ORAL SESSION
ABSTRACTS

SESSION 1. BACTERIOLOGY I: Molecular Aspects

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Genomic Subtyping of Edwardsiella ictaluri Isolated from Diseased Channel Catfish by Arbitrarily Primed Polymerase Chain Reaction

JA Bader (1)*, CA Shoemaker (1), PH Klesius (1), MA Connolly (2) and JM Barbaree (3)

1	USDA Agriculture Research Service, Fish Diseases and Parasites Research Laboratory, Auburn, AL 36831 USA. jabader@mindspring.com
2	Armed Forces Institute of Pathology, Molecular Pathobiology Laboratory, Washington D.C. 20306 USA. connoly@email.afip.osd.mil
3	Department of Botany and Microbiology, Auburn University, AL 36849 USA. jbarbare@ag.auburn.edu

Arbitrarily primed polymerase chain reaction (AP-PCR) combined with an enterobacterial repetitive intergenic consensus PCR primer (ERIC II) was used to genomically subtype 20 isolates of Edwardsiella ictaluri. Nineteen isolates were derived from channel catfish Ictalurus punctatus (18 from the southeastern United States and an American Type Culture Collection reference strain), and 1 isolate was derived from a walking catfish Clarias bactrachus from Thailand. From these isolates, four major subgroupings of E. ictaluri were observed. These subgroups were distributed with the following prevalences: subgroup 1, 55%; subgroup 4, 20%; subgroup 2, 15%; and subgroup 3, 10%. Biochemical analysis showed homogeneity among isolates and was not useful for discriminating between isolates.

Sequencing and Molecular Analysis of Edwardsiella ictaluri Plasmids

DH Fernandez* and RL Thune

Department of Veterinary Science, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center and Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803. fernandez_d@vt8200.vetmed.lsu.edu.

Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish, is a Gram negative, oxidase negative rod belonging to the family Enterobacteriaciae. Isolates of E. ictaluri from catfish are biochemically and serologically homogenous and are known to harbor two plasmids of approximately 4.8 and 5.7 kilobase pairs in length. Although plasmids are known to carry antibiotic resistance determinants, virulence factors, metabolic determinants and genes for conjugation, functions of the E. ictaluri plasmids are unknown. To facilitate the evaluation of their role in E. ictaluri pathogenesis, we have cloned and sequenced both plasmids. The smaller plasmid is 4,808 base pairs and the larger is 5,643 base pairs. A database search using the BLAST and FASTA programs (Wisconsin Sequence Analysis Package, Genetics Computer Group) has identified no known antibiotic resistance genes. The smaller plasmid appears to have an insertion element, a region that controls copy number, and a gene with homology to the ipaH gene of S. flexneri. A region containing the origin of replication was also identified. The larger plasmid appears to contain a region that controls the partitioning of plasmids into daughter cells, a gene for a Rep like protein and direct repeats of approximately 45 and 140 nucleotides. In addition, an open reading frame with homology to the InvB protein of S. typhimurium and the Spak (Spa15) protein of S. flexneri was identified. Both plasmids are present in all E. ictaluri strains and preliminary attempts to cure them failed, indicating that they are physiologically significant. To date, however, no region of either plasmid appears to encode significant factors.

Comparative Analysis of Flexibacter, Flavobacter, and Cytophaga Species of Bacteria Isolated from Rainbow Trout

SE LaPatra (1)*, R Walker (2), AW Morton (1), and JM Groff (3)

1	Clear Springs Foods, Inc., Research Division, P.O. Box 712, Buhl, Idaho 83316 USA scottl@clearsprings.com; andy@clearsprings.com
2	California Veterinary Diagnostic Laboratory System, P.O. Box 1770, Davis, California 95617 USA. rwalker@cvdls.ucdavis.edu
3	Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, California 95616 USA. josephvmd@aol.com

Five isolates of Flexibacter psychrophilus were isolated from various tissues of rainbow trout that had a systemic clinical infection or spinal compression deformities. Two additional unidentified yellow-pigmented bacteria were also isolated from rainbow trout with similar lesions. The bacteria were evaluated by biochemical typing, fatty acid analysis, and antibiotic sensitivities. These isolates were compared to the type strain of F. psychrophilus (NCMB 1947) and ATCC strains Cytophaga johnsonae (17061), C. aquatilis (29551), and Flavobacterium branchiophilum (35035). In addition, a recent isolate of F. columnaris from rainbow trout was also compared to the ATCC type strain (23463). The results indicated that the five F. psychrophilus isolates from rainbow trout appeared identical to the type strain NCMB 1947, which has been reclassified as Flavobacterium psychrophilum, and appeared unrelated to any of the other isolates. The two Cytophaga strains appeared closely related to each other and the yellow- pigmented bacteria obtained from rainbow trout are most likely Cytophaga or Cytophaga-like species. Flavobacterium branchiophilum and F. columnaris appeared unrelated to each other or the other two clusters of bacteria.

Characterization of Danish Flavobacterium psychrophilum Strains Isolated from Rainbow Trout (Oncorhynchus mykiss) by Ribotyping and Plasmid Profiles

L Madsen* and I Dalsgaard

Danish Institute for Fisheries Research, Fish Disease Laboratory, BÀG Àlowsvej 13, DK-1870 Frederiksberg C, Denmark. lom@kvl.dk; ida@kvl.dk

A total of 298 strains of Flavobacterium psychrophilum were examined with respect to rRNA gene restriction patterns (ribotypes) and plasmid profiles. The strains were isolated during a two-year period at five different farms. F. psychrophilum was not only isolated from diseased fish in connection with Rainbow Trout Fry Syndrome (RTFS) or cold-water disease but also from fish without clinical symptoms. Ribotypes were separated into one major cluster and six minor clusters. The majority of the strains had the same profile A. Eight strains had a slightly different profile B. Five other profiles were found but they did not differ much from the A profile. EcoR I was used as the restriction enzyme. The majority of the strains harboured a small plasmid with an approximately size of 3.3 kb, while five strains harboured two small plasmids with sizes around 2.8 and 3.3 kb. Other two strains harboured plasmids with the approximately sizes 2.2 and 3.3 kb. One strain had a plasmid around 2.8 kb and two strains had plasmids around 2.8 and 5.5 kb. There seemed to be no connection between ribotyping and plasmid profiles. Strains with ribotype profile B had all been isolated at the same fish farm where outbreaks of RTFS had not occurred. Virulence studies showed that these strains were less pathogenic than strains with profile A. Strains used in the virulence studies all harboured the small plasmid around 3.3 kb.

Accuracy of the AQUAEIA Kit (BIONOR AS) for the Detection of the Pasteurellosis Agent in Diseased and Carrier Fish

F Lores, B Magariños, CR Osorio, AE Toranzo and JL Romalde*

Departamento de Microbiologia y Parasitologia, e Instituto de Acuicultura.
Universidad de Santiago de Compostela. 15706 Spain. mpromald@usc.es

The availability of accurate, sensitive, and fast detection methods is of great importance in Aquaculture. Recently, a new commercial kit (AQUAEIA) based in the magnetic particle enzyme immuno assay (MP-EIA) has been developed by a Norwegian company (BIONOR AS) for the diagnosis and screening of several fish pathogens. In this work, we present the results of the evaluation of this procedure in the detection of Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida) in both "in vitro" experiments and natural samples. The new kit showed very good results in sensitivity (detection limit of 104-105 cells/g fish) and specificity (no cross reactions with other bacterial species). In addition, it was able to detect carrier fish in apparently healthy seabream populations. One of the most important findings of this study was the demonstration of vertical transmission for pasteurellosis, since the AQUAEIA kit detected the pathogen in seabream gonads and also in the eggs obtained from the same broodstock.

PCR-based Detection of Fish Pasteurellosis

CR Osorio (1)*, JL Romalde (1), JL Barja (1), MD Collins (2) and AE Toranzo (1)

1	Departamento de Microbiología y Parasitología, e Instituto de Acuicultura.Universidad de Santiago de Compostela. 15706 Spain. mpaetjlb@usc.es mpromald@usc.es
2	Institute of Food Research.Reading RG6 6BZ U.K. david.collins@bbsrc.ac.uk

16S rDNA gene sequencing of 20 isolates of Photobacterium damselae subsp. piscicida and 6 isolates of Ph. damselae subsp. damselae was carried out in order to assess the phylogenetic relationship among both taxa. Subsequently, a PCR-based diagnosis method was designed with the data obtained. In addition, two different primers were designed on basis of a previously published DNA sequence from a genomic library of Ph. damselae subsp. piscicida. The suitability of both sets of primers for the detection of fish pasteurellosis was tested. Our results have shown a high degree of genetic relationship between both subspecies. By means of a Nested-PCR approach, 16S-based primers proved to be efficient for the detection of fish pasteurellosis in mixed-plate cultures and are currently being assayed with tissues from different fish species and in environmental samples.

Characterization of Streptococcus iniae from Cultured Fish by Pulsed-Field Gel Electrophoresis

CA Shoemaker and PH Klesius

USDA-ARS Fish Diseases and Parasites Research Laboratory, PO Box 0952, Auburn, AL 36831 USA cshoemak@acesag.auburn.edu; klesiph@vetmed.auburn.edu

Streptococcal disease results in severe losses in intensively cultured fish throughout the world. Streptococcus iniae, a Gram-positive facultative anaerobic bacterium that infects tilapia and hybrid-striped bass, has recently gained attention because of its apparent association with human infection. Human infections were reported to be a result of handling live fish and/or were associated with skin wounds. Researchers suggest a clone of S. iniae identified by Pulsed-field gel electrophoresis (PFGE) exists which is responsible for invasive infection in humans. We obtained isolates of S. iniae from distinct geographic regions of the United States. All isolates were similar by biochemical analysis. PFGE analysis using standard methods and the restriction enzymes ApaI and SmaI was performed to determine if differences in DNA profiles of the isolates existed. We found DNA profiles were different between regions. Results of PFGE suggest that the human isolate and some fish isolates tested may be closely related or possibly related (i.e., genetic changes resulting in two to three or four to six band differences, respectively).

SESSION 2. EMERGING DISEASES I

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Overview of Emerging Diseases in Coral Reef Ecosystems of the World

EC Peters (1)* and DL Santavy (2)

1	Tetra Tech, Inc., 10306 Eaton Place, Suite 340, Fairfax, VA, 22030 USA peteres@tetratech-ffx.com
2	US Environmental Protection Agency, Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze, FL 32561 USA. santavy.debbie@epamail.epa.gov

Destruction and death of living coral tissue has been studied for several decades, but the nature and role of diseases in the ecology of tropical coral reefs are far from being understood. Biotic and abiotic diseases of corals and other reef organisms, recognized since the 1970s, threaten the biodiversity of these ecosystems in many localities around the world. The black-band and red-band diseases of corals result from infections of microbial mats with cyanobacteria as the key component. Several diseases of corals (white-band disease, white plague, and white pox) are characterized by the sudden disappearance of tissue from the skeleton in specific patterns on different species, with no grossly observable cause. More recently, yellow-blotch and yellow-band diseases are causing coral tissue loss in the western tropical Atlantic and Arabian Gulf, respectively, and coralline algae, which also contribute to the structure and function of these ecosystems, are succumbing to mysterious maladies. Progress is being made in identifying pathogens and other factors contributing to diseases of ecologically valuable reef organisms. Additional vigilance is required to bring reports of morbidity and mortality promptly to the attention of researchers, so that multidisciplinary studies can begin before information is lost. One example in which this did not happen is that of the mass mortalities of long-spined sea urchins, which occurred throughout the Caribbean in the most extensive epizootic of a marine invertebrate. The cause has never been determined.

Brucellosis in Harbor Seals (Phoca vitulina richardsii) and Sea Lions (Zalophus californianus) from Puget Sound, Washington, USA: Evidence of Transmission Through Infected Parafilaroides sp. Lungworms

MM Garner (1)*, DM Lambourn (2), SJ Jeffries (2), PB Hall (2), JC Rhyan (3), DR Ewalt (4), LM Polzin (5), NF Cheville (6)

1	Northwest ZooPath, 18210 Waverly Drive, Snohomish, Washington 98296-4815 USA zoopath@aol.com
2	The Washington Department of Fish and Wildlife, 600 Capitol Way N., Olympia, Washington 98501-1091 USA LAMBODML@dfw.wa.gov, jeffrsjj@dfw.wa.gov
3	National Wildlife Research Center, 1716 Heath Parkway, Fort Collins, Colorado 80524 USA jack.c.rhyan@aphisnotes.usda.gov
4	National Veterinary services Laboratory, PO Box 844, Ames, Iowa 5001o USA. dewalt@aphis.usda.gov
5	Washington Department of Agriculture, 3939 Cleveland Avenue, S.E., Olympia, Washington 98301 USA
6	Department of Veterinary Pathology, Iowa State University, Ames, Iowa 50011 USA nchevill@iastate.edu

The Washington Department of Fish and Wildlife and National Marine Mammal Laboratory have been conducting annual blood testing on Pacific harbor seals on Gertrude Island and California sea lions at Shilshole Bay in Puget Sound, WA. Forty-eight of 200 harbor seals (12%) and 4 of 84 California seal lions (5%) had antibodies to Brucella.abortus by the Brucella card test, Brucella buffered plate hemagglutination test, Rivanol test and complement fixation procedure. The National Veterinary Services Laboratory (NVSL) isolated a Brucella sp. from 4 seropositive stranded harbor seals. This isolate is similar to a strain isolated from a seal in the United Kingdom. Necropsy of most affected seals revealed emaciation and lungworm infection. Histopathology revealed severe verminous pneumonia with intralesional Parafilaroides sp. lungworms. In Giemsa-stained sections, large numbers of minute bacterial coccobacilli were detected along the inner membrane of the uterus and within the gut lumen of some of the Parafilaroides sp. adult worms. Immunohistochemistry using a Brucella abortus antibody revealed large quantities of antigen within the gut lumen and/or uterus of several of the Parafilaroides sp., corresponding to areas where bacteria were detected within the worms. Labeled Brucella antigen was also detected within the cytoplasm of leukocytes in the surrounding pulmonary parenchyma. Bacteria within the uterus and gut lumen of the worms had ultrastructural morphology typical of Brucella sp. The significance of Brucella sp. infection to the lungworm and to the seal is unknown. Because seals frequent habitats shared by terrestrial mammals, and because seals are occasionally used for human consumption, there may be some risk of transmission to terrestrial wildlife, domestic livestock and humans.

Cardiomyopathy Syndrome in Scotland: Investigations into a Disease of Concern

HD Rodger (1)*, T Turnbull (2) and M Bonniwell (3)

1	Veterinary Diagnostic Service, Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, UK hdmr1@stir.ac.uk
2	Hydro Seafood GSP, South Shian, Connell, Argyll, PA37 1SB, UK
3	The Veterinary Surgery, Tweeddale Street, Oban, Argyll, PA34 5DD, UK

In 1997 and 1998 a disease condition emerged in two Atlantic salmon (Salmo salar) farms in Scotland which resulted in significant mortalities in harvest-sized fish. Clinically the moribund fish appeared lethargic, exhibited marked exophthalmos, oedematous skin, ventral congestion and internally had ascites, an increase in pericardial fluid and, in some fish, congested livers. Enlarged cardiac atria or ventricles were also observed. Recent mortalities presented with haemopericardium and ruptured atria. Histopathology revealed haemorrhage, necrosis and myodegeneration in the ventricles and atria of the hearts, some of which had small thrombi and others focal endocardial cell proliferation and fibrosis. Skin sections had oedematous changes in the dermis with focal areas of haemorrhage and inflammatory cell infiltration and on some fish the livers had multifocal areas of haemorrhage and necrosis. Blood biochemistry confirmed the extensive cardiomyopathies with greatly raised creatine phosphokinase levels. Immunohistochemistry conducted using antisera to the seabass nodavirus revealed a negative reaction and attempts to isolate a causal agent on various cell lines, including the striped snakehead line (SSN-1), were not successful. Electronmicroscopy of sections of cardiac tissue was also conducted but no virus or virus-like particles were observed.

The Epidemiology and Dynamics of Shell Disease in the Edible Crab, Cancer pagurus (L).

CL Vogan*, PJ Llewellyn and AF Rowley.

Biomedical and Physiological Research Group, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea, Wales, UK. SA2 8PP.

The incidence and severity of shell disease in the edible crab, Cancer pagurus, was investigated in animals collected from Langland Bay, U.K. at monthly intervals from 1997-1998. Shell disease is the progressive degradation of exoskeletal chitin accompanied by melanisation of the region. Over 50% of the crabs sampled had one or more black-spot lesions. The proportion of exoskeleton affected increased with crab age. For both sexes, the dorsal carapace was the area most affected. Examination of the dorsal carapace revealed that the areas most commonly affected were located to the posterior of the animal. Occasionally, lesions were observed on the internal surface of the carapace that showed no corresponding external manifestation, suggesting that the disease may be retained through moult. Male crabs showed significantly higher levels of the disease than females, a higher incidence of black-spot lesions (63% in males, 40% in females) and a higher mean percentage of body covered by lesions (1% in males, 0.2% in females). This difference between the sexes corresponded to an increase in ventral carapace and chelae infection in males. There was no difference, however, in the total and differential haemocyte counts and haemocyte-derived antibacterial activity between diseased and control animals. Chitinoclastic bacteria were isolated from both healthy and infected regions of the exoskeleton. One such Gram negative isolate, when injected into the haemocoel, caused mortality within an hour. The animals cleared the bacteria and mortality resulted from exotoxin damage. The nature and dynamics of exotoxin generation by this isolate will be discussed.

Tumors of Gray Snapper (Mangrove Snapper) in the Florida Keys (Dry Tortugas to Biscayne Bay)

RM Werner (1)*, GR Stanton (2), MC Schmale (3) and J Bernstein (4)

1	Laboratory Animal Resources, Florida State University, 101 Biomedical Research Facility, Tallahassee FL 32306-4341. rwerner@mailer.fsu.edu
2	Academic Diving Program, Florida State University, 36 Montgomery Bldg.., Tallahassee FL, 32306-2310,USA. gstanton@mailer.fsu.edu
3	Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Cswy., Miami, FL 33149, USA. mschmale@rsmas.miami.edu
4	Dept of Envir. Protection, Aquatic Health Program, FMRI, 100 8th Ave, SE, St Petersburg, FL 33701-5095 BERNSTEIN_JW@epic66.dep.state.fl.us

Subcutaneous tumors, primarily single but occasionally multiple, have been tentatively diagnosed as neurofibroma in Gray Snapper (Mangrove Snapper) Lutjanus griseus, from Florida waters, however the distribution of the disease has not been described and reports of prevalence have not been supported by observational data. Data on the prevalence of tumors with the characteristic appearance of neurofibroma were collected at sites from the Dry Tortugas to Triumph Reef off Biscayne Bay by divers using chance encounter, direct count technique. During the summer of 1997, 27,709 fish were observed at 61 sites. Prevalence of tumors in populations of fish ranged from 0 to 14.3% and populations of fish with tumors were found throughout the range studied with an overall prevalence of 0.9%. The percentage of populations affected with tumors was higher in the Dry Tortugas than the more northeasterly sections of the Florida Keys. A moat surrounding Fort Jefferson in the Dry Tortugas harboring a semi-isolated population of Gray Snapper was monitored every six months from August 1995 to August of 1997. The tumor prevalence at this site ranged from 0 to 4.9% over the 3-year study. Tagging studies in the moat site resulted in relocation of 20% of tagged subjects after 4 months and 5% after 18 months.

An Outbreak of Spondylolisthesis (Vertebral Dorsal Osteochondrosis) in an Alligator, Caiman croccodilus, Farm in a Closed Environment

V Bermùdez, I Pineda, J Moreno, M Rossini and G Garcìa

FCV-UCV, Cat. Clin. Bàsuca, El Limòn, Marncay, AP-4563, Edo, Aragua, Venezuela, South America

An outbreak of spondylolisthesis (overridding of the 6th and 7th dorsal vertebra) was observed in alligators, Caiman croccodilus, between 4 and 6 months of age, kept in a closed environment fashion since hatching was observed. The lesions gave a "hump" appearance to the alligators with deformity of the vertebral facettes and body chondrodystrophy and displacing the long axis of the dorsal vertebrae, causing wallerian degeneration of the white matter of the spinal cord with terminal down alligator syndrome or ataxia. This condition has appeared in this farm twice two years apart. The diet in this farm is based on concentrate comercial feed, and ground chicken wings and feet as only 4% of the total feed prepared daily for n population of 6,000 gators distributed in four sealed tanks. The outbreak came about in 10% (n=600) of the total population when the concentrated feed temporarily disappeared from the market and chicken parts were unconsultatedly increased to 20%. A second crisis resulted from another crop of gators about the same age in 20% (n=1400) out of 7,000 gators that were exclusively fed on donkey meat because of no concentrate feed in the market. Liver, kidneys and femur bone homogenates and serum fractions from normal and humped gators were studied by mass spectometry, finding Ca:P ratio significantly elevated in humped versus normal gators in the two different situations. This is the first report of this pathology in gators. Spondylolisthesis is a common feature in locomotor abnormality in broiler chickens associated with changes in Ca:P ratio and condrodystrophies. The present situation deserves attention when alligator farms managed in intensive fashion suffer from extreme changes in the diet.

Leukemia in Salmon Culture in Chile

VR Enriquez* (1), M Monrás (1), JP Núñez (1), M Soto (1), D Suárez (2) and VL Cubillos (2)

1	Ictiopatologia, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia. renrique@valdivia.uca.uach.cl.
2	Anatoma Patologica, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia. 20 (Fondecyt 1960501)

A new emerging disease of salmonid fish was detected during the period of 1993-1994 in Southern Chile. The mortality associated with this pathology was 1.3% per week. Based on the histopathological findings, it was termed "Lymphoblastic Leukaemia". Diseased fish showed proliferation and infiltration of kidney mainly by early stages of lymphoblastic blood cells. Lately, other organs have shown to be involved, producing a Leucosis - like condition. Fish showed intense pale gills, exophthalmia and severe increase in size of the internal organs. Electron microscopy showed the presence of retrovirus-like particles, and different stages of the intranuclear parasite Nucleospora salmonis confirm their similarity with the Plasmocytoid Leukaemia affecting Chinook salmon in British Columbia and Washington and California State. During 1995-1996, the disease was detected in 3.09% of the samples tested: 2.18% in the autumn/winter and 3.7% in the spring/summer period. The distribution of the disease was 2.18%, 4.1% and 3.6% in Atlantic salmon, Coho salmon and rainbow trout, respectively. Nucleospora salmonis was detected in 2.2% of the fish tested, but only 20% of the histologically positive fish showed the parasite in the tissues. Some of fish were also positive for piscirickettsiosis (46.7%) and BKD (26.7%). The disease was only detected in the 10th region of the country.

SESSION 3. IMMUNOLOGY I: Salmonids

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Stimulation of Rainbow Trout Immune Cells with Oligodeoxynucleotides Bearing CpG Motifs

J Heppell (1)*, J Sanchez-Dardon (1), AM Krieg (2) and HL Davis (1)

1	Loeb Research Institute, Ottawa Civic Hospital, 725 Parkdale Avenue, Ottawa. Ontario, K1Y 4E9, Canada. jheppell@civich.ottawa.on.ca; jsanchez@lri.ca; hdavis@lri.ca
2	2 University of Iowa, Rheumatology Division, 200 Hawkins Drive, 540G EMRB, Iowa City, Iowa 52242-1081, USA arthur-krieg@uiowa.edu

Bacterial DNA can stimulate the humoral and cellular immune system of > mammals. This was shown to be due to unmethylated CpG dinucleotides in a specific context (CpG motifs). Thus, oligodeoxynucleotides (ODN) bearing such motifs can be used as strong adjuvants to regulate and/or increase the immune response of animals to a specific protein. In fish, activation of immune cells with CpG motifs has never been shown. As a first attempt to demonstrate the stimulatory effect of these short DNA sequences in trout, we have tested a panel of 12 ODNs. Leukocytes isolated from rainbow trout (15 cm average size) blood or head kidney were stimulated in vitro for 24 hours with ODNs, then the phagocytic, oxidative burst and NK activity was determined. Some ODNs significantly increased the activity of leukocytes, when compared to that of control cells which were not incubated with ODNs. The CTL activity was also determined and was shown to be higher with lymphocytes activated for 3 days with some ODNs. These results show that CpG motifs can stimulate rainbow trout immune cells in vitro. We are now testing immunostimulatory ODNs in vivo, to determine if they can increase the nonspecific immune response in fish, as is the case for mammals.

The Type I Interferon Response in Salmonids

MC Johnson*, CH Kim, K Suzuki and JC Leong

Department of Microbiology, Oregon State University, Corvallis OR 97331
johnsmar@bcc.orst.edu, kimc@bcc.orst.edu, leongj@bcc.orst.edu.

Type I interferons are viral-induced cytokines which upregulate the production of several anti-viral proteins. It has been shown in mice that deletion of the type I interferon receptor results in an acute susceptibility to viral infection. While interferon is also believed to be important in the anti-viral defenses of fish, no interferon has been cloned from a fish and little is known about the genes which are induced by type I interferons in fish. In our laboratory we have cloned and characterized several genes involved in the type I interferon response including interferon regulatory factor (IRF-1), signal transducers and activators of transcription 1 (STAT 1), and Mx. In addition, we have cloned the promoters for IRF-1 and Mx. Work with these genes has led to several conclusions about type I interferon signaling in fish. First, the promoter of the interferon-inducible Mx gene is upregulated in chinook salmon embryo (CHSE) cells by exogenous interferon, but not by double-stranded RNA, a potent inducer of interferon. Second, the Mx promoter contains a conserved interferon-stimulated response element (ISRE) which is responsible for promoter induction. Third, transfection of CHSE cells with IRF-1 results in upregulation of interferon inducible genes. Finally, the IRF-1 promoter is induced by double-stranded RNA in epithelial carp cells (EPC) but not in (CHSE) cells and contains a conserved gamma activated sequence motif (GAS). Together these data indicate that interferon signaling in fish is similar to that found in mammals.

Trout IgM Assembly Processes Yield Multiple Structural Forms

SL Kaattari (1)*, DA Evans (2) and JV Klemer (1)

(1) Dept. of Environmental Sciences and (2) Physical Sciences, School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062 USA. kaattari@vims.edu; david@vims.edu; jklemer@vims.edu

An essential quality control mechanism of human and murine plasma cell ER is their ability to retain and direct the degradation of partially disulfide-linked IgM molecules. However, trout IgM assembly normally produces varied degrees of disulfide crosslinking among the monomeric subunits. The inter-monomeric bonds of secreted and intracellular trout Ig were examined using non-reductive, denaturing composite gel electrophoresis. Secreted Ig was found to be composed of either completely crosslinked tetramers or non-covalent tetrameric associations of crosslinked monomers, dimers, and trimers. Isoelectric focusing revealed that each clonal antibody product displays the same heterogeneity as is observed for whole serum Ig. The molar ratios of these constituent forms were also found to be identical in all individuals, suggesting a relatively stringent assembly process that could be mathematically modeled. The ratios of the various disulfide-bonded isomers predicted by these models were compared to the ratios observed in vivo. The data were found to be most consistent with a random process of disulfide bond formation between monomeric subunits which is terminated either immediately prior to, or during the secretory process. Intracellular Ig was primarily composed of covalent monomers and dimers, and not the more mature, covalent trimers or fully cross-linked tetramers, thus corroborating this model. Examination of the C-terminal amino acid sequences from a variety of mammals and fish species exhibiting this property strongly suggests that heterogeneity of disulfide crosslinking is determined by a common amino acid motif.

Analysis of Anti-IHNV Antibodies from Thymectomized and Sham Thymectomized Rainbow Trout Using Rotofor Isoelectric Focusing

JV Klemer (1)*, SE LaPatra (2) and SL Kaattari (1)

1	Virginia Institute of Marine Science, Department of Environmental Science, School of Marine Science, College of William and Mary, Gloucester Point, VA 23062, USA. jklemer@vims.edu; kaattari@vims.edu
2	Clear Springs Foods, Inc., P.O. Box 712, Buhl, ID 83316, USA. scottl@clearsprings.com

Rainbow trout antibodies specific for infectious hematopoietic necrosis virus (IHNV) have been characterized by isoelectric focusing using the Bio-Rad Rotofor isoelectric focusing system. This apparatus allows isoelectric focusing of intact, functional antibodies in a liquid phase. This minimizes manipulation of the sample; facilitating the antibody's use in other assays. Juvenile rainbow trout were either thymectomized by cautery or sham thymectomized. Both groups were later challenged with IHNV and the serum was periodically examined for the presence of anti-IHNV antibodies. These antibodies were characterized by isoelectric focusing and subsequent ELISA analysis. The serum antibody of the two groups showed distinct differences in the isoelectric profiles of anti-IHNV antibodies. Thymectomized trout serum antibodies demonstrated restricted antibody profiles compared to the profiles of the sham-thymectomized group. This analysis indicates that the thymus may play a critical role in the development of a broad, fully developed antibody repertoire to an infectious agent.

Analysis of CDR2 Regions of Antigen-Specific Trout Antibody Genes

TD Lewis* and SL Kaattari

Department of Environmental Sciences, School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062 USA. tdlewis@vims.edu; kaattari@vims.edu

The specificity of an antibody response to a pathogen ultimately determines the effectiveness of this response. This specificity may be fine-tuned on the molecular level to facilitate improved antigen-antibody interaction. These changes occur predominantly in the complementarity-determining regions (CDRs) of the genes encoding the variable regions of the heavy (VH) and light (VL) chains of the antibody molecule. Historically, hybridoma technology has allowed for the investigation of this process with exquisite detail in mammalian systems, however, the development of a suitable substitute for use with piscine systems has impeded comparable investigations. The development of new panning techniques provides the means to study this process in fish. Rainbow trout were immunized with purified glycoprotein of infectious hematopoietic necrosis virus (IHNV gp) or trinitrophenylated-keyhole limpet hemacyanin (TNP-KLH) to initiate a specific antibody response. Blood samples were collected at weeks 0, 5, and 10 and analysis of specific antibody titers and affinity were performed by ELISA. Specific B lymphocytes were panned using antigen-conjugated magnetic beads, cultured in the presence of lipopolysaccharide (LPS) to expand cell numbers, and the mRNA was isolated. RT-PCR (reverse transcriptase PCR) and 5U RACE (rapid amplification of cDNA ends) were performed to amplify the antigen specific CDR2 of Ig heavy chain variable regions, and the resulting PCR products cloned. Sequence analysis of the various clones and comparison to germline sequences can provide insight into the molecular processes which occur during the maturation of a specific immune response in rainbow trout.

Effect of VYS-2 and Glucan on Immune Response of Rainbow Trout (Oncorhyncus mykiss) Immunosuppressed with T-2 Toxin

NV Guseva (1) and LA Ford (2)*

1	All-Russian Research Institute of Freshwater Fisheries (ARRIFF), Rybnoe, Dmitrov region, Moscow province, 141 821, Russia, root.dmtrv@rex.iasnet.ru
2	Department of Fisheries & Wildlife Resources, CFWR, University of Idaho, Moscow, ID, 83844-1136 USA, lford@uidaho.edu

Yeast glucan in a dosage of 0.6 mg/fish or vaccine against Motile Aeromonads Septicemia ­ VYS-2 (ARRIFF, Russia) in a dosage of 50 mg/fish were used as immunostimulators for rainbow trout. T-2 toxin was used as an immunosuppressor and fish were exposed to the toxin 2 days after fish were injected with the respective immunostimulator. A dose of 0.325 mg/kg of fish body weight (1/20 of LD50) was either injected intrapertioneally (i.p.) into fish or fish were fed the toxin in a dose of 0.325 mg/ fish/day in their diet, for a period of 36 days. Blood, serum, spleen, skin, head kidney and last gut segment were sampled on days 4, 7, 14, 21 and 36 after first exposure to T-2 toxin. Erythrocytes and leukocyte counts, differential white blood cells counts, total serum proteins, hematocrits, phagocytosis assays with spleen cells, and histology of skin, head kidney and last gut segment were performed. Results of the study showed that injection of T-2 toxin induced a decrease in all parameters tested during the first 14 days after exposure. Using T-2 toxin as a feed additive induced the same changes, but was compounded by the accumulation of T-2 toxin in the fish. In fish injected with glucan, the decreases in tested parameters were not as strongly expressed as in the control groups, and fish recovered shortly after injection with T-2 toxin. In the vaccinated groups, changes in the tested parameters were statistically less than those noted in the fish that received glucan.

SESSION 4. PARASITOLOGY I: Myxozoa

[TOP]

Application of Ribosomal DNA Sequences to the Study of Myxosporean Parasites of the Genus Myxobolus

KB Andree (1)*, C Székely (2), K Molnár (2), M El-Matbouli (3), TS McDowell (1) and RP Hedrick (1)

1	Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616 U.S.A
2	Veterinary Medical Research Institute, Hungarian Academy of Sciences, 1143, Budapest, Hungaria Krt. 21, Hungary
3	University of Munich, Institute of Zoology, Fish Biology and Fish Diseases, Munich, Germany 80539

Ribosomal DNA (rDNA) sequences were used to study the life cycle, epidemiology, phylogeny and for improvement of diagnostic approaches of Myxobolus cerebralis, the myxozoan parasite causing salmonid whirling disease. Small subunit ribosomal RNA (SSU rRNA) gene sequences from the actinosporean and myxosporean stages of the parasite from a single source were 99.8% identical. SSU and ITS-1 rRNA sequences enabled us to compare different geographic isolates of the parasite from N. America and Europe. The SSU rDNA sequence was also used to develop a highly specific and highly sensitive PCR diagnostic test for M. cerebralis. Lastly, SSU rDNA of M. cerebralis was compared to SSU rDNA sequences we obtained from 10 Myxobolus spp. from salmonid and nonsalmonid fish from N. America and Europe. These comparisons, representing ~1700 bp of the 18S rRNA gene, grouped them into three clusters that showed little correlation with spore morphology and size or host specificity, criteria currently used for both higher and lower taxonomic placements in the Myxozoa. Spores of similar size and shape (M. cerebralis vs. M. squamalis) were distantly related in some instances, while spores with divergent morphology and size were sometimes found to be closely related (M. cerebralis and M. insidiosus). These initial investigations into the phylogenetic relationships of putative members of the genus Myxobolus clearly indicate the potential limitations of groupings based on size and morphological properties of the spores and host species infected. We propose that 18S rRNA gene sequences, combined with standard myxosporean taxonomic criteria (when and if known), be given greater consideration in taxonomic placements of myxosporeans.

Whirling Disease: Detection of Early Developmental Stages of Myxobolus cerebralis in Fish and Oligochaete Hosts by In Situ Hybridization

DB Antonio(1)*, M El-Matbouli(2), TS McDowell (1) and RP Hedrick (1)

1	School of Veterinary Medicine, Department of Medicine and Epidemiology, University of California, Davis, CA 95616 USA. dbantonio@ucdavis.edu; tsmcdowell@ucdavis.edu; rphedrick@ucdavis.edu
2	University of Munich, Institute of Zoology, Fish Biology and Fish Diseases, Munich, Germany 80539 melmatbouli@ucdavis.edu

Myxobolus cerebralis, the causative organism of whirling disease, has become a major worldwide pathogen among farmed salmonids. A critical impact of this myxosporean parasite has been recently demonstrated as contributing to the decline of wild trout populations in the U.S. The infective forms of the parasite, the myxosporean and actinosporean spores, develop through many stages in the two hosts, the salmonid fish and the tubificid oligochaete, respectively. The radically different structural forms of the parasite throughout its development compelled us to develop a DNA-based test, the in situ hybridization, as useful research and diagnostic tool to detect the exact location of M. cerebralis in each of the two hosts. Using a modified nonradioactive in situ hybridization (ISH) protocol, early, developmental and sporogonic stages of the parasite could be detected in paraffin-embedded fixed tissues of both fish and oligochaetes. In fish, earliest forms of the parasite penetrate the epidermis, then into the peripheral nerve and finally cartilage where the parasite sporulates. A unique application of the ISH procedure is its ability to detect and localize forms of the parasite in its oligochaete host. These developmental stages cannot be identified by any existing extraction/concentration procedure used for the spore stages in fish. Developmental stages of the parasite can be seen sequentially through their development in oligochaete gut epithelium. This new DNA-based method provides tool to further study the factors that influence the impact of whirling disease on cultured and feral trout populations.

Laboratory Experiments on Some Exogenous Factors Influencing the Development of the Triactinomyxon Stages of Myxobolus cerebralis, the Causative Agent of Whirling Disease

M El-Matbouli (1)*, TS McDowell and RP Hedrick

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis CA 95616, USA
(1) Address after September 1998: Institute of Zoology, Fish Biology and Fish Diseases, University of Munich, Germany

Whirling disease caused by the triactinomyxon stage of Myxobolus cerebralis has become the most widely known disease of salmonids in the 1990s. The parasite has a complex, two-host life cycle, beginning when the spores of M. cerebralis are released or reach the aquatic environment. This occurs only when infected fish die and autolyse, or when they are consumed and excreted by predators. In both cases, M. cerebralis released into the water can be ingested by the oligochaete T. tubifex that then hatches in the gut lumen releasing a binucleated sporoplasm to the gut epithelial cells. Afterwards they develop interepithelially in about 3 months through four phases (schizogony, gametogony, gametogamy, and sporogony) into the actinosporean triactinomyxon, which are the only stages infectious for salmonid fish. In this study the influences of light and different temperatures (5, 10, 15, 20, 25 and 30 C) on the development of M. cerebralis spores to the triactinomyxon stages in the oligochaete host has been studied. The result indicates that 15C is the optimal temperature for regular production of triactinomyxon spores in infected T. tubifex. A lower temperature seems to slow down the development and maturation of the pansporocysts and triactinomyxon spores. It could also be shown that temperatures between 5 and 10C extend the period of spore production. The possibility of reinfection of T. tubifex with M. cerebralis spores will be presented and discussed. Also the relationship between temperature and longevity in respect to infectivity of the waterborne triactinomyxon stages of M. cerebralis have been studied. The triactinomyxon spores survive more than 15 days at a water temperature of up to 20C. Therefore, it can be concluded that the triactinomyxon spores in ponds and streams can remain virulent for more than 15 days.

Development of Molecular Markers to Identify Susceptible Oligochaete Hosts for Myxobolus cerebralis

KA Beauchamp (1)*, TS McDowell (1), RD Kathman (2), C Rasmussen (3), A Colwell (3), JR Winton (3) and RP Hedrick (1)

1	Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616 U.S.A. kabeauchamp@ucdavis.edu
2	Aquatic Resources Center, 4410 Peytonville Rd., Franklin TN 37064 U.S.A
3	Western Fisheries Research Center, Biological Resources Division, United States Geological Survey, Seattle, WA 98115 U.S.A.

Tubifex tubifex serves as the alternate host for Myxobolus cerebralis, the causative agent of whirling disease in salmonid fish. Accurate identifications of T. tubifex using solely morphological criteria may not be adequate, yet distinguishing the true host for M. cerebralis from related oligochaetes is critical to understanding and controlling whirling disease. Molecular markers used in concert with morphological criteria may provide the level of distinction needed. Molecular genetic approaches provide data on evolution and phylogenetics including quantitative estimates of genetic relatedness among species and the order of divergence among groups of species. We are exploring the use of a variety of genetic markers that reflect different forms of genetic variation, and thus may respond differently to the processes that lead to genetic divergence between populations of tubificid worms. Conserved nuclear ribosomal 18S genes separated morphologically identified tubificid worms from other families of oligochaetes while more rapidly evolving mitochondrial 16S genes differentiated between Tubifex spp. The use of RAPDs has already begun to demonstrate variability among potential strains of T. tubifex. The development of markers from more variable sequences of the nuclear ITS rDNA, mitochondrial protein coding genes and hypervariable microsatellites should further identify ecological races, strains and subspecies of tubificid worms. Once developed these markers will provide definitive identifications of worms serving as hosts of M. cerebralis and other myxosporean parasites. This will aid in determining the distribution, genetic population structure and systematics of the hosts for these important parasites.

Host Specificity of Myxobolus cerebralis Spores and Their Triactinomyxon Stage, the Causative Agent of Whirling Disease

M El-Matbouli (1)*, TS McDowell and RP Hedrick

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis CA 95616, USA
(1) Address after September 1999: Institute of Zoology, Fish Biology and Fish Diseases, University of Munich, Germany

Whirling disease (WD) is an economically devastating parasitic disease of salmonid fish, especially rainbow trout (Onchorhynchus mykiss) caused by the triactinomyxon stage of Myxobolus cerebralis. It is typically a chronic disease that can cause high moralities in fry and fingerlings of hatchery-reared and recently also, feral rainbow trout. A potential negative impact of WD on wild trout populations, such as the disappearance of young wild rainbow trout and population decline, has been reported. Investigations on the interaction between M. cerebralis and oligochaete worms other than Tubifex tubifex demonstrate that the spores hatch in the gut lumen of these worms. However the released sporoplasm of M. cerebralis failed to develop further in the investigated oligochaete worms compared to that in the susceptible T. tubifex. Similar investigation on the interaction between the triactinomyxon stage of M. cerebralis and non-salmonid fish were conducted. Exposure experiments of the triactinomyxon spores to non-salmonid fish such as goldfish (Carassius auratus), carp (Cyprinus carpio), nose (Condrostoma nasus), medaka (Oryzias latipes), guppy (Poecilia reticulata) and also the amphibian tadpole (Rana pipiens) as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucous prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results of both investigations and the possibility to develop a biological control method to inactivate stages, M. cerebralis spores and triactinomyxon spores, from enzootic aquatic environments will be discussed.

The Case for Naming Actinosporeans

RJG Lester (1)*, SL Hallett (1) and M El-Matbouli (2)

1	Department of Parasitology, The University of Queensland, Brisbane, Australia 4072 Lester@mailbox.uq.edu.au
2	Institute of Zoology, Fish Biology and Fish Diseases, University of Munich, Kaulbachstr. 37, 80539 Munich, Germany elmatbouli@irz.uni-muenchen.de

The discovery that some myxosporeans develop through an actinosporean stage has led to the suppression of the Class Actinosporea in favour of the Class Myxosporea. It has been further proposed that names of existing actinosporean genera be declared invalid, existing species be considered species inquirendae and that a new system for referring to actinosporeans be adopted. This may be premature as it is far from certain that all species of actinosporeans (which are found in invertebrates) develop into a myxosporean (in a vertebrate). Five myxosporean species (with unknown life cycles) are reported from invertebrates, and one species from a vertebrate has a life cycle that is apparently direct, vertebrate to vertebrate, demonstrating that the myxosporean life cycle may not be as clear cut as proposed. Probably less than half of the world's myxosporeans have been described and of these the life cycle is known for < 1%. Our recent rDNA sequences from 4 marine actinosporeans do not show clear affiliation with sequences from known Myxosporea. We suggest caution in naming Actinosporea but if there is no clear link to a recognised myxosporean species, it may be best to follow the International Code of Zoological Nomenclature until the Commission makes a formal ruling to the contrary.

Studies on Whirling Disease Prevention and Control

J Lifan and Z Wanghua

Freshwater Fisheries Research Centre, Chinese Academy of Fisheries Sciences, Wuxi 2214081 China

As far back as the beginning of the 19th century, whirling disease was recorded in Central Europe. It was then gradually introduced to much of Europe, portions of North America, Africa and Asia. In the United States, this disease was detected in several states (Pennsylvania and Nevada) in 1955, 18 states in 1992, then 21 states in 1997, and it is still spreading. It is found both in artificial culture and in the wild. R.B. Nehring et al. have researched this disease in 11 sections of the Colorado River with a detection rate of 50-100%. The number of parasitic spores in individual trout ranged from 50,000 to 125,000, averaging 84,400. At the Rome hatchery, 570,000 trout were infected resulting in mortality in 1995. Trout and salmon have suffered irreparable declines in population.
Whirling disease is a parasitic infection of cold water trout and salmon caused by the myxosporean protozoan Myxobolus cerebralis (Myxosoma cerebralis). It selectively buries deep into the cartilage of the brainpan and has a strong spore. Histological observation shows that it is specialized from plasma and its fine structure prohibits the invasion of toxin. This is the main reason why general drugs, such as furazolidone, furoxone, benomyl, fumagillin, proguamil, clamoxyquin, aureomycin, allicin and atrabrine, cannot kill them. The scientists at University of California-Davis developed a special DNA- based test as a detection for whirling disease in 1997. So far, there is still no chemical drug that can treat infected fish. A series of chinese herbs went through surveys and screenings by combining the theory of traditional herb medicine with modern technology.
It has been proven that prevention and control of Myxosporidiasis cyprini, M. dermatobia and other spores in fish is very difficult. So that we consider that may control same family whirling disease, Myxobolus cerebralis in trout and salmon.

Laboratory and Field Trials on the Efficacy of Fumagillan to Control Whirling Disease

M El-Matbouli* and RW Hoffmann

Institute of Zoology, Fish Biology and Fish Diseases, University of Munich, Germany
* Current Address until September 1999: Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis CA 95616, USA

Myxosporean infections have been considered non-treatable diseases for a long time, and control is primarily restricted to avoidance and prophylaxis. Recent experimental studies have shown the potential use of the relatively long known antibiotic, Furnagillin against several myxosporeans. To examine the efficiency of Fumagillin against whirling diesease, the antibiotic was applied orally to experimentally infected rainbow trout fry in three concentrations (0.25, 0.5 and 1.00 g Fumagillin/kg feed) for a period between two weeks and four months. The best effect was found at a concentration of 0.5g Fumagillin/kg feed, used immediately after presumed infection for two weeks. In a field study from 1992-1996, we tested Fumagillin at 0.5g per kg diet in three trout farms in Bavaria enzootic for whirling disease. In these farms, each with 40,000 rainbow trout fry, Fumagillin was applied at 0.5g per kg diet for two weeks immediately after transfer of trout from breeding station to the ponds. After three years of treatment, only about 1% of the fish in each farm has shown clinical signs of whirling disease.
In light and electron microscopic studies of the samples, considerable alterations in the spore morphology of the parasite were detected after Fumagillin therapy. Totally vacuolated spores have been observed in addition to undeveloped polar capsules and disintegrated polar filaments as well as solitary polar capsules and spore values without any visible content. All Fumagillin concentrations that were tested for longer than two weeks had a toxic effect on the fish.

SESSION 5. PARASITOLOGY II: Perkinsus, Part 1

[TOP]

Identification of Carbohydrates on the Surface of Perkinsus marinus

GD Brown* and M Faisal

Virginia Institute of Marine Science, College of William and Mary, Department of Environmental Sciences, Gloucester Point, Virginia 23062 USA. gbrown@vims.edu; faisal@vims.edu

Perkinsus marinus is the causative agent of Dermo, a serious disease of the eastern oyster, Crassostrea virginica. Since sugar moieties on the surface of many protozoans have been found to contribute in host-parasite recognition, we identified several carbohydrates on the surface membrane of P. marinus using a variety of lectins known to bind specific sugar residues. Perkinsus marinus cells from a cloned isolate, Perkinsus-1, were incubated separately with eight fluorescein labeled lectins. Concanavalin A (specificity for mannose and glucose), wheat germ agglutinin (N-acetylglucosamine) and peanut agglutinin, galactose) all exhibited strong fluorescence whereas soybean agglutinin (N-acetyl galactosamine) and Vivia villosa (N-acetyl galactosamine) showed weak, almost non-specific binding. Lectins specific for alpha-L-fucose (asparagus pea and UEA-I) and sialic acid (Maackia amurensis agglutinin) did not bind to P. marinus cells. The specificity of concanavalin A, wheat germ agglutinin, and peanut agglutinin was tested by inhibition studies employing the carbohydrate specific for the lectin as well as control carbohydrates. These findings suggest the presences of mannose, glucose, galactose and N-acetylglucosamine residues on the surface of P. marinus cells. The role these carbohydrate residues contribute to the pathogenicity of P.marinus is currently being investigated.

Relationships Between Land-Use Patterns and Dermo Disease in Oysters from Two South Carolina Estuaries

D Bushek*, D White, D Porter, B Jones, J Keesee and D Edwards

Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, SC 29208 USA bushek@sc.edu, dwhite@marsh.geol.sc.edu, dporter@hugo.geol.sc.edu, bjones@marsh.geol.sc.edu, jkeesee@belle.baruch.sc.edu, edwards@math.sc.edu

South Carolina estuaries are relatively pristine but many are being rapidly developed. They support extensive intertidal populations of the eastern oyster Crassostrea virginica. Perkinsus marinus which causes Dermo disease in C. virginica is prevalent, but rarely produces widespread epizootic mortalities as described elsewhere (e.g. Chesapeake Bay and the northern Gulf of Mexico). Increased contaminant and nutrient loadings due to human development and encroachment are often regarded as the major factors contributing to declining aquatic animal health. We examined the spatial and temporal patterns of P. marinus in two South Carolina estuaries, one pristine and one undergoing continuing development, to identify potential relationships between development and disease. Using GIS and spatio-analytical tools, continuous spatial distributions of P. marinus were developed from discrete sampling data and integrated with patterns of adjacent land-use, contaminant and nutrient concentrations, and existing geomorphology. Disease intensity followed typical seasonal cycles throughout both estuaries. Relationships between spatial patterns of disease intensity, adjacent land use and hydrology were most discernible during peak infection intensity. In the pristine system, outbreaks were localized and associated with tidal nodes that are likely to be poorly flushed. In the developed system, outbreaks were widespread. Intensities generally increased adjacent to concentrated urbanization, but highest intensities were observed along the primary boating waterway. Results support a correlation between contaminant load and disease, but also indicate the importance of hydrological alterations. A better understanding of the natural ecological processes controlling host-parasite interactions in aquatic environments is needed to design effective regulatory tools that permit human development and protect aquatic animal health.

Acid Phosphatase and Superoxide Dismutase Activities in Different Strains of the Oyster Protozoan Parasite, Perkinsus marinus

F-LE Chu (1)*, S Armknecht (2) and AK Volety (3)

1	Virginia Institute of Marine Science, School of Marine Science, College of William and Mary, Gloucester Point, VA 23062 chu@vims.edu
2	Orbimage Inc., Dulles, Virginia
3	NRC, EPA, Gulf Ecology Division, 1 Sabine Island Dr., Gulf Breeze, FL 32561

The oyster protozoan parasite, Perkinsus marinus, is one of the two important parasites causing severe mortality in the eastern oysters (Crassostrea virginica) on the US East Coast. Our recent study suggests that P. marinus cells and its extracellular products (ECP) could scavenge the reactive oxygen intermediates produced by oyster hemocytes or inhibit their production. The parasites' acid phosphatase (AP), superoxide dismutase (SOD), and other antioxidant enzymes are believed to play a role in scavenging or inhibiting hosts' respiratory burst. Recent studies also suggest that the virulence of P. marinus varies with strains. The extra- and intra-cellular activities of AP, SOD, catalase, and glutathione peroxidase (GP) were examined in six different P. marinus strains/isolates, i.e., Delaware Bay, New Jersey (DB-NJ), Mobjack Bay, Virginia (MB- VA), Barataria Bay, Louisiana (BB-LA), Laguna Madre, Texas (LM-TX), Oxford, Maryland (OX-MD), and York River, Virginia (YR-VA). It was found that no calatase or GP was detected in P. marinus and its ECP. The YR-VA strain has significantly higher extracellular AP activities (units/mg cell protein) than all other strains. Intracellular AP activity was low (<1.0 unit/mg total cell protein) in all strains. LM-TX strain had the greatest intracellular AP activity. The mean SOD activity (ng SOD/mg total cell protein) was higher in the YR-VA strain, but statistically insignificant from the other strains. SOD activity was detected only in the culture media of 97 days old P. marinus culture. Results will be reported and discussed in relation to the virulence of this parasite.

Use of Riboprinting in Discriminating Between Perkinsus Species Isolates from the Chesapeake Bay

GD Brown*, S Kotob and M Faisal

Virginia Institute of Marine Science, The College of William and Mary, Department of Environmental Sciences, Gloucester Point, Virginia 23062 USA. gbrown@vims.edu; kotob@vims.edu; faisal@vims.edu

Perkinsus species are parasitic protozoans infecting molluscs worldwide. Several isolates of Perkinsus originating from bivalves of the Chesapeake Bay have been found to differ morphologically and biochemically. We employed a genetic approach, riboprinting, to determine genotypic diversity, or relatedness, between Perkinsus isolates from the Chesapeake Bay. This technique has been applied recently and successfully to protozoans. Isolates of Perkinsus species were collected from infected oysters and from infected clams in the Chesapeake Bay. The small subunit ribosomal RNA gene (SSUrRNA) and the Internal Transcribed Spacers (ITS) and 5.8S region of the ribosomal gene unit were amplified by polymerase chain reaction (PCR) and cut with restriction enzymes. RFLP analysis of the SSUrRNA gene of clam Perkinsus isolates, H49 and G117, and an isolate collected from an infected oyster, P-1, generated different restriction maps. We are currently utilizing riboprinting to compare the ITS-5.8S region and the SSUrRNA gene unit of eight Perkinsus marinus isolates, including P-1. Our data suggest riboprinting is valuable in determining relatedness between isolates of Perkinsus.

Antibodies to the Protozoan Oyster Pathogen Perkinsus marinus (Apicomplexa) Bind to Some Dinoflagellates (Dynophyceae): Pragmatic and Phylogenetic Implications

CF Dungan (1)*, D Bushek (2) and AJ Lewitus (2)

1	1 Maryland Department of Natural Resources, Cooperative Oxford Laboratory, 904 S. Morris St., Oxford, Maryland USA 21654. cdungan@hatteras.bea.nmfs.gov
2	University of South Carolina, Baruch Marine Field Laboratory, P.O. Box 1630, Georgetown, South Carolina, USA 29442. dbushek@belle.baruch.sc.edu; lewitus@belle.baruch.sc.edu

Destructive impacts on aquatic habitats and living aquatic resources by toxic algae, parasitic dinoflagellates, and pathogenic protozoans in estuarine environments have all been increasingly recognized, yet the taxonomic status of some agents remain controversial, and objective methods for their rapid identification unavailable. Nucleotide sequences from several genes of the apicomplexan protozoan oyster pathogen Perkinsus marinus suggest phylogenetic affinities with dinoflagellate taxa. To test for phenotypic evidence of such affinities and to locate possible shared epitopes, we probed a variety of parasitic, toxic, and autotrophic dinoflagellates with polyclonal rabbit antibodies raised against P. marinus, and analyzed results by fluorescence microscopy. Many, but not all, syndinean dinoflagellate parasites of marine crustacea were labeled, as were several autotrophic and toxic species. Dinoflagellate epitopes apparently recognized by anti-P. marinus IgG included nuclear, cytoplasmic, and cell wall/membrane elements. These results provide serological phenotypic support for recent molecular evidence suggesting genetic affinities between these protistan groups, and provide cautionary information to efforts generating diagnostic antibodies for differentiation of organisms in these groups.

Riboprinting and Sequence Analysis of the Small Subunit Ribosomal RNA Genes of Two Perkinsus spp. Isolates from the Softshell Clam, Mya arenaria

SA Kotob*(1), SM McLaughlin (2), and M Faisal (1)

1	Department of Environmental Sciences, Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, Virginia 23062, USA. Kotob@VIMS.edu; Faisal@VIMS.edu
2	National Marine Fisheries Service, Cooperative Oxford Laboratory, 904 South Morris St. Oxford, Maryland 21654. shawn.mclaughlin@noaa.gov

Sporozoans of the genus Perkinsus have been associated with high mortalities in bivalve molluscs worldwide. The taxonomy and speciation of Perkinsus species, however, remains controversial. Most recently, our laboratory has isolated and cloned two distinct Perkinsus spp. from the gill (G117), and the hemolymph (H49) of the softshell clam, Mya arenaria. This study was undertaken to differentiate between the two isolates and reported Perkinsus species. Riboprinting (PCR/RFLP), molecular cloning, and sequence analysis of the small subunit ribosomal RNA genes were used to characterize G117 and H49. The amplified genes of G117 and H49 isolates were 1,803 and 1,796 nucleotides in size respectively. Restriction fragment length polymorphism (RFLP) analysis of the SSU rRNA genes from these isolates showed genetic variations. Direct nucleotide sequencing confirmed the existence of variations at 47 positions. A sequence similarity of 97.5% was found between the two isolates. Comparison of the SSU rRNA sequence of G117 with the published gene sequence of Perkinsus marinus infecting the eastern oyster (Crasstosrea virginica), and Perkinsus sp. infecting the blood cockle from Anadara trapezia, indicated a sequence diversity of 2.9% and 1.3%, respectively. However, the H49 isolate showed a sequence diversity of 1.8% when compared to Perkinsus sp., and 2.4% when compared to P. marinus. The genetic variations between G117 and H49 suggest that these isolates are probably two distinct species. To our knowledge this is the first case where two Perkinsus spp. have been isolated from one host.

SESSION 6. PROPHYLAXIS / BIOLOGICAL CONTROL / NATURAL TREATMENTS

[TOP]

The Use of Natural Products in the Treatment of EUS (Epizootic Ulcerative Syndrome)

RE Campbell, JH Lilley and RH Richards

Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA UK. rec1@stir.ac.uk

Epizootic ulcerative syndrome (EUS) is a serious disease of wild and farmed tropical freshwater fish which has probably been occuring in Asia for more than twenty years. It is characterised by the presence of invasive Aphanomyces infection and necrotising ulcerative lesions, typically leading to a granulomatous response. It can be of profound economic significance, particularly in poorer regions. As yet there is no vaccine for EUS, nor is there an effective chemical treatment, largely due to lack of knowledge and the widespread occurrence of the disease in the wild population. The disease can be managed to a certain extent by farm level intervention, including exclusion of wild fish, use of saline groundwater to maintain higher salinity, use of resistant species and treatment of ponds with lime. There have been a number of reports of local plant or herbal treatments proving effective against the disease, and these have been investigated using a simple MIC (minimum inhibitory concentration) test, along with a variety of commercial fungicides and other natural products such as propolis, tea tree oil, garlic and a range of plant extracts used in Chinese traditional medicine. Promising results have been obtained from preparations containing tea tree oil and also propolis, and a surfactant initially added to emulsify the tea tree oil demonstrated a synergistic effect in combination with tea tree oil and with other products. The activity of these products against zoospores is under investigation with a view to field testing in Thailand in 1999.

Inhibition of Fish Pathogenic Bacteria by Non-Pathogenic Aquatic Bacteria: Development of a Probiotic Concept.

L Gram (1)*, T Nielsen (2), B Spanggaard (1) and I Huber (3)

1	Danish Institute for Fisheries Research, Department of Seafood Research, Danish Technical University bldg. 221, DK-2800 Lyngby Denmark. gram@dfu.min.dk; bsp@dfu.min.dk
2	BioMar A/S, Mylius Erichsensvej 35, DK-7330 Brande, Denmark. aquavet@post3.tele.dk
3	Biotechnological Institute, Kogle alle 2, DK- Hørsholm, Denmark. ih@bio.atv.dk

The concern about development of bacterial resistance against antibiotics has increased during recent years due to the widespread use of antibiotics and chemotherapy in the aquaculture sector. Therefore alternative, environmentally friendly methods of disease prevention have to be developed. The antagonism of bacteria isolated from water and fish against fish pathogenic bacteria has been evaluated with the purpose of finding bacterial cultures with potential as probiotics in fish farming. Particularly pseudomonads are strong antagonists and our model experiments show that these bacteria inhibit growth of fish pathogenic bacteria like Aeromonas salmonicida and Vibrio anguillarum. This inhibition is seen both in well-diffusion assays and when testing the antibacterial effect of culture supernatants from potential probiotic strains. Also, it was shown in co-culture experiments that growth of the pathogenic bacteria can be almost completely inhibited. The inhibition is particularly effective at high ratios of probiont to pathogen (e.g. 1000:1) but also nutrient composition is important as iron limitation facilitates inhibition of the pathogen. The latter factor is probably due to the production of iron-chelating siderophores by the probiotic cultures. Finally, infections trials indicate that probiotic treatment can significantly reduce mortalities from bacterial disease.

Immunostimulation by the Leaf Extract of the Indian Medicinal Plant, Ocimum sanctum in Oreochromis mossambicus (Peters)

DR Michael*, SM Logambal, S Venakatalakshmi, and YC Viji

Centre for Fish Immunology, Postgraduate Department of Zoology, The American College, madurai 625 002, Tamil Nadu, India. rdmichael@yahoo.com

An immunostimulant is a chemical or a drug that elevates nonspecific and/or specific immune mechanisms. Immunostimulants have been routinely or experimentally used in association with vaccines or vaccination protocols. These immunostimulants include bacterial derivatives, vitamins, glucans and heavy metals. The immunostimulants alone (without vaccine) can also be used to elevate nonspecific immune mechanisms and to potentiate specific immune responses as well. In India, extracts of medicinal plants have been traditionally used for preventing and curing many human infectious and other ailments. The aim of the present study was to look for possible immunomodulatory effect of the extract of the Indian medicinal plant, Ocimum sanctum on the antibody response, neutrophil activity and host resistance in the tilapia, Oreochromis mossambicus (Peters). Leaf extracts were prepared in water and administered intraperitoneally prior to immunization with sheep erythrocytes, or challenge with virulent Aeromonas hydrophila. Antibody titres were measured by haemagglutination in microtitre plates and activated neutrophils were enumerated by a nitro-blue tetrazolium assay. Degree of protection was estimated by relative percent survival (RPS). The results revealed that the leaf extract of O. sanctum had immunostimulatory properties as indicated by increased primary and secondary antibody titres, neutrophil activity and RPS. The easy availability of the medicinal plant, the simple extraction procedure and the biodegradability and the significant immunostimulatory effect of the extract suggests its use as an immunostimulant or as an immunoprophylactic agent in intensive freshwater finfish culture.

Expression of an Insect Cecropin Gene in Channel Catfish

Q Zhang (1)*, TR Tiersch (2), and RK Cooper (1)

1	1 Department of Veterinary Science, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803 USA. qzhang@agctr.lsu.edu; rcooper@agctr.lsu.edu
2	School of Forestry, Wildlife, and Fisheries, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803 USA. ttiersch@agctr.lsu.edu;

We performed in vitro and in vivo studies of transfer and expression of an insect cecropin B gene (CecB) in channel catfish. The objectives of these studies were to produce an antibacterial lytic peptide in an inducible fashion to defend against bacterial pathogens such as Edwardsella ictaluri. Expression vector pQZ-1 was constructed containing the CecB promoter and a cDNA derived from green fluorescent protein (GFP). The construct was delivered using lipofection to fibroblasts, and cultured and freshly isolated leukocytes. Expression was induced by addition of irradiated bacteria (Flavobacterium columnare) or lipopolysaccharide (LPS). We found by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry that GFP was expressed in all cell types. Expression was increased after challenge with bacteria for 24 h in fibroblast cells, and for 10 h in fibroblasts and leukocytes after challenge with LPS. We transferred another construct (pPC-6), consisting of the entire CecB gene and promoter into channel catfish. The plasmid DNA was partially linearlized, mixed with lipososme, and injected peritoneally into channel catfish. Fish were challenged with LPS 14 days after injection. On average 35% of fish from treatment groups revealed presence of the CecB gene that was expressed in these individuals as shown by RT-PCR. Further experiments will be conducted to evaluate the effects of LPS dosage and challenge time on the expression of the CecB gene in vivo.

Biological Method of Struggle Against Diplostomoses in Fish Farm of Kazakhstan

D Zhatkanbayeva

Institute of Zoology and Genofond of Animals, Ministry of Science-Academy of Sciences of Republic of Kazakhstan, 480060 Almaty, Kazakhstan. common@ZOOL2.academ.alma-ata.su

Under conditions of intensive pisciculture the negative role of the parasitic diseases, including diplostomoses, increases sharply. The agents of the letters are especially pathogenic for young fish in the first days and weeks of their life. In fish farms of Kazakhstan up to 50% of young herbivorous fishes perish yearly from diplostomoses. The search and trial of the measures of struggle against fish diplostomoses show high effectiveness of the ecologically safe biological method. The main point of this method is in the considerable enrichment of natural nutritive base of young fishponds by currying of Cladocera (Moina macrocope, M.weberi, Daphnia pulex, D.magna) culture in them. Being the eliminators of the cercarae of the trematodes they significantly reduce the number of parasites in ponds biocenoses. These Cladocera, being the organic part of the young fishes nutrition, are the growth-promoting, favour the augmentation of the mean mass of fishes and, in the end, increased fish productivity in young fish ponds. The experimental use of this method in the ponds of Chilik fish farm near Almaty City in Kazakhstan increased the fish productivity (young of the current year Hypophthalmichthys molitrix) up to 4,6 centers per hectare of the water surface. The use of the biological method promotes the sanitation and growth of the young.

Reduction in Artemia nauplii Mortality with the Use of a Probiotic Bacterium

B Gomez-Gil (1), A Rogue (1), JF Turnbull (2) and V Inglis (2)

1	CIAD/Mazatlán Unit for Aquaculture and Environmental Management. AP. 711 Mazatlán, Sin. México 82000. Bruno@cascabel.unam.mx; roque@cascabel.ciad.mx
2	Institute of Aquaculture, University of Stirling. Stirling FK9 4LA, Scotland. Jftl@stir.ac.uk; vbmi@stir.ac.uk

Probiotic microorganisms have been used to enhance the performance of aquatic animals with variable results. Few studies prove the efficacy of probionts to prevent the pathogenic effect of bacterial strains. This study aims to demonstrate that a probiotic bacterial strain can successfully prevent mortalities caused by a pathogenic Vibrio strain in Artemia franciscana nauplii. The methodolgy proposed by Gomez-Gil et al. (in press) was employed to aseptically bioencapsulate bacteria in sterile Artemia nauplii. The pathogenic strain HL58, a green colony in TCBS agar identified as V. parahaemolyticus, and the probiotic strain C14, a yellow colony in TCBS agar identified as V. alginolyticus, were employed in these experiments. The inoculation of strain HL58 reduced significantly the survival of Artemia nauplii in all the experiments compared to the control. This strain was clearly pathogenic to the nauplii even when they were exposed for 1 hr to a density of 6.5 x 105 CFU m1-1. Stain C14 significantly prevented Artemia nauplii from dying when it was inoculated before the challenge with HL58, its role as probiotic bacteria was established under some conditions. Artemia nauplii were successfully colonized by the probiotic strain C14, although HL58 was detected in the nauplii; the colonization was observed in the results of the bacteriological analyses of the nauplii at the end of the experiments. The nauplii had on average, 1.21 x 105 yellow colonies forming units per nauplius against 5.27 x 104 green CFU per nauplius. The capacity of the probiotic strain C14 to prevent the mortality caused by a pathogenic bacterium was proved.

Probiotic activity of Aeromonas media on the Pacific oyster, Crassostrea gigas, when challenged with Vibrio tubiashii

LF Gibson

Department of Cell & Molecular Biology and the Cooperative Research Centre for Aquaculture, University of Technology Sydney (UTS), P O Box 123, Broadway, New South Wales 2007, Australia

Three strains of Aeromonas media (A161, A164 and A199) were shown to be active in vitro producers of bacteriocin-like inhibitory substances (BLIS). For example, the producer strain, Aeromonas media A199, displayed antagonistic activity against all strains tested of Aeromonas caviae, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii var. sobria, Listonella anguillarum, Photobacterium damsella, eight species of Vibrio and Yersinia ruckeri. Because of this wide-ranging activity against fish/shellfish pathogens, A199 was chosen for the probiotic work. By contrast, however, the BLIS produced by A199 did not inhibit the growth of Enterococcus seriolicida. The aim of the project was to ascertain whether or not the activity observed in vitro could be repeated in vivo.
The ability of BLIS-producing strain A199 to act as a probiotic was assessed on the host animal, Crassostrea gigas, by testing whether or not strain A199 could prevent death of the oyster larvae when challenged with Vibrio tubiashii. Whereas larvae, challenged with the Vibrio, died within five days, the presence of both the pathogen and the probiotic strain, together, did not affect the viability of the larvae over the same time period; the viability of larvae challenged with A199 alone was also unaffected when compared with the viability of unchallenged larvae (controls). These findings have important, economic implications for those engaged in the oyster producing industry where heavy losses can be experienced as a result of an infectious outbreak.
At this stage, the association between BLIS activity and probiotic activity is circumstantial and, hence, future work will involve the use of non-BLIS-producing strains of Aeromonas media and BLIS-negative variants of the producer. Moreover, extension of the project will involve the use of other BLIS-producing strains (A161, and A164), hosts (salmon, crayfish, scallops and abalone) and pathogens.

SESSION 7. VIROLOGY I: Salmonids and Sturgeons

[TOP]

The Effect of Fish Density on the Survival of Rainbow Trout Fry During an Infectious Pancreatic Necrosis (IPN) Epidemic

J Bebak (1), PE McAllister (2), R Boston (3) and G Smith (3)

1	Freshwater Institute, P.O. Box 1746, Shepherdstown, WV 25443 USA. jbebak@ix.netcom.com
2	National Fish Health Research Laboratory, 1700 Leetown Rd., Kearneysville, WV 25430 USA phil_mcallister@usgs.gov
3	382 West Street Rd., New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, PA 19348 USA boston@cahp.nbc.upenn.edu; garys@vet.upenn.edu

One consequence of the intensive culture of animals is an increased risk for outbreaks of infectious disease. Few studies have characterized or quantified this problem as it relates to the intensive culture of finfish. In controlled, replicated experiments, susceptible rainbow trout fry were exposed to infectious pancreatic necrosis virus via the introduction of fish infected with the virus. One, two or three infectious fish were introduced to tanks containing varying densities of susceptible fish. The mortality data was subjected to survival analysis, which epidemiologists often use to investigate "time to an event" problems affecting human and animal health. For susceptible fish, the probability of survival declined with fish density. Characterization of the relationship between fish density and infectious disease epidemics will facilitate the development of management strategies that optimize fish density in intensive aquaculture systems.

Preservation of IHNV Infectivity in Sediments

T Kimura, T Yoshinaka, M Yoshimizu and Y Ezura

Laboratory of Microbiology, Faculty of Fisheries, Hokkaido University, Minato, Hakodate, Hokkaido 041-0821 Japan takak@pop.fish.hokudai.ac.jp

Infectious hematopoietic necrosis (IHN) is one of the major viral diseases causing high mortality among farm-reared salmonids. For health control of salmonid fish, it is important to know where IHNV exists in the environment. In a river, there are many fishes, macro- and microorganisms and sediment. Since there are reports that human viruses, bacteriophages etc adsorb to clay mineral, we studied the adsorption of IHNV to sediment: seasand, Japanese acid clay, diatomaceous earth, kaolin, bentonite, quartz sand, chitin, cellurose powder, ion exchange hydrophobic (TOYOPEARL, Cellulofine), alundum, active carbon, silica gel, glass and plastic, and also 4 kinds of bacteria. IHNV was adsorbed to clay mineral (Japanese acid clay, kaolin, bentonite) and diatomaceous earth. Japanese acid clay, kaolin and diatomaceous earth-associated IHNV showed CPE when specimens were inoculated onto RTG-2 cells. Bentonite showed cytotoxic effects. On the Japanese acid clay, kaolin and diatomaceous earth, IHNV was able to survive and retain infectivity for as long as 9 weeks. Furthermore, cumulative mortality of rainbow trout bathed with the Japanese acid clay, kaolin, bentonite and diatomaceous earth associated IHNV, and free IHNV reached more than 73%.

Seasonal Variation in Prevalence of Infectious Pancreatic Necrosis (IPN) Virus in Atlantic salmon (Salmo salar) Broodstock

K Ross and TS Hastings*

Marine Laboratory, P O Box 101, Victoria Road, Aberdeen AB11 9DB, Scotland UK

Infectious pancreatic necrosis (IPN) can be associated with significant mortalities in Atlantic salmon (Salmo salar) fry and post-smolts. In Scotland, Atlantic salmon broodstock on IPN-infected farms are individually tested at time of stripping and the ova from infected parents are destroyed in order to prevent possible vertical transmission of virus from parent to progeny. In this study, the prevalence of detectable virus infection was measured using a virus isolation technique in different broodstock populations at intervals prior to and at time of spawning. Virus was isolated from Atlantic salmon by inoculation of 0.45µm-filtered extracts of kidney tissue onto monolayers of CHSE-214 cells and incubation at 15oC. In each of the populations the prevalence of detectable infection was found to be lowest at the time of spawning. In one population, levels of infection in individual fish were measured in terms of virus titre using a plaque assay, and levels of circulating antibody to virus were measured using an ELISA. There was no apparent relationship between level of infection and presence or absence of circulating antibody in these fish. The implications for broodstock testing and disease control are discussed.

Viral Diseases in Sturgeon: Challenges for a New Industry

MA Adkison* and RP Hedrick

School of Veterinary Medicine, Department of Medicine and Epidemiology,
2108 Tupper Hall, University of California, Davis, California 95616 USA maadkison@ucdavis.edu

Sturgeon aquaculture is a relatively new but steadily expanding industry worldwide. The introduction of sturgeon into intensive aquaculture environments has been accompanied by the recognition of important pathogens. White sturgeon (Acipenser transmontanus) have been cultured since 1979 in the state of California. Significant losses annually have been attributed to infections with one of four viruses; an adenovirus (WSAV), two herpesviruses (WSHV type1 and 2) and an iridovirus (WSIV). Losses range from 50% to 95% and may result from infections among fish on commercial farms. Three of the agents (WSAV, WSIV, and WSHV-2) have also been identified in feral stocks of white sturgeon. Due to the problems caused by these viral pathogens we initiated studies of these viruses and of the sturgeon immune response. The immune response studies include the characterization of white sturgeon Ig, the ontogeny of the immune response, the kinetics of the antibody response to defined antigens and viral pathogens and development of markers to study the cells of the immune system. We recently began investigating reports of unexplained mortality of young sturgeon on farms in Europe and Russia. Routine microbiological examinations of these fish have failed to identify parasites or bacteria as the etiological agents (personal communications). Samples from a farm in Northern Europe with fish experiencing episodes of high mortality have yielded microscopic (light and electron) evidence of an iridovirus with properties identical to those of the white sturgeon iridovirus. We will discuss this first identification of an iridovirus infection in Russian sturgeon (Acipenser guldenstadi) or any sturgeon from outside the U.S.A. We will also discuss efforts to have resources in place for virus isolations when future outbreaks occur in Europe.

Applications of Epidemiology in Aquaculture: Controlling White Sturgeon Viruses

MP Georgiadis (1)*, IA Gardner (1), TE Carpenter (1), WO Johnson (2) and RP Hedrick (1)

1	Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, California 95616 USA. mmgeorgiadis@ucdavis.edu; iagardner@ucdavis.edu; ecarpenter@ucdavis.edu; rphedrick@ucdavis.edu
2	Division of Statistics, University of California, Davis, California 95616 USA wojohnson@ucdavis.edu

Intensification of fish production has increased the risk of viral and other infectious disease outbreaks. There are two main obstacles to controlling viral diseases in aquaculture. First, non-infectious risk factors for clinical disease are mostly unknown. Were these factors known, control of the viral diseases could be possible even in endemically infected farms. Second, available diagnostic tests frequently are inadequately sensitive and cannot identify carrier fish or infected eggs and sperm, which can introduce a virus or spread it within a farm. White Sturgeon Iridovirus (WSIV) and Herpesvirus-2 (WSHV-2) are the most frequent causes of elevated mortality in white sturgeon. Using epidemiologic methods, the source, transmission patterns and risk factors for these diseases were investigated in three Northern California sturgeon farms. Temporal and spatial statistical analysis of outbreaks in the farm hatcheries failed to detect evidence of tank-to-tank transmission and provided support for a vertical transmission hypothesis. Furthermore, cumulative mortality for followed grow-out tanks ranged from 1 to 60% even though WSIV and WSHV-2 were identified in fish from 92% and 100% of the tanks, respectively. This finding supports the importance of non-infectious risk factors in determining the severity of these viral outbreaks. Multivariable analysis identified significant correlation (p<0.001) of cumulative mortality with stocking density, previous exposure to the virus(es) and water quality characteristics, but not with weight and source of the fish. The presented approaches can provide the knowledge essential for formulating disease prevention and control schemes and can be implemented for other diseases and aquaculture settings.

Adaptive Viral Disease Management Strategies for an Endangered Strain of White Sturgeon Acipenser transmontanus

SE LaPatra (1)*, S Ireland (2), JM Groff (3) and K Clemens (4)

1	Clear Springs Foods, Inc., Research Division, P.O. Box 712, Buhl, Idaho 83316 USA. scottl@clearsprings.com; andy@clearsprings.com
2	Kootenai Tribe of Idaho, P.O. Box 1269, Bonners Ferry, Idaho 83805 USA. ireland@kootenai.org
3	Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, California 95616 USA. josephvmd@aol.com
4	United States Fish and Wildlife Service, Dworshak Fish Health Center, P.O. Box 18, Ahsahka, Idaho 83520 USA. kathy_clemens@mail.fws.gov

Conservation aquaculture was identified as a prudent and necessary recovery tool due to the biological status of the endangered Kootenai River white sturgeon population and the demonstrated uncertainties of other recovery efforts. Potential impacts of disease need to be assessed prior to the implementation of conservation aquaculture programs. The white sturgeon iridovirus (WSIV) is the most prevalent viral pathogen of white sturgeon relative to its distribution and frequency of occurrence and may be endemic to wild white sturgeon populations throughout the Pacific Northwest. This case study illustrates the importance of conservation aquaculture programs in certain situations and the necessity that the management strategies remain flexible and adapt to the current available scientific information for maximum benefits.

SESSION 8. PARASITOLOGY III: Helminths

[TOP]

Pathogenicity of an Exotic Heterophyid Trematode Infecting the Gills of the Endangered Species Etheostoma fonticola (Fountain Darter)

AJ Mitchell (1)*, AE Goodwin (2), MJ Salmon (3), TM Brandt (3) and DG Huffman (4)

1	Stuttgart National Aquaculture Research Center, U. S. Department of Agriculture, Agricultural Research Service, P. O. Box 860, Stuttgart, Arkansas 72160 USA. ncapps@futura.net
2	Aquaculture and Fisheries Center, University of Arkansas at Pine Bluff, P. O. Box 4912, 1200 North University Drive, Pine Bluff, Arkansas 71611 USA. goodwin@seark.net
3	United States Fish and Wildlife Service, National Fish Hatchery and Technology Center, 500 East McCarty Lane, San Marcos, Texas 78666 USA. r2ffa_sm@mail.fws.gov
4	Southwest Texas State University, 601 University Drive, Room 384, New Science Building, San Marcos, Texas 78666 USA. dho9@swt.edu

A heterophyid trematode associated with an exotic aquatic snail (Melanoides tuberculata) caused massive gill infections in the endangered fountain darter (Etheostoma fonticola) living in the Comal River near San Marcos, Texas. Every darter (n=150) examined was infected. A host response encased and killed the trematodes leaving hardened cysts (70-100__ X 150-200__) that numbered in excess of 200 per gill arch in some fish (25 to 35 mm - total fish length). Wet mount microscopy demonstrated extensive proliferation of gill epithelium and cartilage displacement and deformity. Histological examination revealed parasites adjacent to the cartilage in the central sinus of gill filaments. The parasites were encased in masses of cartilage that swelled affected filaments to several times their normal diameter and completely disrupted the normal gill structure. Affected filaments were short, thick, distorted and had few if any lamellae.

The Potential for an Exotic Heterophyid Trematode to Invade and Cause Disease in Four Major Aquaculture Species

AJ Mitchell (1)*, AE Goodwin (2), MJ Salmon (3) and TM Brandt (3)

1	Stuttgart National Aquaculture Research Center, U. S. Department of Agriculture, Agricultural Research Service, P. O. Box 860, Stuttgart, Arkansas 72160 USA. ncapps@futura.net
2	Aquaculture and Fisheries Center, University of Arkansas at Pine Bluff, P. O. Box 4912, 1200 North University Drive, Pine Bluff, Arkansas 71611 USA. goodwin@seark.net
3	United States Fish and Wildlife Service, National Fish Hatchery and Technology Center, 500 East McCarty Lane, San Marcos, Texas 78666 USA. r2ffa_sm@mail.fws.gov

A heterophyid trematode reported from wild fish in Texas and tropical fish in Florida was examined to determine if it had the potential to spread to cultured fish species in other states. Cercariae of this unnamed trematode established infection in channel catfish (Ictalurus punctatus), sunshine bass (Morone chrysops female X Morone saxatilis male), golden shiners (Notemigonus crysoleucas) and fathead minnows (Pimephales promelas). Penetration of the cercariae occurred in less than three minutes and the trematodes positioned themselves along the cartilage support of the gill filament. A strong host response, host cyst production, cartilage displacement and proliferation of epithelial tissue on the filament edges, was observed in sunshine bass and fathead minnows. Host responses in golden shiners and catfish, although similar, were slower and much less pronounced. Histological examination showed the parasites were encased in a cyst-like structure composed of cartilage and there was displacement and distortion of adjacent filament cartilage. There were no inflammatory cells present. The gill surface adjacent to the cysts exhibited moderate epithelial hyperplasia. The strong host response to this trematode indicates that it could cause serious disease problems in sunshine bass and fathead minnows and to a lesser extent in golden shiners and channel catfish.

Preleminary Integrated Study of the Effects of Cadmium and Ammonium on the Immunological and Histo-Pathological Responses of Carp (Cyprinus carpio), Infected with Sanguinicola inermis.

P-MM Schuwerack (1), D Hoole (1) and JW Lewis (2)

1	Centre for Applied Entomology and Parasitology, Biological Sciences, Keele University, Keele ST5 5BG UK M.Schuwerack@Keele.ac.uk; D.Hoole@Keele.ac.uk
2	Environmental Biology, Royal Holloway, University of London, Egham, Surrey, TW20 0EX. J.Lewis@rhbnc.ac.uk

Thirtysix carp (0+) specimens were individually infected with 500 cercariae of the blood parasite, Sanguinicola inermis, which were obtained from the freshwater snail, Lymnaea peregra. After 28-34 days post-infection at 20 oC and a photoperiod of 12D:12L specimens were transferred into separate tanks (at 20 oC) containing ammonia (0.5 mg l-1) or cadmium (0.1 mg l-1). A flow-through-system was used with a rate of 12 ml sec-1 and a turnover time of 6 h 21 min. The pronephros, spleen and thymus were removed from controls (infected carp, not subjected to Cd or NH4+) and exposed infected specimens after 48 or 168 h for histo-pathological and immuno-toxicological analysis. Leucocyte suspensions obtained from the pronephros of individual fish were exposed to ConA and PWM (0.2 mg 100ml-1 in sterile Leibovitz L-15 culture medium) or cercarial homogenate (0.16mg protein 100 ml-1 in L-15 medium). Blastogenic response was monitored for 72 h using [C6-3H] thymidine at 20 C. Responses of infected carp to the pollutants Cd2+ and NH4+ over time were expressed in terms of stimulation of lymphocyte proliferation. Lymphocyte stimulation occurred in the presence of Cd but was suppressed with NH4+. Histo-pathological effects in specimens, subjected to Cd, included a granular dense cytoplasm, concentric whorls and disintegrated cistae in mitochondria. Increased pinocytotic activity, disruption of lymphocytes, scattered disrupted organelles were observed in the presence of NH4+. Differential cell counts are in progress. The application of a multiple pollutant-specific biomarker response assay are evaluated.

Heavy Metal Bioaccumulation by Acanthocephalans Parasitizing Fish and Their Role in Environmental Impact Studies

B Sures* and H Taraschewski

Zoologisches Institut I - Ökologie, Geb. 30.43, Universität Karlsruhe, 76128 Karlsruhe, Germany, Bernd.Sures@bio-geo.uni-karlsruhe.de

Attempts at using parasites as biological indicators in environmental impact studies have been the subject of several recent reviews. The majority of investigations have examined the effects of various forms of pollution on the abundance and distribution of parasites and the combined effects of pollution and parasitism on the health of the hosts. In contrast to this still increasing number of articles only a few of them deal with quantitative analysis of certain toxins in parasites. Thus, common fish species have been sampled from different moderately polluted sites and the concentrations of various heavy metals were determined in the fish tissues (muscle, liver and intestinal wall) and their respective intestinal parasites. The most conspicuous bioconcentration factors were found for acanthocephalans which contained e.g. up to 3 ¥ 103 fold more lead than the muscle of their fish hosts and up to 1.1 ¥ 104 more lead than the water surrounding the fish. Seventeen different elements including essential elements like Ca, Fe, K, Mg, Mn and toxic elements like Ag, Cu, Tl were analyzed by inductively coupled mass spectrometry in Acanthocephalus lucii. Again, this acanthocephalan showed for nearly all elements significantly higher concentrations than the tissues of its host perch (Perca fluviatilis) and than the zebra mussel Dreissena polymorpha which is a commonly used bioindicating organism in Europe. Experimental studies demonstrate a clear time and dose dependent accumulation of lead for Pomphorhynchus laevis in its final host chub. Interestingly, by comparing the lead burden of the inner organs of chub it could be seen that the lead concentration was significantly lower in the intestinal wall of infected fish than of uninfected ones. This presentation summarizes the present knowledge on element accumulation in acanthocephalans.

Pathobiology Associated with the Acanthocephalan Southwellina hispida in the Alimentary Canal of Phalacrocorax carbo (Aves)

B S Dezfuli*, S Capuano, M Manera, C Barbieri and S Volponi

Department of Biology, University of Ferrara, Via Borsari 46, 44100 Ferrara, Italy

During 1997 on several occasions a total of 23 specimens of Phalacrocorax carbo were obtained from the Comacchio lagoons (northern Adriatic Sea, Italy). Sixteen (69.5%) birds were infected with Southwellina hispida and the intensity of infection ranged from 1 to 50 acanthocephalans per host. The most parasitized segments of waterbird digestive tract were respectively mid-gut and foregut. The histopathology induced by S. hispida in the intestine of P. carbo was examined. This investigation comprises observations carried out by light and transmission electron microscopy on the histopathology caused by this parasite. At its site of attachment, S. hispida induced complete destruction of the columnar epithelia. Both male and female parasites penetrated deeply through all the layers of the host digestive tract by means of their neck and proboscis. The praesoma of the acanthocephalan caused flask-shaped ulcers in the intestinal wall. In some instances, the proboscis emerged in the coelom. The most pronounced reactions in the site of attachment to the presence of S. hispida were an intense host cellular reaction and accumulation of collagenous fibres.

Rodlet Cells in the Digestive Tract of Different Species of Fish and Their Relationship with Parasitic Helminths

B S Dezfuli*, M Manera, S Capuano and R Rossi

Department of Biology, University of Ferrara, Via Borsari 46, 44100 Ferrara, Italy

Rodlet cells, characterized by a cell cortex or capsule and conspicuous inclusions, the "rodlets", have been described in different organs of freshwater and marine teleosts. Some authors have thought these cells to be protozoan parasites; others have maintained that the rodlet cells are integral constituents of fish epithelia. The present investigation was undertaken to gain information on these cells in the alimentary canals of acanthocephalan-infected freshwater fish (Anguilla anguilla, Leuciscus cephalus, Gasterosteus aculeatus) and of trematode-infected A. anguilla from brackish water. The cell cortex (capsule) was composed of thin microfilaments and embraced several rodlets. Its thickness ranged from 0.4 to 0.9 µm. The rodlet had a central core of highly electron dense material with less dense substances surrounding it. Within the mature rodlet cell the occurrence of organelles was rare. According to our observations the rodlet cells secrete their contents in a holocrine manner into the lumen of fish intestine. In a comparison between uninfected and parasitized eels from brackish water, rodlet cells were counted in 150 microscopic fields; the intestine of infected fish had significantly more rodlet cells (ANOVA, P < 0.01). Detail on the ultrastructural features and function of rodlet cells of the digestive tract of the above mentioned fish will be provided.

SESSION 9. VACCINES

[TOP]

Development of Vaccines Against Bacterial Diseases in Salmonid Fishes Using DNA Immunization

M Gomez-Chiarri (1)*, MJ Mauel (2), SH Marshall (3), C Orrego (4) and RP Levine (5).

1	Department of Fisheries, Animal, and Veterinary Science, University of Rhode Island, Kingston, RI 02881 gomezchi@uriacc.uri.edu
2	Center for Vector-Borne Disease, University of Rhode Island, Kingston, RI 02881 mauelm@uriacc.uri.edu
3	Instituto de Biologia, Universidad Catolica de Valparaiso, Casilla 4059, Valparaiso, Chile dginv@ucv.cl
4	Department of Biology, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132 cob@sfsu.edu
5	Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950 plevine@leland.stanford.edu

The farming of salmonid fishes constitutes a major sector of U.S. aquaculture. Bacterial kidney disease (BKD) and piscirickettsiosis are two bacterial diseases that have a serious impact on the health of salmonid fishes and pose a severe problem for the development of salmonid aquaculture. Our goal is the development of effective, safe, and economic vaccines against BKD and piscirickettsiosis using DNA to immunize rainbow trout. We are using two different approaches for the development of DNA vaccines against bacterial diseases: single-antigen DNA immunization and expression-library immunization. We have established the optimal conditions for gene delivery into the tissue of live rainbow trout, constructed expression vectors specially designed for the DNA immunization of fishes, and constructed single-antigen and expression-library DNA vaccines specially designed for the prevention of BKD and piscirickettsiosis in salmonid aquaculture. We are currently investigating the potential of these DNA vaccines to confer protection in challenge experiments. DNA immunization may provide safe, efficient, and economic vaccines for aquaculture and a tool to study the role of cellular and humoral immune responses in clearing bacterial infections.

The Expression of the Mx Protein and Vaccine Efficacy in Rainbow Trout

CH Kim*, M Johnson, E Thomann, B Simon and JC Leong

Department of Microbiology and the Center for Salmon Disease Research, Oregon State University, Corvallis OR 97331 kimc@bcc.orst.edu; ohnsmar@bcc.orst.edu; thomanne@bcc.orst.edu; simonb@bcc.orst.edu; leongj@ccmail.orst.edu

The Mx gene is a type I interferon (IFN a/b)-inducible gene that appears to be present in all vertebrate species including human, rat, sheep, pig, duck, chicken, perch, and trout. The trout Mx gene has been cloned, characterized, and a rabbit anti-Mx polyclonal antibody reagent has been produced. Mx proteins are synthesized in response to infectious hematopoietic necrosis virus (IHNV) infection or poly I:C treatment in both cultured fish cells and rainbow trout. In this study, fish were vaccinated with formalin-killed virus or the E.coli produced subunit vaccine, pXL3. These fish were moderately protected against virus challenge with 50% survival. When the liver and kidney tissues were examined by western blot analysis, there was no Mx induction until challenge with IHNV 30 days post vaccination (dpv). For fish vaccinated with the DNA vaccine, pCMV-G, Mx was expressed early after vaccination (2 dpv), but disappeared upon IHNV challenge. The genetically immunized fish were well protected against the lethal effects of the virus and exhibited a 98% rate of survival. These results suggest that Mx may be used as a marker for IHNV vaccine efficacy in rainbow trout. In addition, the effects of viral glycoprotein in inducing an Mx response and providing specific protection was examined.

Development and Use of Vaccines for Fish Cultured in Warm-water

PH Klesius (1)*, CA Shoemaker (1) and JJ Evans (2)

1	USDA-ARS Fish Diseases and Parasites Research Laboratory, PO Box 0952, Auburn, AL 36831 USA klesiph@vetmed.auburn.edu; cshoemak@acesag.auburn.edu
2	Maryland Department of Natural Resources, Cooperative Oxford Laboratory, 904 S. Morris Street, Oxford, MD 21654 USA

Vaccines that induce protective immunity to pathogens result in prevention of disease. Most animal husbandry industries have relied on vaccines as a management tool to prevent disease and reduce use of expensive and ineffective antibiotics. The development and use of vaccines in the catfish industry has been slow due to lack of information on the fish immune system. Fish have the ability for both humoral (antibody) and cell mediated immune responses. The nature of the disease process determines which branch of the fish immune system will result in acquired immunity. Acquired immunity to facultative intracellular pathogens (i.e. Edwardsiella ictaluri) is cellular more than humoral. Live attenuated vaccines are more effective in stimulating cellular immunity than are bacterins. We developed an E. ictaluri vaccine that is safe and efficacious against enteric septicemia of catfish (relative percent survivals about 90-95 %). The attenuated vaccine E. ictaluri RE-33 is safe (even at 200 times the dose), easily administered by bath immersion, and stimulated long-lasting (6 months without booster) protective cell­mediated immunity in fry as young as 10 days post hatch. The RE-33 vaccine is being field-tested with permission from USDA, Animal Plant Health Inspection Service (APHIS) and state veterinarians of Alabama and Mississippi for approval as an ESC vaccine. We believe that safe attenuated vaccines that are low cost, easy to administer, effective in the youngest life stages and able to provide long lasting protection without booster are the best answer to infectious disease management.

Advances in Vaccine Development for Chilean Bacterial Diseases

DB Powell*, RC Palm, Jr., RM Brown, EA Greger and MP Peña

Aquatic Animal Heath Division, Alpharma NW Inc., 1720 - 130th Ave. NE, Bellevue, WA 98052 USA dpowell@alpharmaaah.com

Chilean salmon aquaculture has grown rapidly in recent years to produce millions of tons of high quality food fish for the world market. Emerging bacterial diseases including Piscirickettsia salmonis, Yersinia ruckeri, atypical Aeromonas salmonicida and various gill bacteria are economic threats to the stability and growth of an increasingly competitive industry. The objective of this work is to further the development and use of new vaccines to allow fish farmers to reduce their reliance on antibiotic treatments for disease control. Our initial research efforts have been to collect, identify and compare many of the pathogenic strains from the region. The gill bacteria isolated by local veterinarians appear to be a diverse collection of strains and species. Recently, we have made advances in the development of challenge models and the formulation of new vaccines. Depending on the disease, safety and efficacy experiments were conducted with coho salmon, atlantic salmon or rainbow trout. The selection of immersion, oral or injection vaccine delivery systems was related to the level of protection required and husbandry limitations. A combination of two strains of Yersinia in a vaccine was not equivalent to the use of either strain alone. The results of experiments with other Chilean bacterial species will be presented.

Exposure of Goldfish Carrasius auratus (L.) and Koi Cyprinus carpio to Live Atypical Aeromonas salmonicida, a Causal Agent of Bacterial Ulcer Disease

LE Swaim (1)(3), ML Landolt (1) and DB Powell (2)

1	School of Fisheries, University of Washington, Box 357980, Seattle, Washington, 98195-7980 USA landolt@u.washington.edu
2	Alpharma Aquatic Animal Health Division, 1720-130th Avenue NE, Bellevue, Washington, 98005-2203 USA dpowell@alpharmaaah.com
3	Aquatic Consulting Services, 401 NE 92nd Street, Seattle, Washington, 98115-2722 USA leswaim@ix.netcom.com

Three exposure models were developed in goldfish and koi to test the virulence of a goldfish isolate of atypical Aeromonas salmonicida: intramuscular injection, bath challenge of healthy fish and bath challenge of scarified fish. The results showed a host-specific virulence difference among the bath-challenged fish compared to the injected fish. There was no difference in the proportion of 14-day mortalities when healthy goldfish (mean±sd = 79.00 ± 7.12%) and koi (mean±sd = 76.67 ± 3.31%, Fisher's Exact test P2,0.05 = 0.858) were injected with equal concentrations of live, virulent bacteria. Following bath exposure to equal concentrations of virulent bacteria, healthy (unscarified) goldfish morbidity (mortalities plus ulcerated survivors) was significantly higher (mean±sd = 84.30 ± 5.13%) compared to healthy koi morbidity (mean±sd = 33.33 ± 6.51%, P2,0.05 = 1.08x10-12). Likewise, mortality of scarified goldfish (mean±sd = 89.00 ± 10.15%) exceeded that of koi (mean±sd = 16.33 ± 4.93%, P2,0.05 = 2.62x10-24). Goldfish and koi were immunized with a live avirulent strain of atypical A. salmonicida and subsequently bath-challenged with the live virulent strain. The live vaccine protected immunized healthy goldfish against mortality (RPS = 82.4), but afforded less protection against ulceration (RPS = 25.9). This investigation found ulcer size, number, morphological location and stage of development to be important criteria for assessing vaccine protection. The ability of a vaccine to protect the external mucosa and prevent ulcer disease was equally important to the prevention of mortality. Ornamental fish farmers require that a vaccine prevent or significantly reduce ulceration to maintain the high value of their fish.

Evaluation of a Live-Attenuated Vaccine Against Edwardsiella ictaluri in Fry and Fingerling Channel Catfish

RL Thune*, DH Fernandez and L Lumbard

Department of Veterinary Science, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center and Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803. thune@vt8200.vetmed.lsu.edu

A genetically defined strain of E. ictaluri was constructed that contained insertion/deletion mutations in the aroA gene of E. ictaluri. Bacterial pathogens with stable aroA defects are avirulent because they require the aromatic metabolites para-aminobenzoate and dihydroxybenzoate, which are not available in vertebrate tissues. An initial study was conducted in which replicated groups of fingerling catfish were vaccinated by immersion in the E. ictaluri aroA mutant and challenged with the wild-type strain. Results indicated excellent protection against disease, with Relative Percent Survivals (RPS) ranging from 54 to 94. A second study was conducted to evaluate the feasibility of vaccinating channel catfish fry. Briefly, one group of catfish fry was vaccinated by immersion 16 days after hatching, at a weight of 290 mg/fish, one group after 24 days, at 570 mg/fish, and a third group after 32 days, at 1152 mg/fish. Fish from each of the groups were immersion challenged 6 weeks post-vaccination, given an immersion booster vaccination 90 days post-hatching at a weight of 19.4 g/fish, and challenged again by immersion in the virulent wild-type strain 6 weeks after the booster vaccination. Fish given a single vaccination as fry demonstrated little or no protection from disease. Fish vaccinated as fry and given a booster dose as fingerlings had RPS values ranging from 78 to 86, while fish given a single vaccinated as fingerlings had an RPS of 64. Vaccination was also demonstrated to significantly reduce the rate of chronic carriers from 32% to 5% 65 days after the challenge.

SESSION 10. HUMAN HEALTH AND ZOONOSES

[TOP]

Detection of Atypical Mycobacterium spp. in Fish and Man Using Immunohistochemistry, ELISA, PCR and in situ Hybridization

A Adams(1)*, KD Thompson(1), S Puttinaowarat(1), DJ Morris(1), T Somsiri(2), W Knibb(3) and A Kolk(4)

1	1 Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland. aa2@stir.ac.uk
2	Aquatic Animal Health Research Institute, Kasetsart University, Bangkhen, Bangkok,Thailand. aahri@ksc15.th.com
3	Israel Oceanographic & Limnological Research Ltd., National Centre for Mariculture, Elat, Israell. knibb@agri.huji.ac.il
4	Royal Tropical Institute, Mauritskade 63, Amsterdam, The Netherlands. bo@mail.support.nl

Mycobacteria isolated from ornamental fish (Siamese fighting fish, Beta splendens) and food fish (snakehead, Channa straitus) in Thailand and sea bass (Dicentrarchus labrax) from Israel were identified as M. marinum, however photochromogenic, biochemical and antigenic differences indicate that three sub species may exist. These mycobacteria were found to thrive at 37C. This, and the fact that mycobacteriosis can be transmitted from contact with infected water through skin abrasions highlights the potential risk to man. Some mycobacteria from fish are extremely slow growing and difficult to isolate from infected tissue despite the presence of large numbers of acid fast bacilli. Diagnosis is therefore both difficult and time-consuming using conventional methods. Polyclonal and monoclonal antibody probes to a variety of mycobacteria strains were produced and utilised in the development of ELISA and immunohistohemistry tests which are specific for Mycobacterium marinum. A reverse cross blot PCR was also developed both for the detection of mycobacteria in fish tissue and human biopsies. PCR and an in situ hybridisation test were developed to detect mycobacteria in fish tissue. These methods are presently being used to screen ornamental fish for export from Thailand and to identify mycobacteria in food fish and man. Water samples, sediment, faeces and blood samples are also being analysed to confirm sources of infection. These results will be discussed with reference to the control of mycobacteriosis.

Oral Mycoplasmal Infections in Canadian Pinnipeds

LN Measures*

Fisheries and Oceans Canada, Maurice Lamontagne Institute, P.O. Box 1000, Mont-Joli, Qc G5H 3Z4 CANADA. MeasuresL@dfo-mpo.gc.ca

Mycoplasma phocacerebrale has been implicated in human infections known as "seal-finger". Human infections can be acquired by being bitten by infected seals or by handling live or dead infected seals. Bacterial swabs of the tooth-gum interface in the oral cavity of four species of pinnipeds (N=162) from eastern Canadian waters were collected and cultured for Mycoplasma spp. Three species of Mycoplasma and unidentified species of Mycoplasma and Ureaplasma were detected. In juvenile or adult seals: 23 of 29 (79%) grey seals (Halichoerus grypus), 10 of 19 (53%) harbour seals (Phoca vitulina), 32 of 36 (89%) hooded seals (Cystophora cristata) and 8 of 27 (30%) harp seals (Phoca groenlandica) were infected with Mycoplasma phocacerebrale. Mycoplasma phocidae and M. phocarhinis were also detected in some seals. Seals held captive in salt water or fresh water aquaria were also infected. Young-of-the-year (Age=0) harbour (N = 5), harp (N =10) and hooded seals (N = 10) from a few days to six months old were negative. However, 9 of 12 (75%) young-of-the-year grey seals were infected with M. phocacerebrale (infected seals were 4 - 5 weeks old). Some recent "seal-finger" infections in biologists and successful treatment are described.

Aquacultural Hazards: A Chapter from the Textbook, Safety and Health in Agriculture, Forestry, and Fisheries

RM Durborow*

Cooperative Extension Program Facility, Kentucky State University, Frankfort, KY 40601 USA. bdurborow@gwmail.kysu.edu

Consumption of fish is considered to have many positive health benefits in humans, and the demand for fish is expected to increase by over 70% in the next 35 years. This chapter reviews the hazards associated with feral and farm-raised aquatic organisms such as zoonotic infections, diseases from ingestion of toxins in fish and shellfish, sewage in ponds, hydrogen sulfide gas on pond bottoms, adverse reactions from shrimp preservatives, and the dangers of handling antibiotics and vaccines used in aquaculture. Many of the zoonotic conditions are avoided by consuming cultured fish rather than those caught from the wild, and by properly cooking fish. Safety hazards around fish production facilities include electrical shock when in the culture water, drownings, musculoskeletal strains from feeding and cleaning activities, tractor rollovers and PTO-related injuries. Accidents occurring during fish processing include spine punctures, band saw lacerations and amputations.

An Investigation of the Self-Reported Occupational Health Concerns of Aquaculture Workers in the Northeast of Scotland.

KJ Sharp*

The Robert Gordon University, Associate Faculty of Nursing, Midwifery and Community Studies, Foresterhill Campus, Westburn Road, Aberdeen, Scotland, UK, AB25 2XG. k.sharp@rgu.ac.uk

Observational work in fish farming enabled the researcher to undertake a risk assessment profile for aquaculture workers, and salmon farmers in particular. An interview schedule/questionnaire was developed and adminstered to 200 aquaculture workers in the Orkney and Shetland Isles. The data collection tool recorded the self-reported incidence and prevalence of ill-health symptoms and problems in aquaculture. Anecdotal evidence was also collated in order to begin to identify areas thought to require further investigation for possible association with occupational health and safety. The sample population data was compared with data gathered for the control populations of 119 ferry workers and 104 general population workers. The principle findings of this investigation will be presented.

"Yo-yo" Diving on Scottish Salmon Farms

SE Wilcock*

The Robert Gordon University, Aberdeen, United Kingdom. S.wilcock@rgu.ac.uk

Salmon farming at inshore sites in the United Kingdom often involves regular diving activity to perform maintenance duties, e.g. removing dead fish, checking the integrity of predator nets, sampling the sea bed, or even recovery of submerged equipment. Diving to remove dead fish can involve multiple descent-ascent cycles of short duration. This, it has been claimed, allows gas loading of the divers tissues, without time between cycles for the gas to be off-loaded; it has been argued that this "yo-yo" diving presents a hazard for the diver which could lead to decompression sickness. The investigation described here gathered data to assess the extent of this problem by estimating the incidence of decompression sickness relative to the number of dives undertaken and the number of divers completing the work. The difficulties addressed by the investigation are numerous and the approach taken had to be sensitive to these issues. For example, due to the high cost, some organizations did not employ or contract professional divers who had been trained in accordance with the current health and safety requirements; some divers did not dive with appropriate stand-by provision. The confidence of the respondents had therefore to be gained and the independence of the researchers from the government Health and Safety Executive body, or any other policing department, had to be assured. The techniques used to acquire valid information on what is essentially a sensitive issue will be discussed along with the implications of these issues for other areas of research.

SESSION 11. EMERGING DISEASES II: Bacteria

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Characterization of Vibrio viscosus (Moritella viscosa) and V. wodanis Associated with Winter Ulcers in Salmonids in Iceland, Norway and Scotland

E Benediktsdóttir (1)*, L Verdonck (2), C Spröer (3), S Helgason (4) and J Swings (2)

1	Institute of Biology, Mircobiology Laboratory, University of Iceland, Ármúli 1A, IS-108 Reykjavík, Iceland. eben@rhi.hi.is
2	Laboratory of Microbiology, University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium Linda.Verdonck@rug.ac.be, Jean.Swings@rug.ac.be
3	DSMZ, Mascheroder Weg 1b, D-38124 Braunschweig, Germany ckc@GBF-Braunschweig.De
4	Institute for Experimental Pathology, Fish Disease Laboratory, Keldur, University of Iceland, IS-128 Reykjavík, Iceland. siggih@rhi.hi.is

V. viscosus and V. wodanis are recently described species of psychrotrophic bacteria that have been found associated with a disease called winter ulcers, affecting salmonid fish reared in saline water in Norway, Iceland, and recently in Scotland. V. viscosus and V. wodanis strains initially isolated from fish in Iceland and Norway were subjected to characterization using biochemical tests, SDS-PAGE of whole cell proteins, and a novel DNA fingerprinting method, AFLP. According to analysis by AFLP, the strains of V. viscosus form two definite clusters: one uniform cluster is formed by strains isolated in Norway, and the other by strains isolated in Iceland, divided into two subclusters and one single strain. Difference is found between AFLP-clusters in one to three biochemical tests. The SDS-PAGE protein prophiles show that the strains isolated in Norway, Scotland and in SW-Iceland form one homogenous cluster, other strains differ in one major protein band. Challenge experiments performed in Atlantic salmon parr using V. viscosus revealed that representatives of V. viscosus groups isolated from salmonids are virulent. According to all three methods used, the V. wodanis strains are heterogenous genotypically and phenotypically, and do not group according to geographical origin. Sequencing of almost complete 16S rRNA genes of V. viscosus and V. wodanis revealed that V. viscosus showed 99.1 % sequence similarity to Moritella marinus, and V. wodanis showed 98.8 % sequence similarity to V. logei CIP 103204. A proposal is made that Vibrio viscosus should be reclassified and renamed Moritella viscosa.

Recent knowledge on the Taxonomy of Gram-Positive Cocci Pathogenic to Fish

C Ghittino (1), A Eldar (2) and RP Hedrick (3)

1	1 Fish Disease Laboratory, IZS (State Veterinary Institute), Via Bologna 148, 10154 Turin, Italy (EU) izstoghi@ipsnet.it
2	Fish Disease Laboratory, Kimron Veterinary Institute, P.O.Box 12, Beit-Dagan 50250, Israel vvshark1@volcani.agri.go.il
3	Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, CA 95616 (USA) rphedrick@ucdavis.edu

"Streptococcal" diseases of fish are worldwide emerging pathologies. Heavy economic losses due to infections caused by different genera and species of Gram-positive cocci have been recently reported in several continents (Asia, North America, Africa, Australia, and Europe). The application of molecular approaches has led to a more refined taxonomy of the various Gram-positive cocci pathogenic to fish, demonstrating that at least 4 species are associated with disease conditions: Lactococcus garvieae (jun. syn. Enterococcus seriolicida), Vagococcus salmoninarum, Streptococcus iniae and S. agalactiae. In some instances, these fish pathogens are also a direct threat to human health (S. iniae and L. garvieae have been isolated from diseased patients, associated with cellulitis, meningitis, UTIs and bacteremias). Molecular epidemiology (RFLP ribotyping) revealed the existence of various L. garvieae and S. iniae ribotypes. L. garvieae Japanese strains, although phenotypically resembling Italian isolates, have evolved along several epidemiological routes. One of the Japanese isolates is presumably the source of infection in Italy. In a similar fashion, it was demonstrated that Israeli and American S. iniae isolates, although sharing identical phenotypic traits, belong to two distinct epidemiological groups and the disease has evolved along two separate tracks.

Comparison of Virulence of Piscirickettsia salmonis Isolates

ML House (1)*, JL Bartholomew (1), JR Winton (2) and JL Fryer (1)

1	1 Center for Salmon Disease Research, Dept. of Microbiology, Oregon State University, Corvallis, OR 97331-3804. Marcia_House@usgs.gov; bartholj@bcc.orst.edu; fryerj@bcc.orst.edu
2	Western Fisheries Research Center, Seattle, WA 98115 Jim_Winton@usgs.gov

Piscirickettsia salmonis is known to cause up to 90% mortality in pen-reared coho salmon (Oncorhynchus kitsutch) in Region 10, Chile. Since the initial isolation of this rickettsia in 1989, similar gram-negative intracellular organisms have been described from areas of the world distant from Chile. In British Colombia, Canada, P. salmonis was reported to cause 8% mortality in Atlantic salmon (Salmo salar), and negligible losses in chinook salmon (O. tshawytscha). In 1992, Piscirickettsia salmonis causing low cumulative mortality was recovered from Atlantic salmon cultured in netpens in Norway. A comparison of representative isolates (LF-89T, ATL-4-91 and NOR-92, respectively) was conducted in the Biosafety Level-3 containment facility at the Western Fisheries Research Center, Seattle, Washington, to determine if the virulence of these organisms varied, or if other conditions dictated the severity of the disease. For each of the three isolates, 90 coho salmon per group were injected intraperitoneally with 105, 104, or 103 TCID50 P. salmonis. The control fish were injected with MEM-10. Mortalities were sampled daily for 41 days post-injection. Piscirickettsiosis was observed for each of the three isolates. LF-89T was the most virulent, with losses reaching 97% in the group injected with 105 TCID50, and 91% in the group injected with 104 TCID50. ATL-4-91 caused losses of 92% in the group injected with 105 TCID50, and 76% in the group injected with 104 TCID50. NOR-92 was the least virulent causing 70% mortality in the group injected with 105 TCID50.

Piscirickettsia salmonis Inactivation by Chlorine and Freshwater

RC Palm, Jr.*, DB Powell and MP Peña

ALPHARMA, Aquatic Animal Health Division, 1720-130th Avenue N.E., Bellevue, Washington 98005-2203 USA. fishdoc@aa.net

Piscirickettsiosis can be a devastating disease of cultured salmonid fishes and has been a particularly serious problem in Chile. Efforts are underway in several locations around the world to discover effective treatments and vaccines. These research efforts must rely on live challenges with virulent Piscirickettsia salmonis to demonstrate protection. However, regulatory authorities typically require the research facility to provide proof that the pathogen can be contained and completely killed in all effluent water. In the present study, the use of chlorine and freshwater to inactivate P. salmonis was examined. The bacterium was grown in CHSE-214 cells for 14 days and the resulting supernatant was treated with four chlorine concentrations (0.5, 2, 4, and 10 ppm) at four different exposure times (0.5, 2, 10, and 20 min). In addition, the effect of freshwater dilution on survival was examined. Titers were determined for all treatments and controls by plaque assay. Parameters such as pH and temperature were controlled to match wet laboratory conditions. Results indicate that P. salmonis may be more easily inactivated and contained by common hatchery conditions and effluent treatment than some other important fish pathogens.

Entry Portal of Piscirickettsia salmonis in Rainbow Trout (Oncorhynchus mykiss)

PA Smith, P Ojeda, P Pizarro, JR Contreras and JJ Larenas

Faculty of Veterinary Sciences. University of Chile. Casilla 2 Correo 15. Santiago. Chile.
psmith@abello.dic.uchile.cl

Since 1989, Piscirickettsia salmonis, the causal agent of piscirickettsiosis, has been killing virtually millions of fish every year, in cultured populations of salmonid species in southern Chile. In this work, the route of entry for the pathogen was investigated by the use of various challenge methods in juvenile rainbow trout (12g). The methods employed were patch contact of skin (filter paper soaked in bacterial suspension placed for 1 min), patch contact with gills, subcutaneous injection (SC), intraperitoneal injection, gastric intubation and anal intubation. Cumulative mortalities for the different exposure methods at day 33 post-inoculation were 52% in skin, 24% in gills, 100% subcutaneous injection, 98% intraperitoneal injection, 2% in gastric intubation and 24% in anal intubation. Results suggest that the main routes of entrance would be through skin and gills. P. salmonis can invade the fish in the absence of vectors or superficial wounds, but the high mortality of fish injected SC also suggests that vectors could play a role in the transmission of piscirickettsiosis.
Supported by Grant FONDECYT 1960976

Aerobic Bacterial Flora Associated with Tropical Ornamental Fish Imported into Scotland

RE Del Rio-Rodriguez (1), JF Turnbull (1), RH Richards (1) and A Murray (2)

1	Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, Scotland, UK red2@stir.ac.uk, jft1@stir.ac.uk, rhr1@stir.ac.uk
2	Murray Aquatics, 53-63 Washington Street, Glasgow, G3 8AZ, Scotland, UK

Over a six-month period the aerobic bacterial flora of imported tropical ornamental fish species and their transportation water was examined. Consignments arriving in Scotland from Singapore and South America were each sampled once a month. Up to 97% of the sample population were found to be bacteraemic in some consignments from Singapore and 95% from South America. Monomicrobic infections were rare in bacteraemic fish. Bacterial loads were higher in the tissues of fish and in the transportation water from Singapore. The most prevalent organism from both geographic origins were Pseudomonas spp., Alcaligenes faecalis and motile Aeromonas spp., although the relative prevalence varied between samples. The significance of these organisms and the role of prophylaxis for fish importation will be discussed.

SESSION 12. EMERGING DISEASES III: Viruses

[TOP]

Report on the First Occurrence of Infectious Salmon Anemia (ISA) in Atlantic Salmon (Salmo salar) in Scotland, United Kingdom

IR Bricknell, DW Bruno, C Cunningham, TS Hastings, AH McVicar, PD Munro, R Raynard and RM Stagg

FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen AB11 9DB UK. staggr@marlab.ac.uk

In May 1998, a suspect outbreak of Infectious Salmon Anemia (ISA) occurred in two fish farms in Scotland. The disease was diagnosed by the mortality patterns, clinical signs, histopathology and putative viral identification using IFAT on tissue imprints and RT-PCR. The fish affected were Atlantic salmon smolts (S1/2's) transferred to sea in November 1997. This paper will describe the pathology of the fish, the tests used to diagnose the disease and the subsequent containment and eradication program. Clinical signs were consistent with the pathology described previously in Norway. Many slow-moving fish near the water suface were observed and individuals were easily netted. Significant scale loss over the lateral flank was recorded with bilateral exophthalmos and occasional hemorrhage noted in the eye chamber. The gill lamellae were pale with darker areas near the gill arch. Hematocrit in many cases was <10. At necropsy, blood coloured ascites were recorded. The livers were pale to dark and ranged in colour from burgundy to a deep black-brown, sometimes with mottling. The heart was anemic in appearance. In some fish the spleen and/or kidney were swollen. Petechiae were observed on the pyloric caeca. Microscopically, the liver showed multifocal hemorrhage and congestion of the hepatic sinusoids with diffusing necrosis, associated single cell necrosis and pyknotic nucleii. The cells surrounding the central veins were largely intact. The splenic parenchyma was congested and associated with a generalized necrosis. Putative viral identification has been carried out using tissue imprints stained with FITC labelled goat anti mouse secondary antibody against anti ISA monaclonal antibody 3H6F8 (generous gift from Falk) and RT-PCR amplification of a 155 nucleotide fragment of the viral genome using RNA extracted from tissue homogenates as a template. However to date, the virus has not been isolated in salmon head kidney cells. All fish at the two sites in which ISA has been confirmed have been subject to compulsory slaughter and the farms and all associated equipment disinfected. The containment exercise and surveillance program in the surrounding areas will be described together with an account of the epizootiological studies so far initiated.

First Identification of Infectious Salmonid Anaemia Virus (ISAV) in North America: Initial Isolation and Appraisal of Diagnostic Tests.

SG Griffiths, MD Cook*, KJ Melville, B McDonell and LA Hutchin

RPC, Dept. Food, Fisheries and Aquaculture, 921 College Hill Road, Fredericton, NB, E3B 6Z9, Canada SGriffit@rpc.unb.ca

During the summer of 1996, elevated mortalities of Atlantic salmon were believed to be caused by an unknown aetiological agent. The sole diagnostic lesion of this disease, referred to as haemorrhagic kidney syndrome (HKS), was identified by histological analysis. Samples were collected from an affected site in February 1997 and subjected to bacteriology, cell culture and PCR analysis. Cytopathic effects (CPE=s) were quickly apparent following application of tissue samples to the salmon head kidney (SHK) cell line. In a virulence assessment, a cell free lysate of these cultures could kill smolt. SHK lesions were produced from all of the sites known as "HKS" positive during the summer of 1997. Subsequently, electron micrographs of viral particles within the SHK cell lysates suggested a strong superficial resemblance to published micrographs of ISAV. Through the cooperation of colleagues at the Central Veterinary Laboratory in Oslo, Norway, it was determined that the particles were indeed ISAV based on strong immunofluorescence with a monoclonal antibody and the amplification of ISAV specific cDNA fragments by RT-PCR. During the current presentation, an overview of ISAV identification in Atlantic Canada will be given in addition to comparison of culture, RT-PCR and IFAT methods and their significance in the screening of clinical and sub-clinical manifestations of the disease.

Infectious Salmon Anaemia Virus: Pathology and Transmission in Fresh and Salt Water

LL Brown (1)*, AM MacKinnon (2) and HW Ferguson (3)

1	National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, NS, B3H 3Z1, Canada. mailto:laura.brown@nrc.ca
2	Fisheries and Oceans Canada, Maritimes Region, P.O. Box 5030, Moncton, NB, E1C 9B6, Canada MacKinnonA@mar.dfo-mpo.gc.ca
3	Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada mailto:hferguso@uoguelph.ca

High mortalities within farmed Atlantic salmon in the Bay of Fundy in 1996 and 1997 were attributed to Hemorrhagic Kidney Syndrome (HKS). Tissues from 7 infected fish were homogenized, diluted to 10, 5, and 2.5 % w/v in saline and injected into (n =10 per concentration) Atlantic salmon (200-400 g), held in seawater at 10C. Mortalities were seen in all groups by day 39. All mortalities were confirmed as positive for HKS. No mortalities were seen in the control fish (injected with saline only). Tissues from these experimentally infected fish were inoculated onto Salmon Head Kidney (SHK) cells. A virus was isolated from the SHK cells and characterized as the Infectious Salmon Anaemia Virus (ISAV) by RT-PCR. In order to establish a causal relationship between ISAV and HKS, three selected concentrations of this ISAV isolate were injected i.p. into 3 groups of Atlantic salmon parr (50 g each) held in freshwater. Additional salmon parr were injected i.p. with the ISAV isolate and placed in tanks with non-injected fish in order to evaluate the possibility of horizontal transmission of ISAV in freshwater. We are presently monitoring mortalities and assaying tissue samples at selected times post-infection for pathological examination. We will compare the efficacy of selected virus detection methods: IFAT, culture on SHK cells, and histology. Antibody titres will be determined using a mAb against Atlantic salmon Ig.

Pathogenesis of White Spot Virus (WSV) in Penaeid Shrimps from South Carolina

RM Krol (1)*, WE Hawkins (1), RM Overstreet (1), KK Vijayan (2) and JM Lotz (1)

1	Gulf Coast Research Laboratory, Institute of Marine Sciences, University of Southern Mississippi, Ocean Springs, Mississippi 39566 USA. rkrol@seahorse.ims.usm.edu
2	Central Institute of Brackishwater Aquaculture, Madras, India.

White spot virus (WSV) is a significant disease of cultured shrimp in Asia. WSV has been reported rarely in the U.S. In 1996, a presumed WSV outbreak occurred in cultured shrimps from South Carolina. One month after the outbreak began, gill, cuticle, and lymphoid organ from five Penaeus vannamei and five P. setiferus overwintering together in raceways were collected and examined by transmission electron microscopy (TEM). No virions were found, but three P. setiferus were positive for the virus in a bioassay in which a filtered inoculum prepared from cephalothorax tissues was injected into the tail of specific-pathogen-free P. vannamei used as the sentinel animal. One of the P. setiferus was the WSV source for further experimental feeding and injection studies. In several experimental exposures, gill, epidermis, lymphoid organ, foregut, heart, muscle, and antennal gland were examined by TEM and found positive for WSV. It was a rod-shaped, enveloped, non-occluded virus that replicated in hypertrophied nuclei containing a central virogenic stroma and marginated chromatin. The virogenic stroma contained membranous profiles, stacks of tubules, small empty capsids surrounded by open-ended envelopes, and large, dense virions, with a nipple-like projection, sometimes organized in parallel arrays. Lysed cells released virions. Clumps of virions occured in the cytoplasm of lymphoid organ cells. This study showed that the pathogenesis and ultrastructure of the South Carolina virus resembled Chinese and Indian WSV-infected P. vannamei from experimental infections. Supported by USDA, CSREES Grant 96-38808-2580.

Histopathology and Epizootiology of the North American Strain of Viral Hemorrhagic Septicemia Virus in Pacific Herring in Prince William Sound, Alaska

GD Marty (1)* and TR Meyers (2)

1	VM:APC, Univ. of California, 1 Shields Ave., Davis, CA 95616-8732 USA. gdmarty@ucdavis.edu
2	Alaska Dept. of Fish and Game, P.O. Box 25526, Juneau, AK 99802 USA. Fishpath@FishGame.state.ak.us

In years when the Pacific herring (Clupea pallasi) population of Prince William Sound (PWS), Alaska, is dominated by young fish, expression of the North American strain of viral hemorrhagic septicemia virus (VHSV) may contribute to mortality significant at the population level. Between fall 1992 and spring 1993, the biomass of mature Pacific herring in PWS declined nearly 80%, and VHSV was the most significant pathogen isolated. From 1994 to 1998, Pacific herring were sampled annually in the spring (before and during spawning, n = 230-260/year) and in the fall (n = 80-160/year). Among spring samples, VHSV prevalence steadily declined from 4.7% (1994) to 1.8% (1995) and 0.0% (1996), but unexpectedly increased to 15% in 1997. VHSV was never isolated from Pacific herring sampled in the fall. Expression of VHSV was more common in young fish, females, and fish with severe external lesions. Microscopic lesions significantly associated with VHSV included hepatocellular coagulative necrosis, submucosal lymphocytic gastritis, decreased numbers of hematopoietic cells in the trunk kidney, and an absence of inflammatory cells in the heart and liver. Absence of inflammatory and hematopoietic cells in multiple organs provides evidence that immunosuppression is associated with VHSV expression. Immunosuppression during early spring is also consistent with Pacific herring life history: early spring is when fish are nearing the end of their winter fast and are coming into spawning condition.

Susceptibility of Seabream Sparus aurata Fry and Adult Fish to Nodavirus Disease

GP Skliris (1)*, RH Richards (1), SE Papoutsoglou (2) and G Fleris (2)

1	The Institute of Aquaculture,University of Stirling, Stirling FK9 4LA, Scotland,UK george.skliris@stir.ac.uk ; r.h.richards@stir.ac.uk
2	Agricultural University of Athens, Laboratory of Applied Hydrobiology, Votanikos, Athens, 11855, Greece; sof@auadec.aua.ariadne-t.gr

Seabream (Sparus aurata) and seabass (Dicentrachus labrax) are the most important species for Mediterranean Aquaculture. The difference between them is that the latter has suffered extensive loss in the past due to Nodavirus infection while the former appears to be refractory to the disease. However in a recently published report, nodavirus-like particles were observed in the retina of seabream larvae. The aim of this study was to investigate the susceptibility of fry and adult fish to two nodavirus isolates. Therefore a series of experiments was conducted in order to investigate the above hypothesis. Waterborne, intraperitoneal (i/p) and intramuscular (i/m) challenges for the fry and only intraperitoneal (i/p) for the adult fish were carried out and evaluated as different challenge methods. Samples for virology were collected on a weekly basis and blood samples were taken from adult fish for serological studies. Brains of adult fish were also collected for examination by transmission electron microscopy. The effect of temperature was also examined (21-23C and 24-26C). Appropriate controls were included. Mortality rates were higher when the temperature was increased to 24-26C. Clinical signs were observed in some fry experiments while adult fish were uninfected. Intraperitoneal and intramuscular challenges were more unsuccessful than waterborne infections. Nodavirus was re-isolated from the majority of the examined samples (55/57) while control samples were negative. Nodavirus was also confirmed by transmission electron microscopy. Moreover serological studies showed that adult fish produced antibodies (80-160 mean titre) against both isolates. In conclusion the above experiments suggest that fry and adult seabream can be infected with nodavirus under laboratory conditions. Further work will be needed in order to confirm the above preliminary results and assess their field significance.

An Unusual Subcellular Agent Associated with Damselfish Neurofibromatosis

MC Schmale(1)*, PDL Gibbs (1), C Campbell (1), S Kamper (2), SD Baribeau (1) and SM Cacal (1)

1	Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149
2	Department of Microbiology and Immunology, School of Medicine, University of Miami, Miami, FL 33101

Damselfish neurofibromatosis (DNF) is a disease affecting bicolor damselfish (Pomacentrus partitus) which is characterized by multiple neurofibromas, neurofibrosarcomas and chromatophoromas. These tumors can be transmitted via intramuscular injection of cell-free tumor homogenates (0.22m filtrates) as well as by tumor cell lines maintained for many years in vitro. Injection of tissue homogenates from healthy fish or cells cultured from healthy fish does not induce tumor formation. These findings suggest that DNF is induced by a sub-cellular agent. We have recently identified a group of extrachromosomal DNA forms ranging in size from 1.4 to 7 kb in DNF tumors. We have tentatively termed this closely related group of DNAs the unknown DNF agent or DNFX. Southern blot analyses have demonstrated an apparently identical pattern of DNFX DNA in essentially all spontaneously occurring and experimentally induced tumors as well as tumor-derived cell cultures and cell lines. These DNAs also occur in other organs of DNF affected fish but are relatively rare in healthy fish. Preliminary analysis of the most common, 1.4 kb form of DNFX has indicated that this DNA exists in an unusual structure, consisting of a 1 kb closed loop hairpin and a 0.5 kb partially single stranded loop region. The larger forms may be replicative intermediates or byproducts. Preliminary northern analyses have indicated the presence of at least 11 transcripts in infected cells. Although DNFX may be a virus, we have not detected an association of DNFX DNA with viral particles. This disease appears to be the only naturally occurring, transmissible cancer affecting any neuroectodermally derived cell type (Schwann cells and chromatophores in the case of DNF) and thus may provide a unique and important model for investigating viral carcinogenesis in these cell types. Supported by USPHS grants NS21997 and ES05705.

SESSION 13. IMMUNOLOGY II: Tilapia and Catfish

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Lytic Peptide Expression in Channel Catfish (Ictalurus punctatus)

RK Cooper (1)*, BE Smith (1), QZ Zhang (1) and TR Tiersch (2)

1	1 Dept. of Veterinary Science Louisiana Agricultural Center, Louisiana State University Baton Rouge, LA 70803. rcooper@agctr.lsu.edu; bsawyer@agctr.lsu.edu; .qzhang@agctr.lsu.edu.
2	School of Forestry Wildlife and Fisheries, Louisiana Agricultural Center, Louisiana State University, Baton Rouge, LA 70803, ttiersch@agctr.lsu.edu.

Bacterial diseases account for more than half of the disease mortality in the channel catfish industry with few available treatment tools at the farmers' disposal. Currently, there are only 2 antibiotics approved for use in channel catfish, and resistant strains of Edwardsiella ictaluri and Flexibacter columnare have been observed in an effort to enhance the immune system of catfish to resist bacterial infection, a gene encoding a lytic peptide, cecropin B, under the control of an acute phase promoter was delivered to fish. Presence of the gene was determined using PCR (on genomic DNA) and ISPCR (on cells from known transgenic fish), and in at least one group, has been stable for 4 years. To demonstrate expression, channel catfish fingerlings were lipofected with the cecropin construct and held for two weeks. The potentially transgenic fish and the controls were challenged with Edwardsiella ictaluri, held 3 days, and the spleen and hind kidneys sampled for presence of the bacteria. Catfish receiving the cecropin gene demonstrated either no bacteria or reduced numbers compared to the controls, of which all were positive for E. ictaluri. In this study, we have demonstrated stable maintenance of a gene encoding a lytic peptide and shown an increased clearance rate of E. ictaluri in the channel catfish.

Humoral Antibody Responses of Channel Catfish Fry Exposed to Live Edwardsiella ictaluri

L Petrie-Hanson* and J Ainsworth

College of Veterinary Medicine, Mississippi State University, P.O. Box 9825, Mississippi State, MS 39762 USA. lora@cvm.msstate.edu

We evaluated the ability of catfish fry and fingerlings to develop antibody titers to Edwardsiella ictaluri. Channel catfish fry and fingerlings were exposed to live bacterial suspensions at weekly intervals starting at hatch. Specific antibody titers were determined at 2 weeks post-exposure by ELISA. Specific antibody titers were first detected in fry that were exposed to live bacteria at 3 and 4 weeks post-hatch. In a study involving secondary exposures, a heightened secondary response was first demonstrated in fry that received a primary exposure at 4 weeks post-hatch and a secondary exposure at 8 weeks post-hatch. Fry receiving primary and secondary exposures at 3 and 7 weeks post-hatch did not demonstrate secondary responses. The lack of a response upon secondary exposure in fish that were of an age that could produce a detectable primary response implies induction of pre-development immunological tolerance. Antibody titers were correlated with age. The development of humoral immunity was directly related to the development of the lymphoid organs. These studies suggest that the majority of a channel catfish population may not be considered immunologically developed until 3 to 4 weeks of age and vaccination before that time may be ineffective and potentially detrimental to the fish upon natural re-exposure.

A Comparison of the Immune Response of Clonal Lines of Nile Tilapia (Oreochromis niloticus L)

MRI Sarder, KD Thompson*, DJ Penman, A Adams and BJ McAndrew

Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA UK. kdt1@stir.ac.uk

The importance of genetic variation on the specific and non-specific immune responses of Nile tilapia (Oreochromis niloticus L) clonal lines was examined. Homozygous inbred lines of Nile tilapia were successfully produced using mitotic gynogenesis and then clonal lines were produced from mitotic gynogenetic females by meiotic gynogenesis. Homozygocity, based on differences in Major Histocompatibitiy Complex (MHC) class II B genotype, was assessed in the clonal lines using polymerase chain reaction (PCR) amplification. Two primers specific for a MHC class II B gene identified multiple loci on the chromosome, with the number of amplified loci varying between individuals. PCR products showed that the mitotic gynogens were homozygous for one or other of the maternal alleles and the clones were identical to their mitotic mother. Multifocal DNA fingerprinting also showed parental inheritance in the gyngenetic offspring, with both meiotic and mitotic gynogens possessing bands of inheritance from their mothers, but not their fathers. Specific and non-specific immune responses were compared between the clonal groups of tilapia. Firstly, scale grafting was used to examine MHC-restriction of transplantation immunity, and secondly a variety of non-specific immune responses were compared, including serum lysozyme activity, respiratory burst and phagocytosis. Significant differences in these tests were observed between the clonal groups. Their natural resistance to Aeromonas hydrophila infection was also assessed by bacterial challenge. A correlation was observed between the level of infection obtained and non-specific immune parameters measured. Cumulative mortalities of fish obtained in the study showed that when a clonal line susceptible to A. hydrophila was crossed with a resistant clonal line, the resulting progeny exhibited intermediate levels of resistance to that of their parents.

Immunoglobulin, Antibody and Ig Positive Cells in the Epidermis of Channel Catfish, Ictalurus punctatus

D Zilberg (1,2)* and PH Klesius (1)

1	U.S Department of Agriculture, Agricultural Research Service, Fish Diseases and Parasites Research Laboratory, 990 Wire Road, Auburn, AL 36830 USA. agross@acesag.auburn.edu
2	Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36830 USA

Cutaneous mucus immunoglobulin (Ig), antibody (Ab) and their relation to serum Ig and Ab were studied in channel catfish. Ig levels in serum of healthy fish were much higher than in their cutaneous mucus (~0.5 mg ml-1 and ~0.5 ng cm-2, respectively). The kinetics of immunoglobulin production following infection with Edwardsiella ictaluri showed an increase in serum Ig 13 days post infection. The increase in mucus Ig concentration occurred 14 days later. Ab response to E. ictaluri was detected in serum, but not in the mucus of infected fish. Catfish infected with Ichthyophthirius multifiliis had an increase in mucus, but not in serum Ig concentration 18 days post infection. Serum and mucus Ig levels in both E. ictaluri and I. multifiliis infected fish did not correlate. Fish immunized with dinitrophenylated human serum albumin (DNP-HuSA) had much higher serum than mucus anti-DNP Ab titers following parenteral immunization (1:5120 vs. 1:16, respectively). An indirect immunofluorescence method was used to identify Ig-producing cells in catfish skin. Cells were located in the 3-4 top and bottom epidermal cell layers, in the epidermal-dermal junction and occasionally in the middle, between alarm substance cells. Cell diameter was 5-7mm, within the size range of catfish blood lymphocytes. Skin sections from fish infected with I. multifiliis had Ig producing cells surrounding the parasite's trophonts. This study suggests that in channel catfish, cutaneous mucus Ig is locally produced in the epidermis.

Social Stress Suppresses Neutrophil Activities in Tilapia

T Iida* and J Kurogi

Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-Nishi, Miyazaki 889-2192, Japan a0c203u@cc.miyazaki-u.ac.jp

Neutrophil activities were assessed under social stress in tilapia, Oreochromis niloticus. We made five pairs of large (average weight of 144g) and small fish (average weight of 88g). For control, similar-sized fish were held individually. Within a few hours post-pairing, large fish became dominant in all pairs, and charged, chased and rammed subordinated small fish. Cortisol and glucose levels in plasma of subordinates were significantly higher than in either the dominants or the controls, indicating that subordinates were under stressful conditions. Both fish were injected with formalin-killed Escherichia coli (1 mg/fish) into swim bladder just before pairing, and after 24 hours neutrophils exuded into swim bladder were collected and defense activities of the neutrophils were measured. Neutrophil activities from subordinates were significantly decreased in number of cells filtrated into the swim bladder, phagocytosis (phagocytic rate and phagocytic index) and respiratory burst by chemiluminescence. This result indicates that stressed fish will be easily infected with even facultative pathogens. Respiratory burst of dominants was higher than controls, suggesting that light stress stimulates the defense activity.

Immunodiagnosis: Immunological Status of Infested and Immunized Catfish Clarias lazera

NM El-Bahy*

Faculty of Veterinary Medicine, Tanta University, Department of Parasitology, KAFR El-Shikh, Egypt.

The immune status of fish infested with helminth parasites was studied. Crude antigen prepared from some nematodes and some crested worms were used in immunization of parasite-free catfish. Comparison between serum proteins of infested and immunized fish on the basis of Gel electrophoresis were done. Agar gel precipitation tests were used to clarify the diagnostic value of the prepared antigens. There were similar bands in the sera of infested and immunized fish and the crude antigen. These bands are not found in the serum of parasite-free catfish. The results hold promise for the early diagnosis of parasites of zoonotic importance.

SESSION 14. PARASITOLOGY IV: Ciliates, Dinoflagellates, Flagellates

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Identification of Ciliated Protozoans in the Respiratory Tract, Skin, and Somatic Lymph Nodes of Bottlenose Dolphins (Tursiops truncatus) from California, USA

KD Arkush (1)*, WG Van Bonn (2) and SL Poynton (3)

1	University of California, Bodega Marine Laboratory, P.O. Box 247, Bodega Bay, CA 94923 USA kdarkush@ucdavis.edu
2	Upstream Associates, P.O. Box 60680, San Diego, CA 92166 USA. vanbonn@nosc.mil
3	Division of Comparative Medicine, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205 USA. spoynton@welchlink.welch.jhu.edu

Few reports of commensal or parasitic protozoa in marine mammals exist. The apparent paucity of protozoa in cetaceans is not necessarily an indication of the rarity of their occurrence, but rather of the rarity of reporting. We report here on the identification of two ciliated protozoans in the respiratory tract of apparently healthy bottlenose dolphins held at the Space and Naval Warfare Systems Center San Diego facility at San Diego Bay, California. The organisms are frequently collected by introducing a swab into the spiracular cavity, vestibular sac(s), or nasal passage(s) of the dolphins during routine physical examination. Bouins fixed material was stained by Protargol impregnation to reveal details of the ciliature. Kyaroikeus cetarius was commonly found, and has previously been reported from cetaceans from the Atlantic Coast of the USA. We believe this to be the first report of K. cetarius from the Pacific Coast. An organism similar to a Kyaroikeus sp. was detected in histological sections of lymph nodes of a geriatric bottlenose dolphin from the same facility. The primary cause of death was bronchointerstitial pneumonia. However, lymphadenitis with intralesional ciliated protozoa and focal dermal abscessation and ulceration with intralesional protozoa were noted. This is a rare finding and one of potential clinical significance. The chilodonellid ciliates, occasionally recovered from the respiratory tract, are newly reported from marine mammals. Chilodonellids are known pathogens of fish and similar host-parasite interactions may be expected in cetaceans.

An Unusual Parasitic Dinoflagellate in Prawns, Pandalus platyceros, in British Columbia

SM Bower and GR Meyer

Department of Fisheries and Oceans, Pacific Biological Station, Nanaimo, B.C. V9R 5K6, Canada BowerS@dfo-mpo.gc.ca

A parasitic dinoflagellate with large plasmodia characteristic of Syndinium (parasitic in radiolarians, amphipods and copepods) and numerous trophonts typical of Hematodinium (parasitic in crabs and lobsters) occurred in up to 27% of the prawns from Malaspina Strait, British Columbia. Infections in most prawns were cryptic but of sufficient duration to affect secondary sexual characteristics and castrate the host. Cryptic infections consisted of plasmodia containing numerous pleomorphic nuclei that lacked condensed chromatin characteristic of Syndinium. Examination via electron microscopy revealed that the outer membrane in some locations along the surface of the plasmodium was indistinct and the cytoplasm of the parasite appeared to coalesce with the cytoplasm of lysed haemocytes. The plasmodia invaded the haemal sinuses of all tissues then broke up into trophonts with single nuclei. Prawns with gross evidence of infection (body discolouration, lethargy and haemolymph milky with a plethora of either mainly round or mainly discoid trophonts) rarely exceeded a prevalence of 2%. In a few prawns with round trophonts, about 25% of the trophonts were dividing and either had one nucleus containing mitotic figures in which the chromosomes were attached by microtubules (spindle fibers) to centriole-like structures at the nuclear membrane or had two nuclei. Attempts to transmit the infection between prawns in the laboratory were unsuccessful. Trichocysts in the cytoplasm, a feature typical of at least one life stage of most Syndinida, were not found in the prawn parasite. Also, a flagellated stage (proposed to be the free-living infectious stage for some species) was not observed.

"Bumper car" Disease - A Model of Health and Infectious Disease Processes in American Lobsters

RJ Cawthorn*, RJ MacMillan and B Despres

Lobster Health Research Centre, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, C1A 4P3 CANADA. cawthorn@upei.ca, rmacmillan@upei.ca, bdespres@upei.ca

"Bumper car" disease, induced by the scuticociliate Anophryoides haemophila, is an excellent model to study health, and other infectious diseases, in American lobsters, Homarus americanus. In vitro, ciliates are maintained in a cell-free, chemically defined medium at 5C, or stored in the liquid phase of liquid nitrogen refrigerators (11% dimethylsulfoxide as cryoprotectant) for extended periods. The efficacies of potential disinfectants and therapeutants against A. haemophila are rapidly screened with a tetrazolium-based metabolic assay. These methodologies have recently been applied to the pathogenic scuticociliates Mesanophrys pugettensis (from Dungeness crabs) and M. chesapeakensis (from blue crabs), and an unidentified ciliate from hermit crabs. In vivo, Anophryoides haemophila is readily transmitted from lobster-to-lobster by intrahaemocoelic injection. The Aquatic Animal Facility, with a saltwater recirculation system, is highly controlled and allows manipulation of many environmental parameters to determine their effect(s) on the developmental cycle and pathogenesis of A. haemophila in lobsters. Utilizing immunological and molecular techniques, we determined that the ciliates initially develop in gills, heart and muscle tissues. Subsequently, the presence of ciliates in haemolymph indicates that lobsters are terminally ill. Overall, the model of "Bumper car" disease in lobsters, with multidisciplinary and multifaceted approaches, facilitates examination of health and infectious diseases in these and other crustaceans.

Protozoan Ectoparasites of Fish from the Salton Sea, California

B Kuperman* and V Matey

Center for Inland Waters, San Diego State University, Biology Department, 5500 Campanile Dr., San Diego, California 92182-4614 USA. kuperman@sunstroke.sdsu.edu

The Salton Sea is a great endangered lake in southern California with an extremely high levels of water salinity - up to 45 ppt, temperature - up to 37C in the shore area, eutrophication, and pollution. These factors are considered as the main reasons for recent massive fish die-offs. We have carried out a parasitological investigation on 221 tilapia Oreochromis mossambicus, the dominant fish species in the Salton Sea. For the first time three species of protozoan parasites Amyloodinium ocellatum (Dinoflagellida), Cryptobia sp. (Kinetoplastida), and Ambiphrya ameiuri (Peritrichida) have been revealed in young tilapia (L 1.8 - 5.3) in June - October, 1997. The ultrastructure of these protozoan has been studied with SEM. Infestation of fish with these species of protozoan occurred not at the same time and varied from 89% to 100%. Massive infestation of fish gills with trophonts of A. ocellatum has caused a severe distortion of general gill structure and erosion of epithelium in the sites of parasites attachment. Under an extremely high level of infection with Cryptobia sp. the swellin and local fusion of gill lamellae and filaments were typical for infected fishes. Flagellates have been spread along the gill filaments, arches and between gill rakers. A. ameiuri, attached to the fish body, covered a large area of skin surface and reduced the cutaneous respiration of young fish. We suppose that the parasitological factor associated with some environmental and anthropogenous stressors can be one of the reasons for the disease and death of young fish in the Salton Sea.

Flagellates from Cetaceans: Identification Techniques, Morphology, and Clinical Significance

AB Heinrich (1) *, SL Poynton (1) and B Whitaker (2)

1	1 Division of Comparative Medicine, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205 USA. aheinri@gwgate1.jhmi.jhu.edu; spoynton@welchlink.welch.jhu.edu
2	National Aquarium in Baltimore, Pier 3, 501 East Pratt Street, Baltimore, MD 21202, USA bwhitaker@aqua.org

Extensive surveys have been conducted on metazoan parasites from cetaceans, but there are few studies of protozoans from these hosts. Reports include ciliates from the blowhole, skin, and baleen plates, and apicomplexans from the skeletal muscles and internal organs. The paucity of published information concerning protozoa from cetaceans largely reflects the practical difficulties encountered when studying such small and delicate parasites, infecting large hosts from relatively inaccessible environments. Material for the present study included samples taken from the blowhole of a stranded pygmy sperm whale (Kogia breviceps) and a stranded harbor porpoise (Phocoena phocoena) held at the National Aquarium in Baltimore during rehabilitation. These samples were taken by holding a petri dish over the blowhole while the animal exhaled 3-5 times. Other samples were obtained by taking swabs of the blowhole of several captive bottlenose dolphins (Tursiops truncatus) from San Diego. Live flagellates were documented by photography and video recording, air-dried smears were stained with Giemsa, and Bouin's fixed organisms were stained using protargol (silver protein). All three cetacean species were infected with flagellates, tentatively identified as kinetoplastids. Both the pygmy sperm whale and the bottlenose dolphins carried organisms believed to be Cryptobia spp. Members of this genus are usually reported as ectocommensals and endocommensals of fish; only rarely are they pathogenic. An ectocommensal relationship with the cetacean hosts was also evident in our studies, since the flagellates were commonly present in apparently healthy animals.

SESSION 15. NUTRITION

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Possible Cause for Thiamine Deficiency Associated with Sac-Fry Mortality in Early Mortality Syndrome in Feral Great Lakes Salmonid Species.

DC Honeyfield

Research and Development Laboratory, USGS/BRD RD #4 Box 63, Wellsboro, PA 16901 USA. 1. honeyfie@epix.net

Early mortality syndrome (EMS) results in death of salmonid sac-fry from the Great Lakes, New York Finger Lakes, and the Baltic Sea. Clinical signs in affected species include erratic swimming, hyperexcitability, dark coloration, lethargy, anemia, emaciation, and anorexia prior to death. Early mortality syndrome in affected salmonid species (Atlantic salmon, coho salmon, chinook salmon, steelhead and lake trout) from 1972 to 1992 was generally less than 20%. Since then mortality has risen dramatically (for example 60%-100% in coho salmon). Hypothesized causes of EMS include thiamine deficiency, contaminants, exotic species introduction and food web differences associated with ecosystem changes. The alewife, a non-indigenous forage species within the Great Lakes contains thiaminase, an enzyme that degrades thiamine. Consistently, egg thiamine concentrations are lower in affected fish. Successful egg and sac-fry treatment with thiamine highlights the importance of thiamine in EMS. However, treatment of eggs or sac-fry does not address the underlying cause of EMS. Two microbial strains of thiaminase positive bacteria were recently isolated from alewife viscera. Bacillus thiaminolyticus and an un-named Bacillus species were cultured and identified by fatty acid methyl ester analysis. This is the first evidence linking bacterial thiaminase activity with a fish species known to contain thiaminase. This suggests that thiaminase activity in the alewife may fluctuate and this could be one reason for variability observed in EMS occurrences from year to year.

Effects of ß-Carotene Supplementation on the Growth and Immune Response of Rainbow Trout Oncorhynchus mykiss

V Kiron*, EC Amar, N Okamoto, S Satoh and T Watanabe

Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato, Tokyo 108-8477, Japan

Carotenoids have been found to influence immune function in higher vertebrates and recently in fish. In this study, rainbow trout of average an size of 45g were fed diets supplemented with different levels of ß-carotene for 12 weeks to determine whether this substance modulates growth and immune function. Semi-purified casein based diets were formulated to contain 0, 40, 200 and 400 mg ß-carotene/kg dry diet. At the end of the feeding, trial no marked differences in specific growth rate and feed conversion were detected. Of the immune parameters measured, total plasma immunoglobulin was significantly higher (10.62 mg/ml) in the group fed the diet containing 200 mg ß-carotene/kg diet. Serum complement activity (ACH 50) was highest (314.5 units / ml) in group fed 400 mg ß-carotene, but this was significantly different from that of the carotenoid deprived group. Differences in lysozyme activity noted among the groups were not significant and ranged between 14.2-16.7 mg/ml. Phagocytic activity of head kidney cells was not markedly different among the diet groups though it was greater for the higher carotenoid levels. NBT reduction by peripheral blood leukocytes appeared to be lower at the higher ß-carotene levels (range 0.29-0.38; OD540). Overall, dietary supplementation of ß-carotene influenced some of the immune functions in rainbow trout, but not growth.

Ovarian Ceroidosis in a Cichlid, Prognathochromis periri: A Clinical Assessment

R Klinger*(1), R Francis-Floyd (1) and Vicki Blazer (2)

1	1 Department of Fisheries and Aquatic Sciences and Department of Large Animal Clinical Sciences, University of Florida, 7922 NW 71st Street, Gainesville, Florida 32653 USA. rek@gnv.ifas.ufl.edu; rff@gnv.ifas.ufl.edu
2	National Fish Health Research Laboratory, Biological Resources Division, United States Geological Survey, Kearneysville, West Virginia 25430 USA. Vicki_Blazer@usgs.gov

A clinical case was presented to our laboratory involving Lake Victoria cichlids, Prognathochromis periri. Twelve adults, F1 generation from wild caught fish, had been held in a 55 gallon home aquarium for six years. The client was successfully breeding them every year until six months prior to submission. The females had inappetance, eroded fins and abdominal swelling. Males had curled operculums but appeared healthy otherwise (including good appetites). Four females and one male were examined grossly. Ovaries were enlarged and waxy with intermittant large fluid- filled sacs. Two females had granulomas in the posterior kidney and liver while the male had multifocal granulomas throughout its internal organs. Histological examination of both sexes revealed multifocal ceroid deposits with large areas of necrosis, hemorrhage and inflammation in most internal organs. Ovarian tissue was nearly replaced with ceroid and necrosis. The presentation of ceroid and necrosis and the severity, especially in the ovarian tissue, suggests a nutritional etiology, most likely a chronic reaction to lipid oxidation.

The Impact of Dietary Vitamin E and Dissolved Oxygen Supplementation on Immune Response Hematology and Antioxidant Status in Atlantic Salmon (Salmo salar)

B Lygren*, K Hamre and R Waagbø

Institute of Nutrition, Directorate of Fisheries, P.O.Box 185, N 5002 Bergen, Norway bjarte.lygren@fiskeridir.no; kristin.hamre@fiskeridir.no; rune.waagboe@fiskeridir.no

In this study we examined the effect of low, normal and high dietary levels of vitamin E on immune response, hematological parameters and antioxidant status in Atlantic salmon under normoxic and hyperoxic rearing conditions. The use of dissolved oxygen levels above saturation is believed by some fish farmers to improve fish health. The fat-soluble dietary antioxidant, vitamin E, protects biomembranes against lipid peroxidation and seems to be important for optimal function of the immune system in salmonids. After six weeks of treatment the fish were immunized with Aeromonas salmonicida ssp. salmonicida and Vibrio anguillarum O1. The immune response was evaluated by measuring specific immune parameters including the level of specific antibodies (ELISA) and the number of antibody producing cells (ELISPOT), as well as the nonspecific parameters; serum lysozyme activity, spontaneous -and antibody specific complement mediated hemolytic activities and phagocytic respiratory burst activity. In addition, the hematological health parameters; hematocrit, hemoglobin, and the number of red blood cells were determined. The antioxidant status was evaluated by measuring the dietary antioxidants; vitamin E and vitamin C and the endogenous antioxidants; catalase, superoxide dismutase, glutathione peroxidase, glutathione and total level of thiols. The results from this study will be presented.

Novel Harmless Inert Marker for Digestibility Determinations in Fish

AO Meyer-Willerer*

Centro Universitario de Investigaciones Oceanológicas, Universidad de Colima, Departamento de Acuacultura, km 20 Carretera Manzanillo - Barra de Navidad, Manzanillo, Colima 28860 MEXICO. ameyer@cgic.ucol.mx

The apparent protein digestibility coefficient (APDC) method uses inert markers such as chromic oxide, acid insoluble ash, silicate or radioactive compounds. These coefficients may be similar regardless of the marker used, but in some cases they may represent 1.0% of the feed, an amount that may change the coefficients of variation calculated for APDC. The estimates of absorption efficiency are normally higher when silicate is used instead of ash, except when the proportion of silicate in the food is very low, thus giving low precision with high coefficients of variation. The proposed new markers for APDC are diatom skeletons. They are concentrated from specific cultures, cleaned by sulfuric acid oxidation and washed with distilled water. Diatom skeletons are selected by size and are included in the feeds as low as 0.01% binding with gelatin. A marked feed is offered to a separated tranquilized fish held in a clean aquarium ("pulse"), later it is fed in the usual way ("chase"). The feces are collected periodically and half of them are digested separately for diatoms, the other half is processed for protein determination. Each diatom sample is counted under a light microscope and the total amount in the different fractions is useful for recording accurate digestion times. Simultaneous use of unlike diatom genera may be helpful for better understanding of digestion times. Tilapia were fed isoproteic isocaloric diets prepared with different fishmeals and compared with commercial first-class diets. The growth curves, digestion times and APDC for each feed will be reviewed.

The Use of a Riched Diet Added with Antibiotic Growth Promoter to Improve Hatchability Alligator Syndrome and Growth Retardation in Closed Environment

V Bermùdez, I Pineda, V Blanco, G Garcìa, M Rossini and J Moreno

FCV-UCV, Cat. Clin. Bàsuca, El Limòn, Marncay, AP-4563, Edo, Aragua, Venezuela, South America

Hatchability Alligator Syndrome (HAS) considerably decreased the gator population at hatching period negatively impacting on the year crop with significant economic loss for the farm. Another important situation at intensive units is the growth retardation that small percentage of the crop can suffer for many reasons. The latter is responsible for an increase of mortality rate between the first week of age until they reach to six month which become the stage of greater long axis development of large bones meaning less weight and development of the gators at market age; translating into more economic loss for a highly demanding cost operation in Venezuela nowadays. For experimental purposes and due to the fact that the mortality rate was so high at HAS period and first six month of age at one of the most highly developed alligator farm operations in Central Northern Venezuela, the following design was proposed. Takin into account that the mortality rate was high between 1994 (26%; 1,551 out of 5,966 gators) and 1995 (24.7%; 1130 out of 4,575 gators) crops, in 1996 a field case-control designwas done using four experimental groups (n=100 gators each) that were randomly assigned and located in one roofed water tank (Australian model) each group had a replicate of similar number and diet. The diet was commercial concentrate feed (Group A), commercial feed plus pecutrin (mineral from Bayer's lab; Group B), comercial feed plus pecutrin and Virginiamycin (Stagac, Pfizer/Smith & Kline at 10 ppm; Group C) and beef plus Virginiamycin 10 ppm and pecutrin (GroupD) given from birth to six months of age. Temperature in the tank was around 42ºC and humidity 70% average. Total mortality rate and growth retardation were evaluated during the experiment. The overall rate 20.1% (1,370 out of 6,816 gators). The lowest mortality rate was signigicantly found to be in groups C (p<0.05) and D where there was no growth retardation as compared to the other groups. These results demonstrate the beneficial impact that growth promoter antibiotics (Virginiamycin) can offer not only to swine operations but to the gator farms in which HAS and growth retardation can be significantly controlled.

SESSION 16. EMERGING DISEASES IV: Parasites

[TOP]

Amoeba-like Organisms on the Gills Associated with Mortality of Atlantic Salmon (Salmo salar L.) in Fresh Water in Norway

H Bleie (1)*, A Levsen (2), T Alvik (2), OB Dale (3), K Nyberg (4) and AE Broederud (5)

1	National Veterinary Institute Bergen, PO Box 40, N-5032 Minde, Norway. hogne.bleie@vetinst.no.
2	Department of Zoology, University of Bergen, Allegt. 41, N-5007 Bergen, Norway. arne.levsen@zoo.uib.no.
3	National Veterinary Institute Oslo, PO Box 8156 Dep., N-0033 Oslo, Norway. ole.bendik.dale@vetinst.no.
4	National Veterinary Institute Harstad, PO Box 652, N-9401 Harstad, Norway. kjell.nyberg@vetinst.no
5	Tretten, N-9080 Storslett, Norway

During the winters of 1996-97 and 1997-98 a sudden onset of high mortality occurred in several populations of fry, parr and pre-transfer smolt of Atlantic salmon (Salmo salar L.) in several geographically separated Norwegian hatcheries. In some cases the mortality reached almost 100% in a few tanks, while fish in neighbouring tanks appeared to be unaffected. Moribund fish showed signs of asphyxiation with brown mucus secretion on the gills. Light microscopy of fresh gill tissue revealed slow-moving pleomorphic cells with pseudopodia-like protrusions extending in the direction of the movement on the surface of the gill epithelium of both moribund and apparently healthy fish from the same population. Numerous degenerating cells, bacteria, fungi, and, algae were also observed on the gills of moribund fish. Histological examination of gill tissue from affected fish in the acute phase showed a single layer of hypertrophic epithelial cells covered by larger, pleomorphic cells, some of which appeared to be attached to the underlying epithelium by multiple projections. In haematoxylin and eosin stained material several basophilic bodies of variable size, frequently arranged in a circle, were observed in the eosinophilic cytoplasm of the pleomorphic cells. These cells were assumed to be trophozoites as no cysts were found. Scanning and transmission electron microscopy of gill tissue revealed cells which corresponded in structure and size to the cells found in histological examination. The size of the cells was approximately 20x10x5mm.

Microsporidiosis in Leopard Sharks

MM Garner (1)*, TJ Miller-Morgan (2), SR Brown (3), R Reimschuessel (4), SL Poynton (5), TJ Baldwin (6) and RE Olson(2)

1	Northwest ZooPath, 18210 Waverly Drive, Snohomish, Washington 98296-4815 USA. zoopath@aol.com
2	Oregon State University, Department of Fisheries and Wildlife, Laboratory for Fish Disease Research, Hatfield Marine Science Center, 2030 Marine Science Drive, Newport, Oregon 97365 USA. millermt@ccmail.orst.edu
3	Animal Medical Center of Newport, 159 NE 10th, Newport, Oregon, 97365 and Oregon Coast Aquarium, 2820 SE Ferry Slip Road, New Port, Oregon 97365 USA. anmed@pioneer.net
4	Aquatic Pathobiology Center, University of Maryland School of Medicine, 10 S. Pine Street, Baltimore, Maryland 21201 USA. rreimsch@umabnet.ab.umd.edu
5	Division of Comparative Medicine, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore Maryland 21205 USA. spoynton@welchlink.welch.jhu.edu
6	Washington Animal Disease Diagnostic Laboratory, PO Box 2037-College Station, Pullman, Washington 99165-2037 USA. tjb@vetmed.wsu.edu

Although microsporidia are among the best known and most serious pathogens of marine teleosts, they have not previously been reported from elasmobranchs. Three captive adult leopard sharks (Triakis semifasciata) initially presented with disorientation and ventral cutaneous erythema. Two sharks did not respond to supportive care and died. Treatments had included enrofloxacin (Bayer) 13mg/l, bath; triamcinalone acetonide (Solvay) 0.2mg/kg, IM; dexamthazone sodium phosphate (Steris Laboratories) 2mg/kg, IP; and amikacin (Fort Dodge) 3mg/kg, IM. Gross lesions included ventral cutaneous erythema and meningeal congestion in two sharks and left unilateral hyphema in one shark. Histopathology revealed moderate to severe systemic inflammation and necrosis involving brain, pancreas, kidney, liver, gills and blood vessels. These processes were associated with the presence of numerous parasites in the cytoplasm of macrophages and unidentified cells in most affected tissues. These parasites had morphologic and ultrastructural features consistent with microsporidia. The organisms were pyriform spores 2 - 4 um long, containing a prominent vacuole and a coiled polar tube. A gill biopsy from the sick surviving shark revealed microsporidia. The shark responded to treatment with toltazuril (Bayer AG) 10mg/kg, PO, q 24 h x 5d, and is currently healthy. Preliminary evidence suggests that microsporidosis can be a fatal disease in sharks, infected animals can be screened by gill biopsy, and that the disease may respond to Toltazuril treatment.

Distribution and Pathology of QPX, Quahog Parasite Unknown, in Hard Clams, Mercenaria mercenaria, in Virginia, USA

LM Ragone Calvo and EM Burreson*

School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, P. O. Box 1346, Gloucester Point, Virginia 23062 USA. ragone@vims.edu; gene@vims.edu

In July 1996, the Virginia Institute of Marine Science initiated a sampling program to examine wild and cultured hard clams, Mercenaria mercenaria, for QPX, Quahog Parasite Unknown, a protistan parasite associated with severe mortalities of hard clams in localized areas in maritime Canada and Massachusetts, USA. Initially, the program monitored wild clams from one site within Chesapeake Bay, and cultured clams from two sites, one along the Atlantic coast and one within Chesapeake Bay. Samples revealed 8% prevalence of the parasite in 1-2 year old cultured clams at the coastal site; clams at the other sites were not infected. To ascertain the distribution of the parasite in Virginia, the survey was broadened to include 24 additional sites within Chesapeake Bay and along the Atlantic Coast of Virginia where harvest and culture of hard clams occur. Over 1700 clams were examined. QPX was not found in Chesapeake Bay or in wild clams, but was present in cultured clams from three coastal embayments-Chincoteague Bay, Burton Bay and Quinby Inlet. During 1997 prevalence of QPX reached 72% in Chincoteague Bay and 68% in Burton Bay where notable mortality was observed. Infections were generally light to moderate intensity and were most often observed in mantle and gill tissues. To date QPX has not significantly impacted Virginia's hard clam fishery and aquaculture industry; however, the presence of the pathogen in three of the state's most productive hard clam growout areas and the increasing prevalence and intensity of the parasite is cause for concern.

QPX, A Protozoan Parasite of Hard Clams

R. Smolowitz*

Laboratory for Aquatic Animal Medicine and Pathology, University of Pennsylvania, Marine Biological Laboratory, 7 MBL St., Woods Hole, MA, USA 02543. rsmol@mbl.edu

Hard clams, (Mercenaria mercenaria, quahog) is an important commercial species in the United States. Quahog Parasite Unknown (QPX), tentatively identified as belonging to the Phylum Labyrinthomorpha, was first identified as a cause of disease and mortality in hard clams in Canada in 1969 and again in 1989. In 1995, hard clams planted in leases in Provincetown and Duxbury, MA, USA, began to experience heavy mortalities. Examination of these cultured populations of hard clams showed the mortality was due to QPX infections. Since that time QPX had also been found in Virginia and New Jersey, although significant mortalities have not yet been directly associated with QPX infections of hard clams from those areas. In Massachusetts, mortalities are first noted in cultured clams just before they reach market size. At this stage approximately 30% of the animals show large swellings and nodules in the mantles and mantle edges. Squash preparation of these nodules demonstrate the parasites well. Histologically, the organisms appear as thalli (uninucleate round cells from 5 to 12mm in diameter) that mature into sporangia (approximately 10 to 25mm in diameter). Each sporangium contains approximately 40 endospores. Eventually the sporangia lyse and the endospores are released to mature into new thalli. The thalli and sporangia are loosely connected to each other by a mucofibrillar material. The clams' inflammatory response to the organism is intense. Culturing of QPX organism was accomplished in 1997. Several investigations are presently underway to increase our understanding of the QPX and the disease in hard clams.

Pathogenesis of Loma salmonae, a Gill Microsporidian Pathogen of Emerging Significance to Pacific Salmon Aquaculture

DJ Speare (1)*, RJF Markham (1), HJ Beaman (1), GJ Sanchez (1), J Daley (1) and SRM Jones (2)

1	Department of Pathology & Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI. Canada CIA 4P3. speare @upei.ca
2	Aqua Health Ltd, West Royalty Industrial Park, Charlottetown, PEI. Canada CIA 4P3.

Loma salmonae is a gill microsporidian parasite recently recognized as causing significant economic losses, through morbidity and mortality, for marine netpen farmers of Pacific salmon. Based on studies conducted at 15 oC in rainbow trout, we have found that after oral exposure to spores, parasite DNA is detectable by in situ DNA hybridization in gill endothelial cells two weeks post exposure (PE). Spore-wall assembly, as determined by immunohistochemistry using of a spore-wall specific monoclonal antibody, begins by week 3 PE. Most xenomas develop in size until week 9 PE and upon their dissolution trigger dramatic gill damage. An unexpected finding was that growth suppression and an early wave of branchitis began during week 4 PE in infected fish resulting from dissolution of a subset of small xenomas. In a series of infection and re-challenge studies we found that fish were protected from experimental re-infection after (1) recovery from disease, or (2) intraperitoneal exposure to non-viable spores, or (3) oral exposure to viable spores at water temperatures outside of the permissive range for the parasite. Developing methods of inducing resistance, which by-pass xenoma formation on the gills, offers key economic advantages to fish farmers rearing Pacific salmon in regions endemic for Loma salmonae.

Characterization of Achlya sp. Associated with Diseased Soft-Shelled Turtle (Trionyx sinensis) from Hong Kong

EM Leano*, LLP Vrijmoed and EBG Jones

Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, P.R. China. 95410742@plink.cityu.edu.hk; bhlilian@cityu.edu.hk; bhgareth@cityu.edu.hk

In September 1996, a disease outbreak was reported in one turtle farm in the New Territories, Hong Kong. Soft-shelled turtle (Trionyx sinensis) cultured in tanks showed white spots on the body surface and carapace resulting in gradual but high mortalities. Microscopic examination of the affected tissues revealed the involvement of thick, non-septate fungal mycelia. A fast-growing Straminipilous fungus was isolated from the infected animals and identified as Achlya sp. Physiological studies were undertaken and results show that the isolate can grow from pH 4 to 10, with strong growth at pH 5 and 9. Growth was also observed at 0-20 ppt salinities at temperature of 10-40C with optimum at 0 ppt salinity at 35C. Abundant zoosporangia were produced and primary zoospores released at 0-5 ppt salinities, but secondary zoospores were only released at 0 ppt. A wide temperature range was observed for sporulation (10-40C) with an optimum at 15-40C. This is the fist report of a fungal infection in cultured soft-shelled turtles in Hong Kong.

SESSION 17. BACTERIOLOGY II: Virulence

[TOP]

Inducible Catalase in Aeromonas salmonicida subsp. salmonicida: Possible Role in Protection Against Killing by Activated Rainbow Trout Macrophages

AC Barnes (1)*, TJ Bowden (1), MT Horne (2) and AE Ellis (1)

1	FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, Scotland.
2	Aqua Health (Europe) Ltd., Unit 31 Enterprise House, Springkerse Business Park, Stirling FK7 7UF, Scotland.

Aeromonas salmonicida subsp. salmonicida expresses a single cytoplasmic catalase. This enzyme was found to be inducible by exposure to 20µM hydrogen peroxide in exponential phase, resulting in a 4-fold increase in activity. Cultures pre-exposed to 20µM peroxide and then subsequently treated with 2mM peroxide in stationary phase exhibited a further increase in catalase activity, 20 fold higher than in uninduced cultures. Cultures exposed solely to 2mM peroxide in stationary phase were killed. Induced catalase protected A.salmonicida against exogenous sources of peroxide: Exponentially induced cultures were protected against subsequent exposure to 10mM peroxide with 22% of the culture surviving 60 min exposure. Bacteria subjected to induction in both exponential and stationary phase were resistant to 100mM peroxide, although viability was reduced to 0.001%. There was a slight increase in resistance to peroxide in stationary phase cultures compared to exponential cultures, even though there was no significant difference in the amount of catalase expressed. Effect of catalase activity on resistance of A. salmonicida to rainbow trout macrophages was also investigated. A.salmonicida MT423 (A+) was resistant to killing by normal rainbow trout macrophages, but was susceptible to killing by activated macrophages. However if catalase was induced by prior exposure to 20µM peroxide in exponential phase, A.salmonicida was also resistant to killing by activated macrophages. This suggests that catalase induction may be important to survival of A. salmonicida in the early stages after entering the host.

Effect of Temperature and Exposure to Renibacterium salmoninarum on Healing of Descaling Injuries in Juvenile Chinook Salmon Oncorhynchus tshawytscha

DG Elliott (1)*, CM Aiwohi (1) and JL Congleton (2)

1	Western Fisheries Research Center, Biological Resources Division, US Geological Survey, 6505 Northeast 65th Street, Seattle, Washington 98115 USA. diane_elliott@usgs.gov; carla_aiwohi@usgs.gov
2	Idaho Cooperative Fish and Wildlife Research Unit, Department of Fish and Wildlife Resources, University of Idaho, Moscow, Idaho 83844 USA. jconglet@uidaho.edu

Descaling is the primary criterion used to assess physical damage to salmonid smolts passing through fish collection systems at dams on the Snake and Columbia Rivers of the Pacific Northwest USA. Despite reliance on descaling to quantify physical damage associated with collection and bypass systems, little is known about the effects of descaling on health and viability of juvenile salmonids. It is also uncertain that evaluation of descaling by gross observation provides an accurate representation of the degree of tissue damage or stage of healing in a fish. Histological examination of fish collected at the uppermost dam suggested that at least 25% of fish classified as descaled by gross observation had suffered the injury before they entered the collection system. A laboratory study investigated healing of descaling injuries in chinook salmon at three temperatures (8C, 13C and 18C) within the range experienced by outmigrating smolts. Following a standardized descaling injury, integument samples were taken during 9 weeks for light and scanning electron microscopic analyses of the regeneration of the epidermis, mucous cuticle, and scales. Preliminary results indicated that epidermal closure occurred as early as 12 h after descaling at 18C, but required 24 h to 96 h at lower temperatures. Scale regeneration was grossly visible within 2 weeks at 18C, 3 weeks at 12C, and 6 weeks at 8C. Descaled and non-descaled fish challenged with waterborne Renibacterium salmoninarum (Rs) were monitored for integumentary changes and Rs infection.

A Virulence Comparison of Wild-type ATCC 33209 and Attenuated MT239 Renibacterium salmoninarum Isolates in Chinook Salmon Oncorhynchus tshawytscha

CL O'Farrell (1)*, DG Elliott (2) and ML Landolt (3)

1	School of Fisheries, University of Washington, Box 357980, Seattle, Washington 98195 USA ofarrell@fish.washington.edu
2	Western Fisheries Research Center, BRD, US Geological Survey, 6505 Northeast 65th Street, Seattle, Washington 98115 USA. diane_elliott@usgs.gov
3	Dean, The Graduate School, University of Washington, 200 Gerberding Hall, Box 351240, Seattle, Washington 98195 USA landolt@grad.washington.edu

One putative virulence factor of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), is the cell surface protein p57. A non-agglutinating isolate MT239 reportedly produces p57 that does not remain anchored on the cell surface. Experiments with chinook salmon compared onset and progression of BKD and percent mortality among groups of fish exposed to either the wild-type ATCC 33209 or the MT239 isolate of R. salmoninarum. Fish were exposed to either isolate by an intraperitoneal injection of about 1 x 103 or 1 x 106 bacteria per fish, or by a 24 hour immersion in about 1 x 105 or 1 x 107 bacteria/mL. Fish that died were analyzed for the presence of R. salmoninarum by ELISA, FAT, and bacteriological culture. Twice monthly, fish were sampled for histological examination of disease progression. Fish challenged by immersion and injection with the high dose of 33209, but not MT239, showed clinical signs of BKD. At ten weeks post-injection, BKD granulomas were prominent in the kidneys of fish exposed to 33209, but fish exposed to MT239 did not show evidence of BKD by histological examination. Total mortality was higher in groups injected with the high dose of 33209 (73%) than fish injected with the same dose of MT239 (12%) and was negligible in all of the low dose groups.

Virulence Properties of Aeromonas Species Isolated from Cultured Frogs

MD Pearson (1), N Tange (2), I Hirono (2), T Aoki (2), R Miranda (3) and V Inglis (1)

1	Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland. mdp1@stir.ac.uk
2	Laboratory of Genetics and Biochemistry, Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4-5-7, Minato-ku, Tokyo 108, Japan.
3	Bureau of Fisheries and Aquatic Resources, 860 Quezon Avenue, Quezon City, metro Manila 3008, Philippines

A collection of motile aeromonads from septicaemic and clinically normal frogs farmed in Thailand, was screened for haemolytic and cytolytic properties. Haemolysin activities against frog RBC were significantly different within the collection of aeromonads. Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A. hydrophila) and low haemolytic activity (A.veronii biovar sobria, A. veroni biovar veronii, A. caviae, A. schubertii) were noted. The isolates with high haemolytic activity all originated from internal organs of septicaemic animals, whereas the other isolates were all found externally. The haemolytic and cytolytic activity of the Aeromonas isolates varied according to the source of cells used in the assays. Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species. In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A. hydrophila. Selection of suitable assay substrates is therefore very important. Cells from homeotherms may be insensitive to cytotoxins associated with pathogenic processes in poikilotherms. DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any of the other Thai aeromonads or type strains tested. Previous work with haemolysin gene probes had indicated that almost all motile aeromonads possess either the sobria gene ASA1 (or one of its homologs) or the hydrophila gene AHH4. Au is unusual among haemolytic motile aeromonads in possessing neither of these genes although both were present in other motile aeromonads from Thailand.

Development of Challenge Test for Penaeus vannamei Post-Larvae: Diet Composition and Resistance to Disease

R Robles* and P Sorgeloos

Laboratory of Aquaculture and Artemia Reference Center, University of Ghent, Rozier, 44, B- 9000 Gent, Belgium rocio.robles@rug.ac.be; Patrick.Sorgeloos@rug.ac.be

The significance of a suitable diet in preserving the health of living organisms is widely recognized. Nutritional status is considered one of the most important elements which determines the competence of the animal to resist diseases. This study assessed the effect of dietary vitamin C and phosphatidylcholine (PC) on the resistance of penaeid shrimp post-larvae to artificial infection. The first series of experiments aimed at the development of a suitable challenge technique and selection of the pathogen to be used. Two bacterial strains (pathogen and non-pathogenic strain: P and NPS) and one control (blank, saline solution) were used as experimental treatments. The infection technique was abdominal injection; the pathogen a Vibrio harveyi strain isolated from Penaeus vannamei and the non-pathogenic strain identified as Vibrio sp. The dose was 103 to 104 CFU/shrimp and the challenge period five days. A second series of experiments showed no positive effect from an increased dietary supply of vitamin C (2000 ppm versus 200 ppm) on disease resistance in shrimp post-larvae. Challenge survival ranged from 9 to 82 %. In a third series of experiments, the effect of two types of dietary PC (marine and soybean PC at 1.5%) were compared with a diet lacking phospholipids. No significant PC effect on disease resistance could be revealed in the challenge test. More information is required on the pathogenesis and the specific resistance mechanisms involved in disease resistance, on the specific effect of several nutrients and how these effects are modulated by other dietary components and environmental factors.

Changes in the Gut Microflora of Intensively-Reared Atlantic Halibut (Hippoglossus hippoglossus L.) Larvae in a British Hatchery

DW Verner - Jeffreys (1)*, RJ Shields (2), IR Bricknell (3) and TH Birkbeck (1)

1	Division of Infection and Immunity, Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ Scotland UK. djeffreys@mblab.gla.ac.uk; gbma05@udcf.gla.ac.uk
2	Seafish Aquaculture, Seafish Industry Authority, Ardtoe, Acharacle, Argyll, PH36 4LD Scotland r.shields@sfia.uhi.ac.uk
3	FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, AB11 9DB Scotland bricknellir@frs.marlab.ac.uk

Intensively reared Atlantic halibut larvae and juveniles were monitored for the presence of gut-associated aerobic bacteria. Water from the tanks the halibut were being grown in, and some of the possible sources of microbial inputs into the rearing system, were simultaneously sampled and analysed. For the characterisation, a combination of traditional biochemical tests and the BIOLOG GN system were utilised. A gut microflora appeared to be seeded towards the beginning of the non-feeding yolk-sac stage, by the onset of first feeding this had reached in excess of 102 CFU/ larva. First feeding saw a rapid increase in bacterial numbers present within the gut to more than 104 CFU/ larva, a level which was reached within a week of the start of feeding. There were apparently structural differences between the bacterial communities making up the tank water and gut-associated microfloras, particularly after first feeding. Some of the bacterial phenotypes isolated from samples of freshly fertilised halibut eggs and the live food (highly unsaturated fatty acid enriched Artemia) resembled isolates later recovered from the guts of developing halibut larvae.

SESSION 18. PARASITOLOGY V: Perkinsus, Part 2

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The Effects of Perkinsus marinus Proteases on the Eastern Oyster, Crassostrea virginica

JL Oliver, M Faisal and SL Kaattari

Department of Environmental Sciences, School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062 USA. jloliver@vims.edu, faisal@vims.edu, kaattari@vims.edu

Perkinsus marinus is a pathogenic protozoan that causes serious mortalities in the eastern oyster, Crassostrea virginica. Virulence factors of Perkinsus marinus have not been fully identified, however, the pathogen is known to secrete a variety of extracellular proteins (ECP) including serine proteases both in oysters and in axenic in vitro P. marinus cultures. Using SDS-PAGE (reducing conditions) and immunoblot techniques, we have identified and physically characterized a 35 kDa protein (p35) within the plasma of C. virginica that is targeted by P. marinus serine proteases. However, in the presence of a low molecular weight fraction of the plasma, p35 is preserved. Other serine proteases such as trypsin and chymotrypsin also target p35, however, inhibition of proteolysis in the presence of the low molecular weight fraction of plasma does not occur. These results suggest that protease inhibitors may be present in eastern oysters and that they are specific against P. marinus proteases. The Pacific oyster, Crassostrea gigas, is resistant to P. marinus infections and the mechanisms of resistance were investigated. C. gigas plasma proteins showed less susceptibility to proteolysis than C. virginica when exposed to P. marinus proteases. Analysis of anti-proteolytic activity in the low molecular weight fraction of C. gigas showed no apparent inhibitory activity against P. marinus proteases. Ongoing research in our laboratory is focusing on the function and importance of the p35 molecule.

Effects of Protease Inhibitors on the Oyster Pathogen Perkinsus marinus and Oyster Cells in vitro

JF La Peyre*, HS Kristensen, KC McDonough and RK Cooper.

Department of Veterinary Science, Louisiana State University, Baton Rouge, LA, 70803 jlapeyre@agctr.lsu.edu, rcooper@agctr.lsu.edu

Perkinsus marinus is a protozoan that causes extensive mortalities in oysters (Crassostrea virginica) along the Atlantic and Gulf coasts. This pathogen secretes potent serine proteases which are believed to play an important role in pathogenesis. Blocking of parasite proteases by synthetic or naturally occurring protease inhibitors can significantly reduce the ability of parasites to invade host tissues and cells, inhibit their growth and interfere with their development. Therefore, the objectives of this study were to determine the effects of serine protease inhibitors on the growth and viability of P. marinus in vitro as well as on the viability of oyster cells. Perkinsus marinus was incubated with increasing concentrations of serine protease inhibitors (AEBSF, aprotinin, 1-antichymotrypsin, 1-antitrypsin, 2-macroglobulin, chymostatin, elastatinal, potato chymotrypsin inhibitors (PCI) I and II, and soybean trypsin inhibitor) and its propagation was measured by three methods: 1) turbidimetric, 2) cell counts and 3) intracellular reduction of tetrazolium salts. Viability of P. marinus was measured by the uptake of neutral red. Oyster primary cell cultures were exposed to increasing concentrations of protease inhibitors and cell viability was estimated by measuring intracellular reduction of tetrazolium salts using MTS/PMS reagents. We found that protease inhibitors which strongly inhibited P. marinus protease activity (AEBSF, 1-antitrypsin, 2-macroglobulin, chymostatin, PCI I and II) significantly suppressed the growth rate of P. marinus in a dose-dependent manner. The three methods used to quantify P. marinus proliferation gave similar results. Only PCI II caused significant mortality of P. marinus at the highest concentration used (i.e., 36%). Finally, except for AEBSF none of the protease inhibitors were toxic to oyster cells. It is clear that certain protease inhibitors can be detrimental to P. marinus in vitro and do not appear to be toxic to oyster cells. Their potential effects in vivo remain to be determined.

Molecular Genetic Analysis of Perkinsus marinus, a Protozoan Oyster Pathogen

KS Reece (1)*, D Bushek (2), KL Hudson (2), JE Graves (1)

1	Virginia Institute of Marine Science, School of Fisheries, College of William and Mary, Gloucester Point, VA 23062 USA. kreece@vims.edu; graves@vims.edu
2	Baruch Marine Field Laboratory, Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, PO Box 1630 Georgetown, SC 29442 USA. bushek@belle.baruch.sc.edu; khudson@belle.baruch.sc.edu

Perkinsus marinus is a major protozoan pathogen of the eastern oyster Crassostrea virginica. Recently developed in vitro culture and cloning methods have enabled us to examine the population structure of P. marinus using molecular genetics. Genetic relatedness of 47 geographic isolates and 8 monoclonal cultures derived from these isolates was examined at six polymorphic nuclear loci by restriction fragment length polymorphism analysis. Distance analyses indicated two major groups. One cluster was comprised predominantly of isolates from the mid-Atlantic and northeastern coastal areas of the United States. The other group was dominated by isolates from southeastern Atlantic and Gulf coast regions. DNA sequence analysis of two alleles at an anonymous locus in heterozygous monoclonal cultures indicated diploidy. There were thirteen nucleotide differences between the two alleles. Allele sequences were identical in isolates from New Jersey and South Carolina. Additional isolates are being collected and examined. In addition, we are looking for variation in microsatellite loci. These data will be used to help elucidate the population structure of P. marinus.

Characterization of Two Perkinsus spp. from the Softshell Clam, Mya arenaria

SM McLaughlin (1)* and M Faisal (2)

1	National Marine Fisheries Service, Cooperative Oxford Laboratory, 904 S. Morris St., Oxford, MD 21654 shawn.mclaughlin@noaa.gov
2	Virginia Institute of Marine Science, School of Marine Science, The College of William and Mary, Gloucester Point, VA 23062. faisal@vims.edu

Significant losses of bivalves reported worldwide have been associated with infections by protozoa of the genus Perkinsus spp. Routine epizootiological studies show Perkinsus spp. infections of softshell clams in the Chesapeake Bay appear to be on the rise based on thioglycollate assays and histopathology. In general, the gills appear to be the major site of infection but the parasites may become systemic with lesions occurring in other tissues including the digestive gland, gonads, and kidneys. Attempts to culture the organism from infected softshell clams resulted in two continuous, axenic cultures of Perkinsus spp. (H49 and G117). Isolate H49 was obtained from hemolymph and G117 from a combined gill/palp sample. Except for their larger size, H49 trophozoites and schizonts exhibited the characteristic morphology of P. marinus and divided by schizogony. Unlike H49, vegetative forms (trophozoites and schizonts) of G117 were present along with prezoosporulation stages in the same culture. In culture, G117 cells multipled by both schizogony and successive bi-partition. Both H49 and G117 cells reacted positively with anti-Perkinsus marinus polyclonal serum and formed hypnospores upon incubation in Ray's fluid thioglycollate medium. Biochemical studies showed that H49 cells produce two serine proteases of 112 and 40 kDa molecular weights. On the contrary, no proteolytic activities were detected in culture supernatants of G117. Moreover, a clear difference between the two isolates was observed in terms of optimal temperature and osmolarity for their in vitro propogation. Studies to determine the infectivity and pathogenicity of the two isolates are ongoing.

SESSION 19. VIROLOGY II: Invertebrates, Fish, Cetaceans

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Genomic Variation Among Aquatic Birnaviruses Based on Nucleotide and Deduced Amino Acid Sequences of cDNA of Genome Segment A

S Blake, J-Y Ma, D Caporale, S Jairath and BL Nicholson*

University of Maine, Orono, Maine, 04469 USA

Aquatic birnaviruses, such as IPN virus, cause serious diseases in a variety of fish species used worldwide in aquaculture. These viruses exhibit a high degree of antigenic heterogeneity and variation in biological properties such as pathogenicity, host range, and temperature of replication. The larger segment (A) of the bi-segmented dsRNA viral genome contains a large ORF encoding a polyprotein which is posttranslationally cleaved into the outer capsid protein, VP2, and an internal protein, VP3. To better understand genetic and biological diversity among these viruses, we determined the nucleotide and deduced amino acid sequences of cDNA of the ORF of genome segment A of the 9 type strains of Serogroup A, the ORF of a variety of representative isolates of the major USA serotype, and the VP2 coding region of a variety of isolates representing all other major serotypes. RT-PCR was used to amplify the 2,970 bp ORF cDNA fragment or a 1643 bp fragment representing the VP2 coding region. PCR products were sequenced by the dyedeoxy terminal cycle method with an automated DNA sequencer. Sequences were compared using several computer programs. From a phylogenetic tree constructed from the sequence comparisons, these viruses were clustered into 6 genogroups each comprised of several genotypes, which generally correlated with geographical origin and serological classification. In addition, within genotypes, specific amino acid variations distinguished avirulent variants. These data provide the most comprehensive analysis to date of genetic variation among aquatic birnaviruses and the first insights into the genetic basis for variations in virulence.

Disseminated Herpesviral Infection in a Bottlenose Dolphin

T Blanchard, T Libscomb, W McFee and R Garber

Armed Forces Institute of Pathology, Washington, D.C. 20306; National Marine Fisheries Service, Charleston, SC 29412; and Pathogenesis Corporation, Seattle, WA 90119

An immature female bottlenose dolphin was found stranded on Hilton Head Island, SC. Necropsy findings included thymic enlargement, pericardial hemorrhage and hydrothorax. Histologically, there was necrosis and acute inflammation in adrenal gland and uterus, lymphoid necrosis in spleen and lymph nodes, lymphohistiocytic myocarditis and necrotizing interstitial pneumonia. The thymus was hemorrhagic and contained syncytial cells and necrotic lymphocytes; both often contained 4-6 µm diameter homogeneous eosinophilic intranuclear inclusion bodies often surrounded by heart, spleen, lymph nodes, and glomeruli. An immunohistochemical test for human cytomegalovirus and a PCR test for morbillivirus were negative. Ultrastructure examination revealed that thymic intranuclear inclusions contained 90-110 nm diameter viral particles with dense cores and icosahedral capsids. Rare cytoplasmic viral particles were surrounded by a variably electron-dense envelope; enveloped virions were 140 nm in diameter. PCR reactions targeting the DNA polymerase and terminase genes of herpesviruses were carried out on unfixed lung tissue extract and sequencing with both DNA products indicated the presence of a novel alphaherpesvirus. Histologic, ultrastructural and PCR findings are consistent with herpesvirus infection.

A Review of Freshwater Crayfish Viruses

B F Edgerton*

Queensland Department of Primary Industries, Oonoonba Veterinary Laboratory, PO Box 1085, Townsville, QLD, 4810. AUSTRALIA. edgertb@dpi.qld.gov.au

Four crayfish viruses have been described since the recent first description of a natural virus infection in freshwater crayfish. There have been additional reports of viruses infecting freshwater crayfish, but these viruses have not been properly described. Experimental or natural infections of freshwater crayfish by viruses important in the aquaculture of other organisms have also been reported. Freshwater crayfish viruses have been tentatively grouped with virus families based predominantly on morphology. Intranuclear bacilliform viruses (IBV) have emerged as the most important virus group in freshwater crayfish as distinct IBVs infect three species of freshwater crayfish on four continents. Crustacean IBVs are currently unclassified viruses, however they are morphologically similar to penaeid baculoviruses. Other viruses which naturally infect freshwater crayfish have been tentatively assigned to the Parvoviridae, Totiviridae and Picornaviridae virus families. The current state of knowledge on crayfish viruses will be reviewed, with particular emphasis on the need for caution when translocating crayfish.

Grouping and Identification of VHSV-Strains by Comparison of G- and NV-Genes

PJ Enzmann (1)*, TH Meyers (2), J McArdle (3), G Geoghegan (3), H Schutze (1), D Fichtner (1) and G Walliser (1)

1	Federal Research Centre for Virus Diseases of Animals, Paul-Ehrlich-Str. 28, D-72076 Tubingen, Germany enzmann@tue.bfav.de
2	Alaska Dep. Fish & Game, CFMD, P.O.Box 25526, Juneau, AK 99802-5526, fish-path@fishgame.state.ak.us
3	Marine Institute, Malahide Road, Dublin, Ireland. jmcardle@frc.ie

VHSV strains differ in pathogenicity and sensitivity for different host species. Strains from marine species seem to be less pathogenic for freshwater species. But nevertheless, the role of marine species in epizootiology of VHS is unclear. The glycoprotein G, as a component of the viral envelope, is supposed to determine host specificity of a virus by specific attachment to the cell surface, i.e. it determines cellular tropism and affects virus penetration into susceptible cells. By this fact one can speculate that species specificity (as well as attenuation of a live vaccine strain) is founded on the G-gene. For molecular epidemiological studies the G- and NV-genes of a series of VHSV-strains were reverse transcribed, amplified by PCR and sequenced. The amino acid and nucleotide differences between the G-genes of VHSV-strains did not correlate with differences in host specificity and pathogenicity, but confirmed earlier estimates of genetic variability of VHSV in contrast to IHNV, especially a most variable region within the G-gene is demonstrated. But NV-genes show considerable potency for grouping of VHSV-strains. Several genotypes could be distinguished. Representatives of strains F1, He, 23,75, F3 (Kl), and F4 (Ri) were examined in addition to a series of isolates from various regions, in total 19 strains, including those from marine species in the pacific region as well as the Atlantic region.

Green Fluorescent Protein Gene is an Efficient Reporter in Recombinant CCV Research

LA Hanson* and S Marinovic

College of Veterinary Medicine, Mississippi State University, PO Box 9825, Mississippi State, MS 39762 USA hanson@cvm.msstate.edu

Thymidine kinase (TK) negative channel catfish virus recombinants are attenuated, express foreign inserted genes, and induce immunity to these expressed products in vaccinated catfish, thus having potential as vaccine vectors for the catfish industry. In previous research we used the Escherichia coli lacZ gene as a reporter gene to evaluate expression and immune response. However, this gene is relatively large (3.1 Kb), providing some diffuculty in selection of recombinants, and the product is not properly processed in the endoplasmic reticulum for excretion or surface expression. Therefore, we evaluated the use of green fluoroscent protein (GFP) gene from the jellyfish Aequorea victoria, the products of which can be directly analyzed by fluoroscent emmision in blue light. The red shift GFP gene amplified from plasmid pS657-C1 (Clonetech) using Vent polymerase was cloned into the TK region of two different CCV transfer plasmid constructs. These constructs were co-transferred with CCV DNA and recombinant TK-negative CCV were dilution cloned and evaluated. The clones contained the proper GFP insert with no TK sequence amplification in PCR using primers flanking the TK site. Conventional and confocal fluorescent microscopy revealed greeen fluorosing cells in culture by 24 hours post-infection. The evaluations could be done on fixed or live cells. However, analysis of tissues from infected fish was hindered by auto-fluoroscence. Also, level of expression and intensity of fluoroscence was not sufficient for GFP quantification using a multiwell fluorometer. We are not evaluating human optimized-enhanced GFP and a blue mutant gene.

Pathogenicity of a Wild Marine European Isolate of Viral Haemorrhagic Septicaemia for Atlantic Cod (Gadus morhua)

M Snow (1)*, IR Bricknell (1), DA Smail (1), CO Cunningham (1), WT Melvin (2) and R Shields (3)

1	FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, AB11 9DB, Scotland snowm@marlab.ac.uk; bricknellir@marlab.ac.uk; cunninghamc@marlab.ac.uk.
2	Department of Molecular and Cell Biology, Zoology Building, University of Aberdeen, Tillydrone Avenue, Aberdeen. wtmelvin@aberdeen.ac.uk
3	Sea Fish Industry, Marine Farming Unit, Acharachle, Argyll. r_shields/sfia@uhi.ac.uk

Viral haemorrhagic septicaemia virus (VHSV) is a rhabdovirus which is responsible for significant losses in European cultured rainbow trout populations. Although previously considered to be confined to rainbow trout in fresh water, VHSV has been increasingly isolated from both wild and cultured, marine and freshwater fish species. In this study, a European strain of VHSV isolated from wild caught cod (Gadus morhua) was shown to cause clinical disease and mortality in excess of 80% in juvenile Atlantic cod when administered by the intra-peritoneal (I.p.) route. Specific pathogen free animals were reared under laboratory conditions, and challenged with a dose of 108 TCID50 per fish, a dose known to induce mortality in rainbow trout, the reference susceptible fish. No virus was recoverable from uninfected fish cohabited with experimentally infected fish at a ratio of 1:1. External signs of disease were generally limited, although one of six surviving i.p. injected fish exhibited cutaneous haemorrhaging. Virus identified as VHSV was recovered from both the brain and organ pools (kidney, liver and spleen) of 100% of the i.p. injected dead or moribund cod. No mortality or recoverable virus was observed in control fish. The susceptibility of non-salmonid marine species to VHSV raises important questions surrounding the potential propagation and maintenance of a naturally occurring reservoir of virus in the marine environment. The existence of such a reservoir of VHSV poses significant implications with respect to the potential losses of economically important species such as cod.

SESSION 20. IMMUNOLOGY III: Invertebrates, Fish, and Marine Mammals

[TOP]

Nitric Oxide Synthase Activity in Oyster Hemocytes

RS Anderson, LM Mora and ML Haasch

Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, Post Office Box 38, Solomons, Maryland 20688-0038 USA anderson@cbl.umces.edu; mora@cbl.umces.edu; haasch@cbl.umces.edu

The ability of oyster (Crassostrea virginica) hemocytes to generate nitric oxide (NO) is currently under investigation, in order to better understand the antimicrobial defense mechanisms that contribute to resistance to infectious diseases. These cells are known to produce superoxide which can react with NO to produce peroxynitrite, a more cytotoxic, more long-lived product. In mammals, NO, superoxide, and/or peroxynitrite have been show to kill intra- and extracellular parasites; the role(s) of NO in oyster responses to parasitic pathogens has yet to be evaluated. NO is produced by the oxidation of L-arginine to L-citrulline by nitric oxide synthase (NOS), a family of isoenzymes often named for their tissue location or whether they are constitutive or inducible. Here we report the presence of NOS in C. virginica hemocytes and that its activity may be stimulated by administration of a Listonella anguillarum bacterin. Western blots of SDS-polyacrylamide gel electrophoretic separations of oyster hemocyte lysates were probed with an anti-mouse iNOS polyclonal antibody. A hemocyte protein of ~130 KDa showed strong cross-reactivity with iNOS of similar molecular weight in a control mouse macrophage lysate run simultaneously. The result was reconfirmed using another commercial immunoglobulin preparation directed against "universal" NOS; i.e. all NOS isoforms. In mammalian macrophages, inducible NOS (iNOS) activity is elevated by exposure to bacterial endotoxins. A bacterial (L. anguillarum) vaccine was injected into the hemolymph of C. virginica. Within 48 hours of injection, stimulation of iNOS was detected in hemocytes from treated oysters compared to controls.

Activity of Plasma Protease Inhibitors in Sarcomatous Softshell Clams, Mya arenaria

E Elsayed (1)*, S McLaughlin (2) and M Faisal (3)

1	1 Faculty of Veterinary Medicine, Cairo University, Guiza, Egypt. ehab@intouch.com
2	National Marine Fisheries Service, Cooperative Oxford Laboratory, 904 S. Morris St., Oxford, MD 21654 shawn.mclaughlin@noaa.gov
3	Virginia Institute of Marine Science, School of Marine Science, The College of William and Mary, Gloucester Point, VA 23062. faisal@vims.edu

Disseminated sarcoma causes significant mortalities in softshell clam (Mya arenaria) populations along the East Coast of the United States. Reduced cellular defense mechanisms have been reported in sarcomatous clams; however, the biochemical basis of this decreased activity is not fully understood. In the present study, we provide evidence on the presence of protease inhibitors in softshell clam hemolymph. Levels of protease inhibitory activity varied greatly from one enzyme to another, e.g. 1 ug of plasma protein inhibited 595 ± 175 ng of pepsin (aspartic protease), 5 ± 2 ng of papain (cysteine protease) and 3 ± 1 ng of trypsin (serine protease). Significantly lower levels of anti-metalloprotease activity was displayed in softshell clam plasma. The effects of sarcoma progression on plasma protease inhibitory activities was investigated. Clams with early and intermediate stages of sarcoma showed a non-significant decrease in the levels of protease inhibitors. However, clams with advanced sarcomas showed a marked decrease in the ability to inhibit trypsin, papain, and pepsin while the protease inhibitory activity levels against metalloprotease were completely exhausted. The levels of inhibition against chymotrypsin (also a serine protease) showed, however, a significant increase. The mechanism leading to this suppression is being investigated.

Induction of Cell-mediated Immunity by Dimerized Lysozyme (KLP-602) After Iridovirus-induced Immune Suppression in Sheatfish (Silurus glanis)

AK Siwicki (1,4)*, M Morand (2), P Klein (3), F Pozet (2), E Terech-Majewska (4)

1	Department of Fish Pathology and Immunology IFI, 05-500 Piaseczno, Poland. irs@atos.warman.com.pl
2	Laboratoire Departemental d'Analyses, Conseil General du Jura, 39016 Lons le Saunier, France
3	Nika Health Products Limited, Lawrenceville, New Jersey 08648 USA
4	Department of Epizootiology, Faculty of Veterinary Medicine, Agricultural University in Olsztyn, 10-957 Olsztyn-Kortowo, Poland. irs@atos.warman.com.pl

The iridovirus-like agents associated with systemic infection has recently been isolated from sheatfish (Silurus glanis) and catfish (Ictalurus melas) in Europe. Our in vitro study showed that iridovirus-like agent decreased the macrophages activity and proliferative ability of lymphocytes in sheatfish. Also the in vivo study demonstrated a strong suppressive influence of iridovirus-like agent on macrophage and lymphocyte activity in sheatfish. This study reported the immunomodulating influence of dimerized lysozyme (KLP-602) on the cell-mediated immunity after in vitro and in vivo suppression induced by iridovirus-like agent in sheatfish. The in vitro study showed that dimerized lysozyme activated the respiratory burst activity and potential killing activity of macrophages suppressed by iridovirus. The lymphocyte proliferation analysis by MTT assay indicated a similar pattern. The results showed that dimerized lysozyme increased the lymphocyte proliferation suppressed by iridovirus. The in vivo study presented that dimerized lysozyme (KLP602) modulated of cell-mediated immunity after iridovirus-induced immune suppression in sheatfish.

The Immunobiology of the Gulf Killifish, Fundulus grandis: A Model for Assessing Estuarine Environmental Health.

CD Rice*, MM Banes and AZ Andol

College of Veterinary Medicine, Mississippi State University, P.O. Box 9825, Mississippi State, MS 39762, USA. rice@cvm.msstate.edu

The gulf killifish, Fundulus grandis, is common in salt marshes along the northern Gulf of Mexico, USA. The home range of this fish is limited, consequently it is chronically exposed to local environmental conditions, including pollution and disease stress. As with the mummichog, F. heteroclitus, a very closely related species from the east coast of USA, F. grandis adapts well to laboratory rearing practices and is routinely used in reproductive, developmental, carcinogenesis, and chemical adaptation studies. However, little is known of it's immununophysiology beyond innate immune responses measured in vitro. We developed monoclonal antibodies (mAb) against circulating and lymphocyte-bound immunoglobulins (Ig) of F. grandis that, in turn, allows us to quantitate antibody responses of this fish. We also developed a mAb specific to eosinophillic granular cells (EGC), the major lymphoid phagocyte in F. grandis (and F. heteroclitus). Using these mAbs and leukocyte function assays, we find (1) that F. grandis mount vigorous antibody and cellular immune responses to Vibrio anquillarum, (2) that there are diel, seasonal, and gender-related differences in immune functions in this fish, and (3) that 10, 1, 1, and 10 ppm of aroclor 1254, TBT, 3-MC, and nonyl-phenol, respectively (in mixture as a 90 day dietary exposure), alters their immune responses. These compounds also induce Hepatic CYP1A. Efforts are underway to determine if our mAbs recognize F. heteroclitus Ig and EGC. If so, then the two species may be comparable as sentinel models for assessing environmental health in their respective habitats. (MS-AL SeaGrant NA16RG0129-R/ER-37PD)

Selective Loss of High and Low AffinityTrout Antibody During Purification Procedures

HL Zhang* and SL Kaattari

Department of Environmental Sciences, School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA23062 USA. hlzhang@vims.edu; kaattari@vims.edu

Antibody affinity is an important parameter of humoral immunity and has been demonstrated crucial in protecting from some viral diseases. During the process of resolving the delicate affinity maturation in fish and its related molecular characteristics, we found that commonly employed antibody purification procedures can exclude high or low affinity antibodies from an immunoadsorbed antibody pool. Specifically, hapten elution of trout-anti-TNP antibody from an TNP-BSA conjugated immunoadsorbent Sepharose column can mask high affinity subpopulations by retaining hapten in their binding sites. This binding activity is high enough that the bound hapten can not be removed by extensive dialysis. Consequently, the highest affinity subpopulations generated during affinity maturation are blocked from being detected in subsequent affinity assays, thereby inaccurately portraying a lower affinity profile. However, by passing the eluant through a Dowex anion exchange column, the tightly bound hapten can be absorbed out and the high affinity binding sites are freed, as reflected by the recovery of high affinity subpopulations and the restoration of the original intact affinity profile. Secondly, it has also been found that phosphorate buffered saline or Tris buffered saline, formerly considered inert in their desorbing activity on specific antibodies, can selectively remove low affinity antibodies. We concluded that immunopurification procedures should incorporate our modifications in order to conduct affinity analysis of the complete pool of purified antibody. The selective elution of high and low affinity subpopulations can further be utilized in in vitro antibody affinity selection and fractionation.

Restoration of Cell-mediated Immunity After Suppression Induced by Xenobiotics - in vitro and in vivo Study

M Studnicka (1), AK Siwicki (2,3), A Rymuszka (1), A Bownik (1), I Krakowska (1) and E Terech-Majewska (3)

1	Department of Biology and Toxicology, Catholic University, 20-35 Lublin, Poland
2	Department of Fish Pathology and Immunology IFI, 05-500 Piaseczno, Poland. irs@atos.warman.com.pl
3	Department of Epizootiology, Faculty of Veterinary Medicine, Agricultural University in Olsztyn, 10-957 Olsztyn-Kortowo, Poland. irs@atos.warman.com.pl.

Understanding the human and animal health risk associated with environmental exposures involves defining the cascade of events between exposure to environmental xenobiotics and the resulting health effect. Increasing concentrations of xenobiotics in the environment has direct effects on the defense mechanisms and immunocompetent cell activity. In our investigation we examined the possibility of restoring cell-mediated immunity after suppression induced by chemotherapeutics (oxytetracycline, oxolinic acid) and pesticides (pyrethroids, triazins, phosphoroorganics) in fish. Restoration of suppressed cellular defense mechanisms have been attempted using natural immunomodulators: chitosan (SFI Poland) and dimerized lysozyme (Nika Helath Products, USA). The in vitro and in vivo study showed that chitosan and dimerized lysozyme (KLP-602) increased the macrophage and lymphocyte activities and antibody secreting cell (ASC) levels after suppression induced by chemotherapeutics and pesticides. The results showed that natural immunomodulators could be used to restore cellular and humoral immunity and eliminate the immunosuppressive effects of xenobiotics in fish.

Purification and Serological Characterization of Florida Manatee Immunoglobulin G

SA Kania (1)*, D Murphy (2) and LND Potgieter (1)

1	Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Knoxville, Tennessee 37996. skania@utk.edu
2	Lowry Park Zoological Garden, 7530 North Boulevard, Tampa, Florida 33604

The Florida manatee (Trichechus manatus latirostris) is endangered and suffers a high annual rate of mortality. It is, therefore, important to identify the cause of disease outbreaks to facilitate medical intervention whenever possible. Diagnostic tests necessary to acquire this information are limited, due in part to a lack of detecting reagents for serological studies. To address this problem we isolated manatee IgG and prepared anti-manatee IgG in rabbits. For the issolation of IgG, sera from five T. Manatus latirostris were pooled and applied to a recombinant protein A/G column. Eluted IgG was further purified using diethylaminoethyl cibacron blue dye resin. Manatee IgG was characterized by SDS-PAGE using 7.5% resolving gels followed by Coomassie blue staining. Antisera reactive with manatee IgG was produced in New Zealand White rabbits. The specificity of the anti-manatee IgG was confirmed with western blots. An IgG fraction of the anti-manatee IgG was coupled to biotin for use as a detecting antibody in capture ELISAs. Manatee IgG was detected in a linear fashion over the range of 5 to 100 ng/ml. Purified IgG and sera from other mammalian species had poor reactivity with anti-manatee IgG. Peroxidase conjugated antisera prepared against cat, bovine, dog, chicken, rabbit, and deer IgGs reacted weakly with manatee IgG compared to the reactivity with their homologous IgG. The reagents developed in this study should be useful to study the exposure of manatees to infectious organisms and to measure IgG concentrations in manatee sera to monitor passive transfer and immune status.

SESSION 21. LEGISLATION

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Licensing and Regulation of Veterinary Biologics for Fish in the United States

NG Birnbaum*

U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Center for Veterinary Biologics © Licensing and Policy Development, 4700 River Road, Unit 148, Riverdale, Maryland 20737©1231

The United States Department of Agriculture (USDA) regulates biologics (including vaccines, bacterins, and diagnostic test kits) for fish, produced in, imported into, or exported from the United States. The regulatory process is designed to ensure that biologics under USDA jurisdiction are not contaminated, worthless, dangerous, or harmful. The Animal and Plant Health Inspection Service (APHIS), an agency within the USDA, licenses and inspects biologics production facilities, and licenses and tests veterinary biological products. Veterinary biological products offered for sale should be pure, safe, potent, and efficacious. A biologics producing firm located in the United States may sell biological products it manufactures provided the firm possesses a valid U.S. Veterinary Biological Product License for each biological product, as well as a valid U.S. Veterinary Biologics Establishment License. The permittee (i.e., legal representative in the United States of a biologics producing firm located outside the United States) may import biologics into the United States provided the permittee possesses a valid U.S. Veterinary Biological Product Permit. Biologics available in the United States specifically for fish are manufactured by Alpharma NW Inc., Aqua Health Ltd., and Diagxotics.

Minimizing the Spread of Disease Agents Due to Transport of Cryopreserved Germplasm of Aquatic Species

JA Jenkins (1)* and TR Tiersch (2)

1	1 National Wetlands Research Center, Biological Resources Division, US Geological Survey, US Department of the Interior, 700 Cajundome Blvd., Lafayette, Louisiana 70506 USA. jill_jenkins@usgs.gov
2	School of Forestry, Wildlife and Fisheries, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803 USA. ttiersch@agctr.lsu.edu

Selective breeding and genetic management in livestock industries routinely employ cryopreserved germplasm, and legislative guidelines have been developed for its use and transport. However, no comparable approach has been formulated for an integrated program of collection, handling, shipping, and banking of cryopreserved germplasm from aquatic species. Approaches for limiting the spread of microorganisms associated with the increasing global transfer of germplasm exist within national and international regulations, guidelines, and programs currently in place. For example, Title 50 of the Code of Federal Regulations presents guidelines for importation of fish, crustaceans, mollusks, and their gametes. The National Fish Health Policy addresses issues of concern for salmonids, provides policy and implementation guidelines for fish health, and advocates those standards recommended by the Fish Health Section of the American Fisheries Society. The Fish Disease Commission of the Office International des Epizooties (OIE) has published an International Aquatic Animal Health Code and Diagnostic Manual for Aquatic Animal Diseases, and the OIE has previously developed guidelines to work with disease-free mammalian sperm. The Fish Health Blue Book presents acceptable detection methods for disease agents, and the National Fish Hatchery System is developing a National Fish Disease Database. With respect to agriculture, the Veterinary Services of the Animal and Plant Health Inspection Service of the USDA is currently defining its role with aquatic animal health. Critical points in the processes involved with the cryopreservation of germplasm from aquatic animals should be identified, and working guidelines generated to minimize the spread of potentially harmful microorganisms.

Use of Molecular Biology Techniques in Abalone Fishery Management

AP Sierra-Beltrán (1)*, NY Hernández-Saavedra (1), JL Ochoa (1), L Fueyo-McDonald (2) and A Fuentes-Montalvo (2)

1	Marine Pathology Unit, The Center for Biological Research, Northwest. (CIBNOR) P.O. Box 128, C.P. 23000, La Paz, B.C.S., México. asierra@cibnor.mx; nhernan@cibnor.mx; jlochoa@cibnor.mx
2	Environmental Protection Attorney, PROFEPA, Periférico Sur 5000, C.P. 04530, México City, D.F., México

The high price attained by abalone products in the international market, made this a highly profitable target for smugglers. Under strict regulations of size and season of harvest, only two of the four species inhabitants of México are allowed to go into the market. In late summer, 1997, collaborative efforts between Mexican and US Authorities detected a suspect traffic of abalone through the border. Some anomalies were found in the records of authorized processing facilities. The industry alleged they were importing frozen blacklip abalone (Haliotis rubra) from Australia and canning the product in México. Three molecular techniques were used to try to fingerprint abalone species to have knowledge of the origin of the products: RFLP, RAPD and PCR with specific primers. In general, to recover good quality DNA from cooked (canned) abalone is extremely difficult and an improved extraction protocol has been developed. RFLP and RAPD results were not enough to resolve the relatedness among the different specimens. Primers were designed for sequences of the initial (variable) and final (conserved) portions of the gene coding for the protein lysin involved in sperm species recognition during mating. Specific primers for the Australian blacklip abalone Haliotis rubra, and the Mexican species Haliotis fulgens, H.corrugata, H.cracherodii and H.sorenseni, allow for discrimination of specimens belonging to each of the species. A useful molecular tool has been developed which will help recognize the origin of the products and, thus, will enforce the management and control of abalone trading.

Import Health Risk Analysis: Salmonids for Human Consumption

MAB Stone* (1), SC MacDiarmid (2) and HJ Pharo (3)

1	1 Ministry of Agriculture, Regulatory Authority, PO Box 2526, Wellington, New Zealand. stonem@maf.govt.nz
2	As above. macdiarmids@maf.govt.nz
3	As above. pharoh@maf.govt.nz

Evisceration of salmonid fish traded for human consumption is accepted by most countries as a measure which provides an appropriate level of protection from transfer of fish diseases. However, until 1995 New Zealand required all imports of table salmon to be cooked to reduce disease risks. Trading partners formally requested New Zealand provide justification for this measure. A risk analysis has been conducted examining the likelihood and the consequences of disease being introduced into New Zealand through imports of eviscerated salmonids, as well as the options for managing these risks. Qualitative and quantitative risk assessment methods were used. The risk analysis document and process is consistent with the recommendations within the Office International des Epizooties International Aquatic Animal Health Code. Prior to publication the document was reviewed by international fish health experts, New Zealand experts, and an expert in quantitative risk analysis. The document is undergoing a period of public comment. Following analysis of submissions, a decision will be made on the risk management measures which are considered will provide an appropriate level of protection from disease risk.

SESSION 22. PARASITOLOGY VI: Infections in Crabs and Fish

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Proliferation of Hematodinium perezi, a Parasitic Dinoflagellate, in the Blue Crab, Callinectes sapidus.

JD Shields*

Department of Environmental Sciences, Virginia Institute of Marine Science, P.O. Box 1346, Gloucester Point, VA 23062 USA. jeff@vims.edu

Hematodinium perezi is a lethal parasitic dinoflagellate that lives in the hemolymph of brachyuran crabs. The parasite is found along the eastern seaboard of the USA where it occurs in epizootics in the commercially important blue crab, Callinectes sapidus. Crab mortalities associated with the disease occur in moderate to high salinity waters. The parasite proliferates to extremely high densities in the host (virtual monocultures of up to 100 million parasites/ml of hemolymph) over 3 to 6 weeks. In aquaria, naturally infected crabs with light infections (0.33 to 1 plasmodium/100 host cells) developed heavy infections (>100 trophonts/100 host cells) over two to three weeks. In some cases, heavily infected crabs survived as long as lightly infected crabs (up to 40 days). Needle injections successfully transmitted both plasmodia and trophont stages to naive hosts; and the parasite has been maintained through at least a dozen serial passages with no apparent loss of pathogenicity. In crabs held at 24 psu and 20C, parasites were not visible in the hemolymph during the first 6-12 days post injection. Prepatent, occult infections were not effectively diagnosed at densities less than 1.0 x 104 parasites/ml. The prepatent period was at least 8 to 10 days in naturally infected and injected crabs. Light infections, characterized by plasmodial stages (1-3 x 104 plasmodia/ml), were observed after approximately 13 days. Moderate infections (105 parasites/ml) occurred after 16 days, and heavy infections (106 parasites/ml) occurred after 30 days. Host mortalities to the infection generally occur after 17 days, and peak between 21 to 35 days, but time to death is related to the inital inoculum. The proliferation of the parasite is in sharp contrast to that repor ted for the cold-water species of Hematodinium in Tanner crabs and Norway lobsters that may take 5 to 18 months to develop.

Effects of Salinity and Temperature on Intensity of Hematodinum sp. Infections in Blue Crabs, Callinectes sapidus.

GA Messick (1)*, W Van Heukelum (2) and S Jordan (3)

1	National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Cooperative Oxford Laboratory, 904 S. Morris Street, Oxford, Maryland 21654-9725 USA. Gretchen.Messick@noaa.gov
2	University of Maryland, Center for Environmental Science, Horn Point Laboratory, P.O. Box 775, Cambridge, Maryland 21613. heukelem@hpel.cees.edu
3	Maryland Department of Natural Resources, Cooperative Oxford Laboratory, 904 S. Morris Street, Oxford, Maryland 21654. Sjordan@remote.dnr.state.md.us

The parasitic dinoflagellate, Hematodinium sp. infects and causes mortalities in blue crabs from Gulf of Mexico and Atlantic coast embayments. A seasonal infection cycle in Maryland coastal bays peaks in the late autumn and early winter but is diminished to 0% prevalence by spring. No infected crabs have been collected from salinities below 11 ppt, although crabs from lower salinites have been assayed. To study how temperature and salinity affect infection intensity, a series of experiments held infected crabs at various water temperatures and salinities. Temperature effects: crabs held in flow-through seawater tanks for 52 d had a significant increase in infection intensity, when temperature varied between 15-8C, when temperature dropped to 1-4C, infection intensity was reduced. Under controlled parameters, where water temperature was held at 9C, infection intensity was reduced in crabs held in artificial seawater. In another controlled experiment, infection intensity increased when crabs were held at either 12 or 16C. Salinity effects: Infected crabs held in 10 ppt had a statistically greater reduction in infection intensity than those held in 29 ppt. Crabs held in artificial seawater at 12 or 16C had a decrease in infection intensity whereas crabs held in raw seawater at 12 or 16C had an increase in infection intensity. Salinity appears to limit the disease to high salinity coastal bays, sparing highly productive fishery areas such as upper portions of Chesapeake Bay. Cold winter temperatures reduce infections allowing juvenile crabs to survive initial fall-winter infections and enter the commercial fishery the following summer as young adults.

Bitter Crab Syndrome in the Bering Sea, 1988-1996

JF Morado (1)*, LL Mooney (1), TR Meyers (2) and RS Otto (3)

1	1 National Oceanic & Atmospheric Administration, National Marine Fisheries Service, Alaska Fisheries Science Center, 7600 Sand Point Way NE, Seattle, Washington 98115-0070 USA. Frank.Morado@noaa.gov; Lisa.Mooney@noaa.gov
2	Alaska Dept. of Fish & Game, Commercial Fisheries Management & Development Division, Fish Pathology Section, PO Box 25526, Juneau, Alaska 99802-5526 USA. fishpath%fishgame@state.ak.us
3	National Oceanic & Atmospheric Administration, National Marine Fisheries Service, Alaska Fisheries Science Center, Kodiak Laboratory, PO Box 1638, Kodiak, Alaska 99615 USA. Robert.Otto@noaa.gov

Between 1988 and 1996, hemolymph from Chionoecetes bairdi (N=5180) and C. opilio (N=9184) were examined for Hematodinium sp., the causative agent of Bitter Crab Syndrome (BCS). For this period, total prevalences of BCS in C. bairdi and C. opilio were 1.76% and 3.57%, respectively. In C. bairdi, highest prevalence was observed in 1996, reaching 9.93% in females and 2.74% in males. In C. opilio, highest Hematodinium prevalence was observed in 1988 (8.45%) reaching 7.62% in males and 10.00% in females. In general, BCS prevalence in C. opilio increased with increase in latitude. Despite the fact that prevalences in C. opilio were generally lower in the Chukchi Sea than in Norton Sound and west of St. Lawrence Island, a greater percentage of sampled stations were positive for BCS in the Chukchi Sea. For C. bairdi, infections were rare in the Eastern Bering Sea, but increased markedly along the shelf edge west and north of the Pribilof Islands. Infections in C. opilio were more frequent in shell condition-1 crabs (8.9%); females (15%) were more frequently infected than males (6.7%). In contrast, infections in C. bairdi were more common in shell condition-2 crabs (2%) with male and female prevalences being 1.7% and 2.6%, respectively. For both C. opilio and C. bairdi, infections were more common in small crab less than 60mm. In C. bairdi, the highest prevalence was observed in 20mm crab (64.3%) while in C. opilio, highest prevalence was observed in 35mm crab (16.1%).

Parasites of the Turkish Wrasse, Thalassoma pavo (Labridae) from Madeira: Preliminary Results

G Costa (1)*, AA Rego (2) and JC Chubb (3)

1	Dept. Biologia, Universidade da Madeira. Lg. do Colégio, 9000 Funchal, Portugal. costag@dragoeiro.uma.pt
2	Dept. Helmintologia, Fundação Oswaldo Cruz, Av. Brasil, 4365 Manguinhos, CEP 21045-900 Brasil arego@gene.dbbm.fiocruz.br
3	School of Biological Sciences, Derby Building, University of Liverpool, Liverpool L69 3BX, UK j.c.chubb@liverpool.ac.uk

The Turkish wrasse, Thalassoma pavo, is a small coastal fish species, not exceeding 20 cm in length. Although not commercially exploited, the study of its parasite fauna is of vital interest to understand the life cycles of helminth parasites, infecting commercial fish species, to which this and others small coastal fish species are frequent feeding items. In 7 out of 13 Turkish wrasse with lengths from 8 to 18 cm, several larvae of the cestode Scolex pleuronectis Müller 1788, (Tetraphyllidea) were found in the digestive tract. These larvae, a developmental stage of a variety of tetraphyllids, are quite common in marine invertebrates and fishes (Rego et al. 1983; Rohde 1984 in Kinne, 1984). It is hoped scanning electron microscopy will differentiate the species in Madeira. In one out of the 13 fish examined the myxosporean Ceratomyxa sp. resembling in shape with Ceratomyxa sparusaurati (Sitjà-Bobadilla, A., Palenzuela, O. and Alvarez-Pellitero, P. 1995.), infected the gall-bladder and bile. Spore dimensions were, however, larger than those recorded for C. sparusaurati. Mature spores of Ceratomyxa sp. had a mean length of 8.621±0.135 (8-10) µm and a mean thickness of 29.45±0.534 (23-32)µm (mean ± std. error) (n=29 spores) compared with the 5.65±0.74 (4.5-7.5) µm in length and 15.76±1.01 (14-17.5) µm thickness of C. sparusaurati.

The Susceptibility and Immunity of Paralabrax maculatofasciatus (Pisces: Serranidae) to Neobenedenia sp, a Monogenetic Trematode

SF Martinez-Diaz*, M Moreno-Legorreta, M Contreras-Holguin and JL Ortiz-Galindo

Centro Interdisciplinario de Ciencias Marinas, IPN, Departamento de desarrollo de tecnologias, Laboratorio de Microbiologia, Playa el Conchalito S/N, La Paz, Baja California Sur. CP 23000, Mexico. Sdiaz@vmredipn.ipn.mx

The monogenean Neobenedenia sp. is reported as a risk factor in the culture of cabrilla bass Paralabrax maculatofasciatus (Pisces: Serranidae). The parasite was recovered from the body surfaces fins and eyes during an outbreak. Eggs were collected and development was followed under laboratory conditions. At 24C, the eyespots are apparent at 5-6 days and the eggs began to hatch at 7-8 days of incubation. The resistance of eggs to treatments was investigated. Primary infection was experimentally induced in juvenile fish and the immune response was analyzed. The mucus and sera of infected fish can provoke the paralysis of oncomiracidia. An acquired protection of fish against secondary infection was demonstrated by a reduction in the number of parasites established on primed fish recovered from a primary infection. The passive immunization was tested by injection with sonicated oncomiracidia antigen. The sera and mucus changes were discussed.

Effect of Parasitism on Immunity of Cultured Fishes

MA El-Seify (1), MK Soliman (2) and BM Nadia (3)

1	Parasitology Department, Faculty of Veterinary Medicine, Kafr El-Sheikh, Tanta University, Egypt
2	Department of Avian and Aquatic Animal Medicine, Faculty of Veterinary Medicine (Edfina), Alexandria University, Egypt
3	Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Kafr El-Sheikh, Tanta University, Egypt

There has been increased interest in the intensive breeding of freshwater fishes in commercial production programs. Parastitic infections constituted a major problem among freshwater fishes investigated in this present study. The results obtained revealed that the cultured fish were parasitized with protozoa (Trichodina sp., Chilodonella hexasticha, Scyphidea sp., Glossatella sp., Trichophrya sp., and Ichthyophthirius multifiliis), Monogenea (Dactylogyrus sp. and Gyrodactylus sp.), digenitic encysted metacercariae and Lernae cyprinacea. The parasitic prevalance and the antagonism between the different species among some cultured fishes in Kafr El-Sheikh Provence, Egypt were studied. The effects of parasitism on some haematological aspects were also studied and the results indicated that the R.B. cs count, HB%, PCV, total protein, albumin and liver function enzymes were decreased, while the leukocytic count as well as the eosinophiles and globulins were increased. The study revealed also that the parasitism increased the phagocytic cell count. The effect of parasitism on agglutinating antibody response, electrophoretic pattern, and histochemical aspects were also studied.

Pollution Effects on the Ectoparasite Fauna of Mullets

R Madhayi* and K Ruchika

Department of Zoology, Andhra University, Visakhapatnam 530003, India

An investigation was undertaken during 1995-'96 on the ectoparasite fauna of three species of mullets, Mugil cephalus, Liza macrolepis and Valamugil cunnesius collected from 3 different sites, including an unpolluted region in Gosthani Estuary (site 1) and 2 polluted regions (sites 2 and 3) in Visakhapatnam harbour, with a view to determine the effects of pollution on the species composition and diversity of the parasite fauna and the occurrence and abundance of individual parasite species. In all 12 species of ectoparasites, inluding 2 sp. of Monogenea and 10 sp. of Copepoda infected the mullets. The ectoparasite assemblage on mullets comprised 9 species at site 1 and 10 and 6 species at sites 2 and 3 respectively. Five species were common to all the 3 sites. While there were no species specific to site 1, it was found that 2 species were specific to site 2 and one to site 3. The diversity of ectoparasite fauna at site 1 was high where as it was very low in the two polluted regions. Many parasite species showed high prevalence and abundance at site 1. The parasite fauna of mullets from the two polluted regions also exhibited differences especially in the species composition. The study revealed that certain ectoparasite species exhibit a high degree of host as well as site specificity.

SESSION 23. IMMUNOLOGY IV: Salmonids and Marine Fish

[TOP]

The Immunological Defences of Halibut Eggs

TJ Bowden and IR Bricknell*

FRS Marine Laboratory, Immunology Section, PO Box 101 Victoria Road, Torry, Aberdeen, AB53 6UL, UK. bowdent@marlab.ac.uk, bricknellir@marlab.ac.uk

It has been previously reported that many species of fish incorporate immunologically active proteins in their eggs. These defence proteins are hypothesised to provide non-specific defences against pathogens throughout the yolk sac stage until the larvae's immune system is sufficiently developed to take on this role. This concept is particularly attractive as it may offer a way of manipulating the immune system of broodstock halibut so they will transfer immunoglobulins, or other defensive proteins, into their eggs which can be targeted towards any pathogens the larvae are likely to encounter during their long larval period. The preliminary results of a project to investigate the immunological investment made by female halibut in their eggs will be presented here, with special reference made to the transfer of maternal immunoglobulins. The initial findings of this study strongly suggest that halibut do not transfer immunoglobulins into the eggs in contrast to reports for other pleuronectiform fish. These findings will be discussed in the light of current knowledge of egg immunology and its' implication on larval husbandry will be considered.

Molecular Analysis of Complement Component C7, C8 Beta and C9 of Japanese Flounder Paralichthys olivaceus

T Katagiri*, I Hirono and T Aoki

Laboratory of Genetics and Biochemistry, Tokyo University of Fisheries, Konan 4-5-7, Minato, Tokyo 108-8477, Japan ad95217@s4201.tokyo-u-fish.ac.jp

This study details the cloning and molecular analysis of the C7, C8 beta and C9 complement components of Japanese flounder Paralichthys olivaceus. The same sequence motifs reported here are found in human terminal components C6, C7, C8 alpha and beta and C9, and these proteins are thought to have evolved from a common ancestor of the terminal complement gene. The deduced amino acid sequences of C7, C8 beta and C9 of Japanese flounder all exhibit the following domains: thrombospondin, LDL receptor, EGF precursor and a second thrombospondin. The human counterparts also share these domains, with the exception of C9 from which the second thrombospondin is absent. Japanese flounder C7, like human C6 and C7, was, in addition, found to possess two SCRs and two factor I domains. However, only one of the flounder C7 factor I domains was complete, with several amino acids deleted from the C-terminus of the second. Two thrombospondin domains are present in Japanese flounder C9, a feature which provides it with more resemblance to human C8 alpha and beta than to human C9. Cysteine residues were highly conserved between all components regardless of flounder or human origin. Collectively these features emphasise the great similarity among the terminal complement family.

Characteristics of the Leukocyte-like Cell Line, That Derived from Leukemic Red Sea Bream

M Miyata*, T Kageyama, K Iinuma, T Kobayashi and T Miyazaki

Laboratory of Aquacultural Management, Faculty of Bioresources, Mie University, Kamihama 1515, Tsu, Mie 514, Japan. miyata@bio.mie-u.ac.jp

The suspension cultured cell line, that derived from spleen in leukemic red sea bream, Pagrus major was established (red sea bream spleen-leukemia: RSL) and characterized. The spleen was removed from leukemic fish, and meshed through nylon sieve. Adherent and erythroid cells were discarded by centrifugation with Ficoll 400. The cells recovered from the Ficoll 400 surface were cultured at 20C in RPMI1640 media that included FBS, Red Sea bream serum, and antibiotics. Since May 1997 to present (March 1998), stable and vigorous cell multiplication was observed in the media suspension. The light microscopy and the electron microscopy revealed that, there were three or more different shaped cells in RSL. In esterase staining, there were several cells that were positive for alpha-NB and N-ASDCLA. Also in the RSL, the phagocytosis of latex beads was observed. From the above results, the RSL must be a fish leukocyte like cell line. To investigate the natural resistant mechanisms in fish, Northern analysis on several leukocyte-specific genes (EST clones) is in progress, comparing the healthy to the bacteria infected RSL cells.

Structural and Functional Characterization of a Mannose-Binding Lectin from the Serum of Atlantic Salmon (Salmo salar)

KV Ewart (1), CA Ottinger (2), NW Ross (1), LL Brown (1) and SC Johnson(1)*

1	National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada, B3H 3Z1 mailto:vanya.ewart@nrc.ca; vanya.ewart@nrc.ca; mailto:neil.ross@nrc.ca; neil.ross@nrc.ca; mailto:laura.brown@nrc.ca; laura.brown@nrc.ca; stewart.johnson@nrc.ca
2	National Fish Health Laboratory, 1700 Leetown Road, Kearneysville, West Virginia, USA mailto:chris_ottinger@usgs.gov chris_ottinger@usgs.gov

Lectins are sugar-binding proteins that have been postulated to play several major roles in the innate immune system of invertebrates and vertebrates. We have isolated a number of mannose-binding proteins from Atlantic salmon serum by affinity chromatography. One of these, a calcium-dependent lectin, exists as a disulfide-linked multimer of a polypeptide having a relative molecular mass (Mr) of 17,000. This lectin binds to the surfaces of Vibrio anguillarum and Aeromonas salmonicida. Its ability to modulate macrophage responses was assessed in vitro. Wild-type A. salmonicida was selected as a target for the functional characterization studies of the lectin. Binding of the lectin in the absence of other factors did not significantly reduce the viability of A. salmonicida. However, lectin binding to A. salmonicida did result in significant dose-dependent increases in phagocytosis, respiratory burst and bactericidal activity of macrophages. The structure and activity of the lectin are reminiscent of those of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. It may be especially important when fish are exposed to gram-negative bacteria such as A. salmonicida. If it is determined that the production of this lectin can be induced, there may be potential for artificially stimulating its production to provide protection against important bacterial pathogens of Atlantic salmon. Such an immunomodulatory approach could provide a useful alternative or supplement to vaccination and/or chemotherapy in fish culture.

Multiparameter Cell-Cycle Analysis of Atlantic Salmon Peripheral Blood Leukocytes

CA Ottinger (2)*, LL Brown (1), NW Ross (1) and SC Johnson (1)

1	1 National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada, B3H 3Z1 mailto: laura.brown@nrc.ca; laura.brown@nrc.ca; mailto:neil.ross@nrc.ca; neil.ross@nrc.ca; stewart.johnson@nrc.ca
2	National Fish Health Laboratory, 1700 Leetown Road, Kearneysville, West Virginia, USA chris_ottinger@usgs.gov

In order to improve our understanding of fish immunological responses, there is an increasing need for techniques that yield highly detailed information on leukocyte function. FACS-based cell cycle analysis was applied to mitogen-stimulated Atlantic salmon peripheral blood leukocytes (PBLs). Cell cycle analysis using only the nucleic acid stain propidium iodide revealed an increased percentage of cells at the G2/M stage in mitogen-stimulated cultures. However, problems of high background staining of mitogen-induced mRNA necessitated a multiparameter method, which used both the presence of proliferating cell nuclear antigen (PCNA/cyclin) and the amount of DNA in the cells as quantified by propidium iodide to identify proliferating cells. PCNA/cyclin is a highly conserved protein and it is known to be expressed in salmonids. Using Western blot analysis of reducing SDS-PAGE gels, we demonstrated the ability of a commercially available anti-PCNA/cyclin mAb developed against mammalian PCNA/cyclin to recognize this protein in the PBLs. The occurrence of PCNA/cyclin within salmon PBLs was confirmed by epifluorescence microscopy, comparing mAb-associated FITC activity in the nucleus of PBLs from mitogen-stimulated and non-stimulated cultures. The multiparameter method consists of cell fixation followed by Zwittergent detergent permeablization of the cells. Prior to the addition of the propidium iodide, the PBLs were treated with RNAase to reduce background staining. This multiparameter method allows for traditional proliferation measures (mitogenic indices based on the percentage of PBLs expressing PCNA/cyclin in mitogen treated and non-treated cultures), detailed analysis of cell cycle position, and evaluation of apoptosis.

The Cell Mediated (CMI) and Humoral Immune Response Developed by Piscirickettsia salmonis in Atlantic Salmon

CS Farias (1)*, A Adams (2), K Thompson (2), M Monras (3) and E Landskron (4)

1	Aquatic Biotechnology and Immunology Unit, Faculty of Veterinary Sciences, Universidad Austral de Chile, Casilla 567 Valdivia Chile Email: cfarias@valdivia.uca.uach.cl
2	Aquatic Vaccine Unit, Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK Email: aa2@stir.ac.uk
3	Fish Pathology Unit, Faculty of Veterinary Sciences, Universidad Austral de Chile, Casilla 567 Valdivia Chile
4	Marine Harvest, Av. Diego Portales 860 Puerto Montt Chile

The immune response of Atlantic salmon experimentally infected with the fish pathogen Piscirickettsia salmonis was studied. The role of the cell mediated immunity (CMI), particularly fish phagocytic cells was determined by seven different techniques which included hematocrit levels, adherence/NBT cell number, Leukocyte count (by hemocytometer and NBT/spectrophotometric reading), potential killing activity, phagocytic index and myeloperoxidase activity count. The humoral immune response to the pathogen was determined by measuring the total protein levels, total and specific IgM levels and lysozyme levels in the sera. Prior to the experiments the TCID50 and LD50 of Piscirrickettsia salmonis was determined. Fish were challenged by ip injection and groups of ten fish per tank were taken every 48 hours in order to determine the cellular and humoral immune response. The role of the immnune system in the pathogenesis of the Piscirickettsiosis will be discussed.

SESSION 24. BACTERIOLOGY III

[TOP]

A Bacteriological Study of Oysters, Crassostrea virginica and Ostrea edulis, in the Damariscotta River, Maine, USA

MA Crosby* and BJ Barber

School of Marine Sciences, University of Maine, Orono, Maine 04469. USA mcrosb61@maine.maine.edu

This experiment compared the bacteria associated with healthy and diseased oysters, Crassostrea virginica and Ostrea edulis, in the Damariscotta River, Maine, where Juvenile Oyster Disease (JOD) is endemic. Three replicates of each of three juvenile oyster cohorts (C. virginica (Flowers); C. virginica (Piscataqua); and O. edulis) were deployed in June 1997. Three animals from each replicate were sampled weekly for associated bacteria by swabbing the inner surfaces of the valves and tissues and washing the swab in sterile seawater. These samples were pooled and plated on low nutrient seawater agar. The resulting colonies were enumerated and isolated for identification using standard biochemical tests, the API 20E* system, and the Biolog* system. C. virginica experienced significant mortality (80-100%) due to JOD, while O. edulis was unaffected by JOD. Total viable counts of bacteria varied seasonally and between oyster and seawater samples, but there was little difference between species. Oysters affected by JOD had greater numbers of associated bacteria than healthy oysters. Differences in specific bacterial populations (e.g., Vibrio spp., Aeromonas spp., and Cytophaga/Flexibacter spp.) occurred between species.

Isolation of Anaerobic Intestinal Bacteria, Pathogens and Normal Flora, from Fish and Shellfish

BA Dixon *, DV Straub and J Truscott

Department of Biological Science California State University, Hayward, California 94542 USA bdixon@csuhayward.edu

The normal intestinal anaerobic bacterial flora of fish and shrimp for the most part remains unknown. The purpose of this on-going research is to isolate and identify the anaerobic intestinal flora of cultured fish and shrimp species, including, Tilapia (Oreochromis spp.), Channel catfish (Ictalurus punctatus), ornamental fish and shrimp (Penaeus vannemai). Samples for bacterial culture were anaerobically collected and grown in a Bactron II anaerobe chamber at 25C on pre-reduced anaerobically sterilized media. Isolated anaerobes were identified by cellular and colonial morphology, Gram stain, aerotolerance testing, biochemical reactions, and inoculation into the An-Ident multitest system. Enzyme analyses were performed on 19 different substrates using the API-ZYM panel. To date, several anaerobic genera including Bacteroides, Clostridium, Fusobacterium, and Peptostreptococcus were identified from fish and shrimp intestines. Enzyme analyses of bacteria from shrimp were found to produce esterase, galactosidase and fucosidase. Enzymatic profiles of bacteria from fish were found to produce trypsin, lipase, and alpha and beta glucuronidase. The pathogenic bacterium, Clostridium difficile, was isolated and identified from African cichlids (Nimbochromis venustus) with the condition known as Amalawi bloating.

Bioencapsulation of Antibacterial Drugs in Adult Brine Shrimp, Artemia franciscana

RF Ramirez (1)*, BA Dixon (1), and DK Kawahigashi (2)

1	Department of Biological Sciences, California State University, Hayward, California 94542 USA rramire2@haywire. csuhayward.edu; bdixon@csuhayward.edu
2	San Francisco Bay Brand Inc., 8239 Enterprise Dr., Newark, California 94560 USA baybrand@shellx.best.com

A promising method for the treatment of bacterial diseases in cultured fish and shrimp is the administration of live food supplemented with therapeutic agents via bioencapsulation. This study was initiated to determine if effective levels of antibacterial drugs could accumulate in brine shrimp, Artemia franciscana. Efficacy was assayed in vitro against bacterial isolates of Aeromonas and Vibrio from diseased fish and shrimp. Artemia were enriched for 2 hrs with either, Romet 30®, erythromycin, minocycline, sarafloxacin in 0.6g/1 of Super Selco or lecithin. After enrichment, sensitivity assays were performed using a modification of the Kirby-Bauer disk diffusion method against test strains. Bacterial suspensions were adjusted to a density standard of 1.5 x 108 CFU/ml, and swabbed onto Mueller-Hinton agar plates. Sterile filtrates from enriched Artemia were applied to blank disks on the swabbed plates. Following 24 hr incubation at 30°C, the zones of inhibition were compared to standards. Optimal concentrations for bacterial sensitivity differed among the drugs tested with Sarafloxacin achieving the highest efficacy at the lowest concentration. The results indicate that the oral delivery of antimicrobial drugs can be facilitated through bioencapsulation of Artemia.

Microbial Control of the Intensive Culture of Artemia Juveniles

L Verschuere (1), G Rombaut (1), J Dhont (2), G Huys (3), P Sorgeloos (2), J Swings (3) and W Verstraete (1)

1	Lab of Microbial Ecology, University of Gent, Coupure L 653, B-9000 Gent, Belgium ; laurent.verschuere@rug.ac.be
2	Laboratory of Aquaculture & Artemia Reference Center, University of Gent, Rozier 44, B-9000 Gent, Belgium ; patrick.sorgeloos@rug.ac.be
3	Laboratory of Microbiology, University of Gent, K.L. Ledeganckstraat 35, B-9000 Gent ; geert.huys@rug.ac.be

Gaining control over the microbiology becomes essential for the economic viability of modern intensive aquaculture ventures. Recently, new approaches have been proposed, taking into consideration some principles of microbial ecology and the present knowledge about the interactions between bacteria and the cultured organisms (probiotics, matured water,...). This work focusses on the intensive culture of Artemia juveniles for aquaculture and aquarium purposes. A first series of experiments showed that the microbial community that colonizes the culture tanks seems to be determined by both deterministic factors and stochastic factors. Based on these observations, it was assumed that the variability of the microbial community associated with the culture of Artemia juveniles could be restricted through the early application of a probiotic strain or a mixture of bacterial strains, provided they are well adapted to the prevailing conditions. In a second stage, the effect of a number of bacterial isolates on the Artemia juveniles was assessed in monoxenic test tube cultures. From these tests, 9 strains were selected which had a positive or neutral effect on the zootechnical results. In a third stage, the effect of autoclaved seawater preemptively inoculated with the selected strains was assessed on the survival and the growth of the Artemia juveniles under holoxenic rearing conditions. The microbial communities of the culture water and those occurring at the surface and in the gut of the shrimps were analysed and compared by means of the Biolog system. Isolates were characterized with GC-FAME and were compared to the inoculated strains.

Pathogenic Bacteria in a Shrimp Hatchery and Backwaters in Andhra Pradesh, India

C Kalavati*, N Aravindan and AV Raman

Division of Protozoology, Department of Zoology, Andhra University, Visakhapatnam - 530003, India. pcmavr@md2.vsnl.net.in

Pathogenic bacteria infecting shrimp post larvae were examined from three select brackish water locations namely, Gosthani estuary (lat. 17 19'N; long. 83 30' E), Gambhera Gedda (lat. 17 18' N; long. 83 28' E) and Kakinada Bay (lat. 16 44'N; long. 82 31' E) and a hatchery in Andhra Pradesh in India. The predominant species were Aeromonas, Pseudomonas and Vibrio with varying abundance in water (Gosthani estuary, 9x108 nos. per ml; Gambera Gedda, 5x108 nos. per ml and Kakinada, 7x108 nos. per ml) and sediments (Gosthani estuary, 5x108 nos. per ml; Gambhera Gedda, 8x108 nos. per ml and Kakinada, 5x108 nos. per ml). Twenty bacterial cultures isolated from the hepatopancreas of shrimp Penaeus indicus, P. monodon, Macrobrachium rosenbergii and M. malcomsonii), water and sediments were identified as Vibrio parahaemolyticus, V. anguillarum and V. damsela. In the laboratory, threshold levels of Vibrio species in the ambient water causing 50% mortality in early post larvae (PL 22) among P. indicus, P. monodon, M. rosenbergii and M. malcomsonii were studied. Results obtained on the incident profiles emphasize the significance of these organisms and autocthonous microflora in hatchery systems.

Dermal Ulceration of Nile Tilapia, Oreochromis nilocticus, in Freshwater Ponds

F Palisoc Jr. (1)*, R Macion and W Baraquio

1	SEAFDEC Aquaculture Dept., 17 Times St., West Triangle, Quezon City 1004, Philippines

Missing abstract

SESSION 25. PARASITOLOGY VII: Infections in Shellfish and Gastropods

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Results of Trials to Determine the Relative Resistance of an Irish Population of Flat Oysters to the Parasite Bonamia ostreae

SC Culloty* and MF Mulcahy

Department of Zoology and Animal Ecology, University College Cork, National University of Ireland Cork, Lee Maltings, Prospect Row, Cork, Ireland. s.culloty@ucc.ie; mmulcahy@ucc.ie.

The population of flat oysters Ostrea edulis in Cork harbour on the south coast of Ireland has been known to be infected with the parasite Bonamia ostreae since 1986. Mortalities of up to 90% were associated with the parasite infection. To increase production and minimise losses due to bonamiasis, selective breeding for resistance against this disease has been in progress for more than ten years. Only oysters that survive for four years or more are used as broodstock. In recent years production of flat oysters has increased significantly and losses decreased. Trials were undertaken in both laboratory and field to determine if this population has built up some resistance to the parasite. The Cork harbour population was compared to two other Irish populations: Tralee and Lough Foyle; and two Scottish populations: Mull and Loch Eriboll, all of which are from areas where the disease has never been detected. Prevalence of infection and cumulative mortalities were compared in all populations following both natural field based transmission experiments and experimental transmission by injection of the parasite in the laboratory. Results of these trials to date indicate that the Cork harbour population developed lower prevalence of infection and experienced lower mortalities over the trial period than the other populations tested. Larger scale trials are now being undertaken on a European basis to investigate the relative resistance of this population and other European oyster populations to Bonamia ostreae.

Isolation of a Labyrinthuloides sp. (Protozoa: Labyrinthomorpha) with Unique Properties from the Softshell Clam (Mya arenaria)

M Faisal (1)*, SM McLaughlin (2), E Elsayed (3), EA MacIntyre (1), SI Kotob (1) and H El-Gawady (4)

1	Virginia Institute of Marine Science, School of Marine Science, The College of William and Mary, Gloucester Point, VA 23062 faisal@vims.edu
2	National Marine Fisheries Service, Cooperative Oxford Laboratory, 904 S. Morris St., Oxford, MD 21654 shawn.mclaughlin@noaa.gov
3	Faculty of Veterinary Medicine, Cairo University, Giza, Egypt ehab@intouch.com
4	Department of Parasitology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt

Thraustochytrids are ubiquitous to the marine environment worldwide and a few of them have been associated with serious diseases in bivalve molluscs. Most recently, we isolated a Labyrinthuloides sp. from a softshell clam with disseminated sarcoma. In this study, we describe several unique properties of this protozoan. Most characteristically, the organism produced copious amounts of exopolysaccharides that formed a relatively thick layer surrounding the protozoal cells. Scanning electron microscopy revealed the presence of pores in the exopolysaccharide layer and the number and position of these pores correspond to the presence of enveloped cells. The vegetative stages showed vestigial sagenogentosomes and most of their cytoplasm was occupied with secretory vacuoles containing either crystals or polysacharrides. In vitro, the parasite secretes two high molecular weight serine proteases (120 and 90 kDa) with extraordinarily high specific activities. Morphological characteristics and life cycle stages observed in this study suggest the organism is a Labyrinthuloides sp. although similar biochemical properties have not been reported in other Thraustochytrids. We are currently studying the genetic relationship of this parasite to other related protozoal species.

MULTIPLEX PCR: a Rapid Method for Screening of Oyster Pathogens Perkinsus marinus (Dermo), Haplosporidium nelsoni (MSX) and Haplosporidium costale (SSO)

S Penna (1)*, J Volk (2), J Karolus (2), I Sunila (2) and RA French (1)

1	University of Connecticut, Department of Pathobiology, 61 N. Eagleville Rd., Storrs, CT 06269. map96006@uconnvm.uconn.edu; french@uconnvm.uconn.edu
2	Connecticut Dept. of Agriculture, Bureau of Aquaculture and Laboratory, Milford, CT 06278

New England, and specifically, Connecticut have seen an increase in oyster production while most of the Atlantic coast has experienced a decline during the past decades. The decline along the Atlantic coast has been due mainly to disease, particularly Haplosporidium nelsoni (MSX) and Perkinsus marinus (Dermo). Presently, the monitoring of oyster populations for pathogens is infrequent and no routine surveillance is performed due to the dependence on histopathology for H. nelsoni, Haplosporidium costale (SSO) and the Ray/Mackin assay for P. marinus. The continued success of the Northeast and mid-Atlantic states as producers of shellfish products requires a practical means of diagnosing and monitoring oyster diseases. This requires diagnostic aids which are sensitive, rapid, cost effective, and convenient. In the present study, we investigated the development of the multiplex PCR for application in the screening and surveillance of disease agents of the eastern oyster (Crassostrea virginica). Multiplex PCR will allow for the simultaneous testing of two or more pathogens in a single test reaction with a total assay time of approximately 12 hours or two working days. Preliminary multiplex PCR results have identified three PCR primer sets which amplify 564-bp, 150-bp, and 304-bp products of control cloned small subunit rDNA of H. nelsoni, H. costale, and P. marinus, respectively, which were differentiated by using agarose gel electrophoreses. Further testing is in progress to optimize the assay for field use and quantitative measure. The assay would be a comprehensive, sensitive and rapid tool for routine screening of seed, disease surveillance, inspection and certification, and research application.

DNA-based Molecular Diagnostics for Haplosporidium nelsoni (MSX) Life Cycle Studies

NA Stokes (1)*, BS Flores (1), KA Ashton-Alcox (2), JR Pharo (2), SE Ford (2) and EM Burreson (1)

1	Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062 USA stokes@vims.edu; crazybsf@vims.edu; gene@vims.edu
2	Haskin Shellfish Research Laboratory, Rutgers University, Port Norris, NJ 08349 USA kathryn@hsrl.rutgers.edu; jessica@hsrl.rutgers.edu; susan@hsrl.rutgers.edu

The oyster pathogen Haplosporidium nelsoni, the agent of MSX disease, has caused extensive oyster mortality in the eastern United States since 1957. The life cycle stage infective to oysters and the source of that stage have not yet been identified. Attempts to infect oysters directly with H. nelsoni spores have been unsuccessful, thus leading to speculation that parasite transmission between oysters occurs via an obligate intermediate host. We have developed H. nelsoni-specific polymerase chain reaction (PCR) and in situ hybridization (ISH) diagnostic assays. These assays have been optimized for use with environmental samples and are being used in the search for the putative intermediate host(s). Samples of water and sediment fractions and of macroinvertebrates have been taken from MSX-endemic areas of York River, VA and Delaware Bay since March 1996. Total genomic DNA has been extracted from each sample and subjected to PCR amplification. Samples that have yielded H. nelsoni PCR product have been much more frequent from the York River than from the Delaware Bay, corresponding to the MSX disease prevalence in oysters from these locations. ISH is being used to screen PCR-positive macroinvertebrate samples to discriminate between true infections and those where H. nelsoni is simply adhering to the external surface or passing through the gut.

Molecular Detection of Marteilia sydneyi, Pathogen of Sydney Rock Oysters

SN Kleeman (1)* and RD Adlard (2)

1	1 Department of Parasitology, University of Queensland, Brisbane Qld 4072 Australia S.Kleeman@mailbox.uq.edu.au
2	Protozoa Section, Queensland Museum, P.O. Box 3300, South Brisbane Qld 4101 Australia robertad@qm.qld.gov.au

Marteilia sydneyi, the aetiological agent of QX disease, is recognised as the most pathogenic parasite of the Sydney rock oyster on the East Coast of Australia, where stock mortalities in excess of 90% have been recorded. Patterns of infection in the oyster host are well described, however the origin of the life-cycle stage infective to the oyster is unknown. We have developed and optimised two diagnostic assays based on the polymerase chain reaction (PCR) and in situ hybridisation for use in investigating the role of possible alternative hosts in the life-cycle of this pathogen. PCR primers, designed within the internal transcribed spacer region (ITS) of the rDNA gene cluster of M. sydneyi, were found to amplify selectively M. sydneyi DNA from isolates of mixed oyster and parasite genomic DNA. Sensitivity of the PCR assay was assessed using DNA extracted from known numbers of sporonts both in isolation and in the presence of host DNA. A DNA probe was constructed using the M. sydneyi unique primers and found to be sensitive and specific in dot-blot hybridisations. The probe hybridised with M. sydneyi stages in paraffin sections of oyster digestive gland and there was no non-specific binding. While the high sensitivity and specificity of the PCR test will allow rapid screening of large numbers of potential alternative hosts for the presence of parasite DNA it does not identify the infective stages themselves. In situ hybridisation conducted on paraffin sections will determine the locality of the parasite within the host and allow morphological characterisation.

Pathological Changes Caused by Parasites in Some of the Commercially Important Marine Gastropods from Southeast Coast of India

BA Venmathi Maran (1)* and K Ayyakkannu (2)

1	Research Student, Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai, 608 502 Tamil Nadu, India.
2	Professor, Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai, 608 502 Tamil Nadu, India.

Two trematode larval cercariae and a nematode were identified from the marine predatory gastropods Chicoreus ramosus, Rapana rapiformis and Chicoreus virgineus respectively. Numerous cysts of trematode parasites cause extensive damage to the digestive gland which appears to be the preferred site of infection. Most of the infected snails showed heavily bored and severely damaged shell morphology. Sporocysts of trematodes, cestodes and nematodes generally infest the digestive gland, gonad, foot and mantle of the molluscs. Histopathological examination of digestive glands revealed severe obliteration in the organization of the acinar cells, their shape and structural integrity, occurrence of pycnotic nuclei, autolysis of acinar cells and shrinkage of foot evidenced. Acute infection was observed in adult males than the juveniles.

SESSION 26. TOXICOLOGY

[TOP]

Metabolism and Biochemical Constituents in an Intertidal Gastropod, Turbo intercostalis Exposed to Cadmium.

SL Pandeswara and PR Yallapragada

Division of Animal Physiology and Toxicology, Department of Zoology, Andhra University, Visakhaptam - 530 003, INDIA. E-mail : vijaya_vizag/bangalore@dartmail.dartnet.com

Molluscs have the potential to act as biomonitors of pollutants in the marine environment. Earlier investigations on cadmium toxicity in an intertidal gastropod, Turbo intercostalis revealed an LC50 of 6-18 ppm for 96 hrs. Further, laboratory experiments were conducted to study the oxygen consumption and biochemical constituents in T. intercostalis exposed to different concentrations of cadmium (2.8 to 13.6 ppm). A significant decrease in oxygen consumption was observed from LC50 onwards. The biochemical constitutents were estimated in different tissues such as gonad-digestive gland (GDG) complex, mantle, foot and viscer of T. intercostalis on exposure to above concentrations of cadmium. The total carbohydrates, glycogen and total proteins exhibited a depletion in GDG complex and mantle. An erratic trend was noticed for total lipids in all the tissues. The depletion was more for glycogen than for protein levels. Glycogen/protein ratios decreased in GDG complex at all concentrations of cadmium. A significant decrease was noticed for glycogen/lipid ratio from LC25 onwards in GDG complex. These results indicate that decrease in biochemical constituents in T. intercostalis on cadmium exposure might be either due to utilisation of these constituents under metal stress or their less synthesis due to inhibition of pathways by the metal.

Effects of Cadmium Exposure on Brown Cells of the Quahog Mercenaria mercenaria in vivo

EC Peters (1)*, G Zaroogian (2) and DJ Borsay (3)

1	Tetra Tech, Inc., 10306 Eaton Place, Suite 340, Fairfax, VA, 22030 USA. peteres@tetratech-ffx.com
2	US Environmental Protection Agency, Atlantic Ecology Division, 28 Tarzwell Drive, Narragansett, RI 02882 USA. zaroogian.jerry@epamail.epa.gov
3	US Environmental Protection Agency, Atlantic Ecology Division, 28 Tarzwell Drive, Narragansett, RI 02882 USA. borsay.dodi@epamail.epa.gov

Brown cells of marine bivalves are important in the detoxification and excretion of soluble substances, particularly metals. Glutathione appears to operate in the uptake and detoxification processes, perhaps by transporting the metal through the brown cell cytoplasm to bind with other proteins or into lysosomes. Partial mortality of brown cells in the quahog Mercenaria mercenaria occurred during in vivo studies following injection of the clam with metal solutions, suggesting that some brown cells are more resistant to metal toxicity. Histopathological examinations of the pericardial glands were made during and after seawater exposures of quahogs to cadmium (0.5 and 1.0 ppm) for 20 days. Brown cells exhibited variable responses both within each gland and between clams. Fewer and less extensive or severe degenerative and inflammatory changes were observed in the 0.5 ppm-exposed clams than in the 1.0 ppm-exposed clams. Severity of lesions increased until approximately 6 days, then declined. These results support the hypothesis that the pericardial gland contains a heterogeneous population of brown cells with the capability of developing a more toxicant-resistant population of brown cells after cytotoxicity and sloughing of affected cells, which could explain the variable response of these bivalves to metal pollution in the field.

Formation of Benzo(a)pyrene-DNA Adducts in Hemopoietic Tissues and Blood of the Mummichog (Fundulus heteroclitus)

WL Rose (1)*, BL French (2), WL Reichert (2), E Elsayed (1) and M Faisal (1)

1	Virginia Institute of Marine Science and School of Marine Science, College of William and Mary, Gloucester Point, VA 23062 USA. wrose@vims.edu; faisal@vims.edu
2	Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Boulevard East, Seattle, WA 98112-2097 USA. barbara.french@noaa.gov; william.l.reichert@noaa.gov

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that alter immune responses in all vertebrates. In fish, biochemical and pharmacological pathways of PAH-induced immunotoxicity have received little attention. We have shown that the potentially carcinogenic PAH, benzo(a)pyrene (BP), forms associations with DNA of hemopoietic tissues and blood of the mummichog (Fundulus heteroclitus). These DNA associations may be DNA adducts (BP covalently bound to nucleotides) or non-covalent associations. DNA adducts have the potential to induce changes in immunocompetent cells. Consequently, we examined the hemopoietic tissues and blood of BP-exposed mummichog for the presence of BP-DNA adducts. Mummichog were injected intraperitoneally with a sublethal dose of BP and sampled at predetermined intervals until 96 days. Anterior kidney, spleen, and liver were removed and blood collected. 32P-postlabeling (PPL) and thin layer chromatography (TLC) were used to determine if BP-DNA adducts formed in vivo. Our results indicate the formation of BP-DNA adducts in the hemopoietic tissues and blood of exposed mummichog. Because these tissues are integral parts of the fish immune system, the potential exists for PAH-DNA adducts to interfere with the immune response.

Lead Toxicity on Growth, Oxygen Consumption and Ammonia-N Excretion in Postlarvae of Penaeus indicus

S Chinni (1)* and RY Prabhakara (2)

Division of Toxicology, Department of Zoology, Andhra University, Waltair, Visakhapatnam -530 003, India. Email: pcmavr@md2.vsnl.net.in

The estuaries and backwaters which are potential breeding grounds of penaeid shrimps, are subject to heavy metal pollution through industrial effluents and domestic sewage. In the present investigation, laboratory experiments were conducted to study the effect of lead on growth, oxygen consumption and ammonia-N excretion in postlarvae (PL) of Penaeus indicus. The PL were subjected to sublethal concentration of lead (1.44 ppm) for a period of 30 days. Parallel controls were maintained without the toxicant. The growth of PL was determined by measuring the length as well as their weight. The oxygen consumption measurements were made by using a respiratory chamber equipped with an oxygen electrode and ammonia-N was determined with trione (dichloro-S-triazine-2, 4, 6 (1H, 3H, 5H-trione)). Lead exposure resulted in the retardation of growth and a significant decrease in the length and weight was observed from 10th day onwards. There was a gradual and significant decrease in oxygen consumption as well as ammonia-N excretion on exposure to lead. The decrease was more for ammonia excretion (79.5) than oxygen consumption (56.5) after exposure for 30 days. The data suggest that lead exposure results in reduction of growth together with a decrease in respiratory and excretory metabolism. This might be due to interaction of the metal with the metabolic pathways.

Pathologic Disorders and Reproductive Disturbance in Periwinkle (Littorina littorea) from the German Coast in Relation to Tributyltin Contamination

B Watermann (1)*, M Wölm (2), I Ide (1) and S Liebe (1)

1	Laboratory for freshwater/marine research and comparative pathology (LimnoMar), Bei der Neuen Münze 11, D-22145 Hamburg, Germany. limnomar@aol.com
2	Office for veterinarian pathology, Bei der Neuen Münze 11, D-22145 Hamburg, Germany, woelmpatho@aol.com

Tributyltin (TBT) is known as a relevant cause to induce pseudohermaphroditism or intersexuality in prosobranch gastropods. Yet little information exists about TBT associated effects along the German coast. In 1994 and 1995 an investigation was performed to study the effects of TBT contamination along the North Sea and the Baltic Sea coast. Several marine snails were checked for the occurrence of intersexuality or pseudohermaphroditism. 611 periwinkles (Littorina littorea) from 19 stations were histologically investigated. They exhibited several degrees of intersex stages in relation to TBT body burden. Parallel histologic investigations of the female gonads revealed disturbance in the reproductive cycle and an enhancement in the resorption of gametes. Different types of resorption by haemocytes and the epithelial cells in the follicles could be identified. In contrast to the changes according to the normal reproductive cycle, the follicle epithelium degenerated. In contrast to a positive correlation between morphological changes and the TBT contamination, pathological alterations in the gonad did not correlate positively. Several other factors have to be considered.

Study on Intoxication Mechanism of Microcystins on Fish

L Xu (1), B Zhou (1), KSL Paul (2), J Chen (1), G Chen (1), Y Zhang (1) and H Ken-ichi (3)

1	The State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan, Hubei, China, xulh@lily.whihb.ac.cn
2	Centre for Environmental Science and Technology, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, bhpksl@cityu.edu.hk
3	Faculty of Pharmacy, Meijo University, Nagoya 468, Japan, kiharada@meijo. u..ac.jp

Microcystins are a group of toxic secondary metabolites produced by certain genera of cyanobacteria. The presence of microcystins arising from frequent and wide occurrence of cyanobacterial blooms in water bodies is a global problem. The intoxication of rodents exposed to microcystins is characterised by an inhibition of protein phosphatases. In vitro inhibitory effects of microcystin-LR, -YR and -RR on protein phosphatases type 1 and 2A isolated and purified from grass carp, Ctenopharyngodon idellus, as well as protein phosphatases present in fish tissue homogenates were investigated in this study. Results showed that microcystins had specific and potent inhibition on fish protein phosphatases. Liver protein phosphatase activity was completely inhibited in fish injected (i.p.) with microcystin-LR, and this was accompanied by a corresponding decrease in the GSH content in the fish liver. When GSH was given to the fish before microcystin administration, the ultrastructure of the fish liver was less affected by microcystins as compared to fish that were not pre-injected with GSH. Hepatocytes of fish not pre-injected with GSH showed complete dissociation and severe damage in their ultrastructure, suggesting that GSH could offer protection to the fish hepatocytes against the toxins. The possible role of GSH in toxin detoxification is discussed.

*This work was funded by the Natural Science Foundation of China (Project No.:39400024) and the Hong Kong Research Grants Council (Project No.:9040278).

Chromium Toxicity on Size, Salinity and Oxygen Consumption in a Fouling Dreissinid Bivalve Mytilopsis sallei

UD Venigalla and PR Yallapragada

Division of Animal Physiology and Toxicology, Department fo Zoology, Andhra University, Visakhapatnam ­ 530003, India. pcmavr@md2.vsnl.net.in

Recent reports on the harbor waters of the Visakhapatnam on the East Coast of India have shown a tremendous increase in heavy metals which pose a great threat to a number of organisms that inhabit the area. Therefore, the effect of size and salinity on chromium (Cr) tolerance and oxygen consumption in relation to different concentrations of the metal have been investigated on a gouling dreissinid bivalve Mytilopsis sallei. In M. sallei, three size groups namely big (2-2.5 cm), medium (1-1.5 cm) and small (0.5-1 cm) individuals were tested by exposing each group to Cr for 96 hours. The test solutions of Cr were prepared by adding appropriate amounts to the medium. A concentration range of 5 to 16 ppm of Cr was used. The tolerance of M. sallei varies with different sized individuals. The older and younger bivalves appeared to be less sensitive with high LC50 values of 10.10 and 10.13 ppm, respectively, while medium sized individuals showed more sensitivity with a low LC50 value of 6.18 ppm. Since salinity is one of the major fluctuating environmental parameters in the harbor waters, these studies were made in relation to different salinities 10, 15, 25, 32 and 35 . The LC50 values were found to increase with increasing salinities indicating a decrease in toxicity. However, low salinities did not affect the toxicity of Cr while high salinities decreased the Cr toxicity in these bivalves. The metabolic rate was monitored by employing a respiratory chamber equipped with an oxygen electrode. The metabolic rate showed in increasing trend up to 32 followed by a decrease at 35. In general, different concentrations of Cr inhibited the metabolic rate at all salinities studied.

SESSION 27. PARASITOLOGY VIII: Apicomplexa, Microsporidia, Myxozoa

[TOP]

Life Cycle of Calyptospora funduli (Apicomplexa: Calyptosporidae)

JW Fournie (1)*, WK Vogelbein (2), RM Overstreet (3) and WE Hawkins (3)

1	U.S. Environmental Protection Agency, Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze, Florida 32561 USA. fournie.john@epamail.epa.gov
2	Department of Environmental Science, Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, Virginia 23062 USA. wolf@vims.edu
3	Gulf Coast Research Laboratory, PO Box 7000, Ocean Springs, Mississippi 39564 USA roverstr@seahorse.ims.edu; whawkins@seahorse.ims.usm.edu

Calyptospora funduli, an extraintestinal, piscine coccidium transmitted by a true intermediate host, naturally infects the liver of at least 7 atheriniform fishes. The sporozoite of C. funduli apparently reaches the hepatic parenchyma of its killifish definitive hosts via the peripheral blood. Sporozoites were seen in blood smears from the longnose killifish, Fundulus similis, 4 hours after being fed experimentally infected grass shrimp (Palaemonetes pugio). Additionally, the coccidium trophozoites were seen in hepatocytes from several longnose killifish at 48, 72, and 96 hours postinfection. Light microscopically, large numbers of sporozoites were seen suspended in the intestinal contents of experimentally infected grass shrimp. No sporozoite was apparent in intestinal epithelial cells or other cells of the grass shrimp. Ultrastructurally, however, sporozoites were seen in the cytoplasm of the basal cells located proximal to the nuclei of the columnar epithelial cells. Cross sections of up to 13 sporozoites were seen within a single cell, and the sporozoites appeared to be situated in individual membrane-bound vesicles rather than in a single parasitophorous vacuole. Whether these resulted from asexual reproduction or represent maturing individuals is not known. Nevertheless, without a developmental period of about 5 days in the grass shrimp, the "sporozoite" is not infective to killifishes. Consequently, the crustacean invertebrate host is necessary to complete the life cycle, making the cycle different from that for members of the family Eimeriidae. We therefore accept the family Calyptosporidae on both morphological and life cycle features.

Loma spp. Infection in Fishes from British Columbia, Canada

ML Kent (1)*, RW Shaw (1,2), SC Dawe (1), MJ Higgins (1), AMV Brown (2) and ML Adamson (2)

1	Department of Fisheries and Oceans, Pacific Biological Station, Nanaimo, British Columbia V9R 5K6 Canada
2	Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4

For the past 10 years Loma salmonae has been recognized as an important pathogen of pen-reared chinook salmon (Oncorhynchus tshawytscha) in the Pacific Northwest, whereas its was considered relatively non-pathogenic to salmonids in fresh water. Our recent investigations have shown that Loma salmonae is more prevalent than previously thought in ocean-caught and sexually mature salmon that have returned to fresh water to spawn. Furthermore, we have found Loma spp. in several new hosts caught in marine waters of British Columbia: lingcod Ophiodon elongatus (17%), sablefish Anoploma fimbria (12%). Pacific cod Gadus macrocephalus (33%), Pacific tomcod Microgadus proximus (7%), walleye pollock Theragra chalcograma (40%) and shiner perch Cymatogaster aggregata (12%). Severe infections were observed in some Pacific cod. Most of these isolates probably represent undescribed species based on transmission studies and rDNA comparisons, and we recently described the shiner perch parasite as L. embiotocia. We have also seen Loma salmonae in all five species of ocean-caught Pacific salmon Oncorhynchus spp. that we have examined, with a combined prevalence of 5 %. The infection was also associated with high prespawning mortality of sockeye salmon (Oncorhynchus nerka) in the Babine system in northern British Columbia. These observations demonstrate that Loma spp. infections are more common in the Province than previously thought.

In situ Hybridisation of PKX, the Causative Organism of Proliferative Kidney Disease (PKD)

DJ Morris*, A Adams and RH Richards

Institute of Aquaculture, University of Stirling, Stirling, Scotland. FK9 4LA

Proliferative kidney disease (PKD) is a commercially important disease of salmonid culture both in North America and Western Europe. Little is known of the life cycle of the causative organism referred to as PKX. The parasite appears in the fish kidney 2-3 weeks post-exposure to infective waters. From here it proliferates evoking a strong immune response. The initial developmental stages inside the salmonid host remain unknown, as do infective stages and any intermediate hosts. Monoclonal antibodies (MAbs) raised to PKX have demonstrated that the parasite alters its antigenic properties during its development in the fish. This means that these MAbs are restricted in their use as diagnostic tools and for studying the life cycle of the parasite. Recently, oligonucleotide probes have been developed towards PKX DNA. These have been used to identify the presence of the parasite by using the polymerase chain reaction PCR. An in situ hybridisation technique is described that utilises these probes to visualise the parasite in paraffin embedded sections. This technique detects PKX, regardless of its developmental stage and location within the host. Studies investigating the development of PKX in rainbow trout, using this technique, are described.

Comparative Study of PKX and Myxosporeans of Wild Fish Using Monoclonal Antibodies and Lectins

J O'Flynn (1)*, R Hoare (2), C Hanjavanit (2), S Acevedo (2), A Adams (1) and M Mulcahy (2)

1	Institute of Aquaculture, Stirling University, Stirling FK9 4LA, SCOTLAND. janet.o'flynn@stir.ac.uk; alexandra.adams@stir.ac.uk
2	Department of Zoology and Animal Ecology, University College, Cork, IRELAND. mmulcahy@ucc.ie

Availability of specific antibody and lectin probes for PKX have been invaluable for the study of possible alternate stages and alternate hosts of the parasite. In this study, the seasonal incidence of myxosporeans of Atlantic salmon (Salmo salar), brown trout (Salmo trutta), minnow (Phoxinus phoxinus) and eel (Anguilla anguilla) from a river with enzootic PKD was investigated over two years. Extrasporogonic and sporogonic stages of Myxidium giardi, Myxobolus cyprini, Chloromyxum sp. and an unidentified myxosporean were observed in wild fish. Typical PKX and sporogonic PKX was detected in brown trout. Using the specific probes, antigenic relatedness of the myxosporeans to PKX was investigated. Extrasporogonic and sporogonic stages of myxosporeans were tested with two monoclonal antibodies (MAb C5, MAb B4) directed against PKX. MAb C5 cross-reacted with typical PKX only. MAb B4 cross-reacted with sporogonic stages of Myxidium giardi in eel and sporogonic stages of Chloromyxum sp. in Atlantic salmon. Parasites were also tested with two lectins (Bandieraea simplicifolia, GS-I; Glycine max, SBA) which were previously shown to bind specifically to PKX and Sphaerospora sp., respectively. GS-I cross-reacted slightly with sporogonic stages of myxosporeans, while SBA cross-reacted strongly with spore stages of all myxosporeans.

Molecular Tools for the Study of Ceratomyxa shasta and Other Myxozoans.

O Palenzuela (1)* and JL Bartholomew (2)

1	Instituto de Acuicultura Torre la Sal (CSIC). Ribera de Cabanes, Castellon, Spain ES12595 oswaldo@iats.csic.es
2	Department of Microbiology, Oregon State University. 220 Nash Hall, Corvallis, Oregon 97331 USA bartholj@bcc.orst.edu

The phylum Myxozoa remains a controversial group of parasites, after more than a century of study. In the last 15 years, since the discovery of an heteroxenous life cycle in Myxobolus cerebralis, this situation is starting to change, at least for a few freshwater species. Molecular techniques, from immunochemistry to DNA cloning and sequencing, have been successfully used for the study of different aspects of the biology of myxozoans. Ceratomyxa shasta is a good example of a species in which a wide variety of these techniques have been used to describe the pathogenesis of infection, provide diagnostic assays and infer phylogenetic relationships with other myxosporeans. In this presentation, the authors review the different approaches used for the study of C.shasta and other myxosporeans: development of polyclonal and monoclonal antibodies, lectin histochemistry, AP-PCR, DNA cloning and sequencing, molecular phylogeny, PCR and in situ hybridization. The utility, advantages, information obtained and problems of each technique will be discussed.

Experimental Occurrence of Hemorrhagic Thelohanellosis of Color Carp

H Yokoyama (1)*, YS Liyanage (1), H Matoyama (2), H Hosoya (2) and H Wakabayashi (1)

1	Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. ayokoh@hongo.ecc.u-tokyo.ac.jp
2	Niigata Prefectural Freshwater Fisheries Experimental Station, Ohkawara-machi, Nagaoka, Niigata 940-11, Japan

Hemorrhagic thelohanellosis of color carp (Cyprinus carpio) caused by Thelohanellus hovorkai Achmerov, 1960 (Myxozoa: Myxosporea) was investigated at a culture pond and in experimental tanks. Spore purification by 2% trypsin digestion from infected tissues of the diseased carp demonstrated that the outbreak of the disease required an intensity of infection with more than 50,000 T. hovorkai spores / g-tissue. Field survey of the alternate oligochaete host (Branchiura sowerbyi) revealed a high density of B. sowerbyi at the culture pond (3.6 worms / kg-mud) and a high prevalence of infection with the corresponding actinosporean (max. 81%). In experimental tanks with the specific-pathogen free water, carp which were cohabitated with B. sowerbyi exhibited the typical signs of the disease 4 weeks post-cohabitation. Mortalities and high intensities of infection with more than 50,000 spores / g-tissue were noted in both color carp and common carp which were cohabitated with B. sowerbyi. During the cohabitation period, numbers of oligochaetes fed by one carp were estimated to be 22 - 23. On the other hand, no morbidity and a low intensity of infection with T. hovorkai were found in carp reared with the effluent from the B. sowerbyi tank (exposed only to the waterborne actinospores). These results suggest that feeding of the oligochaete host may be one of the critical factors responsible for the outbreak of hemorrhagic thelohanellosis.

SESSION 28. CLINICAL INVESTIGATIONS AND PATHOGENESIS

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Comparative Serological Studies of Tiger Shrimp (Penaeus monodon) Broodstock Hemolymph in Thailand

N Chansue*

Veterinary Medical Aquatic Animal Research Center, Faculty of Veterinary Science, Chulalongkorn University, Henri Dunant Road, Patumwan, Bangkok 10330, Thailand. cnantari@.chula.ac.th

Fifty-three shrimp broodstocks were collected from the Andaman Sea in Trang and Satool province situated in the southern part of Thailand. The average length of shrimp sample was 32.4 cm with average weight of 150 g. Serological analysis was performed from hemolymph collected from ventral sinus of the shrimp. Average levels of glucose, creatinine (Cr), blood urea nitrogen (BUN), phosphorus (P), serum GOT, serum GPT, albumin (Alb), alkaline phosphatase (ALP), and total protein were determined. The results showed that average values of serum glucose, Cr, BUN, and P were 12.82 (SD 4.67), 0.22 (SD 1.17), 5.5 (SD 1.36) and 3.94 (SD 2.57) mg%, respectively. Average SGOT, SGPT, and ALP were 19.71 (SD 9.26), 8.32 (SD 7.61), and 1.15 (SD 0.55) U/L, respectively. Average Alb and total protein levels were 1.11 (SD 0.30) and 4.97 (SD 1.37) g%. There was a statistically significant correlation among SGPT, ALP, P, and total protein levels (p<0.05). Serum glucose level showed no significant relationship with other parameters.
As a conclusion, there was a significant difference in serum BUN, P, SGOT, and SGPT values between shrimp broodstock from Trang and Satool province. But there was no significant difference in glucose, Cr, Alb, ALP, and total protein levels between the two groups (p< 0.05).

Whale Stranding Investigations in the United States

CP Driscoll (1)* and B Mase (2)

1	Cooperative Oxford Laboratory, NOAA/MD DNR 904 South Morris Street Oxford, Maryland 21654 USA. Cindy.Driscoll@noaa.gov
2	Southeast Fisheries Science Center National Marine Fisheries Service 75 Virginia Beach Drive, Miami, FL 33149. Blair.Mase@noaa.gov

Of the species of large whales under US jurisdiction nearly all are listed as endangered or threatened. Currently the northern right whale is the most critically endangered whale is US waters with an estimated 250-350 remaining in the northwest Atlantic Ocean. Since the late 1980's humpback whale strandings have dramatically increased along the eastern seaboard. An investigation into the causes of all large whale mortalities began in 1992. A systematic protocol for examination and sample collection of whale carcasses was subsequently developed and implemented in the Southeastern coastal states. Recent efforts to expand the use of consistent methods and protocols have been met with wide acceptance. Multiple deaths of right whale calves and other high profile cetacean species emphasize the critical need for in depth evaluation of stranding causes. Thorough necropsy, tissue collection and analyses are necessary to rule in/out disease, human interaction, etc., and determine cause(s) of death. By following a nationwide set of protocols for large whale examination more useful information can be collected forming databases of biological and health information for scientific as well as management use.

Ultraviolet-B Radiation Effects on Freshwater Fish: Epithelial Responses of Three Species

MS Ewing (1)*, VS Blazer (2), DL Fabacher (3), EE Little (3) and KM Kocan (4)

1	Zoology Department, Oklahoma State University, Stillwater, Oklahoma 74078 USA msewing@okway.okstate.edu
2	US Geological Survey Biological Resources Division, National Fish Health Research Laboratory, Leetown Science Center, 1700 Leetown Road, Kearneysville, West Virginia 25430 USA
3	US Geological Survey Biological Resources Division, Midwest Science Center, 4200 New Haven Road, Columbia, Missouri 65201 USA
4	Department of Anatomy, Pathology and Pharmacology, Oklahoma State University, Stillwater, Oklahoma 74078 USA

Channel catfish (Ictalurus punctatus), Lahontan cutthroat trout (Oncorhynchus clarki henshawi) and razorback suckers (Xyrauchen texanus) have been found to vary widely in their tolerance of ultraviolet-B radiation (UV-B). The UV-B absorbance of methanol extracts of skin from these species was compared as was the cellular response of skin to exposure to an average simulated solar UV-B dose of 3.0-5.0 J/cm2/day for up to 72 h. The skin extract UV-B absorbance, minimal in cutthroat trout and abundant in razorback suckers, was undetectable in channel catfish. Cutthroat trout, sensitive to simulated UV-B exposure, survived 72-h exposure, but channel catfish did not. Razorback suckers, in contrast, were comparatively tolerant of this treatment. Signs of sunburn such as edema of the epidermis and pycnotic nuclei of epidermal cells occurred by 48 h in trout and by 24 h in catfish. Sloughing of mucous cells in trout and of the outer epidermal layer in catfish occurred in the next 24-h interval. Razorback suckers did not show any gross signs of sunburn during 72 h of experimental exposure. Although cell necrosis occurred in suckers, severe necrosis and sloughing were not observed. Instead, epidermal thickness in the suckers increased, apparently due to hypertrophy and hyperplasia of club cells, which may contain the substance that appears to protect razorback suckers against UV-B radiation. In channel catfish, club cells that were initially prominent in the epidermis were sloughed as part of the outer epidermal layer.

Ultrastructural and Mineral Component Analysis of Nephroliths in Rainbow Trout

JM Groff (1), SE LaPatra (2)*, P Schiffman (3), RJ Munn (4) and JG Zinkl (1)

1	Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, California 95616 USA. josephvmd@aol.com; jgzinkl@ucdavis.edu
2	Clear Springs Foods, Inc., Research Division, P.O.Box 712, Buhl, Idaho 83316 USA scottl@clearsprings.com
3	Department of Geology, University of California, Davis, California 95616 USA pschiffman@geology.ucdavis.edu
4	Department of Pathology, School of Medicine, University of California, Davis, California 95616 USA rjmunn@ucdavis.edu

Nephrolithiasis (nephrocalcinosis) is not an uncommon condition in various cultured teleosts including rainbow trout. Affected kidneys often have multiple, coalescent, chalky-white foci on capsular and cut surface with gross enlargement and distortion secondary to the intratubular accumulation of mineral. Microscopically, nephroliths may result in destruction of the renal tubules with an associated chronic, granulomatous, tubulointerstitial nephritis dependent on the progression and severity of the condition. Scanning electron microscopy revealed that the nephroliths were generally amorphous structures with irregular surface profiles that were often polygonal. Bright- field transmission electron microscopy further revealed that the nephroliths were composed of mineralized concretions generally arranged in concentric arrays of variable electron-density often with the formation of spicules within the concentric arrays. Occasional nephroliths were not arranged in concentric arrays but were amorphous, globular accumulations of mineral. Electron-diffraction analysis of the nephroliths did not indicate a crystalline structure. Electron-probe microanalysis revealed that the nephroliths were composed of (Na, Mg, Ca)3P2O.H2O similar to the mineral collinsite.

Gill Conditions in Cultured Atlantic Salmon in Australia

BF Nowak* and A Clark

School of Aquaculture, University of Tasmania, PO Box 1214, Launceston, Tasmania 7250 Australia

Amoebic gill disease (AGD), generalised epithelial hyperplasia, clubbing necrosis gill syndrome (CNGS), epitheliocystis, post-transfer nodules and plaques were the main conditions found during two seasons of histological survey of fish until 8 months post-transfer. Trichodina sp. and metazoan parasite were found only in single individuals. The survey focused on identification of environmental factors predisposing the fish to gill problems. It included four salmon farms in southern Tasmania. The farms use different husbandry procedures and the relationship between the procedures and the gill conditions was also investigated. The prevalence of AGD was greater at full salinity sites. Month also had significant effect on its prevalence, which was greatest during summer. Similarly, epitheliocystis was more common in summer, however the site salinity had no effect on its prevalence. There was no evidence that adding levamisole to freshwater bath improved protection against AGD. The farm environmental records were sometimes incomplete and often incompatible, which reduced their usefulness in the interpretation of the results of the gill histology survey. The survey clarified the effects of some of environmental factors such as salinity on the prevalence of different gill conditions. Laboratory trials will complement the results of the survey by providing more detailed information. For infectious conditions such as AGD, the reservoirs of the pathogen in the natural environment should be identified.

Hypersensitivity Type I (Anaphylactoid) Reaction Causing Sudden Death Syndrome in an Amazonian Manatee (Trichechus inunguis)

V Bermùdez, G Castro, G Conroy and J Moreno

FCV-UCV, Cat. Clin. Bàsuca, El Limòn, Marncay, AP-4563, Edo, Aragua, Venezuela, South America

Sudden death syndrome was observed in a Mantee (Order Sirena, Family Dugongidae, Trichechus inunguis) captured from the Amazon River Delta and held in captivity at the National Aquaruim of Valencia City in Venezuela. Clinically, the Manatee was suddenly noticed very stressed in a pond where it lived for approximately four years. It showed rolling and respiratory distress and died. At necropsy, it was found a severe facial-nasal edema at the dorsal lateral aspect extending from the left anteriornasal fossa to the periocular rims bilaterally. There was nasal passage obliteration due to marked edema and congestion with severe cellulitis of the nasal subcutis. The trachea revealed abundant reddish froth and water down to the bifurcation and lung parnchyma. The lungs were very rubbery and reddish. The liver and kidneys showed a light generalized tanned discoloration. Postmortem exam and histopathology confirmed a Type I hypersensitivity anaphylactois reaction causing sudden death possibly associated to biological toxin (bees or other insects) was very likely the casue but not confirmed, since foreign solutions or poisonouss criminal innoculation were ruled out by toxicological analyses. Copper toxicity lesions were obseved in liver and kidney due to residues after repeated skin copper sulfate treatments.

SESSION 29. TREATMENTS

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Health Monitoring and Treatment of Laboratory Reared Cephalopods

JM Scimeca (1)* and PG Lee (2)

1	Animal Resources Center, University of Texas, Medical Branch, Department of Pathology, 301 University Blvd., Galveston, Texas 77555-0621 USA. jscimeca@utmb.edu
2	National Resource Center for Cephalopods, Marine Biomedical Institute, University of Texas, Medical Branch, 210 League Hall, Galveston, Texas 77555-0863 USA. pglee@utmb.edu

Cephalopods have been used as aquatic animal models in biomedical research worldwide. In addition, many institutional and aquarium exhibits contain one or more cephalopods. Our objectives are to discuss the optimal environment for cephalopods especially with recent advances in design of seawater filtration equipment. These advances have also increased the long-term maintenance success of laboratory reared cephalopod populations. Additionally along with an appropriate environment frequent observation of animal behavior is one of the most important management tools in a quality animal health program, a brief understanding of their biology and life history as well as their environmental needs will be included. General water quality parameters are more narrowly defined for maintenance of cuttle fishes and squids than for most marine fishes. Cephalopods are sensitive to rapid PH, salinity, low dissolved oxygen concentrations, and nitrogenous wastes. Common health problems can be grouped within three categories: traumatic lesions, lesions related to their environment and infectious diseases. Although prevention is the key success for healthy populations an overview of treatment regiments for health related problems will be presented. Our results compiled over a ten-year period include monitoring of key water parameters and frequent observations daily of animals including a complete necropsy of unexpected deaths. In conclusion, the success of long term husbandry of cephalopods should include a high quality environment, monitoring individual animal health, and when appropriate treat specific problem conditions with appropriate medications.

Killing of the Oyster Pathogen Perkinsus marinus with Synthetic Antimicrobial Peptides in vitro and Modulation by the Pathogen Proteases

JF La Peyre*, KC McDonough and RK Cooper.

Department of Veterinary Science, Louisiana State University, Baton Rouge, LA, 70803 jlapeyre@agctr.lsu.edu, rcooper@agctr.lsu.edu

Antimicrobial peptides are fast acting, widespread in animals and kill microbes including protozoa while having limited effect on host animal cells. They are however, readily degraded by proteases. Recent studies have shown that Perkinsus marinus can be killed in vitro by a variety of natural antimicrobial peptides including magainins, tachylepsins and polyphemusin but only at high concentrations. However, the potential degradation of antimicrobial peptides by P. marinus serine proteases has not been addressed. In this initial study the lytic activity of nine amphipathic -helical peptides against P. marinus was investigated and the effect of P. marinus proteases on the efficacy of these peptides were determined. These synthetic peptides included Agni-14 (KLAKKA)2 , Agni-21 (KLAKKLA)3, Gagni-14 (KLGKKG)2 , Gagni-21 (KLGKKLG)3, Phor-14 (KFAKFAK)2, Phor-21 (KFAKAK)3, Thor-14 (KLAKLAK)2, Thor-21 (KLAKLAK)3 and Thor-28 (KLAKLAK)4. Perkinsus marinus viability was determined by two methods: 1) intracellular reduction of tetrazolium salts using MTS/PMS reagents and 2) uptake of neutral red. The most effective synthetic peptides against P. marinus cultured cells were Agni-21 and Phor-21. A concentration of 50 µM of either peptide killed 100% of cultured P. marinus cells at a density of 106 cells/ml. In comparison, 6.25 µM of the cytotoxin melittin used as a reference peptide was needed to kill 100% of the protozoan cells. Pre-incubation of the peptides with P. marinus proteases significantly reduced their lytic activity. However, preliminary evidence indicates the efficacy of the peptides can be enhanced through the use of inhibitors of P. marinus proteases.

Antibiotic Treatment of Abalone Infected with Rickettsiales-like Organisms.

JD Shields (1)*, TT Hibbard-Robbins (2) and CS Friedman (2)

1	Department of Environmental Sciences, Virginia Institute of Marine Science, P.O. Box 1346, Gloucester Point, VA 23062 USA. jeff@vims.edu
2	Shellfish Diseases Laboratory, California Fish & Game, Bodega Marine Laboratory, P.O. Box 247, Bodega Bay, CA 94923 USA. ttrobbins@ucdavis.edu, csfriedman@ucdavis.edu

Withering syndrome (WS) is a debilitating and fatal disease of black abalone (Haliotis cracherodii) that is caused by a rickettsia-like organism (RLO). Foci of the RLO are found infecting the digestive tract (intestinal epithelia, and digestive tubule epithelia). The RLOs occur at extreme levels in early infections, with less intense infections occuring in seriously afflicted abalone. The major sign of the syndrome, the withered and weakened foot, is an end-stage symptom of the disease. Populations of black abalone have been decimated by WS. Red abalone (H. rufescens) exhibit lower prevalences of the disease. Laboratory observations suggest that they are more resistant to its effects. We undertook controlled laboratoy studies to examine the efficacy of several antibiotics in treating afflicted abalone. Treatments consisted of injecting naturally infected abalone with low, moderate or high levels of specific anti-rickettsial antibiotics. Control groups of naturally infected abalone were injected with diluents. In daily doses over a two-week course, we tested the following drugs for efficacy against the disease: chloramphenicol, tetracycline, sarafloxacin, and clarithromycin. Intramuscular injections were given to insure delivery. Tetracycline at 25 mg/kg and 50 mg/kg (body weight) was moderately to completely successful in ridding black and red abalone of RLOs. Surprisingly, chloramphenicol, sarafloxacin, and clarithromycin were not effective when given intramuscularly. Thus, tetracycline or one of its derivatives may provide a potential treatment against rickettsial diseases of abalone.

Bactericidal Efficacy of Disinfectants Against Mycobacterium Fortuitum

SA Smith* and C Tate

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 USA. stsmith7@vt.edu

Mycobacteriosis, commonly known as "piscine tuberculosis" or "granuloma disease", is a chronic, progressive localized or systemic bacterial infection of both foodfish and aquarium fish. Infections with Mycobacterium spp., especially M. marinum, M. fortuitum and M. chelonae, have been reported worldwide from over 150 different species of marine, brackish and freshwater fishes representing at least 40 piscine families. While clinical manifestations of emaciation and generalized "wasting" are often readily apparent in fish, subclinical carriers are probably more common than realized. Transmission of the disease among fish is generally thought to be by ingestion of infected fish tissues or through contaminated food or debris in the water. Water may become contaminated by the feces or decomposing carcasses of infected fish, through the rupture of skin or gill lesions, or through the release of infectious gonadal fluids during spawning. This project examined the efficacy of standard wastewater disinfectants to inactivate one species of aquatic mycobacteria. Stock cultures of M. fortuitum were exposed to various concentrations and exposure times of sodium hypochlorite. The results of this assay found that an increased amount of time was necessary to inactivate this species of Mycobacterium than what is commonly recommended. Additional studies using ethanol, quartanary ammonia and phenol are in progress to compare the relative inactivation of each chemical. The results of this study will provide research-based information on the most effective solution, concentration and treatment duration required to disinfect a Mycobacterium-infected aquaculture facility or aquarium.

Hydrogen Peroxide as a Therapeutic Compound for Bacterial Gill Disease in Fish.

MJ Tort (1)*, GA Wooster (2) and PR Bowser (3)

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

1	mjt16@cornell.edu
2	gaw5@cornell.edu
3	prb4@cornell.edu

Hydrogen peroxide and its primary decomposition products, oxygen and water, are considered environmentally compatible. The apparent lack of adverse environmental impacts of hydrogen peroxide combined with its effectiveness as an antimicrobial agent makes this compound an excellent candidate for further evaluation in aquaculture. Hydrogen peroxide is currently being tested for its potential as a chemotherapeutant for a variety of external disease organisms of fish. Research in our laboratory has focused on delineating effective hydrogen peroxide treatment regimes for treating bacterial gill disease in salmonids (rainbow trout as a model) and walleye. Results from target animal studies indicate that toxicity of H2O2 is dependent on species, age, dose and water temperature. Acute toxicity is evident from histopathologic inspection of gills (e.g. extensive epithelial lifting, necrosis) of fish treated with high doses of H2O2. The value of hydrogen peroxide as a therapeutic compound for use in walleye culture has been questioned due to the high sensitivity of walleyes to this compound at suggested treatment concentrations. Studies indicate that exposure of walleye to low concentrations of hydrogen peroxide result in increased tolerance to subsequent exposure. Induction of catalase activity in such fish could be one of the mechanisms involved to provide greater protection. Ultimately, this phenomenon of increased tolerance may be of value in the development of management strategies that will permit the use of hydrogen peroxide with walleyes and other sensitive species.

Experience with Acid-Fast Bacterial Infections in Cultured Ornamental Fish in Venezuela

DA Conroy (1) and G Conroy (2)

1	Sección de Patobiología Acuática, Facultad de Ciencias Veterinarias, Universidad Central de Venezuela, Apartado de Correo No. 4563, MARACAY 2101-A, Estado Aragua, Venezuela. FAX: (58) (43) 45.68.10. e-mail : anig@telcel.net.ve
2	Unidad de Diagnóstico y Asesoría Técnica en Patobiología Acuática (UDATPA), Pharma-Fish S.R.L., Apartado de Correo No. 406, MARACAY 2101-A, Estado Aragua, Venezuela. FAX: (58) (43) 45.68.10. e-mail : anig@telcel.net.ve

Acid-fast bacterial infections, including mycobacteriosis and nocardiosis, commonly occur in fish species of interest to aquaculture and aquariology, and are now recognised as of zoonotic importance to Man. Several outbreaks of spontaneous mycobacteriosis were diagnosed in common and fancy varieties of goldfish (Carassius auratus) and guppies (Lebistes reticulatus) reared on fish farms in Aragua, Carabobo and Miranda States, Venezuela, and in a population of three-spot gouramies (Trichogaster trichopterus) produced on a fish farm in Colombia and imported to Venezuela for sale as pet fish. The clinical signs of the disease are described and illustrated. Acid-fast bacteria identified as Mycobacterium sp. were detected in all of the outbreaks investigated. The condition was successfully controlled in practice by the use of kanamycin sulphate, administered by intra-muscular injection (goldfish), as a bath (guppies and gouramies) or as a combination of a bath and the use of medicated feed (guppies).