SPECIAL SESSION
ABSTRACTS

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SPECIAL SESSION 1: INFECTIONS IN ESTUARINE FISHES/HARMFUL ALGAL BLOOMS

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Lesions Observed in Menhaden Collected During Fish Kills and During Other Collections in Tributaries of the Chesapeake Bay

VS Blazer (1)*, CL Densmore (1), E May (2) and W Vogelbein (3)

1	National Fish Health Laboratory, BRD/USGS, 1700 Leetown Road, Kearneysville, WV 25430
2	Maryland Department of Natural Resources, Cooperative Oxford Laboratory, 904 s. Morris Street, Oxford, MD 21654
3	Department of Environmental Sciences, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062

During 1997 there was a great deal of public interest in the increased incidence of fish lesions observed in fish from the Pocomoke River and other tributaries of the Chesapeake Bay. These lesions and a number of fish kills, primarily involving menhaden, have been attributed by some to toxic dinoflagellates such as Pfiesteria or Pfiesteria-like organisms. This report will present histologic findings in menhaden collected during two fish kills in the Pocomoke as well as those collected in a variety of tributaries throughout the summer and fall 1997. Many of the fish had "typical Pfiesteria lesions" - shallow to deep ulcers, often around the anal area. Most of these lesions contained a chronic granulomatous reaction which extended deep into the musculature. Within this chronic reaction were fungal hyphae, believed to be Aphanomyces sp. Often there was a more acute reaction around these focal granulomatous areas, indicating an active, ongoing infection. The possible relationship of these lesions to Pfiesteria-like toxins as well as the similarity of this infection to epizootic ulcerative syndrome seen in the Pacific/Asia area will be discussed.

The Role of Fungi in Menhaden Mortality: How to Narrow Epidemiological Searches

MJ Dykstra* and JF Levine

Microbiology, Pathology and Parasitology Department, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606 USA Michael_Dykstra@ncsu.edu; Jay_Levine@ncsu.edu

In 1984, large numbers of ulcerated Atlantic menhaden (Brevoortia tyrannus) were observed in East Coast waters of the United States. Oomycete fungi (Aphanomyces sp., Saprolegnia sp.) have been repeatedly isolated from lesions of moribund fish. Once Pfiesteria piscicida was isolated, described and implicated in fish kills in our estuaries, there has been a marked de-emphasis of the complexity of the disease process in menhaden, attributing ulcerations in menhaden to Pfiesteria alone. In addition, there has been a tendency to suggest that all ulcerative lesions in estuarine finfish in our East Coast waters are due to Pfiesteria toxins. We maintain that ulcerative mycosis in Atlantic menhaden is a disease syndrome distinct from ulcers on other finfish and that fungi are a significant aspect of the pathology and mortality in menhaden. The specific involvement of Pfiesteria in menhaden fish kills, while likely, is not yet proven, and the entire disease process may be more complicated than has been previously stated in recent years. Knowledge of the behavior of opportunistic fungal infections in menhaden may allow us to predict where diseased fish are located at a specific point in time, which should help in the analysis of this complex disease process.

Mycobacteriosis in Striped Bass, Morone saxatalis, from Chesapeake Bay

WK Vogelbein*, DE Zwerner, H Kator, M Rhodes, SI Kotob, and M Faisal

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

The striped bass, Morone saxatilis is an anadromous fish prized by recreational anglers and commercial fishermen of Chesapeake Bay. Between July and December 1997, the VIMS Aquatic Animal Disease Diagnostic Laboratory (AADDL) received about 30 striped bass (30-66 cm total length) exhibiting prominent ulcerative dermatitis. This condition appeared widespread in certain areas of the southern Chesapeake Bay with anecdotal information (provided by recreational fishermen) suggesting that prevalence of the disease approached 30-40%. Histopathologic evaluation of the skin lesions and internal organs such as spleen, kidney and heart identified prominent granulomatous inflammation. Ziehl-Neelsen stains of histologic sections confirmed the presence of acid-fast bacilli within granulomas. To date culturing of lesions and internal organs has yielded two isolates of Mycobacterium spp., one from a skin ulcer belonging to the M. fortuitum/chelonae complex and a M. marinum from the spleen of another fish. Definitive identifications of these and additional isolates are underway using traditional biochemical tests and PCR analysis. Reports of skin ulcers associated with mycobacteriosis in wild fishes are uncommon. Studies are needed to determine the spatial distribution and prevalence of this disease in striped bass populations. In addition, the presence of skin lesions may pose a human health risk to fishermen exposed to infected striped bass. Mycobacterium spp. pathogenic to fish have been documented from human infections.

Pathogenic Amoebae Affecting Fish in Tidal Freshwater James River

SR Webb (1)*, BL Brown (1), SP McIninch (1,2) and GC Garman (1,2)

1	Department of Biology, Virginia Commonwealth University, PO Box 842012, Richmond, VA 23284 USA blbrown@atlas.vcu.edu; spmcinin@atlas.vcu.edu; ggarman@atlas.vcu.edu
2	Center for Environmental Studies, Virginia Commonwealth University, PO Box 843050, Richmond, Virginia 23284 USA

During summer and fall of 1997 there were numerous reports of moribund and dead fish in Maryland and Virginia coastal tributaries. Although some of these reports have been attributed to toxic algae of the Pfiesteria-complex organisms (PCO), most incidents have no documented etiology. Coincident observations of fish with lesions were also made in the tidal freshwater James River near Richmond, Virginia. The affected fish were collected by boat electrofishing and represented several families, including cyprinids (e.g.,common carp), catostomids (Moxostoma spp.), ictalurids (blue catfish), centrarchids (largemouth bass) and percichthyids (white perch). The identities of amoebae cultured from selected fish lesions were confirmed to be Acanthamoeba spp. by light and scanning electron microscopy and PCR amplification of specific rDNA sequences. Other fish were infected by an amoeba identified tentatively as Naegleria based on morphology alone. Both Acanthamoeba and Naegleria can cause fish kills and members of both groups are human pathogens. Acanthamoeba and Naegleria represent opportunistic, free-living pathogens that must be considered in evaluating outbreaks of lesions in fish taken in freshwater and Acanthamoeba should be considered in both fresh and saltwater environments.

Molecular Approaches for the Detection and Identification of Parasitic and Toxic Dinoflagellates

GR Vasta (1)*, DW Coats (2), DK Stoecker (3), A Li (3), AC Wright (1), JAF Robledo (1) and CA Coss(1)

1	Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD
2	Smithsonian Environmental Research Center, Edgewater, MD
3	University of Maryland Center for Environmental Science, Cambridge, MD

Parasitic and toxic dinoflagellates constitute detrimental factors for aquaculture, fisheries and human health in the Chesapeake Bay region and other coastal areas of US. Mass mortalities due to parasitic infections by Perkinsus marinus ("dermo") have caused a critical reduction of existing oyster populations and have affected commercial harvests. Impaired human health has been associated to recent fish kills in Chesapeake Bay tributaries, and have been attributed to the toxic dinoflagellate Pfiesteria piscicida. Current methods for detection and identification of both P. marinus and P. piscicida involve some form of cultivation and morphological characterization, with detailed examination of thecal plates, and confirmation of toxicity in a fish bioassay for the latter. Identification and classification of microorganisms can be enhanced by the vast genetic and structural information available in DNA, RNA, and protein sequences. We are currently applying biochemical and recombinant DNA approaches to the basic questions of detection, identification and enumeration of parasitic and toxic dinoflagellates in their hosts, and the environment, and to gain insight in their mechanisms for proliferation and life cycle transformations. We developed a semi-quantitative polymerase chain reaction (PCR)-based assay for the diagnosis of P. marinus in oyster tissues selecting an intergenic non-transcribed spacer in the rRNA locus as the target nucleotide sequence. The assay is species-specific and can detect a single P. marinus trophozoite in 30 mg of oyster tissue, indicating that this diagnostic methodology is not only faster, but also more sensitive than current assays. We are currently developing quantitative competitive PCR assays for this oyster parasite, and similar tests for the environmental detection of toxic dinoflagellates.

An Outbreak of Domoic Acid Toxicity in California Sea Lions Off the Central California Coast

FMD Gulland (1)*, M Haulena (1), T Rowles (2), LJ Lowenstine (3), T Spraker (4), T Lipscomb (5), V Trainer (6), F Van Dolah (7) and C Sholin (8)

1	The Marine Mammal Center, Marin Headlands, Sausalito, CA 94965
2	National Marine Fisheries Service, 1315 East West Highway, Silver Spring, MD 20910
3	Department of Veterinary Pathology, Microbiology and Immunology, University of California at Davis, Davis, CA 95616
4	Colorado State University, College of Veterinary Medicine, Fort Collins, CO 80523
5	Armed Forces Institute of Pathology, Washington DC 20306-6000
6	Marine Biotoxin Group, NOAA/NMFS/ECD, 2725 Montlake Blvd, Seattle WA 98112
7	National Oceanic Services, 219 Fort Johnson Road, Charleston, S. Carolina 29412
8	Monterey Bay Aquarium Research Institute, Moss Landing, Monterey, CA 95039

Between 15th and 30th May 1998, 67 California sea lions (Zalophus californianus) stranded along the central California coast, the majority off Oceano Dunes and Monterey Bay, with severe neurological signs. Fifty-six animals were adult females, eight were subadult and juvenile females and ten were subadult and juvenile males. Characteristic clinical signs were scratching, dullness ataxia and intermittent seizures. Fifty-six animals died or were euthanized due to progressive increase in seizure frequency and severity. Sea lions that died within the first two days of stranding had pale myocardium on gross post mortem, while the majority of other animals had no gross lesions. The predominant histologic lesion in affected animals was neuronal necrosis in the hippocampus. Domoic acid was detected in urine and serum from four affected animals by both a microplate receptor assay method and HPLC. A bloom of Pseudonizchtia australis producing domoic acid was detected over the same time frame in the Monterey Bay. Domoic acid was also detected in anchovies and sardines from the Monterey Bay. Although described in humans, domoic acid toxicity has not been previously documented in marine mammals.

SPECIAL SESSION 2: INTEGRATED MONITORING OF DISEASE, MORTALITY AND THE ENVIRONMENT: DEVELOPMENT OF A GLOBAL DATABASE

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Integrated Monitoring of Disease, Mortality and the Environment: Development of a Global Database

Coordinator: WS Fisher
Speakers: WS Fisher (1), P Epstein and J Geraci (2)

1	US EPA National Health and Environmental Effects Laboratory, Gulf Ecology Division, One Sabine Island Drive, Gulf Breeze, FL 32561. 850 934 9200. Fisher.william@epamial.epa.gov
2	National Aquarium in Baltimore, Pier 3, 501 E. Pratt St., Baltimore, MD 21202 USA jgeraci@aqua.org

Session abstract: This session introduces the concept and potential for world-wide reporting and documentation of marine disease and mortality events and the value of this evidence for determination of cause and for tracking global change. There have been apparent increases in disease and mortality of marine mammals, fish, shellfish, corals and other marine organisms, presumably due to higher levels of anthropogenic stresses generated in watersheds and coastal zones. Most often, these events are investigated from a local perspective even though they may equally have been driven, or at least influenced, by regional or global conditions. To hypothesize which conditions are most closely linked to marine events will require examination of data that has been compiled from local sources into a regional and global context. In the United States, this process has been initiated through regional mortality response groups. Compiled data can be integrated into a relational database that includes reports of harmful algal blooms, physical and chemical disturbances, human diseases from marine organisms, biophysical characteristics (water quality) and hydrographic characteristics that can all be displayed in a Geographic Information System. Ultimately these relationships can be incorporated into ecosystem and economic models. Achievement of this potential requires a consistent format for collection of data and a global network of responsible scientists and resource managers.

SPECIAL SESSION 3: THE USE OF ADVANCED TECHNOLOGIES IN AQUATIC HEALTH MANAGEMENT

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The Use of Advanced Technologies in Aquatic Animal Health Management

Coordinator: S LaPatra
Speakers: L Hanson (1), S LaPatra (2), TJ Baldwin and JR Winton (3)

1	Mississippi State University, College of Veterinary Medicine, P.O. Box 9825, MS 39762 hanson@cvm.msstate.edu
2	Clear Springs Food, Inc., P.O. Box 712, Buhl, ID 83316 scottl@clearsprings.com
3	Western Fisheries Research Center, 6505 NE 65th St., Seattle, WA 98115 jim_winton@usgs.gov

Session abstract: Advanced technologies for the detection of nucleic acids or other components of aquatic animal pathogens are being developed and utilized in diagnostic and inspection procedures. These tests have the potential to provide valuable scientific information but could also have serious regulatory implications. Recent applications of these advanced technologies have raised concerns about the use and interpretation of the results of nonvalidated / nonstandardized techniques. Additionally, the use of nonvalidated methods could cause inappropriate devaluation or condemnation of aquatic animal stocks that could have serious legal and political ramifications. Topics to be addressed include:
1) The use of nonvalidated techniques in the diagnosis, inspection and certification of aquatic animals; 2) development of a process to validate and standardize diagnostic methods; 3) consistent use of diagnostic methods and requirements for confirmation; 4) the use of results obtained by nonvalidated techniques and the potential legal and political ramifications; and 5) Management implications of detecting nucleic acids or other components of aquatic animal pathogens.

SPECIAL SESSION 4: NEOPLASIA, CARCINOGENESIS, FIBROPAPILLOMATOSIS

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Gonadal Neoplasms in Softshell Clams, Mya arenaria: A Summary of Knowledge

BJ Barber*

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

Gonadal neoplasms (germinomas), first reported in softshell clams, Mya arenaria, in 1975, are characterized by the presence of monomorphic, basophilic, undifferentiated germ cells that appear initially as small foci in one or more follicles (Stage 1), but which apparently multiply to completely fill most to all follicles (Stage 2) and exhibit invasion and metastasis (Stage 3). Although the condition appears to be progressive, it is unclear to what extent mortality results. Neoplasms do, however, have a negative impact on gametogenesis; female M. arenaria with neoplasms produce significantly fewer and smaller oocytes than normal clams. Since 1993, clams from 24 locations in Maine have been histologically examined. Neoplasms occur more frequently in females than males. Prevalence ranges from 0 to 43%, with no apparent seasonal pattern. Even though M. arenaria is distributed from Labrador to North Carolina on the east coast of North America, gonadal neoplasms have only been found in clam populations located between Penobscot Bay and Cobscook Bay, Maine. This restricted distribution may provide clues as to the cause of the disease.

Prevalence of Walleye Dermal Sarcoma by Age Class in Walleyes in Oneida Lake, New York

RG Getchell (1), GA Wooster (1), LG Rudstam (2), AJ Van De Valk (2), TE Brooking (2) and PR Bowser (1)*

1	Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401 USA. rgg4@cornell.edu; gaw5@cornell.edu; prb4@cornell.edu
2	Cornell University Biological Field Station, Department of Natural Resources, Cornell University, 900 Shackelton Point Road, Bridgeport, New York 13030-9747. lgr1@cornell.edu; ajv6@cornell.edu; teb1@cornell.edu

Walleye dermal sarcoma is an external benign neoplasm caused by a retrovirus. The lesion has a seasonal prevalence on walleyes (Stizostedion vitreum) in Oneida Lake, New York. Prevalence is high during the cooler months of the year and low during the summer. In an earlier study, we demonstrated that the tumor does regress from spring to summer on individually identified adult fish. The objective of this study was to determine if a tumor-positive fish would again become a tumor positive fish in the following year. Because a mark and recapture effort was seen as an impractical approach to this question, an indirect approach was initiated in which prevalence of walleye dermal sarcoma was determined by year class of fish and those year classes were examined for several successive years. Approximately 1000 adult walleyes (50:50; male:female) from Oneida Lake were aged and examined for tumors each year during the spring spawning run. The data has allowed the prevalence of walleye dermal sarcoma to be followed in several year classes since they have joined the spawning run (3+ yrs for males; 4+ yrs for females). At this point in time, we have conducted such examinations for four consecutive years. Upon preliminary examination of the data, it appears that tumor-positive fish in one year do not return in later years as tumor-positive fish.

Cellular Proliferation and Carcinogenesis Following Short-term Exposure to N-Nitrosodiethylamine

WE Hawkins (1)*, NJ Brown-Peterson (1), Y Zhu (2) and RA Krol (1)

1	1 Institute of Marine Sciences, University of Southern Mississippi, PO Box 7000, Ocean Springs, MS 39566 USA. whawkins@seahorse.ims.usm.edu; nbrownp@seahorse.ims.usm.edu; rkrol@seahorse.ims.usm.edu
2	Department of Epidemiology and Biostatistics, College of Public Health, University of South Florida, Tampa, FL 33612. yiliang@yl.coph.usf.edu

Relationships among carcinogen exposure concentration cell proliferation and carcinogenesis are poorly understood in fish. To better understand these relationships, 14 day post-hatch medaka were exposed to 0, 10, 25, 50 and 100 ppm N-nitrosodiethylamine (DEN) for 48 hrs under static renewal conditions. Following exposure, medaka were held in clean water until sampling at three and six months. The frequency of neoplastic and other lesions was determined from paraffin sections stained with hematoxylin and eosin. To determine patterns of cell proliferation in fish sampled at three months, an immunohistochemical assay for proliferating cell nuclear antigen was applied to whole fish specimens. Overall, no DEN exposure level significantly altered fish growth. DEN effects appeared to be limited to the liver. A significant (p < 0.0001) concentration-related increase in vacuolated foci, suggesting a toxic response, occurred in three and six month samples. A dose-related increase in hepatic cysts and spongiosis hepatis, probable degenerative lesions, was significant in fish sampled at three months (p=0.0038) but not at six months and there was a significant (p=0.0014) concentration-related occurrence of macrophage aggregates at six months post-DEN exposure. With respect to carcinogenic effects, basophilic foci were significantly related (p < 0.0001) to DEN concentration at three months post-exposure. At six months post-exposure there was a significant concentration-related increase in both basophilic foci (p=0.02) and hepatocellular carcinoma (p=0.0003). A probit dose-response model showed a significant positive correlation (p=0.015) of proliferating hepatocytes and DEN exposures at three months post-exposure. These results confirm that short-term exposure to DEN initiates carcinogenic effects in medaka and describes parameters of initiation-promotion studies.

The Effects of Polycyclic Aromatic Hydrocarbons (PAH) on the Common Dab (Limanda limanda): Mutations in the Ras-2 Gene as a Potential Biomarker of Pathology

RM Stagg (1)*, JM Rotchelle (2), DW Bruno (1) and JA Craft (3)

1	FRS Marine Laboratory, P O Box 101, Victoria Road, Aberdeen AB11 9DB, United Kingdom. staggr@marlab.ac.uk
2	Faculty of Applied Sciences, UWE, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, United Kingdom
3	Department of Biological Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, United Kingdom. j.a.craft@gcal.ac.uk

Exposure of fish to PAH results in a pleiotropic response which includes the induction of CYPIA, the appearance of PAH metabolites in bile and the occurrence of cytotoxic, genotoxic and carcinogenic pathology. In both national and international monitoring programmes to assess the effect of sedimentary PAH exposure on the health of flatfish a variety of these responses have been utilised to collectively diagnose PAH induced impacts. The assessment of PAH induced genetic damage in these programmes is mainly through measurement of DNA adducts. In this paper we investigate whether mutations in the ras gene of the dab (Limanda limanda) can provide an alternative link between PAH induced DNA damage and pathology. Two dab ras genes (ras-1 and ras-2) have been identified and partially cloned including those regions of the gene containing codons recognised as being sensitive to PAHs in mammalian studies. Dab were exposed to 7, 12-dimethyl benz (a) anthracene (DMBA) alone or in combination with a tumour promoter 12-o-tetradecanoyl 13-acetate (TPA) and denaturing gradient gel electrophoresis (DGGE) was used to screen amplification products for mutations at critical codons of the dab ras-2 gene. Liver samples were also processed for histological analysis and screened for the presence of neoplastic and preneoplastic pathology. Evidence is presented for the presence of DMBA and TPA induced mutations in codon 13 and the relationship of occurrence of these mutations to the pathology observed is discussed.

Metastatic Carcinoma of California Sea Lions: Evidence of Genital Origin

TP Lipscomb (1)*, FMD Gulland (2) and DP Scott (1)

1	1 Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC 20306 USA lipscomb@email.afip.osd.mil; scott@email.afip.osd.mil
2	The Marine Mammal Center, Golden Gate National Recreation Area, Sausalito, California 94965 USA

Metastatic carcinoma was reported in 66 of 370 (18%) adult California sea lions (Zalophus californianus) admitted to a rehabilitation center on the central California coast. Neoplastic masses were present in lumbar lymph nodes of all reported cases; other frequently affected sites included lung, omentum, liver, kidney, mediastinum, urinary bladder and reproductive tract. Histologically, the masses were composed of epithelial cells arranged in nests. Squamous differentiation was commonly present while glandular differentiation was noted rarely. The primary site of neoplasia was not detected in any of the reported cases. The neoplasms were considered to be of probable transitional cell origin based on histologic appearance. In an attempt to identify the site of origin of these neoplasms, we performed detailed histologic examinations of the genitourinary tracts of 5 California sea lions that had metastatic carcinoma with gross and microscopic features similar to those previously reported. Three were female and two were male. Intraepithelial neoplasia was identified in the genital tracts of all 5. The intraepithelial neoplasia was histologically similar to the metastatic carcinomas. In one case, an area of transition from intraepithelial neoplasia to invasive carcinoma was identified histologically. In no case was intraepithelial neoplasia observed in transitional epithelium. These findings support genital origin for the metastatic carcinoma of California sea lions. The search for the etiology should focus on agents known to cause cancer of the genital tract.

Preliminary Evidence Links Sea Turtle Fibropapillomatosis to Tumor-promoting Microalgae

JH Landsberg (1), GH Balazs (2), KA Steidinger (1), DG Baden (3), TH Work (4) and DJ Russell (5)

1	Florida Department of Environmental Protection, Florida Marine Research Institute, St. Petersburg, Florida 33701, USA. landsberg_j@epic7.dep.state.fl.us; steidinger_k@epic7.dep.state.fl.us
2	Southwest Fisheries Science Center, Honolulu Laboratory, National Marine Fisheries Service, Honolulu, Hawaii 96822, USA. gbalazs@honlab.nmfs.hawaii.edu
3	NIEHS Marine and Freshwater Biomedical Sciences Center, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, Florida 33149, USA. dbaden@rsmas.miami.edu
4	United States Geological Survey, Biological Resources Division, National Wildlife Health Center, Honolulu Field Station, Honolulu, Hawaii 96850, USA. thierry_work@usgs.gov
5	Biology Department, University of Alaska Southeast, Juneau, Alaska 99801, USA. JFDJR@acad1.alaska.edu

Sea turtle fibropapillomatosis (FP) is a debilitating, neoplastic disease, that has reached worldwide epizootic levels with up to 90% prevalence in green turtles, Chelonia mydas, in the Hawaiian Islands. Fibropapillomas are commonly found in green turtles, Chelonia mydas, although loggerhead turtles, Caretta caretta, and olive ridley turtles, Lepidochelys olivacea, are also affected. The tumors are benign, but fibropapillomas can adversely affect locomotion, vision, swallowing, and breathing. Visceral fibromas can disrupt normal organ function to the extent that death ensues. Possible causes of FP include infectious agents such as oncogenic viruses, parasites, genetic predisposition, toxicants, ultraviolet radiation, and other undefined synergistic environmental factors. Although strongly linked to oncogenic viruses the etiology still remains unproven. While marine biotoxins cause acute human and animal poisonings, and suspected chronic effects or tumor induction in aquatic animals, their potential role as tumor promoters in FP has not been considered. Benthic dinoflagellates, Prorocentrum spp. produce okadaic acid (OA) that promotes the development of skin papillomas in mice. Prorocentrum spp. are epiphytic on macroalgae and seagrasses that are normal components of green turtle diets. In this study, we show that in the Hawaiian Islands, green turtles consume Prorocentrum, and that high-risk FP areas are linked to areas where P. lima and P. concavum are both highly prevalent and abundant. The presence of OA in the tissues of Hawaiian green turtles further demonstrates exposure and a potential role for this tumor promoter in the etiology of FP.

Marine Turtle Fibropapillomatosis: Experimental and Epizootiologic Evidence for an Infectious Etiology

LH Herbst*

Department of Pathology and the Institute for Animal Studies, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 USA. herbst@aecom.yu.edu

This presentation will provide a synopsis of findings obtained from a series of transmission experiments and associated studies that have helped to characterize the nature of fibropapillomatosis in green turtles (Chelonia mydas) and pointed toward an infectious agent, most probably an enveloped virus, as the cause of this disease. Research findings from both experimentally-induced and spontaneous fibropapillomas of free-ranging marine turtles that tend to support an hypothesis that a novel alphaherpes virus may be the transmissible fibropapillomatosis agent in several turtles species will be presented and the management implications for the involvement of varies viruses in this disease will be discussed.

SPECIAL SESSION 5: INFECTIONS IN ESTUARINE FISHES/HARMFUL ALGAL BLOOMS

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Epizootic Ulcer Disease Syndromes and Mass-Mortalities of Estuarine Fishes

AS Hargis*

Department of Environmental Sciences, Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, Virginia 23062 USA. WJHARGISJR@aol.com

Epizootic ulcerative syndromes in certain temperate and subtropical estuarine fishes of the North American Atlantic coast attract great interest. Sometimes accompanied by noticeable mortalities, and even affecting human health some of these epidemics have been attributed to toxic dinoflagellates of the genus Pfiesteria. First found in aquarium-held fishes and then in estuarine populations in North Carolina they were later observed in some Chesapeake populations. Though Pfiesteria is new, epizootic ulcerative syndromes are not! Epidemics of dermal lesions appeared among young menhaden (Brevoortia tyrannus) in Chesapeake Bay in 1984. General sampling from 1984 through 1989 revealed them in menhaden and numerous other estuarine species from Delaware to Florida. No definitive causative organism was discovered and the disease was called Ulcer Disease Syndrome (UDS) or Ulcerative Mycosis (UM). Scientific interest waned as this epizootic appeared to disappear around 1989. Renewed attention due to recent outbreaks of lesions and mortalities attributed to the toxic dinoflagellate, Pfiesteria, reveals otherwise. The ulcer syndrome persisted in the Chesapeake and North Carolina and, probably, coastwide. The widespread epizootics and related mortalities of the 1980's (UDS/UM) were associated by some with unfavorable environmental conditions, including man-made pollution. So have the Pfiesteria related ones. River closures have been enforced. Suggested ameliorative environmental actions which would affect shoreline agricultural and development practices cause keen economic and political interest. Questions regarding the actual role of toxic dinoflagellates in recent, and earlier, epizootics have arisen among scientists. These and related matters are discussed.

Mycobacteria - A Possible Emerging Pathogen of Striped Bass Caught in the Maryland Portion of the Chesapeake Bay

A Baya (1), EB May (2), T Li (3), C Weedon (2), L Peiper (2), J Huang (3), V Blazer (4) and R Reimschuessel* (5)

1	Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland 20740
2	Department of Natural Resources, Oxford, Maryland
3	Maryland Department of Agriculture, Animal Health Diagnostic Laboratory, College Park, Maryland
4	National Fish Health Lab, Kearneysville, WV 25430
5	Aquatic Pathobiology Center, University of Maryland, Baltimore, MD 21201.

During the summer and fall of 1997, there was much media attention given to ulcerative lesions and fish mortalities among menhaden in several Maryland tributaries of the Chesapeake Bay. During this period, other fish species were also reported to have skin lesions, including the striped bass (Morone saxatilis). A survey conducted in 1997 by Maryland Department of Natural Resources on striped bass from pound and gillnets found that 12% of fish had skin lesions, primarily ulcers. These ulcers ranged from 1-2 small sores to large lesions extending along the body wall. Necropsy examination of a subset of these fish showed all fish had many Pomphorhynchus bulbocolli parasites in their intestines. Numerous fish had grayish-white nodules in the spleen, cranial kidney, liver, heart and gonads. Affected spleens were frequently twice their normal size. Mycobacterium spp. were isolated from splenic cultures of these fish and have been submitted for species identification. Other bacteria were isolated from some of the fish. One fish had a white fibrinous opaque fluid surrounding the peritoneal organs from which Photobacterium damsela was isolated. A non-hemolytic Streptococcus type Ib was isolated from some of the fish. Grossly, these fish had corneal opacity and whitish meninges. Edwardsiella tarda was isolated from 3 other fish. Histopathology of the lesions from the fish with mycobacteriosis showed epidermal necrosis and a chronic inflammatory infiltrate in the underlying dermis. Granulomata were present in the dermis, underlying scales in areas adjacent to the ulcers. Granulomata were also found in liver, kidney, spleen, peritoneum and heart. Acid fast staining was variable. Since mycobacteriosis is a zoonotic disease, it is important to monitor the incidence of this disease in Maryland striped bass.

Fish Kills, Disease, and Associated Harmful Algal Blooms (HABs) in the Gulf of Mexico and Eastern United States

JH Landsberg* and KA Steidinger

Florida Department of Environmental Protection, Florida Marine Research Institute, 100 Eighth Avenue SE, St. Petersburg, Florida 33712 USA. landsberg_j@epic7.dep.state.fl.us; steidinger_k@epic7.dep.state.fl.us

In the Gulf of Mexico and eastern United States, HABs cause massive fish kills and animal mortalities, shellfish poisonings, and respiratory irritation in humans. At least 40 species of toxic or harmful marine microalgae, including 10 ichthyotoxic species, have been identified; all potentially have varying impacts on natural resources and public health. Recently, episodes that include fish kills, ulcerated fish disease events, and public health threats have highlighted many small (< 25µm), potentially harmful dinoflagellates. These include Heterocapsa, Gymnodinium, Gyrodinium, Pfiesteria, a newly described cryptoperidiniopsoid, and other new heterotrophic dinoflagellates (Steidinger & Landsberg, in preparation). Many dinoflagellates have morphologies that are superficially similar and can lead to misidentification and inaccurate reporting. We have identified as many as 10 potentially harmful species associated with fish kill or disease events and therefore caution that some existing records, including those of Pfiesteria piscicida and Pfiesteria-like species, are suspect. The fact that several species can co-occur necessitates accurate identification since the risk of exposure of animal resources or the public to dinoflagellates will depend upon which species are involved and their potential threat. We have consistently documented the presence of a cryptoperidiniopsoid at sites where fish with lesions have been reported. The role of this cryptoperidiniopsoid, Pfiesteria piscicida, and other species that may produce bioactive compounds leading to the potential initiation of fish lesions needs to be examined. A review of fish kill and disease events since the 1940s indicates various etiologies and while these events do not appear to be related to HABs they can confuse interpretations about HAB effects. Care should be taken to investigate all potential etiologies in fish kill and disease events.

The Light Microscopic Appearance of the Pfiesteria-like dinoflagellate "Shepherd's Crook" with Hematoxylin and Eosin

R Reimschuessel (1)*, AS Kane (1) and G Vasta (2)

1	Aquatic Pathobiology Center, University of Maryland School of Medicine, Department of Pathology, 10 S. Pine St., Baltimore Maryland 21201-1192 USA. rreimsch@umaryland.edu, akane@umaryland.edu.
2	Center of Marine Biotechnology, University of Maryland, Suite 236, Columbus Center, 701 E. Pratt St., Baltimore, Maryland 21202. vasta@umbi.umd.edu

Pfiesteria-complex dinoflagellates are speciated using scanning electron microscopy to evaluate the configuration of the armored plates. Light micrographs of the organisms have been published showing their appearance using bright field, phase-contrast and differential interference optics. The appearance of these organisms using standard histological techniques for tissue sections has, however, not been reported. We present here, the morphology of Pfiesteria-complex dinoflagellates using Hematoxylin and Eosin, the stain most commonly used to evaluate tissue sections.

Fish Lesions in the Chesapeake Bay: Pfiesteria-like Dinoflagellates and Other Etiologies

AS Kane*, D Oldach and R Reimschuessel

Aquatic Pathobiology Center, University of Maryland School of Medicine, Department of Pathology, 10 S. Pine St., Baltimore Maryland 21201-1192 USA. akane@umaryland.edu

Ulcerative lesions and mass mortalities of Atlantic estuarine fish, particularly menhaden (Brevoortia tyrannus), have been associated with exposure to Pfiesteria-like dinoflagellates and their toxins. We collected fish from the Chicamacomico River, Maryland, and observed solitary ulcerative lesions on the majority of menhaden sampled. One striped bass (Morone saxatilis) had an area of reddening around the base of the dorsal fin. Bluegill (Lepomis macrochirus), channel catfish (Ictalurus punctatus), yellow perch (Perca flavescens) and carp (Cyprinus carpio) were externally non-remarkable. Histologically, ulcerative menhaden lesions demonstrated a marked chronic inflammatory infiltrate in large areas of exposed necrotic muscle. The ulcers contained granulomata with fungal hyphae in the necrotic tissue. Gram negative rod-shaped bacteria were also observed in the lesions, a common finding in ulcers of aquatic organisms. Our data suggest that "typical" ulcerative lesions observed on fish from areas of Pfiesteria-like dinoflagellate blooms are reflective of dermatosis which may be related to a variety of individual or combined environmental stressors. Exposure to dinoflagellate toxin(s) potentially represents one such stressor. The role of Pfiesteria-like dinoflagellate toxin in fish primary lesion development, and the role of nutrient enrichment on opportunistic fish pathogens, is currently under investigation. These efforts are supported, in part, by the US Environmental Protection Agency; the State of Maryland, Maryland Department of the Environment, and Maryland Department of Natural Resources.