M. A. Mahmoud^2^, and N. Ezz El Dien^3^

Department of Fish Diseases and Management^1^, Department of
Pathology^2^

Department of Parasitology^3^, College of Veterinary Medicine, Cairo
University, Egypt

Ichthyophthiriasis is one of the most devastating diseases that affects
freshwater ornamental fish. An outbreak of Ichthyophthiriasis erupted in
one of the aquaria holding Siamese Shark (*Pangasius sutchi*) and
Goldfish (*Carrasius auratus* var. *bicausatus*) at the College of
Veterinary Medicine, Cairo University, Egypt. Initial observation of the
outbreaks showed that only *Pangasius sutchi* was affected with typical
white spots associated with mortalities. However, *Carrasius auratus*, a
species susceptible to Ichthyophthiriasis, in the same aquarium showed
only mild erythema that disappeared during the course of infection with
no mortalities. To confirm the previous observation an experimental
infection with Ichthyophthirias was induced in *Pangasius sutchi* and a
similar number of goldfish was introduced to the aquarium one day later.
Three days after the induction, *Pangasius sutchi* started showing
typical clinical signs. Mortalities associated with severe infection
were recorded in *Pangasius sutchi* by the seventh day after infection.
*Carrasius auratus* showed only mild erythema that disappeared by the
end of experiment. Histopathological examination of skin from both
species in natural and experimental infections was performed and will be
addressed. Substantial numbers of typical large size trophonts
surrounded by layers of fibrous tissue, melanophores and hemorrhages
were detected in dermal and epidermal layers. Underlying myodegeneration
was also associated the skin lesions in *Pangasius suchi*, but *C.
auratus* skin showed only few numbers of small trophont with prominent
hemorrhage in dermal and epidermal layers. No fibrous layer formation or
myodegeneration was detected in *C. auratus* skin.

**From Wild To Exhibit: Notes On Caribbean Collecting, Shipment And
Quarantine**

[Scott Weber]{.underline} ^1^, Holly Martel-Bourbon ^2^, and Don Stark
^3^

^1^ Animal Health Department, ^2^ Fishes Department, New England
Aquarium, Boston, MA 02110-3399; ^3^ ScubaVision Productions, Boston,
MA, USA and Providenciales, Turks & Caicos Islands, BWI

Because of various constraints and limited knowledge on reproduction for
marine aquatic animals, public aquaria often conduct collecting trips to
stock exhibits with live specimens for display purposes. Although these
displays are designed for public education, collecting animals poses a
mixed bag of issues that must be addressed. Many issues are logistical,
environmental, and/or political. At the New England Aquarium, we conduct
one or two Caribbean collecting trips annually in order to diversify
species in our largest premier exhibit tank, the Giant Ocean Tank (GOT).
This tank resembles a Caribbean coral reef, showing animals that inhabit
Caribbean waters. The GOT exhibits one of the most diverse Atlantic
collections in the world with over 120 species and 600 + specimens of
fish and 3 species of sea turtles. These inhabitants vary in size (cm to
m) and in age ranges (span 1- 25 + years). Some animals in this tank
such as the Pacific batfish, *Platax orbicularis*, are marquee for
educational programming, as they illustrate the dangers of releasing
non-native species into Atlantic tropical waters. The prominence of the
New England Aquarium in conservation, research and education, have lead
staff in the husbandry department to constantly improve collecting
techniques for obtaining specimens. This presentation follows Caribbean
fishes through wild collection, during transport, in quarantine, and
onto exhibit to illustrate various protocols, techniques, apparatus,
programs, and methodologies that are required to successfully maintain
and stock exhibits in public aquaria. Collaboration between our Fishes
and Animal Health Departments, as well as with other institutions,
serves to continually adapt, evolve, and improve this program, which has
achieved minimal mortalities (1-3%) and provided a remarkable
educational centerpiece for the New England Aquarium over the last 35
years.

**Diagnostic Microbiology For Small Laboratories: A Practical Guide For
The Identification Of Bacterial Isolates From Fish**

[Jill E. Arnold^1^]{.underline} and Ana Baya^2^

^1^Animal Health Department, National Aquarium in Baltimore, 501 E.
Pratt St., Baltimore, MD 21202; ^2^Virginia-Maryland Regional College of
Veterinary Medicine, 8077 Greenmead Drive, College Park, MD 20740

Diagnostic microbiology is an important health assessment tool for
veterinarians in aquatic medicine. However many aquaculture facilities,
zoo, and aquarium laboratories are limited in staff and microbiology
expertise. Time and budget constraints may prohibit submission of
cultures to a reference lab. Biochemical tests have been the standard
for bacterial identification for years, but results may yield incorrect
identifications, especially for oxidase-positive, non-lactose
fermentors. Analyzers developed to automate biochemical techniques for
identifying bacteria from humans are of limited use with isolates from
fish and method validation is lacking. There is a need for the
combination of biochemical tests with more advanced techniques to offer
accurate, efficient results. In 2001, the authors reviewed the
identification data from over 700 bacterial isolates found in clinical
cases at the National Aquarium in Baltimore (NAIB) and developed a
protocol, organized in a series of easy-to-use flowcharts, to group the
bacteria into clinically useful categories. The bacteria were grouped as
follows: Gram-Positive Coccus (*Staphylococcus* sp*., Micrococcus* sp*.,
Enterococcus* sp*., Streptococcus, Non-Enterococcus*); Gram-Positive
Bacillus (*Listeria* sp*., Corynebacterium* sp*., Erysipelothrix,
Bacillus* sp*., Clostridium* sp*., Carnobacterium* sp*., Actinomyces,
Mycobacterium/Nocardia/Tsukamurella*); Gram-Negative Cocco-Bacillus
(*Moraxella* sp*., Acinetobacter* sp*.*); and Catalase- and
Oxidase-Positive Gram-Negative Bacillus
(*Vibrio/Plesiomonas/Sphingobacter, Aeromonas* sp*., Shewanella
putrefaciens, Pseudomonas aeruginosa, Pseudomonas
(*non*-aeruginosa)/Flavobacter* ). Oxidase-negative, Gram-negative
bacteria were identified by biochemical tests and commercially available
test strips. Isolates were frozen at --70 °C and, when further
identification was required, subcultured for submission to a reference
laboratory specializing in aquatic microbiology. This strategy has been
in place at NAIB since 2001; it offers an efficient and inexpensive
option for preliminary identification of isolates that is easily taught
to laboratory staff with basic microbiological skills.

**Molecular Approaches To The Diagnosis Of Infectious Disease Concerns
In Commercial Aquaria**

[Salvatore Frasca Jr^1^]{.underline}, Kathleen R. Nevis^1^, Andrew
Draghi II^1^, Thomas E. Mullen^1^, Spencer Russell^1^, Allison
Kamens^1^, Akinyi Nyaoke^1^, E. Scott Weber^2^,

and Christian J. Keller^3^

^1^Department of Pathobiology and Veterinary Science, University of
Connecticut, Storrs, CT; ^2^New England Aquarium, Central Wharf, Boston,
MA; ^3^Tennessee Aquarium, Chattanooga, TN

Infectious diseases are among the most significant and costly threats to
the health of individual fish species and tank populations in commercial
aquaria. As attempts are made to maintain collections of greater numbers
and more diverse species of aquatic animals, the incidence of infectious
disease in aquarium populations is bound to increase. A potential
complicating factor in this equation is the concern that newly
encountered or emerging diseases in aquarium-held fish may be caused by
microbial agents that are difficult, if not impossible, to isolate by
routine microbiological techniques. Specimens for microbiological
analysis may not be easily obtained in some instances because of
limitations imposed by species rarity, specimen size, or husbandry
conditions. Molecular biological techniques have provided valuable
insights into identification and characterization of unculturable
infectious agents in human medicine. Such approaches can be useful in
identification and characterization of fastidious or yet uncultured
infectious agents in host species economically relevant to commercial
aquaria. Members of the Syngnathidae, e.g. seahorses, pipefish, and
seadragons, have been subjects of intensive aquaculture initiatives in
aquaria nationwide. These are prominent exhibit species in which several
infectious diseases have emerged and threaten their long-term
maintenance in aquaria. A concerted approach coordinating
histopathologic analysis of tissues with consensus sequence-based PCR,
targeting conserved genes, has provided partial gene sequence data from
which genus- or species-level identifications of infectious agents can
be made in the context of anatomic correlates of disease. Conscientious
and controlled application of molecular approaches using consensus
sequence-based PCR may advance identification and characterization of
certain fastidious or yet uncultured infectious agents and contribute to
their epidemic control.

**Apatite Concretions In Spiral Valve Of Yellow Stingray**

**(*Urobatis \[Urolophus\]* *Jamaicensis*) Fetal Pups**

[Craig A. Harms]{.underline}1, Keith E. Linder2, Michael H. Fatzinger3,
Barbara A. Wolfe4, Kathy A. Spaulding2, Gregory A. Lewbart2

1North Carolina State University, College of Vet. Med., Center for
Marine Sciences and Technology, 303 College Circle, Morehead City, NC
28557; 2North Carolina State University, College of Vet. Med., 4700
Hillsborough Street, Raleigh, NC 27606; 3North Carolina Aquarium at Fort
Fisher, 900 Loggerhead Road, Kure Beach, NC 28449; 4North Carolina
Zoological Park, 4401 Zoo Parkway, Asheboro, NC 27205

Yellow stingrays conceive in captivity, but rarely proceed to normal
parturition. Retained fetuses can mummify and cause life-threatening
complications. During clinical management of five yellow stingray
females housed intermittently with one male, radiography and
ultrasonography were used to stage fetal development. One striking
radiographic feature of the fetal pups was a beehive-shaped radio-opaque
spiral corresponding to the spiral valve. By contrast, the spiral valve
was not radiographically detectable in the adults. The spiral valve was
the only radiographic feature visible in early-term fetuses. In larger
fetuses, the barb, vertebral column and pectoral girdle became more
radiographically apparent, as the mineralized opacity of the spiral
valve diminished asymmetrically. The fetal spiral valve was a useful
ultrasonographic landmark that was identified by its oval shape and
alternating thin wavy hyperechoic and hypoechoic bands. Assisted
parturitions were performed twice by manual dilatation and extraction,
resulting in two premature pups and a normal-appearing full-term pup
that died 24 h later. Pharmacologic induction was performed using
intracloacal misoprostol, resulting in delivery one hour later of five
pups that thrived at 2 months. Confirmed or presumed spontaneous
abortions occurred three times and caused concern about the stress of
handling, transport and anesthesia. In formalin-fixed specimens, the
spiral valve was gritty and difficult to cut, particularly in smaller
fetuses. On cut surface, grey concretions with varying amounts of soft
pale green gelatinous material were interleavened within folds of the
valve. After routine processing for histology, the organ was easily cut
with the microtome. Histologically, grey granular material and
homogeneous pale pink glycoprotein-like material filled the spaces
between spiral valve folds. Cytoplasm of the epithelial cells was
uniformly distended with pink material. Stone analysis of the
concretions revealed that they were composed of 100% apatite
(Ca~5~\[PO~4~\]~3~\[OH\] and related compounds).

# Problems With Protozoa: Treating A Large Reef Tank For *Cryptocaryon irritans*

[Susan Goodridge]{.underline}, Andrew Routh , and Scott Weber

Animal Health Department, New England Aquarium, Central Wharf, Boston,
MA 02110

Since the New England Aquarium opened its Giant Ocean Tank in 1970,
there have been unavoidable outbreaks of various pathogens including
*Cryptocaryon irritans, Neobenedenia*, and *Amyloodinium* despite
vigorous quarantine efforts. Some previous and historical treatments
have included copper sulfate (various forms), trichlorfon, and
praziquantel. Preventive measures have included the addition of cleaner
wrasse fish; careful disinfection of wetsuits, nets, food prep
materials, and other dive gear; stricter quarantine procedures; monthly
surveillance of sentinel fish using freshwater dips, skin scrapes and
gill clips; sediment filtering; and skimming with an egg catching
device. In recent years, *C. irritans* has caused the most frequent
outbreaks of disease in the Giant Ocean Tank. Such outbreaks have
required three tank treatments in the last two years. Possible sources
of infection include newly introduced fish, cross contamination with
human or equipment vectors from other galleries or quarantine areas, the
water supply from Boston Harbor, the presence of a copper-resistant
strain of *C. irritans*, or aging infrastructure and deferred
maintenance that has prevented theraputants to penetrate all the filters
and gravel beds. The most recent treatment with Cupramine^©^ (Seachem
Laboratories Inc.), a copper product complexed with an amine molecule,
although safe, was least efficacious. Although no mortalities were
observed when Cupramine^©^ was applied, subsequent reinfection after six
weeks suggested that stages of the parasite persisted well beyond the
termination of the treatment. This discussion hopes to demonstrate the
trials, tribulations, and environmental quality management required to
treat a 200,000-gallon system for a disease outbreak.

Number range CHAPTER**Characterization Of *Photobacterium damselae*
Subspecies *damselae* Isolates From Teleosts And Elasmobranchs Housed In
An Aquarium Exhibit**

[J. Arnold]{.underline}^3^ J. Abbott^1^, S. Zhao^1^, M. H.Kothary^1^, S.
R. Monday^2^, P. Whittaker^2^, J. Trinidad-Perez^1^, R.P.
Gonzalez-Nieves^1^, M. Marrero-Moran^1^ V. Velez-Vega^1^, E.
Ayala-Diaz^1^, M. Ramos-Valle^1^, G. Modderman^1^, A. Nyarko^1^, I.
Agba^1^, T. DeSiano^1^, S. Taherkhani^1^, N.C. Flores^1^, and
B.D.Tall^1^

^1^Food and Drug Administration, Laurel, MD 20708 and ^2^College Park,
MD 20740, and ^3^National Aquarium, Baltimore, MD 21202

*Photobacterium damselae* subsp. *damselae* (Pdd) is a marine vibrio
that causes extraintestinal infections and gastroenteritis in humans and
fatal septicemia and wound infections in cartilaginous and bony fish.
Nine isolates, obtained from various fish species and sources, were
identified using the API20E and MIDI identification systems and by a
duplex PCR assay targeting concomitantly, a species-specific 16s rRNA
gene sequence and the phospholipase D hemolysin (damselysin) gene. All
of the isolates were positive for the 16s rRNA gene amplicon; none of
the isolates were positive for the damselysin gene amplicon; yet 7 of
the 9 isolates were hemolytic on sheep blood agar. All were urease
positive indicating that these isolates were Pdd not *P. damselae*
subsp. *piscicida*. Plasmid profile analysis showed that several of the
isolates possessed plasmids; some had more than one. Pulsed field gel
electrophoresis (PFGE) showed that the isolates comprised 8 different
subtypes with Dice relatedness coefficients ranging from 40% to 100%.
However, 2 of 9 isolates had indistinguishable PFGE banding patterns.
These two isolates were obtained from ill fish housed in the facility,
15 months apart. Together, these data suggested that isolates causing
infections in the aquaria exhibits arose with one exception, from
different bacterial clonal linages; further suggesting that derivatives
of no one clone dominated any ecological niche or aquarium location.
Lastly, a non-damselysin hemolysin was isolated from one Pdd strain. The
hemolysin was heat labile, had a molecular weight of 64,000 and an
isoelectric point of ca 4.7. In summary, these studies also showed that
the strains causing infections in the fish represented a diverse group,
several isolates possessed plasmids, and that a second hemolysin that is
different than the already described damselysin was isolated. It is not
known whether plasmid(s) play a role in disease. Other studies will
further characterize the hemolysin's role in disease.

# Further Investigation Into The Brain Parasite In Yellow Perch

[Lester H. Khoo]{.underline}^1^, Frederick A. Rommel^2^, Stephen A.
Smith^3^, and Linda M. Pote^4^

^1^University of Pennsylvania, School of Vet. Med., 382 West Street Road
Kennett Square, PA 19348, ^2^Pennsylvania Veterinary Laboratory, 2305
North Cameron Street, Harrisburg, PA 17110, ^3^VA/MD Regional College of
Veterinary Medicine, Duck Pond Drive,Virgina Tech, Blacksburg, VA 24061,
^4^Mississippi State University, College of Vet. Med., PO Box 9825,
Mississippi State, MS, 39762

Myxozoan parasites found in brains of yellow perch (*Perca flavacens*)
presented to the Pennsylvania Animal Diagnostic Laboratory System in
2000 were compared to a case in September 2003 that affected juvenile
yellow perch. In 2000, the affected fish were raised in recirculating
systems and came from states where they were grown in earthern ponds.
The fish showed hyperexcitability when introduced into the recirculating
systems that changed to lethargic abnormal swimming and drifting with
the water flow. Mortalities occurred 3-5 months post introduction to the
recirculating systems. Gross and postmortem examinations performed were
not remarkable except once when *Flavobacterium columnare* was isolated,
but resolved with antibiotics. Viral isolation was negative.
Histopathological examination revealed large localized clusters of
spores beneath the ependymal lining of the central canal of the brain
with little or no inflammatory response. Based on spore location and
morphology, the parasite was identified as *Myxobolus neurophila.* In
the 2003 case, fish did not show any clinical signs or mortality.
Histological comparisons were performed using fresh and formalin fixed
tissue and the parasite was only found in yellow perch and not in four
other species (large mouth bass, fat head minnows, golden shiners, and
walleye) that were raised in the same pond or shared the water supply.
Spore visualization was enhanced with Diff-quick stain on touch
impressions and Giemsa stain for histological preparations. Spores in
unfixed tissues measured 15.4-16.8μm long and 5.6-7.0μm wide with two
prominent polar capsules. Presence of an iodopholius vacuole with
Lugol's iodine was not confirmed but spores picked up the stain, which
facilitated differentiation from tissue in the touch impressions of the
brain. As in 2000, spores of this histozoic myxosporea were found in the
multiple locations in the brain including the meninges. A mild
mononuclear inflammation was often present when spores occurred outside
parasitic cysts. Spores (often immature) were also found in the spinal
cord. The histopathological examination was performed on cross-sections
of the fish and spores were not readily evident in other organs.
Currently, other techniques (EM, PCR) are being employed to provide
further information on this parasite.

**The Discovery Of Four Cryptic Species Of Oligochaete Hosts (*Tubifex
tubifex*) Provides Insights Into The Spread Of *Myxobolus cerebralis*
Among Wild Trout Populations**

[Katherine A. Beauchamp]{.underline}^1^, R. Barry Nehring^2^, Garry O.
Kelley^3^ and

Ronald P. Hedrick^3^

^[1]{.underline}^United States Geological Survey, National Fish Health
Laboratory, 11649 Leetown Road, Kearneysville, WV 25430; ^2^Colorado
Division of Wildlife, 2300 Townsend Avenue, Montrose, CO 81401;
^3^Department of Medicine and Epidemiology, School of Veterinary
Medicine, University of California, CA. 95619

Two studies in Colorado examined river drainages where whirling disease,
caused by *Myxobolus cerebralis,* has or has not had significant impacts
on wild populations of trout. The distribution of oligochaete hosts
resistant or susceptible to infections with *M. cerebralis* was
determined at each site with newly developed mitochondrial 16S rDNA (mt
16S rDNA) lineage specific markers. We tested the hypothesis that
certain cryptic species (mt 16S lineages) of *Tubifex tubifex*
preferentially support *M. cerebralis* infections in a given geographic
region. In the first study, worms from seven sites were experimentally
exposed to the parasite in the laboratory prior to genetic screening. In
the second study, worms collected from thirty sites were not exposed to
*M. cerebralis* in the laboratory and these samples were screened
shortly after field collection. Results from both studies demonstrated
that genetically heterogeneous populations of *T. tubifex* or cryptic
species coexisted at most sites, however non-susceptible *Tubifex sp.*
(lineage V and VI) predominate at low impact sites. This supported the
conclusion that when these non-susceptible *Tubifex* worms were in
greater abundance the potential for more severe effects of whirling
disease on wild trout populations may be ameliorated. Furthermore, the
distribution and abundance of particular *T. tubifex* may in part be
responsible for defining high compared to low impact whirling disease
sites.

***Bonamia* Sp. (Haplosporidia) Found In Non-Native Oysters,
*Crassostrea ariakensis*, In Bogue Sound, North Carolina**

[Eugene M. Burreson]{.underline}^1^, Nancy A. Stokes^1^, Ryan B.
Carnegie^1^ and Melanie J. Bishop^2^

^1^Department of Environmental and Aquatic Animal Health, Virginia
Institute of Marine Science, College of William Mary, PO Box 1346,
Gloucester Point, VA 23062; ^2^Institute of Marine Sciences, University
of North Carolina at Chapel Hill, 3431 Arendell Street, Morehead City,
NC 28557

The Suminoe oyster, *Crassostrea ariakensis*, is being considered for
introduction into the middle Atlantic coast region of the United States,
where diseases have decimated native *Crassostrea virginica* stocks.
Triploid *C. ariakensis* produced at the Virginia Institute of Marine
Science (VIMS) and transferred to Bogue Sound, North Carolina
experienced high mortality in the summer of 2003. Histopathological
examination of oysters collected after the peak mortality revealed the
presence of intrahemocytic inclusions suggesting a *Bonamia*
species-like parasite infecting hemocytes in 9.1% (2/22) of the oysters.
Sampling of a subsequent, October 2003 deployment to Bogue Sound
revealed 47% of the oysters were infected by a *Bonamia*-like parasite.
Diagnosis of a second sample by *Bonamia*-specific PCR primers revealed
a 60% prevalence and confirmed that the parasite was *Bonamia* sp.
Sequence analysis of the PCR fragment suggested that the parasite was
not one of the described species of *Bonamia*. No infections were
detected in *C. ariakensis* from Pamlico Sound, North Carolina or from
the York River, Virginia that originated from the same hatchery cohort,
suggesting that the parasite originated in Bogue Sound and not from the
VIMS hatchery. Additionally, no *Bonamia* sp. infections were detected
in *C. virginica* from Bogue Sound, indicating specific susceptibility
of *C. ariakensis* for the parasite. The natural host for this *Bonamia*
sp. is unknown. How, or if, this parasite will affect the suitability of
*C. ariakensis* for resource restoration or aquaculture in the middle
Atlantic region must await studies on temperature and salinity
tolerance, transmission and distribution of the natural host.
Nonetheless, *C. ariakensis* appears to be highly susceptible to a
previously unknown local pathogen.

**Protozoan Infections Of The Eastern Oyster (*Crassostrea virginica*)
In The Upper Chesapeake Bay: A Potential Ecological Forecast**

[Shawn M. McLaughlin]{.underline}^1^, Stephen J. Jordan^2^, and Fred G.
Kern^1^

^1^NOAA NOS Center for Coastal Environmental Health and Biomolecular
Research/Cooperative Oxford Laboratory, 904 South Morris St., Oxford, MD
21654; ^2^Environmental Protection Agency, Gulf Ecology Division, Gulf
Breeze, FL 32561

*Perkinsus marinus* and *Haplosporidium nelsoni* cause devastate
populations of the eastern oyster, *Crassostrea virginica*, along the
United States Atlantic coast and Gulf of Mexico. Salinity and
temperature are major controlling factors in the prevalence and
infection intensity of these two parasites. Fishery managers and oyster
growers use this relationship to predict potential outbreaks of disease
in oyster populations and to determine the best time, or sites, for
planting and harvesting of oysters. Predicting medium or long term
fluctuations in oyster disease in the Chesapeake Bay is limited by the
inability to accurately predict medium and long term fluctuations in
weather patterns such as occurrence and duration of drought conditions.
Several mathematical models have been developed to assess the health of
oyster populations in the Gulf of Mexico and Chesapeake and Delaware
bays. An impediment to the accuracy of mathematical models is the
current gap in knowledge regarding the parasites, the host, and their
interactions. A prototype population dynamics model was recently
developed by Jordan and Coakley to predict outcomes of management
options for resource restoration and fisheries enhancement. Sixteen
years of Chesapeake Bay oyster disease and population data collected by
the Maryland Department of Natural Resources provided the basis of the
model. The potential for using the Jordan and Coakley model to predict
the effects of disease on oyster populations in the upper Chesapeake Bay
was examined in this study. Alternatives to the stock assessment or
population dynamics modeling approach to predicting oyster disease are
also discussed.

**The Depression Of Oxygen Uptake In Blue Crabs In Response To Bacterial
Challenge**

Jeremy D. Holman^1^, Louis E. Burnett^1^, [Karen G.
Burnett]{.underline}^1^,

and Darwin D. Jorgensen^2^

^1^Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, SC
29412; ^2^Department of Biology, Roanoke College, Salem, VA 24153

We investigated the respiratory responses of the blue crab, *Callinectes
sapidus*, to an injection of bacterial pathogen *Vibrio* *campbellii*.
Current results suggested that bacteria were removed from the hemolymph
in a process associated with a reduction in circulating hemocytes. It
was thought that the gill was an important site of accumulation and
ultimate removal of the bacteria. We tested the hypothesis that the
accumulation of bacteria at the gills disrupted gas exchange. Whole
animal oxygen uptake was measured in well-oxygenated water (25 ppt,
25°C) in control animals injected with saline and compared with animals
injected with 2.5 x 10^4^ live *Vibrio*/g crab weight. Injections were
made directly into the lumen of the heart to promote rapid distribution
of *Vibrio*. Crabs injected with *Vibrio* had a significantly (p\<0.037,
t-test) depressed oxygen uptake (2.56 μmol/g-h, 0.23 sem, n=6) compared
with controls (4.53 μmol/g-h, 0.59 sem, n=6) in a period of 1 to 2 hours
after injection. In separate experiments we compared pre- and
postbranchial hemolymph Po~2~ and pH in sham-injected controls with the
same variables in *Vibrio*-treated crabs 30 min after treatment.
Postbranchial Po~2~ was significantly (p=0.006, t-test) depressed in
*Vibrio*-treated crabs (99 torr, 5.8 sem, n=9) compared with the
controls (122 torr, 3.4 sem, n=7). There were no significant differences
in any of the other variables. Hemolymph pressure was measured in the
infrabranchial sinus and the pericardial space unilaterally before,
during and after sham and *Vibrio* injection, allowing for calculation
of hemolymph pressure drop across the gill circuit. Ventilatory
function, monitored as pulsatile hydrostatic pressure in the branchial
chamber, did not change significantly after either sham or bacteria
injection. Resistance to hemolymph flow increased across the gill
circuit after bacterial injection. These results suggested that
injection of crabs with *Vibrio* causes a disruption of gas exchange at
the gills, supporting our hypothesis. The precise mechanisms involved
were not yet clear and are currently under investigation. (Supported by
NSF IBN-0212921.)

**Effects Of Environmental Stressors On Mortality Rates In Lobsters: A
Controlled Laboratory Study**

[Diane Kapareiko^1^]{.underline}, Richard A. Robohm^1^, Andrew F. J.
Draxler^2^, Daniel Wieczorek^2^, Steven Pitchford^1^, and Lori Davias^2^

^1^NOAA, National Marine Fisheries Service, The Milford Laboratory, 212
Rogers Avenue, Milford, Connecticut 06460; ^2^James J. Howard Marine
Sciences Laboratory, 74 Magruder Road, Sandy Hook, Highlands, New Jersey
07732

The cause of unprecedented lobster mortalities in western Long Island
Sound (LIS) in 1998 and 1999 is under investigation by scientists who
are conducting numerous studies on paramoebiasis, pesticide poisoning,
and environmental stressors. The objective of our contribution to this
effort is to determine whether increased (but environmentally realistic)
conditions of temperature, hypoxia, sulfide and ammonia, alone or in
combination, can increase susceptibility to microbial infections, and
thus, affect mortality rates. A study completed in 2003 indicated that
lobsters exposed to levels of hypoxia and hydrogen sulfide that are
known to occur in LIS became more susceptible to the bacterial lobster
pathogen *Aerococcus viridans.* Further experiments to evaluate
high-temperature stress in the absence of bacterial infection showed
that lobsters obtained from eastern LIS survived at 24^o^C indefinitely
under normoxia. Survival dropped to three days at a lower oxygen level
of 95 µM, and to only one day when dissolved oxygen levels were further
dropped to 55 µM, and combined with 9 µM sulfide and 17 µM ammonium.

**Immunodiagnostics In Theory And Practice**

[Stephen L. Kaattari]{.underline}

Department of Environmental and Aquatic Animal Health, Virginia
Institute of Marine Science, College of William and Mary, Gloucester
Point, VA 23062

Current capabilities in immunodiagnosis transcend those most routinely
exploited by fish health professionals. Although some diagnoses can be
sensitively ascertained by molecular techniques, a number of prognostic
indicators of immunity or immunopathology are determined via
immunoassays. However, as with all diagnostic assays, optimal
utilization of immunoassays requires in-depth knowledge of the system
being examined. Often antibodies are developed with the purpose of being
diagnostic of a disease or physiological condition without knowledge of
the specific antigen or the specificity of the antibody or how either
relates to the disease. For example, the relevance of the level of
antibody activity generated or even the presence of the antibody itself
has been confused with a prophylactic state, without consideration of
the fact that antibodies may, at times, be contraindicative of a
prophylactic response. Currently immunoassays have progressed beyond the
simple assessment of antibody activity to the determination of the form
of antibody induced, its specificity, and whether a cellular response
has been generated. Thus the role immunodiagnostics has not only
expanded in its sensitivity and specificity, but now also includes more
circumspect analyses of other parameters of the immune response.

**Molecular Diagnostics In Theory And Practice**

[Kimberly S. Reece]{.underline}

Department of Environmental and Aquatic Animal Health, Virginia
Institute of Marine Science, The College of William and Mary, Gloucester
Point, VA 23062

Developments in molecular genetic technology and the proliferation of
sequence information during the past 20 years have facilitated progress
in several areas of aquatic disease research. Advances have been made in
pathogen detection and identification, as well as the use of molecular
tools to enhance studies of transmission, host/parasite interactions and
mechanisms of virulence. There is an expanding database of molecular
information available, which can be used to develop sensitive and
specific assays. Molecular diagnostics, including DNA probes for in situ
hybridizations and primers for polymerase chain reaction (PCR) are
available for a variety of pathogens found in the aquatic environment.
These methods greatly facilitate disease diagnoses, especially when it
is difficult and/or time-consuming to isolate and identify pathogens,
and in situations when pathogens are not easily distinguished
morphologically. In addition, molecular diagnostics are invaluable when
a particular species within a genus or even certain strains of the same
species may be pathogenic, while closely related species or strains are
harmless. Although molecular techniques are powerful, caution must be
employed to ensure that assays are based on adequate molecular
information and that the information and techniques are used
appropriately. It is essential that molecular probes and primers are
reliable, accurate and sensitive. Development of genus-specific,
species-specific and/or strain-specific DNA probes and PCR primers
entails obtaining sequence data for an intended target locus from as
many different strains and species within a genus as possible. In
addition, DNA sequences from targeted loci are needed from closely
related taxa. Intra- as well as inter-specific sequence variation must
be characterized. This approach optimizes the chance of developing a
genus-specific probe that works for all members of the genus and that
does not cross-react with members of other genera. To minimize the
chances of a species-specific probe failing to detect a particular
strain of a species, as many strains as possible from a wide geographic
and in some cases the host range should be examined. Finally, in order
to confidently employ molecular diagnostics, the assays should undergo
rigorous validation against established diagnostic protocols. Several
examples of how these techniques are used to detect aquatic pathogens
will be discussed.

**Issues Associated With An OIE Reference Laboratory For Shellfish
Disease Diagnosis**

[Eugene M. Burreson]{.underline}

Department of Environmental and Aquatic Animal Health, Virginia
Institute of Marine Science, College of William and Mary, PO Box 1346.
Gloucester Point, VA 23062

The Office International des Épizooties (OIE) is a Paris, France-based
organization whose aim is to assure the sanitary safety of international
trade in aquatic animals including fish, molluscs and crustaceans. This
is accomplished by identifying diseases of concern, called \"reportable
diseases,\" and by developing health measures implemented by member
countries that prevent the transfer of reportable diseases while
avoiding unjustified barriers to trade. The OIE approves a Reference
Laboratory for each reportable disease, which is responsible for
developing and standardizing diagnostic tests and providing diagnostic
support for international colleagues. The OIE also appoints a Reference
Expert for each reportable disease, who is responsible for providing
advice to the OIE. The OIE publishes three documents---1) the Aquatic
Animal Health Code that lists all reportable diseases and explains the
steps necessary to be designated a disease free country, and reporting
requirements should a disease outbreak occur; 2) the Manual of
Diagnostic Tests for Aquatic Animals, which includes OIE-approved
diagnostic methods developed by the Reference Laboratories; and 3)
Annual Reports of OIE Reference Laboratories, which document all
research and diagnostic support provided by reference laboratories.
Accurate and rapid diagnosis for reportable diseases is critical to
prevent spread of pathogens and also because of the economic
implications associated with possible trade restrictions. DNA-based
diagnostics are increasingly being used, but interpretation can be
problematic. Most countries require confirmation of identification of
reportable disease outbreaks by the Reference Laboratory for that
disease agent. Some case examples will be discussed.

**USDA/APHIS And Veterinary Diagnostics**

[Janet V. Warg]{.underline} and David A. Miller

National Veterinary Services Laboratories, P.O. Box 844, 1800 Dayton
Avenue

Ames, Iowa 50010

One role of the Animal Plant Health Inspection Service (APHIS) in
support of the aquaculture industry is to facilitate the movement of
aquatic animals and products in interstate and international commerce by
providing health certification services. In many cases the importing
country or facility requires that the surveillance or diagnostic testing
procedures be conducted in a USDA, APHIS approved laboratory. Any
Federal, state, university, or private laboratory is eligible for APHIS
approval. The procedures for designating laboratories as qualified to
conduct diagnostic testing of aquatic animals, embryos, ova and semen
intended for export are outlined in Veterinary Services memorandum
567.2. In brief, the approval process requires consultation with your
state's Federal Area Veterinarian in Charge, including a laboratory site
visit, submission of laboratory documentation (specifics outlined in
memorandum), and review of the laboratory's testing protocol(s) for
scientific merit and conformity to internationally recognized standards.
Internationally recognized standards can be found in the Office
International Des Epizooties (OIE) Diagnostic Manual for Aquatic Animal
Diseases and the OIE International Animal Health Code. In accordance
with the OIE International Animal Health Code, the United States is
required to report to the OIE any aquatic diseases that are on the list
of OIE notifiable diseases. The official contact point for reporting to
the OIE on notifiable diseases in the United States is APHIS. Any
diagnostic laboratory that identifies or detects OIE notifiable diseases
should report them to the Federal Area Veterinarian in Charge in their
state.

**Standardization And Implementation Of Molecular Diagnostics Used In
The United States Fish And Wildlife Service's National Fish Health
Program**

# [Patricia A. Barbash]{.underline}

U.S. Fish and Wildlife Service, Northeast Fishery Center, Fish Health
Center, P.O. Box 155, Lamar, PA16848

Until recently, fish disease inspection and diagnosis performed by the
U.S. Fish and Wildlife Service (USFWS) relied on standard protocols
published in the American Fisheries Society- Fish Health Section's
"Bluebook". When the USFWS implemented the National Wild Fish Health
Survey in 1997, however, the decision was made to include recent
advances in molecular diagnostics as corroborative tests to accepted
detection methods. This deviation from standard "Bluebook" protocol
presented the nine USFWS fish health laboratories with the opportunity
to field test molecular techniques developed for detection of pathogen
genome segments from fish tissues. The necessity to impose strict
standardization was realized and accomplished with the publication of
the USFWS Wild Fish Health Survey Laboratory Procedures Manual in 2000:
a detailed set of standard operating protocols for every procedure and
assay employed in the Wild Survey. The impressive quality of this manual
lead the USFWS Fish Health Program to evolve a complete set of protocols
and procedures employed at all nine fish health centers in every aspect
of operation. In addition, it has led to a successful collaboration with
the AFS-FHS to revise the Bluebook with the addition of Standard
Procedures for Aquatic Animal Health Inspections (2003, 5^th^ edition).
Molecular techniques have now become a Bluebook standard for
confirmatory testing. This presentation will discuss the advantages and
limitations of using polymerase chain reaction (PCR) technology as a
corroborative test in addition to previously established standard
protocols for aquatic animal health inspection and diagnostics. In
addition, the good and bad implications of using PCR beyond the scope of
these standards is discussed, with particular reference to the use of
PCR to screen fish samples for pathogens such as Infectious Salmon
Anemia virus in wild fish.

**Species Specific Molecular Probe Investigations Of**

# \"White\" Diseases Of Corals

[Laurie L. Richardson]{.underline}, Elizabeth Remily, and DeEtta Mills

Department of Biological Sciences, Florida International University

Miami, FL 33199

A fluorochrome-labeled molecular probe specific to *Aurantimonas
coralicida*, the bacterial pathogen of the coral disease white plague
type II (WPII), was constructed for use in the investigation of coral
disease etiology. The probe was optimized and validated in the
laboratory using pure cultures. It was designed based on a unique subset
of the complete16S rRNA gene sequence obtained using a pure culture of
the pathogen. The pathogen specific probe was labeled with the
fluorochrome fluorescein isothiocyanate. As a control, a second,
universal eubacterial probe (EUB 338) was constructed and labeled with a
different fluorochrome (tetra-methyl-rhodamine isothiocyanate). The
probes were used in analyses in conjunction with fluorescent *in situ*
hybridization (FISH). We are currently using the probe to investigate
the identification of pathogens associated with the "white" diseases of
corals. "White" diseases are a suite of diseases characterized by coral
tissue lysis that is associated with either a thin band of advancing,
completely bleached (i.e. white) coral tissue, or a thin line that
separates apparently healthy coral tissue from freshly exposed (white)
coral skeleton. This suite of diseases includes three forms of white
plague (types I, II, and III); two forms of white band (types I and II);
white pox; patchy necrosis; shut down reaction; and bacterial bleaching.
Of these, associated primary pathogens have been isolated and
characterized for only three (white plague type II, white pox, and
bacterial bleaching). It has been postulated that the same microorganism
may cause some of these diseases, particularly those for which no
pathogen has been identified. Through use of the probe on coral tissue
samples collected from corals infected with each of the white diseases
we are investigating the potential involvement of *A. coralicida,*
either to document it's involvement or to rule it out as a potential
causative agent.

# Assessment of bacterial communities associated with white plague-Infected *Montastraea annularis* corals from St Croix

# 

# [J. Paige Rothenberger]{.underline}, Esther C. Peters, Masoumeh Sikaroodi, Patrick M. Gillevet and Robert B. Jonas

Department of Environmental Science and Policy, George Mason University,
MSN 5F2, 4400 University Drive, Fairfax, VA 22030

The coral disease white plague type II (WPII) threatens scleractinian
corals and the reefs they support in the Caribbean and Western Atlantic.
The disease is particularly virulent and kills coral tissue at a rate of
2 cm per day. It has been documented in the waters of the Bahamas,
Florida, Puerto Rico and the U.S. Virgin Islands (USVI), and affects at
least 17 species of coral. Due to the virulence of WPII, it can kill
entire coral colonies within days and can alter the structure,
composition and function of coral reef ecosystems. Although studies have
concentrated on identifying the etiological agent, until recently the
microbial community structure of WPII- infected tissue and healthy coral
tissue had not been investigated. Furthermore, most investigations into
the disease have utilized culture-based techniques, which may not
provide an accurate assessment of the entire community. The goal of this
research was to compare bacterial communities between healthy and
diseased coral, and to determine if the communities associated with the
coral disease WPII are the same among different infected *Montastraea
annularis* coral colonies. Samples were collected from the water column,
apparently healthy, and infected *M. annularis* corals at Sprat Hole
reef in St. Croix, USVI. Samples were cultured for enumeration and to
provide cellular characteristics and bacterial isolates. Additionally,
whole-community DNA was extracted from samples for use in the molecular
fingerprinting technique, Length Heterogeneity Polymerase Chain Reaction
(LH-PCR). The LH-PCR technique provides information on the composition
of the bacterial community and abundance estimates for each of the
community members. The combination of culture-based approaches and
molecular tools such as LH-PCR, provides a more complete assessment of
the bacterial communities associated with the apparently healthy tissue
and the diseased coral state.

**Amplicon Length Heterogeneity - A Tool To Investigate Coral Microbial
Communities**

[DeEtta Mills]{.underline}, Joshua Voss, and Laurie Richardson

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

A microbial community is an assemblage of organisms, genes and gene
functions that are linked in a complex web that sustains them as a unit.
It is unlikely that one microbe has all the genes necessary to survive
in any environment or adequately represents the total microbial
community function. Many of the basic concepts for developing a rational
ecological approach to microbial ecology have been difficult due to
methodological limitations. However, these limitations are rapidly
decreasing with expanded use of molecular methods combined with new
computational tools. There is an urgent need to understand what happens
inside complex interactions that exist in the \"black box\" of bacterial
diversity because shifts in bacterial communities can signal onset of
ecological stress or episodic diseases. Development of new genomic tools
allows for design of community-level studies that will interrogate the
finer details of the biological components of a given habitat. One such
molecular tool is amplicon length heterogeneity (ALH) DNA profiling of
bacterial communities. This technique interrogates the variable domains
of the ribosomal small subunit genes (SSU rDNA), and separates these
amplicons on high-resolution genetic analyzers. The assays are based on
natural variation in sequence lengths of specific regions of DNA.
Lengths are independent of restriction enzyme recognition sites thus
eliminating problems associated with other techniques. Data are
phylogenetically relevant because the amplicon lengths can be associated
with specific taxonomic sequences archived in databases. Black Band
Disease (BBD) of corals consists of a microbial biofilm community
dominated by cyanobacteria. The disease has impacted the world's coral
reefs. It was suggested that the BBD community included
disease-associated microbes and community members associated with corals
in the non-disease state. Preliminary data from ALH profiling supported
this idea and indicated that the BBD community is dominated by amplicons
associated with members of the alpha-proteobacteria. Amplicons
representing the members present in the non-disease state community are
present but at lower abundance. With bioinformatics tools, we are
conducting a 'virtual' molecular analysis of BBD. Cloning and sequencing
of BBD samples from different geographical regions and coral species
will verify these *in silico* inferences.

**Recent Studies On Yellow Band Disease Of Scleractinian Corals**

James M. Cervino and [Garriet W. Smith]{.underline}

Marine Science Program, University of South Carolina, Columbia SC, 29208

Yellow band disease (YBD) affects massive corals in the Caribbean,
particularly species in the genus *Montastrea*. Surveys in Bonaire
indicated that as many as 90 % of the *Montastrea* species were affected
with the disease. Measurements of the progression of tissue lesions were
about 0.6 cm/month, but this can increase with temperature and nutrient
loads. Within tissue affected with YBD, fewer zooxanthellae, lower
chlorophyll concentrations and fewer dividing zooxanthellae cells were
reported. Bacteria isolated from YBD affected tissue and inoculated onto
healthy tissue produced typical signs of YBD. Different bacterial
strains, all in the genus *Vibrio*, could induce YBD signs in healthy
tissue, but a combination of strains were more effective. This evidence
indicated that a differential zooxanthellae strain response to specific
*Vibrio* strains might be responsible for YBD.

**Differential Response Of Zooxanthellae To Bacterial Toxins That
Produce Bacterial Bleaching In Stony Corals**

[Diego L. Gil-Agudelo]{.underline}^1,4^, Yael Ban-Haim^2^, Xavier
Hernández^3^, Garriet W. Smith^4^, Eugene Rosenberg^2^, Roberto
Iglesias-Prieto^3^.

^1^Department Marine Science, University of South Carolina, Columbia,
SC, 29208; ^2^Department of Molecular Microbiology and Biotechnology,
Tel-Aviv University, Ramat Aviv, 69978 Israel; ^3^Instituto de Ciencias
del Mar y Limnología, Universidad Nacional Autónoma de México, Unidad
Académica Puerto Morelos, Quintana Roo, México; ^4^Department of Biology
and Geology University of South Carolina at Aiken, Aiken, SC, 29801,

*Vibrio shiloi* is the species responsible for bacterial bleaching in
some stony corals in the Mediterranean Sea. This bacterium produces
extracellular toxins that inhibit photosynthesis of zooxanthellae and
ultimately, lyse the symbiotic algae. It has been hypothesized that this
bacterium might play a role in bleaching of stony corals from the
Caribbean and other areas. To evaluate the possible effects of *V.
shiloi* species in bleaching events from different parts of the world,
zooxanthellae were extracted from corals of the Mediterranean Sea, Red
Sea, and the Caribbean. These zooxanthellae were then exposed to the
extracellular toxins produced by this microorganism. Our results showed
that the zooxanthellae extracted from different corals differed in their
responses to the extracellular materials produced by *V. shiloi*. For
example, *V. shiloi*'s Toxin P more strongly affected zooxanthellae
extracted from *Oculina patagonica* than from zooxanthellae isolated
from Acroporid corals from the Red Sea and Milleporid corals from the
Caribbean. These differences might be the result of variation in the
tolerance to ammonia of different zooxanthellae clades or even species.
It might also be a result of differences in the specific binding
capacity of Toxin P to the algal membranes. These results might have
implications in the tolerance of some corals to bacterial bleaching.
Tolerance can also be the result of host-specificity of the bacteria and
the coral, because the bacteria have to overcome the barrier of the
coral before adhering to the zooxanthellae.

**Histopathology Of The White Pox Disease Of The Caribbean Elkhorn Coral
*Acropora palmate***

[Kathryn P. Sutherland]{.underline}^1^, Esther C. Peters^2^, and Randi
Rotjan^3^

^1^Department of Environmental Health Science, University of Georgia,
Athens, GA 30602; ^2^Tetra Tech Inc., Fairfax, VA 22030; ^3^Department
of Biology, Tufts University, Medford, MA 02155

White pox disease of the Caribbean elkhorn coral *Acropora palmata* is
caused by the enterobacterium *Serratia marcescens.* The disease has
contributed to catastrophic declines in elkhorn coral populations in the
Florida Keys, with losses averaging 88% between 1996 and 2002. White
pox-affected and apparently healthy coral tissues were collected from
diseased colonies of *A. palmata* in August 1998 at Looe Key Reef,
Florida. The tissues were processed for histopathological examination
with light microscopy. Cellular and tissue degeneration was observed in
both apparently healthy and diseased coral tissues. White pox disease is
associated with (1) rounding of granular gland cells in the mesenterial
filaments, (2) atrophy of the coenosarc, (3) necrosis of the coenosarc,
oral disk, gastric cavity, and mesenterial filaments, and (4)
disruptions in the gastric cavity. Most lesions were concentrated in the
coenosarc tissue. There was no significant difference in the number or
type of lesions present in diseased versus apparently healthy samples.
While the frequencies of 41% of the abnormalities showed a significant
difference between apparently healthy and diseased tissues, half of
these abnormalities were more common in diseased tissues. The overall
extent and severity of lesions was significantly different between
apparently healthy and diseased tissues. However, only seven of the 26
individual lesion types showed a significant difference in extent and
severity between apparently healthy and diseased tissues. The
similarities between apparently healthy and diseased tissues suggest
that colonies affected by white pox sustain a whole-colony reaction to
infection.

**Update On The International Registry Of Coral Pathology**

[Shawn M. McLaughlin]{.underline}^1^ and Kathy L. Price^2^

^1^NOAA NOS Center for Coastal Environmental Health and Biomolecular
Research/Cooperative Oxford Laboratory, 904 South Morris St., Oxford, MD
21654; ^2^Jardon and Howard Technologies, 12424 Research Parkway,
Orlando, FL 32826

The International Registry of Coral Pathology (IRCP) was recently
established by the National Oceanic and Atmospheric Administration at
the Center for Coastal Environmental Health and Biomolecular Research in
Oxford, MD in affiliation with the Coral Disease and Health Consortium
(CDHC). The coral registry is designed to offer a safe, permanent
repository where the scientific community can deposit, retrieve, and
study archived materials. The IRCP has expanded its tissue archives to
over 140 specimens this year through the accession of healthy and
diseased coral samples representing sixteen species and eleven
locations. The coral registry has also provided histology and
histopathology support in collaborative coral disease investigations.
For example, the coral registry participated in the development of
collection and fixation protocols during a coordinated CDHC response to
a summer coral mortality event in the Florida Keys. The IRCP prepared
microslides from collected samples and organized an expert
histopathology review panel. Similar research support is being provided
in an ecotoxicology investigation of coral reefs in Bermuda. Further,
IRCP has initiated several technical studies to develop and/or apply new
methods to the study of corals and to help develop standard coral
histology protocols. Six fixatives, four decalcification solutions, and
several stains are being compared in a study to determine optimal
preservation of coral tissues. Petrography methods are being evaluated
in other studies to permit the examination of calcium carbonate
skeletons routinely removed during decalcification. The archives and
investigations of the coral registry are designed to support CDHC goals
to identify coral disease etiologies, develop diagnostic criteria, and
standardize terminology and descriptive pathology.

**Shifting the Paradigm For Coral Reef Health Assessment**

[Cheryl M. Woodley^1^]{.underline} , Craig A. Downs^2^, John E.
Fauth^3^, John Halas^4^, Judy Halas^5^, Elizabeth Fisher^6^ and Pamela
Hallock^6^

^1^NOAA NOS CCEHBR, Hollings Marine Laboratory, 331 Ft Johnson Rd.,
Charleston, SC 29412; ^2^EnVirtue Biotechnologies, Inc., 35 W.
Piccadilly St., Winchester, VA 22601; ^3^Dept. of Biology, University of
Central Florida, 4000 Central Florida Blvd., Orlando, FL 32816; ^4^FL
Keys NMS, P. O. Box 1083, Key Largo, FL 33037; ^5^Environmental
Moorings, Intl, Inc., 172 Lorelane Place, Key Largo, FL 33037;
^6^College of Marine Science, University of South Florida, 140 7th Ave.
S., St. Petersburg, FL 33701

In the last five years a new paradigm has evolved for coral health
assessment, which embraces the use of science and technologies that can
move us from a descriptive science (e.g. a phenomenon has occurred) into
the realm of mechanistic science (e.g. how has the phenomenon occurred).
Coupling the principles of biomedical science (e.g., biochemistry, cell
physiology and functional genomics) with traditional measures of
ecosystem health, produces integrated strategies that can (1) identify a
stressor(s) affecting marine biota, (2) distinguish among different
physiological conditions (3) elucidate mechanisms leading to adverse
health effects resulting from interactions between organisms and their
environment, and (4) predict the consequences of these interactions.
This approach provides a means to understand mechanisms of action at a
molecular, cellular, organismal or population levels that determine
responses of organisms to their environment, and allows marine
scientists to address questions such as: How do marine resources respond
to a changing environment? How can one determine when a system has
exceeded its plasticity in responding to a changing environment and
determine its fate? How can the factors affecting coral reefs be
identified? Answers to these questions will enable identification of
'cause and effect' relationships so that recognition of the 'smoking
gun' of coral decline may give new insights for devising wise management
options.

**The Prophenoloxidase Activating System Of The Lobster**

***Homarus americanus***

[Robert S. Anderson]{.underline} and Tracey M. Luskey

University of Maryland Center for Environmental Science, Chesapeake
Biological Laboratory, P.O. Box 348, Solomons, MD 20688-0038

Previous research has implicated the prophenoloxidase (proPO) system in
non-self recognition and host defense mechanisms of certain anthropods.
This system has been most thoroughly described in insects and
crustaceans, particularly shrimp, crabs and crayfish. However, proPO has
not previously received much study in the commercially important
American lobster. *In vivo* proPO and serine proteases are localized in
hemocyte granules and are released from the cells during phagocytosis or
other situations that trigger degranulation. In the plasma, the proPO
system is activated, producing factors that enhance phagocytosis,
encapsulation, coagulation, fungistatic activity and melanization. Using
hemocyte lysate supernatant (HLS) and trypsin as activator, the presence
of a proPO activating system was shown for *Homarus americanus*. A
low-level proPO activity found in HLS was probably due to endogeneous
serine protease. *In vitro*, proPO activation was strongly stimulated by
trypsin in a dose- and time-dependent manner. However, microbial
non-self determinants such as bacterial lipopolysaccharides (LPS) and
fungal β1,3 glucans failed to activate lobster proPO in HLS. Other
potential activating procedures, such as increasing incubation
temperature or preheating HLS, were not effective. Studies are in
progress to determine if LPS and/or β1,3 glucans promote hemocyte
granulation rather than directly activating cell-free components of the
proPO system. It seems that the proPO system of lobsters differs from
that described in other crustaceans; however, such differences in
mechanistic details are to be expected, based on comparative studies of
proPO activation in marine crabs and freshwater crayfish.

**Identification Of Tumor Necrosis Factor (TNF) And TNF-Receptor
Superfamily Members Expressed In Rainbow Trout (*Oncorhynchus mykiss*)**

# [Gregory D. Wiens]{.underline}

USDA-ARS, National Center for Cool and Cold Water Aquaculture, 11871
Leetown Rd, Kearneysville WV 25430

Tumor necrosis factor (TNF) and its receptor are each prototype members
of large gene superfamilies. Since the discovery of tumor necrosis
factor and lymphotoxin α, 17 additional ligands and 29 cognate receptors
have been identified in mammals. Many members of the TNF and
TNF-receptor superfamilies play important roles in coordinating the
proliferative and protective functions of pathogen-reactive cells. Some
receptors function as decoy receptors as they lack a transmembrane
domain and interfere with ligand signaling. These large superfamilies
may exist in rainbow trout as two ligands, one receptor, and one decoy
receptor have been identified. We searched a database of rainbow trout
expressed sequence tags to identify additional genes that may belong in
the ligand or receptor superfamilies. We have identified and sequenced
two cDNA clones that have homology to TNF ligands and nine cDNA clones
that share homology with TNF receptors. Of the putative receptor genes,
four cDNA clones may be decoy receptors as they lack a predicted
transmembrane domain. RNA expression analysis of these genes in
*Yersinia ruckeri* bath vaccinated and challenged fish is underway.
These data indicated that a number of TNF ligand and receptor
superfamily members were expressed in rainbow trout and suggested that
some of these genes might have arisen early in vertebrate evolution.

**Striped Bass (*Morone saxatilis*) *Nramp* Is Inducible *In Vitro*
After Exposure To LPS And *Mycobacterium marinum***

[Erin J. Burge^1^]{.underline}, David T. Gauthier^2^, and Peter A. Van
Veld^2^

^1^College of Charleston, Hollings Marine Laboratory, 331 Fort Johnson
Road, Charleston, SC 29412; ^2^College of William and Mary, Virginia
Institute of Marine Science, 1208 Greate Road, Gloucester Point, VA
23062

Mycobacteriosis in Chesapeake Bay striped bass (*Morone saxatilis*) is
an ongoing disease problem with potentially important economic
implications for the commercial and recreational fishery. The natural
resistance-associated macrophage protein 1 gene, *Nramp1,* is
responsible for innate resistance to mycobacterial infections in mice.
*Nramp1* is inducible by treatment with antigenically unrelated
intracellular macrophage parasites and by synergistic activity of
bacterial lipopolysaccharide (LPS) and interferon-γ. The striped bass
homolog*, MsNramp*, is strongly induced in peritoneal exudate cells (PE)
after intraperitoneal injection with *Mycobacterium* spp. *in vivo*.
Unlike its mammalian homolog, *MsNramp* is expressed ubiquitously, but
at variable levels depending on tissue type. The purpose of the present
study was to investigate short-term *in vitro* *MsNramp* expression and
reactive oxygen intermediate production in primary cultures of adherent
anterior kidney cells (AK) and PE after exposure to LPS, or live- or
heat-killed (HK) *Mycobacterium marinum*. Peritoneal exudate cells
expressed significantly higher levels of *MsNramp* at 4 and 24 hours
post-treatment with live and HK *M. marinum.* *MsNramp* response to LPS
was dose-dependent in these cells, with maximum expression at 4 hr and
20 μg/ml LPS. Treatment of PE with LPS caused an increase in
intracellular superoxide anion (O~2~^-^) levels, whereas treatment with
live *M. marinum* caused a significant depression. Adherent AK responded
to LPS with increased ROI and *MsNramp* production, but were not induced
or suppressed relative to controls by mycobacteria. These results
suggested that *MsNramp* might be regulated similarly to mammalian
*Nramp1.* This study represents the first report of induction of an
*Nramp* gene by mycobacteria *in vitro* in a poikilothermic vertebrate,
and supports reports of *Nramp* induction by LPS in teleosts.

**The Response Of The Immunome Of Haddock (*Melanogrammus aeglefinus*)
To Vaccination**

[Andrew Dacanay]{.underline}^1^, B. Erin Bentley^1^, Darren Sarty^1,2^,
Laura Brown^1^, Stewart Johnson^1^

^1^ National Research Council of Canada -- Institute for Marine
Biosciences, Halifax, Nova Scotia, B3H 3Z1 Canada; ^2^ Department of
Biology, Dalhousie University, Halifax, Nova Scotia, Canada

Atlantic cod (*Gadus morhua*) are reported to have an absent
immunoglobulin (Ig) response to vaccination. We have found that another
gadid, haddock (*Melanogrammus aeglefinus*), also does not produce
specific Ig to a variety of T~D~ antigens. To determine the mechanisms
of this we investigated the haddock immunome (Ig variable (V~H~),
Diversity (D) or Joining (J~H~) heavy chain gene Ig rearrangements), in
vaccinated and control animals. We obtained \>60 sequences from each
group of naïve or vaccinated fish and these were translated at EXPASY
and aligned by CLUSTAL-W. Identity matrices and entropy plots were
constructed using BioEdit. Almost every sequence had a unique VDJ
structure indicating that they were derived from independent B-cell
rearrangements. Identity matrices showed most of the V~H~ shared \>80%
identity at the nucleotide level and therefore belonged to a single V~H~
family, which showed greatest homology to human V~H~ family II. There
was no preferential usage of any V~H~, D or J~H~ in vaccinated haddock
compared to unvaccinated controls. The immunomes of unvaccinated and
vaccinated haddock were more diverse than that reported for Atlantic
salmon (*Salmo salar*) survivors of an *Aeromonas salmonicida*
challenge. Entropy plots showed a greater diversity in all three
Complementarity Determining Regions (CDRs) and in all groups CDR3 was
relatively short (8 amino acids), a feature shared with surviving
salmon. It appears that, *in lieu* of an antigen-specific Ig response
upon vaccination haddock, and perhaps all gadids, utilise a pre-existing
pool of Ig that has diverse CDR1 and CDR2 as well as a short CDR3,
characteristics seen in Atlantic salmon survivors of *A. salmonicida*
infection, but not in naïve controls. These data suggest that this Ig
pool may be somewhat specific and not "sticky", as has been suggested in
Atlantic cod. Furthermore as haddock have a relatively diverse immunome,
they must also have a relatively diverse genome, yet a limited proteome
has been described, and functional studies have shown limited, if not
absent, Ig function. Our data suggest that the paucity of specific Ig
production upon vaccination is due to an inability to access the B-cell
repertoire, rather than a poor B-cell repertoire itself.

**Potency And Shelf-Life Of A *Streptococcus agalactiae* Vaccine In Nile
Tilapia (*Oreochromis niloticus*)**

[David J. Pasnik]{.underline}^1^, Joyce J. Evans^1^, Phillip H.
Klesius^2^, and Craig A. Shoemaker^2^

Aquatic Animal Health Research Laboratory, United States Department of
Agriculture, Agriculture Research Service, ^1^151 Dixon Drive,
Chestertown, MD 21620; ^2^P.O. Box 952, Auburn, AL 36832

Group B streptococcal infections have been associated with significant
morbidity and mortality in freshwater, estuarine, and marine fish
species. Currently, no commercial fish vaccines are available that
prevent disease caused by the emerging group B streptococcal fish
pathogen, *Streptococcus agalactiae*. Thus an effective vaccine for *S.
agalactiae* could help decrease associated mortalities due to *S.
agalactiae* infection. Juvenile Nile tilapia (*Oreochromis niloticus*)
were used to test the efficacy of a killed vaccine composed of
formalin-killed *S. agalactiae* cells and concentrated extracellular
products. Previous studies showed that fresh vaccine provided
significant protection against *S. agalactiae* challenge. Our objective
was to determine whether vaccine stored at 4ºC for one year remained
efficacious. Tilapia were inoculated with one year-old vaccine by
intraperitoneal (IP) injection, and control tilapia were inoculated IP
with tryptic soy broth (TSB). All fish were then held at approximately
32ºC. At 31 days post-inoculation, fish were challenged IP with
1.7x10^4^ CFU *S. agalactiae*/fish. Fish were monitored for clinical
signs and mortality for 14 days. Blood was also sampled from control and
vaccinated fish on days 0 and 31 for antibody production. In our
previous studies, inoculation with fresh vaccine significantly helped
prevent development of clinical signs and reduced post-challenge
mortalities, yielding a relative percent survival (RPS) of 80. The
vaccine held for one year only produced an RPS of 29 and allowed
development of clinical signs. The year-old vaccine induced
significantly increased antibody production in vaccinates as compared to
controls. However, this production was significantly lower than that
produced with fresh vaccine. This decreased antibody production may
account for the decreased RPS. This study suggested that shelf life of
the vaccine \<1 year when stored at 4ºC, indicating that a fresh
preparation of the vaccine should be utilized to obtain the best
efficacy. Because it is desirable to have vaccine available for multiple
tilapia production cycles, other vaccine preparations (lyophilized) or
storage considerations (freezing) should be assessed.

**Immune Responses Of Channel Catfish Against *Ichthyophthirius*
m*ultifiliis* After Immunization With Live Theronts And Sonicated
Trophonts**

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

USDA, Agricultural Research Service, Aquatic Animal Health Research
Laboratory, P.O. Box 952, Auburn, AL 36831

The ciliated protozoan *Ichthyophthirius multifiliis* Fouquet (Ich)
causes high mortality in fish and leads to heavy economic loss in
aquaculture. The Ich infection is difficult to control with chemical
treatments because the parasite penetrates into fish skin and gill. Fish
that survived an Ich infection acquire immunity against reinfection by
the parasite. Vaccination against the parasite can be used as an
alternative to chemical treatments. This study determined the humoral
immune responses and host protection of channel catfish *Ictalurus
punctatus* (Rafinesque) against *Ichthyophthirius* *multifiliis* (Ich)
after immunization with live theronts and sonicated trophonts, with an
emphasis on assessing the cutaneous immune response. Immunizations with
live theronts or sonicated trophonts were done by both bath immersion
and intraperitoneal (IP) injection. Cutaneous and serum immunoglobulin
(Ig) levels and anti-Ich antibodies were measured 12 and 21 days post
immunization with the enzyme-linked immunosorbent assay and theront
immobilization assay. The level of Ich infection and survival of catfish
were determined after theront challenge at a dose of 15,000 theronts per
fish for 1 hour. Cutaneous and serum anti-Ich antibodies were
significantly higher in fish immunized with live theronts by immersion
or IP injection or with sonicated trophonts administered by IP injection
as compared to fish immunized with sonicated trophonts by immersion,
with bovine serum albumin by IP injection, or non-immunized controls.
Host protection was noted only in fish immunized with live theronts by
immersion or IP injection or with sonicated trophonts by IP injection.
There was a positive correlation between higher levels of anti-Ich
antibodies and host survival in the immunized fish. Specific anti-Ich
antibody levels appear to be a better indicator of Ich tolerance than
total non-specific Ig.

***Bothriocephalus acheilognathi*: The Australian Experience**

[Alistair D.M. Dove]{.underline}

Department of Microbiology and Immunology, Cornell College of Veterinary
Medicine, C/o Marine Sciences Research Center, Stony Brook University

Stony Brook NY 11794-5000

The Asian fish tapeworm, *Bothriocephalus acheilognathi*, was originally
a parasite of carp and related cyprinids in the Amur River basin of
China. It has now been distributed around the world due to the
translocation of its hosts for aquaculture, ornamental display, and weed
control. In the process, the list of fish species that it infects has
grown to over 50, representing six orders of fish and one amphibian. It
is this extraordinarily low host-specificity at all life history stages
that has allowed the worm to establish in non-native habitats, despite
the complexity of its life cycle. Australia has a depauperate freshwater
fish fauna of just 180 species with a high degree of familial and
generic endemism. I collected over 1400 fish from a range of sites in
coastal and inland drainages of eastern Australia. *Bothriocephalus
acheilognathi* was recorded from all well-sampled native hosts that were
collected sympatrically with carp. The worm was not recorded from any
sites where carp were absent. These patterns, combined with the fact
that *B. acheilognathi* rarely matures in native fishes, suggested that
a reservoir of infection in carp was required for infections to persist
in native fishes. Given the potential pathogenesis to hosts (stenosis,
eosinophilia) it appears that parasite-mediated competition may occur
between exotic and native fish species. *Bothriocephalus acheilognathi*
appears to be a silent and underestimated mechanism by which carp can
damage non-native ecosystems.

**Bolbophorus Infections In Catfish: Identification, Impact and
Treatment**

[Andrew J. Mitchell]{.underline}

HKDSNARC/ARS/USDA, PO Box 1050, Stuttgart, AR 72160

*Bolbophorus* is a genus of trmatodes that infects pelicans, rams-horn
snails and fish. The trematodes have a hind- and forebody, lateral
pseudosuckers around the oral sucker, a holdfast organ, interconnecting
excretory channels, and a clear parasite cyst within the host cyst. The
taxonomy of these parasites is in transition and there are at least four
species. Prior to 2002, most of these trematodes were identified as *B.
confusus* , but that nomenclature is now restricted to one species from
Europe. In the United States, *B. damnificus* and other distinct species
(*B*. spp.) are found in fish. *Bolbophorus damnificus* occurs in the
flesh of channel catfish and fathead minnow, while other *B.* spp. occur
in cyprinids, centrarchids, and percichthyids. *Bolbophorus*
*damnificus* causes serious losses in the catfish industry. The
trematode first showed up on a catfish farm in Louisiana in 1994 and has
since been found on \>50 catfish farms in Arkansas, California,
Louisiana, and Mississippi. The trematode continues to spread and losses
continue to occur but, in the last two years, the number of case reports
dropped. This was partially attributed to the ability of farmers to
privately diagnose and implement management and control measures. Pond
shoreline treatments of copper sulfate and hydrated lime as well as the
stocking of black carp have lessened the impact of the parasite. With
the probable listing of black carp as an injurious species, greater
impetus is placed on chemical treatment. Copper sulfate treatment
effectively reduces rams-horn snails that host *B. damnificus.* Ten
pounds (4.5 kg) of copper sulfate applied along 250 ft (76.3 m) of
shoreline in a 6 ft (ca 2 m) wide band reduced snail survival to about
5%. The treatment lost effectiveness below 21°C, but was very effective
even at ½ the copper sulfate rate at temperatures of 31°C and higher.
The hydrated lime treatment, although reported as effective, is not well
defined or tested: recommended treatment rate per shoreline length and
treatment width varied, influence of temperature on the treatment was
unknown, and treatment results were not published. Tests were initiated
to define the effectiveness of the hydrated lime treatment. Initial
results indicated that treatment was effective if 90 lbs (27.5 kg) of
hydrated lime was applied along 100 ft (30.5 m) of shoreline in a 3 ft
(ca 1 m) wide band. The effectiveness of hydrated lime, when applied at
31°C, was dramatically higher than when the same treatment was applied
at 25±2°C. Other tests are planned.

**Preliminary Study On The Mitochondrial Genome Of The Tapeworm
*Bothriocephalus acheilognathi***

[Deborah D. Cartwright]{.underline}^1^, Vicki S. Blazer^1^, William B.
Schill^1^, Katherine A. Beauchamp^1^, and Andrew J. Mitchell^2^

^1^USGS, National Fish Health Research Laboratory, 11649 Leetown Road,
Kearneysville, WV 25401; ^2^H. K Dupree, Stuttgart National Aquaculture
Research Center, 2955 Hwy 130 East, Stuttgart, AR 72160

The tapeworm, *Bothriocephalus acheilognathi* has been a problem with
aquacultured grass carp and golden shiners for about 30 years and has
recently been reported in many other cyprinids**.** Several states
restrict the importation of fish with *B. acheilognathi,* therefore,
identification and detection of these worms are very important. Because
definitive identification methods vital to the inspection process do not
exist, a study was initiated to develop such methods. Molecular genetic
approaches using both nuclear and mitochondrial genes have been used in
studies on the phylogenetic relationship of closely related organisms
including several species of cestodes. Past studies on the internal
transcribed spacer of ribosomal DNA (ITS rDNA) have determined genetic
variation in *B. acheilognathi* in different fish hosts. It was found
that there were three closely related genotypes with certain degrees of
host-specificity. Metazoan mitochondrial sequences are known to evolve
rapidly, but gene arrangements will remain unchanged for long
evolutionary periods of time. Therefore, information on the
mitochondrial genome is important in understanding the evolutionary
relationships of cestodes. In this study we will attempt to look at the
mitochondrial sequences of *B. acheilognathi* using the cytochrome B
region and several primers designed for long chain PCR. Samples of *B.
acheilognathi* from were collected from grass carp at a private
aquaculture facility in Arkansas.

# Cestodes Of Elasmobranches: Friends Or Foes

## [Janine N. Caira]{.underline}

Department of Ecology and Evolutionary Biology, University of
Connecticut, Unit 3043, 75 N. Eagleville Rd., Storrs, CT 06269-3043

Cestodes of sharks and rays exhibit amazing diversity of morphological
forms represented by four orders and approximately 900 species. Three
orders, the Diphyllidea, Lecanicephalidea and Trypanorhyncha, are known
only from elasmobranches; the fourth and most diverse order, the
Tetraphyllidea, primarily parasitize sharks and rays, but a few species
parasitize holocephalans. Essentially all adult cestodes in these orders
parasitize their host's spiral intestines. Neither the evolutionary
relationships among the tapeworms of elasmobranches, nor the
relationships between the tapeworms of elasmobranchs and the 10 orders
of tapeworms that parasitize other vertebrates are well understood. It
seems clear that the four major tapeworm lineages that parasitize
elasmobranches are not each other's closest relatives. Rather, it
appears that several unrelated groups of tapeworms have become
associated with sharks and rays. Second, it is likely that the
associations between the majority of these groups of tapeworms and their
elasmobranch hosts are remarkably ancient, which possibly accounts for
their incredible host specificity. Investigation of the mode of
attachment of a diversity of tetraphyllidean tapeworms reveals that the
interface between each tapeworm and the spiral intestine mucosal surface
is extremely intimate. The morphology of the tapeworm's primary
attachment structure, or scolex, corresponds closely to the surface of
the mucosa at the site of attachment. However, these tapeworms exhibit a
variety of strategies for attachment. Whereas some species insert
themselves into existing spaces offered by the normal configuration of
the mucosa, other species grasp the various surface elements of the
mucosa. Yet others induce changes in the mucosa that facilitate their
attachment. Although it remains to be confirmed, field observations
suggest that larger tapeworm species are more destructive than smaller
species. This observation is interesting given that our field work has
shown that some stingray species routinely host thousands of small
tapeworms, but large tapeworms are uncommon parasites of these animals.
Unfortunately, several major aspects of the biology of these parasites,
which might provide insight into these observations, remain poorly
understood. For example, a complete life cycle has yet to be determined
for any of these tapeworm species.

**Histopathological Examination Of Wild American Eels, *Anguilla
rostrata,* Infected With *Anguillicola crassus***

[Mark S. Sokolowski]{.underline}^1^ and Dr Alistair D.M. Dove^2^

^1^Marine Science Research Center, Stony Brook University, Stony Brook
NY, 11794; ^2^College of Veterinary Medicine, Cornell University, Stony
Brook, NY 11794

*Anguillicola crassus* is a parasite that is native to the Japanese eel
(*Anguilla japonica*) and has been introduced in the European eel
(*Anguilla anguilla*) and American eel (*Anguilla rostrata*). Typically
the introduction of a parasite will have a greater effect on the naïve
rather than its native host. This has been the case in wild and cultured
European eels and cultured American eels. Pathogenesis of *A. crassus*
in eels has included enlarged abdomens, skin ulcers on the posterior
part of the abdomen, red and swollen anus, thickening of the entire swim
bladder wall through the proliferation of epithelial cells and fibrosis,
lymphocytic infiltration of the swim bladder tissue, dilation of blood
vessels in the swim bladder wall, hemorrhaging in the swim bladder, and
swim bladder rupture. We describe the histopathology of *A. crassus* in
wild American eels from the Carlls River in Babylon, NY and compare it
with these previous studies. This is the first examination of the
pathogenesis of *A. crassus* in wild American eels.

**The State Of Science Of Leech Parasites Of Fish**

### Eugene M. Burreson

Department of Environmental and Aquatic Animal Health, Virginia
Institute of Marine Science, College of William and Mary, P. O. Box
1346, Gloucester Point, VA 23062

All fish leeches are in the family *Piscicolidae*. Very few phylogenetic
analyses have been conducted on the fish leeches, but recent molecular
phylogenetic analyses, based mainly on European taxa, suggested that the
family was monophyletic. A larger analysis that will include taxa from
around the world is underway at VIMS. Fish leeches occur in both
freshwater and marine environments, but they are far more diverse in
marine environments where they occur worldwide. Approximately 95% of the
65 recognized genera occur in the ocean. Leeches are not considered to
be important pathogens of fish themselves, although in unusual
circumstances they can cause problems. However, fish leeches are
well-documented vectors for pathogenic haematozoa. Fish leeches are
difficult to identify and this has hindered research on the biology of
many species, including their vector role. Taxonomic research on fish
leeches, funded by the National Science Foundation PEET program
(Partnerships to Enhance Expertise in Taxonomy), is underway at VIMS.
The intent of this program is to produce web-based keys to the world
fauna and to train students in leech taxonomy. Such keys should help
other researchers identify leeches and this may result in increased
attention to the group. The freshwater leech *Myzobdella lugubris* often
causes lesions in the mouth of a variety of freshwater fish, including
recent problems in largemouth bass in Michigan.

**Molecular Characterization Of The Chlamydia-Like Bacterium Associated
With Epitheliocystis In Arctic Char (*Salvelinus alpinus*)**

[Andrew Draghi II^1^]{.underline}, Julie Bebak-Williams^2^, Gregory J.
Tsongalis^3^,

A. Brian West^4^, Vsevolod L. Popov^5^ and Salvatore Frasca Jr.^1^

^1^Department of Pathobiology and Veterinary Science, University of
Connecticut, Storrs, CT 06269-3089; ^2^Freshwater Institute,
Shepherdstown, WV 25443; ^3^Department of Clinical Chemistry and
Molecular Pathology, Hartford Hospital, Hartford, CT 06102;
^4^Department of Pathology, New York University Medical Center, NY
10016; ^5^Electron Microscopy Laboratory, Department of Pathology, The
University of Texas Medical Branch, Galveston, TX 77555-0609

Epitheliocystis is used to describe gill disease associated with
infection by gram-negative, intracellular, chlamydia-like or
rickettsia-like bacteria. Histologically, the agent forms
intracytoplasmic, basophilic, granular inclusions in gill epithelial
cells. For Arctic char to become a viable commodity, disease
identification and control needs to continue to be a major focus of the
industry. As the bacteria associated with epitheliocystis are yet
uncultured, molecular characterization was begun by amplification of 16S
rRNA genetic sequences directly from infected gill. Samples were taken
for histopathologic, ultrastructural analysis and nucleic acid studies
from Arctic char during outbreaks of epitheliocystis. Histopathology of
epitheliocystis was characterized by intracytoplasmic inclusions in gill
epithelial cells with varying degrees of proliferative branchitis.
Special stains, e.g. Gimenez, Macchiavello and Lendrum's techniques, all
reacted positively with inclusions, which was not the case in Atlantic
salmon. Transmission electron microscopic analysis of infected gill
epithelial cells revealed intracytoplasmic inclusions containing round
to elongate reticulate bodies embedded in a granular to fibrillar
electron-dense matrix without distinct dense-core cells. Immunogold
labeling using an antibody to chlamydial lipopolysaccharide demonstrated
moderate labeling of membranes and cytoplasm of reticulate bodies.
Order-specific chlamydiales primers were used in PCR-based experiments
to amplify a 300-bp 16S rRNA gene sequence considered to be a
"signature" region. The PCR products were sequenced directly and
assembled to yield an overall consensus for future phylogenetic studies.
This characterization is the first step to better understand the biology
of this agent.

**Prevalence Of Walleye Discrete Epidermal Hyperplasia And Walleye
Dermal Sarcoma By Age Class In Walleyes (*Sander vitreus*) From Oneida
Lake, New York**

Rodman G. Getchell1, Gregory A. Wooster1, Lars G. Rudstam2, Anthony J.
VanDeValk2, Thomas E. Brooking2, and [Paul R. Bowser1]{.underline}

1Aquatic Animal Health Program, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
NY 14853; 2Cornell Biological Field Station, Department of Natural
Resources, Cornell University, 900 Shackelton Point Road, Bridgeport, NY
13030

Walleye discrete epidermal hyperplasia has been described as a
seasonally limited skin disease caused by two closely related
retroviruses, walleye epidermal hyperplasia virus type 1 and type 2.
Like walleye dermal sarcoma, which is also caused by a retrovirus, the
in-lake transmission patterns of this disease are unknown. We followed
the prevalence of visible proliferative lesions on walleye aged 3 to 8+
years from 1997 to 2003 in Oneida Lake, New York. The percent of fish
developing walleye epidermal hyperplasia lesions increased slowly with
age. The observed prevalence of discrete epidermal hyperplasia does not
support the hypothesis that walleye with visible hyperplastic lesions in
the spring will not develop the disease again the following year. It is
unlikely that all walleye of a given year class will contract discrete
epidermal hyperplasia in their lifetimes as less than 20% of the fish
age 8 and older were infected. This is in contrast to changes in
prevalence of walleye dermal sarcoma with age, a disease that we believe
eventually affects almost all exposed walleye and where prevalence
decreases with age after age 6 suggesting that walleye develop immunity
to the disease.

**Maine Strain II: A Case History Of The Multidisciplinary Analysis Of
An Emerging Infectious Salmon Anemia Virus Strain Variant**

[Peter L. Merrill]{.underline}^1^, Lori L. Gustafson^2^ and Stephen K.
Ellis^2^

^1^ Micro Technologies, Inc., Richmond ME; ^2^United States Department
of Agriculture-Animal and Plant Health Inspection Veterinary Services
(USDA-APHIS VS): ISA Program, Eastport, ME

Infectious Salmon Anemia (ISA) is an important disease of wild and
farmed Atlantic salmon. Strains from Norway, Scotland and North America
have been characterized by genomic sequencing of segments 2, 8 or 6. The
pathogenicity of different ISAV strains was studied by infectivity
trials, vaccine trials, field observations and epizootiology. Recent
publications forwarded the hypothesis that varying levels of
pathogenicity exist among strains, and a model has been presented to
explain how host-adapted, non-pathogenic 'wild' strains may repeatedly
mutate into pathogenic forms. The 2001 ISA epizootic in Maine, like
simultaneous epizootics in contiguous New Brunswick, Canada, was
attributed exclusively to infection with the New Brunswick strain (Maine
Strain I). Recent routine surveillance detected a 'new' strain of ISAV
at a farmed Atlantic salmon site about 100 km from Cobscook Bay,
Maine; - site of the previous epizootic. When this abstract was
prepared, this strain (Maine Strain II) was not associated with elevated
mortality or typical clinical disease. Maine Strain II shared genomic
and epizootiological characteristics with a strain from Nova Scotian
farmed salmon (1997), which was less pathogenic and more closely aligned
with European strains. The Maine Strain II, while similar to the Nova
Scotian strain in some respects, had a novel genomic segment 6 sequence,
which shared homology with both European and North American strains.
Thus far, Maine Strain II has resisted cell culture attempts. This
presentation describes the events that were used to study the Maine
Strain II virus. A brief history of the known strains of ISAV is given
and techniques used to determine the identity and physical
characteristics of the new strain are described. Important epi factors
such as the determination of environmental presence of virus, monitoring
the potential spread of the virus, and putative transmission vectors
such as sea lice are presented. A discussion of biosecurity as it
relates both to site-level and zone-level management is included. A
planned infectivity trial comparing the New Brunswick and Maine Strain
II of ISAV is also summarized.

**Environmental Persistence Of Infectious Salmon Anemia Virus**

[Cem Giray]{.underline}, Deborah A. Bouchard, Keith A. Brockway, Peter
L. Merrill

# Micro Technologies, Inc., 41 Main Street, Richmond, ME

The transmission of Infectious Salmon Anemia virus (ISAV) among
populations at aquaculture sites is not fully understood. Carrier fish
species such as brown trout (*Salmo trutta*) and sea lice (*Caligus
elongatus*, *Lepeophtheirus salmonis*) are implicated as vectors. The
major route of entry is believed to be the gills and seawater is
suggested as a source for its dissemination. It is presently unclear
where and for how long ISAV can survive once it is shed outside of its
host into the natural environment. This study examined the ability to
detect the presence of ISAV in seawater, freshwater, suspended and
bottom sediments, sea lice and blue mussels (*Mytilus edulis*). We also
assessed the ability of ISAV to survive within some of these sample
sources. Cell culture assays and reverse transcription polymerase chain
reaction (RT-PCR) were used to test samples collected from ISAV-affected
sites in Canada and Maine. Detection of ISAV in recently collected
seawater samples was possible by RT-PCR following virus concentration
using an inexpensive seawater filtration system with a sensitivity of 30
virus particles per liter of seawater. The virus was also detected on
pontoon and boat surfaces, and in sea lice and mussels by RT-PCR but not
by cell culture assays. Sequencing of genomic segments from various
isolates indicated that different viral strains were present at the
sites that were tested. Sea lice, which tested positive for ISAV but
were collected from Atlantic salmon (*Salmo salar*) that had tested ISAV
negative, also indicated that there was some possibility of recent host
switching. Seawater and freshwater samples were inoculated in the
laboratory with ISAV at 10^3^-10^4^ TCID~50~/mL final concentration in
order to follow virus loss over time under varying conditions of
temperature and microbial activity through the use of TCID~50~ assays
and RT-PCR. In sterile seawater, ISAV cytopathic effect (CPE) was
observed for up to 3 weeks incubation at 16^o^C and 4^o^C; detection by
RT-PCR was possible for up to 9 weeks after inoculation at both
temperatures. In non-sterile seawater, ISAV CPE was not observed after
24 hours and 2 weeks incubation at 16^o^C and 4^o^C, respectively; ISAV
was detected by RT-PCR after incubation for up to two weeks at 16^o^C
and up to 5 weeks at 4^o^C. Determinations of ISAV longevity in
freshwater, in suspended and bottom sediments, and in blue mussels are
ongoing.

# Critical Factors For The Establishment Of Biosafety Level-3 Aquaria

[Raghunath B. Shivappa]{.underline}, Flavio Corsin, Jay F. Levine

Department of Population Health and Pathobiology, College of Veterinary
Medicine, North Carolina State University, 4700 Hillsborough St.,
Raleigh NC 27606

During the past five years, two viruses never reported previously in the
United States, Infectious Salmon Anemia virus and Spring Viremia of Carp
virus, have been isolated from both cultured and wild populations. These
incidents emphasize the need for active disease surveillance, rapid
detection methods to detect their presence, and vaccines to prevent
their introduction spread, and minimize their impact on aquatic
populations. The study of foreign pathogens and other pathogens
identified in the List A of the World Organization for Animal Health
(OIE) requires biosecure laboratories and aquaria. However, currently
there is only one USDA certified biosafety level 3 (BSL-3) aquarium
located on the West Coast in Washington State. This limitation seriously
hampers research on pathogen diagnostics, transmission and vaccine
development. In addition, lack of guidelines to establish BSL-3 aquaria
further complicates future efforts to develop similar facilities. In
this presentation we will present the results of a review of practices
followed worldwide by BSL-3 facilities. Information was collected using
a structured survey and unstructured data gathering activities. Critical
factors including disinfection practices required by BSL-3 aquaria to
ensure strict biosecurity and containment will also be discussed.

**The Acute Effects Of A Handling Stressor On Select *Oreochromis
aureus* Plasma Components**

[Nora E. Demers]{.underline}

College of Arts & Sciences, 19501 FGCU Blvd. S. Florida Gulf Coast
University, Fort Myers, FL 33965

Numerous plasma components in teleost fishes are useful in demonstrating
stressors and include potentially immuno-enhancing or suppressing
activity. In this preliminary investigation outbred blue tilapia
(*Oreochromis aureus*) were anesthetized with 2-phenoxy-ethanol and
bled. Then the experimental group were revived and stressed by being
held in the air and placed in a shallow bucket of water for
approximately 10 minutes, and bled again. The control fish were kept
under anesthesia until bled again 10 minutes after the initial bleed.
Plasma samples were evaluated for lysozyme, iron, cholesterol, glucose,
and ion levels. Significant differences were found between the control
and experimental groups in some plasma ions and glucose. Plasma iron
levels increased in all fish between the initial and final bleed with a
greater increase in the control fish than in the experimental fish.
Plasma cholesterol levels were significantly decreased in both groups
between the initial and final bleed. No differences were found in plasma
lysozyme levels. The data will be presented and possible implications
related to innate immunity discussed.

**Absence Of Intestinal Histologic Changes In Fingerling Channel Catfish
(*Ictalurus punctatus*) Fed Raw Soybean Meal**

Joyce J. Evans^1^, [David J. Pasnik]{.underline}^1^, Helena Peres^2^,
Chhorn Lim^2^, and Phillip H. Klesius^2^

Aquatic Animal Health Research Laboratory, United States Department of
Agriculture, Agricultural Research Service, ^1^151 Dixon Drive,
Chestertown, MD 21620, and ^2^P.O. Box 952, Auburn, AL 36831

Considerable effort has been made to utilize alternative protein sources
to replace fishmeal. Soybean meal (SBM) is a commonly utilized plant
protein in fish feeds, because it is readily available and inexpensive,
and has a high nutritional value. However, the inclusion of high levels
of SBM in fish diets has usually produced unfavorable effects, such as
reduced growth rates, reduced feed utilization efficiency, and adverse
intestinal histologic changes. Many of these negative effects were
attributed to the heat-labile anti-nutritional factors of SBM, and thus
SBM is commonly heat-treated before it is used. The histopathologic
effects of feeding heat-treated or non-heat-treated raw SBM to
fingerling channel catfish (*Ictalurus punctatus*) had not been
previously assessed, but were studied here. Fish were fed one of six
diets: a diet containing 45% commercial soybean meal (CSBM), or diets
with the CSBM replaced by non-heat-treated raw soybean meal (RSBM0), or
RSBM heated in an autoclave at 130 °C and 22 PSI for 5 (RSBM5), 10
(RSBM10), 20 (RSBM20), and 40 (RSBM40) min. After 10 weeks, tissue
samples were taken from the stomach, proximal intestine, distal
intestine, liver, pancreas, and spleen from fish in each group for
histologic examination. Mild necrotic lesions were found in the gastric
glands, pancreas, and liver of fish in all the groups, including the
control group. Hepatic glycogen deposition was also observed in all the
groups, as was moderate hyperplasia of the bile ducts. Spleen samples
had considerable brown-black pigment deposition around the splenic
corpuscles and diffuse mild-to-moderate congestion in all of the groups.
Generally, these histological effects appeared to be equivocal between
all of the groups, and abnormalities were not noted in the proximal or
distal intestines. These findings suggested that unheated 45% RSBM could
be used in channel catfish feed without causing severe histological
changes routinely associated with SBM utilization in fish.

**Histopathological And Physiological Evidence Of Thyroid Disruption In
Common Carp (*Cyprinis carpio*) From The Middle And Lower Rio Grande
River Basin**

[Luke R. Iwanowicz]{.underline} ^1^ and Vicki S. Blazer ^2^

^1^University of Massachusetts Amherst, National Fish Health Research
Laboratory, Kearneysville, WV 25430; ^2^United States Geological Survey,

National Fish Health Research Laboratory, Leetown Science Center, 11649
Leetown Road, Kearneysville, WV 25430

In mammals the thyroid is a discrete endocrine gland comprised of
thyroid follicles surrounded by a capsule of connective tissue. The
teleost thyroid lacks a capsule, and follicles are often diffusely
distributed along the dorsal aorta and ventrally along the branchial
arches. Ectopic thyroid follicles are occasionally observed in some fish
species; however, they are often present in low numbers and follicles
are morphologically similar to those in the expected regions. Here we
present the presence of hypertrophic, ectopic follicles located in the
posterior kidney of common carp (*Cyprinus carpio*) collected from
middle and lower Rio Grande River Common carp were collected from
multiple sites in the upper, middle, and lower Rio Grande between
mid-October and mid-November of 1997. Occasional solitary or small
clusters of follicles were found in posterior kidney samples from carp
in the upper Rio Grande. They were not observed in all kidney samples,
however, and were generally of expected size for thyroid follicles.
Conversely, follicles observed in carp from the middle Rio Grande were
numerous and generally hypertrophic. They often occurred as foci
containing eight or more follicles. Follicle size was highly variable,
and the columnar follicle epithelium of some follicles was hypertrophied
and vacuolated. Similar observations were made in carp from the lower
Rio Grande, but of lesser severity. Average size and number of thyroid
follicles were determined from individual carp via image analysis (Image
Pro 3.0) and this data will be presented. Total plasma thyroid hormones
(T~3~ and T~4~) were measured using a radioimmune assay validated for
common carp. The T~3~ /T~4~ ratios (an indicator of deiodinase activity)
were less than one at most sample sites. A T~3~ /T~4~ ratio of 5.12 (n =
20, SE = 1.65) was determined from an aberrant site in the middle Rio
Grande, which indicated that there was some physiological thyroid
disruption. The putative role of contaminants is discussed.

**Copper Sulfate Target Animal Safety In Channel Catfish**

# [Ahmed M. Darwish]{.underline}, David L. Straus, and Billy R. Griffin

Harry K. Dupree-Stuttgart National Aquacutlure Research Center

United States Department of Agriculture, Agricultural Research Service

P. O. Box 1050, Stuttgart, AR 72160

A histological study was performed to evaluate the effect of waterborne
exposures of channel catfish (*Ictalurus punctatus*) to copper sulfate
(CuSO~4~) as a therapeutant. Channel catfish were exposed daily for 11
consecutive days to three concentrations of CuSO~4~ representing one,
three and five times the recommended therapeutic level (2.91, 8.71 and
14.55 mg CuSO~4~/L) based on a total alkalinity of 291 mg/L (CaCO~3~).
Copper sulfate was applied in flow through tanks and more than half the
copper was reduced in 1 hour similar to the elimination rates in ponds
(this is due to the rapid uptake of copper by aquatic plants or algae
and the binding to organic and inorganic material). The exposed fish did
not exhibit any mortality, clinical signs or gross or histological
lesions in the following tissues: gill, liver, anterior and posterior
kidney, spleen, axial muscle with attached skin, pyloric intestine,
heart, swim bladder and cornea. The absence of histological lesions was
attributed in part to the brief daily exposure to Cu^++^ (the toxic form
of copper), the use of alkalinity (\> 50 mg/L) to calculate of the
therapeutic dose, and the moderate hardness of the well water used which
is essential to maintain a normal gill function. The results of this
study suggested that the use of CuSO~4~ as a therapeutant is safe
provided that the dose of copper applied is considered in relation to
the total alkalinity of the water. This target animal safety study
followed an approved protocol by the Food and Drug Administration (FDA)
to support the data package required for the future FDA-approval of
copper sulfate as an aquaculture therapeutant in the United States.