***Mycobacterium sp*. Infection in Cultured Cobia (*Rachycentron
canadum)***

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

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

Juvenile cobia, *Rachycentron canadum*, were presented to the Aquatic
Medicine Laboratory of the Virginia-Maryland Regional College of
Veterinary Medicine with the complaint of chronic losses over a two-week
period. Fish, approximately 15 to 20 cm in length, were representative
of the affected population and presented with emaciation, lethargy,
ulcerative dermal lesions, exophthalmia, hyperpigmentation and
hypopigmentation. Necropsies were performed on the fish and bacterial
cultures were taken from the posterior kidney and skin for culture on
TSA and BHI media. Representative tissues were obtained for
histopathology, preserved in 10% neutral buffered formalin and processed
for standard H&E staining. Bacterial cultures from the skin grew
*Aeromonas hydrophila* and *Citrobacter* sp., but bacterial cultures
from the posterior kidney grew only *Aeromonas hydrophila*.
Histopathology of the gill tissue demonstrated mild to moderate lamellar
hyperplasia, while skin lesions showed epithelial ulceration and deeper
dermal necrosis with a few well-formed granulomas. Numerous granulomas
were also observed throughout the spleen, liver, and anterior and
posterior kidney. A modified acid-fast stain (Fites) revealed numerous
acid-fast bacteria within the granulomas and granulomatous inflammation
of the tissues, suggesting a *Mycobacterium* sp. infection. In addition,
bacterial cultures grown on Middlebrook agar developed small, white
colonies in seven days that stained acid-fast positive. Though the fish
were externally and systemically infected with several other species of
bacteria, it was thought that these were secondary to the underlying
*Mycobacterium* sp. infection. The producers were immediately advised to
depopulate and disinfect the systems holding the fish.

**Mycobacterium Infections In New York Striped Bass And Bluefish**

[Mark S. Sokolowski]{.underline} and Alistair D. M. Dove

Marine Science Research Center, Stony Brook University, Stony Brook NY,
11794

Due to the epizootic of mycobacteriosis in Chesapeake Bay striped bass
over the past several years and the mixing of the Chesapeake Bay and
Hudson River striped bass stocks, a survey for mycobacteriosis in the
New York bight area (n=26) and Hudson River (n=53) was conductedHudson
River H. Spleen, liver, and kidney samples were collected for
histopathology and the remaining spleen tissue was homogenized for
bacteriological culture. The prevalence of acid-fast positive bacteria
in the spleens (n=2, 2.5%) was low, with one positive from each site.
Granulomas that were suggestive of mycobacterial infections but were
acid-fast negative were found in the New York bight striped bass spleens
(n=3, 11.5%) and liver (n=11, 42.3%) and in Hudson River striped bass
spleens (n=6, 11.3%), liver (n=22, 41.5%) and kidney (n=1, 1.9%). Thus
the prevalence of definitive and suggestive lesions was very similar in
fish from both locations. *Mycobacterium lentiflavum*, a pathogen known
previously only from people, was the only isolate to be successfully
cultured, although mold contamination hampered culture efforts. The
biology of striped bass and bluefish is similar in several respects
including distribution, prey, and parasite assemblages. For this reason
spleen, liver, and kidney samples were also collected for histopathology
from bluefish (n=51) in the mid-Atlantic bight area. The prevalence of
acid-fast positive bacteria in splenic granulomas was low (n=2 or 3.8%)
but higher than for striped bass; no other granulomas were observed that
were acid-fast negative. Bluefish and striped bass in NY waters are both
infected by mycobacteria, although the species identities of isolates in
each fish remain to be determined.

**Mycobacterial Infections In Striped Bass (*Morone saxatilis*) From
Delaware Bay**

[^1^C. Ottinger]{.underline}, ^2^J. Brown, ^1^C. Densmore, ^1^C.
Starliper, ^1^V. Blazer^­^, ^1^K. Beauchamp, ^3^D. Gauthier, ^3^M.
Rhodes, ^3^H. Kator, and ^3^W. Vogelbein.

U. S. Geological Survey, Leetown Science Center, National Fish Health
Research Laboratory, 11649 Leetown Road, Kearneysville, WV 25430.
^2^U.S. Fish and Wildlife Service, 2610 Whitehall Neck Road, Smyrna, DE
19977. ^3^Department of Environmental and Aquatic Animal Health,
Virginia Institute of Marine Science,College of William and Mary,PO BOX
1346, Gloucester Point, Virginia, 23062

A study was initiated in 2003 to determine if mycobacteriosis is
occurring in Delaware Bay striped bass (*Morone saxatilis*). Eighty
striped bass were obtained from gill-nets off Woodland (n = 70) and
Bowers (n = 10) Beach in December of 2003. Data obtained from these
samples were compared to those obtained in November 2003 from striped
bass captured in pound nets located in the Chesapeake Bay at the mouths
of the Potomac (n = 50) and Nanticoke Rivers (n = 50). Tissues were
examined by selective culture for the presence of mycobacteria and
associated pathology by histology. Pooled data from Delaware Bay
indicated an infection rate of approximately 17% and infection intensity
(Log mean ± S.E.) of 3.398 ± 0.576 Log CFU g^-1^ of spleen in the
infected striped bass. Infection rates in Delaware Bay were
significantly lower (*p* ≤ 0.025) than those observed at the Chesapeake
Bay sites where the rates were 80.4% and 60.4% for the Potomac and
Nanticoke Rivers respectively. Infection intensity in impacted striped
bass was significantly lower than observed in the Potomac but not the
Nanticoke River where the infection intensities (Log mean ± S.E.) were
5.054 ± 0.318 and 4.772 ± 0.384 Log CFU g^-1^ of spleen respectively.
The fish were in good condition overall with only one sampled fish
exhibiting a relatively heavy infection (over 10,000 CFU g^-1^ of
spleen). The isolate from this heavily infected fish, identified as
*Mycobacterium chelonae,* was the only isolate to clearly fit current
species descriptions. Other isolates appear to be similar to recently
described species obtained from Chesapeake Bay striped bass.

Coldwater Disease Caused By Flavobacterium psychrophilum In Ontario;
Characterization Of Strains And Antimicrobial Resistance

S. Hesami, K. Welsh, J. Parkman, [J.S. Lumsden]{.underline} and J.
MacInnes.

Fish Pathology Laboratory, Department of Pathobiology, Ontario
Veterinary College, University of Guelph, Guelph, ON N1G 2W1

*Flavobacterium psychrophilum* is the causative agent of bacterial
coldwater disease in North America and rainbow trout fry syndrome in
Europe. BCWD has a considerable economic impact in Ontario aquaculture
operations; however our limited understanding of bacterial population
structure and epidemiology is an impediment to improved management
strategies. Eighty-five isolates of *F. psychrophilum* were collected
during an 8 year period from farmed salmonids with tail rot, necrotic
myositis and cephalic/scleral osteochondrosis. These isolates were
characterized phenotypically, biochemically and serologically. Two
distinct biovars were identified by API-ZYM, however the isolates were
relatively serologically homogeneous, with 80% recognized by antiserum
raised against *F. psychrophilum* ATCC 49510. To assist identification
of *F. psychrophilum* from clinical samples, a multiplex PCR based on
intergenic 16S rRNA and DNA gyrase was adapted. Antimicrobial
susceptibility profiles of Ontario isolates were determined using a
commercial multi-well plate system. We plan to monitor temporal trends
in sensitivity as antimicrobial use evolves in the Ontario aquaculture
industry. Management strategies used to reduce the impact of bacterial
coldwater disease will also be discussed.

**Detection Of Flavobacteria On Gills And Skin Of Salmonids By PCR And
DGGE**

[Virginia Harper]{.underline} and Roselynn Stevenson

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

Skin and gill diseases involving species of the genus *Flavobacterium*
present major and constant problems in freshwater aquaculture. Routine
diagnosis of Bacterial Gill Disease (BGD) is almost entirely by
microscopic examination of gill tissues for long rods, as culture of *F.
branchiophilum* is difficult. PCR amplification with primers for 16S
rRNA sequences was used to identify species of flavobacteria present on
gills and skin. We tested DNA isolated from 82 gill samples from fish
with bacterial gill disease, representing 10 separate cases. Of these,
27 showed an amplification product with the PCR primers for *F.
branchiophilum,* BRA1 and 1500R. There was no amplification with PSY
primers for *F. psychrophilum.* DNA from bacterial colonies cultured
from gill samples on cytophaga agar did not amplify with either primer
set. DNA from 32 of 70 gill samples was amplified with eubacterial rRNA
GM5 primers (with GC clamp). Denaturing Gradient Gel Electrophoresis
(DGGE) analysis showed 26 samples had bands coincident with the product
from DNA of *F. branchiophilum*. Bands resembling *F. psychrophilum*
were in two samples. Amplifying DNA from cytophaga agar cultures from
gills with GM5 primers gave a product in 69 of 70 samples. On DGGE gels,
the bands indicated mixed cultures of bacteria, including 9 samples with
bands coincident with *F. branchiophilum*, and 32 with patterns similar
to *F. psychrophilum*. With PSY-primers and DGGE, *F. psychrophilum* was
detected in skin scrapings of diseased fish.

**Application Of DGGE Techniques As A Potential Approach To Non-Lethal
Testing For Fish Pathogens**

Matt Baker, Virginia Harper, and [Roselynn Stevenson]{.underline}

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

Non-lethal sampling procedures are desirable for detecting pathogen
carriers among broodstock fish. Using the method of Cipriano *et
al.*(1992), *Aeromonas salmonicida* could be cultured from the skin
mucus of Ontario freshwater Chinook and coho salmon and rainbow trout.
To broaden the range of bacteria that could be detected in mucus
samples, we used denaturing gradient gel electrophoresis (DGGE) to
analyze 16S ribosomal RNA obtained from PCR-amplification of bacterial
cultures and from mucus directly. First, 25 cultures were isolated from
the mucus of salmon and trout from the Credit River, Dorion Hatchery,
Chatsworth Hatchery and Hagen Aqualab to assess diversity of bacterial
communities associated with fish mucus. Physiological tests carried out
with Biolog GN2 and GP2 plates identified 18 species of bacteria,
predominantly gram negative. These isolates were used to establish the
biodiversity of mucus isolates. No bacterial species were common to all
sources. Eubacterial GM5 primers with a GC clamp were used to amplify a
200 bp segment of 16S rDNA directly from fish mucus and also from
cultures of *Aeromonas salmonicida*, *Flavobacterium branchiophilum*,
and *Yersinia ruckeri.* Mucus of fish from two sources did have bands
corresponding to those of *A. salmonicida* and *Y. ruckeri,* but
sequences of the bands are required for confirmation, as related
non-pathogens may share conserved sequences.

**Signature-Tagged Mutants For Studies Of Pathogenic Mechanisms Of
*Yersinia ruckeri* In Rainbow Trout**

[Indervesh]{.underline} and Roselynn M.W. Stevenson

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

The virulence factors and the pathogenic mechanisms that enable
*Yersinia ruckeri* to cause Enteric Red Mouth disease in salmonid fish
are still unclear. To identify possible virulence-related genes, a
library of 1056 transposon mutants of a serotype 1 strain of *Y.
ruckeri,* RS1154, was prepared and screened for mutants unable to
survive in rainbow trout for 7 days after a bath-immersion infection.
For efficient screening, the mutants were prepared using 11 signature
tags present on pUT miniTn5 km2 transposon vectors. Groups of 11 mutants
with different tags were screened by infecting two adult rainbow trout
by bath immersion for 10 min in 10^8^-10^9^CFU/ML of each mutant in
challenge dose. At 7 days post-infection, fish kidney tissue was
cultured, and recovered mutants were screened by PCR for missing tags.
In initial screening, 132 mutants were missing in both fish. New pools
of these mutants were re-screened in fish, and 24 mutants (2.2% of the
library) were selected for further characterization. Southern blots with
the kanamycin marker indicated each had only a single mutation. Cloning
and sequencing the mutations allow homology searches for genes that may
help explain the persistence of *Y. ruckeri* in carrier fish*,* and
identify the key pathogenic processes that are triggered in the stressed
host.

**Estimation Of The Genome Size Of *Renibacterium salmoninarum* ATCC
33209 By Pulsed-Field Gel Electrophoresis.**

[Gregory D. Wiens]{.underline}^1^ and Mark S. Strom^2^

^1^USDA-ARS, National Center for Cool and Cold Water Aquaculture, 11861
Leetown Rd, Kearneysville, WV 25430; ^2^Northwest Fisheries Science
Center, National Marine Fisheries Service, 2725 Montlake Blvd. E.
Seattle, WA 98112

*Renibacterium salmoninarum* is a salmon and trout pathogen that belongs
to the high G + C subgroup of gram-positive bacteria. Little is known
about the molecular genetics of this microorganism. Here, we used
pulsed-field gel electrophoresis to estimate the genome size of the ATCC
33209 type strain. Digestion of genomic DNA with *Pme*I generated four
bands containing five restriction fragments with a cumulative size of
3.4 [+]{.underline} .1 Mb. Seven previously identified *Renibacterium*
genes were mapped to the *Pme*I fragments by Southern blotting. Both
*hly* (metalloprotease) and one copy of *msa* (major soluble antigen or
p57) were mapped to *Pme*I fragment I, while the other copy of *msa*,
*lysB* and three IS994 insertion-element flanking-genes were mapped to
*Pme*I fragments II/III. Unlike the duplicated *msa* gene, only single
copies of *hly*, *lysB* and the three IS994 insertion-element
flanking-genes were identified by Southern blotting. In summary, these
data suggest that the genome size of *R. salmoninarum* ATCC 33209 is
close to the average for prokaryotes (3.2 Mb), and that most *R.
salmoninarum* genes are not duplicated.

**An Introduction To Canadian Aquaculture**

[David B. Groman]{.underline}

Aquatic Diagnostic Services, Atlantic Veterinary College, University of
Prince Edward Island, 550 University Avenue, Charlottetown, Prince
Edward Island, Canada. C1A 4P3

This presentation will highlight the current status of commercial
aquaculture in Canada, providing information on production data and
commercial focus for future development. A comparison to the current
status of the United States aquaculture industry will be provided.
Information regarding key emerging and re-emerging infectious disease
problems facing commercial fin-fish and shellfish (molluscan)
aquaculture in Canada will be detailed.

**Number range CHAPTERThe Current Status Of *Loma salmonae*, Disease And
Control In Farm Raised Chinook Salmon, *Oncorhynchus tshawytscha***

[Jan Lovy]{.underline}^1^, David J. Speare^1^, and Glenda M. Wright^2^

Departments of ^1^Pathology and Microbiology and ^2^Biomedical Sciences,
Atlantic Veterinary College, UPEI, Charlottetown, PEI Canada

Microsporidial Gill Disease (MGD), caused by the pathogen *Loma
salmonae* is an important disease affecting the culture of Pacific
salmon in British Columbia. The industry, which currently produces
16,000 metric tonnes annually and has a value of \$40 million dollars,
undergoes seasonal losses of up to 13% of the stock attributed to MGD.
In addition to research into vaccination and treatment, an important
consideration is understanding the nature of the host inflammatory
response to the disease. This is particularly relevant for MGD because
morbidity and mortality begin late in the course of infection, prompted
by the severe host reaction elicited by the rupture of the spore-filled
xenomas. A study of the ultrastructural pathology of MGD has revealed
several novel findings including (1) the discovery of dendritic-like
cells within acute lesions, (2) arterial thrombosis, and (3) chronic
inflammatory lesions with neovascularization. This suggests that
rupturing xenomas cause an inflammatory cascade that start as tissue
destructive acute lesions which persist and develop into chronic lesions
characterized by macrophage infiltration and neovascularization. Based
on this, future work into treatment will also focus on determining the
therapeutic benefit of anti-inflammatory agents during xenoma rupture
and resolution.

> **Emerging Infectious Diseases Of Atlantic Cod (*Gadus morhua*),
> Haddock (*Melanogrammus aeglefinus*) And Halibut (*Hippoglossus
> hippoglossus*)**
>
> [Daryl S. Whelan]{.underline}
>
> Newfoundland and Labrador Department of Fisheries and Aquaculture,
> Aquaculture Health Unit, PO Box 8700, 30 Strawberry Marsh Road, St
> John's, Newfoundland and Labrador, Canada A1B 4J6
>
> Alternative marine species culture of cod, haddock and halibut has
> resulted in the detection and investigation into disease issues not
> commonly encountered in salmonid production. Nodavirus and Loma have
> been detected in stocks cultured in eastern Canada. Fish health
> practitioners have adapted terrestrial mammal and salmonid biosecurity
> practices to mitigate or negate these threats to the continued
> commercialization of these species. Extensive applied research
> endeavors are underway to remove or control these pathogenic agents.
> This presentation will highlight current and emerging infectious
> viral, bacterial and parasitic threats to these coldwater marine
> finfish species in Atlantic Canada.

**Multiple Drug Resistance In *Aeromonas salmonicida* Infected Salmon
Fry And An Update On Infectious Salmon Anemia In The Bay Of Fundy
Region, Atlantic Canada**

[Michael J. Beattie]{.underline}

New Brunswick Dept Fisheries & Aquaculture, 61A Wallace Cove Road,
Blacks Harbour, New Brunswick, Canada. E5H 1G9

In May 2005, the New Brunswick Department of Agriculture Fisheries and
Aquaculture (DAFA) was informed of an *Aeromonas salmonicida*
(furunculosis) outbreak at a hatchery in the northeast section of the
province. Investigation indicated that *A. salmonicida* had massive
resistance to most known antibiotics. including all potentiated
sulfonamides, erythromycin, naladixic acid, amoxicillin, enrofloxacin,
florfenicol, oxytetracycline, oxylinic acid, streptomycin, ceftiofur,
and chloramphenicol. The DAFA immediately generated a plan to prevent
persistence or spread of this bacterium into the surrounding
environment. The plan consisted of five major pillars; risk to workers
health, risk to human health (consumers), environmental risk, risks
associated with depopulation, and needs for immediate and future
studies. The facility was depopulated within 7 days of notification and
the case was submitted to DFO, CFIA and Health Canada. Since the
outbreak, DAFA determined, with the help of personnel at RPC
laboratories, that the *A. salmonicida* was infiltrated with a first
class fully integrated integron. Integrons are defined as potentially
mobile DNA elements that comprise a site-specific recombination system
capable of capturing and expressing genes contained within cassette
structures. The integron is over 100 kbp's in size and further DNA
analysis has uncovered the genetic sequence of the resistance packages
within each of the cassettes. The presence of such a diverse array of
resistance genes has not been reported previously for *A. salmonicida*.
This is the first isolation of this class of beta-lactamase in *A.
salmonicida* and is identical to the plasmid pSCR1 identified in
*Salmonella* isolated in China and the United States. The most
significant finding was that the MDR strains possessed a multiple gene
cassette, which demonstrated a very high degree of sequence homology and
structural organization with the antibiotic resistance cassette of SXT
element of *Vibrio cholerae* found in India. The resistance pattern was
not associated with irresponsible or indiscriminant use of antibiotics.
Resistance packages in many bacteria could be the result of untreated
effluent from hospitals being discharged into local river systems, in
addition to the ease at which these integrons are accepted by
nonpathogenic aquatic bacteria. An update on the status of Infectious
Salmon Anemia (ISA) will also be presented. In addition new information
will be provided on the typing of segments 5 and 6 of the ISA structure.

**Identification Of A New HPR Group Of Infectious Salmon Anemia Virus In
New Brunswick: What Does It Mean?**

[Molly Kibenge]{.underline}^1^, Biao Qian^1^, Sandi McGeachy^2^, Shebel
Hariharan^1^, and Frederick Kibenge^1^

^1^Department of Pathology and Microbiology, Atlantic Veterinary
College, University of Prince Edward Island, Charlottetown, PEI; ^2^New
Brunswick Department of Agriculture, Fisheries and Aquaculture,
Fredericton, New Brunswick, Canada

Infectious salmon anemia (ISA) is a highly fatal disease of
marine-farmed Atlantic salmon caused by ISA virus (ISAV). The virus also
infects several marine salmonid and non-salmonid fish species resulting
in asymptomatic carriers of the virus. Such benign infections in the
wild fishery are considered to be the source of virulent strains that
cause clinical disease in marine-farmed Atlantic salmon. It has been
hypothesized that the ISAV variant without any deletion in the
haemagglutinin-esterase (HE) protein, designated HPR0 to indicate a
full-length highly polymorphic region (HPR) in HE, represents the
avirulent archetype of the virus from which the virulent strains arise
by deletion of several nucleotides in the HPR. Because HPR0 virus has
not been isolated in cell culture, it remains a source of diagnostic
confusion, particularly in surveillance programs with ISAV
RT-PCR-positive fish that are negative on virus isolation. This is
compounded by the variations in susceptibility of the different salmonid
cell lines used to detect ISAV. Our laboratory has been typing ISAV
isolates from disease outbreaks on Atlantic salmon farms in New
Brunswick, Canada, in order to gain detailed knowledge about the
epidemic strains of ISAV. We report here the identification of a new
European HPR group of ISAV which replicates poorly in the ASK-2 cell
line. Of all HPR groups reported to date, this new HPR group is the
closest to HPR0 in terms of the pattern of deletion associated with this
unique phenotype, and indicates a direct molecular relationship between
cytopathogenicity of ISAV in cell culture and ability to cause clinical
disease in farmed Atlantic salmon.

**Current Status Of Canada's National Aquatic Animal Health Program
(NAAHP)**

[Rod Penney]{.underline}

Aquaculture Management Directorate, Fisheries and Oceans Canada, Ottawa,
Ontario. K1A 0E6

The Office of Aquatic Animal Health (AAH) of the Canadian Food
Inspection Agency (CFIA) in conjunction with Science Branch of Fisheries
and Oceans Canada (DFO) are implementing the federal component of the
National Aquatic Animal Health Program (NAAHP). On February 23, 2004,
\$59 Million in funding over 5 years was announced in the federal budget
for the NAAHP, which, when implemented, will secure seafood production
and trade, improve governance on aquaculture that meets domestic and
international standards, protect Canadian aquatic resources (wild and
farmed) from serious infectious diseases and help ensure access to
international markets on an on-going basis. The federal NAAHP will apply
to all reportable diseases of aquatic animals (currently of finfish,
molluscs, crustaceans), and will cover all sectors that use fisheries
resources in Canada (e.g. aquaculture, the capture fishery, seafood
processing, and live fish and seafood trade. The program will add
legislative and infrastructure support for aquatic animal health and
seafood certification that is consistent with other food sectors. Roles
and responsibilities of the CFIA include program direction, trade
certification, international negotiation, risk analysis, aquaculture
surveillance, contingency planning, zonation, and the incorporation of
new aquatic animal health requirements into the existing Regulations
under the federal Health of Animals Act (HAA). Roles and
responsibilities of DFO include management of a national diseases
database, diagnostics, technology development, disease research and wild
surveillance. An Aquatic Animal Health Committee, co-chaired by DFO and
CFIA, has been established to coordinate broad NAAHP initiatives.

**The National Aquatic Animal Health Plan: Briefing And Update**

[Jill Rolland]{.underline}

U.S. Department of Agriculture, Animal & Plant Health Inspection
Service, National Center for Animal Health Programs, 4700 River Rd. Unit
46, Riverdale, MD 20737

The Joint Subcommittee on Aquaculture's National Aquatic Animal Health
Task Force has been charged with the mission to develop and implement a
national aquatic animal health plan (NAAHP) for aquaculture in
partnership and cooperation with industry, regional organizations,
State, local, tribal governments and other stakeholders. The purpose of
the NAAHP is to foster and support effective and efficient aquaculture;
to protect the health of our nation's wild and cultured aquatic
resources; and to meet our national and international trade obligations.
The NAAHP is being developed via input from a series of Task
Force-associated working groups. Each working group consists of 10-20
experts, each representing a sector of the aquaculture community. Each
work group focuses on a specific element of the NAAHP such as: roles and
responsibilities of health professionals, laboratory methodologies and
species-specific working groups that address issues for that sector.
Several work groups have met and recommendations are considered when
drafting the NAAHP. The first complete draft of the NAAHP is expected to
be completed in the spring of 2006, with refining and implementation to
follow. The NAAHP in itself will not be codified into regulation;
however, implementation of certain elements, such as import
requirements, may require revisions to existing laws, regulations or
policies.

> **The Proposed Use Of The Non-Native Oyster *Crassostrea ariakensis*
> In Chesapeake Bay**

# Eugene M. Burreson

> Virginia Institute of Marine Science, College of William and Mary,
> Gloucester Point, Virginia 23062

The abundance of the native oyster in Chesapeake Bay, *Crassostrea
virginica*, has declined drastically over the last four decades because
of mortality caused by two protistan diseases, *Haplosporidium nelsoni*
(MSX) and *Perkinsus marinus* (Dermo). Annual harvest levels in
Chesapeake Bay between 1930 and 1959 averaged about 5.5 million bushels,
but have declined to less than 50,000 bushels in the last decade.
Interest in the use of a non-native oyster to rehabilitate the fishery
and to provide ecological services began in the 1990s. Field trials with
sterile, triploid Pacific oysters, *Crassostrea gigas*, revealed that
they did not grow well in the low to moderate salinities of Chesapeake
Bay. Interest shifted to the Suminoe oyster, *Crassostrea ariakensis*,
and field trials using sterile triploid oysters demonstrated faster
growth and greater survival compared with the native oyster.
*Crassostrea ariakensis* seems to be totally refractory to *H. nelsoni*
as no infections of this pathogen have ever been observed. Infections of
*P. marinus* are acquired, but intensity levels remain low and no
mortality has been observed caused by this parasite. Taste tests have
demonstrated wide acceptance of the product in the marketplace. Virginia
had originally proposed the use of sterile, triploid *C. ariakensis* as
an aquaculture product to prevent inadvertent introduction of
reproducing non-native oysters. Maryland was originally opposed to any
use of *C. ariakensis*, but a new governor elected in 2004 has reversed
this position and now Maryland is proposing the introduction of diploid,
reproducing *C. ariakensis* to rehabilitate the public oyster fishery in
Maryland. A decision has recently been delayed from March 2005 until
July 2005 to allow more research. Maryland Department of Natural
Resources and NOAA have provided over \$3 million for research.

**Detection And Identification Of Oyster Herpes Viruses (OsHV) And
*Perkinsus* Species From Oysters In Asia: Relevance To Chesapeake Bay**

[Kimberly S. Reece]{.underline}^1^, Jessica A. Moss^1^, Christopher F.
Dungan^2^, Ryan B. Carnegie^1^, and ^1^Eugene M. Burreson^1^

^1^VIMS, The College of William and Mary, Gloucester Point, VA 23062;
^2^Maryland DNR, Cooperative Oxford Laboratory, Oxford, MD 21654

Decline of native *Crassostrea virginica* populations in the Chesapeake
Bay region due to overfishing, disease, and habitat destruction, along
with continued heavy disease pressure has led to interest in using the
relatively more disease tolerant Asian oyster, *Crassostrea ariakensis,*
for aquaculture development, fishery resource enhancement and habitat
restoration. *Crassostrea ariakensis* oyster samples from China, Japan,
Korea, and from US hatcheries were surveyed for OsHV and *Perkinsus*
species using molecular diagnostics. Although several oysters were also
examined for parasites and potential pathogens by histology, very few
were found. Two cases of *Perkinsus* spp. were found at Beihai, in
southern China, by histopathology. PCR screening revealed *Perkinsus
spp.* and/or OsHV DNA in many additional samples. Of 18 Asian sites
surveyed, 9 showed evidence of OsHV and 13 evidence of *Perkinsus* spp.
None of the hatchery samples contained OsHV DNA, but local *Perkinsus*
spp. were detected in VIMS hatchery groups. Sequencing of PCR products
from Asian oysters positive for *Perkinsus* indicated that two species
of *Perkinsus* not currently found in Chesapeake Bay were present; *P.
olseni*, and a new, undescribed species. We are conducting studies to
determine the potential impact these non-native pathogens could have on
local bivalve species in case of accidental introduction. Live oyster
samples from southern China were recently brought into quarantine at our
laboratory in order to further characterize the pathogens through
challenge and transmission studies and to develop in vitro cultures of
*Perkinsus* species.

**Epizootiology Of *Bonamia* sp. In Non-Native Oysters, *Crassostrea
ariakensis*, In North Carolina, And Implications For Aquaculture**

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

^1^Virginia Institute of Marine Science, Gloucester Point, Virginia
23062; ^2^University of North Carolina Institute of Marine Sciences,
3431 Arendell Street, Morehead City, North Carolina 28557

At Bogue Sound, North Carolina, in July and August 2003, very high
mortality (\> 85%) was observed in experimental lots of
hatchery-produced *Crassostrea ariakensis*. A microcell parasite was
observed at high prevalence and intensity in moribund oysters fixed
during the height of the epizootic, and in subsequent samples from an
October deployment to the same area. PCR amplification confirmed this
parasite to be a *Bonamia* sp., and DNA sequencing established it as a
possible new species. This *Bonamia* sp. has been monitored in
experimental *C. ariakensis* and wild *Ostrea equestris* in Bogue Sound
(the only area in which it has yet been detected) since November 2003.
It has persisted in *C. ariakensis*, and has been found also in *O.
equestris*, which may be a reservoir of the parasite; it has been
transmitted, with new infections established, from at least May (2004
data) through October (2003, 2004); and it has shown a greater
infectivity towards smaller (\< 40 mm) oysters. The latter finding,
combined with information on parasite salinity tolerance, may allow
aquaculturists to avoid the parasite or mitigate its impact by using
separate nursery and grow-out locations: maintaining oysters through
their window of susceptibility in more growth-marginal but parasite-free
environments, then transferring to more growth-optimal locations for
culture to market size.

**Salinity Effects On The Persistence Of *Bonamia* sp. In C*rassostrea
ariakensis***

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

^1^ Virginia Institute of Marine Science, College of William and Mary,
Rt. 1208 Greate Road, Gloucester Point, VA 23062;^2^ Institute of Marine
Sciences, University of North Carolina at Chapel Hill, 3431 Arendell
Street, Morehead City, NC 28557

The Asian oyster *Crassostrea ariakensis* is a candidate for
introduction to Chesapeake Bay in part because it resists diseases
caused by the parasites *Haplosporidium nelsoni* and *Perkinsus
marinus*. Its susceptibility to *Bonamia* parasites, however, emerged as
a key issue in 2003 after the finding of a new *Bonamia* sp. in *C.
ariakensis* dying in Bogue Sound, North Carolina. This parasite has been
observed in high salinity environments and it is not clear whether it
would be infective in lower salinity waters of Chesapeake Bay. To
address this issue, experimental trials were designed to investigate the
persistence and potential transmission of *Bonamia* sp. under three
salinities: 30, 20 and 10 psu. Salinity tolerance was also investigated
*in situ* by deploying *C. ariakensis* along a salinity gradient from
Bogue Sound to Neuse River through Core Creek Canal. Field and
experimental trials results have suggested that salinity plays a
significant role in *Bonamia* sp. dynamics. Field sites that experienced
salinities lower than 30 ppt were characterized by an absence of
infection acquisition, while in the laboratory trials oyster mortality,
parasite prevalence, and salinity were positively correlated.

Polydora-Associated Shell Disease: Recent Distribution In Native And
Non-Native Oysters, And Implications For A Crassostrea ariakensis
Introduction.

[Ryan B. Carnegie]{.underline} and Eugene M. Burreson

Virginia Institute of Marine Science, Gloucester Point, Virginia 23062

The Asian oyster species *Crassostrea ariakensis* is favored by some for
introduction to Chesapeake Bay because it is "disease resistant". This
oyster's "disease resistance" does not necessarily extend beyond native
oyster parasites *Haplosporidium nelsoni* and *Perkinsus marinus*,
however, and other results suggest *C. ariakensis* may not even be more
resistant than *Crassostrea virginica* to all disease agents active in
Chesapeake Bay. The shell-burrowing polychaete *Polydora websteri* is
very active in mesohaline environments like Chesapeake Bay and a common
pest of native *C. virginica*. Its burrows impair oyster shell
structure, leaving the oyster potentially more vulnerable to crab
predation; when the inner shell surface is penetrated, the host responds
with production of conchiolin blisters, an energetic cost that could
leave the oyster more vulnerable to parasitic infection. Side-by-side
trials since 2003 at eight sites in and around Chesapeake Bay have
compared *P. websteri* infestation and conchiolin shell blistering in
*C. ariakensis* and *C. virginica*. While infestation levels, heaviest
inside Chesapeake Bay, are similar between hosts, conchiolin blistering
is much more intense in *C. ariakensis*, a probable function of this
oyster's production of a thin and easily penetrated shell. The
repercussions of this for aquaculture are profound. Besides potential
losses to crab predation, a serious question is whether enough *C.
ariakensis* may be produced and sold to high-end markets to make
aquaculture of this species in Chesapeake Bay a viable enterprise.

> **Recent Trends In The Distribution And Abundance Of *Haplosporidium
> nelsoni* (MSX) And *Perkinsus marinus* (Dermo) In Native Oysters In
> Chesapeake Bay**
>
> [Eugene M. Burreson]{.underline} and Ryan B. Carnegie
>
> Virginia Institute of Marine Science, College of William and Mary,
> Gloucester Point, Virginia 23062

*Haplosporidium nelsoni* is an introduced pathogen that began causing
mortality in lower Chesapeake Bay in 1959; it rapidly spread up the Bay
throughout areas where salinity was greater than 15 psu. *Perkinsus
marinus* has probably always been an associate of oysters in the Bay,
but intensified and spread throughout the Bay during drought periods of
the late 1980s. These two pathogens combined have caused a drastic
reduction in the abundance of the native oyster *Crassostrea virginica*.
Within Chesapeake Bay, *H. nelsoni* distribution and abundance are
controlled by salinity with few infections below 10 psu. *Perkinsus
marinus* is distributed throughout all oyster beds in Chesapeake Bay,
but its abundance and impact fluctuate with salinity. Generally, for
both pathogens, their abundance and impact decrease in wet years and
increase in dry years. Beginning in summer 1998 the Chesapeake Bay
watershed experienced over four years of consecutive drought conditions.
During only three months over that entire period was streamflow above
average. These drought conditions and resultant increased salinity
caused a dramatic increase in abundance of both *H. nelsoni* and *P.
marinus*, and widespread oyster mortality. Intensity of *P. marinus*
infections was the highest ever observed in 40 years of monitoring.
Unfortunately, funds for restoration of native oysters also increased
during this period, so we were trying to restore native oysters under
the worst possible disease pressure. 2003 was an extremely wet year, as
was the summer of 2004. With the resultant decline in salinity,
abundance of both *H. nelsoni* and *P. marinus* declined significantly
and mortality from these pathogens was low during 2003 and 2004.

**Coral Diseases: An Overview And Update On Black Band Disease In The
Bahamas And The Florida Keys**

[D. K. Mills]{.underline}, S.Raju, J. D. Voss, E. Remily, J. Myers and
L. L. Richardson

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

Over the past twenty years, a dramatic increase in coral diseases has
caused massive mortalities in many species of corals worldwide. The
species and the number of coral infected can vary from year to year, but
an overall increase is evident. Black band disease (BBD) is one of
several coral diseases that contribute to coral reef degradation. Since
1972, BBD has spread throughout the western Atlantic, Indo-Pacific, the
Red Sea, and the Great Barrier Reef. We have been studying the
prevalence, disease incidence, and severity of BBD on reefs near Lee
Stocking Island, Bahamas and the Florida Keys. It known that the biofilm
responsible for BBD is composed of a diverse microbial community. As the
disease consortium moves across the coral surface it creates an
"extreme" environment of anoxia and sulfide that destroys the host
tissue. Earlier characterization of BBD was based on bacterial
morphology, selective media and light microscopy that identified the
cyanobacteria, sulfate-reducers and other members of the microbial mat.
More recently, we have cultured an assortment of heterotrophs and
cyanobacteria from different BBD sampled in both Lee Stocking Island,
Bahamas and the Florida Keys. In addition to molecular community
profiling, we have started to clone and sequence many of the isolates
from BBD samples. The preliminary results of the members of the
community, including a comparison of cultivative and non-cultivative
approaches, will be presented. Insight as to which populations of
microbes are members of this pathogenic consortium will provide more
definitive assessment of the disease and its dominant members.

**Comparison Of Bacterial Communities Between Geographically Separated
Corals Infected With White Plague Type II**

[Geoffrey Cook]{.underline}^1^, Patrick Gillevet^1^, Esther Peters^2^,
J. Paige Rothenberger^1^, Masoumeh Sikaroodi^1^, and Robert B. Jonas^1^

^1^Dept. of Envr. Sci. and Policy, George Mason Univ., Fairfax, VA;
^2^Dept. of Envr. Sci. and Policy, George Mason Univ., Fairfax; ^2^Tetra
Tech, Inc., and George Mason Univ., Fairfax, VA

White plague type II (WPII) is a disease of scleractinian corals which
characteristically destroys tissue beginning at the edge of the colony.
This investigation probed fundamental questions about the bacterial
community associated with WPII and the tissue/cell responses by
comparing WPII-diseased corals with apparently healthy controls. We
collected tissue cores from healthy and diseased *Montastraea annularis*
(complex) from the U.S. Virgin Islands (USVI) and the Bahamas. The
tissues were analyzed using a combination of molecular (amplicon length
heterogeneity -- LH-PCR) fingerprinting, microbial culturing, 16S rRNA
gene sequencing, and histological examination. We hypothesized (1) that
the causative agent(s) is/are opportunistic pathogen(s) normally present
in the host or its environs rather than a novel, obligate pathogen; (2)
that corals exhibiting WPII disease signs from different geographical
regions harbor differing microbial communities in healthy and diseased
tissue; and (3) the WPII disease process is the result of a broad shift
in the microbial community (dysbiosis). The LH-PCR fingerprints indicate
that the bacterial diversity in healthy *M. annularis* tissue from the
Bahamas was significantly greater than in samples from the USVI. In the
Bahamas bacterial diversity was lower in the diseased tissue compared to
healthy tissue, but that was not true for the USVI. Principal component
analysis of LH-PCR data from pairs of corals suggest that the microbial
community on control corals and apparently healthy areas of diseased
corals were similar at each geographical location, but the microflora on
the Bahamian corals differed markedly from that on the USVI corals.
Similarly, the fingerprints of the active diseased samples from each
geographic location clustered together, but there was little clustering
between the different locations. An amplicon corresponding to that of
the bacterium *Aurantimonas coralicida* was found in tissue from
diseased and healthy coral from the Bahamas but not from the USVI.

**Prevalence And Potential Origin Of The Acroporid Serratiosis Pathogen
In The Florida Keys**

[Kathryn P. Sutherland]{.underline}^1,\ 2^, E. K. Lipp^1^, and J. W.
Porter^2^

^1^Department of Environmental Health Science, 206 Environmental Health
Science Bldg., University of Georgia, Athens, GA 30602; ^2^Institute of
Ecology, University of Georgia, Athens, GA 30602

In the Florida Keys, populations of elkhorn coral, *Acropora palmata,*
have been decimated by white pox. The fecal enteric bacterium, *Serratia
marcescens,* has been identified as a cause of the disease. To reflect
its etiology, when white pox lesions are associated with *S.
marcescens*, the disease is referred to as acroporid serratiosis. While
*S. marcescens* is common in clinical settings and in the guts of
vertebrates and invertebrates in terrestrial and freshwater
environments, little is known of the prevalence and ecology of this
bacterium in the marine environment. Terrestrial and reef environments
in the Florida Keys were surveyed for *S. marcescens*. Samples were
collected from *A. palmata, Coralliophila abbreviata*, *Hermodice
carunculata*, parrotfish feces, reef water, beach water, canal water,
wastewater influent and final effluent (Key West Wastewater Treatment
Plant), and seabird guano. Samples were plated onto MacConkey Sorbital
agar (MCSA). Colonies appearing pink to red on MCSA were tested for
DNase activity. Isolates were confirmed to species level using PCR
directed at 16S rDNA. More than 250 *S. marcescens* isolates were
confirmed, and the majority (90%) originated from human sewage and
polluted nearshore sources. Molecular fingerprints for each confirmed
isolate were generated by restriction enzyme digestion followed by
pulsed field gel electrophoresis. Band patterns between isolates were
compared using presence and absence of specific DNA fragments to
determine the potential origin of the acroporid serratiosis pathogen.

**The Role Of *Vibrio* spp. In Coral Disease: From Normal Microbiota To
Pathogens**

Kiho Kim^1^ and [Garriet W. Smith]{.underline}^2^

^1^American University, Hurst Hall 101, 4400 Massachusetts Ave NW,
Washington DC 20016; ^2^University of South Carolina Aiken, Department
of Biology and Geology, Aiken, SC 29801

Various species within the genus *Vibrio* have been reported as
associates of corals for some time. Members of this genus were indicated
as normal inhabitants of the surface mucopolysaccharide layers from a
number of coral species. In addition, the relative population levels of
bacteria with carbon source utilization patterns consistent with
*Vibrio* changed with the health of certain corals. More recently, a
number of *Vibrio* isolated have been identified as pathogens for
specific coral diseases. In some cases more than one species was
required to cause significant disease signs. This paper discusses the
range of *Vibrio* species shown to play a role in disease and proposes
testable mechanisms of how this may take place, primarily, through
environmental induction of toxin genes.

**Use Of Fluorescence *In Situ* Hybridization For The Identification Of
*Aurantimonas coralicida* In Environmental Samples**

[E. R. Remily]{.underline}^1^, D. K. Mills^1^, R. Sekar^1^, J.
Pinzon^1^, J. E. Foley^2^, L. L. Richardson^1^

^1^Florida International University, Miami, Fl, 33199, ^2^ Center for
Vector-borne Disease Research, University of California, Davis, CA 95616

One of the most virulent coral diseases, white plague type II, was first
described in 1995 on coral reefs of the Florida Keys. The pathogen for
this disease has been identified as *Aurantimonas coralicida*. A
fluorochrome-labeled probe was designed based on the full sequence of
its 16S rRNA gene, and used with a fluorescence *in situ* hybridization
protocol. The probe was optimized using pure cultures of *A. coralicida*
and tested for specificity against closely related bacteria. A
sensitivity test was conducted to determine the concentration of *A.
coralicida* needed in a sample to result in a positive identification.
Results revealed that 4 out of 10 cultures tested hybridized with the
probe in question. One of the four may be present in the reef
environment. The concentration of *A. coralicida* cells necessary for a
positive identification varied with the method used. One method, which
concentrated cells by settling and centrifugation, required at least
2250 cells/ml for detection by the probe, while a modified filter method
could detect *A. coralicida* down to 10 cells/ml. Currently the probe is
being tested on environmental samples from the disease line of infected
colonies, healthy tissue on diseased colonies, healthy colonies, and
sediment from the reefs. Results are being used to determine not only
the presence/absence of the pathogen in coral populations, but also in
the determination of the pathogen's reservoir.

Coral Mucous And Indigenous Bacteria: A Possible First-Line Defense
Against Coral Disease Pathogens

[John T. Lisle]{.underline}

U.S. Geological Survey, Florida Integrated Science Center for Coastal &
Watershed Research, St. Petersburg, FL

Coral reefs worldwide are undergoing a significant decline in species
abundance and changes in community structure. A contributing factor to
this decline is bacterial infections of scleractinian corals. Recently,
a bacterial pathogen, *Serratia marcescens*, has been identified as the
etiological agent for acroporid serratiosis (white pox disease). The
current hypothesis for the development of this disease is based on a
dose-response relationship between *S. marcescens* and the affected
host, *Acropora palmata*. However, mechanisms of natural resistance
associated with coral systems have not been determined. To address this
issue, bacterial isolates (n=150) recovered from mucus samples collected
from corals in the Florida Keys were screened for their ability to
inhibit or prevent growth of *S. marcescens* and *E. coli*, using a soft
agar overlay method. Isolates that demonstrated inhibition or prevention
of growth of either pathogen were further characterized using Biolog GN2
plates and sequencing of the 16S rRNA gene. Approximately 5% (n=7) of
the isolates demonstrated pathogen inhibition. All isolates were
biochemically identified as *Vibrio alginolyticus*. However, their
relative 16S rRNA sequence data positioned two isolates as closely
related to *V. alginolyticus*, while the remaining isolates were related
to *V. harveyi* (n=1), *V. proteolyticus* (n=2), and *V. fisheri* (n=2).
All of the characterized *Vibrio* species were capable of preventing or
inhibiting the growth of *S. marcescens* and *E. coli*. These results
indicate bacterial disease mechanisms in coral reef systems are much
more complex than the current model indicates and that the coral mucus
layer acts as a primitive immune system, possibly providing a first-line
defense against the establishment of pathogens.

**Customs-Confiscated Corals: Gifthorses Requiring Oral Examination**.

[Andrew Routh]{.underline}^1^, Rachel Jones^2^ and Ashley Sharp^3^

^1^Senior Veterinary Officer, ^2^Deputy Team Leader, Aquarium,
^3^Aquarist, Zoological Society of London, Regents Park, London NW1 4RY,
UK

Corals are currently exhibited in the Aquarium at the London Zoo and are
propagated in anticipation of the 2008 opening of "BIOTA!" a new
aquatic-themed facility in east London. Live corals will feature
strongly in "BIOTA!" exhibits to highlight coral reef conservation. The
Zoological Society of London (ZSL) has policy statements for the
acquisition of all animal species. At irregular intervals, ZSL is
offered animals that have been seized by HM Customs. Their accession to
the collection falls within ZSL acquisition policies. A large proportion
of ZSL's coral specimens have been thus acquired. Many of the coral
species traded legitimately are the hardier, more easily propagated
species. The care of confiscated corals can be challenging. Many of them
are the more delicate hermatypic species and have been almost invariably
taken, often illegally, from the wild. During subsequent quarantines and
holding, individual colonies of *Euphyllia* spp. and *Goniopora* spp.
have been found with burdens of the protozoan *Helicostoma* sp. Areas of
tissue death have also been noted on a number of colonies of *Montipora*
spp. and *Porites* spp. caused by nudibranch predation, all believed to
be members of the genus *Phestilla*. Treatment of these coral colonies
was to be further complicated by the desirable presence on the *Porites*
of the commensal Christmas tree worms *Spirobranchus* spp.
(tube-dwelling serpulid polychaetes). Treatment regimes for these
conditions will be described. In the light of ZSL's experience with
these confiscated corals, protocols for examination and management are
being reviewed. Quarantines and routine quarantine treatments will in
the future be more proactive, in particular as coral holding and
propagation capacity will increase in the run-up to the opening of
"BIOTA!" The London Zoo Aquarium is land-locked. Above and beyond risks
to corals within the quarantine holding and, more broadly, within the
zoological collection, the potential future risk of the release of
potential pathogens, via waste water, to the wild has to be addressed.
This risk is going to be greatest at coastal institutes where identical
or similar species of coral occur in the wild.

Preparation Of Coral Tissues For Histological Examination: A Primer On
Fixation And Decalcification

[Kathy L. Price]{.underline}^1^, Esther C. Peters^2^, Shawn M.
McLaughlin^3^

^1^Jardon and Howard Technologies Inc., Orlando, Florida; ^2^Tetra Tech,
Inc., Fairfax, Virginia; ^3^NOAA NOS Center for Coastal Environmental
Health and Biomolecular Research/Cooperative Oxford Laboratory, Oxford,
Maryland

Microscopic examination of tissues and cellular structure has long been
a component in the diagnostic process of human and veterinary pathology,
and there is a critical need to investigate the cellular alterations
associated with coral diseases. Achieving optimal fixation of coral
tissue is the essential first step in this diagnostic process. Numerous
chemical formulations are available which preserve tissue morphology,
yet each has a varying impact on the integrity of tissues with
implications for interpretation of normal histology and the description
of lesions in corals. Selection of a fixative is determined primarily by
the objectives of the study; however, the logistics of specimen
collection often require a balance between achieving optimal
preservation of tissues with ease of use in the field. Selection of a
decalcifying solution for traditional paraffin processing will also
affect the final product and success of subsequent procedures. In this
study, the quality of tissue fixation in *Porites astreoides*,
*Siderastrea radians*, and *Plexaurella fusifera* was compared using six
fixative formulations and three decalcification solutions. Evaluation
included effect on tissue architecture, staining quality, nuclear
morphological characteristics, and impact on zooxanthellae. In addition,
some field situations necessitate the temporary storage of collected
coral specimens in ambient seawater prior to fixation. The length of
time between collection and subsequent immersion in fixative has a
negative impact on tissue quality, as observed in samples of *Porites
compressa*, *Montipora capitata*, and *Pocillopora damicornis* fixed in
seawater-diluted Z-Fix concentrate at intervals of 10, 100, and 200
minutes after collection.

**The Control Of Fish Parasites And The Eradication Of Asian Tapeworm
Infections In Grass Carp Using Praziquantel Bath Treatments**

[Andrew J. Mitchell]{.underline}

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

There are few studies on the control of fish parasites and even fewer
effective or accepted methods for the direct or indirect control of
these parasites. When parasite eradication from fish stocks is the goal,
the probability for success is very low. Condition of host, life cycle
of parasite, water quality, temperature, and chemical formulation, dose,
and contact time are some of the factors that affect the outcome of
control methods. In this session, presentations will be made on the use
of chemicals to control alternate hosts, pre-parasitic phases, and
parasites on the hosts. Presentations will also be given on management
techniques to limit the number of pre-parasitic phases in a water
system, to prevent parasite infection through disinfection, and to
potentially enhance the production of natural host-produced substances
that kill invading parasites. A number of states require that imported
fish be free of the Asian tapeworm *Bothriocephalus acheilognathi*.
Eradication of this tapeworm from the intestines of infected fish can be
achieved by Praziquantel bath treatments. To obtain successful
treatment, consideration must be given not only to dose, but also to
treatment duration, fish holding concentration, and the intensity of the
tapeworm infection. At a low fish density (6 g fish/L of water) a 1.5
mg/L Praziquantel bath treatment for 24 h is completely effective in
eliminating tapeworm infections from heavily infected grass carp
*Ctenopharyngodon idella* (30-150 tapeworms/fish).

**Sea Lice Control In The Maine Aquaculture Industry**

[Mike Pietrak]{.underline}

Maine Aquaculture Association, P.O. Box 148, Hallowell, ME 04347

The first epidemic outbreak of sea lice, *Lepeophtheirus salmonis* and
*Caligus elongatus*, in Maine occurred in 1994. Since that time the
Maine aquaculture industry initiated a program to control the sea lice
population on farmed Atlantic salmon. From 1996-2000 the control program
relied on the use of Excis which has cypermethrin as the active
ingredient under an Investigative New Animal Drug (INAD) application.
From 2001 to the present, the program switched to SLICE, again under an
INAD, which has emamectin benzoate as the active ingredient. In addition
to chemotherapeutant use, a strong emphasis has been placed on
continuous refinement of industry best management practices (BMP). These
include regular use of practices such as site and/or bay fallowing and
single year class sites and zones. Both the Excis and SLICE INADs have
relied on the establishment of a monitoring program on all farms which
desire to treat their animals under the INAD. This monitoring has
provided almost a decade of information on sea lice populations around
Maine's salmon farms. Continuous improvement in industry best management
practices and the use of Slice have helped to bring the management of
sea lice populations under control.

**The Role Of Antimicrobial Polypeptides In Resistance To Parasitic
Diseases Of Fish**

[Edward J. Noga]{.underline}^1^, Angelo Colorni^2^, and Umaporn
Silphaduang^3^

^1^Department of Clinical Sciences, College of Veterinary Medicine,
North Carolina State University, 4700 Hillsborough Street, Raleigh, NC
27606 USA; ^2^National Center for Mariculture, Israel Oceanographic and
Limnological Research, Ltd., P.O. Box 1212, Eilat 88112 Israel;
^3^Department of Food Science, University of Guelph, Guelph, Ontario N1G
2W1 Canada

Antimicrobial polypeptides (AMPP) are host-produced innate defenses that
typically have potent, broad-spectrum activity against many serious
pathogens. We have identified several types of AMPP in fish. One group
of these AMPP comprise the histone-like proteins (HLPs), small (\~12-25
kDa), highly basic proteins that have been isolated from channel catfish
(*Ictalurus punctatus*), rainbow trout (*Oncorhynchus mykiss*) and
hybrid striped bass (*Morone saxatilis* x *M. chrysops*). HLPs are
highly lethal to the important marine ectoparasite *Amyloodinium
ocellatum* and interestingly target the trophont (feeding) stage, which
is highly resistant to conventional drug treatment. Piscidins are small
\~2.5 kDa AMPP initially isolated from hybrid striped bass. We now have
evidence that piscidin-like AMPP are probably present in a taxonomically
wide range of fish species, especially members of the Perciformes. Both
HLPs and piscidins are expressed in high concentrations in healthy fish
at portals of entry for potential pathogens such as skin, gill and
gastrointestinal tract. Piscidins display highly potent activity against
a number of fish pathogens, including viruses, bacteria (gram-positive
and gram--negative) and parasites. Unlike most AMPP isolated from other
animals, piscidins are also effective in the presence of significant
salt concentrations, suggesting that they function in both marine and
freshwater environments. The sensitivity of certain AMPP to chronic
stress indicates their potential usefulness as health biomarkers in many
fish species.

**Further Elucidation Of The Life Cycle And Pathology Of The Digenetic
Trematode, *Bolbophorus damnificus***

[L. M. Pote]{.underline}^1,^ M. C. Yost^1^, C. M. Doffitt^1^, B. S.
Dorr^2^, D. T. King^1^, A. Camus^1^, and D. Wise^1^

^1^Department of Basic Sciences, College of Veterinary Medicine,
Mississippi State University, Mississippi State, MS 39762; ^2^USDA/WS
National Wildlife Research Center, Mississippi State, MS 39762

The digenetic trematode *Bolbophorus damnificus* has been associated
with high mortalities in channel catfish in the Mississippi Delta. The
American white pelican and the snail *Planorbella trivolvis* have been
identified as hosts for this parasite based on life stages found in each
host. In this research, a series of studies were done to: confirm stages
of this life cycle in a single study; determine if other fish- eating
birds and snail species could serve as hosts; and confirm the
pathogenicity of this parasite. An artificial infection in which
American white pelican and *P*. *trivolvis* were exposed to *B.*
*damnificus* metacercariae and ova, respectively, demonstrated that the
American white pelican shed *B*. *damnificus* ova for 4 months
post-infection; snails shed cercaria 23 days post-infection, and fish
mortality occurred 13 days post-exposure to cercariae. In a series of
studies where the snails *Physella gyrina* and *P*. *trivolvis*
(Mississippi Delta) and *P. trivolvis*, subspecies (North Dakota) were
exposed to *B. damnificus* ova, only *P*. *trivolvis* became infected. A
pen study in which American white pelicans, cormorants, great blue
herons, and great egrets were artificially infected with *B*.
*damnificus* metacercaria, demonstrated that the American white pelican
was the only bird to develop a patent infection. A series of studies
where fish were exposed to 200 *B*. *damnificus* cercariae resulted in
mortalities at 6 d post-infection. These studies confirmed: stages of
the *B*. *damnificus* life cycle; cormorants, great egrets and blue
herons and *P*. *gyrina* are not hosts for this parasite; and 200 *B*.
*damnificus* cercaria can cause high mortalities in catfish 6 days
post-infection. Research supported by grants: USDA/NRI:
2002-35204-11678; USDA/IPM: 2002-34103-11858; USDA/SCRAC:
2002-38500-11805.

**Evaluation Of Copper Sulfate To Control Snail Numbers In Catfish Ponds
Affected By *Bolbophorus* Trematodes**

David Wise, Chuck Mischke, Todd Byars, and [Al Camus]{.underline}

National Warmwater Aquaculture Center, 127 Experiment Station Road,
Stoneville, MS 38776

*Bolbophorus damnificus* emerged as a threat to channel catfish
aquaculture in 1995. Fingerlings rapidly succumb to heavy infections
while sublethal infections decrease feed consumption and increase
susceptibility to *Edwardsiella ictaluri* in fish of all sizes. The
digenetic trematode's life cycle involves ram's horn snails *Planobella
trivolvis* and catfish as first and second intermediates, respectively,
and American white pelicans *Pelicanus erythrorhynchos* as final
hosts*.* Control has focused on eliminating snails from ponds. Chemical
methods rely on hydrated lime or copper sulfate applied in high
concentrations along pond edges. These treatments are effective,
provided snails are concentrated on pond margins, but fail if they are
dispersed away from the bank. A 24-hr LC50 of 2.4 mg/L CuSO~4~^.^5H~2~O
was determined for snails, independent of water alkalinity. Studies were
conducted to evaluate the efficacy and safety of 2.5-5.0 mg/L treatments
applied to entire pond volumes with alkalinities averaging 239 mg/L as
CaCO~3~. In 0.25-10-acre ponds, greater than 95% of caged snails were
killed with only minor fish losses. In a heavily infested commercial
pond treated with 5.0 mg/L CuSO~4~^.^5H~2~O, 96.5% of caged snails and
99% of snails along the bank died. Prior to treatment, 20,000 lbs of
fish had been lost to enteric septicemia and columnaris disease. An
additional 2,000 lbs died within 24 hrs of treatment. Although some risk
of fish losses exists, in high alkalinity waters typical of the
Mississippi Delta, results suggest copper sulfate applied above
traditional guidelines (alkalinity ÷ 100) effectively salvages
production in ponds heavily impacted by *Bolbophorus* trematodes.
Research supported by grants: USDA/IPM: 2002-34103-11858; USDA/SCRAC:
2002-38500-11805.

**Integrated Control Program For The Sea Lamprey, *Petromyzon marinus*,
Macroparasite Of The Great Lakes**

[Michael A. Boogaard]{.underline} and Cynthia S. Kolar

U.S. Geological Survey, Upper Midwest Environmental Sciences Center,
2630 Fanta Reed Road, La Crosse, Wisconsin 54603

The sea lamprey (*Petromyzon marinus)*, an ancient parasitic eel-like
fish native to the Atlantic Ocean, was first discovered in Lake Ontario
in 1835. Construction of the Welland Canal in 1919 provided sea lampreys
access to the remaining Great Lakes and by 1947 they were prevalent
throughout the entire Great Lakes basin. Sea lampreys prey on
commercially important species such as lake trout (*Salvelinus
namaycush*) by living off blood and body fluids of adult fish. During
its life as a parasite one sea lamprey can kill 40 pounds or more of
fish. Coupled with commercial over-harvest, sea lampreys were a major
cause of the collapse of the Great Lakes fishery in the 1940s and 1950s.
In response to this collapse, an extensive bi-national effort by the
United States and Canada and headed by the Great Lakes Fishery
Commission was launched in the late 1950s to control the sea lamprey.
Today, sea lamprey populations are maintained at about 10% of their peak
level in the 1960s by an integrated pest management control program that
includes the use of selective chemicals, mechanical and electrical
barriers, traps, and sterilization and release of spawning males. In
this talk, we will provide background on sea lamprey, evolution of the
control program, and new control methods now under development.

# **Control Measures For *Heterosporis* Species**

Peggy Stelzig^1^, Daniel Sutherland^1^, [Susan
Marcquenski]{.underline}^2^

^1^ Biology Department, University of Wisconsin-La Crosse, La Crosse WI
54601; ^2^Wisconsin Department of Natural Resources, Box 7921, Madison
WI 53707

*Heterosporis* sp*.* is a newly identified microsporidan parasite that
infects muscle cells of fish. Although first discovered in wild yellow
perch, *Perca flavescens*, in Wisconsin, Minnesota, Michigan and the
province of Ontario, other natural infections have since been confirmed
in walleye, northern pike, rock bass, pumpkinseed, burbot, sculpin and
trout-perch. Laboratory studies have shown that rainbow trout, Coho
salmon, brook trout, brown trout, lake trout, white suckers, mosquito
fish, channel catfish, fathead minnow, and largemouth bass are also
susceptible to infection. Bluegill, lake sturgeon, smallmouth bass and
golden shiners exposed to spores remained uninfected. Fish are infected
by consuming live, infected fish or pieces of infected muscle, and by
contact with spores suspended in water. Until its appearance in North
America, *Heterosporis* sp. infections were described in cichlids and
angelfish from Europe, bettas from Thailand and Japanese eels from
Taiwan. There is potential for routine fisheries activities to
accidentally spread the parasite to new lakes. Several control measures
have been tested and the following were found to inactivate
*Heterosporis* spores: 1) Complete desiccation for 24 hours; 2) Freezing
at -20^o^C for 24-hours; and 3) immersing spores in a chlorine solution
for 5-minutes (3 cups household bleach \[6% chlorine\] in 5-gallons of
water). Nets, raingear, live wells and boats can be treated using these
protocols to reduce the risk of moving spores to new locations. This
parasite has the potential to infect multiple species wherever it is
introduced. Although there is no evidence that *Heterosporis* sp*.* can
infect people, it is likely that anglers will discard infected fillets
due to changes in texture and quality.

**Some Effects Of *Philometra* s*altatrix* On Bluefish Ovaries**

[Walter R. Burak]{.underline}, Alistair D.M. Dove, David O. Conover

Marine Sciences Research Center, State University of New York, Stony
Brook, New York 11794

The prevalence and intensity of an ovarian nematode parasite,
*Philometra saltatrix*, and its effect on bluefish (*Pomatomus
saltatrix*) reproductive potential was investigated. Prevalence of live
*Ph. saltatrix* in ovaries of adult bluefish (*n* = 168) captured in
2004 was highest in July at the height of the spawning season, when 39 %
of sampled fish were infected with live worms. Intensity of infection
was also highest in July at an average of 2.4 worms per female fish.
Standard histological examinations of ovaries revealed pathologies
including hemorrhage, inflammation, and follicular atresia associated
with the infection. Damage to ovarian tissues may affect bluefish
fecundity. The highest prevalence of live worms in young-of-the-year
(YOY) fish obtained in 2004(*n* = 243) was 22.2%, which occurred in
August. All infections of YOY were in the pericardial cavity except for
two fish in which worms were found in the body cavity. The first
intermediate hosts in all known philometrid life-cycles are cyclopoid
copepods, and it is possible that YOY bluefish, while still
zooplanktivorous, first become infected by feeding on these copepods.
Alternatively, bluefish may acquire the parasite once they have become
piscivorous through either a paratenic or a second intermediate fish
host. This is supported by the fact that the smallest infected specimen
was 59 mm in length, and certainly piscivorous.

**Gross Observations From A Chesapeake Bay Fish Survey**

[Cynthia Stine]{.underline}^1^, Andrew Kane^1,2^, Madeline Sigrist^1^,
Ana Baya^1,3^

^1^Department of Veterinary Medicine, University of Maryland, College
Park, MD; ^2^University of Maryland, Department of Epidemiology and
Preventive Medicine, Division of Environmental Epidemiology and
Toxicology, Baltimore, MD; ^3^Maryland Department of Agriculture, Fish
Health Laboratory, College Park, MD

A variety of fishes, including striped bass (*Morone saxatilis*) and
Atlantic menhaden (*Brevoortia tyrannus*), were sampled during 2004 from
the Chesapeake Bay. External lesions were observed including areas of
reddening, fin erosion, as well as ulcers. Skin scrapes and gill
biopsies for parasitology were performed, and a variety of protozoan and
metazoan parasites were found. Striped bass were more heavily
parasitized than menhaden, and consequently, parasites of striped bass
represented more taxa than those of menhaden. The most common parasites
observed from striped bass were ciliates (*Trichodina*, *Ambiphyra)* and
copepods (*Ergasilus*) on the skin and gills*,* acanthacephalans
(*Pomphorhynchus bulbocolli*) in the distal gut, and nematodes in the
viscera and peritoneum. Other parasites observed from striped bass
included *Ichthyobodo* and *Trichophyra* in the gills and on the skin,
and red nematodes in the gut. Leeches and the branchiuran *Argulus* were
also observed from the skin of several species. Interestingly, the
majority of menhaden sampled were free of parasites, except for
observations of isopods (*Olencira praegustator*) commonly found in the
oral cavity of fish from multiple river systems. While parasitology
concentrated on observations from striped bass and menhaden, findings
from other fish species included an abdominal mass in spot, cataracts in
striped bass and white perch, and lordosis in white perch. These
findings illustrate the breath of observations that can be found in wild
fish populations.

**Spinal Deformity In Triploid Grass Carp *Ctenopharyngodon* *idella***

[Stephanie G. Grimmett^1^]{.underline}, H. J. Chalmers^2^, J. C.
Wolf^3^, P. R. Bowser^1^

^1^Aquatic Animal Health Program, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
NY 14853, ^2^Department of Clinical Studies, Cornell University Hospital
for Animals, College of Veterinary Medicine, Cornell University, Ithaca,
NY 14853, ^3^The Registry of Tumors in Lower Animals, Experimental
Pathology Laboratories, Inc., 22900 Shaw Road, Suite 107, Sterling,
Virginia  20166

In mid-2004, grass carp, *Ctenophayngodon idella (*Valenciennes*),*
showing evidence of spinal deformity were presented to the Aquatic
Animal Health Program, Cornell University. The carp were from two
separate locations in New York State. The first case, involving several
fish, was submitted by the New York State Department of Conservation
(NYSDEC) staff. The second was a single affected individual from a
backyard pond in the Ithaca region. In early 2005, another fish showing
similar signs was submitted from a hatchery in a third location. In all
cases, the fish were at least seven years of age and had been stocked
for weed control. In the first case, there was concern that
electroshocking for biological surveys may have caused the spinal
lesions however fish from the backyard pond and hatchery had never been
shocked. Radiographs and Computer Tomography (CT) scans revealed the
deformities to be of bony origin. This paper discusses general causes of
spinal deformity in cultured and wild fish, and the methods by which
spinal deformity may have occurred in the submitted fish.

**Deaths associated with vascular pathology in hybrid striped bass**\
\
[Lester Khoo]{.underline}^1^, Jeff Wolf^2^, Chris Weaver^1^ and Leon
Weiss^1^

^1^ University of Pennsylvania, School of Veterinary Medicine, New
Bolton Center, 382 West Street Road, Kennett Square, PA 19348; ^2^
Registry of Tumors in Lower Animals, Experimental Pathology
Laboratories, 22866 Shaw Rd., Sterling, VA  20166

Low level mortalities (about 10 of 3600 fish) began appearing in
approximately one year old hybrid striped bass that had been raised in
recirculating systems consisting sixteen 1136-L tanks configured in
clusters of four. These fish were purchased as approximately 1-cm
fingerlings and were part of a feed trial consisting of four commercial
diets that varied in the level of protein and fat. Diets did not appear
to be of consequence since there were affected fish from different tanks
on each of the diets. Larger fish were most often affected. There was an
acute onset of clinical signs with affected fish appearing listless and
separating from the rest of the fish in the morning. They were sometimes
unable to maintain equilibrium and hold an upright position in the water
column. By the afternoon, these fish had developed exophthalmia (usually
bilateral), pendulous abdomens and were sometimes dead. Upon necropsy,
significant gross findings included a coelomic cavity that was filled
with clotted (sometimes unclotted blood). Source of the blood could not
be grossly determined. These fish also had pale, friable livers and pale
gills. Bacterial cultures of the kidneys did not reveal any known
pathogens. Histologic examination of the formalin fixed tissues revealed
irregular proliferative growths on the adventitial surfaces of the heart
(bulbus arteriosus, ventricle and pericardium), serosal surface of the
GI tract, and the capsular surface of the liver. These lesions consisted
of incompletely blood filled channels lined by variably plump spindle
shaped cells arranged haphazardly. These cells had large condensed or
open faced nuclei (the latter with prominent nucleoli) and scant
cytoplasm. Mitotic figures were rare. Tumor cells were supported by a
stroma consisting of fine reticulin fibers. These changes were
consistent with hemangiosarcoma. The cause of the neoplasms in these
fish was unknown but there are reports of similar chemically induced
neoplasms in medaka and zebrafish using
N-methyl-N'-nitro-N-nirtosoguanidine. Other significant lesions in these
fish included erythrocyte depletion in the spleen and hyperplasia of
hematopoietic tissue in the kidney. The changes in the liver were
consistent with hepatocellular degeneration and pancreatic metaplasia
with general loss of hepatocellular fat and glycogen.

**Widespread Deformities In An Inland Yellow Perch Population In
Michigan**

[Mohamed Faisal]{.underline} and Elaine Holmes

Michigan State University, East Lansing, Michigan 48824

Widespread skeletal deformities in a yellow perch (*Perca flavescens*)
population have been noticed in a major inland lake in Michigan. The
deformities involved all adult fish examined (300 fish). Fishermen
report that 100% of adult yellow perch, but not other fish species, in
this lake are affected. Radiographic analysis revealed the presence of
an unusual complex spinal column deformity consisting of consecutive
repetition of lordosis and, scoliosis from head to tail. Most spinal
curvatures occurred in two specific sites along the vertebral column.
Lordiosis was characterized by V-shaped dorso-ventral curvature;
however, all lordotic fish possessed inflated functional swimbladders.
Additional malformations such as vertebral fusion, absence of one or
both operculum, and bent-jaw were also observed albeit at much lower
prevalence. The cause of these deformities and their impact on the
population are currently unknown.

An Extreme Example Of Common Seahorse Diseases

[Salvatore Frasca Jr]{.underline}.^1^, Akinyi Nyaoke^1^, Lynn
Hinckley^1^, Sybren de Hoog^2^, Brian Wickes^3^, Deanna Sutton^4^, E.
Scott Weber^5^, and Christian Keller^6^

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

Sygnathid fishes, e.g. seahorses, seadragons and pipefish, have become
popular and commercially important exhibit animals. As a testament to
the value of these fish to the aquarium industry, the pathology service
of the Connecticut Veterinary Medical Diagnostic Laboratory (UConn,
Storrs, CT) has seen 296 sygnathid fish from 2000-2005, representing at
least 11 different seahorse species, 2 species of seadragon, and 5
separate pipefish species. From this submission experience, several
pathological conditions have been encountered with frequency in
aquarium-maintained sygnathid fish and include mycobacteriosis, erosive
to ulcerative bacterial dermatitis, uronemosis, and more recently
cutaneous or systemic phaeohyphomycosis. This case presentation involves
an aquarium-maintained male potbellied seahorse (*Hippocampus
abdominalis*) that exhibited several of these frequently seen pathologic
conditions concurrently. Histopathologic findings included focal
phaeohyphomycotic dermatitis, locally extensive bacterial ulcerative
dermatitis with uronema, and mycobacteriosis. Fungal cultures and DNA
sequence analysis have determined the fungus to be *Exophiala
pisciphila.* Albeit these several conditions do not constitute the
entirety of diseases seen in aquarium-maintained sygnathid fish and
presentations may vary significantly between specimens, pathologists and
fish health clinicians should be aware of them and be cognizant that
their occurrence may be concurrent.

**Pathology Associated With Large Cavernous Lesions In Chinook Salmon
From Lake Ontario**

Emily K. Meseck^1^, Tracy W. French^2^, Stephanie G. Grimmett^3^, Susan
L. Bartlett^3^, Gregory A. Wooster^3^, Rodman G. Getchell^3^, John H.
Schachte, Jr.^4^, and [Paul R. Bowser]{.underline}^3^

^1^Department of Biomedical Sciences, ^2^Department of Population
Medicine and Diagnostic Sciences, ^3^Aquatic Animal Health Program,
Department of Microbiology and Immunology, College of Veterinary
Medicine, Cornell University, Ithaca, New York 14853-6401,^4^Fish
Disease Control Unit, New York State Department of Environmental
Conservation, Fish 8314 Hatchery Road, Rome, New York 13440

Chinook salmon (*Onchrhynchus tshawytscha*) from Lake Ontario have been
observed since 1999 to have large focal cavernous, fluid-filled lesions
in their muscle. The disease appears to be limited to adult Chinook
salmon. Fish were presented for evaluation by the sport fishing public
or by the staff of the Salmon River Fish Hatchery (Altmar, New York,
USA) of the New York State Department of Environmental Conservation (NYS
DEC) during the spawning season (October to December). All of the
examined fish originated from Lake Ontario, and we are not aware of fish
presenting with this lesion from any other body of water in New York
State. The affected fish ranged in size from 4.5 -- 9.1 kg. Fish
presented for evaluation typically had large raised, poorly demarcated,
oval areas in their dorsal musculature near and posterior to the dorsal
fin. These lesions ranged in size from 70 to 100 mm long x 40 to 60 mm
wide x 20 to 30 mm deep. Some contained as much as 300 mL of variably
clear to slightly turbid, yellow to yellow-brown odorless fluid. The
ability to culture bacteria from the fish was variable, with *Aeromonas
salmonicida*, *Klebsiella* sp., or no bacteria being isolated. In some
cases a parasite similar to *Spironucleus* was found in the fluid from
the cavernous lesions and in the blood.

**A Case Of Mistaken Sexual Identity And Function: Pitfalls Of
Methyl-Testosterone Sex-Reversal In Aquaculture**

[Gregory A. Wooster]{.underline}, Stephanie M. Grimmett and Paul R.
Bowser

Aquatic Animal Health Program, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
New York, 14850-6401

The compound, 17-Methyl-testosterone (MT) is sometimes used in Tilapia
aquaculture to produce mono-sex all male populations of fish, which
results in better production efficiency. However care should be
exercised in proper handling and labeling of MT-medicated feed when used
in aquaculture. Accidental exposure of young Tilapia destined for use as
brood stock to MT-treated feed during the early development of the fish
resulted in a case of mistaken sexual identity during later brood-stock
selection. The subsequent failure of a recirculation aquaculture
facilities brood-stock to meet production expectations resulted in the
case presented here.

**Mass Mortalities In The Brook Stickleback (*Culaea inconstans*)
Exhibiting Abnormal Abdominal Tumor**

Ehab A. Elsayed and [Thomas Loch]{.underline}

Michigan State University, East Lansing, MI, 48824

Widespread tumor-like lesions along with 100% mortalities were reported
from a Brook stickleback population residing in a 4-acre stocking pond
in northern Michigan. No other species were affected at the time of
outbreak. Affected fish exhibited relatively large, symmetrical
abdominal swellings that disabled fish swimming. Microscopic examination
of the tumor contents revealed the presence of large numbers of oval
spores that measured 5 um length and 2 um width with a prominent
posterior vacuole. Electron microscopy indicated that the tumor-like
lesion is a typical xenoma microsporidians and that the spores and
developmental stages are those of *Glugea anomala*.

**Life Is Never Dull At College Park**

[Ana Baya]{.underline}^1,2^, Cindy Stine^1^, John Abel^2^, Karl
Roscher^2^,Ruby Paramadhas^1^, Andrea Ferrero-Perez^1^, and Andrew
Kane^1^

^1^University of Maryland, College of Veterinary Medicine and
^2^Maryland Department of Agriculture, College Park

Three case reports and one on-going project will be presented. The first
case involved tilapia from an aquaculture facility presented with
bulging eyes that literally fell from their respective sockets. A second
case, also involving tilapia, presented with gills filled with
*Leptospira*. The source of infection is believed to be fish food
contaminated with rat urine. The third case involved sturgeon with a
severe case of mites. In our on-going project, we have also isolated
more than 450 Mycobacteria over the last few years from both cultured
and wild caught fish. Mycobacteria were cultured and submitted to fatty
acid methyl ester (FAME) analyses using gas chromatography. Bacteria
were grown on Middlebrook agar and incubated at 28 C until there was
sufficient growth for analysis. A dendrogram of these isolates was
produced to allow cluster analysis. This dendrogram, which measures the
relatedness of isolates based on several characteristics, allows
identification of strains or species, some of which appear novel. The
dendrogram groupings will be supported by polymerase chain reaction
results and sequencing. Life is never dull.

**Effects Of Elevated Suspended Sediment On Stress, Growth And Gill
Condition Of The Spotfin Chub And The Whitetail Shiner**

[Andrew B. Sutherland]{.underline}

Institute of Ecology, University of Georgia, Athens, GA 30602

North America's freshwater fish fauna continues to decline, in part due
to excessive sedimentation in streams and rivers. The objective of this
research was to investigate the effects of elevated suspended sediment
concentration (SSC) on the spotfin chub (*Erimonax monachus*), an
imperiled southern Appalachian minnow, and on a surrogate species, the
whitetail shiner (*Cyprinella* *galactura*). I investigated the effects
of SSC (0, 25, 50, 100, and 500 mg/L) on the stress response (cortisol
concentration), specific growth rate (percent change in mass per day),
and gill condition (i.e., lamellar thickness and interlamellar area) of
young-of-year (YOY) spotfin chubs and whitetail shiners. Exposure of YOY
to elevated SSC caused a significant increase in cortisol levels, in
both species and a significant decrease in growth rate at three life
stages. Increased SSC elicited a stress response in spotfin chubs 3-fold
higher than controls; this response was similar to previous accounts of
rainbow trout exposed to acute handling stress. For spotfin chubs, a
15-fold decrease in specific growth rate occurred at the highest SSC
(500 mg/L). Gill damage observed by quantitative confocal microscopy was
minimal at 0, 25, and 50 mg/L, moderate at 100 mg/L, and severe at 500
mg/L. Specific growth rate was significantly and inversely related to
increasing gill lamellar thickness. The sublethal effects documented
here support the hypothesis that elevated suspended sediment contributes
to the imperilment of southeastern native fishes.

**Infectious Hematopoietic Necrosis Virus In Atlantic Salmon Seapens In
British Columbia**

[Evi Emmenegger]{.underline}^1^, Garth Traxler^2^, Eric Anderson^3^, and
Gael Kurath^1^

^1^Western Fisheries Research Center, USGS, 6505 NE 65th Street,
Seattle, WA 98115; ^2^Deparment of Fisheries & Oceans, Pacific
Biological Station, Nanaimo, BC V9R5K6 ; ^3^Maine BioTek, 259 Main St.,
Winterport, ME 04496

Atlantic salmon were introduced to British Columbia (BC) for aquaculture
production in the mid 1980\'s. In 1992, infectious hematopoietic
necrosis (IHN) was first diagnosed in pen-reared Atlantic salmon, and
over the next 5 years spread to 14 net-pen sites in a very localized
area on Vancouver Island. IHN was not reported again until August 2001
at a location near the 1992 index case. Since then the virus has been
detected in over 25 net-pen sites over a wide geographic area around
Vancouver Island. IHNV isolates were collected from Atlantic salmon in
seapens, and from Pacific salmon in Vancouver Island and mainland BC
watersheds. Sequence analysis of the midG region, a 303 base sequence in
the middle of the glycoprotein (G) gene, separated the recent seapen
IHNV isolates into two sequence types within the U genogroup of IHNV.
Comparisons of historical IHNV isolate midG sequences from throughout BC
from 1974 to 2001 with the recent Vancouver Island Atlantic salmon sea
pen outbreaks will be presented.

**Virulence, Monoclonal Epitope, And Genome Analyses: A Conjoined
Assessment Of Infectious Pancreatic Necrosis Virus Isolates Endemic And
Exotic To The Great Lakes Basin**

[Philip E. McAllister^1^]{.underline}, Christine L. Densmore^1^, Vikram
N. Vakharia^2^, and Paul W. Reno^3^

^1^U.S. Geological Survey, Leetown Science Center, National Fish Health
Research Laboratory, 11649 Leetown Road, Kearneysville, West Virginia
25430; ^2^University of Maryland Biotechnology Institute, Center for
Biosystems Research, 5115 Plant Sciences Building, College Park,
Maryland 20742; ^3^Oregon State University, Hatfield Marine Science
Center, 2030 SE Marine Science Drive, Newport, Oregon 97365

Attendant to the "Strategic Vision of a Healthy Great Lakes Ecosystem,"
the Great Lakes Fish Health Committee is developing a risk assessment
decision-making process to estimate the impact on the ecosystem of
various biota, including fish pathogens. An appraisal of the virulence
of isolates of infectious pancreatic necrosis virus (IPNV), endemic and
exotic to the Great Lakes Basin, is a component of this program. We
began with a collection of 129 IPNV isolates, representing historic and
contemporary recoveries from epizootics and routine fish health
inspections involving diverse species of fishes and invertebrates.
Conjoining analyses of virulence (mortality ranging from \<10% to near
100%), monoclonal epitope (19 Birnadent groups) and genome sequencing
(phylogenetic analyses of nucleotide and deduced amino acid sequences of
the VP2 variable region), we winnowed our collection to evaluating 40
IPNV isolates. Virulence estimates are being developed in 8 species of
salmonid fishes using standard immersion challenges, IPNV-carrier
excretion exposures, and defined chronic waterborne exposures.

**Largemouth Bass Virus In New York State: An Update**

[Vanessa L. Kirsipuu]{.underline}, Stephanie G. Grimmett, Rodman G.
Getchell, Courtney Grandner, and Paul R. Bowser

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

We have undertaken an effort to determine the distribution of Largemouth
Bass Virus (LMBV) in New York State. The virus had been found in Lake
Champlain, on the New York--Vermont border, in 2002 through the efforts
of the Vermont Fish and Wildlife Department. Our efforts began in the
summer of 2004. Largemouth bass were provided by the New York State
Department of Environmental Conservation and by the Cornell Biological
Field Station, Oneida Lake. The fish were collected in connection with
previously scheduled fish population monitoring efforts. Our goal was to
test a minimum of 5 fish per source. The samples were screened using the
fathead minnow cell line. In addition, a quantitative PCR test for the
LMBV was developed and used to conduct parallel screening of all
samples. During 2004, 107 largemouth bass were collected from 12 lakes
in New York State. Based on cell culture efforts in FHM cells, all fish
were negative. All cell cultures were maintained for 28 days and had
gone through 2-3 blind passages before being scored negative.
Quantitative PCR testing indicated a positive largemouth bass from Hyde
Lake. In a related effort, we are developing a cell line from the
largemouth bass. We hypothesize that this cell line may be a more
sensitive cell culture system for the culture of the largemouth bass
virus.

**First Report Of Koi Herpesvirus In Wild Common Carp In The Western
Hemisphere**

[Jeffery S. Terhune]{.underline}^1^, John M. Grizzle^1^, Karl J.
Hayden^1^, Shasta D. McClenahan^1^, Scott D. Lamprecht^2^, and Miller G.
White^2^

^1^Southeastern Cooperative Fish Disease Project, Department of
Fisheries and Allied Aquacultures, Auburn University, Alabama 36849;
^2^South Carolina Department of Natural Resources, Bonneau, South
Carolina 29431

Koi herpesvirus (KHV) is a recently discovered virus infecting koi and
common carp *Cyprinus carpio.* Most cases of KHV disease have been
reported in the ornamental fish industry and farmed common carp, but KHV
has been reported in wild populations of common carp in the United
Kingdom and Japan. During April and May 2004, thousands of wild common
carp died in the Santee-Cooper Reservoir system in South Carolina. Three
moribund fish were captured having ventrally located reddened areas of
skin and severe necrosis of the gills. *Flavobacterium columnare* was
observed in both wet mounts and histological preparations of gills.
Additionally, *F. columnare* was isolated on agar plates from both from
the gills and internal organs. Histological lesions characteristic of
KHV disease were not observed in any organs in any of the fish. Cell
culture of samples from each fish on fathead minnow cells, epithelioma
papulosum cyprini cells, and koi fin cells also failed to produce any
cytopathic effect. Organ samples were tested for KHV with a polymerase
chain reaction assay. All samples from the 3 fish collected during the
outbreak were positive for KHV. To our knowledge, this is the first
report of KHV infection in wild common carp other than in the Western
hemisphere.

**Diagnosis Of Goldfish Hematopoietic Necrosis (*Cyprinid herpesvirus
2*) By Quantitative PCR**

[Andrew E. Goodwin]{.underline} and Gwenn Merry

University of Arkansas at Pine Bluff, 1200 N. University Drive, Pine
Bluff, AR 71601

Koi and goldfish producers and hobbyists are very concerned about koi
herpes virus (KHV) and spring viremia of carp virus (SVCV), but have
overlooked other viruses that may be equally important. The CyHV-2 virus
(associated with hematopoietic necrosis disease in goldfish) produces
high mortality and has been reported in Japan, Taiwan, Australia and the
United States, but this pathogen is rarely reported. This is because
diagnosis of the disease has been based only on electron microscopy and
histological findings. Recently, degenerate PCR of viral DNA polymerase
genes and sequencing showed that tissues from moribund goldfish, sampled
during three epizootics in North America, carried the CyHV-2 genome.
This work established that the disease is widespread in the United
States and demonstrated the need for a sensitive and specific assay for
CyHV-2. In our laboratory, we have developed a TaqMan PCR method for
CyHV-2 that targets the viral DNA polymerase gene. Using a cloned
version of the PCR product and North American cases as controls, we have
validated the assay and determined that it is specific for CyHV-2,
detects as few as 200 copies of the viral genome per reaction, is
quantitative over 8 logs of template number, and detects the virus in
asymptomatic fish and brood stock.

**Spring Viremia Of Carp Virus In Rainbow Trout: Possible But Uncommon**

[Igor S. Shchelkunov]{.underline}^1^, Tatiana I. Shchelkunova^1^,
Svetlana F. Oreshkova^2^, and Nadezhda N. Blinova^2^

^1^All-Russia Research Institute of Freshwater Fisheries, Rybnoe,
Dmitrov Region, Moscow Province, 141821, Russia; ^2^Research Institute
of Bioengineering of The State Research Center for Virology and
Biotechnology \"Vector\", Koltsovo, Novosibirsk Region, 630559, Russia

Generally, the cyprinid vesiculovirus SVC (genogroup I) does not readily
infect salmonids; however, there are no epizootiological constraints
that prevent such transmission and the susceptibilities of salmonid cell
lines (RTG-2 and ASE) to SVCV suggests that this is feasible. We
repeatedly isolated SVCV from a cohort of rainbow trout held in our
laboratory that were bath infected with VHS virus (\>2 years earlier) to
monitor viral cycling. The isolation was originally believed to be
contamination from SVCV-M2 infected common carp. Isolations were done
from skin, fins, gills, brains, and pooled kidneys, spleens, and livers,
which yielded an isolate (FB1/03) from internal organs. It was not
neutralized with IHNV or VHSV antisera. The CPE was delayed but not
neutralized by M2-SVCVantiserum and by M2-SVCV mixed with VHSV antisera,
suggesting that the virus mutated in its novel host. Using two primer
pairs to viral N-gene, RT-PCR identified the agent as SVCV. Data from
N-gene based RT-PCR and RFLP suggested that the isolate was a mix of two
genetically distinct strains. Independent G-gene sequencing at CEFAS
provided similar evidence indicating that one strain belonged to the
"parent" genomic subgroup Id and another belonged to subgroup Ic.
Interestingly, FB1/03 caused low morbidity absent mortality when
injected in yearling common carp, but produced acute disease (70%
mortality) in rainbow trout (\~1g) compared with 55% mortality produced
by M2-SVCV. Both strains were recovered from moribund fish indicating
that SVCV multiplied in the rainbow trout (10^7.85^--10^8.1^ TCID~50~/g
tissue). After a two week bath challenge in M2, only one of 50 rainbow
trout (\~0.3g) died absent clinical signs but with high virus load
(10^8.85^ TCID~50~/g tissue). Neither disease nor mortality was evident
among common carp cohabited with these trout. Another SVCV strain from
common carp, KU/0204, killed 2 of 96 bath infected rainbow trout
producing hydrocephalus and skin hemorrhages with moderate viral titers
in tissues (10^4.35^--10^7.6^ TCID~50~/g). Carp injected with virus
re-isolated from the dead rainbow trout fry had reduced morbidity and
mortality compared with the original KU/0204 strain. By injection, this
strain produced 50% mortality in 3-mo trout (\~1.3g) with signs of acute
exsudative hemorrhagic syndrome and high virus titers (10^8.35^
TCID~50~/g tissue). The virus did not cause disease in 4-mo rainbow
trout (\~4g) and was not recovered from occasional mortalities or
surviving fish, however, semi-nested RT-PCR detected it in 20--50%
pooled tissue samples, 34 days post injection.

***In Vitro* Sensitivity And Specificity Analysis Of Spring Viremia Of
Carp Virus Diagnostic Tests**

[Raghunath Shivappa]{.underline}, Shannon Kozlowicz, Flavio Corsin, and
Jay Levine

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

Spring viremia of carp (SVC), which was considered a foreign animal
disease in the US, was first detected in April 2002. The viral disease
continues to pose a major threat to the carp industry, as well as free
ranging cyprinid species. There are several diagnostic tests that are
available for the detection of SVCV. However, the tests have not been
validated. *In vitro* sensitivity and specificity for several SVCV
diagnostic tests were evaluated. Serial dilutions of SVCV virus as low
as 10^1^ TCID~50~/mL were used to evaluate the *in vitro* sensitivity of
SVCV diagnostic tests. *In vitro* specificity was evaluated by serially
diluting five different strains of fish rhabdoviruses and testing using
SVCV diagnostic tests. The study showed that a minimum viral titer of
10^5^ TCID~50/mL~ is required to detect the virus in cell culture within
48 hours. Molecular and serological based techniques (RT-PCR, ELISA and
IFAT) proved to be more sensitive and were able to detect the virus in
less time than virus culture. Among these tests, RT-PCR was the most
sensitive and detected the virus at 10^4^ TCID~50~/mL. RT-PCR and ELISA
assays did not detect any fish rhabdovirus other than SVCV. *Although In
vivo* studies are pending, the preliminary findings of this study
indicate that RT-PCR could be recommended as a test of choice for rapid
and accurate diagnosis of SVCV.

**Cutaneous Infection Of Channel Catfish By Microscopic Nematodes**

[Joanne Sadler]{.underline} and Andy Goodwin

University of Arkansas at Pine Bluff Aquaculture/ Fisheries Center, 1200
N. University Dr., Pine Bluff, AR 71601

In January 2005, two samples of channel catfish (*Ictalurus punctatus*)
were submitted by an Arkansas fish farmer. Focal, raised, firm 3-5 mm
lesions were scattered over the entire tegument of the fish. Fish that
were 3-years-old (1.5 kg) had many lesions (20 up to 400) while younger
fish in the same holding facilities were not affected. The farm manager
reported that the lesions ruptured and became hemorrhagic over the
winter months and then healed over during summer. In two of the larger
fish submitted, many of the lesions were ulcerated and hemorrhagic. The
farmer reported no changes in feeding behavior and no mortality.
Necropsy revealed that the lesions were located within the dermis layer
of the skin and that live microscopic nematodes were present.
Histopathology showed cross sections of nematodes associated with
necrotic muscle debris, lymphocytes and macrophages. No other nematode
life stages or nematode species were found in any other fish tissue. To
our knowledge this nematode species has not been previously identified.
Historically, there have been twelve cases of this skin condition
reported in Arkansas since 1965 and two cases in Mississippi. We will be
working in collaboration with Dr. Al Dove, of Stony Brook University, to
identify the nematode species using molecular techniques.

**Widespread Skin Ulcerations In The Yellow Perch Population Of The Lake
St. Claire**

Ehab E. Elsayed and [Tom Loch]{.underline}

Michigan State University, East Lansing, MI, 48824

Widespread skin ulcerations were reported exclusively in Yellow perch
collected from Lake St. Clair, Michigan, during summer 2004. The case
has directed public attention and concerns about zoonotic potentials
have been raised. In almost all fish submitted for analysis, shallow
ulceration in posterior side of their body surrounded by hemorrhagic rim
were observed. The affected fish were emaciated exhibiting sunken eyes
and rough, dry skin. Bacteriological examination revealed consistent
isolation of *Pseudomonas fluorescens* from skin lesions and kidneys of
affected fish.

**A Moribund Lake Sturgeon From The Niagara River, New York**

[Paul R. Bowser]{.underline}, Rodman G. Getchell, and Rachelle E. Kosoff

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

A moribund lake sturgeon *Acipenser* *flavescens* was found on 1 January
2005 in shallow water along the edge of the Niagara River near
Wheatfield, NY, upstream from Niagara Falls. The fish was still alive on
2 January, but died on the morning of 3 January and was transported by
the New York State Department of Environmental Conservation to Cornell
University for examination. Upon presentation, the fish was 229 cm and
95.3 kg (7' 6" long, weighed 210 lbs). Evaluation of a cross-section of
the anterior fin ray of the left pectoral fin revealed that the fish was
75 years old. Gross examination of the fish revealed some petechiation
of the ventral body surface, which was likely due to it resting on the
bottom of the river for several days. No other gross pathology was
noted. Bacterial cultures from the posterior kidneys were negative for
growth. An attempt at virus isolation on the white sturgeon cell line
(courtesy of R. Hedrick, UC Davis) was negative. Histopathology of major
organs was unremarkable. A primary rule-out was botulism type E as this
condition has become problematic in the Lower Great Lakes. Gut contents,
liver, kidney, spleen and blood were evaluated for presence of botulism
type E with a quantitative PCR test. The results of the botulism testing
were negative. The fish was found in a location known to have been
heavily contaminated with hexachlorobenzene.

**A Fish Kill Of Common Carp In New York State And An Unusual Koi
Herpesvirus**

^1^[Stephanie G. Grimmett]{.underline}, ^2^J.V. Warg, ^2^D.J. Johnson,
and ^1^P.R. Bowser

^1^ Aquatic Animal Health Program, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
New York 14853-6401;^2^ National Veterinary Services Laboratories, USDA,
APHIS, P.O. Box 844, Ames, Iowa 50010

Koi herpesvirus (KHV), a highly contagious and lethal virus affecting
both koi (*Cyprinus carpio koi*) and common carp (*Cyprinus carpio*) was
isolated in 1998 from two outbreaks of koi suffering mass mortality in
the New York State, USA and in Israel. However, the disease was
described as early as 1996 in Europe. In July 2004, this virus was found
to be associated with a mass mortality event in wild common carp in the
Chadakoin River in western New York State. Affected fish typically
showed marked hyperplasia of gill tissues and severe multifocal to
diffuse external haemorrhages. The viral isolate found in this outbreak
was somewhat unusual in that it grew well on fathead minnow FHM cells
giving initial rise to the fear that the virus was Spring Viremia of
Carp Virus (SVCV). Testing at the National Veterinary Services
Laboratories, Ames, Iowa, USA, confirmed the virus's identity to be that
of KHV. KHV is not currently on the OIE (Office International des
Epizooties) list of notifiable diseases however is capable of causing
mass mortality in susceptible fish at permissive temperatures.

Encounters With Fungi In Sygnathid Fish

[Salvatore Frasca Jr.]{.underline}^1^, Akinyi Nyaoke^1^, Lynn
Hinckley^1^, Allison Kamens^1^, Andrew Draghi II^1^, Mindy Barnett^2^,
Timothy Gorton^2^, Donald Stremme^3^, Christian Keller^4^, E. Scott
Weber^5^, Sybren de Hoog^6^, Amy Grooters^7^, Brian Wickes^8^ and Deanna
Sutton^9^

^1^Department of Pathobiology and Veterinary Science and ^2^Center for
Excellence in Vaccine Research, University of Connecticut, Storrs, CT
06269-3089; ^3^Adventure Aquarium, Camden, NJ 08103-1060; ^4^Tennessee
Aquarium, Chattanooga, TN 37401-2048; ^5^New England Aquarium, Boston,
MA 02110-3399; ^6^Centraalbureau voor Schimmelcultures, Uppsalalaan 8,
3584 CT Utrecht, The Netherlands; ^7^Department of Veterinary Clinical
Sciences, School of Veterinary Medicine, Louisiana State University,
Baton Rouge, LA 70803; ^8^Department of Microbiology and ^9^Fungus
Testing Laboratory, Department of Pathology, The University of Texas
Health Science Center at San Antonio, San Antonio, TX 78229-3900

From 2000-2005, sygnathid fish represent the largest number of
submissions from aquaria to the pathology service of the Connecticut
Veterinary Medical Diagnostic Laboratory (UConn, Storrs, CT). With
greater frequency in recent years, phaeoid fungi have been identified as
agents of cutaneous lesions or systemic infections. A desire to
characterize these phaeoid fungi has been the incentive to culture an
increased number of specimens suspicious of having fungal lesions. The
result has been morphologic and molecular identification of several
*Exophiala* species, a novel one isolated from systemic lesions of
seadragons (*Phyllopteryx taeniolatus* and *Phycodurus eques*), and
another (*E. pisciphila*) infecting potbellied seahorses (*Hippocampus
abdominalis*). More frequent attempts at fungal culture have led to
isolation of several notable hyaline hyphomycetes, e.g. *Paecilomyces
lilacinus*. Active fungal testing is necessary to understand both the
range of fungi, as well as the range of clinical and pathological
manifestations, encountered in certain aquarium-maintained fish.

**Furunculosis In The Sea Lamprey (*Petromyzon marinus*) In Lake
Ontario**

[Alaa Eissa]{.underline}, Kristi Jones, Thomas Loch, Ehab Elsayed, and
Mohamed Faisal

Michigan State University, East Lansing, Michigan 48824

For the last six decades, the Laurentian Great Lakes have been plagued
with parasitic sea lamprey that invaded the basin causing devastating
losses in salmonid fisheries. To reduce the number of sea lampreys, the
Great Lakes Fishery Commission began a large-scale program based on
trapping male sea lamprey, sterilizing them, and subsequently releasing
the sterile males back into streams where they compete with fertile
males for spawning females. These transfers of lamprey to different
locations may additionally transfer various pathogens concurrently,
which have raised major concerns regarding the possibility of resident
fish populations becoming infected. During health inspection of a group
of sea lampreys, collected from Lake Ontario, for their suitability to
translocation within the basin, lampreys with obvious furuncles (1-2 cm
in diameter) were noticed. Most of the furuncles occupied the
dorso-lateral musculature of affected lampreys. From the furuncles,
spleens, and kidneys of affected fish, typical *Aeromonas salmonicida*
subsp. *salmonicida* isolates were retrieved. Additional isolates were
retrieved from clinically asymptomatic fish. Biochemical profiles and
variations between the isolates were compared among each others and
among those isolated from other fish species in Michigan. It is highly
probable that sea lamprey play a role in the temporal and spatial spread
of *A. salmonicida* in the Great Lakes basin.

**Lesions Caused By *Pomphorhynchus* spp. (Acanthocephala) In Bluefish
And Striped Bass**

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

Marine Sciences Research Center, Stony Brook University, Stony Brook NY
11794-5000

In New York waters, both striped bass (*Morone saxatilis*) and bluefish
(*Pomatomus* *saltatrix*) are infected by acanthocephalans of the genus
*Pomphorhynchus*, but whether or not the parasites are the same species
has not yet been determined. *Pomphorhynchus* spp. prefer the rectum,
where they can occur in very large numbers. Striped bass and bluefish
were examined for pathologies associated with pomphorhynchid infection
and to investigate a potential role for this parasite in the
epidemiology of mycobacteriosis. In both hosts, worms penetrate all
layers of the rectum with a narrow extension of the trunk, which is
inflated in the coelom and thereby acts as a permanent anchor (the spiny
proboscis probably plays little part in attachment). Presence of worms
was associated with complex mixtures of pathologies and host responses.
The former included interstitial hemorrhage, necrosis and sloughing of
the rectal epithelium, whereas the latter include thickening of the
*lamina propria,* multiple layers of epithelioid cells, and fibrous
connective tissue encapsulation of parasites. Sites of attachment were
also associated with multifocal granulomatous inflammation. In one
bluefish these granulomas were acid-fast positive and acid fast bacilli
were observed in the spaces between the parasite and the layers of host
response. The penetration of the host rectum by *Pomphorhynchus* spp.
may play an important role in providing mycobacteria in the digestive
stream with a patent conduit to the coelom.

> **An Outbreak Of Phaeohyphomycosis In Hatchery-Reared Chinook Salmon
> Fingerlings**
>
> [Scott D. Fitzgerald]{.underline}^1^, Ehab Elsayed^1^, Martha
> Wolgamood^3^, Victor Silva^2^, Leonel Mendoza^2^, and Mohamed
> Faisal^1^

^1^Dept. of Pathobiology and Diagnostic Investigation, and ^2^Medical
Technology Program, Michigan State University, East Lansing, Michigan,
^3^Wolf Lake Hatchery, Michigan Dept. of Natural Resources

A hatchery experienced a major die-off of fingerling Chinook Salmon
(*Oncorhynchus tschawytscha*) during Feb. and March, 2003. Clinical
signs included abnormal swimming behavior, loss of appetite,
exophthalmia, distended abdomens, and redness surrounding the vent.
Internal examination revealed pale livers, distended stomachs filled
with yellow fluid, swim bladders which were collapsed and contained
white necrotic material. Mortality was estimated at 8-12%, with nearly
100,000 presmolts dying. Wet mount smears of the swim bladder contents
revealed large numbers of branching, septate fungal hyphae. Mycotic
growth appeared in 3 days when swim bladder contents were inoculated
onto Sabouraud's dextrose agar media. The small subunit ribosomal DNA
(18S ssu-rDNA) was amplified by PCR, sequenced, and compared with
sequences in GenBank, and found 100% homology with of *Phoma herbarum*.
Histologically, the fungus appeared to enter the host swim bladder
forming a dense mat of septate, irregular branching, non-pigmented
hyphae. These hyphae obliterated the swim bladder wall, and extended
into the stomach, kidney, gonads, coelom, blood vessels and mesentery.
Normally, *P. herbarum* is a plant saprophyte; it has been reported from
the water, soil, diseased and dead plants worldwide. It has rarely been
associated with disease in fish; a single previous report from a die-off
of hatchery chinook and coho salmon occurred in Washington state in
1975. Interestingly, the hatchery in this report was free of the disease
in 2004, but experienced a recurrence in March, 2005. Phaeohyphomycosis
due to *P. herbarum* may be an emerging disease of hatchery-reared
salmon, and fish health personnel should be on the alert for this
pathogen.

**Livid Lesions In Lumpfish (*Cyclopterus lumpus*) Associated With A
Fungus *Exophiala pisciphila***

[Jeffrey C. Wolf]{.underline}^1^, Sea Rogers William^2^, Lynne
Sigler^3^, Michael Dykstra^4^, Marilyn J. Wolfe^1^

^1^The Registry of Tumors in Lower Animals, 22900 Shaw Road, Suite 107,
Sterling, VA 20166; ^2^Woods Hole Science Aquarium, NOAA: Fisheries,
Northeast Fisheries Science Center, NMFS, 166 Water Street, Woods Hole,
Massachusetts 02543; ^3^University of Alberta Microfungus Collection and
Herbarium, Devonian Botanic Garden, Edmonton, AB, Canada T6G 2E1; ^4^The
Laboratory for Advanced Electron and Light Optical Methods, College of
Veterinary Medicine, North Carolina State University 4700 Hillsborough
Street, Raleigh, NC 27606

During the preceding 40 years, The Registry of Tumors in Lower Animals
(RTLA) has been the fortunate recipient of many excellent diagnostic
cases. Quite often, submitted specimens contain tumor-like lesions that
are ultimately discovered to be non-neoplastic. The present case
involves two captive lumpfish (*Cyclopterus lumpus* Linnaeus 1758) from
the Woods Hole Science Aquarium, Woods Hole, MA, that were originally
collected in the Gulf of Maine during routine biological sampling by
NOAA Fisheries Service vessels. While in captivity, these fish were
maintained in a cold water (average T = 49-50^o^F) aquarium system.
Signs of disease in the submitted fish included skin ulcers and sudden
death. Skin scrapings yielded fungal elements and paramecium-type
protozoa. Necropsy revealed abundant peritoneal fluid, and black, red,
or green nodules (0.5-2.0cm) in multiple organs, including the kidney,
cardiac ventricle, spleen, liver, gill, stomach, and skin.
Microscopically, the nodules were determined to be infarcts (areas of
necrosis caused by vascular obstruction) in various stages of
progression. Thrombi associated with the infarcts contained abundant
narrow, branching, septate, brown-pigmented, angiocentric, fungal
hyphae. Cultures from two lumpfish on CM+ yielded colonies of the same
fungus. The fungus was identified as *Exophiala pisciphila* based on
colonial and microscopic morphology, and comparison with reference
isolates of this species.

**Coldwater Disease Outbreak At A West Virginia Rainbow Trout
(*Oncorhynchus mykiss*) Farm: An Unusual Presentation**

[Julie Bebak-Williams]{.underline}^1^, Tim Welch^4^, Cliff Starliper^3^,
Ana Baya^2^, and Michael Garner^5^

^1^Freshwater Institute, Shepherdstown, WV; ^2^Maryland Dept. of
Agriculture, College Park, MD; ^3^USGS National Fish Health Research
Laboratory, Leetown, WV; ^4^USDA National Center for Cool and Coldwater
Aquaculture, Leetown, WV; ^5^Northwest ZooPath, Monroe, WA

In 2004, a WV trout farmer reported mortality in three month old rainbow
trout fingerlings reared in flow-through single-pass tanks. The
groundwater supply was 12°C and good quality for trout culture.
Mortality was \>100 fish per tank per day in a population of 35,000. The
problem was noticed two weeks before when fish stopped feeding in one
tank and then fish in 15 other tanks went off feed. Fish were lethargic
with exophthalmia, thin, had pale red gills and kidneys, red-tinged
coelomic fluid and pale brown livers. Some were laying on the bottom of
the tank with bilateral pigmentation. The clinical diagnosis was
bacterial or viral septicemia. Samples were collected for virology and
bacteriology. The spread from tank to tank was consistent with the
cleaning routine used by personnel. The option of antibiotic-medicated
feed was declined by the farmer. He was instructed to remove all dead
and dying fish twice a day, work on sick fish last, use separate
equipment for each tank, chlorine for equipment, gloves or hand-washing
for fish handlers, block off the area with tape and cones and transfer
fish to clean, disinfected tanks. The mortality rate declined quickly
after changes were implemented. Histologic changes were consistent with
bacterial septicemia, and involved spleen, heart, gills, bone, and
cartilage. The distribution of bone lesions was typical of cold water
disease. The organism stained with Warthin-Starry silver stain, but not
with Brown and Brenn tissue Gram stain. Three *Flexibacter*-like
isolates from three fish were cultured. One was confirmed as
*Flavobacterium psychrophilum* by biochemical characterization and all
three were *F. psychrophilum* as assessed by 16S sequencing.

Probable Branchiomycosis Among A Captive Population Of Horseshoe Crabs,
Limulus polyphemus, From Delaware Bay

[Christine Densmore]{.underline}^1^, Eric Crawford^2^, David Smith^2^,
and Michael Dykstra^3^

^1^National Fish Health Research Laboratory, Leetown Science Center,
U.S. Geological Survey, 11649 Leetown Road, Kearneysville, WV 25430;
^2^Aquatic Ecology Laboratory, Leetown Science Center, U.S. Geological
Survey, 11649 Leetown Road, Kearneysville, WV 25430; ^3^College of
Veterinary Medicine, North Carolina State University, 4700 Hillsborough
Street, Raleigh, NC 27606

A laboratory maintained population of horseshoe crabs, Limulus
polyphemus, experienced an acute high-level mortality event in the
summer of 2004 following the introduction of additional animals taken
from the Delaware Bay. Necropsy of affected animals revealed multifocal,
pale, often circumscribed lesions over the ventrum, particularly
concentrated on the gills. Microscopic examination of affected branchial
tissues revealed the consistent presence of dense aggregates of
branching, aseptate hyphae with associated tissue reaction including
amoebocyte cell infiltration. Treatment was attempted for the remainder
of the population through manipulation of water quality parameters and
freshwater baths, but these measures were not successful, and mortality
continued at a slower rate throughout the fall and winter. An
oomycete-like organism repeatedly isolated from affected branchial
tissues is the presumed pathogen. Work to identify and further
characterize this organism is currently ongoing.

The Role Of The Anterior Kidney In Generating Long-Term Immunity

[Stephen Kaattari]{.underline}^1^, Erin Bromage^1^, Patty Zwollo^2^,
Ilsa Kaattari^1^

^1^Department of Environmental and Aquatic Animal Health, Virginia
Institute of Marine Science, College of William and Mary, Gloucester
Point, VA; ^2^Department of Biology, College of William and Mary,
Williamsburg, VA

Recent work in our laboratories has been dedicated to the resolution of
various stages of antibody secreting cell development, including the
production and dissemination of plasmablasts, long- and short-lived
plasma cells, as well as putative memory precursors. These studies have
demonstrated that long-term antibody production is not uniformly
distributed throughout all immune tissues, but exclusively resides
within a pool of long-lived plasma cells that are maintained in the
anterior kidney. It is striking that two developmentally distinct
functions which reside in the bone marrow of mammals (hemopoiesis and
plasma cell maintenance) both solely reside within the anterior kidney
of teleost fish. Such a dual occurrence prompts questions as to the
underlying reasons for the preservation of dissimilar functions with the
same tissue. Using markers for transcription factors such as Pax-5,
Blimp-1, and surface and cytoplasmic Ig we plan to resolve the various
developmental and trafficking processes as well as the role that
critical niches play within the teleost anterior kidney for the
production of these differentiated lymphocytes.

**Development Of *In Vitro* Methods To Determine Antibody Gene
Specificity In Salmonids**

[Ilsa Kaattari]{.underline}, Gwynne Brown, and Stephen Kaattari

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

Salmonids utilize at least eleven different families of VH
immunoglobulin genes in the production of antigen binding sites.
Individual VH gene families, however, have yet to be correlated with
their antigenic specificities. To determine whether family specificity
occurs, we are developing *in vitro* immunization techniques that result
in production of antibody with high specificity and affinity.
Concurrently, we are examining the mRNA from cultured cells secreting
antibody, utilizing such molecular techniques as 5'RACE with primers for
secreted antibody, cloning, and sequencing. Currently employed antigens
include *Vibrio anguillarum* lipopolysaccharide (LPS), phosphorylcholine
(associated with streptococcal species), G protein of infectious
hematopoietic necrosis virus (IHNV), p57 of *Renibacterium
salmoninarum*, as well as the common anthropogenic antigen, TNP.
Strategies for B cell stimulation incorporate recent data delineating
the regional development of plasmablasts or plasma cells from specific
tissues. In summary, this work may lead to improved genetic markers for
disease resistance as well as estimates of robustness of resistance
within salmonid populations.

**Unique Mechanisms Employed By Trout To Generate Antibody
Functionality**

[Jianmin Ye]{.underline}, Erin Bromage, and Stephen Kaattari

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

The predominant trout serum Ig is a tetrameric molecule which varies in
the degree of inter-monomeric disulfide crosslinking. This non-uniform
crosslinking leads to considerable structural diversity (redox forms) of
the tetrameric product, with either fully crosslinked tetramers or
tetramers composed of non-covalently associated monomers, dimers, and
trimers. There is limited data available on the role of such structural
diversity amongst teleost antibodies in regard to antigen binding or
effector function. To address this, we have studied the relationship of
affinity distribution and redox structure using selective adsorption of
Ig using a TNP-BSA carrier with varying haptenation ratios. We have
observed that selective adsorption can cause significant skewing of
affinity profiles of antibody subpopulations from the same serum sample.
The average affinity of the low-density hapten-carrier conjugates
(TNP~1~-BSA) adsorbed antibodies possessed the higher apparent affinity
constant (aK) than that of those adsorbed by the high density conjugates
(TNP~13~-BSA). Further, there selective antibodies demonstrated
different redox profiles. TNP~1~-BSA preferentially bound the higher
order redox forms (i.e. those wherein the tetramers were completely
disulfide polymerized, or containing a substantial proportion of
cross-linked trimers). However, TNP~13~-BSA captured significantly less
higher order forms and possessed increased concentrations of lower order
forms (i.e. unlinked monomers and dimers). The linking of the binding
affinity/avidity to redox structure is discussed, and an alternate
structural model of antibody affinity maturation is explored.

**Unique Difficulties In The Development Of Serological Reagents For
Striped Bass Antibody Responses**

[Erin Bromage]{.underline}, Mary Ann Vogelbein, Stephen Kaattari

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

The development of serological reagents against striped bass Ig is
pivotal in the monitoring of humoral immune responses in this species.
Despite extensive effort on the part of a number of investigators, these
reagents have not been forthcoming due to what we believe to be the
unique characteristics of the striped bass Ig. The difficulty in
producing these reagents appears to stem from the degree to which
striped bass Ig is glycosylated. We have found that, originally all Mabs
generated were against carbohydrate epitopes, which exhibit a large
degree of cross-reactivity with other serum components as well as some
Mycobacterial antigens. Deglycosylation of striped bass Ig allows the
production of heavy chain and light chain specific Mabs, which all
appear to be specific for the Fab region of the antibody. Thus such
epitopes become blocked as the antibody binds to antigen, rendering the
mAbs useless for determining antibody titers. A three step process was
therefore developed to generate Mabs specific for striped bass Ig that
would work in various formats. Mice were immunized and boosted with
highly purified, denatured and reduced striped bass light chains. Four
days prior to fusion the mice were IV boosted with affinity-purified
striped bass antibody and fused with standard techniques. All screening
was performed using anti-TNP striped bass antibody bound to TNP-BSA
coated plates. This technique has allowed the development of a suite of
antibodies specific for native striped bass Ig, that has been
successfully used in ELISA, western blotting and immunohistological
applications.

**Mechanisms of Antibacterial Resistance by Nonspecific Cytotoxic
Cells**

[D. L. Evans]{.underline}^1^, H. Kaur^2^, J. H. Leary III^1^, K.
Praveen^1^, and L. Jaso-Friedmann^1^

^1^Department of Infectious Diseases, College of Veterinary Medicine,
University of Georgia, Athens, GA 30602. ^2^Department of Chemical
Engineering, North Carolina State University, Raleigh, NC 27606

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of
mammalian NK cells. NCC kill tumor and viral transformed cells via
receptor mediated target cell recognition. The role of NCC in innate
anti-bacterial immunity has recently been suggested by finding a
scavenger receptor homologue as well as a new pattern recognition
protein on these cells. This new protein (referred to as NCC cationic
antimicrobial protein-1/ncamp-1) binds bacterial DNA,
oligodeoxynucleotides and polyguanosine. Sequence comparisons of this
protein indicated similarity to zebrafish histone family member 1-X and
(to a lesser extent) to trout H1. Inspection for signature sequences or
repeats in ncamp-1 indicated the presence of multiple lysine based
motifs composed of AKKA or PKK repeats. Membrane expression of ncamp-1
on teleost cells was confirmed using a rabbit polyclonal antiserum
raised against the recombinant protein. Binding competition experiments
demonstrated that both the polyclonal and oligodeoxynucleotide bound to
the same protein (i.e. ncamp-1) on NCC. In recombinant form, ncamp-1 not
only binds to the oligodeoxynucleotides, but also kills certain Gram
positive and Gram negative bacteria. We suggest that ncamp-1 may belong
to a new class of pattern recognition receptors. As a membrane protein
it binds bacteria to exacerbate inflammatory responses, whereas in
soluble form it elicits bactericidal activity. Research supported by
BARD US-3159-99C.

**Effector Molecules Of Cell Mediated Cytotoxicity In Catfish And
Tilapia NCC**

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

Department of Infectious Diseases, College of Veterinary Medicine,
University of Georgia, Athens, GA 30602

The existence of cell-mediated cytotoxicity in lower vertebrates has
been well documented but the molecules involved in this process had not
been identified until recently. We have previously reported the
functional significance of non specific cytotoxic cell (NCC) activity in
catfish and tilapia. As part of the innate immune system, these cells
are important in the defense against viral, bacterial and parasitic
infections. We hypothesized that the multiple roles of NCC activity
would require diverse pathways to kill target cells. We now show
molecular evidence that NCC indeed employ several mechanisms to kill
target cells with granule-exocytosis as well as TNF superfamily mediated
effectors. These cells express multiple granzymes along with accessory
molecules like perforin, granulysin and serglycin. The recombinant
granzymes were expressed and substrate specificities were assessed using
various thiobenzyl esters revealing granzymes with tryptase and chymase
activities. We had previously demonstrated functional evidence for the
secreted form of Fas ligand in NCC, which is capable of inducing cell
death in susceptible target cells. Now we also show that NCC express the
membrane-bound form of TNF-alpha, which induces cytotoxicity in
susceptible targets. Recombinant TNF (rTNF) has been expressed and has
been tested for its effects on NCC. rTNF appears to function in the
regulation of NCC activity by protecting from activation-induced cell
death and by increasing the expression of granzymes. These data show
that NCC have pleitropic activities with multiple pathways of target
cell death providing diverse roles in innate immune mechanisms. Research
supported by BARD \# US-3159-99C and by grants from the Veterinary
Medical Experiment Station of the State of Georgia.

**Mannose-Binding Lectin Of Channel And Blue Catfish**

[Donald D. Ourth]{.underline}, M. B. Narra and B. A. Simco

Department of Biology, University of Memphis, Memphis, TN 38152

The channel catfish (*Ictalurus punctatus*) is extensively used in
aquaculture in the Southeast U. S. and is susceptible to bacterial
infections acquired from its pond environment. Research is needed to
understand innate immunity and the catfish defensive response against
bacterial pathogens. We purified by affinity chromatography a
mannose-binding C-type lectin (MBL) from channel catfish serum and also
isolated MBL from blue catfish (*Ictalurus furcatus*) serum. MBL in
mammals acts as an opsonin for phagocytosis by macrophages and also
activates the lectin complement pathway of the innate immune response.
This leads to killing of Gram-negative bacteria and enveloped viruses.
Another objective of the research was a quantitative comparison by the
ELISA technique (using guinea pig anti-MBL IgG) of channel catfish, blue
catfish, and other catfish strains for serum MBL as a resistance marker
for innate bacterial resistance in catfish. Functional studies of
catfish MBL binding to *Edwardsiella* *ictaluri* using the
immunoperoxidase ELISA method were done. This research in catfish
describes for the first time the existence of serum MBL and lectin
complement pathway and adds a new component of innate immunity to
catfish defense. MBL could be used as a molecular resistance marker for
selection of innate bacterial resistance in the various strains of
catfish used in fish farming.

**Plasma Proteomic Analysis Of The Acute Phase Response Of Rainbow Trout
To Intraperitoneal Inflammation And LPS Injection.**

[S. Russell]{.underline}^1^, M.A. Hayes^1^, E. Simko^2^, and J.S.
Lumsden^1^

^1^Fish Pathology Laboratory, Department of Pathobiology, Ontario
Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G
2W1;^2^ Department of Veterinary Pathology, Western College of
Veterinary Medicine, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada S7N 0W2

An acute phase response was induced in rainbow trout (*Oncorhynchus
mykiss*, Walbaum) by inflammation triggered by intraperitoneal
administration of purified *A. salmonicida* LPS emulsified in Freund's
incomplete adjuvant (LPS/FIA) or a commercial oil-based *A. salmonicida*
vaccine. Acute phase proteins were characterized by comparative
densitometry of plasma proteins separated by 2D-PAGE and identified by
MALDI-TOF and ESI MS/MS mass spectrometry. In one experiment, plasma
samples were compared between treatment and control groups in which fish
were terminally bled. In another experiment, individual fish were
sampled repeatedly. Proteins with increased expression were those whose
normalized value increased three-fold or greater between pre- and
post-stimulus. Proteins with reduced expression were those whose
normalized values decreased two-fold or greater. Constitutive proteins
were those that were not altered or did not meet either of these
criteria. Proteins that were absent in pre-stimulus gels but present in
post-stimulus profiles were considered to be induced. Only those
proteins that were altered in all fish for a given treatment were
considered. In both experiments, protein p36 was increased up to 13-fold
and several proteins were detected that had not been previously. In all
fish treated with LPS/FIA, p9.5 was consistently increased an average of
75-fold in plasma. The only proteins similar to previously identified
proteins were pre-cerebellin-like (p24a), transferrin (p37) and
apolipoprotein (p10.5, p24c and p28).

## Cross-matching Of Elasmobranch Blood And A Transfusion Trial Within Atlantic Stingrays (*Dasyatis sabina*)

[Catherine A. Hadfield]{.underline}^1^, Ashley N. Haines^2^, and Brent
R. Whitaker^1^

^1^National Aquarium in Baltimore, Pier 3/501 E. Pratt St., Baltimore,
MD 21202; ^2^Dept. of Microbiology and Immunology, University of
Maryland School of Medicine, 655 W. Baltimore St., MD 21201

Bite wounds have lead to mortalities in several species of shark due to
hemorrhage and sepsis. Whole blood transfusions are a potential
supplement to elasmobranch-adapted isotonic crystalloids and hemoglobin
products. Cross-matching of red blood cells and sera was carried out
between nineteen individuals from seven species: sandbar shark
(Carcharhinus plumbeus), sandtiger shark (Carcharhinus taurus), common
nurse shark (Ginglymostoma cirratum), white-spotted and brown-banded
bamboo shark (Chiloscyllium plagiosum and C. punctatum), zebra shark
(Stegostoma fasciatum) and spotted wobbegong (Orectolobus maculatus).
Prior to the trial, the authors identified a suitable washing solution,
anticoagulant and positive controls. No positive cross-matches were seen
between individuals of the same species within sandbars, sandtigers and
common nurse sharks. Positive cross-matches were seen as hemolysis or
agglutination between several of the species, suggesting these would not
be suitable as donor-recipient combinations. A subsequent trial involved
transfusion of heparinized whole blood between five Atlantic stingrays
(Dasyatis sabina). Two control animals received an auto-transfusion or
crystalloids. No morbidity or mortalities were associated with the
trial.

Reproductive Problems, Diagnoses And Treatments In Teleosts

[Scott Weber]{.underline}^1^ and Paul Gary Egrie^2^

^1^New England Aquarium Veterinary, Central Wharf, Boston, MA 02110;
^2^Marketing and Regulatory Programs, Animal and Plant Health Inspection
Service, Veterinary Services, Riverdale, MD

Many species of fish do not readily breed in captivity. Because of
environmental threats and decreasing habitat, breeding fish in captivity
may become necessary for the survival of several species in the wild.
Fish reproductive problems and anomalies are a common occurrence in
public and home aquaria. Many of these cases had been previously
diagnosed on necropsy. With advances in fish medicine, reproductive
problems are being diagnosed pre-mortem to allow for treatments.
Diagnosis of reproductive related diseases begins with a thorough
history and water quality evaluation, as well as knowledge of the
individual species reproductive physiology. Some diagnostic modalities
that can be used to evaluate the fish patient are CBCs, blood
chemistries, ultrasound, radiology, computer assisted tomography,
general palpation, laparoscopy, cystocentesis, and cloacal endoscopy.
The most common problems encountered at the New England Aquarium in
order of prevalence include: dystocia or egg binding, infectious
disease, organ prolapse, organ trauma, and neoplasia. Specific
treatments for reproductive problems in fish can include hormonal
treatment, surgery, antibiotic or antifungal therapy, environmental
changes, and nutritional adjustments. Some individual cases will be
highlighted to examine diagnostic approach and treatment.

#  

# **Ecology Of The Fish Pathogen *Mycobacterium marinum***

# Mohammad R. Alavi^1^, H. D. Shukla^1^, ^2^J. Arnold, B. Whitaker^2^, and M. Shahamat^1^

# 

# ^1^Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD; ^2^National Aquarium in Baltimore, Baltimore, MD

# *Mycobacterium marinum* poses serious health and economic problems to the fish industries. Biofilters and walls in the aquaculture recirculating systems can be potential sites of colonization by this pathogen once it is introduced into the aquaculture tanks. Colonization of surfaces by bacteria requires initial attachment to the surface and increasing evidence suggests that this process can be modulated at the molecular level by the bacteria's microenvironment. The purpose of this research was to examine attachment of *M. marinum* to hydrophobic surfaces in minimal-defined culture medium and to determine whether the nutrient content and age of the medium plays any role in influencing this interaction. As part of this study we determined the strength of hydrophobic interaction between *M. marinum* cell and the surface of polypropylene by using solvents of different polarities to disrupt this interaction. Only highly non-polar organic solvents were able to efficiently remove the attached bacteria from the surface of polypropylene. It was also found that the ratio of mycobacteria in the population that attached to the polypropylene decreased with the age of the culture and this was correlated with accumulation of capsular material on the cell surface of the bacteria. An examination of the protein profiles of cells from exponential and stationary phases by Two-D gel electrophoresis indicated significant upregulation of a \~ 40 kDa protein in the stationary phase. We identified this protein as the alanine dehydrogenase of *M. marinum* by LC MS/MS analysis. This enzyme is thought to be secreted or associated with the cell surface and may be involved in response to stresses such as hypoxic or microaerophilic conditions. Elucidation of the molecular events that regulate attachment and capsular biosynthesis in *M. marinum* will lead to development of efficient and logical strategies for preventing colonization and spread of this pathogen in fish tanks. 

**Mycobacteriosis In Wild-Caught, Laboratory-Maintained Menhaden: A Case
Study**

# 

[Andrew S. Kane]{.underline}^1,2^, Cynthia B. Stine^1^ and Ana M.
Baya^1,3^

^1^University of Maryland, Department of Veterinary Medicine, College
Park, MD; ^2^University of Maryland, Department of Epidemiology and
Preventive Medicine, Division of Environmental Epidemiology and
Toxicology, Baltimore, MD; Maryland Department of Agriculture, Fish
Health Laboratory, College Park, MD

Mycobacteriosis is a chronic wasting disease found in many species of
fish examined from wild-caught, cultured and aquarium-reared
populations. A menhaden (*Brevoortia tyrannus*) with an open ulcer from
a wild-caught, laboratory maintained population was sampled for
microbiology. *Mycobacterium fortuitum* complex and *Mycobacterium* spp.
were recovered from the spleen and the ulcer of this fish, respectively.
Subsequently, twenty menhaden were sub-sampled from this population to
determine the extent of the infection. Bacteriology samples were taken
from the brain, liver and kidney. Spleens were homogenized and plated on
media enriched for mycobacterial growth, and full necropsies were
performed and samples were taken for histology. Bacteriology results
revealed a 100% rate of mycobacterial infection in this population of
menhaden. Three species of mycobacteria were isolated from the spleen
tissue: *M. marinum*, *M. fortuitum* complex, and *M. gordonae*.
Histology results revealed that granulomas, characteristic of
mycobacteriosis, were most prevalent in livers, but were also found in
spleen, posterior kidney and heart. In addition, *Vibrio hollisae* and
*Photobacterium damsela* were isolated from internal organs. This report
adds menhaden to the list of susceptible wild species to mycobacteria.
Ecologically, menhaden are an important prey species, and their
susceptibility indicates the potential for disease transmission.

Chloramine Removal From Municipal Water Supply For Use In Recirculating
Aquatic Life Support Systems

[Andrew Aiken]{.underline}^1^, Jay Bradley^2^, Dave Cohrs^1^

1 National Aquarium In Baltimore, 501 E. Pratt Street, Baltimore MD,
21202; 2 National Aquarium In Washington D.C., 14th St. and Constitution
Ave., NW, Washington, DC, 20230

> Municipal water authorities are beginning to use chloramine, which has
> greater residual than chlorine. For Fishes and Invertebrates,
> chloramine creates equivalent deleterious health conditions as
> chlorine. Removal of chloramine at point-of-use is more difficult than
> removal of chlorine; it cannot be effectively removed by activated
> carbon. Catalytic activated carbon types break the amine bond but
> leave ammonia in solution. At the National Aquarium in Washington,
> D.C., a chloramine removal method employs catalytic activated carbon,
> zeolite, a nitrate specific resin and aeration. Catalytic activated
> carbon is shown to effectively break the amine bond and subsequently
> remove chlorine. Zeolite is effective at removing ammonia; however
> some ammonia is oxidized to nitrite before reaching the zeolite bed. A
> nitrate specific ion resin effectively removes both nitrite and
> nitrate. Although the combination of activated carbon, zeolite, and
> the nitrate resin effectively remove chloramine and its residual
> nitrogen species, frequent regeneration of the various media beds
> result in occasional spikes of nitrite and/or ammonia into the product
> water stream. Aeration is added as a final step to mitigate these
> spikes in nitrogen species.

**Disaster Planning For Aquatic Animal Facilities**

# Brent R. Whitaker

National Aquarium in Baltimore, University of Maryland, Center of Marine
Biotechnology, Pier 3/501 E. Pratt St., Baltimore, MD 21202

Disasters can cause catastrophic damage and loss of life in aquatic
animal facilities. Preparing for major building damage, loss of support
infrastructure, inaccessibility during a crisis, and utility failure and
subsequent life support challenges can minimize the impact of a
disaster. Developing a detailed contingency plan that provides support
for both staff and animals is key to ensuring a successful outcome---or
at least minimizing the damage. This plan must include a designated
disaster response team with established leadership and a chain of
command, clear roles and responsibilities for each team member, methods
of communication prior to and during a disaster, and a mechanism to
provide essential utilities (power and water). Establishing affiliations
and a working relationship with local emergency management service (EMS)
will increase the likelihood that needed resources will be made
available during the event. Next, resources must be identified, and
equipment and supplies obtained. All equipment, such as generators and
radios, must be tested on a routine basis to ensure it is in working
order. Finally, training and drills must be conducted to ensure that all
staff are prepared for a disaster. In most facilities housing fishes the
loss of dissolved oxygen and temperature control are the most immediate
concerns when power is interrupted. Emergency generators can be used to
power air pumps and chillers. If no power is available, supplemental air
can be added using SCUBA tanks or compressed oxygen. To maintain water
temperatures in chilled systems, fresh or saltwater ice blocks can be
placed in the tank. Propane or oil-fired water heaters can be used as a
back-up to electrical units for warm-water systems. The accumulation of
nitrogenous waste can also be deadly to fish. The risk can be minimized
by reducing or stopping feeding for 1 to 2 days in advance of forecasted
events such as hurricanes. If life support is interrupted, most fishes
can be held for one or more weeks without food, as necessary, to
maintain water quality. Disaster planning should be undertaken by all
aquatic animal facilities. Common sense, coordination, communication,
and training will help minimize the impact of any disaster. When
disasters do occur, and life returns to normal, take the time to review
and improve your plan, and make the most of the lessons learned.

**Cooperative Investigations Of Suspect Disease Cases In Atlantic
Sturgeon (*Acipenser oxyrinchus oxyrinchus*)**

[Patricia Barbash^1^]{.underline}, John Coll^1^, John Fletcher^1^, Wade
Jodun^1^, Jerre Mohler^1^, Vicki Blazer^2^, Phil McAllister^2^, and
Cliff Starliper^2^

^1^USFWS, Northeast Fishery Center, 400 Washington Ave., Lamar, PA
16848; ^2^USGS, National Fish Health Research Laboratory, 11649 Leetown
Rd., Kearneysville, WV 25430

The U.S. Fish and Wildlife Service, Northeast Fishery Center
(NEFC-Lamar, PA) has taken a lead role in researching culture technology
for Atlantic sturgeon (*Acipenser oxyrinchus oxyrinchus*), an anadromous
species which has declined in population size in eastern rivers of the
United States. Beginning in 1991, NEFC developed methods of capturing,
spawning wild adult Atlantic sturgeon and rearing juvenile sturgeon from
the egg stage to age 12. The water supplies used for these efforts have
included a recirculated saltwater system, surfacing spring water, as
well as raw creek water. Circular tanks, ponds and serial use raceway
rearing units have been employed. During the course of the sturgeon
capture and propagation activities, several pathological situations
became evident, some of which required assistance from researchers at
the USGS National Fish Health Research Laboratory (NFHRL -- Leetown,
WV). Infections with *Saprolegnia* and other parasitic micro-organisms
diagnosed and treated by NEFC staff are discussed. Winter kill,
pathology associated with ingestion of *Bufo americanus* (American
toad), and swim bladder inflation syndrome contributed to experimental
culture mortalities. In addition, pathology associated with experimental
yearling sturgeon on raw creek water was investigated. Findings of on
site, small scale pathogenicity studies as well as findings of research
assistance from the NFHRL will be presented.

**Asessing Susceptibility Of Atlantic Sturgeon (*Acipenser oxyrinchus*)
To *Aeromonas salmonicida*, Cause Of Furunculosis**

[Rocco C. Cipriano]{.underline}^1^ and Patricia Barbash^2^

^1^USGS, National Fish Health research Laboratory, 11649 Leetown Road,
Kearneysville, WV 25430; ^2^USFWS, Northeast Fishery Center, Fish Health
Unit, 500 Washington Avenue, Lamar, PA 16848

Restoration of Atlantic sturgeon (*Acipenser oxyrinchus*) is a goal of
the Atlantic States Marine Fisheries Commission and the United States
Fish and Wildlife Service has researched methods at its Northeast
Fishery Center (NEFC-Lamar, PA) to enhance populations stocked into
mid-Atlantic rivers. In two instances, *Aeromonas salmonicida* was
isolated from brood fish from the Hudson River, but the pathogenic
significance was unclear. This study assessed virulence of *A.
salmonicida* for Atlantic sturgeon and attempted to transmit the
pathogen to sturgeon by co-habitation with infected trout. Juvenile
Atlantic sturgeon were found to be specific pathogen free after sampling
60 fish failed to isolate any bacteria systemically and only
*Acinetobacter* sp., *Pseudomonas* sp., *Moraxella* sp., *Enterobacter*
sp. and motile aeromonads were isolated externally. Nine of 12 *A.
salmonicida* isolates injected at 10^5^ cfu/sturgeon (3 fish/isolate)
induced mortality within 14-days. Isolate 3.155, originally obtained
from coho salmon (*Oncorhynchus kisutch*), produced complete mortality
within 5-days and had LD~(50)~ values of 2.3 cfu in brook trout
(*Salvelinus fontinalis*) and 2.3x10^4^ cfu in Atlantic sturgeon.
Therefore, sturgeon were susceptible to infection, but more resistant to
*A. salmonicida* than a typical salmonid host, such as the brook trout.
Brook trout were then injected with the bacterium and cohabited with
Atlantic sturgeon. All trout died within 8-days and Atlantic sturgeon
began to die at day 14. On day 21, surviving Atlantic sturgeon (n=21)
were assayed and the pathogen was cultured from mucous (n=20), gills
(n=17), kidneys (19), hearts (14), livers (n=13), spleens (n=7), and
guts (n=3) of these fish. Thus, *A. salmonicida* was horizontally
transmitted, established infection, and induced mortality in Atlantic
sturgeon cohabited with infected trout. A natural furunculosis epizootic
among juvenile Atlantic sturgeon at the NEFC is also discussed. These
studies indicated that *A. salmonicida* is pathogenic for Atlantic
sturgeon and suggested that these fish can contract furunculosis,
especially when cultured where *A. salmonicida* is enzootic.

**Pathogen Binding Proteins In Plasma Of Salmonid Fish**

S Russell, [K.M. Young]{.underline}, M.A. Hayes, P. Huber, J.S. Lumsden

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

Bacterial surface polysaccharides can be recognized by various humoral
and cell-surface lectins exhibiting direct antibacterial activity or
opsonizing capabilities. Identification, quantification and
determination of the potential importance of lectins as immune-active
proteins in fish, however, are recent activities. To assess specific
binding affinities, plasma proteins from rainbow trout (*O. mykiss*),
Atlantic salmon (*S. salar*) and Arctic char (*S. alpinus*) were
isolated based on calcium and carbohydrate-dependent binding to
synthetic matrices and pathogenic strains of *Aeromonas salmonicida* and
*Flavobacterium pyschrophilum*. Several saccharide- or calcium-elutable
proteins were isolated from all three species including those consistent
with serum amyloid P and C-reactive protein. Three other proteins
(16kDa, 37kDa and 40kDa) were also isolated from each species. The
rainbow trout 37kDa protein exhibits sequence homology to human
intelectin, which recognizes galactofuranose within carbohydrate chains
of bacterial cell walls**.** The 16kDa rainbow trout protein was
isolated by direct bacterial binding assays to both *A. salmonicida* and
*F. psychrophilum*. Western blots have confirmed the specificity of a
rabbit antiserum to the 16kDa protein that will be used for enzyme
immunoassay quantification of individual and temporal variation during
experimental infections. Our program goal is to determine the functional
significance of these plasma lectins in salmonid resistance and/or
susceptibility to infectious disease.

**Laboratory Efficacy Of Amoxicillin For The Control Of *Streptococcus
Iniae* Infection In Blue Tilapia**

[Ahmed M. Darwish]{.underline} and Melissa S. Hobbs

Harry K. Dupree-Stuttgart National Aquaculture Research Center, United
States Department of Agriculture, Agricultural Research Service, P. O.
Box 1050, Stuttgart, Arkansas, 72160, USA

Experimental feeding trials were performed to evaluate the efficacy of
amoxicillin (AMX) in controlling *Streptococcus iniae* infection in blue
tilapia, *Oreochromis aureus*. Doses of AMX tested were 0, 5, 10, 30 and
80 mg active ingredient per kilogram of fish body weight (BW) per day.
Administration of medicated feed started within 22-24 h post challenge
by waterborne exposure to *S. iniae* (after skin scraping) and continued
for 12 consecutive days, followed by a 17 d post treatment observation.
Oral administration of AMX medicated feed for 12 d at 10, 30 and 80 mg
AMX/ BW/d significantly increased (*P*\<0.05) the survival of *S. iniae*
infected tilapia from 3.8 % in challenged non-medicated positive control
to 45, 75 and 93.8 %, respectively. The survival rate was significantly
higher in the 80 mg treatment (93.75 %) than the 10 mg treatment (45%)
but there was no significant difference between the 30 and 10 mg
treatments (75 and 45 %, respectively). At the conclusion of the
experiment no carriers were detected in any challenged group receiving
AMX medicated diet while the bacterium was recovered from the
non-medicated challenged survivors of the infection.

**Development Of A Tuberculosis Model Using The Small Fish Medaka
(*Orysias latipes*) And *Mycobacterium marinum***

[Gregory W. Broussard]{.underline} and Don G. Ennis

Department of Biology, P.O. Box 42451, University of Louisiana, LA 70504

Human infection by *Mycobacterium tuberculosis* (TB) is endemic, with
approximately 2 billion people infected and is the leading cause of
fatality with about 3 million deaths annually. Because of its slow
growth rate (\>24hrs) and infection risk to researchers, other species
of *Mycobacterium* have been employed as alternative model systems for
TB. Nucleotide sequence analysis has shown that *Mycobacterium marinum*
is a closely related species to TB and has proven to be a good model for
tuberculosis infection, conferring TB-like infections in some fish.
Medaka (*Oryzias latipes*) have long been used for toxicological
research, are easily propagated, and offer many strains, good genetics,
and transgenic constructs. These include a variety that carry a shuttle
vector for studying *in vivo* mutagenesis. We are currently working to
test if medaka can provide an alternate model for tuberculosis-like
chronic infections by *M. marinum*. We are able to induce both acute and
chronic infections depending on bacterial dose. Bacterial colonization
of specific organs has been observed by colony counts and can be
visualized within organs using a *M. marinum* strain expressing green
fluorescent protein (GFP). The immunological manifestations of infection
are being surveyed by classical histology. With this new model for *M.
marinum* infection, we will be able to study the possibility of a
connection between chronic infection and mutagenesis, the role played by
the DNA repair network in bacterial survival within its host, and
explore other aspects of mycobacterial infections.

**Localization By In Situ Hybridization Of A Chlamydia-Like 16S
Ribosomal RNA Gene Sequence Within Branchial Epithelial Cells Of
Cultured Arctic Char (*Salvalinus alpinus*) With Epitheliocystis**

[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, USA; ^2^ Freshwater Institute,
Shepherdstown, West Virginia 25443, USA; ^3^ Department of Pathology,
Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, USA; ^4^
Department of Pathology, New York University Medical Center, New York,
NY 10016, USA; ^5^ Electron Microscopy Laboratory, Department of
Pathology, The University of Texas Medical Branch, Galveston, TX
77555-0609, USA

Epitheliocystis refers to a gill disease associated with yet uncultured,
gram-negative, intracellular, chlamydia-like or rickettsia-like bacteria
that causes morbidity and mortality in a wide range of fish.
Histologically, the agent forms intracytoplasmic, basophilic, granular
inclusions in branchial epithelial cells. Attempts to develop Arctic
char aquaculture have been plagued by epitheliocystis. Molecular
techniques have been used successfully to characterize an agent of
epitheliocystis in Atlantic salmon (Draghi et al. *Journal of Clinical
Microbiology*, Nov. 2004, 42 (11):5286-5297). Those same techniques have
been used to elucidate one possible etiological agent of epitheliocystis
in Arctic char. Samples from the Fresh Water Institute were taken for
histopathologic, ultrastructural and PCR studies. Histopathology was
typical of epitheliocystis. Special stains commonly used to detect
chlamydia reacted positively with branchial epithelial inclusions of
char. Transmission electron microscopic studies revealed the existence
of chlamydia-like life stages (reticulate bodies). The 16S rRNA gene
signature sequence amplified from histologically positive gill samples
using chlamydiales order-specific primers branched with members of the
order Chlamydiales as opposed to those of *Rickettsia* and *Ehrlichia*
in molecular phylogenetic studies using parsimony and distance methods.
Amplicons were cloned into a dual-promoter vector and transcribed to
generate digoxigenin-labeled riboprobes of 295-bp. These
digoxigenin-labeled sequence-specific riboprobes when applied to
infected arctic char gills labeled chlamydia-like inclusions within
epithelial cells. No labeling was seen in the same tissue with a
*Mycoplasma gallisepticum* 16S rRNA gene riboprobe, or in those sections
not receiving riboprobe. To the best of our knowledge, this is the first
example of a chlamydia-like sequence identified by in situ hybridization
in char, and confirms earlier data suggesting that a chlamydial-like
bacterium is associated with epitheliocystis in Arctic char.

**Quantitation Of Immobilization Antigen-Specific Antibody Secreting
Cells In The Skin And Head Kidney Of *Ichthyophthirius multifiliis*
Immune Channel Catfish Using ELISPOT Assay**

[Xiguang Zhao]{.underline}, R. Craig Findly, and Harry W. Dickerson

Department of Infectious Diseases, College of Veterinary Medicine, The
University of Georgia, Athens, GA 30605

This study provides the first quantitation of antibody secreting cells
(ASC) isolated from the skin of channel catfish (*Ictalurus punctatus*).
ELISPOT assays for the detection and quantification of ASCs were
developed and the isolation of viable leukocytes from skin is described
in detail. The density of leukocytes in skin was found to be between
2.5×10^4^ and 3.5×10^5^ cells/cm^2^. Preliminary ELISPOT assays indicate
that immobilization antigen-specific ASCs occur in the skin and head
kidney of catfish immune to infection with the ciliated protozoan
pathogen *Ichthyophthirius multifiliis*. The percentages of specific to
non-specific ASCs in skin and head kidney are 1.35% (n=1) and 1.97%
(n=2), respectively. Ongoing studies are focused on the ontogeny and
kinetics of the cutaneous antibody response of fish following infection
with the parasite.

**Intersex In Smallmouth Bass From The South Branch Of The Potomac
River: Implications Of Endocrine Disruption**

[Luke Iwanowicz]{.underline}^1,\ 2^, Vicki Blazer^2^, Chris O'Bara^3^,
Doug Chambers^4^, Tom Leiker^5^

^1^University of Massachusetts, Amherst, MA; ^2^USGS, Leetown Science
Center, National Fish Health Research Laboratory, Kearneysville, WV;
^3^West Virginia Division of Natural Resources, Parkersburg, WV;
^4^USGS, Water Resources Division, Charleston, WV; ^5^USGS, Water
Resources Division, Denver, CO

For the past few years, our laboratory, in collaboration with multiple
agencies has been involved in a multidisciplinary study in the South
Branch (SB) of the Potomac River. This work was initiated in response to
observations of skin lesions, occasional fish kills and a perceived
population decrease of smallmouth bass (SMB) by resource managers,
fishermen and the public. During 2003 SMB were collected from multiple
sites in the South Branch drainage and processed for histological
evaluation. Internally, perhaps the most interesting finding was the
high incidence of intersex in male fish at a number of sites. Intersex
in male gonochoristic fishes is typically an indication of exposure to
estrogens or other endocrine disrupting chemicals (EDCs). Thus this
observation led us to suspect that such chemicals may be present in the
SB. During the 2004 sampling seasons we collected SMB and other species
in order to confirm the observation of intersex male SMB and investigate
whether this condition was unique to SMB. Intersex was identified in SMB
and largemouth bass (LMB), but not suckers during 2004. The frequency of
intersex in SMB was high and ranged from 69-100% in pre-spawn fish.
Preliminary screening of water and sediment samples has identified the
presence known endocrine disrupting chemicals, but specific sources and
seasonal concentrations are currently unknown. The high incidence of
intersex in SMB may be associated with sediment or waterborne EDCs, but
this suspicion has not been confirmed. Ongoing research has been
designed to investigate the possible impacts of environmentally relevant
concentrations of EDCs identified in this watershed on SMB and LMB
reproductive development.

**Bacteriological Studies On Freshwater Mussels: The Flora Present In
Free-Ranging Animals And Pathogen Depuration By Two Species, *Amblena
plicata* And *Fusconaia ebena***

[Clifford Starliper]{.underline}^1^, Richard Neves^2^, Patricia
Barbash^3^, Patricia Morrison^4^, John Coll^3^, and Dean Rhine^4^.

^1^USGS Fish Health Laboratory, Leetown, WV; ^2^USGS VA Coop. Fish and
Wildlife Research Unit, Blacksburg, VA; ^3^USFWS Lamar Fish Health
Center, Lamar, PA; ^4^USFWS Ohio River Islands Nat'l Wildlife Refuge,
Parkersburg, WV

We report on studies to further understand the nature of the bacterial
flora of freshwater mussels. A number of Agencies are involved with
conservation, propagation and population augmentation of listed mussel
species. One dimension is collection and captive-rearing of feral
animals at fish hatcheries. Our effort is to study the potential for
pathogen vectoring (i.e. contamination) to hatcheries and to natural
habitats upon restoration. Seasonal mussel die offs have been observed
in the Holston and Clinch Rivers in Virginia; an infectious agent(s) is
suspected, however, none have been identified. We will report on the
bacterial flora from healthy mussels from these rivers, which was
studied to allow for subsequent comparisons with the flora from diseased
mussels. We have found the bacterial flora to rapidly change in response
to a change in the animals' water supply. However, in a study that
employed a model to transmit *Aeromonas salmonicida* between mussels and
fish, it took between 9 and 15 days for mussels to depurate this
pathogen, emphasizing the need for some quarantine of mussels prior to
their introduction to hatcheries. Another study has begun to develop
techniques for non-destructive examination of mussels for pathogens so
that health inspections and management strategies may be implemented to
reduce the risk for introduction and transmission.

**Roles Of Astaxanthin In The Pathobiology Of Lobsters, *Homarus
americanus***

[Amanda B. Tribble]{.underline}^1^, Bassem Allam^1^, Gordon T.
Taylor^1^, Michael Tlusty^2^, Alistair D. M. Dove^1^

^1^Marine Sciences Research Center, Stony Brook University, Stony Brook
NY 11794; ^2^New England Auqarium, Central Wharf Boston MA 02110

During the summer in western Long Island Sound (LIS), a confluence of
natural and anthropogenic factors often reduce dissolved oxygen
concentrations in the epibenthic water layer, which in turn leads to
increased levels of stress for the American lobster, *Homarus
americanus*. Anecdotal evidence suggested that at these times, the
carotenoid astaxanthin was present in higher concentrations in the
circulating hemolymph of lobsters. We hypothesized that astaxanthin is
mobilized to the plasma of lobsters during periods of stress, because of
its anti-oxidant properties. During the summer/fall of 2004, therefore,
207 lobsters were sampled from three different trap lines in western LIS
to investigate the dynamics of astaxanthin in lobster hemolymph and
tissues. Hepatopancreas, antennal gland, muscle, shell epithelium,
plasma, and hemocytes were all analyzed for astaxanthin concentrations
by spectrophotometry. A strong seasonal signal for plasma, shell
epithelium, and hepatopancreas astaxanthin was evident, with highest
levels observed in early September. Astaxanthin concentration in
hemocytes showed an increase until mid August and then decreased as
plasma, epithelium and hepatopancreas concentrations peaked. Lobsters
taken from the more western trap lines, where environmental stress is
typically greater, showed significantly higher astaxanthin
concentrations in hemocytes but not other tissues. Observations on
dynamics and spatial patterns of astaxanthin in lobsters were consistent
with the hypothesized role of this hemolymph carotenoid as a
stress-coping mechanism. The possibility that astaxanthin release into
the hemolymph is a disease symptom rather than a specific immune
response could not be excluded, but if this is so, then it would
represent a novel disease sign.

**Toxicopathology Of Resmethrin In Larval And Juvenile American Lobsters
(*Homarus americanus*)**

[Ann M. Zulkosky]{.underline}, Anne E. McElroy & Alistair D.M. Dove

Marine Sciences Research Center, Stony Brook University, Stony Brook NY
11794-5000

The American lobster (*Homarus americanus*) population in western Long
Island Sound suffered mass mortality in the fall of 1999. Pesticide
spraying, temperature/ hypoxia anomalies and *Neoparamoeba* infection
(or some combination) were proposed as possible explanations. The focus
of this study was to examine the response of larval (stage I and II) and
juvenile lobsters to insecticides, and determine if temperature stress
enhanced toxicity. Lobster larvae were exposed for 24 to 96 h to nominal
resmethrin concentrations up to 900 ng L^-1^ at both non-stressful
(16^o^C) and stressful (24^o^C) temperatures in a flow-through system.
Resmethrin toxicity was time dependent: LC50s decreased from \>300 ng
L^-1^ in 24-h exposures to 95 ng L^-1^ in 96-h exposures. Significant
pathologies were evident in the hepatopancreas of moribund larvae in the
highest treatments after 48 h, including nuclear and cellular
hypertrophy and epithelial sloughing. Elevated mortality was observed in
all groups at 24^o^C. Resmethrin was significantly more toxic than
malathion or methoprene to the larvae. The 48-h 16^o^C LC50 for
malathion was 3700 ng L^-1^, whereas methoprene exposure of up to 10,000
ng L^-1^ resulted in no mortality. Juvenile lobsters were exposed
continuously for 7 d to resmethrin and malathion at sublethal
concentrations of 30 ng L^-1^ and 1000 ng L^-1^, respectively, at both
stressful (22°C) and non-stressful (16°C) temperatures. Hemocyte
phenoloxidase (PO) activity was measured to assess the immune response.
Activity of PO was significantly reduced at 30 ng L^-1^ of resmethrin in
only one out of three experiments. Exposure to malathion failed to
elicit a significant response. Elevated temperature, however, caused a
65% increase in PO activity (*p* [\<]{.underline} 0.006) separate from
the effect of pesticide exposure. Quantitative histological analyses of
tissues collected from juveniles are in progress.

**Laboratory Culture And Health Care Of The Horseshoe Crab (*Limulus
polyphemus*)**

# Stephen A. Smith

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland
Regional College of Veterinary Medicine, Phase II, Duck Pond Drive,
Virginia Tech, Blacksburg, VA 24061-0442

The "American" horseshoe crab, *Limulus polyphemus*, is a unique marine
organism that has served as a laboratory research model to study the
structure, function, embryology and physiology of invertebrates.
Horseshoe crabs can be maintained in a wide variety of systems ranging
from glass aquaria to large fiberglass tanks with various types of
mechanical and biological filtration systems. Some systems may also
employ protein skimmers, UV filtration units, and ozonation to help
reduce potentially harmful bacteria from the water. Adult horseshoe
crabs are tolerant of a wide range of environmental conditions, ranging
in temperatures from --5^o^C to 35^o^C and salinities from 5-ppt to
35-ppt, with optimal temperatures between 15^o^C and 21^o^C and salinity
around 27-ppt. In captivity, horseshoe crabs are commonly fed dead fish,
squid, small crabs, clams and frozen brine shrimp. Horseshoe crabs also
readily consume commercially-prepared artificial shrimp/fish diets, but
the nutritional value of these synthetic formulations is not known.
Though the horseshoe crab has been used for many years as a laboratory
animal, few reports exist describing their diseases or syndromes.
Non-infectious problems range from water quality problems, developmental
problems and traumatic injuries. Infectious etiologies include algae,
fungus, colonial and filamentous cyanobacteria, Gram (-) negative
bacteria and a variety of protozoan and metazoan parasites. Clinical
evaluation of a horseshoe crab can be problematic as the hard,
non-transparent carapace makes examination and sample collection
difficult. In addition, very little is known concerning the treatment
and medical management of horseshoe crabs, though a few external
treatments have been suggested to remove ecto-commensals and external
parasites. To date, antibiotic treatment of bacterial infections in
horseshoe crabs has not been reported.

**Histological Techniques For Marine Bivalve Mollusks And Crustaceans**

[Earl J. Lewis, Jr.]{.underline}, Dorothy Howard, Jane Keller, and Ceil
Smith

NOAA, NOS, Coastal Center for Environmental Health and Biomolecular
Research, Oxford, Maryland 21654

In 1983 a NOAA Technical Memorandum "Histological Techniques for Marine
Bivalve Mollusks" was published by Howard and Smith. The publication
received worldwide circulation and another 500 copies were reprinted in
1997. Due to the popularity and continued requests for the publication,
a second edition was planned and published in December 2004. Like the
original, the updated publication is designed and written as an
instructional manual for scientists and technicians investigating the
health and diseases of coastal ocean and estuarine invertebrates;
however, many of the techniques may be applied to studies of fish and
other aquatic animals.  The 230-page manual provides detailed
instructions for the handling and processing of many bivalves and
crustaceans for histological examination in a step by step manner and
includes color photographs and illustrations of the processes,
information on invertebrate anatomy, signs of disease, recommended
tissue fixatives, color micrographs of results expected from routine and
specialized stains, and cryosection techniques. Chapters have been added
on histocytology, diagnostic techniques for blue crabs, miscellaneous
techniques and methods for mollusks, diagnostic techniques for the
softshell clam, and special techniques. An overview of the manual will
be presented.

**Plasma Cortisol In White Perch: Associations With Sampling Method,
Dissolved Oxygen, And Water Temperature**

[Catherine Czerwinski]{.underline}

U.S. Geological Survey, National Fish Health research Laboratory, 11549
Leetown Road, Kearneysville, West Virginia 25430

Cortisol is the main corticosteroid in fish released upon exposure to
stress. Plasma concentrations were quantified in white perch *Morone
americana* from the Hackensack River (NJ) and Pocomoke River (MD) by way
of competitive ELISA. Cortisol concentrations in white perch from the
Pocomoke River were significantly greater than in Hackensack River fish.
Fish were sampled using otter trawl, trap nets, gill nets, and seining.
Though differences in cortisol levels were seen based on sampling method
used, there were no significant differences. Although increases in water
temperature are associated with reduced cortisol concentrations, these
results were not significant. There were weak significant negative
correlations of cortisol with dissolved oxygen. Concentrations of
cadmium and lead in muscle were significantly positively correlated with
cortisol, whereas there was a significant negative correlation of
mercury with cortisol. Though pooled results showed no significant
associations of PCBs, organochlorine compounds, or DDT and related
compounds with cortisol, when females were analyzed separately, a
significant negative correlation between DDT related compounds and
cortisol was seen.