Laboratory Efficacy Of Florfenicol Against *Streptococcus iniae*
Infection In Sunshine Bass

[Ahmed M. Darwish]{.underline}

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

Abstract. -- An experimental feeding trial was performed to evaluate the
efficacy of florfenicol (FF) in controlling *Streptococcus iniae*
infection in sunshine bass (SB). Doses of FF tested were 0, 5, 10, 15
and 30 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 virulent *S. iniae*. The FF
medication was continued for 10 consecutive days, followed by a 25 d
post treatment observation. Oral administration of FF medicated feed for
10 d at 5, 10, 15 and 30 mg FF/ BW/d significantly increased (*P*\<0.05)
the survival of *S. iniae* infected SB from 4.2 % in the challenged
non-medicated positive control group to 69.2, 86.7, 94.2 and 94.2 %,
respectively. The survival rate was significantly higher in the 15 and
30 mg treatments (94.2 and 94.2 %, respectively) than the 5 mg treatment
(69.2) but there was no significant difference among the treatments 10
mg and higher. At the conclusion of the experiment no carriers were
detected in any challenged group receiving FF medicated diet while the
bacterium was recovered from the non-medicated challenged survivors of
the infection.

**Comparison of Lipopolysaccharide And Protein Profiles Between
*Flavobacterium columnare* Strains From Different Genomovars**

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

^1^Department of Fisheries and Allied Aquacultures, Auburn University,
Auburn, AL 36849; ^2^Aquatic Animal Health Research Laboratory, USDA,
Agricultural Research Service, AL 36849

Lipopolysaccharide (LPS) and total protein profiles from four different
strains of *Flavobacterium columnare* were compared in this study. These
strains belonged to genetically different groups. *F. columnare* ALG-530
and ARS-1 are highly virulent strains that belonged to genogroup I and
II respectively. *F. colulmnare* FC-RR is genogroup III avirulent
mutant. ALG-03-063 is a clinical strain included in the same genogroup
as FC-RR. Electrophoresis of LPS showed differences among the four
strains. Only low molecular weight LPS bands were displayed. ALG-530
shared a similar banding pattern with ARS-1, while FC-RR and ALG-03-063
LPS profiles were more complex. In addition, anti-ALG-530 and anti-FC-RR
sera were generated from channel catfish to perform immunoblotting
analysis. Analysis of LPS by immunoblotting revealed that the avirulent
mutant lacked the higher molecular bands in the LPS. Total protein
analysis displayed by immunoblotting showed differences between the
strains analyzed. However, some common bands were shared by all the
isolates. ARS-1 displayed the highest number of antigenic proteins.
FC-RR lacked two distinct common bands shared by the other three
strains. For both LPS and total protein analysis, each strain displayed
almost identical immunoblotting banding pattern regardless of antisera
used. Based on the differences of LPS and total protein profiles, it is
possible to discriminate between the avirulent mutant FC-RR from other
clinical *F. columnare* strains. According to our results, LPS may play
a major role in *F. columnare* pathogenicity.

**Evaluation Of Medium Components For Growth Of *Flavobacterium
psychrophilum***

[^1^Clifford E. Starliper]{.underline}, ^2^Susan Marcquenski, ^3^Rod
Penney, ^4^Andrew Noyes, and ^1^Pamela S. Whittington

^1^USGS National Fish Health Research Laboratory, 11649 Leetown Road,
Kearneysville, WV. 25430; ^2^Wisconsin Department of Natural Resources,
101 S. Webster St., Madison, WI. 53707; ^3^Canadian Food Inspection
Agency, 59 Camelot Drive, Ottawa, Ontario, Canada K1A 0Y9; ^4^New York
State Department of Environmental Conservation, 8314 Fish Hatchery Road,
Rome, NY 13440.

Coldwater disease has, and continues to warrant consideration when
rearing and managing important sport and restoration fishes in the Great
Lakes Region. Fish health specialists continue to be plagued with
difficulty in primary isolation of the causal bacterium *Flavobacterium
psychrophilum*, particularly among low-level, asymptomatic fish
encountered in health inspections. The thought is that weak *F.
psychrophilum* colony growth and overgrowth by garden-variety bacteria
and fungi on primary isolation plates often lead to false negative
conclusions. Because of this problem, we are trying to develop a medium
to improve primary isolation. Our approach is two-fold: enhance the
growth of what we desire to isolate, *F. psychrophilum*, and
inhibit/retard what we do not desire to isolate. The recipe for the
medium was extracted from the "best of the literature". We have done 72
h growth curves evaluating (in broth and on agar): cell diluents;
Anacker and Ordal Cytophaga vs. EAOA plus serum vs. experimental medium
"#2"; individual medium components; and four different types of serum.
Compared to Cytophaga medium, #2 and to a lesser extent EAOA plus serum,
yielded superior growth for many *F. psychrophilum* isolates. More
luxuriant colony growth, more quickly was evident on #2 plate medium.

Antimicrobial Resistance Patterns Of Ontario *Flavobacterium
psychrophilum*

Strains

[Shohreh Hesami]{.underline}, Katie Welsh, Julie Parkman, Jeff T. Gray,
John S. Lumsden and Janet I. 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 cold
water disease and rainbow trout fry syndrome. Coldwater disease has a
considerable economic impact in Ontario aquaculture operations
especially during the wintertime. Antibiotics in many operations are
used to reduce the economic impact of disease. Limited information is
available regarding antimicrobial resistance of North American (NA)
strains of *F. psychrophilum*. Ninety-two isolates of *F. psychrophilum*
were collected during an 8 year period from rainbow trout, Arctic charr
and Atlantic salmon. These isolates were characterized phenotypically,
biochemically and serologically and were found to be relatively
homogeneous. A multiplex PCR using primers for 16S ribosomal RNA and
subunit B of DNA gyrase is used to aid in primary isolation and to
confirm strain identity. To determine antimicrobial resistance patterns
of these *F. psychrophilum* strains, the minimum inhibitory
concentrations (MICs) were assessed using TREK SENSITITRE susceptibility
plates for aquaculture. The MIC values of three antibiotics frequently
used on farms in NA were high. For trimethoprime/sulfa-methoxazole
(STX), 90.9% of isolates had MIC\>1/19 (range 0.015/0.3 to 1/19 µg/ mL),
for sulphadimethoxine/ormetoprim (PRI), the MICs of 78% were \>76/4
(range 0.15/0.008 to 76/4 µg/ mL) and for oxytetracycline (OXY), the
MICs of 44% were \>8 (range 0.015 to 8µg/mL). In contrast, the MIC of
flofenicol (FFN) for 63% of isolates was low, between 0.25 and 4.0
(range 0.03 to 16µg/ mL). These results are similar to those in a number
of European studies except that the values for florfenicol for NA
strains are moderately higher. Moderate MICs to florfenicol occurred on
farms that had never used the antibiotic. In addition, 56 to 70% of the
strains tested had high MIC values for gentamicin, oxolinic acid, and
ampicillin, and low MICs for erythromycin, enrofloxacin and
flumequine---although none of these drugs are currently used in NA fish
farms. The *in vivo* significance of these results needs to be studied
with infection trials.

**Application Of Polyphasic Taxonomy To The Study Of Bacterial Fish
Pathogens**

^1^[Cova R. Arias]{.underline}^,\ 2^Craig A. Shoemaker, and ^2^Phillip
H. Klesius

^1^Department of Fisheries and Allied Aquacultures, Auburn University,
Auburn, AL 36849; ^2^Aquatic Animal Health Research Laboratory, USDA,
Agricultural Research Service, AL 36849

Bacteria identification constitutes one of the main challenging areas in
microbiology. Identification of bacterial pathogens at the species level
should be rapid and accurate. Moreover, pathogen identification at
strain level is crucial in most epidemiological studies. The best
approach for strain identification is the combination of fingerprints
generated by both phenotypic and genotypic techniques. In our lab we are
using four techniques based on phenotypic characters and up to five
genomic methods in order to fully characterize each bacterial isolate.
Phenotypic methods include Fatty Acid Methyl Ester (FAME) analysis and
miniaturized commercial systems (API, VITEK). Genomic methods comprise
fingerprinting methods such as Amplified Fragment Length Polymorphisms
(AFLP) and Enterobacterial Repetitive Intergenic Consensus(ERIC)
sequences analysis, as well as sequence-based methods such as 16S rRNA
gene and internal spacer region (ISR) analysis. All information
generated is compiled into a centralized database which allows data
integration from different typing methods. Practical examples of
polyphasic taxonomy applied to fish pathogens (*Edwardsiella ictaluri*,
*Flavobacterium columnare* and *Yersinia ruckeri*) are presented. *E.
ictaluri* behaves as a clonal species although high resolution
genotyping methods revealed differences between catfish and madtom
isolates. *F. columnare* is a phenotypically uniform but genetically
diverse species. Atypical *Y. ruckeri* isolates were identified as
biogroup 2 by polyphasic taxonomy methods.

**Using DNA Microarrays** **To Compare Clinical Isolates Of The Fish
Pathogen *Aeromonas salmonicida***

[^1^Laura L. Brown,]{.underline} ^2^W.A. Findlay, ^2^C.C. Luebbert,
^2^O. L. Mykytczuk, ^1^M. Reith, ^1^J. Boyd, ^2^E.N. Taboada, ^2^C.D.
Carrillo, ^1^S. Sperker, ^2^S. Foote, and ^2^J.H.E. Nash

^1^National Research Council of Canada (NRC) Institute for Marine
Biosciences, 1411 Oxford Street, Halifax, NS, B3H 3Z1, Canada; ^2^NRC
Institute for Biological Sciences, 100 Sussex Drive, Ottawa, ON, K1A 0R6

*Aeromonas salmonicida* has been isolated from numerous fish species,
and shows wide variation in virulence and pathogenicity. In order to
compare isolates from select geographic and host locations, an
amplicon-based DNA microarray was constructed using a subset of 2068
genes from the draft genome sequence of an *A. salmonicida* wild-type
strain. The microarray included genes encoding known
virulence-associated factors in *A. salmonicida*, and homologues to
virulence genes of other pathogens. The microarray was used in
comparative genomic hybridizations (CGH) whereby genomic DNA from
selected *A. salmonicida* sbsp*. salmonicida* isolates, as well as other
*Aeromonas* species and subspecies was compared with that of the
sequenced strain. Results showed that variation among the virulence
associated genes increased across sub-species and species boundaries.
Preliminary data showed no correlation between geographic region and
degree of genetic variation. Our data indicate that typing methods that
rely on phenotypes do not always correlate with the CGH data. Genome
variability studies, including microarray CGH, provide essential
information for the selection of targets for candidate vaccines and for
other health management tools.

**From Sequence To Sickness: Pathogenesis In *Aeromonas salmonicida*
Subsp. *Salmonicida***

[Andrew Dacanay]{.underline}, Jessica Boyd, Roger Ebanks, Kirty Solanky,
Leah Knickle, John Walter, Neil Ross, Stewart Johnson, Michael Reith,
and Laura Brown

National Research Council of Canada Institute for Marine Biosciences,
1411 Oxford Street, Halifax, Nova Scotia

*Aeromonas salmonicida* subsp. *salmonicida*, the etiological agent of
furunculosis, an infectious bacteremia of salmonids has been known since
the late 1800s. It was the subject of vaccination trials in the 1940s
and was the first bacterial species to have its outer surface protein
coat described (the eponymous A-layer). After over a century of study
the virulence factors of *A. salmonicida* subsp. *salmonicida* remain
unclear and more than one report in the literature describes the lack of
a relationship between genotype and a virulent phenotype. We used three
different methods to identify putative virulence factors; scanning the
genome manually, comparative genomic hybridization (CGH) and proteomics.
Of the many systems and genes identified in *A. salmonicida* subsp.
*salmonicida* and implicated in virulence in other bacterial pathogens,
three systems were chosen for further study. These systems were Type IV
pili, iron scavenging and sequestration and Type III Secretion. Isogenic
knockout mutants were created and tested for virulence *in vivo* and for
virulence-like behavior *in vitro*. Two adhesins, the Type IV pili Tap
and Flp, were found to be important in, but not absolutely required for
virulence in Atlantic salmon. *In vitro* studies showed that the A-layer
is still an important adhesin. Iron scavenging and sequestration is of
vital importance to pathogenic bacteria as *in vivo* iron levels are
significantly lower than those required to support microbial growth.
Three siderophore receptors; FstA, FstB and HupA were identified. In
contrast to the observations of others at least one of these receptors,
FstB, was required for virulence. In common with other workers the Type
III secretion system (TTSS) was found to be absolutely important for the
virulence of *A. salmonicida* subsp. *salmonicida.* Systematic deletion
of three identified TTSS effectors revealed only subtle effects on
virulence. These data, drawn from a number of systems, have allowed a
more understand more coherently the pathogenesis of furunculosis. This
research was partially supported by the National Research Council of
Canada's Genomics and Health Initiative.

**Bacterial Infections In Representative Lake Whitefish *(Coregonus
clupeaformis)* Stocks In Michigan**

[Thomas P. Loch]{.underline}, Ehab Elsayed, and. Moahmed Faisal

Department of Pathobiology and Diagnostic Investigation, College of
Veterinary Medicine, Michigan State University, East Lansing, MI 48824

Lake whitefish are an indigenous species that are highly sought after in
both the commercial and recreational fisheries within the Great Lakes
region. Within the last four decades, a notable deterioration in the
overall condition of this economically important species has occurred,
with both nutrition and disease being implicated as possible
contributing factors. In order to elucidate the role that pathogens may
play in this decline, wild, adult, Lake whitefish were collected from
four sites in lakes Michigan and Huron and were subsequently subjected
to extensive pathological examinations. Bacteriological cultures were
taken from the kidneys and any internal or external lesions and then
inoculated onto each of the following media; Brain Heart Infusion and/or
Trypticase Soy Agar, Coomassie Brilliant Blue Agar, Modified Kidney
Disease Medium, and Hsu-Shotts Agar. Any growth of interest was then
struck for isolation and subjected to exhaustive biochemical testing.
Concurrently, kidney tissues were sampled and tested via Q-ELISA in
order to assess the level of *Renibacterium salmoninarum* antigens, the
etiological agent of Bacterial Kidney Disease. Q-ELISA results have
illustrated definitive patterns associated with *R. salmoninarum*
infections that fluctuate by sample site, season, and year, yielding a
vivid picture of spatial and temporal prevalence/intensity that at
times, have reached upwards of 80% infection. In addition to *R.
salmoninarum*, other retrieved isolates include multiple species of
gram-negative bacteria belonging to three complexes within the genus
*Aeromonas*, the ubiquitous facultative pathogen known as *Pseudomonas
fluorescens*, and the gram-positive lactic acid bacterium,
*Carnobacterium piscicola*. Thus far, 31 representative *Aeromonas*
isolates, 17 representative *C. piscicola* isolates, and five
representative *Pseudomonas* isolates have been retrieved, some of which
were associated with both internal and external clinical lesions*.* Of
significant interest are the isolates of *C. piscicola,* which are being
reported from this host and region for the first time. In all, this
study is making great strides in elucidating the role pathogens play in
the current declines occurring within the indigenous Lake whitefish
populations of the Great Lakes region.

**Quantification Of A 16kda Defense Lectin (Ladderlectin) In Rainbow
Trout (*Oncorhynchus mykiss*) Plasma**

[Karrie M. Young]{.underline}, Spencer Russell, Mackenzie J.B. Smith,
Paul Huber, John S. Lumsden

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

Extracellular lectins play a critical role in the innate immune response
through recognition of carbohydrate patterns on microbial surfaces.
Soluble, multimeric lectins, such as the mannan-binding lectins (MBL)
and ficolins, enhance pathogen opsonization, directly activate
complement and augment phagocytosis. Although much of the evidence for
defense functions of soluble lectins is based on mammals, several
functionally homologous molecules have been identified in fish. Rabbits
were immunized with a synthetic peptide generated from amino terminal
sequence of the reduced 16kDa subunit of rainbow trout ladderlectin
(RT-LL). Antiserum specificity was confirmed by Western blots of whole
rainbow trout plasma. Similarly, bacterial binding activity of RT-LL to
a variety of fish pathogens including *Aeromonas salmonicida* sbsp.
*salmonicida*, *A.* *hydrophila*, *Yersinia ruckeri* and *Pseudomonas*
spp. was confirmed by Western blotting. Finally, we have developed an
enzyme immunoassay to compare individual and population-level variation
of RT-LL in the plasma of healthy rainbow trout. Similar to
investigations of porcine plasma MBL and ficolin, RT-LL revealed
considerable variation (p\<0.0001) amongst individual fish, as well as
between populations screened to date (p\<0.05). Ongoing investigations
will focus on individual and temporal variations of RT-LL in high and
low abundance fish during experimental infection with *A. salmonicida.*
Our goal is to monitor high and low abundance individuals to determine
if plasma lectin concentration correlates with increased (or altered)
disease resistance.

**Characterization Of Ladder Lectin And An Intelectin In The Plasma Of
Rainbow Trout (*Oncorhynchus mykiss*)**

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

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

To investigate lectin mechanisms in susceptibility and resistance to
disease, we isolated and characterized ladder lectin (RTLL) and
intelectin (RTIntl) from rainbow trout (*Oncorhynchus mykiss*) plasma
based upon their carbohydrate-dependent affinity. Both RTLL and RTIntl
bound to whole *Aeromonas salmonicida subsp. salmonicida*, chitin and
artemia and this activity was calcium-dependant. RTLL was characterized
by reducing 1 and 2D SDS-PAGE as a 16 kDa monomer with three isoforms
between pI 5.0 -5.3. Nucleotide sequence of RT-LL was deduced from the
N-terminal amino acid sequence of the protein and by 3'rapid
amplification of cDNA ends. The deduced amino acid sequence displayed
high homology to the Atlantic salmon mannan-binding lectin. Mannose
binding specificity was confirmed by the presence of and EPN amino-acid
motif in the C-type carbohydrate recognition domain. RTLL was identified
by immuno-histochemistry in the gill epithelium, renal interstitium,
heptic sinusoidal cells, and leukocytes. RTLL was also intimately
associated with *A. salmonicida* microcolonies. Reducing 1 and 2D
SDS-PAGE revealed RTIntl to be a 37kDa monomer with five isoforms
between 5.0 -- 5.6. Nucleotide sequence of cDNA encoding this lectin and
deduced amino acid showed moderate sequence similarity with a human
galactofuranose binding intelectin. RTIntl amino acid sequence does not
display a C-type lectin motif but rather a fibrinogen-like motif that
may involve carbohydrate binding. Immuno-histochemical RtIntl positive
cell were present in the skin, gill, intestine and swim bladder. The
functional significance of these lectins is now being investigated to
determine their roles as innate immune molecules.

**B Lymphocytes And Antibody Secreting Cells In Channel Catfish Skin**

[Xiguang Zhao]{.underline} and Harry W. Dickerson

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

The skin of fish is both the point of entry and site of infection for
many pathogens, and mucosal antibodies play a major role in host defense
against infection by parasites such as *Ichthyophthirius multifiliis*.
To demonstrate that antibody-secreting cells (ASC) and B lymphocytes are
present in fish skin, methods were developed to isolate and culture
leukocytes from the skin of channel catfish (*Ictalurus punctatus*). ASC
were identified by ELISPOT. Immediately after isolation of leukocytes
from fish unexposed to *I. multifiliis*, ASC were found to comprise
approximately 0.06% of these cells. As previously shown, in fish
vaccinated against *I. multifiliis*, 1.35**%** of skin ASC recognized
i-antigen, the major surface protein of *I. multifiliis*. This suggests
that ASC residing in skin are the source of mucosal antibodies that
confer protection against infection by *I. multifiliis*. To determine if
skin contains functional/mature B cells, leukocytes were isolated from
skin, placed into cell culture media [+]{.underline} LPS for 5 days, and
ASC numbers determined by ELISPOT. Initial results showed that LPS
treatment stimulated polyclonal activation, i.e. replication of B cells
(measured by BrdU incorporation) and differentiation into ASC, as the
number of ASC per 10^6^ cells increased significantly after 5 days in
culture. These data demonstrate that both ASC and B cells are resident
in skin of fish.

**The Impacts Of Redox Structure On Trout Antibody Affinity**

[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

Studies to date indicate that although trout serum antibody is comprised
predominantly of large molecular weight (800kDa), tetrameric Igs, these
antibodies possess considerable structural diversity (redox structure)
due to non-uniform disulfide cross-linking of the monomeric subunits.
There is limited data available on the possible functionality of these
different antibody structural forms. We have begun explore the effects
of this redox diversity on antibody affinity by the selective adsorption
of trout Igs onto a TNP-BSA carrier with varying ratios of hapten. This
selective adsorption revealed significant skewing of affinity profiles
of antibody subpopulations from the same anti-serum. The average
affinity of the low-density hapten-carrier conjugates (TNP1-BSA)
adsorbed antibodies possessing a higher apparent affinity constant (aK)
than that of those adsorbed by the high density conjugates (TNP13-BSA).
Further, subsets of antibodies demonstrated different redox profiles.
TNP1-BSA preferentially bound the higher order redox forms (i.e. those
wherein the tetramers were completely disulfide polymerized). However,
TNP13-BSA captured significantly fewer higher order forms and possessed
increased concentrations of lower order forms (i.e. less disulfide
cross-linked). Redox / affinity associations were also observed with
antibodies produced from *in vitro* culture of trout lymphocytes with
the antigen, TNP-LPS. Antibodies secreted by the lymphocytes assumed the
typical redox structure observed with serum antibodies; however, the
intracellular antibody tetramers are strictly composed of halfmer
subunits (100kDa). This intracellular form is surprisingly restricted in
affinity, compared to the secreted antibody which expresses a broader
range of affinities. This is surprising in that the repertoire of
affinities should be identical between both forms as 1) they arise from
the same cells and 2) affinity is supposedly dependent only upon the
antibody binding site. These distinctive affinity repertoires are highly
suggestive that oligomerization of the antibody molecule leads to
complex allosteric effects upon the antibody binding sites. In other
words oligomerization confers much of the affinity heterogeneity
observed in the antibody repertoire. The linking of the binding affinity
to redox structure is discussed.

**Characterization And Utilization Of Affinity-Purified Atlantic
Sturgeon (*Acipenser oxyrinchus*) Immunoglobulin**

# Stephen A. Smith and Mary E. Mainous

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

Immunoglobulin (Ig) was isolated from the serum of healthy Atlantic
sturgeon (*Acipenser oxyrinchus)* by affinity purification and
concentrated by centrifugation. The molecular weight of the reduced and
alkylated heavy chain was determined to be 71.1 kD by SDS-page
chromatography, while the two light chains were determined to be 22.5
and 27.7 kD. Therefore, the calculated molecular weight of the unreduced
native tetrameric IgM-like immunoglobulin using the predominant 22.5 kD
light chain was estimated to be approximately 750 kD. A rabbit antiserum
against the affinity-purified Atlantic sturgeon Ig was produced and
shown to react by gel diffusion with the serum Ig of numerous species of
North American sturgeon (gulf sturgeon, *A. oxyrinchus desotoi*; white
sturgeon, *A. transmontanus*; shortnose sturgeon, *A. brevirostrum*;
lake sturgeon, *A. fulvescens*; green sturgeon, *A. medirostris*;
shovelnose sturgeon, *Scaphirhynchus platorynchus*; pallid sturgeon, *S.
albus*) and paddlefish (*Polyodon spthula*), while serum from
taxonomically unrelated species of fish showed no reaction. The
specificity of the rabbit anti-Atlantic sturgeon antiserum recognized
both the heavy and light chains of the Ig by Western blot analysis. The
affinity-purified Ig was further used in an indirect enzyme-linked
immunosorbent assay (ELISA) to assess serum Ig concentrations in three
different species of cultured sturgeon (Atlantic sturgeon, shortnosed
sturgeon and white sturgeon) It is anticipated that this ELISA will be
useful in assessing the health status of wild populations of sturgeon.

# **The Health Condition Of Impinged And Open Water Fish From The Mobile River**

^1^[Jeffrey L. Baker]{.underline}, ^1^Jeffery S. Terhune, and ^2^William
E. Garrett

^1^Department of Fisheries and Allied Aquacultures, Auburn University,
AL. 36849; ^2^Alabama Power Company Environmental Affairs, Calera, AL.
35040

Studies investigating the general health status of impinged and
open-water fish were conducted at Barry Steam Plant located on the lower
Mobile River, Al. Species studied, due to high rates of impingement and
mitigation value, were threadfin shad, freshwater drum, blue catfish,
and channel catfish. Fish were sampled for the health studies during the
spring and fall of 2005 over a five to six week period in each season.
Fish suitable for necropsy were measured for total length, weighed, and
wet mounts prepared of gill samples and skin scrapes. Additionally,
trunk kidney and liver samples were removed and streaked on to bacterial
growth media to determine prevalence of bacterial pathogens. Impinged
fish were compared to open-water fish that were obtained from within 5
km of the vicinity of the intake area. A total of 938 fish representing
the impinged (N=457) and open water (N=481) populations were sampled.
Results from this study support the general hypothesis that impingement
may select compromised fish. Lower body weight was demonstrated to be
significant (at equivalent lengths) for all species except for channel
catfish. Both protozoan parasites (*Icthyobodo necator* and
*Chilodonella* sp.) and bacterial pathogens (*Flavobacterium columnare*
and *Aeromonas* sp.) were found at a significantly higher prevalence in
impinged fish versus fish from the open water population.

**The South Carolina Aquarium Sea Turtle Rescue Program**

^1^[Kelly J. Thorvalson]{.underline}, ^1^Thomas J. Sheridan, ^1^Jason T.
Crichton, ^1^Bruce G. Hecker, ^2^Rachel E. Metz

^1^South Carolina Aquarium, 100 Aquarium Wharf, Charleston, SC 29403;
^2^Woods Hole Science Aquarium, NMFS, Northeast Fisheries Science
Center, 166 Water Street, Woods Hole, MA 02543

Just after opening its doors in May 2000, the South Carolina Aquarium
responded to a call from the South Carolina Department of Natural
Resources to care for a sick loggerhead sea turtle. That turtle was the
start of a new endeavor for the Aquarium. With strandings on the rise in
the Southeast, there exists a strong regional need for a stranding
center. Although sea turtle rescue and rehabilitation was not in the
initial design of the Aquarium, a stranding program seemed to fit
naturally with its conservation mission. For several years, lack of
space, staff and resources limited the numbers of sick sea turtles the
Aquarium could house. Each year, holding tanks would reach maximum
capacity and additional stranded turtles would be transported a minimum
of 6 hours to the nearest rehabilitation center. The animals washing up
on the beaches are in critical condition and immediate treatment
increases their chances of survival. In response, the SCA Sea Turtle
Rescue Program has grown in its capacity to house and care for these
threatened and endangered animals and in 2005 reached its goal in taking
in every turtle that needed medical attention. Standardized treatments
administered to stranded turtles include taking blood for complete blood
counts, chemistries, cultures and sensitivities, performing radiographs,
analyzing fecal samples and removing ectoparasites. Additional
procedures may include blood transfusions, fluid therapy, tube feeding,
surgeries and physical therapy. Recovered turtles are released off the
coast of South Carolina.

**Characterization Of A Systemic Iridovirus From The Banggai
Cardinalfish (*Pterapogon kauderni*)**

^1^[Devon A. Young]{.underline}, ^2^Thomas Waltzek, ^1^Erica L.
Twitchell, ^3^Alejandro Vagelli, ^4^E. Scott Weber, ^1^Guillermo
Risatti, ^2^Ronald P. Hedrick, and ^1^Salvatore Frasca Jr.

^1^Department of Pathobiology and Veterinary Science, University of
Connecticut, Storrs, CT 06269-3089; ^2^Department of Medicine and
Epidemiology, School of Veterinary Medicine, University of California,
Davis, CA 95616; ^3^New Jersey Academy for Aquatic Sciences, Camden, NJ
08103; ^4^New England Aquarium, Boston, MA 02110-3399

During the period from 2003-2005, specimens of Banggai cardinalfish
(*Pterapogon kauderni*) were submitted to the Connecticut Veterinary
Medical Diagnostic Laboratory (Storrs, CT) to investigate mortalities of
multiple cohorts of fish after transport and apparent acclimation.
Histopathologic examination revealed large cells with basophilic
granular cytoplasmic inclusions located beneath endothelium throughout
the tissues of affected specimens, and transmission electron microscopy
revealed inclusions to consist of paracrystalline arrays of viral
particles with features characteristic of previously described
iridoviruses causing systemic infections of ornamental freshwater and
marine fish. Subgenetic fragments of the DNA polymerase (DNApol) and
adenosine triphosphatase (ATPase) genes were amplified, cloned and
sequenced from total genomic DNA extracted from fresh frozen tissues by
the Fish Health Laboratory (University of California, Davis, CA), and,
later, a segment of the major capsid protein (MCP) gene was amplified,
cloned and sequenced using DNA extracted from formalin-fixed
paraffin-embedded tissues. Molecular data will allow probe generation
toward in situ detection of virus in research and diagnostics, as well
as molecular phylogenetic studies to gain insight into the epidemiology
and evolution of these viruses.

***Coryphaena hippurus:* The Challenges Of Collecting And Exhibiting
This Pelagic Species**

[Arnold Postell]{.underline}

South Carolina Aquarium, 100 Aquarium Wharf, Charleston, SC 29401

During the winter of 2002 the South Carolina Aquarium started planning
to diversify the collection of pelagic species in our Great Ocean Tank,
a 385,000 gallon exhibit. The species we decided to try first was
*Coryphaena hippurus* (aka. dolphin, mahi, or dorado) due to its
availability offshore and proven success in a handful of other aquariums
and aquaculture facilities. The acquisition process was broken down into
5 main areas of focus: collecting, transport, acclamation, quarantine
and exhibiting. Each phase of this project had unique challenges that
would be vital to the success of our efforts. Collecting, quarantine,
and exhibiting the animals ended up being the easiest aspects while
transport and acclimation were the hardest. To date we still are fine
tuning the methods that work for mahi and we have begun to experiment
with other pelagic species.

***Amyloodinium* In Elasmobranchs**

[Christian J. Keller]{.underline}

The Tennessee Aquarium, One Broad Street, Chattanooga, TN 37401

Protozoal infections in aquarium fishes have always been a significant
problem. In particular, *Amyloodinium ocellatum* is persistent and
difficult to remove from marine systems. The life cycle of
*Amyloodinium* consists of an active form that attaches to the fish's
tissue called a trophont, a free swimming form known as a dinospore, and
a tomont or cystic form that retires to the bottom. The tomont is
resistant to chemical medical prophylaxis and treatment.

Copper is traditionally the therapy that helps control *Amyloodinium* in
systems and on teleost fish. Previously, there had been little written
on the subject of this protozoon infecting elasmobranchs. Copper, of
course, is a threat to elasmobranches. Recently, *Amyloodinium* was
diagnosed and treated in two public aquaria. The vector for the
infection appears to be three shipments of stingrays, *Dasyatis
americana,* from the same source. The fish were successfully treated
with 10ppm chloroquinone disphosphate.

Laparoscopic Liver Biopsy In Fish: Equipment, Technique, And Preliminary
Assessment Of Biopsy Quality

^1^[Charles J. Innis]{.underline} and ^2^Salvatore Frasca Jr

^1^Animal Health Department, New England Aquarium, Central Wharf,
Boston, MA 02110-3399; ^2^Department of Pathobiology and Veterinary
Science, University of Connecticut, 61 North Eagleville Road, Storrs, CT
06269-3089

As an initial investigation of the utility of laparoscopy in fish,
laparoscopy and laparoscopic liver biopsy were performed post-mortem or
peri-mortem in six fish that died or were euthanized due to advanced age
or illness. In addition, these procedures were performed in two living
fish for diagnostic reasons. A 2.7mm diameter, rigid endoscope with a
surgical sheath and cup biopsy instruments were used. Insufflation of
the coelom was achieved using 0.9% sodium chloride solution delivered
via the surgical sheath. Anesthesia was induced and maintained with
MS-222 at 90ppm. In all cases, good visualization of the coelomic
viscera was achieved and biopsies were easily performed. In two terminal
cases, the spleen was biopsied in addition to the liver. A size 5 French
biopsy instrument provided better purchase and biopsy size than the
smaller 3.5 French instrument. Histopathologic evaluation of biopsies
revealed good correlation between biopsies and tissues obtained at
necropsy. In two cases, the target organ was incorrectly identified, and
biopsy of testicle and granulomatous ovary were obtained. Surgical
incisions in the two living patients were closed with a single cruciate
nylon suture. Recovery of these patients was uneventful, although they
were later euthanized due to persistence of their illness.

**Challenges Of Managing Horseshoe Crabs (*Limulus polyphemus*) In An
Interactive Exhibit**

[Stacey R. Gore]{.underline}, Catherine A. Hadfield, Leigh A. Clayton,
and Ashleigh E. Clews

National Aquarium in Baltimore, Pier 3/ 501 E. Pratt Street, Baltimore,
MD 21202

The Children's Discovery Corner at the National Aquarium in Baltimore is
a 'touch tank' housing horseshoe crabs, channeled whelk, knobbed whelk,
bay sea stars, green crabs, and pencil urchins. It is an 800-gallon
recirculating artificial saltwater system, which is divided into a
primary exhibit tray and four back up trays. Since June 2005, the
following lesions were identified in horseshoe crabs: soft shells, gill
erosion, abraded compound eyes, and torn telson musculature.
Differentials for these problems include conspecific trauma, improper
handling, infectious diseases, poor water quality, and malnutrition. A
review of lesions, histopathology, and medical treatments will be
presented. Challenges identified in adequately managing this species
included: inadequate record keeping and communication regarding animal
rotation, diseases, and mortalities; inability to perform routine water
changes; inappropriate handling of animals by volunteers and members of
the public; and lack of sufficient numbers of animals for program needs.
Medical and husbandry staffs have addressed these concerns by increasing
water change frequency, improving all aspects of record keeping and
communication, and educating volunteer staff on proper animal handling.
Details regarding these issues will be presented. Invertebrates should
not be considered "disposable" resources. Every effort should be made to
ensure that all animals, vertebrate or invertebrate, are provided with
the same standards of care.

Medical Management Of Cold-Stunned Sea Turtles Stranded On Cape Cod

^1^[E. Scott Weber III]{.underline} and ^2^Constance Merigo

^1^Animal Health and ^2^Rescue/Rehabilitation Departments, New England
Aquarium, Central Wharf, Boston, MA 02110-3399

Although most species of sea turtles inhabit warm waters, over the last
decade increasing numbers of stranded sea turtles have occurred annually
in the late autumn and early winter on the northeastern shores of Cape
Cod. Most of these animals are juvenile Kemp's Ridleys, but
representatives of several other sea turtle species are also recovered.
Because the pelagic stage of Ridleys is unknown, the reason animals
strand on Cape Cod during this time of year can only be conjectured as
related to changes in ocean currents, tides, wind trajectory, and
weather/storm patterns coupled with the geographical anomaly of the
Cape's formation. In autumn, the Massachusetts Audubon Society monitors
the beaches for stranded animals based on weather conditions, prevailing
wind, and high tides. Live animals are then transported to the New
England Aquarium's (NEA) Rescue and Rehabilitation Department. At NEA,
animals receive an initial health assessment and veterinary care to
treat the most immediate and life-threatening conditions of hypothermia,
dehydration, pneumonia, and trauma. From 1999 -- 2005, NEA has responded
to 563 stranded sea turtles and has had over an 88% survival rate in
individuals that are warmed to ambient temperature in an Intensive Care
Unit (ICU) over the first three days post arrival. The following offers
a brief overview of initial triage, diagnostic findings, treatments, and
release of rehabilitated cold-Stunned sea turtles at NEA.

**Vaccine Design**

[Erin S Bromage]{.underline} and Stephen L Kaattari

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

Reduction in catches in commercial fisheries operations is putting
ever-increasing demands on aquaculture to satisfy market demand. This
emphasis on increased farm production has led to a dramatic expansion in
aquaculture operations globally. One of the key factors that can define
the success or failure of an aquaculture operation is control or
prevention of disease. For many years aquarists have turned to
antibiotics as a means to control disease, but there is considerable
resistance developing against their continued use. Vaccines, however,
are seen as one of the most cost effective and efficacious ways to
prevent disease outbreaks in commercial operations; however successful
commercial vaccines are rare. Recent research in our laboratory has
found that the induction of a certain cell type, the long-lived plasma
cell, is important for the long-term maintenance of humoral immunity. In
the absence of these cells, antibody titer diminishes, and humoral
immunity is lost. Furthermore, within the logistics of an aquacultural
setting, often a single immunization is all that can be afforded. In
such cases it is critical that this single immunization produce titers
of the greatest longevity. Therefore it is important when designing a
vaccine that the researcher not only determines the protective epitope
to generate the vaccine, but also administers the vaccine via a route
and at the correct dose to optimize the production of long-lived plasma
cells. This seminar will discuss the new research being conducted on
long-lived plasma cell generation, which will allow more efficient
design, dose and delivery of vaccines.

**Beneficial Use Of Attenuated Vaccines In Aquaculture**

[Victor S. Panangala]{.underline}, Phillip H. Klesius, Joyce J. Evans
and Craig A. Shoemaker

USDA, Agricultural Research Service, Aquatic Animal Health Research
Laboratory, Auburn, AL 36830 and Chestertown, MD 21620

The success of safe and effective vaccines in controlling disease and
enhancing profitability in livestock production has been profound.
Because fish farmers operate on narrow profit margins, cost
effectiveness is of paramount importance in any vaccination program. In
aquaculture, the best vaccines should offer ease of administration with
the minimum of stress, long-term protection, and lowered production
cost. Within these parameters, the vaccines should be efficacious in
protecting fry that are most vulnerable to disease-causing agents until
the pond-stock is ready for the market. With these objectives in view,
two new vaccines for immunization of catfish against *Edwardsiella
ictaluri* \[the cause of enteric septicemia in catfish (ESC)\] and
*Flavobacterium columnare* (cause of columnaris disease) were developed,
field tested, and licensed for immunization of 7-10 day post-hatch fry
by immersion exposure. The USDA, Agricultural Research Service developed
vaccines licensed to Intervet and marketed under the trade names
AQUAVAC-ESC^®^ and AQUAVAC-COL^®^ are used for protection of fish
against ESC and columnaris disease respectively. The vaccines have
augmented survival rates and provided protection up to periods of 1 to 2
years. The cost benefits of vaccination with AQUAVAC-ESC alone, is
estimated by Intervet to be \$1,706 per pond acre compared with
similarly farmed non-vaccinated catfish. Notably, attenuated vaccines
have a proven record of safety and efficacy for vaccination of poultry,
swine and cattle. Since many bacterial pathogens are ubiquitous in the
aquatic environment, attenuated vaccines should afford adequate
long-term protection until fish are ready for market distribution.

**DNA Vaccines And Environmental Safety**

^1^[Nathalie C. Simard]{.underline} and ^2^Diane Moores

^1^Novartis Animal Health Canada Inc., 797 Victoria Road, Victoria,
Prince Edward, Canada C0A 2G0; ^2^Research and Productivity Council, 921
College Hill Road, Fredericton, New Brunswick E3B 6Z9

In 2005, Novartis Animal Health (NAH) was issued a license by the
Canadian Food Inspection Agency (CFIA) for the commercial release of
APEX IHN^©^, a plasmid DNA vaccine targeting the infectious
hematopoietic necrosis (IHN) virus of salmonids. Safety evaluation of
APEX IHN^©^ included a study examining the potential for plasmid DNA
transfer to the intestinal aerobic microbial flora of vaccinated
Atlantic salmon. Microorganisms isolated from the feces of APEX IHN^©^
vaccinated (N=40) and non-vaccinated (N=10) Atlantic salmon were
selected on the basis of their resistance to the aminoglycoside
kanamycin (kan) owing to the presence of a phosphotransferases (3')I
selective marker within the APEX IHN^©^ vector. A total of 109 and 58
Kan+ microorganisms were isolated from the feces of vaccinated and
non-vaccinated fish respectively. Further characterisation based on
morphology, Gram stain, and total protein profiling, indicated that the
isolates belong to nine microbial species. Presence of the APEX IHN^©^
plasmid in the isolates was assessed by polymerase chain reaction (PCR)
using plasmid specific primers. Only DNA extracts with positive
amplification for 16S rRNA confirming the quality of the DNA template
were considered for PCR analysis. The expected 406 bp amplicon could not
be detected in 64 DNA extracts from vaccinated or 48 extracts from
control fish. The results therefore indicated that resistance to
kanamycin observed in the isolated microorganisms was derived from
innate or acquired resistance mechanism(s) and not due to the
transferred of APEX IHN^©^.

**IPNV Cohabitation Challenge Of Atlantic Salmon Post-Smolts**

^1^[Linda M. Bootland]{.underline}, ^1^Malcolm Lizama, ^1^Lisa Phillips,
^2^Andrew C. Barnes, and ^1^Kira Salonius

^1^Novartis Animal Health Canada Inc, Aqua Health, Business, PO Box 17,
Victoria, PE, Canada C0A 2G0; ^2^Novartis Animal Health, PO Box
101,Victoria Rd, Aberdeen, Scotland AB11 9D8

Infectious pancreatic necrosis virus (IPNV) causes high mortality in
Atlantic salmon post-smolts. An experimental saltwater IPNV cohabitation
challenge model using a Canadian strain of Atlantic salmon post-smolts
was developed. Virus isolate and fish stock both affected mortality.
Injection of a Scottish IPNV isolate caused 36-45% mortality in Scottish
salmon. In a new trial using Canadian St. John River salmon, the
Scottish IPNV isolate was avirulent, but a Norwegian IPNV isolate caused
50-80% mortality when given by injection. Naive fish cohabited with
Norwegian IPNV-injected fish had a delayed onset of mortality but a
cumulative mortality of 44-60%. The number of injected fish (15-25)
placed with 25 naive fish did not appear to alter mortality of the naive
fish to a great extent. To evaluate new vaccine constructs, cohabitation
of five Norwegian IPNV-injected fish with fifteen vaccinated fish
resulted in more variable mortality in the injected fish (40-100%), but
the model was adequate for assessing the efficacy of some of vaccines.
In the third trial, 20 injected fish cohabited with 25 vaccinates
resulted in more consistent mortality in the injected fish and allowed
for a realistic evaluation of vaccine dose. Critical parameters for
cohabitation IPNV challenge include fish stock, virus isolate and the
ratio of injected:vaccinated fish. Precocious males should be removed.
The effect of injected virus dose and fish density will be evaluated in
future studies to develop a fully reproducible IPNV challenge for
post-smolts.

***Ichthyophthirius* *multifiliis*: Challenges In Vaccine Development**

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

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

The ciliate *Ichthyophthirius multifiliis* is a highly virulent obligate
parasite that infects freshwater fish. Channel catfish (*Ictalurus*
*punctatus*) are very susceptible to infection, but can be routinely
immunized in the laboratory by exposure to sub-lethal infection.
Immunity develops within 2 to 4 weeks and is long-lived, lasting for at
least one year. Following infection, fish mount an antibody-mediated
immune response directed primarily against a class of abundant 40- to
60- kDa *I. multifiliis* surface membrane proteins, referred to as
immobilization antigens, or i-antigens. Antibodies recognizing
i-antigens are present both in blood and skin, where they are produced
by resident antibody secreting cells. Vaccination with purified
i-antigen also confers protective immunity against parasite challenge.
Thus, two important criteria for the development of a vaccine against
this parasite exist: the development of a naturally acquired adaptive
immunity following infection and the identification of specific
protective antigens. A number of practical challenges remain to be
resolved before a commercial vaccine becomes available. These include:
the presence of different serotypic strains of *I. multifiliis*, the
production of antigen in quantities sufficient for vaccination, and a
method of vaccine delivery that optimizes the induction of cutaneous
mucosal antibodies and stimulates long-term immune memory.

Structural Analysis And Epitope Mapping Of *Ichthyophthirius* I-Antigens

^1^Donna Cassidy-Hanley, ^1^Owen Smith, ^1^Melanie Santspree, ^2^Daniel
Ripoll, and ^1^[Ted Clark]{.underline}

^1^Department of Microbiology and Immunology, Cornell University,
Ithaca, NY 14850; ^2^Theory and Simulation Science and Engineering
Center, Cornell University, Ithaca, NY 14850

The parasitic ciliate *Ichthyophthirius multifiliis*, expresses abundant
GPI-anchored membrane proteins known as immobilization antigens
(i-antigens) that are important in the host immune response. These
antigens vary in natural populations and at least five i-antigen
serotypes (A--E) have now been identified. In connection with efforts at
vaccine development, we are interested in the structure of these
proteins, and have begun to map the determinants responsible for
eliciting immobilizing and protective antibodies in the host. A
characteristic of these proteins is a series of tandem repeats of \~80
aa each that span their length. However, tandem repeat copy number
varies, and the i-antigens thus far analyzed share only \~50% homology
at the level of primary sequence. We have used computational modeling to
deduce a structure in which the tandem repeats are stabilized by
disulfide bonds, and are separated by flexible hinge regions. This in
silico approach further predicts that the N-terminal repeats adopt a
fold similar to the laminin-type epidermal growth factor-like module
(L-EGF) with a high degree of certainty. To experimentally map the
conformational epitopes that interact with protective antibodies, we
have over-expressed the genes for these proteins in the free-living
ciliate *Tetrahymena thermophila*. Cells expressing chimeric gene
products are immobilized by antibodies that are specific for different
i-antigen variants allowing regional mapping of the epitopes responsible
for eliciting protective immunity in the host. In the long term,
identification of such epitopes should contribute to the development of
effective multivalent vaccines.

**Ecological Determinants In Outbreaks Of Bitter Crab Disease
(*Hematodinium* sp.) In Snow Crabs, *Chionoecetes opilio,* From
Conception Bay, Newfoundland, Canada**

^1^[Jeffrey D. Shields]{.underline}, ^2^David M. Taylor, ^2^Steven G.
Sutton, ^2^Peter W. Collins, ^2^Danny W. Ings, and ^2^Amanda L. Pardy

^1^Virginia Institute of Marine Science, The College of William and
Mary, Gloucester Point, VA 23062, USA; ^2^Department of Fisheries and
Oceans, CP5567, White Hills, St. John's, Newfoundland, CANADA A1C 5X1

The parasitic dinoflagellate *Hematodinium* sp. causes bitter crab
disease (BCD) in snow crabs, *Chionoecetes opilio*, and Tanner crabs,
*C. bairdi*. As implied, crabs infected with BCD are unmarketable due to
their bitter flavor. We surveyed the distribution of BCD in Conception
Bay, Newfoundland from 1997 to 2004. The disease has become firmly
established, starting with a prevalence well below 1% in 1997 to an
epizootic in 1999 that persisted through 2000 reaching prevalences of
over 2% to 9% in trapped and trawled male crabs and from 19% to 26% in
trawled and trapped female crabs, respectively. Infections were highest
in females and small males. In 2004, there was a shift in the dynamics
of the disease. An epizootic occurred primarily in adult males. This
coincided with increased temperatures and mass molting events that had
not occurred in previous years. Temperature, benthic substrate, depth,
host size and sex were all correlated with prevalence of the BCD during
outbreaks. Patterns in the molting cycle and prevalence of infection
indicate that transmission occurs during the post-molt condition, and
that overt infections probably develop two to four months after
infection, lasting three to four months thereafter.

**Molecular Diagnostics For *Hematodinium* And Other Protozoa In Water
And Crustacean Hosts**

[Richard F Lee]{.underline} and Marc E Frischer

Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah,
GA 31411

Diseases that effect commercially important crustaceans represent a
threat to commercial fishing, recreational fishing and regional tourism.
However, little is known about many of the diseases, parasites and
pathogens that occur in the estuaries of the South Atlantic Bight.
During a recent drought (1999-2003) we observed an outbreak of
hematodinium disease in coastal Georgia blue crabs caused by the
parasitic dinoflagellate, *Hematodinium* sp. Monitoring of Hematodinium
infection was facilitated by the development of a PCR diagnostic assay
specific for *Hematodinium*. Using this technique we were able to detect
*Hematodinium* sp. in several crab species, including *Callinectes
sapidus, Neopanope sayi, Libinia emarginata, Menippe mercenaria*. The
assay involves extraction and purification of genomic DNA, amplification
of a 187bp 18S rDNA fragment using Hematodinium-specific primers, and
detection by gel electrophoresis. This PCR diagnostic assay is rapid and
more sensitive, by several orders of magnitude, than the histological
assay. In our studies we observed a seasonal pattern of *Hematodinium*
sp. infection with low levels of *Hematodinium* sp. in the winter and
intense infection during the spring and fall. We also used this assay to
detect *Hematodinium* sp.in the water column, providing the first
evidence for a free-living life cycle stage of *Hematodinium* sp. More
recently we developed and validated a quantitative Real Time PCR assay
utilizing the *Hematodinium* sp. primer set. Comparison of quantitative
PCR results to direct hemolymph microscopic counts from heavily infected
crabs indicated a direct relationship between the two techniques
validating the assay. To expand our research into other parasitic
protozoan species infecting crustaceans we plan to use denaturing high
performance liquid chromatography (dHPLC) to detect and identify unknown
protozoan parasites. The protocol involves purification of genomic DNA
from crustaceans, amplification of separated parasite 18S rDNA fragments
using universally targeted primers and identification of parasite 18S
rRNA gene fragments after separation on a DNA binding chromatography
column.

**Lysozyme Gene Expression As A Molecular Marker Of Hemocyte Location In
Pacific White Shrimp, *Litopenaeus vannamei,* After Injection With
*Vibrio***

[Erin J. Burge]{.underline}, Daniel J. Madigan, Louis E. Burnett and
Karen G. Burnett

Grice Marine Laboratory, College of Charleston and Hollings Marine
Laboratory NOAA/NOS, 331 Fort Johnson Rd., Charleston, SC 29412

The purpose of this study was to develop a genetic molecular marker of
hemocyte distribution among tissues of the Pacific white shrimp,
*Litopenaeus vannamei.* We used this marker to investigate the immune
response of shrimp to a bacterial pathogen. We quantified by real-time
PCR the gene expression of lysozyme, an anti-bacterial protein expressed
by shrimp hemocytes. Tissue-level transcript expression also was
detected by *in situ* hybridization of mRNA in circulating hemocytes and
within tissues with high hemocyte concentrations. Lysozyme gene
expression was quantified in 5 tissues and in circulating hemocytes for
48 h following intramuscular injection of shrimp with *Vibrio
campbellii*. Lysozyme mRNA significantly decreased within 4 h in
circulating hemocytes and peripheral tissues after injection of bacteria
due to hemocyte trafficking to the site of injection during the early
course of the immune response. Lysozyme signal increased significantly
in the muscle at the site of injection during this early period and
remained high for the duration of the time-course. These data monitoring
hemocyte localization by lysozyme mRNA expression indicate that after
injection of bacteria, hemocytes accumulate at the injection site, which
supports the contention that the composition of the circulating hemocyte
pool is modified by bacterial injection. Supported by NSF Grant No.
IBN-021292

**Changes In Metabolism And Performance In Crustaceans Fighting
Bacterial Infections**

^1^[Louis E. Burnett]{.underline}, ^1^Lindy Thibodeaux, ^2^David
Scholnick, and ^1^Karen G. Burnett

^1^Grice Marine Laboratory, College of Charleston & Hollings Marine
Laboratory, 205 Fort Johnson, Charleston, SC 29412; ^2^Marine Science
Program, Eckerd College, St. Petersburg, FL 33711

We were interested in determining if mounting an immune defense against
a sublethal dose of pathogenic bacteria affected other functions in
crustaceans. Injecting shrimp (*Litopenaeus vannamei*) with *Vibrio
campbellii* caused a 19% reduction in O~2~ uptake that was rapid and
persisted for 24 h. The blue crab *Callinectes sapidus* experienced a
43% decline in O~2~ uptake following injection of *V. campbellii*. This
decline in blue crabs is associated with lower hemolymph oxygenation and
elevated vascular resistance, consistent with the idea that hemolymph
flow through the gill is occluded by the formation and trapping of
hemocyte aggregates. The low aerobic metabolism resulting from bacterial
injection does not appear to be compensated with anaerobic energy
production. We have begun to assess the impacts that the overall
lowering of metabolism might have on the ability of both shrimp and
crabs to perform sustained periods of activity such as swimming or
walking, activities critical to survival in the field. Shrimp placed on
treadmills sustained swimming activity for hours. Saline-injected shrimp
built up lactate in their tissues (10.6 µmol g^−1^ tissue) and secreted
lactate into the surrounding water (1.4 µmol g^−1^ h^−1^) during 30 min
of swimming. Surprisingly, *Vibrio*-injected shrimp accumulated similar
amounts of lactate in the tissues, but excreted much less (0.5 µmol
g^−1^ h^−1^) than saline controls. After walking on a treadmill for 30
min, saline-injected blue crabs produced higher hemolymph concentrations
of lactate (8 mmol/L) (3.7 mmol/L) than *Vibrio*-injected crabs. Lactate
excretion by saline-injected crabs was much higher (13.9 µmol g^−1^
h^−1^) than by shrimp, and significantly higher than *Vibrio*-injected
crabs. These data suggest anaerobic pathways are important during
exercise, but that bacterial challenge interferes with either the
overall metabolism and/or the excretion of lactic acid. This research
was supported by NSF IBN-0212921 to LEB & KGB.

White Spot Syndrome Virus (WSSV) In Wild *Litopenaeus setiferus* and
*Callinectes sapidus* From The U.S. South Atlantic Coast

^1^[Eleanor F. Shepard]{.underline}, ^1^James Powell, ^1^Craig L Browdy,
^1^Caroline A. Payne, ^1^Deliah Arrington, ^1^Tanya Polon, ^2^Erin J.
Burge and ^1^Robert W. Chapman^1^

^1^South Carolina Department of Natural Resources, Marine Resources
Division, 217 Fort Johnson Rd, Charleston, SC 29412; ^2^College of
Charleston, Hollings Marine Laboratory, 331 Fort Johnson Rd., Charleston
SC 29412

White spot syndrome virus (WSSV) was discovered in shrimp aquaculture
facilities in South Carolina in 1997. The disease is known to cause
devastating mortality in cultured shrimp in Southeast Asia and its
discovery in SC prompted concern for the health of wild populations. An
initial study conducted in 2000 found detectable virus in wild stocks of
the Atlantic white shrimp, *Litopenaeus setiferus* and the brown shrimp,
*Farfantepenaeus aztecus* with incidence and severity levels lower in
the latter species. One *L. setiferus* individual collected offshore
displayed a significant level of acute disease. The current study was
designed to further explore interactions between ovarian development,
spawning and incidence of WSSV in wild populations of *L.setiferus* and
the blue crab, *Callinectes sapidus*, using existing molecular
diagnostics, bioassay, and immunoassays techniques. A total of 1,808
*L.setiferus* and 300 *C. sapidus* specimens at various stages of
reproduction were examined for WSSV infection using a commercially
available immunoassay diagnostic test kit (Shrimple). Shrimple detects
viral infection by recognizing vp28, a viral structural protein. A
subset of the collected specimens was tested for WSSV using real-time
PCR and bioassays. Although 87 of shrimp and 11 of crabs tested positive
with the Shrimple kits, none of the shrimp and only one crab was found
to carry viable virus at levels significant enough to cause infection in
injection bioassays. Thus, although continuing to be present, current
incidence of WSSV in wild shrimp and crab populations along the
Southeastern Atlantic coast is minimal even during the physiologically
rigorous reproductive cycle.

**Diagnosis Of *Panulirus argus* Virus 1 (PaV1) In The Caribbean Spiny
Lobster Using Fluorescence *In Situ* Hybridization**

^1^[Caiwen Li]{.underline}, ^1^Jeffrey D. Shields, ^1^Hamish J. Small,
^1^Kimberly S. Reece, ^2^Carmony L. Hartwig, ^2^Roland A. Cooper, and
^2^Robert E. Ratzlaff

^1^Virginia Institute of Marine Science, The College of William and
Mary, Gloucester Point, VA 23062; ^2^Old Dominion University, Norfolk,
VA 23529

*Panulirus argus* virus 1 (PaV1), the first virus identified in the
Caribbean spiny lobster, *Panulirus argus* from Florida Keys, is highly
pathogenic to juvenile spiny lobsters. The monitoring of the virus in
wild populations and study of its behavior in the laboratory depends
upon reliable diagnostic tools. A sensitive and specific fluorescence
*in situ* hybridization (FISH) assay for diagnosis of PaV1 was developed
in this study. The lower limit of the 110 bp DNA probe in a dot blot
hybridization for PaV1 DNA was 10 pg of cloned template of PaV1 DNA and
10 ng of genomic DNA extracted from hemolymph of diseased spiny lobster.
The fluorescein (FITC)-labeled probe hybridized to PaV1-infected cells
in all tissues tested, producing the unique green fluorescent signal
inside infected cells. Most FITC-stained objects were observed
accumulated around the inner periphery of the nuclear membrane, with a
few distributed in a more dispersed pattern inside the nuclear. The
probe did not hybridize with host tissues of spiny lobsters; nor did it
cross-react with possibly related viruses. This assay will facilitate
understanding of pathogenesis of the viral disease and help in
monitoring efforts directed at determining the prevalence of PaV1 in
juvenile nurseries for the lobster.

**An Overview Of Disease Surveys In Crustaceans From The Chesapeake Bay
And United States Mid-Atlantic Coast**

[Gretchen Messick]{.underline}, John Jacobs, Sue Tyler and Joseph
Filipowicz

Cooperative Oxford Laboratory, Center for Coastal Environmental Health &
Biomolecular Research at Charleston (CCEHBR)USDOC/NOAA/NOS/NCCOS, 904
South Morris St., Oxford, Maryland 21654-1323

NOAA's Cooperative Oxford Laboratory conducts research on health of
crustaceans from the Chesapeake Bay and US Mid-Atlantic coasts to
provide information to researchers and resource managers. Current
projects include investigations on the prevalence of the parasitic
dinoflagellate *Hematodinium* sp. in blue crabs and other crustaceans,
an experiment to assay mortality in crabs infected with *Hematodinium*
sp., the presence of *Vibrio* spp., *Vibrio parahemolyticus*, and
virulent strains in blue crab hemolymph, and a comparison of the general
health of crabs collected from a pristine NERRS site and a site near
Baltimore Harbor in the Chesapeake Bay. Assay methods include histology,
microbiology, electron microscopy and molecular techniques. This
presentation will provide an overview of results to date on these
various research efforts.

**Manganese Induced Immune Suppression In *Nephrops norvegicus***

^1^[Carolina A.M. Oweson]{.underline}, ^1^Susanne P. Baden, ^2^Irene
Söderhäll, and ^1^Bodil E. Hernroth

^1^Deptartment of Marine Ecology, Göteborg University, Kristineberg
Marine Research Station, Kristineberg 566, SE-45034 Fiskebäckskil,
Sweden; ^2^Deptartment of Comparative Physiology, Evolutionary Biology
Centre, Uppsala University, Norbyvägen 18A, SE-75236 Uppsala, Sweden

Manganese (Mn) is one of the most abundant elements in soft bottom
sediments, where the metal is predominantly bound in a four-valent
colloid state (MnO~2~). During hypoxic conditions MnO~2~ can be reduced
and released in its bioavailable state (Mn^2+^). In such situations the
Mn^2+^ concentration can increase by a factor of 1000 and reach 20-22 mg
l^-1^ in the bottom waters. The bioavailable fraction can remain even
after hypoxia, since Mn^2+^ needs to attach to particles to reoxidize.
In bottom living organisms, such as the Norway lobster, *Nephrops
norvegicus*, Mn can reach neurotoxic levels. The main transport of Mn
out of mitochondria occurs via a slow, energy-requiring Na^+^-dependent
efflux mechanism. Thus, the metal accumulates, primarily in the nervous
tissue but also in the hemolymph of *N. norvegicus*. In the hemolymph Mn
accumulated three times the exposure concentration and as much as 80-90%
of the metal was associated to the protein fraction, mainly consisting
of hemocyanin but also including the immune active hemocytes. Recently,
we discovered that a surplus of Mn affected several immunological
processes of *N. norvegicus*. These are the first reports pointing out
Mn as immune toxic. When exposed to relevant concentration of 20 mg
l^-1^ Mn we found that the number of haemocytes of lobsters
significantly decreased by 44-50 %. Despite the great loss of haemocytes
renewal through increased proliferation of the haematopoietic stem cells
did not appear. Additionally, maturation of stem cells to immune active
haemocytes was inhibited in Mn exposed lobsters. The degranulation of
haemocytes, crucial for release of prophenoloxidase system (ProPO), was
also significantly suppressed after Mn treatment. Furthermore, the
activation of the ProPO by the non-self molecule, lipopolysaccharide,
was blocked in Mn treated lobsters. Most recent study have revealed that
apoptosis in both haematopoietic precursor cells and circulating
haemocytes is an important contributing factor to the Mn induced
hemocytepenia observed in *N. norvegicus*. Animals were exposed to
different concentrations and time intervals (0, 5, 10, 20 mg l^-1^
Mn^2+^; 5 and 10 days). Legible results showed a gradual increase in
apoptosis ratio from 0 mg l^-1^ Mn^2+^ to 20 mg l^-1^ Mn^2+^ after both
the time intervals. These results together may increase susceptibility
to bacteria and infectious diseases in naturally living animals.

**Clinical Aspects Of Water Biology And The Effects On Water Quality In
Koi Ponds**

[Julius M. Tepper]{.underline}

Long Island Fish Hospital, 1010 Montauk Hwy., Shirley, NY, 11967

As the new specialty practice of pet fish medicine evolves, important
factors influencing the health care of individual species will begin to
help the practitioner develop a diagnostic protocol and database of
norms. Sufficient information about the chemical aspects of water
quality of koi ponds has been written, yet very little has been
published about the biological aspects of water quality and the
relationship with disease. As a clinician, bacterial disease, from a
point-of-view of both morbidity and mortality, remains one of the most
frequent causes of health problems and reasons to contact the fish
veterinarian. When parasites and water chemistry have been ruled out as
sources of the bacterial disease, problems of water biology should be
considered. As a routine protocol, both the water column and the benthic
zone should be evaluated. The water column is evaluated quantitatively
for turbidity and qualitatively by microscopy using a protocol developed
by the author. The benthic zone is evaluated by direct microscopy of
scrapings. The findings and their effects on water quality will be
discussed.

**Selected Cases In Fish Medicine In Private Practice**

[Sandra F. Yosha]{.underline}

Yosha Veterinary Services, Inc., 6730 Woodside Court, Lakeland, FL 33813

Fish presented to a private practice veterinary clinic are often pets or
are individually important. Owners have often developed a human-pet fish
bond that supersedes estimated dollar value of the animal. As pets, they
have names and are provided a work-up similar to what is provided for
other companion animals. The 20 year old giant gourami named "baby" was
presented with a history of several months of "bloat" and recent
anorexia. This fish was examined, anesthetized, and submitted for x-ray
and ultrasound. Although a pre-mortem diagnosis was not determined, a
poor prognosis prompted the owners to elect euthanasia. Post mortem
analysis was compared to pre-mortem diagnostic tests to arrive at a
probable diagnosis of ileus. The results are discussed and compared to a
previous case of anorexia in a green moray eel.

**Frozen Fish: Private Fish Medicine In Buffalo, NY**

[Helen E. Roberts]{.underline}

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

According to the American Pet Products Manufacturers Association
2005-2006 National Pet Owners Survey (APPMA), Americans own 139 million
freshwater fish and 9.6 million saltwater fish. Treating pet fish is a
unique and challenging combination of herd health and individual pet
medicine in a field where a necropsy is currently the gold standard for
diagnosis. Veterinary care is sought for several reasons including the
deep emotional bond the owners have with their pets and the financial
cost of their fish. Veterinarians can provide diagnostic services that
can ensure a more accurate diagnosis and increased survivability of
these pets. Two detailed case reports of malignant melanoma in
*Carassius auratus* will be presented. The cases will demonstrate the
bond that clients have with their fish and the diagnostic and treatment
options open to private practitioners.

**Dermal Mesenchymal Neoplasms In Goldfish, Koi And Carp**

[Jeffrey C. Wolf]{.underline}, Marilyn J. Wolfe, Richard J. Montali

The Registry of Tumors in Lower Animals, 22900 Shaw Road, Sterling,
Virginia, 20166-4311

Dermal mesenchymal tumors are not uncommon in fish, however, the
prevalence of such neoplasms appears to be especially high in goldfish
Carassius *auratus auratus*, koi *Cyprinus carpio carpio* (ornamental),
and common carp *Cyprinus carpio carpio*. Diagnostic criteria tend to
place these tumors into two major categories: pigment cell neoplasms and
peripheral nerve sheath tumors. However, these piscine neoplasms contain
phenotypic characteristics which do not appear to be mutually exclusive
within each of the tumor categories. Despite previous descriptions in
other studies, differential morphologic criteria for these lesions
remain unclear. Consequently, ancillary tests such as cytochemical and
immunohistochemical and/or transmission electron microscopy may be
required for more definitive diagnoses. The National Cancer Institute's
Registry of Tumors in Lower Animals (RTLA) contains more than 60
examples of pigment cell and nerve sheath neoplasms in goldfish, koi,
and carp. These challenging cases will be reviewed with the salient
features elucidated and illustrated.

**Laser Surgery To Remove Skin Tumors in Goldfish (*Carassius
auratus*)**

[Gregory A. Lewbart]{.underline} and Anthony Blikslager

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

Various types of mesenchymal neoplastic dermal tumors have been
described in goldfish and other teleost species. These tumors rarely
metastasize but can grow quickly and are usually aesthetically
unpleasing. These growths can also put the affected fish at risk of
secondary infection and/or blood loss if the tumor becomes traumatized.
In our experience traditional surgical excision, and even intralesional
chemotherapy in one case, have been unrewarding (the tumors usually
recur). Laser surgery, combined with surgical debulking of the mass, has
promise as a curative treatment regimen for dermal tumors in goldfish.
Laser is an acronym for: Light Amplification by Stimulated Emission of
Radiation. A carbon dioxide (CO~2~) laser was used in the goldfish cases
to be discussed during the presentation. One of the patients has now
been grossly without tumor recurrence for 29 months (since September,
2003). In addition to general anesthesia with tricaine methanesulfonate
(MS-222), a local anesthetic such as 2% lidocaine is recommended.
Post-operative care may include topical silver sulfadiazine cream on the
surgical site and an injectable analgesic agent such as butorphanol or
ketoprofen.

Safe And Effective Control Of Ozone In Aquarium Life Support Systems

^1,\ [2]{.underline}^[Andrew Aiken]{.underline} and ^3^Mark Smith

^1^National Aquarium In Baltimore, 501 E. Pratt Street, Baltimore MD,
21202, USA; ^2^Aqua-Brio, Inc., 8229 Bayside Drive, Pinehurst MD, 21122;
^3^Cosestudi, 302 Dakota Wool Stores, 88 Macquarie Street, Newstead,
4006, Queensland, Australia

> Ozone's chemical properties make it a valuable resource for use in
> managing water quality in aquarium life support systems. As an
> effective disinfectant and micro-flocculent, it provides excellent
> control of microorganisms, dissolved organic molecules, and other
> contaminants that threaten fish health such as heavy metals. When
> applied carefully ozone can remove organic pollutants and disinfect
> aquarium water through two processes: flocculation in protein
> skimmers, in the range 0.01-0.05mg.l^-1^, and oxidation in contact
> chambers, in the range 0.10-1.00mg.l^-1^. When improperly applied and
> controlled, ozone can be severely harmful to species held within the
> system, and can even pose a threat to human health. By understanding
> and using the concepts of applied ozone dose, redox potential and
> redox controllers, and by using best practices for husbandry staff;
> ozone can be effectively applied and controlled to achieve desired
> benefits. Ozone and some of its by-products, collectively referred to
> as total residual oxidants (TRO), are toxic to aquatic life so great
> care must be employed in its use. A safe ozone dosing regime can only
> be achieved by monitoring all of the following parameters: (1) applied
> ozone dose (2) oxidative redox potential; (3) TRO; (4) turbidity; (5)
> husbandry (i.e. feeding and cleaning activities); and (6) animal
> behavior.

**The Microbial Diversity Associated With The Gorgonian Coral,
*Pseudopterogorgia americana***

^1,2^[Christopher Johnston]{.underline}, ^1,2^Carol Savoie, ^2^Cheryl M.
Woodley, ^3^Garriet W. Smith, and ^1,2^Pamela J. Morris

^1^Marine Biomedicine and Environmental Science Center, Medical
University of South Carolina, Charleston, SC 29412; ^2^Center for
Coastal Environmental Health and Biomolecular Research and Hollings
Marine Laboratory, U.S. National Oceanic and Atmospheric Administration,
Charleston, SC 29412; ^3^Department of Biology, University of South
Carolina at Aiken, Aiken, SC 29801

Corals harbor an active microbial community in their surface
mucopolysaccharide layer (SML) that may be specific to the host coral
over spatial and temporal scales. We used both culture-dependent and
culture-independent approaches to characterize bacteria associated with
the SML of a healthy gorgonian coral, *Pseudopterogorgia americana*,
from Rocky Point Reef, San Salvador, Bahamas. Of 53 microbial isolates
obtained, 30 were distinct ribotypes based on 16S rDNA sequence
analysis, and all of the isolates clustered within the *Bacillus*,
*Gamma* and *Alpha Proteobacteria*. Additionally, 10 of the SML isolates
tested positive for quorum sensing signaling molecules and 2 tested
positive for antifungal activity against *Aspergillus sydowii*.
Following extraction of SML DNA, PCR amplification of the microbial
community 16S rRNA genes was followed by denaturing gradient gel
electrophoresis (DGGE) and 16S rDNA cloning. Of the DGGE sequences
obtained, the majority clustered within the *Beta Proteobacteria*. The
16S rDNA clone library was dominated by the genus *Sphingomonas*, with
almost half of the sequences identified belonging to this genus. Thus,
we observed little congruency between the microbial communities observed
in culture-dependent and culture-independent approaches.

**Assessment Of Microbial Communities Associated With An Acroporid Coral
Disease Outbreak**

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

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

Acroporid coral populations in the Western Atlantic have been impacted
by numerous disease events over the past two decades, contributing to
region-wide declines in these formerly dominant reef-building species.
One such mortality occurred in the Florida Keys National Marine
Sanctuary, Biscayne National Park, and Dry Tortugas National Park during
the late spring of 2003. *Acropora cervicornis*, *A. palmata*, and *A.
prolifera* displaying similar disease signs were observed and sampled
from these locations during the mortality event. Aseptically obtained
tissue, mucus, and water samples were used to cultivate bacteria on
non-specific marine media. Samples were also analyzed for total
microbial composition by construction and sequencing of PCR-based 16S
rDNA libraries. The presence/absence of known coral pathogens was
determined by PCR assay using primer pairs designed to specifically
target such organisms. Sequencing of 16S rDNA libraries indicates
co-occurrence of two distinct *Photobacterium* spp. groups
preferentially within diseased corals. Isolation of culturable bacteria
additionally produced several *Photobacterium* spp. from diseased
samples. Ongoing research will further assess potential roles of these
microbes to the disease.

**Investigations Of *Serratia marcescens* As A White Pox Pathogen In The
Florida Keys**

^1^[Erin K. Lipp]{.underline}, ^2^Kathryn P. Sutherland, and ^3^James W.
Porter

^1^Dept. of Environmental Health Science, University of Georgia, Athens,
GA 30602; ^2^Department of Biology, Rollins College, 1000 Holt Avenue,
Winter Park, FL 32789; ^3^Institute of Ecology, University of Georgia,
Athens, GA 30602

*Serratia marcescens* causes white pox disease in the Caribbean elkhorn
coral, *Acropora palmata.* Although *S. marcescens* is common in
freshwater and terrestrial environments and in the intestines of humans
and other animals, the prevalence of the bacterium in the marine
environment and the source of the coral pathogen are unknown. Nearshore
marine and coral reef environments in the Florida Keys and raw sewage
and treated effluent from an advanced wastewater treatment plant were
screened for the presence of the bacterium. *S. marcescens* was
identified using selective agar media and species-specific PCR.
RFLP-PFGE was used to determine the diversity of *S. marcescens* and to
elucidate a potential source of the coral pathogen. A total of 257 *S.
marcescens* isolates produced 118 different pulse-field patterns,
indicating that *S. marcescens* is genetically diverse in environments
of the Florida Keys. While the bacterium was rare in marine environments
(0.3% prevalence), it was common in untreated human sewage (100%
prevalence) and canals (76.5% prevalence). The majority (84%) of *S.
marcescens* was isolated from raw sewage and contaminated nearshore
sources. The source of the known acroporid serratiosis pathogen (strain
PDL100) was not identified, but a second strain of *S. marcescens*
(strain PDR60) was isolated from disease lesions and found to be
identical to a *S. marcescens* strain isolated from human sewage.

**Coral Disease And Genetics Survey In St. John, USVI Using
Non-Destructive Methods**

^1^[William B. Schill]{.underline}, ^1^James B. Murray, ^1^Caroline S.
Rogers, ^2^William J. Miller, and ^3^Anthony S. Spitzack

^1^U.S. Geological Survey, National Fish Health Research Laboratory,
11649 Leetown Road, Kearneysville, WV 25430; ^2^National Park Service
^3^ETI Professionals

In August, 2004, USGS Scientists with the help and cooperation of NPS
personnel, sampled healthy and diseased *Montastrea annularis* along
transects at Tektite Reef, St. John Island, US Virgin Islands.
Importantly, noninvasive, nondestructive sampling methods were employed
to accomplish sampling needs and get statistically significant numbers
with minimal impact on the reef organisms. Sterile, foam swabs were used
to sample healthy and diseased coral, and material was transferred to
Whatman FTA cards for storage and transport to the laboratory. This
card-based sampling method allows room temperature storage of DNA
samples for years and eliminates problems with often-used low
temperature storage and transport from remote locations. Bacterial 16S
ribosomal genes and zooxanthellae ITS-1 genes were readily amplified by
polymerase chain reaction (PCR) from card samples. In particular,
positive PCR signals for *Aurantimonas sp*. (the presumptive causative
agent of White Plague) were widely detected from apparently healthy
corals. Additionally, *Serratia marcescens* (presumptive causative agent
of White Pox) was also detected from healthy as well as diseased
*Acropora* at Hawksnest Bay. The samples analyzed to date preceded the
peak of the large bleaching event that occurred in 2005. The genotypes
of zooxanthellae present before this bleaching event are being
determined and will be compared with post-bleaching recolonizations.
Additional samples have been taken over time and across reef tracts and
are being tested using molecular methodology for the presence of
pathogenic bacteria and examined for possible correlations of pathogen
presence/absence with host (coral) and symbiont (zooxanthellae)
genotypes. The sampling methods developed and tested are easily employed
in the field under a variety of conditions.

**Zooxanthellae Shifts And Yellow Band Disease**

^1^[Diego L. Gil-Agudelo]{.underline}, ^2^Garriet W. Smith, ^3^Ernesto
Weil, and ^4^Todd C. LaJeunesse

^1^Instituto de Investigaciones Marinas y Costeras INVEMAR, Magdalena,
Colombia; ^2^University of South Carolina Aiken;^3^University of Puerto
Rico, Department of Marine Sciences; ^4^Florida International
University, Miami, FL

Many outbreaks of Yellow Band Disease have been reported Caribbean-wide,
affecting mainly the *Montastrea annularis* complex, one of the main
components of the reef framework of the Caribbean. Several authors have
discussed the possibility that the pathogen(s) infect the algal symbiont
rather than the coral tissue. Experiments looking for possible pathogens
yielded a number of *Vibrio* strains. Observations of possible shifts in
the clade composition of corals with the disease support this
hypothesis. In order to study zooxanthellae clade composition and
changes due to the presence of the disease, affected and unaffected
portions of *Montastrea faveolata* corals were extracted from La
Parguera, Puerto Rico and evaluated using Denaturing Gradient Gel
Electrophoresis of the ITS region of the zooxanthellae. Our results
showed changes in zooxanthellae composition in corals affected by Yellow
Band Disease, shifting from C7/C12 and B1 clades to A1.1 in most cases.
Although these patterns do not seem to hold in other parts of the
Caribbean, based on similar studies in other parts of the Caribbean, it
is interesting that changes occur in the zooxanthellae composition. This
supports the suggestion of a zooxanthellae disease. This was the first
time that clade A1.1 was isolated from a coral sample, showing the
possibility that this zooxanthellae is opportunistic.

**White Band Disease In The Caribbean**

^1^[G.W. Smith]{.underline} , ^2^Gil-Agudelo D.L., ^3^E. Weil

^1^University of South Carolina Aiken; ^2^Instituto de Investigaciones
Marinas y Costeras INVEMAR, Magdalena, Colombia; ^3^University of Puerto
Rico, Department of Marine Sciences

The white band disease (WBD) epizootic event of the early 1980's
resulted in significant changes in the structure and composition of
coral communities throughout the wider Caribbean. During the fall months
of 2003 and 2004, samples of *Acropora cervicornis* with signs of white
band disease type II (WBD-II) were collected in Mario reef, La Parguera,
southwest coast of Puerto Rico. Bacteria extracted from these samples
were isolated in TCBS agar, grown in Glycerol Seawater agar, and then
used to infect healthy-looking colonies at the same locality. Signs of
the disease were produced in the inoculated corals, which were
subsequently sampled and the bacterial agent re-isolated to be
identified, thus fulfilling Koch's postulates. Preliminary results
confirmed that the cause of WBD-II is a *Vibrio* species very close to
*Vibrio harveyi*.

**Molecular Diversity Of Microbes Associated With Black Band Diseased
Coral *Siderastrea siderea***

^1^[Raju Sekar]{.underline}, ^1,2^DeEtta K. Mills, ^1^Elizabeth R.
Remily and ^1^Laurie L. Richardson

^1^Department of Biological Sciences; ^2^International Forensic Research
Institute, Florida International University, Miami, FL 33199.

Biodiversity and abundance of reef-building corals are substantially
declining worldwide and coral disease is believed to be a major
contributing factor. Black band disease (BBD) is one of the diseases,
which plays an important role in the decline of coral reefs especially
in Caribbean. The causative agent for BBD has not been identified, and
the earlier studies showed that it is a 'polymicrobial' disease. In the
present study, microbes associated with BBD infected *Siderastrea
siderea* corals collected from two reefs in the Bahamas (Horseshoe and
Rainbow Reefs) and one in the Florida Keys (Watson Reef) were assessed
by 16S rRNA gene cloning, sequencing, and amplicon-length heterogeneity
(LH)-PCR community profiling. Partial and full-length 16S rRNA gene
sequences were retrieved from positive clones and used for comparative
sequence analyses. Results showed that the clone libraries were
dominated by α-proteobacteria (53-87%) followed by δ-proteobacteria
(6-20%), verrucomicrobia (2-11%), cytophaga-flavobacterium-bacteroidetes
(CFB, 6-9%), cyanobacteria (0-6%) and other bacterial groups (0-6%).
These results were compared with LH-PCR whole community profiles to
confirm their presence. Sequences related to bacteria associated with
toxin-producing dinoflagellates, and sulfate-reducing bacteria like
*Desulfovibrio* were consistently present in all the clone libraries.
Sequence types of bacteria associated with juvenile oyster disease (JOD)
and known toxin-producing cyanobacteria were found in at least two
sampling sites. This study provides a better understanding of BBD
associated microbes as compared to previous molecular studies.

**Defining The Reservoir Of The Pathogen *Aurantimonas coralicida* By
Fluorescence *In Situ* Hybridization**

[Jorge H. Pinzón]{.underline}, Raju Sekar, Elizabeth R. Remily, DeEtta
K. Mills and Laurie L. Richardson

Department of Biological Sciences, Florida International University,
11200 SW 8^th^ Street, Miami, FL 33199

The three dimensional structure of coral reefs creates many different
microhabitats that can be used by coral disease pathogens as a
reservoir. In the last decade, the coral disease white plague (WP) has
dramatically reduced the population sizes of its coral hosts. Although
advances in the study of this disease include the isolation and
identification of the causative agent (*Aurantimonas coralicida*), there
are still many unknown aspects to its etiology. These include the mode
of transmission, the reservoir of this bacterium, and the infection
mechanism that initiates the disease. Fluorescence *in situ*
hybridization (FISH) with an oligonucleotide probe specific to *A.
coralicida* has been developed based on the 16S rRNA gene sequence of
the isolate. This probe has been used to diagnose WP in several reefs in
the Caribbean and has proven to be efficient for the identification of
*A. coralicida*. Using FISH and the species-specific oligonucleotide
probe, reef sediment samples, including in association with the surfaces
of the macroalgae *Halimeda* and *Dictyota* *spp*., and water samples,
from three different Caribbean reefs have been analyzed. *Halimeda* was
recently proposed to play a role in WP infection on the reef. Results
showed that *A. coralicida* is present in both the water column and the
sediments around these reefs, and is not exclusively associated with any
algal species. This suggests that the reservoir of *A. coralicida* may
include both the sediment and the water column as well as the surface
mucopolysaccharide layer (SML) of healthy but WP susceptible corals.

**Differential Diagnosis: A Need For The Use Of Multidisciplinary Tools
To Investigate The Coral Disease White Plague Type II**

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

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

Microbial community composition in apparently healthy and white plague
type II (WPII)-diseased colonies of *Montastraea annularis* (complex)
from St. Croix, U.S. Virgin Islands and The Bahamas was characterized
using a combination of molecular fingerprinting, microbial culturing,
16S rDNA sequencing, and histopathology. Principal coordinate analysis
(PCO) of amplicon length heterogeneity (ALH) fingerprints of the
bacterial community on unaffected corals and apparently healthy tissue
from diseased corals were similar within each geographical location but
differed between locations. Diseased samples from both sites were more
similar to each other than to the other samples. The major bacterial
Operational Taxonomic Units from coral genomic DNA extracts proved to be
culturable (32% match) bacteria. 16S rDNA sequencing suggests that this
culturable community is dominated by the *Pseudoalteromonas* and
*Vibrio* groups. *Aurantimonas* *coralicida*, the causative agent of
WPII (amplicon length 313.1 bp), was found in relatively low abundance
on both diseased and healthy tissue on affected corals from The Bahamas.
Tissue changes varied with the sample. These findings conflict with
previous reports on WPII pathogenesis and suggest that the same, or
similar, disease signs could be caused by different organisms, thus
highlighting a need for the application of differential diagnostic
techniques in the identification of coral diseases.

**From Leidy To Snieszko: Pioneers Of Fish Health In The US
(1850-1950)**

[Andrew J. Mitchell]{.underline}

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

The achievements of seven fish health pioneers from Joseph P. Leidy to
Stanislas F. Snieszko are discussed. Leidy (1823-1891), one of the most
prominent 19^th^ century scientists, published more than 600 papers;
working at the fringe of his interests he published 23 papers on fish
parasites making him the most prolific fish health writer of the 19^th^
century. He was one of the first to recognize that stress predisposed
fish to disease. Livingston Stone (1836-1912), clergyman turned premier
fish culturist, described the first fish treatment (salt) and 23
diseases of domesticated trout and drew sketches of microscopic fish
parasites. Stephen A. Forbes (1844-1930), one of the founding fathers of
ecological studies was the first to use histological and bacteriological
techniques in fish diagnostics. Edwin Linton (1855-1939), the father of
fish parasitology in the United States, produced more than 60
publications about fish parasites. Millard C. Marsh (1872-1936), the
first fish pathologist for the U. S. Fish Commission published 19 papers
that were directly beneficial for the control of fish diseases; these
included information on bacterial characterization and diseases
description, disinfection and treatments, blood parameters, immunity,
tumors, and several non-infectious diseases. Herbert S. Davis
(1875-1958), often called the "Father of Fish Health" in the US,
published on viral, bacterial, parasitic, fungal, nutritional, and
environmental diseases and established a disease diagnostic service and
the world renowned fish disease laboratory at Leetown, WV. Snieszko
(1902-1984), the gentleman-ambassador for US fish health, published more
than 200 fish health papers and is best known for his work with
bacterial pathogens, the interaction of the host, pathogen, and
environment, fish health training, and disease diagnostic and
certification programs.

**The Current Status Of The United States National Aquatic Animal Health
Plan**

[Gary Egrie]{.underline} and Jill Rolland

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 must develop and implement a national aquatic animal health
plan (NAAHP) for aquaculture in cooperation with industry, regional
organizations, state, local, tribal governments and other stakeholders.
The NAAHP will foster and support efficient aquaculture; protect the
health of our nation's wild and cultured aquatic resources; and meet
both national and international trade obligations. The NAAHP requires
input from working groups composed of individuals who represent a
certain sectors of the aquaculture industry. A pivotal chapter of the
plan has been drafted on guidelines for the surveillance of fish
diseases. The NAAHP is anticipated to be completed in the spring of
2007. The plan will not be codified into regulation; however,
implementation of certain elements, such as import requirements, may
require revisions to existing laws, regulations or policies. In this
presentation, a summary of key milestones will be presented and
discussed with regard to future goals.

Fish Health Medicine Certificate Program

^1^[Myron J. Kebus]{.underline}, ^2^Michael T. Collins, ^3^Jeannette
McDonald, ^4^Julie Bebak-Williams, ^5^Grace A. Karreman, and ^6^Jill B.
Rolland

^1^Wisconsin Department of Agriculture, Trade & Consumer Protection,
2811 Agriculture Dr., Madison, Wisconsin 53708-8911; ^2^ University of
Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive
Madison, WI 53706-1102; ^3^ WisTEC, University of Wisconsin-Madison
School of Nursing, 600 Highland Avenue, Madison, WI 53792; ^4^
Freshwater Institute, 1098 Turner Rd., Shepherdstown, WV  25443; ^5^
Pacific Marine Veterinary Services, 3000 Andre Road, Nanaimo, BC V9R
6X2, Canada; ^6^ USDA/APHIS/VS/ASEP, 4700 River Road, Unit 46,
Riverdale, MD 20737

The "Fish Health Medicine Certificate Program," was developed by the
Wisconsin Department of Agriculture, Trade & Consumer Protection
(WDATCP), the University of Wisconsin-Madison (UW-Mad), and national
fish health experts. The course is constructed as a series of modules
for veterinarians leading to certification. The full certificate program
involves five online modules and a final hands-on training module. The
online lectures use narrated PowerPoint presentations and supplemental
reading materials delivered using new educational technology software.
The course is available through the continuing education (CE) portal of
the University of Wisconsin, School of Veterinary Medicine,
<http://vetmedce.vetmed.wisc.edu/FishCertificate/>. Each module has a
post-test automatically generated and computer scored. Successful
completion of each module will result in the award of CE credit that can
be used to satisfy state veterinary licensure requirements. The final
module of the certificate program is a hands-on one-day course called
"Aquaculture Veterinary Medicine for Practitioners." It is an intensive
program designed to provide practical training in field techniques for
sample collection and field diagnostics reinforcing the online training.
Selected online training modules will also be made available
individually as well as part of the full certificate program. This
project was made possible by funding from the National Risk Management
Feasibility Program for Aquaculture, a partnership program between the
USDA Risk Management Agency and Mississippi State University. This
certificate program is expected to reduce risk of disease on fish farms
by increasing the number of trained veterinarians available to provide
services to fish producers.

**Natural Content And Processing Of Alternative Protein Sources:
Histologic Effects In Fingerling Channel Catfish (*Ictalurus
punctatus*)**

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

^1^Aquatic Animal Health Research Laboratory, United States Department
of Agriculture, Agricultural Research Service, 118 B Lynchburg Street,
Chestertown, MD 21620; ^2^Aquatic Animal Health Research Laboratory,
United States Department of Agriculture, Agricultural Research Service,
P.O. Box 952, Auburn, AL 36831

Significant effort has been made to utilize alternative protein sources
like cottonseed meal (CSM) and soybean meal (SBM) to replace fishmeal in
channel catfish (*Ictalurus punctatus*) feed. These sources are readily
available and have high nutritional value, but contain anti-nutritional
factors (ANF) and toxins (ex. trypsin inhibitors in SBM and gossypol in
CSM) that may be detrimental to fish. We studied the effects of these
factors by evaluating histopathologic changes in fingerling catfish fed
diets containing various levels of CSM, gossypol-acetic acid, or SBM for
10-12 weeks. Changes associated with ANF/toxins were variable often in a
dose-dependant manner, but included hepatic glycogen deposition,
pancreatic necrosis, pancreatic vacuolization, loss of enterocyte
supranuclear vacuolization, and pigmentation of the spleen. Processing
methods commonly used to inactivate or detoxify CSM or SBM ANF/toxins in
mammalian diets were also studied; iron failed to reduce gossypol
toxicity and heat-treatment failed to significantly reduce trypsin
inhibitor toxicity. These studies indicate that ANF/toxins naturally
present in SBM and CSM may cause histologic changes in fingerling
channel catfish. Further refinement of processing techniques to
inactivate or eliminate these ANF/toxins and minimize their adverse
effects on fish is warranted.

**Growth Response And Acquired Resistance Of *Streptococcus*
*iniae*-Recovered Nile Tilapia, *Oreochromis* *niloticus***

[Craig A Shoemaker]{.underline}, Chhorn Lim, Mediha Yildirim-Aksoy,
Thomas L Welker and Phillip H. Klesius

United States Department of Agriculture-Agricultural Research Service,
Aquatic Animal Health Research Unit, PO Box 0952, Auburn, AL 36831

The growth performance and acquired resistance of *Streptococcus*
*iniae*-recovered Nile tilapia was determined. Tilapia were challenged
with three doses of *S. iniae* (8.8 x 10^3^, 8.8 x 10^4^ and 8.8 x 10^5^
CFU/fish for low, medium and high challenge, respectively). Groups of
non-injected and tryptic soy broth-injected fish were maintained as
controls. Significantly (P \< 0.05) higher mortality (45.0 %) occurred
in the high challenge treatment than in the low challenge treatment
(29.6 %). The medium challenge group had mortality of 36.3%. The *S.
iniae*-recovered tilapia, which were used to assess growth performance,
were selected from survivors without clinical signs of disease. Fish
were randomly stocked at 30 fish per 57-L aquarium in triplicate and fed
to apparent satiation for 8-weeks. No differences were detected in
weight gain, feed intake, feed efficiency ratio or survival between *S.
iniae*-recovered tilapia and the control treatments following the 8-week
performance trial. Following the 8-week feeding study, the tilapia were
challenged with 1 x 10^6^ CFU/fish of *S. iniae* to assess acquired
immunity. Mean cumulative mortality was significantly higher (P \< 0.05)
in the control treatments (41.7 % and 43.3 %) than in the low, medium
and high challenge treatments (7.4, 3.3 and 8.3 %, respectively). The
results suggest that *S. iniae*-recovered tilapia not showing overt
disease signs gained acquired immunity and performed as well as
non-infected tilapia.

**Hurricane Katrina And The Mississippi Catfish Industry, A Little Good
News For A Change**

[Alvin C. Camus]{.underline}

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

In the past five years, commercial catfish aquaculture in Mississippi
has faced a number of challenges, including historically low farm gate
prices, competition from foreign imports, and the ongoing cost of
disease losses. As hurricane Katrina approached the Gulf Coast, dire
predictions were made concerning potential losses to the catfish
industry. In August, oxygen budgets in ponds are critically low and
supplemental aeration, supplied in the form of electric and tractor
driven aerators, is typically required for fish survival. On August 29,
2005, Katrina moved through central Mississippi causing catastrophic
damage in the southern third of the state. Despite widespread power
outages over much of the state's catfish producing areas, through
cessation of feeding ahead of the storm's arrival, a major disaster was
averted. Mississippi's poultry industry was not so fortunate and
suffered major losses. Although there were fuel shortages, damage to the
distribution system for processed fish, as well as temporary losses of
power to processing facilities and feed mills, these problems were
quickly resolved. The major impact of Katrina on the catfish industry
appears to have been the loss of markets in devastated coastal
communities. A number of these areas were supplied with fish donated by
processors to aide in relief efforts. At present, the price of fish
remains strong.

**Bacterial Endocarditis In The Giant Pacific Octopus, *Enteroctopus
dofleini***

[Christian J. Keller]{.underline}

The Tennessee Aquarium. One Broad Street, Chattanooga, TN 37401

Giant Pacific Octopus, *Enteroctopus dofleini,* are extremely popular,
interesting members of many public aquaria's collections. They are
intelligent and easy to fall in love with. Octopus manifest
characteristics of personality, mood, and behavior that attract the
keeper's attachment. Unfortunately, octopuses are relatively short lived
and are sensitive to water quality issues and diseases that we are, just
now, beginning to understand. This presentation refers to the first
octopus that Tennessee Aquarium housed and exhibited. Temperature
changes and water quality associated with starting up a new cold water
marine exhibit may have contributed to its demise. Ultimately, the
octopus exhibited signs of senescence that resembled the typical
geriatric problems that other institutions had experienced. Post-mortem
examination followed by bacteriology and histopathology (Connecticut
Veterinary Medical Diagnostic Laboratory) helped determine that the
octopus succumbed to a bacterial endocarditis associated with coliform
bacteria.

**Enrofloxacin Resistant Meningoencephalitis In A Tiger Shark
(*Galeacerdo cuvier*)**

^1^[Pamela D. Govett]{.underline}, ^1^Natasha Christie, ^1^Andrew
Clarke, ^1^Todd Kemp,

^1^Skishnell Nairn, ^1^Deron Wells, and ^2^Dave Rotstein

^1^Atlantis, One Casino Drive, Nassau, Bahamas; ^2^Department of
Pathobiology, College of Veterinary Medicine, University of Tennessee,
2407 River Drive, Knoxville, TN 37996

A 7 foot 2 inch 163 lb female tiger shark (*Galeacerdo cuvier*) had a
history of abrasions on its dorsal fin due to scraping exhibit rock
work. To prevent further abrasions, the animal was transferred to a new
exhibit and was started on a course on enrofloxacin. A few months later,
the shark started intermittently exhibiting abnormal swimming behavior
consisting of holding its head above and tail below the horizontal
plane. The shark was given several more rounds of enrofloxacin. The
shark became anorexic for 1 week, the veterinarian was notified and a
physical examination was performed. On physical exam, the animal was
extremely cachectic, had exposed cartilage on the cranial edge of the
dorsal fin, and the edges of the pelvic, caudal, and anal fins, and had
severe ventral abrasions. Hematology and chemistries appeared to be
within normal limits. The blood cultured enrofloxacin resistant
*Staphylococcus cohnii*. A fecal wash collected a small amount of stool
in which no parasites were present. The animal was administered vitamin
E, vitamin B complex, ceftiofur sodium, and prednisolone sodium
succinate. The animal continued to deteriorate the following day and
methylprednisolone acetate and oxygen therapy was given. The next
morning, the animal was hyporesponsive, and euthanasia with MS-222 and
quinaldine sulfate was elected. At necropsy, a sterile cerebral spinal
fluid tap was performed. The fluid was clear, transparent, of moderate
tenacity, and contained a few pieces of white flocculent material,
crystals, and other nondescript debris. The fluid cultured enrofloxacin
resistant Streptococcus acidominimus. The GI tract was devoid of food,
and the colon contained numerous tapeworms. Histopathology revealed
meningoencephalitis and nephritis, and branchitis, gastritis, and
coelomitis associated with parasitism.

**Koi Herpesvirus In New York State: The 2005 Common Carp Mortality
Event**

Stephanie G. Grimmett, Geoffrey H. Groocock, Rodman G. Getchell, Gregory
A. Wooster, and [Paul R. Bowser]{.underline}

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

In June, 2005 a mortality event was reported to our Fish Disease
Diagnostic Laboratory by the New York State Department of Environmental
Conservation. The mortality event involved common carp (*Cyprinus
carpio*) on Chautauqua Lake, New York. The mortality event lasted
approximately 2 weeks, during which time an estimated 25,000 adult carp
weighing an estimated 20 -- 30 pounds each died. In consideration of the
number of dead fish floating on the lake as the July 4^th^ holiday
weekend approached, a massive clean-up effort was undertaken by the New
York State Department of Environmental Conservation and the Chautauqua
Lake Association. According to a local newspaper, "The rafts of dead
carp are being buried in trenches next to the local landfill." Our
diagnostic processing of the specimens submitted revealed the Koi
Herpesvirus (KHV) by both cell culture in Fathead Minnow (FHM) cells and
by quantitative PCR. Because the virus grew in FHM cells, we forwarded a
sample of virus to the USDA APHIS National Veterinary Services
Laboratories (NVSL) in Ames, Iowa to rule out the potential of Spring
Viremia of Carp Virus. Results of testing at NVSL were consistent with
our diagnosis of KHV. This was the second consecutive year that KHV has
been found in this watershed. In 2004 approximately 8,000 adult common
carp died in the Chadakion River, the river that drains Chautauqua Lake.

**Pancreatic Amyloidosis In Captive Tricolour Sharks (*Balantiocheilus
melanopterus*)**

Spencer Russell, David McLelland, [Arman Yazdanpanah]{.underline},
Karrie Young, Paul Huber, and John S. Lumsden

Department of Pathobiology, University of Guelph, Guelph, ON, N1G 2W1
Canada

A captive adult tricolour shark from a freshwater display was found
dead. Gross post mortem revealed an enlarged abdomen that was expanded
by approximately 2ml of bloody fluid. Skin scrapes and gill biopsies
were unremarkable. Significant histological lesions were limited to the
peritoneal cavity. The exocrine pancreas comprised an unusually large
proportion of the peritoneal parenchyma. This expanded exocrine
pancreatic tissue was composed of areas of well organized pancreatic
acini interspersed with a population of a poorly differentiated,
basophilic, angular, spindle-shaped cells arranged in cords that
compressed and effaced normal parenchyma. These cells possessed large
pleiomorphic nuclei, prominent nucleoli and basophilic cytoplasm. Some
of these cells appear to have small accumulations of cytoplasmic zymogen
granules. Within mesenteric tissues surrounding the mass, there is
marked, multifocal expansion of collagen fibers by macrophages,
hemorrhage and small numbers of neoplastic cells similar to those
described above. The tunica media of large blood vessels was greatly
expanded by accumulations of a lightly eosinophilic, hyaline,
homogeneous, acellular material. Similar material was present expanding
the fibrovascular connective tissue of the pancreas, liver and
mesenteries. No organisms were identified with a Brown and Brenn
modified gram stain. The homogeneous, acellular material was
light-orange and exhibited birefringence upon polarization when stained
with Congo red and was light-pink with toluidine blue. This material
appeared bright-green upon microscopic examination using 255nm
ultraviolet light after staining with thioflavine-T. Exocrine pancreatic
neoplasia (pancreatic adenocarcinoma in this case) is uncommon in fish
and most reports describe lesions following experimental exposure to
carcinogens. Amyloidosis is a very uncommon finding in fish and to our
knowledge has not been described associated with a neoplastic lesion. In
secondary amyloidosis (secondary to chronic inflammation or some types
of neoplasia) the fibrils are composed of altered serum amyloid A. The
composition of the present deposit is not known but will be investigated
using laser micro-dissection, polyacrylamide electrophoresis and
MALDI-tof mass spectrometry.

**Progressive Erosive Stomatitis In Green Moray Eels: A Case Study**

^1^[Toby L Lowry]{.underline} and .^2^Ueril Blas-Machado

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland
Regional College of Veterinary Medicine, Virginia Polytechnic Institute
and State University, Blacksburg, VA 24061-0442; ^2^ Department of
Pathology, College of Veterinary Medicine, Athens, GA 30602-7388

Two green moray eels (*Gymnothorax funebris)* from a public aquarium
collection were presented to the Oklahoma Animal Disease Diagnostic
Laboratory (OADDL). In this presentation we describe the clinical skin
ulcerations that developed in these two eels. The second eel was
necropsied and samples of skin, gill, tests, kidney, liver and serum
were collected. Skin samples were stained with H&E and Periodic Acid
Schiff and observed with light microscopy. Additional skin samples were
submitted in 2.5% glutaraldehyde for electron microscopy evaluation.
Under light microscopy the skin appeared thickened with scattered foci
of ulceration and erosions with an increased number of goblet cells. No
infectious agents were identified ultrastructurally and no significant
tissue alterations were found in the other tissues examined. Similar
lesions in moray eels have been caused by hypoxic conditions, exposure
to infectious agents or exposure to toxic compounds, however these were
ruled out through water quality tests and histopathology. The absence of
identifiable infectious agents in any of the tissues examined, as well
as, no history of contact with toxins or irritants leave the underlying
etiology undiagnosed. It is believed that stress induced
hypercortisolemia may have been an inciting factor.

**The Big Belly Blues**

[Andrew J. Mitchell]{.underline}

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

Abdominal enlargements (big belly) occur for several reasons: when host
tissues proliferate, when air bladders over-inflate, when fluids fill
the body cavity, when egg production becomes much greater than normal,
when the stomach becomes over-filled with food, and when invading
organisms grow or multiply within the body cavity. Some causes for
abdominal enlargement in fish have not been determined while others have
defined etiologies or associated pathogens including several caused by
bacteria, viruses, and parasites. Several parasites that are associated
with abdominal enlargements in warmwater cultured fish include: a
zygomycete fungi (microsporidian) \-- *Gluglea pimephales*; a
myxosporean called the kidney bloater \-- *Hoferellus carassii*; a
nematode \-- *Eustrongylides* sp.; visceral tapeworms \-- *Ligula
intestinalis*, *Diphyllobothrium sebago* and *Schistocephalus solidus*;
the Asian tapeworm \-- *Bothriocephalus acheilognathi*; and the white
grub \-- *Posthodiplostomum minimum*. These parasites will be briefly
discussed with emphasis placed on the Asian tapeworm and the white grub.
Comments on control measures for these latter two parasites will be
given.

**Pathology Associated With *Coleps* Spp. In Tilapia**

^1^[Lester H. Khoo]{.underline}, ^2^Wilhelm Foissner and ^3^Alistair
D.M. Dove

^1^University of Pennsylvania, School of Veterinary Medicine, New Bolton
Center, 382 West Street Road, Kennett Square, PA 19348; ^2^FB
Organismische Biologie, Universität Salzburg Hellbrunnerstr. 34A-5020
Salzburg, Austria; ^3^Marine Sciences Research Center, Stony Brook
University, Stony Brook NY 11794

Tilapia (*Oreochromis niloticus*) were submitted to the Pennsylvania
Animal Diagnostic Laboratory System from a commercial producer who was
raising the fish in a recirculating system. The major complaint was that
production in the various tanks of the system was not up to expected
standards. No major mortalities were noted at this time. Fish were
submitted live and were subjected to a full diagnostic work up which
included gill biopsies, skin scrapes, necropsy, and microbiological
culture of the kidney and histopathological examination of samples of
all the various tissues collected in 10% neutral buffered formalin.
Microscopic examination of the skin scrapes and gill clips revealed a
large number (approximately 15 to 20 per high power field) of a small
(approximately 75um X 30um), barrel shaped holociliate that was later
identified as *Coleps* sp. The gills also appeared swollen, congested
and slightly blunted. Although *Vibrio angullarum* was isolated from one
fish (out of five), there were no corresponding histopathological
findings that would indicate a bacterial infection. Significant
histopathological findings were renal tubular mineralization as well as
lamellar edema and epithelial erosion of the gills. Occasionally,
sections of organisms consistent with *Coleps* sp. were also present in
between the gill lamella. It was thought that branchial lesions might be
a result of a combination of factors including water quality (the total
ammonia nitrogen was reported to be high), formalin treatments and as
well as possible irritation by the protozoa. Similar appearing organisms
were also rarely seen in the affected renal tissue. *Coleps* sp. are
protozoans that have distinctive protective calcerous plates. They are
mainly regarded as free living scavengers that feed on dead and dying
tissue and microorganisms. However, they have been reported to kill
zebra fish (4-9 day old) larvae in production systems as well tropical
fish and rainbow trout.

**Three Disease Cases: Acid-Sulfate Soil, Largemouth Bass Wintertime
Sudden Death, And Channel Catfish "Pimples"**

[Robert M. Durborow]{.underline}

Aquaculture Research Center, Kentucky State University, 103 Athletic
Drive, Frankfort, KY 40601

Acid-sulfate soils in the watershed of largemouth bass ponds in Ohio
were exposed to air and rain after deforestation. Acid run-off into the
bass ponds decreased pH and caused mortalities ranging from 100% in
ponds closest to the exposed acid-sulfate soil to 0 - 1% in ponds near
the end of the hollow. Red iron oxide and hydroxide precipitate marked
the ponds experiencing high mortalities. In another case, largemouth
bass in Kentucky are experiencing high mortalities this winter
(2005-2006). They display lethargic and disoriented swimming, and when
startled, they thrash vigorously at the water surface and die in less
than a minute with the pectoral fins rigid at a 90º angle from the body.
Daily mortalities range from 20 to 40 out of 4,000 fish. In a third
case, digital photographs of channel catfish with red lesions resembling
pimples or blisters were sent to my lab in January 2006. In previous
similar cases, no cause of this condition was found.

**Unusual Spinal Deformity In Newly Hatched Yellow Perch**

Stephanie G. Grimmett, Geoffrey H. Groocock, and [Paul R.
Bowser]{.underline}

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

In June, 2005 a diagnostic case involving yellow perch (*Perca
flavescens*) was submitted to our Fish Disease Diagnostic Laboratory.
The fish were newly hatched yellow perch that had originated from eggs
collected from Oneida Lake, New York. These fish were to be used in a
research project. The complaint associated with the case was that the
fry from certain egg groups were experiencing moderate to high
mortalities, while fry from other egg groups experienced no such
mortality. Microscopic examination of the fish revealed severe curvature
of the spine. A standard practice of placing used Christmas trees into
the lake to serve as spawning substrate for yellow perch was employed in
this case. A search of the literature revealed studies with the
agricultural pesticide metam, in which similar spinal abnormalities were
observed in young zebrafish exposed to the compound. Metam is considered
to be the third most widely used agricultural pesticide in the United
States. One of the major uses of metam is in the production of
ornamental conifers. While we do not have a direct cause and effect of
metam in this case and we currently consider the diagnosis to be open,
we consider exposure to metam as a cause deserving further
consideration.

**Atypical Clinical Signs Of *Streptococcus agalactiae* In Nile Tilapia
(*Oreochromis niloticus*)**

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

^1^Aquatic Animal Health Research Laboratory, United States Department
of Agriculture, Agricultural Research Service, 118 B Lynchburg Street,
Chestertown, MD 21620; ^2^Aquatic Animal Health Research Laboratory,
United States Department of Agriculture, Agricultural Research Service,
P.O. Box 952, Auburn, AL 36831

After injection challenge with *Streptococcus agalactiae*, a male Nile
tilapia, *Oreochromis niloticus*, exhibited an erratic corkscrew
swimming pattern and assumed a "C"-shaped body posture. The fish did not
die after challenge but developed a grossly-observable "hunchback" at
the level of the cervical spine. Radiographs revealed multiple spinal
curvatures along the length of the spinal column with vertebral lordosis
and kyphosis. The male fish also developed a cranial cavitation on the
dorsal aspect of the head, forming a depression between the eyes. The
fish subsequently mated and helped produce fry that initially appeared
normal. The fry population soon experienced high mortalities, and
moribund fish exhibited reddened gills, presumptively over-inflated
bladders, and spun on their longitudinal axis, often with their head
pointed down (tail-up swimming). Surviving fry had stunted or absent
fins, deformed gill opercula, and/or cranial cavitation, but did not
show vertebral deformities. Gram-positive bacteria identified as *S.
agalactiae* were isolated from the fry. *Streptococcus agalactiae*
appears to be the causative agent of these skeletal anomalies among the
male broodfish and the fry, and this is the first report of hunchback
skeletal abnormalities in *S. agalactiae*-infected fish. Furthermore,
the results demonstrate the vertical transmission of *S. agalactiae* to
the fry.

**A Tale of Two Tumors: The Shrimp's And The Clam's**

^1^[Esther C. Peters]{.underline}, ^2^Kenneth W. Hasson, and
^3^Christopher F. Dungan

^1^Tetra Tech, Inc., 10306 Eaton Place, Suite 340, Fairfax, VA 22030,
and Registry of Tumors in Lower Animals (RTLA), 22900 Shaw Road, Suite
107, Sterling, VA 20166; ^2^Texas Veterinary Medical Diagnostic Lab, 1
Sippel Rd., College Station, TX 77841; Maryland Department of Natural
Resources, Cooperative Oxford Laboratory, 904 S. Morris St., Oxford, MD
21654

Diagnosing neoplasms in the invertebrates presents many challenges.
Solid and disseminated tumors have been recognized in the Arthropoda and
Mollusca; however, the cells involved and changes seen are not always
consistent with vertebrate neoplasm characteristics. A recent
contribution to the RTLA provides further perspectives on rare
crustacean cases in the collection. An epidermal papilloma on
*Litopenaeus vannemei* featured thin, elongated abnormal epidermal cells
with hypertrophied nuclei and prominent nucleoli, mitotically active
areas, abnormal formation of chitin, and cystic, hemolymph-filled stroma
in the verrucose mass. In contrast, disseminated neoplasia (DN), a
proliferation of anaplastic, hypertrophied circulating cells
(hypothesized to be hemocytes, and in many respects similar to
leukemias) can be found in up to 90% of some populations of bivalves,
occurring in 15 species. Histopathological examinations of razor clams,
*Tagelus plebeius*, from Chesapeake Bay, revealed normal-appearing
hemocytes and circulating cells with hypertrophied nuclei and scant
cytoplasm. Mitoses were few but abnormal nuclear morphologies
(binucleate, C-shaped) were common. Razor clams from Delaware Bay also
contain hypertrophic circulating cells with normal-appearing hemocytes.
Is this a normal cell type for this species? Can the binucleated cells
be interpreted based on vertebrate leucocyte variations?

**Molecular Detection And Identification Of *Hematodinium* Species**

[Hamish J. Small]{.underline}, Kimberly S. Reece, Jeffrey D. Shields

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

The parasitic dinoflagellates *Hematodinium* spp. and
*Hematodinium*-like species are economically significant pathogens of
decapod Crustacea. Previous methods used to detect infections included
external assessment for signs of infection, a microscopic index (pleopod
diagnosis) and several immunoassays. The current study describes the
development and application of two sets of *Hematodinium*-specific
polymerase chain reaction (PCR) primers and DNA probes based on
*Hematodinium* ribosomal RNA (rRNA) sequences. The first set of PCR
primers detects *Hematodinium* infections in *N. norvegicus, C. puber,
P. bernhardus* and *C. opilio*. The second set of PCR primers detects
infections in *C. sapidus, L. depurator and P. trituberculatus*. Using
the latter set of primers, a post-PCR restriction enzyme digestion of
the PCR amplification products can be used to differentiate between the
*Hematodinium* species infecting *C. sapidus, L. depurator and Portunus
trituberculatus*. Sequencing and phylogenetic analysis of the first
internal transcribed spacer region (ITS1) of the rRNA region from
several *Hematodinium* isolates indicate two genetically distinct groups
of *Hematodinium/Hematodinium*-like organisms.

**Aspects Of The Pathology Of *Hematodinium* Infections In Snow Crabs
(*Chionoecetes opilio*) From Newfoundland, Canada**

^1^[Kersten Wheeler]{.underline}, ^1^Jeffrey D. Shields, and ^2^David M.
Taylor

^1^Virginia Institute of Marine Science, The College of William and
Mary, School of Marine Science, Gloucester Point, Virginia 23062;
^2^Department of Fisheries and Oceans, St. John's, Newfoundland A1C 5X1
Canada

Bitter crab disease (BCD) of snow crabs, *Chionoecetes opilio*, is
caused by a parasitic dinoflagellate, *Hematodinium* sp. The disease has
increased in prevalence in Newfoundland's commercial fishery since it
was first recorded in the early 1990s. We examined the pathology of
infected snow crabs and documented histopathological alterations to the
tissues in heavy infections and alterations to the gills during
sporulation of the parasite. Infections of *Hematodinium* can lead to
high densities of parasitic cells in the hemolymph (10^8^ cells ml^-1^)
that lead to host death. Pressure necrosis was evident in the soft
connective tissues of the hepatopancreas, the blood vessels in most
organs, and soft connectives in the eyestalks. In heavy infections,
little remained of the soft connective tissues around the
hepatopancreas. Damage to the gills varied, but in some cases it was
severe, involving a loss of host epithelial cells and fusion of the
membranous layer of adjacent gill lamellae. Lamellae exhibited
hypertrophy with the loss of trabecular cells, hemocyte infiltrations,
and clubbing along the distal margins. Large numbers of zoospores were
located along the distal margins of affected lamellae suggesting that
sporulation may result in the lysis or bursting of the lamellar cuticle
to release spores. It is unclear how dinospores transmit the disease to
the next host. Hematodinium infections in the snow crab appear to be
chronic, long-term infections that end in death during sporulation of
the parasite.

**Effects Of Hypercapnic Hypoxia On The Clearance Of *Vibrio*
*campbellii* In The Eastern Oyster, *Crassostrea* *virginica***

Brett M. Macey, Louis E. Burnett, and [Karen G. Burnett]{.underline}

Grice Marine Laboratory, College of Charleston, and Hollings Marine
Laboratory, 331 Fort Johnson, Charleston, South Carolina 29412

In order to understand the role of marine organisms as vectors of human
disease pathogens, we investigated the effects of hypercapnic hypoxia
(HH) on the clearance of live pathogenic bacteria in *Crassostrea*
*virginica*. Oysters were held in normoxic (Po~2~ = 20.0-20.7 kPa or 98%
air saturated, pH 7.8-8.0) or HH (Pco~2~ = 1.8 kPa or 2% CO~2~, Po~2~ =
3.9-4.2 kPa or 19-20% air saturation, pH 6.2-6.5) conditions at 25 °C
for 4 h, then injected in the adductor muscle with 10^5^ live *Vibrio
campbellii.* Culturable *V*. *campbellii* (CFU) were quantified in the
whole oyster, including all tissues and fluids, at 10, 30 and 60 min
post-injection. CFU decreased rapidly in all animals, however, oysters
held in HH retained significantly higher CFU than animals held in
air-saturated water over the test period (2-way ANOVA, p\<0.001). At 60
min post-injection, animals held in normoxic conditions retained less
than 15% of the injected bacteria as compared to animals held in HH,
which retained 30% of the injected bacteria. These results indicate that
HH dramatically reduces the rate at which oysters can render bacteria
non-culturable. We suggest that poor water quality can increase the risk
that oysters will harbor and transmit bacterial pathogens hazardous to
human and ecosystem health.

# **Putative Identification Of Expressed Genes Associated With Attachment Of The Zebra Mussel (*Dreissena polymorpha*) Using Suppression Subtractive Hybridization cDNA Library**

[Wei Xu]{.underline} and Mohamed Faisal

College of Veterinary Medicine, Michigan State University, East Lansing,
Michigan 48824

# Zebra mussels (*Dreissena polymorpha*) continue to cause economical and ecological devastation in the Great Lakes basin. A potentially effective method to control the spread of this invasive species is to prevent its attachment to substrates under water. In this study, a Suppression Subtractive Hybridization (SSH) cDNA library of the zebra mussel foot has been developed. This array contains genes involved in attachment, exocrine excretion, host defense, protease-antiprotease cascades, signal transduction, cell division and development, metabolism, cell structure maintaining, and DNA/RNA synthesis. The relation between those putative genes and zebra mussel attachment is being analyzed. This study provides the first cDNA microarray of zebra mussel and is an important resource toward the bio-control of this invasive species. 

**Recent Advances In Our Understanding Of The Host-Parasite Relationship
Of *Lepeophtheirus salmonis* And Atlantic Salmon (*Salmo salar*)**

[Mark D. Fast]{.underline}, Stewart C. Johnson, and Neil W. Ross

National Research Council of Canada -- Institute for Marine Biosciences,
1411 Oxford Street, Halifax, Nova Scotia, B3H 2Z1, Canada.

The inability of Atlantic salmon to mount a significant inflammatory
response against *Lepeophtheirus salmonis* infection has led to the
belief that immunomodulatory compounds exist in this parasite\'s
secretions. With the use of dopamine stimulation, compounds within *L.
salmonis* secretions have been identified and biologically
characterized. Among those compounds identified, trypsin and
prostaglandin E~2~ have been linked to immunosuppression and
anti-inflammatory actions. More recently, pooled secretions separated by
size-exclusion chromatography have shown differing degrees of
anti-inflammatory activity, measured by down regulation of
lipopolysaccharide-induced IL-1β expression. Multiple *L. salmonis*
genes (SL-0903 and SL-0858), encoding proteins identified in these
secretions have also shown up-regulation following host attachment and
infection. Further isolation of fractions within these pools revealed
that fraction 1-2 could fully account for the inhibition of IL-1β
expression in SHK-1 cells observed in pooled fraction 1. This provides
evidence for the presence of immunomodulatory compounds, not related to
trypsin or PGE~2~, in the secretions of *L. salmonis*, which suppress
expression of Atlantic salmon immune-related genes *in vitro.* The
implications of this data with respect to the host-parasite relationship
and possible development of future vaccine formulations will be
discussed.

**Unusual Trematode "Egg Nests" In The Stomach Tissues Of Goggle-Eyed
Scad, *Selar crumenophthalmus* (Bloch, 1793)**

^1^Jennifer Matysczak, ^2^Tonya Clauss, ^2^Howard Krum, ^2^Theresa
Nietfeld, ^3^Roy P.E. Yanong, and ^4^[Alistair Dove]{.underline}

^1^FDA CVM, HFV-131, 7500 Standish Place, Rockville MD 20850; ^2^Georgia
Aquarium, 224 Baker St., Atlanta GA 30313; ^3^Tropical Aquaculture
Laboratory, Department of Fisheries and Aquatic Sciences, University of
Florida, 1408 24^th^ St. SE, Ruskin FL 33570; ^4^Marine Sciences
Research Center, Stony Brook University, Stony Brook NY 11794

A shipment was received by the Georgia Aquarium of wild goggle-eye scads
from waters off of the Florida Keys and during quarantine 3 mortalities
were presented for necropsy. On gross dissection, brown patches of
discoloration were evident in the stomach wall of all 3 animals
examined. Histological examination showed these to be "nests" of
trematode-like eggs in the lamina propria and muscularis mucosa. This
presentation is extremely unusual for enteric digeneans, which normally
discharge their eggs to the exterior with the host's faeces. Egg nests
were frequently associated with granulomatous inflammation and a
proliferative connective tissue response. Focal and diffuse lymphocytic
infiltrates and blood vessel dilation were also observed, consistent
with more acute inflammatory reaction. Connective tissue necrosis led to
sloughing of the overlying gastric mucosa in at least one case.
Parasitological examination showed infections in the stomach with a
small unidentified hemiurid digenean, and in the intestine with a
fellodsitomid digenean matching the genus *Tergestia* and an
unidentified opecoelid digenean. Histozoic digeneans more often
associated with parenteral egg deposition (basically sanguinicolids)
were not observed and egg nests were not observed in organs other than
the stomach. Egg measurements were made using digital image analysis of
histological sections and of whole mounts of each worm type, in order to
identify the source of the eggs. Eggs from the nests did not match the
dimensions of those from any of the three identified trematodes,
suggesting the existence of a fourth trematode species parasitic in this
host, the identity of which remains unknown. In two cases, digeneans
were seen in histological section in the muscularis mucosa, suggesting
that the responsible agent is truly histozoic. . This presentation is a
result of independent research and does not represent the findings of
the U.S. Food and Drug Administration

**Disease and Adaptive Fishery Management: The Role of the Fish Health
Professional**

[Frank M. Panek]{.underline}

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

Knowledge of fish disease processes, fish disease defense mechanisms and
ecology are important to the development of comprehensive fishery
management strategies. This is especially true where either epizootic
disease or chronic disease influence fish at the population level. In
such instances, successful resource management depends upon the ability
of resource managers to properly diagnosis disease, to predict
population level risks, and to take appropriate management actions.
Adaptive management is a process from which scientific knowledge is
generated and subsequently utilized to formulate management strategies.
It is an iterative strategy that relies upon cooperation, collaboration
and communication. However, fishery managers and fish health
professionals do not always communicate effectively making it often
difficult to integrate aquatic animal health concerns in fishery
management decisions. A review of the peer reviewed literature from
selected journals over the past 10-years suggests that communication and
collaboration between aquatic animal health professionals and fish
culturists is more effective than that between the former and fishery
management biologists. Of the 109 papers reviewed in the Journal of
Aquatic Animal Health approximately 20% specifically provided
recommendations and or applications for fish culture while 14% provided
comparable management recommendations to fishery managers. A similar
trend was noted in 103 papers published in Diseases of Aquatic
Organisms; 16% provided fish culture recommendations and 14% recommended
fishery management actions. Perhaps more interesting is the publication
practices of the fish culturists and fishery managers. Thirty percent of
65 papers in the North American Journal of Aquaculture specifically
addressed a disease issue and provided fish culture disease management
recommendations. However, of the 112 papers reviewed in the North
America Journal of Fish Management only 8% mentioned or discussed fish
disease as a factor in fishery management. The seven (7) components
required to address adaptive fishery management are discussed along with
the recommendations for enhancing the role(s) of aquatic animal health
specialists and veterinarians.

**Drugs For Domestic Aquaculture: Background And Session Strategy**

[Thomas A. Bell]{.underline}

U.S. Fish & Wildlife Service, Aquatic Animal Drug Approval Partnership,
4050 Bridger Canyon Rd., Bozeman, MT 59714

Activities in the U.S. aimed at obtaining new approved drugs for
aquaculture species have been ongoing, in some shape or form, for
several decades. However, true concerted efforts have only occurred in
the last 10-15 years. Historically, and still to this day, developing
new drugs for the U.S. aquaculture industry (private and public) has
been very low on the priority list of nearly all drug companies; the
bottom‑line being that anticipated domestic profits fall far short of
expected R&D costs. Consequently, a significant portion of the
investment in data generation to support new drugs for aquaculture
species has had to come from the public sector. This session will
attempt to not only inform you of progress to date, but also to provide
some insight into why we have progressed as far as we have, but not as
far as most would like. Just as importantly, we hope to also shed some
light on how very well we have done, considering the odds against it.
Our strategy for the session is to very briefly contrast progress
pre-1992 with the present, followed by the FDA process and their view of
our progress. We will then shift gears to provide a glimpse behind the
scenes in two labs conducting pivotal studies. We will close the session
with a realistic view of many of the reasons why we are not as close to
gaining those approvals as all of us would like, but nonetheless
accomplishments for which we can take great pride.

**Drug Approval Status: Then And Now**

[Rosalie (Roz) Schnick]{.underline}

National Coordinator for Aquaculture New Animal Drug Applications,
Michigan State University, 3039 Edgewater Lane, La Crosse, WI 54603

Efforts from 1964 to 1994 produced limited New Animal Drug Application
(NADA) approvals for five aquaculture drugs: formalin, MS-222, oral
oxytetracycline, Romet-30®, and sulfamerazine. These NADA approvals were
gained with very limited funds. In 1990, the U.S. Food and Drug
Administration (FDA) surveyed the aquaculture community and determined
that we lacked proper and broad approvals. That set the stage for
concerted efforts to gain the extensive funding needed to meet the data
requirements of FDA's Center for Veterinary Medicine to prove that a
drug is safe and effective for the intended use. We are currently
pursuing a full spectrum of drugs for NADA approval and have made
tremendous progress. Information comparing the studies completed toward
approval in 1994 as compared to 2006 will be presented for each
aquaculture drug actively being pursued and funded for NADA approval.

**The INAD/NADA Process: That\'s Not A Train, But A Light At The End Of
The Tunnel**

[Donald A. Prater]{.underline}

USFDA, Aquaculture Drugs Team, Office of New Animal Drug Evaluation,
7500 Standish Place, MPN-2, Rockville, MD 20855

Currently there are only a small number of drugs approved for use in
aquaculture. The lack of availability of drugs approved for use in
cultured fish species has significant impact on the health and welfare
of these animals and severely hampers the productivity of this
burgeoning industry. The reasons for a lack of approved drugs for
aquaculture are several and include: the economic value of marketed
products is perceived to be small by major pharmaceutical and chemical
companies, the course of regulatory approval can be long and costly, and
there are additional challenges in designing and conducting the required
safety and effectiveness studies in aquaculture systems. Drugs used in
aquaculture fall into one of several categories, approved, unapproved,
low regulatory priority (LRP), regulatory discretion, or extralabel.
Approved drugs require a new animal drug application (NADA). The NADA is
composed of major and minor technical sections including effectiveness,
target animal safety, environmental safety, human food safety,
chemistry, manufacturing and controls, labeling, and all other
information. Most work drug development work is conducted under an
investigational new animal drug (INAD) exemption file. Once a drug is
approved there is a system for monitoring for adverse reactions,
including lack of effectiveness. Some drugs are available
over-the-counter (OTC) while others are available by prescription or
veterinary feed directive (VFD). Some approved drugs may be used
extralabel, although medicated feeds carry additional restrictions for
extralabel use. The Minor Use and Minor Species (MUMS) Animal Health Act
of 2004 provides new incentives for sponsors of drugs for minor species,
such as aquatic species. The major provisions of the legislation are 1)
designation which qualifies a drug for additional periods of exclusivity
and potential grants, 2) conditional approval which allows a drug to
come to market before completing the required effectiveness section, 3)
indexing which creates a new process for establishing legally marketed,
unapproved new animal drugs. These new incentives should help increase
the availability of new drugs for use in aquaculture.

**Efficacy And Target Animal Safety Studies: Who Said This Was A Cake
Walk?**

[Jim Bowker]{.underline}

Aquatic Animal Drug Approval Partnership Program, U. S. Fish and
Wildlife Service, 4050 Bridger Canyon Road, Bozeman, Montana 59715

The approval of drugs for use in aquaculture is regulated by the U.S.
Food and Drug Administration's (FDA) Center for Veterinary Medicine
(CVM). Although culture and management strategies used typically
minimize the need for drug use, at times, fish culturists and fisheries
managers need access to approved therapeutants, anesthetics, marking
agents, or spawning aids. The process to gain FDA approval is lengthy
and costly, and includes FDA acceptance of data to demonstrate that the
drug is effective, safe to humans, fish, and the environment, and can be
manufactured consistently. Ideally, completion and acceptance of these
"Technical Sections" will result in FDA approval to allow use of a new
drug on all fishes for all purposes (i.e., claims). Although completing
each Technical Section is theoretically straightforward, researchers
designing and conducting studies to demonstrate that a drug is safe to
fish and effective for the specified claim are often faced with unique
biological and logistical challenges. The FDA requires that the
effectiveness of a drug be demonstrated under field and
"quasi-production" conditions and that test fish are "naturally"
infected with the disease pathogen. Variables associated with the onset
of a disease, combined with the necessity of having no secondary disease
pathogen, make successful completion of these pivotal studies
problematic. In addition, logistical challenges, such as trying to get
staff and equipment to the study site in a timely manner, result in a
success rate that is akin to a good major league baseball batting
average. We could boost our "batting average" by utilizing disease
models to initiate disease outbreaks. However, at present, use of such
models can not be used in place of these pivotal studies. Requirements
for successful completion and CVM acceptance of target animal safety
(TAS) studies include different but equally challenging issues. This
presentation will provide an overview of challenges associated with
conducting efficacy and TAS studies in support of a new animal drug
approval, and will demonstrate how CVM acceptance of study results isn't
a cake walk.

**Trials And Tribulations Developing Environmental Safety And Human Food
Safety Data For Aquaculture Drug Approvals**

[Jeffery R. Meinertz]{.underline}, William, H. Gingerich, Theresa M.
Schreier, Mark P. Gaikowski, and Larry J. Schmidt

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

During the aquaculture drug approval process, data are submitted to the
U.S. Food and Drug Administration (FDA) to fulfill data requirements of
six technical sections. The six technical sections include (1) Product
Chemistry, (2) Labeling, (3) Animal Safety, (4) Effectiveness,
(5) Environmental Safety, and (6) Human Food Safety. Studies conducted
to support the Environmental Safety and Human Food Safety technical
sections (aka, data packages) are often the most time and money
consuming projects associated with the drug approval process. Initially,
Environmental Safety data packages were challenging to fulfill primarily
because there was little guidance to draw upon to generate the required
information. Initial submissions required implementing new and
innovative techniques to conduct hatchery surveys, chronic toxicity
studies, and develop drug discharge models. Original submissions
eventually led to the generation of FDA supported templates that will
serve as the basis for future aquaculture drug Environmental Safety
submissions. Human food safety data packages are also challenging to
fulfill primarily because of the scope of data that this data package
requires. This data package draws upon data generated in five scientific
fields including toxicology, microbial food safety, drug
residue/metabolism, analytical method validation, and drug residue
depletion. Studies conducted in these scientific fields are generally
technically complicated and time consuming.

**Drug Approval Status: Why We Are Where We Are And Not Where You
Thought We Should Be**

[Rosalie (Roz) Schnick]{.underline}

National Coordinator for Aquaculture New Animal Drug Applications,
Michigan State University, 3039 Edgewater Lane, La Crosse, WI 54603

The business of New Animal Drug Application (NADA) approvals is
expensive, time-consuming, and difficult to achieve because of specific
factors that are either under our control and others which are not.
Examples of such factors will be given to include (1) new Center for
Veterinary Medicine (CVM) requirements, (2) changes in CVM staffing, and
(3) acquisition of extensive funds to generate the expensive studies
needed for approval. In spite of the many factors that have been an
impediment to the approval process, we have made great strides and have
many label claims on the verge of NADA approval. Information will be
given on the current status of each aquaculture drug label claim being
actively pursued and funded toward NADA approval.

**Monitoring Hawaiian Invertebrates (Crustacea) For White Spot Syndrome
Virus (WSSV)**

^1^[Teresa D. Lewis]{.underline}, ^1^David W. Albert, ^2^Dee
Montgomery-Brock, ^3^Allen C. Riggs, and ^1^Jo-Ann C. Leong

^1^Hawaii Institute of Marine Biology, School of Ocean & Earth Science &
Technology, University of Hawaii at Manoa, P. O. Box 1346, Kaneohe HI
96744; ^2^Oceanic Institute, 41-202 Kalanianaole Highway, Waimanalo, HI
96795; ^3^Aquaculture Development Program -- Disease Prevention, Hawaii
Department of Agriculture, 1039 Sand Island Parkway, Honolulu, HI 96819

April 14, 2004 marked the first identification of white spot syndrome
virus (WSSV) in Hawaii. This outbreak occurred at a commercial shrimp
facility on the island of Kauai. A second confirmation of WSSV on the
island of Oahu occurred in late 2005. This case involved shrimp that
were maintained in an inland primary quarantine facility that received
unfiltered seawater from Kaneohe Bay. WSSV has a wide host range and has
been shown, both experimentally and naturally, to infect many species
found in Hawaiian waters. A preliminary survey for the presence of WSSV
in Hawaiian decapod crustaceans was performed to establish whether a
larger scale survey is warranted. Samples were collected from Oahu and
Kauai and tested for WSSV using a two-step PCR protocol. A low
percentage of samples from Oahu were WSSV positive, to include *Palaemon
debilis* (grass shrimp), *Pachygrapsus* sp. (shore crab), and *Thalamita
crenata* (mangrove swimming crab). These results highlight the
requirement for a larger scale survey to assess WSSV prevalence,
identify virus-susceptible species, as well as the necessity of
experiments to better understand modes of viral transmission.

**Comparative Susceptibility Of Early Life Stages Of The Bullfrog (*Rana
catesbeiana*) To The Tadpole Edema Virus**

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

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

Investigations concerning the worldwide decline of amphibian populations
have revealed a number of potentially related causal factors, including
non-infectious and infectious diseases. Among these, viral pathogens of
the family Iridoviridae (genus Ranavirus) have been isolated from an
increasing number of species of dead and clinically diseased amphibians
over an expanding geographic range, sometimes in association with
significant levels of mortality. These viruses, including Frog Virus 3
and nearly homologous isolates such as the Tadpole Edema Virus (TEV),
represent potentially important pathogens of many amphibians. Still,
there are considerable unknown factors related to their transmission,
virulence, and pathogenesis in and among the host species. Here we
investigate the comparative susceptibility of pre-hatch and post-hatch
life stages of the bullfrog, *Rana catesbeiana*, to TEV. Replicate
groups of fertilized bullfrog eggs or tadpoles were exposed to TEV at 0,
10^5^ or 10^6^ TCID~50~/ml in 50 ml spring water (21^o^C) for 20 hours,
at either 3 days pre-hatch or 3 days post-hatch. Mortality was recorded
daily, dead tadpoles and eggs collected for 7 days following exposure,
and surviving tadpoles were humanely euthanized. Tadpoles and eggs were
evaluated for presence of virus using a quantal viral infectivity assay
to determine 50% endpoint in cell culture. No virus was detected among
any control groups, dead eggs, or tadpoles exposed pre-hatch. Virus was
detected among the post-hatch exposure groups, ranging from 10^7^-10^8^
TCID~50~/g for mortalities and 10^5^-10^7^ TCID~50~/g for select
survivors.

**Largemouth Bass Virus In New York State**

Geoffrey H. Groocock, Rodman G. Getchell, Vanessa L. Kirsipuu, Gregory
A. Wooster, and [Paul R. Bowser]{.underline}

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

During 2004 and 2005, we undertook an effort to identify natural bodies
of water in New York State that contained fish infected with Largemouth
Bass Virus (LMBV). Fish samples collected by the New York State
Department of Environmental Conservation and the Cornell Biological
Field Station during the course of their fisheries studies were made
available to us for testing. A quantitative PCR (Q-PCR) test was used to
individually screen individual fish. Samples that were Q-PCR positive
were subsequently processed for virus isolation using Fathead Minnow
(FHM) cell cultures. In 2004, 91 largemouth bass (*Micropterus
salmoides*) from 13 bodies of water were tested using the Q-PCR. LMBV
was detected in 7 fish from 4 lakes. Cell culture was performed on some
samples, however no cytopathic effect was observed in any inoculation.
In 2005, an additional 54 Largemouth Bass from 6 bodies of water were
tested. LMBV was detected in 15 fish from 3 areas. Inoculations of these
samples in fathead minnow cell produced cytopathic effect in 2 samples.
Additionally, a small sample of 7 smallmouth bass (*Micropterus
dolomieui*) from a single river was tested with the Q-PCR, and 6
positive results were obtained. Subsequently, cytopathic effect was
observed in 2 samples after inoculation in cell culture. This survey
confirms the presence of LMBV within wild fish populations in
geographically diverse areas of New York State, although no mass
mortality events to date have been associated with this virus in this
state.

**Isolation Of Viral Hemorrhagic Septicemia Virus (VHSV) From
Muskellunge (*Esox masquinongy*) From Lake St. Clair, Michigan**

^1^[Mohamed Faisal]{.underline}, ^1^Ehab E. Elsayed, ^2^William Batts
and ^2^James Winton

^1^ College of Veterinary Medicine, Michigan State University, East
Lansing, Michigan 48824;^2^ Western Fisheries Research Center, USGS,
Seattle, Washington 98115

Viral hemorrhagic septicemia virus (VHSV) was isolated from muskellunge
(*Esox masquinongy*) caught from the Clinton River area and the Grosse
Point Yacht site in Lake St. Clair, Michigan, USA. Affected fish
exhibited widespread edema of the skin, fin hemorrhages, sunken eyes,
and congestion of internal organs. The inner wall of the swim bladder
was thicker than usual, with numerous fluid-filled vesicles. A virus was
isolated using FHM and CHSE cell lines inoculated with a homogenate of
kidney and spleen tissues from affected fish. Focal areas of cell
rounding and granulation appeared as early as 24 hr post-inoculation and
expanded rapidly to destroy the entire cell sheet by 96 hr. Electron
microscopy revealed virions having the typical bullet shape morphology
of rhabdoviruses. Viral particles ranged in size from 165-185 nm in
length and 55-75 nm in width. The virus was confirmed as VHSV by RT-PCR
and sequence analysis of the nucleoprotein gene and a portion of the
glycoprotein gene revealed the virus was a member of the North American
genotype of VHSV and nearly identical to an isolate from freshwater drum
in Lake Ontario. This report documents the first isolation of VHSV from
*Esox masquinongy* in Michigan waters. The impact of this virus on the
muskellunge population of Lake St. Clair remains to be determined.

**Isolation Of Viral Hemorrhagic Septicemia Virus Type IV From A
Mortality Event In Lake Ontario Drum (*Aplodinotus grunniens*)**

^1^[John S. Lumsden]{.underline}, ^1^Spencer Russell, ^1^Karrie Young,
^1^Arman Yazdanpanah, ^1^Paul Huber, ^2^Bruce Morrison, ^3^Carmencita
Yason, ^4^David Stone, ^4^Keith Way

^1^Fish Pathology Laboratory, University of Guelph, Guelph, ON, N1G 2W1.
^2^Ontario Ministry of Natural Resources, RR^\#^4 Picton, ON, KOK 2Y0.
^3^Atlantic Veterinary College, University of Prince Edward Island, PEI,
C1A 4P3. ^4^CEFAS Weymouth Laboratory, Barrack Rd. The Nothe, Weymouth,
DT4 8UB, UK

Large numbers of dead and dying freshwater drum were observed within the
Bay of Quinte (Lake Ontario) and later towards the St. Lawrence River
entrance. Moribund fish appeared lethargic, were unable to maintain
proper orientation and were easily caught by hand. Externally, all fish
exhibited multifocal to regionally extensive hyperemia and hemorrhages
at fin bases and ventral surfaces. All fish exhibited mild to moderate
bilateral exopthalmia, and several had hyphema. Internally, there was
copious serosanguanous ascitic fluid and there was extensive serosal and
sub-mucosal gastrointestinal hyperemia. The spleen of several of the
fish was moderately enlarged. Skin scrapes and gill biopsies were
unrevealing. No growth was observed from spleen and kidney on cytophaga,
tryptic soy and blood agar following incubation at 20 and 37°C for
48-72hrs. All drum examined had a similar pattern of histologic lesions
that were consistently oriented on the vasculature. Fibrinoid vasculitis
(arteritis most prominently but also a phlebitis) was most notable in
the kidney and meninges. Consistent lesions noted were as follows;
severe necrotizing myocarditis, epicarditis, and meningoencencephalitis,
mild to moderate multifocal necrotizing splenitis and interstitial
nephritis and diffuse enteritis. No organisms were identified in H&E
sections. All mouse inoculations using pooled tissues were negative for
botulism toxin. There were numerous clusters of viral particles
\~50x150nm present in myocardial tissue fixed in 2% glutaraldehyde and
examined by transmission electron microcopy (TEM). TEM and negative
staining of filtered fathead minnow culture supernatants revealed
similar virions with typical rhabdovirus morphology. RNA polymerase gene
(L) sequence results indicated the virus to be 91% homologous to a viral
hemorrhagic septicemia virus isolate from Japan (type IV strain).

**Kinetics Of The Natural Microbial Populations Of Atlantic Salmon In
Relation To Infection And The Recuperative Process**

[Rocco C. Cipriano]{.underline}

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

Certain bacterial fish pathogens exist in/on affected animals in two
sequential phases; an external phase on the skin, gills or other
external surfaces and an internal or systemic (circulating) phase. As in
the cases of *Aeromonas salmonicida* subsp. s*almonicida* and *Yersinia
ruckeri*, it is typically the systemic phase of the infective process
that initiates extensive pathological changes within vital organs and
produces mortality. Although the external phase is not nearly as
traumatic for the host, it also produces subtle, albeit measurable
changes that can be nonlethally detected by monitoring a pathogen's
interactions with the microbial flora resident within a host's dermal
mucus. As a horizontally transmitted or water-borne pathogen initiates
disease, it becomes more prevalent and displaces the normal
microflora; - most of which are gram-negative, glucose \--inert or
-oxidative bacteria. Replication of the pathogen continues on the dermis
until that bacterium dominates the external microflora, overcomes host
defenses, and thereby initiates systemic infection. During this increase
in pathogen load, the pathogen is shed into the water where contagion,
both in the wild and under conditions of intensive culture, is density
dependent. Depending upon the virulence of the pathogen and host
resistance, a fulminating septicemia develops with resultant mortality
that necessitates antibiotic treatment. In the recovery process, this
microbial situation is reversed and the normal bacterial flora
re-establishes itself to pre-challenge levels. Hence, understanding the
importance of key microbial species and the kinetics of their bacterial
distributions may provide a novel approach toward assessing population
health. It is my hypothesis that the assessment of surface bacterial
flora (the distribution of species and the relative proportion of a
pathogen to other microorganisms) can be used to predict an imminent
disease epizootic prior to development of clinical signs. Similar
measurements are also vital towards understanding the relative
importance of other microbes, often thought of as facultative pathogens
(e.g. -- motile aeromonads and *Pseudomonas fluorescens*), in the
recuperative process. The hypothesis set forth in this abstract will be
explained using the interplay between *A. salmoncida* and the normal
flora of Atlantic salmon (*Salmo salar*) as a model.