PLENARY SESSION
ABSTRACTS

Impact of Environmental Changes on Aquatic Animal Health

[TOP]

Judith T. Zelikoff

New York University School of Medicine, Nelson Institute of Environmental Medicine, 57 Old Forge Road
Tuxedo, NY 10987 USA

Worldwide Perspectives
Many low-level natural or man-made environmental changes are within the normal range of adaptation of living organisms and are well tolerated. More extensive changes, although tolerated for a time, often ultimately result in death of the host.

Adverse effects of the activities of humankind upon the aquatic environment are of growing concern (Malins and Ostrander 1991; Bucke, 1993). While changes in physical factors (i.e., water temperature, oxygen levels, hydrogen ion concentration, and salinity), as well as in biological stressors (i.e., food availability and pathogens), can produce effects upon resident aquatic species, chemical pollutants likely produce the most devastating environmental changes that impact upon aquatic animal health. Moreover, responses evoked by natural environmental factors are likely to be potentiated by chemical exposure (Rouleau et al., 1996; Galvez and Wood, 1997). This presentation will focus primarily upon the impact of chemical pollution on inhabiting aquatic species, particularly finfish.

Chemical pollution is one of the most pronounced consequences of industrialization and of the intensified application of the uses of natural resources in agriculture, forestry, and mining (Pritchard, 1993). Regardless of the source or original intended use, portions of chemicals registered for use in industry and agriculture are released either deliberately or unintentionally into the aquatic environment. In addition, a number of processes can lead to the redistribution of these chemicals and their eventual deposition in aquatic systems. Furthermore, interference by these pollutants with natural cycles, such as those involving carbon, oxygen, sulfur, and phosphorus can lead to incidences where the natural compound becomes detrimental as in the enrichment of methyl mercury in the aquatic biota.

While the most obvious effect of exposure to a chemical pollutant is rapid death, sublethal responses (i.e., those responses that may be induced in one stage of development but expressed in a later stage in terms of reduced survival, increased host morbidity, or decreased reproductive potential) to environmental change are of great concern since they may go undetected until visualized later at the level of the population. Such responses include, but are not limited to: developmental/structural anomalies, altered enzyme function/gene expression, histologic/hematologic manifestations, neoplasia, and altered immunocompetence (Rosenthal and Alderdice, 1976).

The accelerating pace at which man-made changes are occurring in the aquatic environment seems to have channeled substantial interest into the search for immediate and efficacious solutions

Biological/Biochemical Manifestations of Environmental Change
Biological and biochemical effects of aquatic pollutants on inhabiting fish species can be placed into several categories. These include, but are not limited to: (1) community structure modification: (2) habitat alteration; (3) developmental and structural anomalies; (4) altered gene expression/enzyme induction; (5) histopathologic/neoplastic changes; (6) reproductive modifications; and (7) immune dysfunction. Changes in eating patterns, behavioral modifications, and neurodegenerative alterations have also been observed in fish as a consequence of chemical exposure.

Community structure alteration - It has been suggested that pollutant exposure results in changes in aquatic community structure. This may occur as a result of selective mortality among populations exposed to pollutants or from changes in differential resistance to pollutant-induced effects. Either response results in an imbalance in population structure, with one group gaining a selective advantage over the other (O'Connor et al., 1982). A second type of community alteration concerns population structure of ecologically-important species. For example, because older Hudson River tomcod have an increased tumor incidence (>85%), the age-distribution of these fish in the community may change from one which contained 4 age classes to one which is comprised almost exclusively of only younger fish (i.e., 0+, 1+, 2+).

Habitat alteration - Pollution-related modification of habitats has probably occurred at every site where discharged solids contain a different particle-size distribution and contaminant load than in situ sediments. The most thoroughly documented example of such habitat alteration is in the New York Bight, where reported changes in benthic community structure have been related to the disposal of waste materials.

Developmental and structural anomalies - One of the adverse effects of ocean dumping of waste materials is an induction of chromosomal damage in free-floating eggs and larvae of marine fishes (Longwell, 1976). Such genetic damage may lead to developmental abnormalities or increased mortality in the inhabiting aquatic life. Moreover, both laboratory and field studies have shown that pollutant exposure of finfish can produce structural damage, such as spinal and/or vertebral deformities (Bengtsson et al., 1985).

Altered enzyme function/gene expression - Pollutants in aquatic environments may lead to alterations in the concentration or function of a number of different enzyme systems in residing species, including those necessary for oxidative phosphorylation and for mobilization of glycogen reserves (Stegeman and Hahn, 1994). Cytochrome P-450-mediated mixed-function oxidase and metallothionein have also been shown to be particular targets for the effects of organic and metallic pollutants, respectively. Moreover, activation of oncogenes and DNA base modifications have also been attributed to pollutant exposure.

Histopathologic/neoplastic alterations - Although the inability to distinguish changes caused by anthropogenic toxicants from those due to infectious disease, normal physiologic variation, or natural toxins may confound the issue, histopathologic alterations are indicative of adverse acute and chronic effects of exposure in various tissues/organs comprising an individual finfish or shellfish; because of its key function in the activation/detoxification of xenobiotics, pollutant-induced changes in liver histology have been studied in the greatest detail (Hinton et al., 1994). The ultimate histopathological outcome, i.e.,neoplasia, has also been linked with chemical pollution in a variety of finfish (Malins et al., 1988; Moore and Myers, 1994).

Reproductive modifications - A number of studies, performed primarily in vitro have demonstrated effects of exposure to chemicals commonly found as aquatic pollutants upon gamete activity/formation, vitellogenesis, early embryonic development/differentiation, as well as upon hatching time (Malins and Ostrander, 1991).

Immune dysfunction - A number of laboratory and field studies have demonstrated the ability of aquatic pollutants (i.e., metals, polycyclic aromatic hydrocarbons, organophosphate/organochlorine pesticides, etc.) to alter specific immune responses and/or host resistance in a variety of fish species (Bowser et al., 1994; Enane et al., 1993; Zelikoff, 1993, 1994, 1998; Zelikoff et al., 1995). Exposure to polluted aquatic environments has been shown to compromise humoral and cell-mediated immunity by decreasing antibody titers, reducing lymphocyte proliferation, and modulating phagocyte activity. Moreover, while the exact relationship between environmental pollution and host disease in aquatic organisms remains unresolved, studies of disease incidence suggest that exposure to chemical contaminants may cause disease by exerting excessive stress upon the immune system leading, ultimately, to increased host susceptibility to infectious pathogens (Arkoosh et al., 1998). This particular sublethal response to pollution-induced environmental changes will be discussed in greatest detail.

Practical and Theoretical Aspects
The increased influx of xenobiotics into the aquatic environment has contributed to major damage of this ecosystem. The adverse effects of aquatic pollution have evolved from grossly obvious fish kills and barren rivers, as well as from less-easily detected sublethal changes. While stressors such as suspended solids, metals, organic chemicals, and pathogens still threaten aquatic life, they do so primarily through more subtle impairment of biochemical, physiological, and immunological processes.

The successful management of chemical pollutants demands an ability to recognize and measure sublethal responses, to interpret their impact upon piscine rates of growth, reproduction, disease, and mortality, and to establish cause-effect relationships that enable appropriate remedial actions. Laboratory studies are essential to unequivocally relate chemical exposures to specific mechanisms of toxicity. However, field studies are the only way to verify proposed "cause-effect" relationships and to validate the relevance of specific effects to populations (Leubke et al., 1997).

Monitoring the recovery of fish from anthropogenic stressors can include two distinct components: (1) monitoring of contaminant residues in fish tissues and (2) measuring biological injuries (Anderson et al., 1997). While measuring chemical residues is an effective means of evaluating ongoing exposure to contaminants, it is costly and because dose-response relationships between contaminant burdens and biological injuries can be nonlinear (i.e., a change in burden may or may not be associated with a given incremental change in health), it may not effectively monitor recovery of fish. Contaminant monitoring, coupled with direct monitoring of indicators of health (i.e., collectively known as "biomarkers"), offers the best alternative for accurately gauging recovery of fish from anthropogenic injury resulting from environmental changes. This strategy: (1) integrates responses across multiple stressors; (2) facilitates the selection of chemical and biological indicators representative of specific contaminants or contaminant classes; (3) allows for the quantitation of biological injuries and recovery therefrom; and, (4) can be a cost effective long-term monitoring tool since biological indicator measurements may be less costly than analytical chemical measurements (Anderson et al., 1997).

Important Developments
Past - The first and foremost development in our efforts to assess the impact of environmental change (i.e., aquatic pollution) on aquatic animal health has been the recognition that a problem truly exists. From this acceptance has come intensified research priorities, new standards and governmental regulations, and the cooperation of many governments to assess, evaluate, and, hopefully, eradicate the problem.

Studies assessing the effects of toxic substances on aquatic life have increased substantially in the past decade, and this has included and increased emphasis upon understanding/defining mechanisms governing toxic effects at the cellular and subcellular levels. Studies of this type are essential since large gaps in our understanding of mechanistic changes and the processes that govern them often preclude linking exposure to the manifestation of biological events, such as cancer and other disease conditions.

In the past few years, interest has emerged in the use of aquatic organisms as models for higher vertebrates in toxicologic studies. Because of the advantages of using fish over the current rodent models and because their immune system closely resembles that of higher vertebrates (Enane et al., 1993; Zelikoff et al., 1991) one area of research that is actively being pursued is fish immunotoxicology (Zelikoff, 1994, 1996). Studies in this area of research suggest that immune systems of fish respond similarly (and, in many cases, by similar mechanisms) to the toxic effects of many pollutants.

Future - Among the long-term goals of studying biological/biochemical responses to environmental contaminants is the identification of specific responses or biomarkers that might serve as early warning signals for ensuing pathologies in exposed organisms. In selecting appropriate biomarkers, certain key criteria need to be considered, including sensitivity, specificity, applicability, and reproducibility (Anderson et al., 1997). Biochemical, histological, and physiological responses of aquatic species have been widely used in this capacity. However, newly-emerging immunological indicators are thought to represent one of the most sensitive parameters for assessing the effects of low-dose toxicant exposure (Dean et al., 1990). Decreases in immune system competence can result in increased susceptibility to a wide variety of biological stressors, such as bacteria, viruses, and parasites which could, potentially, led to population reductions. More validation studies, as well as studies to better understand the ramifications of pollutant-induced immune dysfunction are necessary goals in the future.

Studies in the area of biomarkers need to focus upon selection of sentinel organisms that provide a key component for the monitoring of environmental quality, as well as a means of detecting the potential impact upon human health arising from environmental contamination. In addition, studies using a comprehensive biomonitoring approach in which biomarkers are employed with techniques designed to specifically assess health of individual organisms and pollutant impacts at higher levels of biological organization are needed.

Determining possible environmental causes of disease still remain an important area of investigation. In particular, research is needed to better understand the conditions where there is strong circumstantial evidence (from epidemiological and experimental studies) of a pollution-related event. A clear association between exposure, uptake and metabolism, and biological effects in individual organisms need to be established.

Finally, considering the increasing political pressure to develop non-mammalian models as alternate species for predicting toxicological risk to higher vertebrates (including humans), validation studies of these models need to be performed.

Overall, the next few years will provide a plethora of information and be one of great accomplishment if fundamental advances are made in the aforementioned areas of research.

References
Anderson, M.J., Barron, M.G., Diamond, S.A., Lipton, J., and Zelikoff, J.T. (1997). Biomarker selection for restoration monitoring of fishery resources. In: Environmental Toxicology and Risk Assessment: Modeling and Risk Assessment. Vol. 6, ASTM STP 1317, (Eds., F.J. Dwyer, T.R. Doane, M.L. Hinman), American Society for Testing and Materials. pp. 333 -359.

Arkoosh, M.R., Casillas, E., Clemons, E., Kagley, A.N., Olson, R., Reno, P., and Stein, J.E. (1998). Effect of pollution on fish diseases: Potential impacts on salmonid populations. J. Aquatic Animal Health, 10, 182-190.

Bengtsson, B-E., Bengtsson, A., and Himberg, M. (1985). Fish deformities and pollution in some Swedish waters. Ambio 14, 32-35.

Bowser, D., Frenkel, K. and Zelikoff, J.T. (1994). Effects of in vitro nickel exposure on macrophage-mediated immunity in rainbow trout. Bull Environ. Contam. Toxicol. 52, 367-373.

Bucke, D. (1993). Aquatic pollution: Effects on the health of fish and shellfish. Parasitology 106, S25-S37.

Dean, J.H., Cornacoff, J.B., and Luster, M.I. (1990). Toxicity to the immune system. Immunopharmacol. Rev. 1, 377-408.

Enane, N.A., Frenkel, K., O'Connor, J.M., Squibb, K.S., and Zelikoff, J.T. (1993). Biological markers of macrophage activation: Applications for fish phagocytes. Immunology 80, 68-72.

Galves, F., and Wood, C.M. (1997). The relative importance of water hardness and chloride levels in modifying the acute toxicity of silver to rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. Chem. 16, 2363-2368.

Hinton, D.E., (1994). Cells, cellular responses, and their markers in chronic toxicity of fishes. In: Aquatic Toxicology. Molecular Biochemical, and Cellular Perspectives. (Eds. D. C. Malins and G.K. Ostrander), CRC Press Inc., Boca Raton, FL., pp 87-206.

Longwell, A.C. (1976). Chromosome mutagenesis in developing mackerel eggs sampled from the New York Bight. Limnol. Oceanogr. Spec. Symp. 2, 337-339.

Luebke, R.W., Hodson, P.V., Faisal, M., Ross, P.S., Grasman, K.A., and Zelikoff, J.T. (1997). Aquatic pollution-induced immunotoxicity in wildlife species. Fundam. Appl. Toxicol. 37, 1-15.

Malins, D.C., and Ostrander, G.K. (1991). Perspectives in Aquatic Toxicology. Annu. Rev. Pharmacol. Toxicol. 31, 371-399.

Malins, D.C., McCain, B.B., Landahl, J.T., Myers, M.S., Krahn, M.M.,

Brown, D.W., Chan, S-L., and Roubal, W.T. (1988). Neoplastic and other diseases in fish in relation to toxic chemicals: An overview. Aquatic Toxicol. 11, 43-67.

Moore, M.J., and Myers, M.S. (1994). Pathobiology of chemical-associated neoplasia in fish. In: Aquatic Toxicology. Molecular Biochemical, and Cellular Perspectives. (Eds., D. C. Malins and G.K. Ostrander), CRC Press Inc., Boca Raton, FL., pp 87-206.

O'Connor, J.M., and Huggett, R.J. (1988). Aquatic pollution problems, North Atlantic coast, including Chesapeake Bay. Aquatic Toxicol. 1, 163-190.

Pritchard, J.B. (1993). Aquatic toxicology: Past, present, and prospects. (1993). Environ. Health Perspect. 100, 249-257.

Rosenthal, H., and Alderdice, D.F. (1976). Sublethal effects of environmental stressors, natural, and pollution, on marine fish eggs and larvae. J. Fish. Res. Board Canada 33, 2047-2065.

Rouleau, C., Tjalve, H., and Gottofrey, J. (1996). Effects of low pH on the uptake and distribution of 54Mn (II) in brown trout (Salmo trutta). Environ. Toxicol. Chem. 15, 708-710.

Stegeman, J.J., and Hahn, M.E. (1994). Biochemistry and molecular biology of monoxygenases: Current perspectives on forms, functions, and regulation of cytochrome P450 in aquatic species. (1994). In: Aquatic Toxicology. Molecular Biochemical, and Cellular Perspectives. (Eds. D. C. Malins and G.K. Ostrander), CRC Press Inc. Boca Raton, FL. pp 87-206.

Zelikoff, J.T. (1993). Metal pollution-induced immunomodulation in fish. Annual Rev. of Fish Diseases. 2, 305-325.

Zelikoff, J.T. (1996). Fish immunotoxicology: A new scientific discipline. Toxicol. and Ecotoxicol. News, 5, 130-132.

Zelikoff, J.T. Biomarkers of immunotoxicity in fish and other non-mammalian sentinel species: Predictive value for mammals. (in press). Toxicology .

Zelikoff, J.T. Fish immunotoxicology. (1994). In: Immunotoxicology and Immunopharmacology (Eds. J. Dean, M. Luster, A. Munson, and I. Kimber), Raven Press, New York. pp. 386-403.

Zelikoff, J.T., Squibb, K., Bowser, D., and Frenkel, K. (1995). Immunotoxicity of low level cadmium exposure in fish: Alternative animal models for immunotoxicological studies. J. Toxicol. Environ. Health, 45, 235-248.

Zelikoff, J.T., Enane, N.A., Bowser, D., Squibb, K.S., and Frenkel, K. (1991). Development of fish peritoneal macrophages as a model for higher vertebrates in immunotoxicological studies. I. Characterization of trout macrophage morphological, functional and biochemical properties. Fundam. Appl. Toxicol. 16, 576-589.

Harmful Algal Blooms and Their Effects on Marine and Estuarine Animals

[TOP]

Jan H. Landsberg (1) and Sandra S. Shumway (2)

1	Florida Department of Environmental Protection, Florida Marine Research Institute, 100 Eighth Avenue Southeast, St. Petersburg, Florida 33712 USA, landsberg_j@epic7.dep.state.fl.us
2	Natural Science Division, Southampton College, Long Island University Southampton, New York 11968 USA, sshumway@sunburn.liunet.edu

Increasing documentation of harmful algal blooms (HABs) in marine and estuarine systems has begun to demonstrate their far reaching impacts on ecosystem integrity, species interactions, aquatic animal health and population growth, human health, economy, industry, and ecology. While some 5000 species of microalgae are known world wide and less than 2% are known to be harmful or toxic, the number of toxic species appears to be increasing (Hallegraeff 1993, Sournia 1995). Much of the increase can be attributed to the study of benthic microalgae and the detection of toxic species or to species previously described that were not known to be toxic. While this increase partially reflects technological improvement in accurate identification of HAB species and their toxins, and enhanced monitoring and surveillance, it is also associated with continued anthropogenic influences and interacting natural processes. Because some HABs tend to be planktonic, visibly obvious, acute in nature, and lead to fast-acting shellfish poisoning events or mass mortalities of aquatic organisms, these are the HABs about which most is known.

Marine and estuarine HABs cause animal mortalities, shellfish and tropical fish poisonings, and respiratory irritation and neurocognitive disease in humans (Steidinger 1993, Burkholder and Glasgow 1997). At least 85 species of toxic or harmful marine microalgae are known worldwide; all potentially have varying impacts on natural resources and public (Sournia 1995, Steidinger pers. comm.). Aquatic animals are typically exposed to toxic or harmful algae when planktonic or benthic species bloom and dominate the food web, but there are also more subtle and insidious exposure mechanisms that are not so obvious. Exposure to toxins occurs through ingestion of cells (filter feeders e.g. sponges, molluscs, crustacea), bioaccumulation by consumption of toxic prey (e.g. crustacea, gastropods, fish, birds, turtles, mammals), aerosolized transport (e.g. respiratory irritation in humans and potentially in mammals, turtles, birds), water-borne toxin after cell lysis (fish, shellfish), sediment sinks (benthic organisms), and possibly through consumption of toxic benthic stages.

Fish or invertebrate kills have been attributed to low dissolved oxygen associated with non toxic, high biomass, plankton blooms such as Ceratium or Noctiluca (Mahoney and Steimle 1979, Adnan 1989). Most blooms of toxic photosynthetic microalgae (Heterocapsa, Gymnodinium, Gyrodinium, Alexandrium, Cochlodinium, Prorocentrum, Chattonella, Heterosigma, Prymnesium, Chrysochromulina, and Nodularia) cause mortalities of marine, estuarine, or associated terrestrial animals, both in the wild and in aquaculture (Gunter et al. 1948, Steidinger et al. 1973, Cross and Southgate 1978, Mortensen 1985, Okaichi 1985,Yuki and Yoshimatsu 1989, Chang et al. 1990; Guzman et al. 1990, Shumway 1990, Kaartvedt et al. 1991, Robertson 1991, Shumway and Cembella 1993, Su et al. 1993, Heidal and Mohus 1995, Horiguchi 1995, Negri et al. 1995, Yongjia et al. 1995, Burkholder 1998, Steidinger et al. 1998).

Recently, a series of shellfish and fish kills, ulcerated fish disease events, and public health threats have also highlighted the presence of small, heterotrophic, lightly-armored dinoflagellates with different life cycle strategies to those of the more typical planktonic photosynthetic blooming forms. Although massive fish kills (mostly menhaden) were prevalent in North Carolina's estuaries for many years, it was not until 1991 that Pfiesteria piscicida was implicated. P. piscicida continues to threaten natural resources and public health along the eastern USA (Burkholder et al. 1992; Burkholder and Glasgow 1997). Other Pfiesteria species are also suspected as being ichthyotoxic (Landsberg et al. 1995, Burkholder and Glasgow 1997) and several other new heterotrophic dinoflagellate species that have recently been described (Steidinger et al. 1996, Steidinger et al. in review) are being investigated for their potential role in fish kill and disease events.

Filter-feeding shellfish accumulate microalgal toxins which in turn become available to consumers, both animal and human, through the food chain. Several toxic dinoflagellates (Alexandrium, Gymnodinium, Pyrodinium, Dinophysis, Prorocentrum), diatoms (Pseudonitzschia) and cyanobacteria (Anabaena) are associated with human shellfish poisonings such as Amnesic Shellfish Poisoning, Diarrheic Shellfish Poisoning, Paralytic Shellfish Poisoning, and Neurotoxic Shellfish Poisoning (Shumway 1989, Steidinger 1993), yet these same toxins can also impact animals through the food chain e.g. Pseudonitzschia (domoic acid) for birds (Work et al. 1996), Alexandrium for fish, birds, and mammals (Armstrong et al. 1978, White 1980, Geraci et al. 1989, Montoya et al. 1996), and Anabaena for mammals (Nehring 1993).

Benthic Gambierdiscus, Prorocentrum, Ostreopsis, and Coolia are toxic dinoflagellates that often inhabit reefs and hard grounds in subtropical and tropical regions (Anderson and Lobel 1987). They are associated with the human tropical fish poisoning, ciguatera (Steidinger, 1993), but are not typically considered to affect other organisms involved in the food chain. It was recently postulated that tropical reef fish disease and mortalities in the USA were triggered by fish immunosuppression associated with the consumption of ciguatera-associated toxins (Landsberg 1995). The species of fish implicated in the ciguatera food chain in Florida (de Sylva 1994) are similar to those species that were affected during the 1980 and 1993/94 reef mortalities and disease outbreaks in Florida (Landsberg 1995).

The majority of HAB events are associated with invertebrate, fish, and bird mortalities, yet there have also been numerous accounts of other unexplained marine animal mortalities, such as dolphins and sea turtles, where biotoxins have been implicated (Geraci et al. 1989, Hokama et al. 1990, Hernández et al. 1998, FDEP unpublished data). The definitive role of biotoxins in marine mammal mortalities has been controversial. For example, although Gymnodinium breve was implicated in mortalities of the endangered Florida manatee, Trichechus manatus latirostris, in 1963 and 1982 (O'Shea et al. 1991), it was only proven in 1996 when 149 animals died from G. breve exposure in southwest Florida (Bossart et al. 1998, Landsberg and Steidinger 1998). Terrestrial organisms (e.g. bears, raccoons, otters, birds) associated with marine or estuarine food webs may also be at risk from toxin exposure. It is already known that terrestrial domestic animals such as cattle are at risk from toxic cyanobacterial blooms in freshwater systems (Falconer 1993).

For obvious reasons, algal species known to be associated with human health risks have received the most attention. Many of the potential longer-term chronic effects associated with these biotoxins are typically not well known, nor is it clear what is the fate of these toxins in the ecosystem. There are, however, many algal species which have had severe impacts on lower vertebrates and invertebrate species (many commercially important) that are also deserving of our attention. Further, even when the impacts on non-human species are considered, it is frequently only the acute or lethal influences which receive recognition. Sublethal or chronic effects of algal species have long been overlooked in both human and non-human species. Only recently has attention been drawn to the fact that microalgal toxins and their chronic effects need to be considered at all biological levels and as major threats to animal health, sustained fisheries, endangered species, and ecosystems (Landsberg 1995, 1996, Burkholder 1998). Chronic dietary exposure of animals to biotoxins can exert lethal or sub-lethal effects at all trophic levels, leading to impaired feeding, avoidance behavior, physiological dysfunction, impaired immune function, reduced growth and reproduction, pathological effects, or mortality (Lesser and Shumway 1993, Luckenbach et al. 1993, Wickfors and Smolowitz 1993, Burkholder 1998). Potential long-term effects of biotoxins on the health of aquatic animals may be expressed in terms of susceptibility to disease, immunosuppression, pathologies, and in the development of neoplasia.

Cyanobacteria produce several toxins that are known to be tumor promoters (Falconer and Humpage 1996) and have been implicated in disease in freshwater/brackish animals (Phillips et al. 1985, Andersen et al. 1993). Rarely are the potential effects of marine biotoxins considered as etiological agents of tumor induction. This is surprising given their defined widespread geographical distribution and the resultant probability that many marine and estuarine animals are chronically exposed to tumor-promoting biotoxins. Recently, the potential role of microalgal toxins in tumor development in marine animals has been explored (Landsberg 1995, 1996, Landsberg et al. in review). Landsberg (1995) recently suggested that there is strong circumstantial evidence for a correlation between the worldwide distribution of biotoxins in molluscs associated with shellfish poisonings in humans and the distribution of tumors in bivalves. In some cases, disseminated neoplasia and germinomas were highly associated with biotoxin components on both a temporal and spatial basis. No experimental assays or field monitoring studies to investigate or corroborate these relationships have yet been done. In another example, fibropapillomatosis (FP) in green turtles is a debilitating, neoplastic disease that has reached worldwide epizootic levels. The etiology of FP is unknown but has been linked to oncogenic viruses (Herbst 1994). Toxic benthic dinoflagellates (Prorocentrum spp.) are not typically considered tumorigenic agents, yet they have a worldwide distribution and produce a tumor promoter, okadaic acid (OA) (Fujiki and Suganuma 1993). Prorocentrum spp. are epiphytic on macroalgae and seagrasses that are normal components of green turtle diets. Landsberg et al. (this conference) recently considered 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 indicates exposure and a potential role for this tumor promoter in the etiology of FP (Landsberg, in press, Landsberg et al. this conference).

The overall effects of HABs on food webs are probably the least understood of all impacts. Not only are pathways of transmission of algal toxins complex and not completely known, long-term impacts of sublethal, chronic effects (e.g. recruitment failure and subsequent loss of species within an ecosystem, reduced filtration of water masses and subsequent impacts on benthic-pelagic coupling) are virtually unknown. Long-term impacts (e.g. toxin accumulation) on higher-level consumers also need to be investigated. While there are many examples of these sublethal, chronic effects, perhaps one of the most revealing in terms of the breadth of impact is the community response to the apparently nontoxic brown tide algae, Aureococcus anophagefferens and Aureoumbra lagunensis (Cosper et al. 1987, Buskey and Stockwell 1993). These algae cause reduced feeding activity in filter-feeding shellfish through ciliary inhibition, depressed egg hatching success in red and black drum, and declines in zooplankton abundance, shellfish and other benthic filter feeders (Tracey 1988, Draper et al. 1990). These species are also responsible for decimation of sea grass beds (critical habitat for a multitude of species) as a result of severe shading (Dennison et al. 1989). Finally, A. anophagefferens is responsible for the demise of the bay scallop fishery on Long Island, New York, USA, due to induced starvation and subsequent recruitment failure (Tettlebach and Wenczel 1993). Similar impacts have been associated with persistent cyanobacterial blooms in Florida Bay, Florida, USA and their association with sponge mortalities and reduction in recruitment of spiny lobsters (Butler et al. 1995). These sublethal, chronic impacts taken in concert have already had far-reaching impacts on the communities, and the continued presence of these algal species is bound to have even more profound effects over the ensuing years.

Although most attention has been focused on the direct effects of toxic algae, there are other indirect effects caused by species that may be harmful to marine animals. For example, blooms of nontoxic diatoms such as Chaetoceros, Skeletonema, Rhizosolenia, or dictyophytes (Parry et al. 1989, Erard-Le Denn and Ryckaert 1990, Albright et al. 1993, Kent et al. 1995, Tester and Mahoney 1995), that have setae, processes, or spines can become trapped in the gills of fish or shellfish, cause mechanical damage, impair respiration, and may lead to mortality. Cyanobacterial blooms of Trichodesmium erythraeum that are not usually considered harmful have been implicated for their effects on zooplankton (Guo and Tester 1994), association with ciguatera-like outbreaks in Australia (Hahn and Capra 1992), mortalities of coral polyps (Endean 1977), and a potential role in coral bleaching (Coles 1994).

In the last 5 years several important developments have occurred in the field of HABs:
1) recognition that genera of small, previously undescribed microalgae are involved in animal mortalities, disease and public health events,
2) technological breakthroughs in verifying and identifying the presence of toxins in animals, and
3) public awareness and political recognition that HABs are a universal problem.

In the next few years we anticipate that there will be:
1) technologies that will improve the diagnosis of toxins in animal tissues, e.g., probes, and that will track blooms and provide better predictive capabilities for the management of wildlife, fisheries, and aquaculture,
2) better integration and interdisciplinary activity between scientists and resource managers across fields to investigate animal mortalities and disease,
3) better integrated management strategies that will look toward "managing HABs",
4) the recognition that chronic effects of biotoxins are likely to be very significant in animal health and may help to explain many currently "unidentified diseases" or "unexplained mortalities", and
5) recognition that the interaction of biotoxins and pathogens needs to be evaluated. For example, mortalities may be attributed to the presence of a virus without necessarily determining if viral expression was enhanced by chronic biotoxin exposure. These aspects may be particularly revealing e.g. in the cases of morbillivirus and potential biotoxin exposure in marine mammals and with oncogenic viruses and tumor promoters in the expression of fibropapillomas in sea turtles. In addition, the potential role of HABs as vectors for aquatic animal pathogens is also an emerging issue.

References
Adnan, Q. 1989. Red tides due to Noctiluca scintillans (MacCartney) Ehrenb. and mass mortality of fish in Jakarta Bay. pp. 53-55. In: Red Tides, Biology, Environmental Science and Toxicology, eds. T. Okaichi, D.M. Anderson and T. Nemoto, Elsevier Press, New York.

Albright, L.J., Yang, C.Z. and Johnson, S. 1993. Sub-lethal concentrations of the harmful diatoms, Chaetocerus concavicornis and C. convolutus, increase mortality rates of penned Pacific salmon. Aquaculture. 117:215-225.

Andersen, R.J., Luu, H.A., Chen, D.Z., Holmes, C.F.B., Kent, M.L., Le Blanc, M., Taylor, F.J.R. and Williams, D.E. 1993. Chemical and biological evidence links microcystins to salmon 'netpen liver disease'. Toxicon 31:1315-1323.

Anderson, D. M. and Lobel, P. S. 1987. The continuing enigma of ciguatera. Biol. Bull. 172: 89-107

Armstrong, I.H., Coulson, J.C., Hawkey, P. and Hudson, M.J. 1978. Further mass seabird deaths from paralytic shellfish poisoning. British Birds 71:58-68.

Burkholder, J. M. 1998. Implications of harmful microalgae and heterotrophic dinoflagellates in management of sustainable marine fisheries. Ecol. Applic. 8:S37-S62

Burkholder, J.M. and Glasgow, H.B. Jr. 1997. Pfiesteria piscicida and other Pfiesteria-like dinoflagellates: Behavior, impacts and environmental controls. Limnol. Oceanogr. 42:1052-1075.

Burkholder, J.M., Noga, E.J., Hobbs, C.H., Glasgow, H.B. Jr. and Smith, S.A. 1992. New "phantom" dinoflagellate is the causative agent of major estuarine fish kills. Nature 358:407-410.

Buskey, E.J. and Stockwell, D.A. 1993. Effects of a persistent 'brown tide' on zooplankton populations in the Lagune Madre of South Texas. pp. 659-666. In: Toxic Phytoplankton Blooms in the Sea, eds. T. J. Smayda and Y. Shimuzu, Elsevier Press, Amsterdam.

Butler, M. J. IV., Hunt, J. H., Herrnkind, W. F., Childress, M. J., Bertelsen, R., Sharp, W., Matthews, T., Field, J. M. and Marshall, H. G. 1995. Cascading disturbances in Florida Bay, USA: cyanobacteria blooms, sponge mortality, and implications for juvenile spiny lobsters Panulirus argus. Mar. Ecol. Prog. Ser. 129:119-125.

Chang, F.H., Anderson, C. and Boustead, N.C. 1990. First record of Heterosigma (Raphidophyceae) bloom with associated mortality of cage-reared salmon in Big Glory Bay, New Zealand. N.Z.J.Mar. Fresh. Res. 24: 461-469.

Coles, S. L. 1994. Extensive coral disease outbreak at Fahl Island, Gulf of Oman, Indian Ocean. Coral Reefs 13:242.

Connell, C.H. and Cross, J.B. 1950. Mass mortality of fish associated with the protozoan Gonyaulax in the Gulf of Mexico. Science 112:359-363.

Cosper, E.M., Dennison, W.C., Carpenter, E.J., Bricelj, V.M., Mitchell, J.G., Kuenstner, S.H., Colflesh, D. and Dewey, M. 1987. Recurrent and persistent brown tide blooms perturb coastal marine ecosystem. Estuaries. 10: 284-290.

Cross, T.F. and Southgate, T. 1980. Mortalities of fauna of rocky substrates in south-west Ireland associated with the occurrence of Gyrodinium aureolum blooms during autumn 1979. J. Mar. biol. Ass. UK. 60:1071-1073.

Dennison, W.C., Marshall, G.J. and Wigand, C. 1989. Effect of "brown tide" shading on eelgrass (Zostera marina L.) distributions. pp. 675-692. In: Novel Phytoplankton Blooms eds. E. M. Cosper, V.M. Bricelj and E. J. Carpenter, Springer Verlag, Berlin.

Draper, C., Gainey, L., Shumway, S. and Shapiro, L. 1990. Effects of Aureococcus anophagefferens ("brown tide") on the lateral cilia of 5 species of bivalve molluscs. pp. 128-131. In: Toxic Marine Phytoplankton, eds. E. Graneli, B. Sundstrom, L. Edler and D. M. Anderson, Elsevier Publishers, New York.

Endean, R. 1977. Destruction and recovery of coral reef communities. pp. 215-254. In:Biology and Geology of Coral Reefs, eds. O. A. Jones and R. Endean, Academic Press, New York.

Erard-Le Denn, E. and Ryckaert, M. 1990. Trout mortality associated with Distephanus speculum. p. 137. In: Toxic Marine Phytoplankton, eds. E. Graneli, B. Sundstrom, L. Edler and D.M. Anderson, Elsevier Press, New York.

Falconer, I.R. 1993. Mechanism of toxicity of cyclic peptide toxins from blue-green algae. pp. 177-186. In: Algal Toxins in Seafood and Drinking Water. ed. I. R. Falconer, Academic Press, London.

Falconer, I.R. and Humpage, A.R. 1996. Tumor promotion by cyanobacterial toxins. Phycologia. 35:74-79

Fujiki, H. and Suganuma, M. 1993. Tumor promotion by inhibitors of protein phosphatases 1 and 2A:the okadaic acid class of compounds. Adv.Cancer Res. 61:143-194.

Forrester, D.J., Gaskin, J.M., White, F.H., Thompson, N.P., Quick, J.A. Jr, Henderson, G. and Woodard, J.C. 1977. An epizootic of waterfowl associated with a red tide episode in Florida. J.Wildl. Disin. 13:160-167.

Geraci, J.R., Anderson, D.M., Timperi, R.J., Staubin, D.J. Early, G.A., Prescott, J.H. and Mayo, C.A. 1989. Humpback whales (Megaptera novaeangliae) fatally poisoned by dinoflagellate toxin. Can. J. Fish. Aquat. Sci.46:1895-1898.

Gunter, G., Williams, R.H., Davis, C.C. and Smith, F.G.W. 1948. Catastrophic mass mortality of marine animals and coincident phytoplankton bloom on the west coast of Florida, November 1946 to August 1947. Ecol. Monogr. 18:309-324.

Guo, C. and Tester, P. A. 1994. Toxic effect of the bloom-forming Trichodesmium sp. (Cyanophyta) to the copepod Acartia tonsa. Nat. Toxins 2: 222-227

Guzman, H.M., Cortes, J., Glynn, P.W. and Richmond, R.H. 1990. Coral mortality associated with dinoflagellate blooms in the eastern Pacific (Costa Rica and Panama). Mar. Ecol. rog. Ser. 60:299-303.

Hallegraeff, G. M. 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32:79-99.

Hahn, S.T. and Capra, M. F. 1992. The cyanobacterium Oscillatoria erythraea - a potential source of toxin in the ciguatera food chain. Food Addit. Contam. 9:351-355.

Heidal, K. and Mohus, A. 1995. The toxic Chrysochromulina - salmon disaster of 1991 in northern Norway with some follow-up monitoring records of 1992 and -93. pp. 163-168. In: Harmful Marine Algal Blooms, eds. P. Lassus, G. Arzul, E. Erard-Le-Denn, P. Gentien and C. Marcaillou-Le-Baut, Technique et Documentation, Lavoisier, Paris.

Herbst, L. H. 1994. Fibropapillomatosis of marine turtles. Ann. Rev. Fish Dis. 4:389-425.

Hernández, M., Robinson, I., Aguilar, A., González, L.M., López-Jurado, L.F., Reyero, M.I., Cacho, E., Franco, J., Lopez-Rodas, V. and Costas, E. 1998. Did algal toxins cause monk seal mortality? Nature 393:28-29.

Hokama, Y., Asahina, A.Y., Hong, T.W.P., Katsura, K., Shang, E., Miyahara, J.T., Sweeney, J.C., Stone, R. 1990. Causative toxin(s) in the death of two Atlantic dolphins. J. Clin. Lab. Anal. 4:474-478.

Horiguchi, T. 1995. Heterocapsa circularisquama sp. nov. (Peridiniales, Dinophyceae): a new marine dinoflagellate causing mass mortality of bivalves in Japan. Phycol. Res. 43:129-136.

Kaartvedt, S., Johnsen, T.M., Aksnes, D.L., Lie, U. & Svendesen, H. 1991. Occurrence of the toxic phytoflagellate Prymnesium parvum and associated fish mortality in a Norwegian fjord system. Can. J. Fish. Aquat. Sci. 48:2316-2323.

Kent, M.L., Whyte, J.N.C. and LaTrace, C. 1995. Gill lesions and mortality in seawater pen-reared Atlantic salmon Salmo salar associated with a dense bloom of Skeletonema costatum and Thalassosira species. Dis. Aquat. Org.22:77-81.

Landsberg, J. H. and Steidinger, K. A. 1998. A historical review of Gymnodium breve red tides implicated in mass mortalities of the manatee (Trichechus manatus latirostris) in Florida, USA, pp. 97-100, Harmful Algae, eds. Reguera, B. et al. Xunta de galicia and Intergovernmental Oceanographic Commission of UNESCO.

Landsberg, J. H. 1995. Tropical reef-fish disease outbreaks and mass mortalities in Florida, USA: what is the role of dietary biological toxins? Dis. aquat. org. 22:83-100.

Landsberg, J. H. 1996. Neoplasia and biotoxins in bivalves: is there a connection? J. Shellfish Res. 15:205-233.

Landsberg, J. H. (in review). The potential role of biotoxins in sea turtle fibropapillomas. Proceedings of the Second Sea Turtle Fibropapilloma Workshop, Hawaii, December 1997. NOAA Technical Memorandum.

Landsberg, J. H., Balazs, G. H., Steidinger, K. A., Baden, D.G., Work, T. M., Russell, D. G. (this conference). Preliminary evidence links sea turtle fibropapillomatosis to tumor-promoting microalgae.

Landsberg, J.H., Steidinger, K.A. and Blakesley, B.A. 1995. Fish-killing dinoflagellates in a tropical marine aquarium. pp. 65-70. In: Harmful Marine Algal Blooms, eds. P. Lassus, G. Arzul, E. Erard-Le-Denn, P. Gentie and C. Marcaillou-Le-Baut, Technique et Documentation, Lavoisier, Paris.

Lesser, M. P. and Shumway, S.E. 1993. Effects of toxic dinoflagellates on clearance rates and survival in juvenile bivalve molluscs. J.Shellfish Res. 12: 377-381.

Luckenbach, M. W., K. G. Sellner, S. E. Shumway and K. Greene. 1993. Effects of two bloom-forming dinoflagellates, Prorocentrum minimum and Gyrodinium uncatenatum, on the growth and survival of the eastern oyster (Gmelin, 1791). J. Shellfish Res. 12:411-415.

Mahoney, J.B. and Steimle, F.W. Jr. 1979. A mass mortality of marine animals associated with a bloom of Ceratium tripos in the New York Bight. pp. 225-230 In: Toxic Dinoflagellate Blooms, eds. D.L.Taylor and H.H. Seliger, Elsevier, New York.

Mortensen, A.M. 1985. Massive fish mortalities in the Faroe Islands caused by a Gonyaulax excavate red tide. pp. 165-170. In: Toxic dinoflagellates, eds. D. M. Anderson, A. White and D. G. Baden, Elsevier, New York.

Montoya, N.G., Akselman, R., Franco, J. and Carreto, J.I. 1996. Paralytic shellfish toxins and mackerel (Scomber japonicus) mortality in the Argentine Sea. pp. 417-420. In: Harmful and Toxic Algal Blooms, eds. T. Yasumoto, Y. Oshima and Y. Fukuyo, IOC/UNESCO, Paris.

Negri, A.P., Jones, G.J. and Hindmarsh, M. 1995. Sheep mortality associated with paralytic shellfish poisoning toxins from the cyanobacterium Anabaena circinalis. Toxicon 33:1321-1329.

Nehring, S. 1993. Mortality of dogs associated with mass development of Nodularia spumigena (Cyanophyceae) in a brackish lake at the German North Sea coast. J.Plankton Res. 15: 867-872.

Okaichi, T. 1985. Fish kills due to the red tides of Chattonella. Bull. Mar. Sci. 37: 772

Parry, G.D., Langdon, J.S. and Huisman, J.M. 1989. Toxic effects of a bloom of the diatom Rhizosolenia chunii on shellfish in Port Phillip Bay, Southeastern Australia. Mar.Biol. 102:25-41.

Phillips, M.J., Roberts, R.J., Stewart, J.A. and Codd, G.A. 1985. The toxicity of the cyanobacterium Microcystis aeruginosa to rainbow trout, Salmo gairdneri Richardson. J. Fish Dis. 8: 339-344

Robertson, A. 1991. Effects of a toxic bloom of Chrysochromulina polylepis on the common dog-whelk, Nucella lapillus, on the Swedish west coast. J. Mar. biol. Ass. UK. 71:569-578.

Shumway, S. E. 1990. A review of the effects of algal blooms on shellfish and aquaculture. J. World Aquacult. Soc. 21:65-104.

Shumway, S. E. and A. D. Cembella. 1993. The impact of toxic algae on scallop culture and fisheries. Rev. Fish. Sci. 1:121-150.

Sournia, A. 1995. Red tide and toxic marine phytoplankton of the world ocean: an inquiry into biodiversity. pp. 103- 112. In: Harmful Marine Algal Blooms, eds. P. Lassus, G. Arzul, E. Erard-Le-Denn, P. Gentien and C. Marcaillou-Le-Baut, Technique et Documentation, Lavoisier, Paris.

Steidinger, K. A. 1993. Some taxonomic and biological aspects of toxic dinoflagellates. pp. 1-28. In:Algal Toxins in Seafood and Drinking Water, ed. I.R. Falconer, Academic Press, London.

Steidinger, K.A., Burklew, M. and Ingle, R.M. 1973. The effects of Gymnodinium breve toxin on estuarine animals. pp. 179-202. In: Marine Pharmacognosy:Action of Marine Toxins at the cellular level. Academic Press, New York.

Steidinger, K.A., Landsberg, J.H., Truby, E.W. and Blakesley, B.A. 1996. The use of scanning electron microscopy in identifying small "gymnodinioid" dinoflagellates. Nova Hedwigia. 112:415-422.

Steidinger, K.A., Landsberg, J.H., Truby, E.W. and Roberts, B.S. 1998. First report of Gymnodinium pulchellum Larsen 1994 (Dinophyceae) in North America and associated fish kills in the Indian River, Florida. J. Phycol. 34:431-437.

Su, H.M., Liao, I.C. and Chiang, Y.M. 1993. Mass mortality of prawn caused by Alexandrium tamarense blooming in a culture pond in southern Taiwan. pp. 329-334. In: Toxic Phytoplankton Blooms in the Sea, eds. T. J. Smayda and Y. Shimuzu, Elsevier Press, Amsterdam.

Tester, P.A. and Mahoney, B. 1995. Implication of the diatom, Chaetocerus convolutus, in the death of red king crab, Paralithodes camtschatica, Captains Bay, Unalaska Island, Alaska. pp. 95-100. In: Harmful Marine Algal Blooms, eds. P. Lassus, G. Arzul, E. Erard-Le-Denn, P. Gentien and C. Marcaillou-Le-Baut, Technique et Documentation, Lavoisier, Paris.

Tettelbach, S.T. and Wenczel, P. 1993. Reseeding efforts and the status of bay scallop Argopecten irradians (Lamarck, 1819) populations in New York following the occurrence of "brown tide" algal blooms. J. Shellfish Res. 12:423-431.

Tracey, G.A. 1988. Feeding reduction, reproductive failure, and mortality in Mytilus edulis during the 1985 'brown tide' in Naragansett Bay, Rhode Island. Mar. Ecol. Prog. Ser. 50:73-81.

White, A.W. 1980. Recurrence of kills of Atlantic herring (Clupea harengus harengus) caused by dinoflagellate toxins transferred through herbivorous zooplankton. Can. J. Fish. Aquat. Sci. 37: 2262-2265.

Wickfors, G.H. and Smolowitz, R.M. 1993. Detrimental effects of a Prorocentrum isolate upon hard clams and bay scallops in laboratory feeding studies. pp. 447-452. In: Toxic Phytoplankton Blooms in the Sea, eds. T. J. Smayda and Y. Shimuzu, Elsevier Publishers, Amsterdam.

Work, T. M., Beale, A. M., Fritz, L., Quilliam, M. A., Silver, M., Buck, K., Wright, J. L. C. 1993. Domoic acid intoxication of brown pelicans and cormorants in Santa Cruz, California. pp. 643-649. In: Toxic Phytoplankton in the Sea, eds. T. J. Smayda and Y. Shimuzu, Elsevier Publishers, Amsterdam.

Yongjia, Z., Munday, B.L. and Handlinger, J. 1995. Mass mortality of flat oysters (Ostrea rivularis) associated with a bloom of Prorocentrum sp. in the port of Zhanjiang, South China. Bull. Eur. Ass. Fish Pathol. 15:61-63.

Yuki, K. and Yoshimatsu, S. 1989. Two fish-killing species of Cochlodinium from Harima Nada, Seto Inland Sea, Japan. pp. 451-454. In: Red Tides, Biology, Environmental Science and Toxicology, eds. T.Okaichi, D.M. Anderson and T. Nemoto, Elsevier Press, New York.

Mass Mortalities of Marine Mammals

[TOP]

JR Geraci* and VJ Lounsbury

National Aquarium in Baltimore, Department of Biological Programs, Pier 3, 501 E. Pratt Street, Baltimore, Maryland 21202 USA jgeraci@aqua org; vlounsbury@aqua.org

Since the late 1980s, there has been an unprecedented series of events in which large numbers of marine mammals died. Some of these die-offs were caused by previously unknown viruses, others by algal toxins, and still others by environmental anomalies. Some have underscored the vulnerability of small populations to such events. Almost all provoked public and scientific concern that the die-offs were a consequence of habitat degradation and growing human pressure on the marine environment.

Marine mammal die-offs are complex events. Their study requires knowledge of the species' biology and normal patterns of distribution and mortality, as well as an understanding of the many environmental factors that may be involved. Even then, cause-and-effect relationships between die-offs and any single factor can be difficult to demonstrate--and links with human activities even more so. As scientists, we must continue to search for answers. As responsible stewards of the earth's resources, we cannot afford to wait for proof.

Historical perspectives
Interest in the health of marine mammals began to grow in the 1960s as a result of both increasing environmental awareness and the developing captive display industry. Through the mid 1980s, studies focused on a fundamental understanding of traditional health indices such as blood and serum, parasites, and detection of microorganisms. By the end of the 1970s, many bacteria and numerous fungal pathogens--but few viruses--had been identified. So poor was our understanding of marine mammal health, that the mere identification of a known bacterial pathogen in a seal or whale was often considered sufficient evidence as to the cause of death or illness. Except for a few "unexplained" die-offs, historical events had been associated largely with storms or unusual ice conditions. Rare outbreaks of bacterial diseases such as leptospirosis or pneumonia, involving perhaps a few dozen pinnipeds, had been reported, but die-offs due to infectious agents--viruses in particular--were not a concern.

The 1980s - 1990s
A new chapter in our understanding of marine mammal health began in 1980, when we first recognized that viruses can cause large-scale mortality. An outbreak of pneumonia caused by Influenza A virus killed about 450 harbor seals (Phoca vitulina) along the New England coast between December 1979 and October 1980 (Geraci et al. 1982). Through the mid 1980s, however, this event seemed unusual--even extraordinary--in terms of known pathogens and patterns of mortality in marine mammals.

The summer of 1987 ushered in a decade of unprecedented events. Between June 1987 and March 1988, more than 740 bottlenose dolphin (Tursiops truncatus) carcasses beached along the US mid Atlantic coast. Based on tissue and stomach samples and environmental evidence--and lack of evidence pointing to a single, common pathogen--investigators concluded that exposure to brevetoxin, produced by the dinoflagellate Gymnodinium breve, left dolphins susceptible to overwhelming infection by a variety of opportunistic pathogens (Geraci 1989). Although this conclusion was controversial, the case for natural toxins as a cause of marine mammal mortality was strengthened during the winter of 1987-88, when 14 humpback whales (Megaptera novaeangliae) died in Cape Cod Bay after eating fish contaminated with saxitoxin (Geraci et al. 1989), the same toxin that causes paralytic shellfish poisoning in humans.

Concurrent with the US Atlantic coast events, unusual mortalities of marine mammals began in Europe with an outbreak of canine distemper (CDV), caused by a morbillivirus, in Baikal seals (Phoca sibirica). Within months, a previously unknown but closely related morbillivirus--phocine distemper, or PDV--swept through European populations of harbor seals, ultimately killing more than 18,000. Also in 1988, another "new" morbillivirus was identified as the cause of death of a few harbor porpoises (Phocoena phocoena) in waters around the United Kingdom. Between 1990 and late 1992, yet another morbillivirus, similar to that found in the porpoises, killed more than a thousand striped dolphins, Stenella coeruleoalba, in the Mediterranean Sea. (See Harwood and Hall 1990, Hall 1995, Geraci et al. in press for reviews of these events).

These epidemics changed our ideas about the role of viruses in marine mammal health. Earlier events were re-investigated from a new perspective. Detection of antibodies to CDV in crabeater seals, Lobodon carcinophagus, (Bengtson et al. 1991) suggested a plausible solution to a 1955 die-off in that Antarctic population. Evidence of morbillivirus infection detected in some bottlenose dolphins during the US Atlantic coast die-off (Geraci 1989) and subsequent studies of preserved tissues suggested that morbillivirus was also a factor in that event (Duignan et al. 1995, 1996).

Indeed, morbillivirus has remained a prime suspect in every subsequent die-off and, for a time, appeared to be one of the greatest threats to marine mammal health. However, infection without associated mortality is now known to be common in many species and populations (Hall 1995, Van Bressem et al. 1998). In those in which the virus is endemic, infection is presumably widespread but of little consequence, because animals have developed immunity through frequent exposure. Outbreaks of illness are more likely to follow introduction of the virus into naive populations.

The Atlantic bottlenose dolphin and humpback whale mortalities of 1987-88 were not the first events for which algal toxins had been proposed as the underlying cause. Suspected mortalities involving several species had been reported over the past few decades (Geraci et al. in press), but events were often poorly investigated or the evidence circumstantial; these toxins are difficult to detect and may leave little trace of their presence. In the past few years, harmful algal toxins have emerged again as a significant cause of large-scale events. In the spring of 1996, about 150 Florida manatees (Trichechus manatus latirostris) died from ingesting and inhaling brevetoxin (Bossart et al. 1998) during a strong red tide in southwestern Florida, the same area in which about 37 manatees died during a red tide in 1982 (O'Shea et al. 1991).

Two recent mass mortalities--of about 60% of the largest remaining colony of Mediterranean monk seals (Monachus monachus) at Cap Blanc, Mauritania in 1997, and of New Zealand sea lions (Phocarctos hookeri) in the Aukland Islands in early 1998--further underscore the vulnerability of endangered populations or species to unusual events. Although investigations are still inconclusive, evidence points to involvement of algal toxins (Hernandez et al. 1998, Madie et al. 1998).

In the past few years we have also witnessed mass starvation in some pinniped populations due to dramatic changes in prey availability associated with sudden changes in oceanic conditions: in Eastern Pacific pinniped populations during the El Niño southern oscillation events of 1982/83 (Trillmich et al. 1991) and 1997/98; in Cape fur seals (Arctocephalus pusillus pusillus) in Namibia in 1994 (Geraci et al. in press). As with El Niño phenomena, such events along Africa's southwestern coast are not without historical precedence. Oceanographic anomalies, red tides, and marine animal die-offs have been reported from this area over the past 170 years (Wyatt 1980).

What have we learned?
Prior to the early 1970s, there was little interest in marine mammal health and no formal mechanisms for reporting unusual mortalities that would allow comparison of recent events with those in the past. We are still limited by the difficulty of studying animals that are largely inaccessible to us until they come ashore, by the remote nature of many shorelines, by differences in reporting efforts among nations, and by the lack of standardized methods that would allow comparisons among different populations. If we consider observations over the past 20 years alone, however, we can say with some certainty that the frequency of unusual events has increased.

Concurrent with advances in techniques for virus identification and isolation, and for detection of toxins in marine mammal tissues, as well as in our understanding of marine mammal pathology, we have learned that viruses and natural toxins are among the greatest threats to the health of marine mammal populations. Conditions that precipitate algal and dinoflagellate blooms are still poorly understood, but their increase in distribution and frequency has been linked to factors including transport of organisms in ballast water, nutrient enrichment of coastal waters, and warmer ocean surface temperatures (Anderson 1994). Thus, human activities may be implicated in some biotoxin-associated marine mammal mortalities, although such relationships are difficult to prove and are likely to remain controversial.

The greatest issue in the die-offs occurring from 1987 through the early 1990s was the possible role of contaminants. Organochlorines such as PCBs are known to influence hormonal balance and immune function in some species, including marine mammals. However, variability in tissue burdens within and between affected populations has not supported a cause-and-effect relationship between exposure to contaminants and any of these events (reviewed in Geraci et al., in press).

Historically, mass mortality due to starvation has been associated with natural factors that cause changes in prey availability. As fisheries have become more efficient and needs of human populations have grown, exploitation of the ocean's living resources has, in many cases, surpassed recognized sustainable yield, and marine mammals are often viewed as competitors for the same dwindling stocks of prey. For individuals and populations, inadequate prey stocks might result in malnutrition and changes in behavior that could increase susceptibility to opportunistic pathogens.

Marine mammal populations, like those of other animals, have always been subject to factors that periodically cause large-scale mortality (Harwood and Hall 1990). Species subject to such catastrophes have generally evolved reproductive strategies that allow populations to recover rapidly. While these populations have been resilient in the past, cumulative, perhaps synergistic effects of multiple insults may change their recovery rates in the future. Small isolated populations, like monk seals and sirenians, are especially vulnerable to these events. For populations that suffer a high level of human-related mortality, the Florida manatee for example, unusual events may further threaten population survival.

What lies ahead?
We are entering yet another era, one in which we recognize our role in degrading marine habitats--from coastal rookeries to deep oceans--through oil spills, marine debris, over-fishing, offshore dumping, and global transport of pathogens and potentially toxic algae in ballast water. We are cutting deeply into marine mammal food resources, at the same time as we encourage world-wide protection of marine mammal species. Predicted changes in climate will influence marine mammal behavior and distribution in ways that may affect how pathogens are transmitted among individuals and populations. In future years, we should expect an increase in the movement of pathogens and intensity of disease. We should also expect more die-offs resulting from exposure to natural toxins, as toxic algal blooms increase in response to both continued nutrient enrichment and, perhaps, unusually warm conditions.

On a positive note, we are beginning to understand the complex effects of both natural phenomena and human-related activities on marine mammal health. Our ability to recognize clinical and pathological effects of disease agents and toxins is growing rapidly. Advances in knowledge are matched by an unflagging determination among marine mammal scientists and conservationists worldwide to develop sound scientific data that can be utilized by managers and policy-makers to bring about changes that will protect the health and survival of many marine mammal populations.

References
Anderson, DM. 1994. Red tides. Scientific American 271:62-68.

Bengtson, JL, P Boveng, U Franzén, P Have, MP Heide-Jørgensen and TJ Härkönen. 1991. Antibodies to canine distemper virus in Antarctic seals. Marine Mammal Science 7:85-87.

Bossart, GD, DG Baden, RY Ewing, B Roberts and SD Wright. 1998. Brevetoxicosis in manatees (Trichechus manatus latirostris) from the 1996 epizootic: gross, histologic and immunohistochemical features. Toxicologic Pathology 26:276-282.

Duignan, PJ, C House, JR Geraci, N Duffy, BK Rima, MT Walsh, G Early, DJ St. Aubin, S Sadove, H Koopman and H Rhinehart. 1995. Morbillivirus infection in cetaceans of the western Atlantic. Veterinary Microbiology 44:241-249.

Duignan, PJ, C House, DK Odell, RS Wells, LJ Hansen, MT Walsh, DJ St. Aubin, BK Rima and JR Geraci. 1996. Morbillivirus infection in bottlenose dolphins: evidence for recurrent epizootics in the western Atlantic and Gulf of Mexico. Marine Mammal Science 12:499-515.

Geraci, JR. 1989. Clinical investigation of the 1987-88 mass mortality of bottlenose dolphins along the US central and south Atlantic coast. Final Report to National Marine Fisheries Service, US Navy (Office of Naval Research) and Marine Mammal Commission. 63 p.

Geraci, JR, DJ St. Aubin, IK Barker, RG Webster, VS Hinshaw, WJ Bean, HL Ruhnke, JH Prescott, G Early, AS Baker, S Madoff and RT Schooley. 1982. Mass mortality of harbor seals: pneumonia associated with influenza A virus. Science 215:1129-1131.

Geraci, JR, DM Anderson, RJ Timperi, DJ St. Aubin, GA Early, JH Prescott and CA Mayo. 1989. Humpback whales (Megaptera novaeangliae) fatally poisoned by dinoflagellate toxin. Canadian Journal of Fisheries and Aquatic Sciences 46:1895-1898.

Geraci, JR, J Harwood and VJ Lounsbury. In press. Marine mammal die-offs: causes, investigations, and issues, chapter 19. In JR Twiss and RR Reeves [eds.] Marine Mammals, vol. 1. Smithsonian Institution Press, Washington, DC.

Hall, AJ. 1995. Morbilliviruses in marine mammals. Trends in Microbiology 3:4-9.

Harwood, J, and AJ Hall. 1990. Mass mortality in marine mammals: its implications for population dynamics and genetics. Trends in Ecology and Evolution 5:254-257.

Hernández, M, I Robinson, A Aguilar, LM González, LF López-Jurado, MI Reyero, E Cacho, J Franco, V López-Rodas and E Costas. 1998. Nature 393(6680):28-29.

Madie, P, P Duignan, J Hunter, M Alley, K Thompson, J Meers, S Fenwick, N Gibbs, N Gales and S Childerhouse. New Zealand sea lion epidemic, January-February 1998. Preliminary report, May 1998. Massey University Cetacean Investigation Centre and Department of Conservation, Palmerston North, New Zealand.

O'Shea, TJ, GB Rathbun, RK Bonde, CD Buergelt and DK Odell. 1991. An epizootic of Florida manatees associated with a dinoflagellate bloom. Marine Mammal Science 7:165-179.

Trillmich, F, KA Ono, DP Costa, RL DeLong, SD Feldkamp, JM Francis, RL Gentry, CB Heath, BJ Le Boeuf, P Majluf and AE York. 1991. The effects of El Niño on pinniped populations in the Eastern Pacific, p. 247-270. In F Trillmich and K A Ono (eds.). Pinnipeds and El Niño: responses to environmental stress. Springer-Verlag, Berlin.
Van Bressem, M-F, K Van Waerebeek, M Fleming and T Barrett. 1998. Serological evidence of morbillivirus infection in small cetaceans from the Southeast Pacific. Veterinary Microbiology 59:89-98.

Wyatt, T. 1980. Morrell's seals. Journal du Conseil International pour l'Exploration de la Mer 39:1-6.

Emerging Crustacean Diseases

[TOP]

Donald V. Lightner and R.M. Redman

Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 85721 USA

Crustacean fisheries and aquaculture are among the most important seafood industries, and because of their high market value, considerable investment has been made in recent decades to develop methods to culture lobsters, crabs, crayfish, prawns, and shrimps. Some of the efforts have been successful, and the aquaculture of freshwater and marine crustaceans is now an expanding industry that accounts for approximately 1.2 million metric tons (t) of production annually (8). While this represents only 6.5% of all aquaculture production, the very high market value of crustaceans ranks them among the most valued aquacultured species (36). The marine penaeid shrimps and prawns (shrimp in this paper) accounted for 86% of the production of crustaceans from aquaculture in 1994, or about 700,000 t (41). The shrimp aquaculture industry grew by 430% during the period of 1985 to 1994, and it is projected to continue to grow at that rate (8, 36). The FAO (8) estimates that approximately 30% of all penaeid shrimp marketed worldwide now come from farms, and U.S. Department of Commerce statistics show that more than half of the marine shrimp now consumed in the United States come from farms (21).

Concomitant with the phenomenal growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. Major epizootics have plagued the world's shrimp culture industries. The most important diseases of cultured shrimp have had viral or bacterial etiologies, but a few important diseases have fungal, protozoans, nutritional imbalances, toxicants, or environmental extremes as their cause (2, 24). Diseases due to virus infections have emerged as the most important diseases of cultured shrimp, and of the ~20 viruses now known to infect shrimp, four (WSSV, YHV, TSV, and IHHNV) stand out as major impediments to the industry (25, 26, 41). Together, these four viruses have been responsible for crop losses that are estimated to have exceed U.S. $6 billion as of the 1998 shrimp growing season.

The Asian WSSV and YHV Pandemics: Two major virus diseases, that emerged during the early 1990's, have resulted in a serious ongoing pandemics in shrimp growing regions of Asia and the Indo-Pacific. Although probably a significant pathogen in cultured Asian Penaeus monodon for at least half a decade before its recognition, yellow head disease (YHD), and its agent, yellow head virus (YHV), was first recognized as a distinct disease during 1991 in cultured P. monodon in Thailand (1, 4). YHV was first described as a baculo-like virus (1), but later YHV was shown to be an RNA virus related to the rhabdoviruses (34, 48). The name for YHD came from gross signs displayed by moribund P. monodon infected with YHV that sometimes display a yellowish cephalothorax and very pale overall coloration (1, 2, 4, 13, 24). YHV has a wide distribution in cultured stocks of P. monodon in SE Asian and Indo-Pacific shrimp farming countries (10, 11, 13, 24, 26). Two very similar viruses, LOV (lymphoid organ virus) and GAV (gill-associated virus) has been reported from P. monodon in Australia (42, 43). Crop losses due to YHV at the peak of the 1992-1993 epizootic in Thailand were estimated to be about U.S.$40 million (12, 13). Although the importance of YHV have been over shadowed by WSSV since its appearance in Asia, YHV has continued to be a problem. YHV commonly occurs as co-infections with WSSV and it has very likely contributed to the huge crop losses credited to WSSV (12, 13, 32, 37).

About the same time that YHV was causing serious epizootics in Thailand, an epizootic due to a systemic, non-occluded baculo-like virus began during 1992 in stocks of P. monodon, P. japonicus, and P. penicillatus being cultured in Taiwan (5). Among the clinical signs of the disease were conspicuous white spots on the inside surface of the cuticle that eventually gave rise to the name of this disease. Viral epizootics accompanied by the same type of cuticular white spots were recognized during 1993 in Japan, Korea, and China (16, 17, 18, 23, 35, 44, 46, 48, 50). By 1994, the disease had spread to Thailand and India, and by 1996, it had severely impacted most of the shrimp farming regions of East Asia, South Asia, and Indonesia (13). While initially given at least five different names by various authors, the etiological agent of this disease is now most commonly known as white spot syndrome virus (WSSV) or white spot baculovirus (WSBV) (6, 7, 13, 22, 24, 28, 29, 46, 50). However, because all non-occluded baculo-like viruses of insects and crustacea were recently removed from the Family Baculoviridae by the International Committee on Virus Nomenclature and are pending re-classification (33), the term white spot syndrome virus (WSSV) may be the most appropriate name for the virus.

The IHHNV and TSV Pandemics of the Americas: Taura Syndrome (TS) emerged in 1992-1993 in Ecuador as a major epizootic disease of Penaeus vannamei that spread rapidly throughout most of the shrimp growing regions of Latin America (3, 15, 19, 24, 25, 27). In 1992 and 1993, P. vannamei accounted for more than 90% (about 132,000 t) of the farmed shrimp production in the Americas or about 15 to 20% of the world's production of farmed shrimp (38-40). Because P. vannamei is the principal species used in aquaculture in the Americas (40), TS has caused serious losses to the shrimp farming industries. The economic impact of TS in the Americas, since its recognition in Ecuador in 1992 and subsequent spread throughout the Americas, exceeds U.S.$2 billion (25).

In Ecuador TS was first recognized in mid-1992 in commercial shrimp farms located near the mouth of the Taura River in the Gulf of Guayaquil, Ecuador (3, 15, 19, 24, 26). Shortly after TS appeared in Ecuador, both toxic and infectious etiologies were proposed, but a viral etiology was eventually proven (3, 15, 24, 25, 27). During 1994, TS occurred in shrimp farms throughout much of Ecuador, as well as in single or multiple farm sites in several South and Central American countries, and in the U.S., occurring at isolated sites in Florida and Hawaii (3, 24, 25, 27). By mid-1996 the disease had expanded its distribution to include virtually all of the shrimp farming regions of the Americas, including the U.S. states of Texas and South Carolina (15, 24-27).

TSV has been documented in wild postlarvae (PLs) and adult P. vannamei on several occasions. The disease has been diagnosed in wild PLs collected during mid-1993 from the Gulf of Guayaquil in Ecuador and in wild adult P. vannamei collected during and since 1994 off the Pacific coast of Honduras, El Salvador, southern Mexico (24-27). The affected adult P. vannamei collected from these sites in 1994 showed high mortalities and developed diagnostic lesions for of the disease (25). Significantly, this occurrence of TSV in wild PLs and adult broodstock documents that virus is established in wild stocks where its potential effect on commercial penaeid shrimp fisheries is unknown. Complicating the situation further have been the discoveries that an aquatic insect and sea birds may be involved in the epizootiology of TSV (14, 15, 25). The salinity-tolerant water boatman, Trichocorixa reticulata and laughing gulls, Larus atricilla have been shown to serve as potential vectors of TSV. Infectious TSV has been found in the feces of the insect and the gull, when collected from shrimp farms during active TSV epizootics TSV (14, 25). Hence, aquatic insects, gulls and other shrimp eating sea birds may transmit TSV within affected farms or to other farms within their flight range.

Compared to WSSV, YHV, and TSV, which emerged as major pathogens after 1990, IHHNV is an "old virus". IHHNV was first recognized in cultured P. stylirostris and P. vannamei imported into Hawaii from several sites in 1980-1981. At that time IHHNV was found to cause catastrophic acute losses often exceeding 90% in cultured populations of juvenile P. stylirostris while P. vannamei appeared to be relatively resistant (24, 25). In P. vannamei, IHHNV infection typically causes the chronic disease, "runt deformity syndrome" or RDS (2, 24, 25), which is an economically important disease in P. vannamei. While not showing significant mortalities due to IHHNV infection, cultured populations of P. vannamei with RDS produce smaller average sized shrimp at harvest, and thus a significantly lower crop value (2, 24, 25).

Studies done on the distribution of IHHNV following its discovery in 1981, showed that the virus was widely distributed in the shrimp culture industries of the Americas, and it was spreading IHHNV is now present in virtually every country or region where either P. vannamei or P. stylirostris is farmed, as well as being present in some shrimp culture regions of the Indo-Pacific where it is not thought to be a significant pathogen (2, 10, 11, 24, 25). IHHNV is, and has been, an extremely important pathogen of cultured penaeid shrimp in the Americas. Because IHHNV often causes an acute disease with very high mortalities in juvenile P. stylirostris, P. vannamei became the preferred species for shrimp aquaculture development in the Americas during the 1970's and 1980's. It was not until TSV arrived, and P. stylirostris was found to be TSV resistant, that significant interest was revived in culturing the species (25).

The Threat of WSSV and YHV to the Americas: PL and/or juvenile stages of the American penaeids P. vannamei, P. stylirostris, P. setiferus, P. aztecus, and P. duorarum have been found to be highly susceptible to experimental infection by WSSV and YHV (32, 37, 45, 51). In November 1995, the first documented cases of WSSV infection was recognized in the Western Hemisphere. Involved in the case were pond-reared P. setiferus from a south Texas farm. From histological lesions presented by the WSSV-infected P. setiferus, co-infection with YHV was suspected but not confirmed. The proximity of the affected farm to shrimp packing plants was suggested to be a plausible source of WSSV because plants in the vicinity of the affected farm at that time were re-processing and re-packing large quantities of shrimp that were imported from affected areas of Asia (21, 24, 37, 51). Since that initial occurrence in 1995, WSSV has been found (and isolated) on at least six occasions from apparently normal or from diseased wild, captive-wild, and cultured stocks of freshwater crayfish and marine penaeid shrimp from sites on the eastern, southeastern, and Gulf of Mexico coasts of the U.S. (37, 51).

The U.S. is a major importer of wild and farm-raised penaeid shrimp. The U.S. imports thousands of tons of cultured shrimp each year from countries where WSSV and YHV are enzootic and causing serious epizootics (9, 21, 37, 51). Emergency harvests are commonly employed in Asia to salvage marketable shrimp crops with developing epizootics due to these viruses (10, 20, 21, 37, 51). P. monodon displaying gross signs of WSSV infection (i.e. cuticular white spots and reddish pigmentation) were found in retail outlets in the U.S. in 1995 and 1996 (21, 34). PCR assays of samples of these shrimp confirmed the presence of WSSV. A bioassay of one sample of WSSV-positive (by PCR) P. monodon, utilizing P. stylirostris as the indicator for infectious virus, showed that the sample was co-infected with WSSV and YHV (34). Imported commodity shrimp are distributed throughout the U.S., and some imported shrimp are re-processed at shrimp packing plants situated on coastal bays and estuaries where native penaeid nursery grounds also occur (21). With infectious WSSV and YHV present, and perhaps, fairly common in imported commodity shrimp, the risk of accidental contamination of wild or cultured stocks of penaeid shrimp may be significant. When the history of outbreaks of WSSV (and possibly YHV) in U.S. coastal areas (in wild and cultured P. setiferus in Texas in 1995 and again in 1997, in crayfish at the U.S. National Zoo in 1996-1997, and in wild and cultured shrimp in South Carolina in 1996 -1997) are considered with the findings of virological studies that have shown the U.S. and Asian isolates of WSSV to be nearly identical (30, 47), the data indicates that WSSV has been introduced to North America. Because of the very high susceptibility of American penaeids to WSSV and YHV, the introduction and establishment of either or both of these pathogens could cause very serious disease epizootics and economic hardships to the shrimp farming industries of the Americas.

Domestication and Development of SPF/SPR Shrimp: Despite its size and importance, the penaeid shrimp culture industry remains largely dependent on the capture of wild shrimp for its "seed stock". Nearly all of the seed stock required to stock the world's 1.3 million ha of ponds is derived either from PLs collected directly from the wild, or produced in hatcheries from spawners which were also taken from the wild (25, 41). This dependence on wild seed stock has left the developing penaeid shrimp culture industry extremely vulnerable to disease. Recent developments in the selection and domestication of shrimp stocks that are either free of ("specific pathogen-free, SPF" or resistant to (SPR) one or more of the major shrimp viruses (WSSV, YHV, TSV, and IHHNV) promise to provide the industry with the same opportunity for disease control and genetic improvement that characterize many other agricultural industries. I predict that by the end of the next decade, most of the penaeid shrimp produced from aquaculture will come from SPF or SPR lines of genetically selected, domesticated lines of three or four species of penaeid shrimp.

Acknowledgments: Research funding has been provided by the U.S. Marine Shrimp Farming Consortium, CSREES, USDA; the National Sea Grant Program, USDC; and the National Fishery Institute.

References

1.	Boonyaratpalin S. et al. 1993. Fish Pathol. 28:103-109.
2.	Brock, J.A., & K. Main. 1994. A Guide to the Common Problems and Diseases of Cultured Penaeus vannamei. Oceanic Institute, HI, 241 pp.
3.	Brock, J.A. et al. 1995. pp. 84-94 in Aquaculture '95. World Aquaculture Society.
4.	Chantanachookin, C. et al .1993. Dis. Aquat. Org. 17:145-157.
5.	Chen, S.N. 1995. pp.29-34 in Aquaculture '95. World Aquaculture Society.
6.	Chou, H.Y. et al. 1995. Dis. Aquat.. Org. 23: 165-173.
7.	Durand, S. et al. 1996. Dis. Aquat. Org. 27: 59-66.
8.	FAO. 1996. FAO Fisheries Statistics Serices No. 46 (Volume 78), 699 p. FAO, Rome, Italy.
9.	Filose, J. 1995. pp. 227-234 in Aquaculture '95. World Aquaculture Society.
10.	Flegel, T.W. et al. 1995a. pp. 65-79 in Diseases in Asian Aquaculture II.
11.	Flegel, T.W. et al. 1995b. pp. 76-83 in Aquaculture '95. World Aquaculture Society
12.	Flegel, T.W. et al. 1996. pp. 126-127 in World Aquaculture '96. Book of Abstracts.
13.	Flegel, T.W. 1997. pp. in New Approaches to Viral Diseases of Aquatic Animals. N.R.I.A., Japan.
14.	Garza, J.R. et al. 1997. J. Aquat. An. Health 9: 156-159.
15.	Hasson, K.W. et al. 1995. Dis. Aquat. Org. 23: 115-126.
16.	Huang, J. et al. 1995. Marine Fish. Res. 16: 1-10.
17.	Inouye, K., S. et al. 1994. Fish Pathol. 29: 149-158.
18.	Inouye, K. et al. 1996. Fish Pathol. 31:39-45.
19.	Jimenez, R. 1992. `Sindrome de Taura (Resumen). Acuacultura del Ecuador', p. 1-16.
20.	Jory, D.E. 1996. Aquaculture Magazine 22 (4):71-78.
21.	JSA. 1997. An evaluation of potential shrimp virus impacts on cultured shrimp and wild shrimp populations in the Gulf of Mexico and southeastern U.S. Atlantic coastal water. Joint Subcom. Aquacult., Shrimp Virus Work Group, Nat. Center Envir. Assessment, W.D.C. 65 p.
22.	Kasornchandra, J. & S. Boonyaratpalin. 1996. Asian Shrimp News 273(3): 4.
23.	Kimura, T., et al. 1996. Fish Pathol. 31:93-98.
24.	Lightner, D.V. 1996a. A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society. 304p.
25.	Lightner, D.V. 1996b. Rev. Sci. Tech. Off. Int. Epiz. 15: 579-601.
26.	Lightner, D.V. et al. 1997a. Rev. Sci. Tech. Off. Int. Epiz. 16: 146-160.
27.	Lightner, D.V. et al. 1997b. pp. 190-205 in New Approaches to Viral Diseases of Aquatic Animals. N.R.I.A., Japan.
28.	Lo, C.F. et al. 1996. Dis. Aquat. Org. 25: 133-141.
29.	Lo, C.F. et al. 1997. Dis. Aquat. Org. 27: 215-225.
30.	Lo, C.F. et al. "In press". Dis. Aquat. Org.
31.	Lu, Y., et al. 1995. J. Fish Dis. 17:649-656.
32.	Momoyama, K., M. et al. 1995. Fish Pathol. 30: 263-269.
33.	Murphy, F.A. et al. 1995. The classification and nomenclature of viruses. Arch. Virol.
34.	Nadala, E.C.B. Jr. et al. 1997. Dis. Aquat. Org. 31: 141-146.
35.	Nakano, H. et al. 1994. Fish Pathol. 29: 135-139.
36.	New, M.B. 1997. World Aquaculture 28(2): 11-30.
37.	Nunan, L.M. et al. "In press". Aquaculture.
38.	Rosenberry B. (ed.). 1993. Shrimp News International, 18(3):6.
39.	Rosenberry B. (ed.). 1994a. Shrimp News International, 19(3):2-4.
40.	Rosenberry B. (ed.). 1994b. World Shrimp Farming 1994. Aquacult. Digest, San Diego. 68 p.
41.	Rosenberry B. (ed.). 1997. World Shrimp Farming 1997. Aquacult. Digest, San Diego. 284 p.
42.	Spann, K.M. et al. 1995. Dis. Aquat. Org. 26: 127-134.
43.	Spann, K.M. et al. 1997. Dis. Aquat. Org. 31: 169-179.
44.	Takahashi, Y. et al. 1994. Fish Pathol. 29:121-125.
45.	Tapay, L.M., et al. 1997. pp. 297-302 in Diseases in Asian Aquaculture III.
46.	Wang, C.S. et al. 1996. Fish Pathol. 31:177-182.
47.	Wang, Q., & D.V. Lightner. 1998. p. 567 in Aquaculture '98, Abstracts. World Aquacult. Soc.
48.	Wang, C.H. et al. 1995. Dis. Aquat. Org. 23: 239-242.
49.	Wongteerasupaya, C. et al. 1995a. Dis. Aquat. Org. 22: 45-50.
50.	Wongteerasupaya, C. et al. 1995b. Dis. Aquat. Org. 21: 69-77.
51.	Lightner, D.V., et al. "In press". J. Aquat. An. Health.

Emerging Molluscan Diseases

[TOP]

SE McGladdery

Department of Fisheries and Oceans, Gulf Fisheries Centre, P.O. Box 5030, Moncton, New Brunswick, E1C 9B6, Canada mcgladderys@mar.dfo-mpo.gc.ca

Global perspective
In the last 30-40 years, most significant diseases of molluscs have been linked to introduction and transfer of infectious agents, e.g., bonamiasis of European oysters (Ostrea edulis) and MSX disease of American oysters (Crassostrea virginica). In the last ten years, however, there have been few examples of new "exotic" diseases, possibly due to increased awareness of the risks associated with transfers of live aquatic organisms, and implementation of "Codes of Practice" to reduce these risks (ICES 1995; OIE 1997). Most disease problems currently appear related to changing culture techniques and diversification of species under culture, although unexplained diseases, which defy categorisation, continue to appear.

Mortalities of juvenile European oysters (O. edulis) and Pacific oysters (Crassostrea gigas) in France have been attributed to Herpes-like viral infections. Virulence appears related to intensive hatchery production and warm water temperatures (Comps and Cochennec 1993; Le Deuff et al. 1996), a situation similar to that with mortalities of hatchery-reared C. gigas and flat oysters (Ostrea angasi) in New Zealand (Hine et al. 1992; Hine and Thorne 1997). Although this appears to be a new problem, the pathogenicity of this type of viral infection has been recognised since its original description from American (Eastern) oysters, Crassostrea virginica (Farley et al. 1972) and summer mortalities of Pacific oysters in Europe included descriptions of Cowdry Type A inclusion bodies (Alderman 1980). Although, not all summer oyster mortalities are linked to Herpes-like viruses, it appears they have a wide host and geographic distribution. Their apparent re-emergence in recent years likely reflects the convergent evolution of rearing technology, conducive to producing disease, and the expertise to detect and identify the viral agent(s) (Elston 1997). Other similar examples include detection of virus-like particles ("VLP") in summer mortalities of the mussels Perna canaliculus and Mytilus galloprovincialis from New Zealand (Jones et al. 1996) and viral-like particles associated with mass mortalities of pearl oysters Pinctada fucata in Japan (Japanese Society of Fish Pathology, March 1998 ). It therefore seems likely that more viruses will be detected in mortalities of previously unexplained aetiology in the near future.

Another problem which has afflicted hatchery-production in recent years is Juvenile Oyster Disease (JOD) of American oysters in the northeastern United States (Bricelj et al. 1992). Oysters 7-30 mm in size can suffer up to 100% mortality at water temperatures over 12-15 °C and salinities over 18 ppt. The disease is frequently characterised by abnormal deposition of conchiolin on the inner surface of the shell and abnormal growth of the cupped valve. Although an infectious aetiology is suggested by experimental studies, the cause is still being investigated (Lewis et al. 1996; Lee et al. 1996). Selective breeding of survivors has proven successful in developing disease-resistance (Farley et al. 1997) which, in addition to early spawning and seed deployment (Davis and Barber 1994), has significantly reduced mortalities.

Recent mass mortalities of cultured hard-shell clams (quahaugs), Mercenaria mercenaria, from Massachusetts, have been linked to infection by Quahaug Parasite Unknown (QPX)(Smolowitz et al. 1998). Infections also occur in wild and cultured populations in Atlantic Canada, where the disease agent was originally described in the early 1960's as a "Chytrid-like Olpidium" (Drinnan and Henderson 1963), and Virginia (Ragone-Calvo et al. 1997). Infections of wild populations are usually sub-clinical, although pathogenic levels of infection have been found in moribund and dying hatchery broodstock (Whyte et al. 1994). Infections appear restricted to older clams, despite direct contact with younger stages of development. Both Canadian and Massachusetts populations only begin to show signs of infection at 12 to 18 months (Whyte et al. 1994; Smolowitz et al. 1998). Following its original description from Gulf of St. Lawrence clams, QPX has been found in Bay of Fundy populations and appears, as with Herpes-like infections, to have a broader geographic range than initially indicated by mortalities. Recent success in culturing the organism should help in determining the exact factors which trigger fatal infections (Kleinschuster et al. 1998).

Another economically important group of molluscan species being brought into culture are the pearl oysters (Pteriidae) of South America, India and Asia. The transition from the wild environment into culture, however, has been plagued by mortality problems which vary with pearl oyster species, culture techniques and climate of operations. One of the most thoroughly investigated cases revealed vibriosis to be a stress related pathogen of gold-lipped pearl oysters (Pinctada maxima) from Western Australia (Pass et al. 1987). Subsequent husbandry adjustments significantly reduced mortalities. As pearl oyster culture develops, however, new disease agents and problems are emerging, as appears to be the case recently, with Japanese pearl oysters (P. fucata). Mass mortalities, originally linked to blooms of the toxic dinoflagellate Heterocapsa circularisquama (Matsuyama et al. 1995), have persisted and may reflect a multifactorial aetiology, including newly isolated viruses and a possible Perkinsus sp. (Japanese Society of Fish Pathology, March 1998). Increasing effort is being placed on hatchery production to reduce pressure on dwindling natural oyster sources, permit greater control over seed availability and enhance selective breeding. This scenario, however, has the disease potential faced by other bivalve species, so it is hoped that pearl oyster production may benefit from their experience.

The progressive spread of Withering Syndrome (WS), since its initial detection in abalones in the Californian Channel Islands in 1985 (Altstatt et al. 1996), shows no apparent correlation to changing culture techniques, transfer of species or environmental changes. It affects black, red and pink abalone (Haliotis cracherodii, H. rufescens and H. corrugata, respectively), but is most pathogenic to black abalone (H. cracherodii). Mortalities of over 95% are associated with water temperatures of 18-20 °C and have led to closure of the black abalone fisheries in the most severely affected Islands (Haaker et al. 1995). Investigations into the aetiology indicate an interaction between warm water temperatures and a rickettsial-like infection (Friedman et al. 1997), but the reason for differential virulence between sympatric Haliotis species is unknown.

Important molluscan disease developments
Recent advances in molecular diagnostic tools for well-known pathogens have set the foundation for future expansion into these areas both for diagnostics and research. One of the most notable advances is the development of DNA probes for Haplosporidium nelsoni (Stokes and Burreson 1995, Burreson 1996), Perkinsus spp. (Yarnall et al. 1997) and other pathogens (Gee and Elston 1996; Carnegie et al 1997). Equal progress has been made with immunodiagnostic assays for Perkinsus spp. (Goggin et al. 1991; Dungan 1997), Bonamia ostreae (Cochennec et al. 1992), and various neoplasias (Noel et al. 1994; Harper et al. 1994). Growing development of pathogen-sensitive diagnostic tools and greater genetic and immunological understanding of the factors associated with disease resistance is also enhancing disease research (Gaffney et al. 1996; Cadoret et al. 1997). Increased diagnostic sensitivity and specificity will permit detection of sub-clinical carriers (normal and abnormal host species) and differentiation between pathogenic and non-pathogenic strains/species, allowing more accurate risk assessment than currently possible.

Priorities
The most enduring and outstanding limitation on molluscan disease research, continues to be the lack of marine molluscan self-replicating cell-lines for intracellular pathogen culture. This lack continues to gain significance in light of the number of viruses and intracellular bacteria now being detected in association with serious molluscan mortalities.

Most emerging disease problems are associated with species for which there is negligible reference material of normal vs. abnormal parasites, pests and diseases. Since the efficiency of pinpointing causative agents in a disease outbreak is greatly enhanced when they can be distinguished from benign and secondary infections, new molluscan candidates for aquaculture need to have "wild" health profiles established, as well as the environmental parameters associated with pathology at the tissue level (eco-physiology). This will enhance our understanding of what triggers opportunistic ("stress") related disease (ecopathology) and is especially important in open water host-pathogen systems where direct disease control and eradication is next to impossible.

References
Alderman, D.J. (1980) Shellfish disease problems - past, present and future. Proc. 11th Ann. Conf.Shellfish Assoc. GB, pp. 30-40.

Altstatt, J.M., Ambrose, R.F., Engle, J.M., Haaker, P.l., Lafferty, K.D. and Raimondi, P.T. (1996) Recent declines of black abalone Haliotidis cracherodii on the mainland coast of central California. Mar. Ecol. Prog. Ser. 142, 185-192.

Briceji, V.M., Ford, S.E., Borrero, F.J., Perkins, F.O., Rivara, G., Hillman, R.E., Elston, R.A. and Chang, J. (1992) Unexplained mortalities of hatchery-reared juvenile oysters, Crassostrea virginica (Gmelin). Res. 11, 331-347.

Burreson, E.M. (1996) 101 uses for the small subunit ribosomal RNA gene: Application to Haplosporidium nelsoni. J. Shellfish Res. 15, 475.

Cadoret, J-P., Gendreau, S., Delecheneau, J-M., Rousseau, C. and Mialhe, E. (1997) of bivalve eggs: Application in genetics. Mol. Mar. Biol. Biotechnol. 6, 72-77.

Carnegie, R.B., Distel, D.L. and Barber, B.J. (1997) Amplification and sequencing of the Bonamia ostreae 18S rDNA gene: Phylogenetic considerations and applications. J. Shellfish Res. 16, 328.

Cochennec, N.; Hervio, D.; Panatier, B.; Boulo, V.; Mialhe, E.; Rogier, H.; Grizel, H. and Paolucci, F. (1992) A direct monoclonal antibody sandwich immunoassay for detection of Bonamia ostreae (Ascetospora) in hemolymph samples of the flat oyster Ostrea edulis (Mollusca: Bivalvia). Dis. Aquat. Org. 12, 129-134.

Comps, M. and Cochennec, N. (1993) A herpes-like virus from the European oyster Ostrea edulis L. J. Invert. Pathol. 62, 1-3.

Davis, C.V. and Barber, B.J. (1994) Size-dependent mortality in hatchery-reared populations of oysters, Crassostrea virginica Gmelin, 1791, affected by juvenile oyster disease. J. Shellfish Res. 13, 137-142.

Drinnan, R.E. and Henderson, E.B. (1963) 1962 Mortalities and a possible disease organism in Neguac quahaugs. Ann. Rept. No. B11 , Biological Sta., St. Andrews, NB.

Dungan, C.F. (1997) Perkinsus marinus: Immunoassay detection in oyster tissues and environmental samples and in vitro experimental systems. J. Shellfish Res. 16, 263.

Elston, R. (1997) Special topic review: bivalve mollusc viruses. World J. Microbiol. Biotech. 13, 393-403.

Farley, C.A., Banfield, W.G., Kasnic, G. Jr. and Foster, W.S. (1972) Oyster Herpes-type virus. Science 178, 759-760.

Farley, C.A.; Lewis, E.J.; Relyea, D.; Zahtila, J. and Rivara, G. (1997) Juvenile oyster disease progress report: Resistance studies, 1996. J. Shellfish Res. 16, 264.

Friedman, C.S., Thomson, M., Chun, C., Haaker, P.L. and Hedrick, R.P. (1997) Withering syndrome of the black abalone, Haliotis cracherodii (Leach): Water temperature, food availability and parasites as possible causes. J. Shellfish Res. 16, 403-411.

Gaffney, P.M., Allen, S.K. Jr. and Pierce, J. (1996) Development of nuclear DNA markers and pedigreed families for disease resistance and genetic mapping in the eastern oyster: Progress report. J. Shellfish Res. 16, 257.

Gee, A. and Elston, R.A. (1996) PCR detection of the bacterial pathogen in oyster nocardiosis. J. Shellfish Res. 16, 264.

Goggin, C.L., Boulo, V., Mialhe, E., Cousin, K., Hervio, D., DePauw, N. and Joyce, J. (1991) In: Aquaculture and the Environment. Spec. Publ. Euro. Aqua. Soc., 14. p. 123.

Haaker, P.L., Harris, S.L., Taniguchi, I.K. and Friedman, C.S. (1995) Withering syndrome in California: Does it occur in subtidal abalones? J. Shellfish Res. 14, 267.

Harper, D.M., Flessas, D.A. and Reinisch, C.L. (1994) Specific reactivity of leukemia cells to polyclonal anti-PCB antibodies. J. Invert. Pathol. 64, 234-237.

Hine, P.M. and Thorne, T. (1997) Replication of herpes-like viruses in haemocytes of adult flat oysters Ostrea angasi: An ultrastructural study. Dis. Aquat. Org. 29, 189-196.

Hine, P.M., Wesney, B. and Hay, B.E. (1992) Herpesviruses associated with mortalities among hatchery-reared larval Pacific oysters Crassostrea gigas. Dis. Aquat. Org. 12, 135-142.

ICES (1995) ICES Code of Practice on the Introductions and Transfers of Marine Organisms 1994. Int. Conc. Expl. Sea, Copenhagen, Denmark. 12pp.

Jones, J.B., Scotti, P.D., Dearing, S.C. and Wesney, B. (1996) Virus-like particles associated with marine mussel mortalities in New Zealand. Dis. Aquat. Org. 25, 143-149.

Kleinschuster, S.J., Smolowitz, R. and Parent, J. (1998) In vitro life-cycle and propagation of quahog parasite unknown. J. Shellfish Res. 17, 75-78.

Le Deuff, R.M., Renault, T. and Gérard, A. (1996) Effects of temperature on herpes-like viral detection among hatchery-reared larval Pacific oyster Crassostrea gigas. Dis. Aquatic Org. 24, 149-157.

Lee, M., Taylor, G.T., Bricelj, M., Ford, S.E. and Zahn, S. (1996) Evaluation of Vibrio spp. and microplankton blooms as causative agents of juvenile oyster disease in Crassostrea virginica (Gmelin). J. Shellfish Res. 15, 319-330.

Lewis, E.J., Farley, C.A., Small, E.B. and Baya, A.M. (1996) A synopsis of juvenile oyster disease (JOD) experimental studies in Crassostrea virginica. Aqua. Living Res. 9, 169-178.

Matsuyama, Y., Nagai, K., Mizuguchi, T., Fujiwara, M., Ishimura, M., Yamaguchi, M., Uchida, T and Honjo, T. (1995) Ecological features and mass mortality of pearl oysters during red tides of Heterocapsa sp. in Ago bay in 1992. Nipp. Suis. Gakk. 61: 35-41. (In Japanese)

Noel, D., Pipe, R., Elston, R., Bachere, E. and Mialhe, E. (1994) Antigenic characterisation of hemocyte subpopulations in the mussel Mytilus edulis by means of monoclonal antibodies. Marine Biology 119, 549-556.

OIE (1997) OIE International Aquatic Animal Health Code 2nd Ed. Office International des Epizooties, Paris, France, 192 pp.

Pass, D.A., Dybdahl, R. and Mannion, M.M. (1987) Investigations into the causes of mortality of the pearl oyster, Pinctada maxima (Jamson), in Western Australia. Aquaculture 65,149-169.

Ragone-Calvo, L.M., Walker, J.G. and Burreson, E.M. (1997) Occurrence of QPX, quahog parasite unknown, in Virginia hard clams, Mercenaria mercenaria. J. Shellfish Res. 16, 334.

Smolowitz, R., Leavitt, D. and Perkins, F. (1998) Observations of the protistan disease similar to QPX in Mecenaria mercenaria (hard clams) from the coast of Massachusetts. J. Invert. Pathol. 71, 9-25.

Stokes, N.A. and Burreson, E.M. (1995) A sensitive and specific DNA probe for the oyster pathogen Haplosporidium nelsoni. J. Euk. Micro. 42, 350-357.

Whyte, S.K., Cawthorn, R.J. and McGladdery, S.E. (1994) QPX (quahaug parasite X), a pathogen of northern quahaug Mercenaria mercenaria from the Gulf of St. Lawrence, Canada. Dis. Aquatic Org. 19, 129-136.

Yarnall, H.A., Stokes, N.A. and Burreson, E.M. (1997) Development of a PCR assay for the quantitation of Perkinsus marinus. J. Shellfish Res. 16, 342-343.

Parasitology: The Myxosporean-Actinosporean Connection

[TOP]

J. L. Bartholomew

Dept. of Microbiology Nash Hall 220, Oregon State University, Corvallis, Oregon, USA
bartholj@bcc.orst.edu

Our understanding of the relationship between the Myxosporea and Actinosporea as well as how these groups fit into the larger taxonomic scheme has changed dramatically in the last 15 years. The Myxosporea, first described by Mller in 1838, were considered to be primarily parasites of fish. Descriptions of new species and hosts proliferated as the pathogenic potential of these organisms was recognized, and 150 years later, the number of known myxosporean species is well over 1,300. The Actinosporea were first discovered at the turn of the century in aquatic oligochaetes and although numerous species have been described, the numbers known do not parallel those of the Myxosporea. A simple explanation for this is the relative economic importance of fish compared with aquatic oligochaetes, however, it is curious that descriptions of actinosporeans are not more numerous in zoological studies.

Pathogenic Potential
Myxosporeans and their fish hosts have a long evolutionary history and many host-parasite relationships have achieved a balance in which the parasite apparently causes little apparent damage. In many infections, it is difficult to detect a tissue response; coelozoic species are generally innocuous and species developing in tissues (histozoic) may be encapsulated by connective tissue. Little evidence of a humoral response has been demonstrated, and it has been suggested that Myxosporea may mimic host antigens, thus avoiding elicitation of an antibody response (McArthur and Sengupta 1982). However, given the large number of myxosporean species, there are exceptions to this pattern. Certain species directly damage their hosts by causing pathological changes, some decrease fitness by reducing fecundity, and others reduce the market value of the fish. The pathologies caused by myxosporeans are varied. Several marine myxosporeans, especially those of the genus Kudoa, are associated with enzymatic degradation of the musculature. When heavily infected fish are harvested and frozen, the flesh turns into a milky, gelatinous substance which renders the fish unmarketable. These infections affect a variety of ocean fishes worldwide. In Europe, Sphaerospora renicola is the cause of swimbladder inflammation in common carp. This condition was described as early as 1904, but because of the diversity of parasite stages in the blood, swimbladder and kidney, the etiology of the infection was only clarified in the last decade (Molnar and Kovacs-Gayer 1986). In the southeastern U.S., the catfish industry is severely affected by proliferative gill disease which is characterized by intense inflammation and swelling of the gills (MacMillan et al. 1989). Worldwide, salmonid culture is plagued by several myxosporeans causing very different pathologies. Myxobolus cerebralis, the agent of whirling disease, is considered one of the most important infectious diseases of freshwater fish. The parasite erodes the cartilage of young fish, resulting in skeletal deformities, neurological impairment, loss of equilibrium, and mortality in heavy infections. Proliferative kidney disease causes hypertrophy of the interstitial kidney tissue which results in impairment of kidney function, osmotic imbalance and erythropoiesis. The causative agent remains unclassified because mature spores fail to develop, possibly because of the strong inflammatory response (Saulnier and de Kinkelin 1996). A more geographically restricted myxosporean, Ceratomyxa shasta, has been responsible for severe losses among salmonid populations in the Pacific Northwest of the U.S. The parasite infiltrates the intestinal tissue, causing hyperplastic changes, hemorrhage, necrosis and ultimately death of the fish (Bartholomew et al. 1989).

Commonality and Diversity
Although Myxosporea and Actinosporea share certain characteristics which define the phylum Myxozoa, in various taxonomic schemes they have comprised different orders, classes and even subphyla. Common attributes include distinctive polar capsules with coiled extrusive filaments, spores of multicellular origin and one or more sporoplasm. However, original descriptions of the groups are quite distinct. Spores of the Myxosporea were characterized by a variable numbers of polar capsules and spore valves and spores of the Actinosporea were defined as having exactly 3 polar capsules and 3 spore valves, but with a variable number of sporoplasms. Within each group, the diversity of forms is truly impressive and can only speak to the highly adaptive nature of these parasites. Spores of both myxosporeans and actinosporeans range from small and compact to elongate with elaborate processes which appear to be specially suited for flotation. The adaptability of the Myxozoa is also reflected in the wide variety of hosts from which they have been described. The hosts of myxosporeans are typically considered to be cold-blooded vertebrates, mostly fishes, although they have also been reported from a variety of amphibia (McAllister and Trauth 1995; Upton et al. 1992) and from reptiles (Lom 1990). The most unusual host for a myxosporean, a freshwater bryozoan, was recently reported by Canning et al. (1996). Actinosporeans are considered parasites of annelids, particularly of freshwater oligochaetes. However, they have recently been reported from a freshwater polychaete (Bartholomew et al. 1997) and from a marine oligochaete (Hallett et al. 1998).

The Connection
Development of both groups of parasites have been described extensively within their respective hosts. In one conventional interpretation of the myxosporean life cycle, the spore hatches in the digestive tract of the fish, the polar filaments extrude and the sporoplasms are released. The sporoplasms undergo autogamy to produce the only uninucleate stage in the life cycle. This cell migrates to the final site of infection where it develops into a sporogonic plasmodium and differentiates to form a pluricellular spore. Because direct transmission was not demonstrable under controlled conditions, this interpretation was controversial. However, most researchers explained the inability to directly transmit the infection by a requirement for the spore to "mature" outside the host, probably in the sediment. This explanation was widely accepted until Markiw and Wolf (1983, Wolf and Markiw 1984) reported some astounding observations. In their investigations on the life cycle of M. cerebralis, they found that a tubificid oligochaete played an essential role as an alternate host. They also discovered that the infectious stage for the fish was an actinosporean spore. Thus, the life cycle consists of two distinct spores, the lenticular M. cerebralis spore which develops in the trout and is infectious for the oligochaete, and the anchor-shaped Triactinomyxon spore which develops in the worm and is infectious for the fish. Confirmation of these findings by El-Matbouli and Hoffman (1989) quelled the skepticism and in the ensuing decade this pattern of alternation between myxosporean and actinosporean developmental stages has been demonstrated for a number of freshwater myxosporeans, all requiring freshwater oligochaetes as alternate hosts (Kent et al. 1994). There have been a few variations on this pattern. Studies on the life cycle of C. shasta identified a freshwater polychaete, Manayunkia speciosa, as the invertebrate host (Bartholomew et al. 1997). Although the host is a freshwater species, it is probable that marine polychaetes serve as hosts for these parasites. There are also reports of direct transmission, most recently by Diamant (1997) who described fish-to-fish transmission of the marine myxosporean Myxidium leei. These observations serve to remind us that a variety of adaptations for transmission may exist in a group as diverse as the Myxozoa.

Molecular Phylogeny - Redefining Relationships
Confirmation of the two-host life cycle, and the discovery that the Myxosporea and Actinosporea are alternate life stages of a single organism have resulted in fundamental changes in the classification of these parasites. One of the first and still most debated questions was raised by Corliss (1985): What is the correct species name? For M. cerebralis, the generally accepted solution has been to maintain the myxosporean designation and refer to the actinosporean stage as the triactinomyxon actinospore. This solution, as proposed by Kent et al. (1994), makes the actinosporean genera invalid but retains the collective-group names to characterize different morphological types. The growing body of knowledge has been accompanied by a number of studies on molecular phylogeny (Schlegel et al. 1996; Siddall et al. 1995; Smothers et al. 1994) which have resulted in higher level taxonomic changes. The long-standing opinion that Myxozoa fit poorly in the protists has been validated by comparison of 18S ribosomal RNA gene-sequences. Based on these data, the Myxozoa are now placed with the Metazoa. Siddall et al. (1995) has proposed that the molecular data coupled with morphological evidence argue that the phylum Myxozoa be abandoned in favor of their classification as a clade of parasitic cnidarians. However, recent analysis of Hox genes by Anderson et al. (1998) argues for closer affinity with the Bilateria. Molecular techniques are also being applied to the problems of inter and intragenic relationships among the Myxozoa. In these comparisons, traditional morphological classifications have not been found to correspond with phylogenetic data. For example, from our own work, species of Ceratomyxa from both marine and freshwater environments appear more closely related to the Multivalvulida than to other genera of Bivalvulida.

Landmark Research - Looking Back Over the Past Five Years
Perhaps the most obvious advance in myxosporean research during the past five years has been the numerous successful efforts to define myxosporean life cycles. The connection between the Myxosporea and Actinosporea is now widely accepted and there has been renewed interest in myxosporean research. One of the most elusive questions is how the parasite penetrates and reaches its target tissue. Studies on the migration of the triactinomyxon-sporoplasm of M. cerebralis from the epidermis into the cartilage of the trout (El-Matbouli et al. 1995) have provided our first insights into understanding the pathogenesis of infection. The reconsideration of the systematics of the Myxozoa has not only changed the way we look at this group, but has fostered the use of new research tools. Molecular technologies developed for phylogenetic comparisons are being used to facilitate determinations of life cycles as well as in the development of sensitive diagnostic assays.

Looking Forward
As life cycles become established under controlled laboratory conditions, researchers can focus on the infectious process. Some of the questions are: What cues attachment and penetration of the parasite? And do routes of migration vary between parasite species and host target organ? Answers to these questions may suggest means for interrupting the life cycle and provide much needed methods for control. Closely related are questions on host resistance. What is the basis for resistance? What is the role of innate versus adaptive responses? Can resistance be linked to specific genes? And can resistance be enhanced or manipulated by stimulating specific responses? Defining relationships will continue to be important. Research on life cycles of marine species and of myxosporeans that infect taxa other than fish should help strengthen our understanding of the connections between the Myxosporea and Actinosporea. The relationship of the Myxozoa among the higher taxa is already being examined by analysis of additional genes, and work in this area will continue to shed light on the evolutionary history of this group and on the basis for the similarity between the myxozoan polar capsule and the cnidarian nematocyst. There is also need for research on the dynamics of host-parasite interactions, both in culture and in natural ecosystems. Some of the most successful efforts to control these parasites in aquaculture inadvertently broke the life cycle by reducing suitable habitat for the annelid host. Confronting problems related to myxosporean infections in wild fish populations will require a better understanding of how disturbances disrupt delicate host-parasite balances.

References
Anderson, C. L., E. U. Canning and B. Okamura. 1998. A triploblast origin for Myxozoa? Nature. 392:346.

Andree, K. B., S. J. Gresoviac and R. P. Hedrick. 1997. Small subunit ribosomal RNA sequences unite alternate actinosporean and myxosporean stages of Myxobolus cerebralis the causative agent of whirling disease in salmonid fish. J. Euk. Microbiol. 44:208-215.

Bartholomew, J. L., M. J. Whipple, D. G. Stevens and J. L. Fryer. 1997. Role of the freshwater polychaete, Manayunkia speciosa, in the life cycle of Ceratomyxa shasta, a myxosporean parasite of salmon and trout. Amer. J. Parasitol. 83:859-868.

Bartholomew, J. L., C. E. Smith, J. S. Rohovec and J. L. Fryer. 1989. Characterization of the host response to the myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy, and immunological techniques. Journal of Fish Diseases 12:509-522.

Canning, E. U., B. Okamura, and A. Curry. 1996. Development of a myxozoan parasite Tetracapsula bryozoides gen. n. et sp. n. in Cristatella mucedo (Bryozoa: Phylactolaemata). Folia Parasitol. 43:249-261.

Corliss, J. O. 1985. Consideration of taxonomic-nomenclatural problems posed by report of myxosporidians with a two-host life cycle. Protozool. 32:589-591.

Diamant, A. 1997. Fish-to-fish transmission of a marine myxosporean. Dis. Aquat. Org. 30:99-105.

El-Matbouli, M., and R. W. Hoffman. 1989. Experimental transmission of two Myxobolus spp. developing bisporogeny via tubificid worms. Parasitol. Res. 75:461-464.

El-Matbouli, M., R. W. Hoffman and C. Mandok. 1995. Light and electron microscopic observations on the route of the triactinomyxon-sporoplasm of Myxobolus cerebralis from epidermis into rainbow trout cartilage. J. Fish Biol. 46:919-935.

Hallett, S. L., P. O'Donoghue and R. J. G. Lester. 1998. Structure and development of a marine actinosporean, Sphaeractinomyxon ersei n.sp. (Myxozoa). J. Euk. Microbiol. 45:142-150.

Kent, M. L., L. Margolis and J. O. Corliss. 1994. The demise of a class of protists: taxonomic and nomenclatural revisions proposed for the protist phylum Myxozoa Grass, 1970. Can. J. Zool. 72:932-937.

Lom, J. 1987. Myxosporea: a new look at long-known parasites of fish. Parasitol Today. 3:327-332.

Lom, J. 1992. Phylum Myxozoa. In Handbook of Protoctista. L. Margolis, J. O. Corliss, M. Melkonian and D. J. Chapman, eds. Jones and Bartlett Pub. Boston, MA. pp 36-52.

MacMillan, J. R., C. Wilson and A. Thiygarajah. 1989. Experimental induction of proliferative gill disease in specific pathogen free channel catfish. J. Aquat. Anim. Health. 1:245-254.

Markiw, M. E. and K. Wolf. 1983. Myxosoma cerebralis (Myxozoa:Myxosporea) etiologic agent of salmonid whirling disease requires tubificid worm (Annelida:Oligochaetes) in its life cycle. J. Protozool., 30:561-564.

McAllister, C. T., and S. E. Trauth. 1995. New host records for Myxidium serotinum (Protozoa:Myxosporea) from North American amphibians. J. Parasitol. 81:485-488.

McArthur, C. P. and S. Sengupta. 1982. Antigenic mimicry of eel tissues by a myxosporidian parasite. Z. Parasitenkd. 66:249-255.

Molnar, K. and E. Kovacs-Gayer. 1986. Experimental induction of Sphaerospora renicola (Myxosporea) infection in common carp (Cyprinus carpio) by transmission of SB-protozoans. J. Appl. Ichthyol. 2:86-94.

Saulnier, D. and P de Kinkelin. 1996. Antigenic and biochemical study of PKX, the myxosporean causative agent of proliferative kidney disease of salmonid fish. Dis. Aquat. Org. 27:103-114.

Schlegel, M., J. Lom, A. Stechmann, D. Bernhard, D. Leipe, I. Dykov and M. L. Sogin. 1996. Phylogenetic analysis of complete small subunit ribosomal RNA coding region of Myxidium lieberkuehni: evidence that Myxozoa are Metazoa and related to the Bilateria. Arch. Protistenkd. 147:1-9.

Siddall, M. E., D. S. Martin, D. Bridge, S. S. Desser and D. K. Cone. 1995. The demise of a phylum of protists: phylogeny of Myxozoa and other parasitic Cnidaria. J. Parasitol. 81:961-967.

Smothers, J. F., C. D. von Dohlen, L. H. Smith and R. D. Spall. 1994. Molecular evidence that the myxozoan protists are metazoans. Science. 265:1719-1721.

Upton, S. J., P. S. Freed, D. A. Freed, C. T. McAllister and S. R. Goldberg. 1992. Testicular myxosporidiasis in the flat-backed toad, Bufo maculatus (Amphibia:Bufonidae), from Cameroon, Africa. J. Wildl. Dis. 28:326-329.

Wolf, K., and M. E. Markiw. 1984. Biology contravenes taxonomy in the Myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science 225:1449-1452.

Emerging Diseases of Fish

[TOP]

RP Hedrick

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616 U.S.A. rphedrick@ucdavis.edu

Worldwide Perspectives
New and recurring diseases are a major concern for fish health worldwide. Diseases are most often detected among captive populations of fish principally those raised in aquaculture facilities. The large number of fish allows observation of diseases of both infectious and non-infectious causes, even when their prevalence may be low. Diseases among wild fish populations may be more difficult to detect unless they are under scrutiny for research or conservation purposes or if the prevalence of disease is sufficient to cause observable mortality. I have attempted to mention certain diseases (emerging) which I believe we will begin to see more and more frequently as greater causes of concern in wild and cultured fish populations. In doing so, I realize that many important diseases that either did not fall into my definition of emerging, or were overlooked, may have been omitted. I will admit also to being partial to those diseases of a microbial etiology.

Emerging Diseases

Diseases of Marine Fish
Diseases due to bacterial and viral agents are being encountered more frequently and in greater numbers of species of marine fish. Members of the gram-positive coccal organisms, Lactococcus and Mycobacterium spp. (Ghittino et al. 1998) are increasing reported as major causes of mortality among fish in net cages and land-based marine aquaculture. The increasing frequency of these gram-positive organisms with traditionally stubborn gram-negative species like Pasteurella and Pseudomonas emerging infections with Piscirickettsia agents now create major impediments in certain growing regions (Fryer and Maule 1997).

Two groups of viral agents, the nodaviruses and the iridoviruses are arising with increasing frequency and among greater numbers of species of marine fish. There is currently no treatment for these viral agents and surveillance and avoidance are the principal tools used to avoid their impacts.

Nodaviruses are the cause of viral nervous syndromes, characterized as the name implies by infection of nervous tissues resulting in behavioral signs of fish prior to death (Castric 1998; Muroga 1997). Species infected include: striped jack, sea bass, barramundi, groupers, parrot fish, porgies, Atlantic halibut, turbot, and Atlantic salmon. Diseases such as cardiac myopathy syndrome (CMS) previously thought to be of nutritional, environmental or non-infectious nature may now be traced to these viruses (Grotmol et al. 1997).
Iridoviruses, characterized by the red sea bream iridovirus (RSIV) are emerging as a major cause of early mortality among several marine fish species including: sea bream, sea bass, yellow tail, amber and striped jack, parrot fish, grouper, flounder, turbot, and puffer (Nakajima 1997; Bloch and Larsen 1993). Some hope for the control of these systemic iridoviral agents comes from recent demonstrations of partial immunity among vaccinated Red Sea bream (Nakajima et al. 1998). The orthomyxovirus-like agent causing infectious salmon anemia or ISA continues to be a major cause of losses in Atlantic salmon culture in Norway. A newly recognized hemorrhagic kidney syndrome among Atlantic salmon in Eastern Canada and the USA (Byrne et al. 1998) may be caused by a variant of the ISA virus.

Non-infectious Diseases
Production of related non-infectious diseases continue to be problematic to most major fish species. This is perhaps best illustrated by the increase in dermatonecrosis, papillomatosis, spinal deformities, cardiopathies, hepatic abnormalities and pancreatic and intestinal anomalies (Vagsholm and Djupvik 1998). These conditions typify the emerging problems that will become of greater significance as microbial diseases are more effectively controlled. The causes of large losses of wild pilchards (Sardinops sagax neopilchardus) in Australia and New Zealand coastal waters remains unknown although a herpesvirus has been found in some affected fish (Hyatt et al. 1997).

Disease of Fresh Water
Bacterial cold water diseases caused by Flavobacterium psychrophilum continue to increase in importance in the major salmonid rearing regions of the North America and most recently in Chile. Potential vertical transmission and poor response for treatments results in large losses in young fish and deformities in older animals. In warm water fish species, particularly in fancy carp, Aeromonas spp. as causes of systematic and ulcerative conditions is on the rise. Unregulated transport of fish between Asia and North America has introduced strains of the bacterium with high virulence and multiple drug resistance. Additionally, viral agents, most recently a pox-like virus known in Japan for over 24 years (Ono et al. 1986) have been introduced to North America with fancy carp or koi (Hedrick et al. 1998). Systemic iridoviruses as the cause of diseases similar to those of epizootic hematopoietic necrosis virus (EHNV) are now appearing with increasing frequency among both food and ornamental fish (Whittington and Hyatt 1997; Hedrick et al. 1995). These viruses as demonstrated by Ariel and Owen (1997) may be moving between amphibians and fish, further compounding attempts to control their spread.

Practical and Theoretical Aspects
The emerging diseases all have several features in common. They represent diseases for which rapid management procedures are ineffective in their prevention or control. This may be a result of a poor identification of the variables that are influencing the severity of the disease or the resistance of a pathogen or its biological properties that make its management difficult. In this respect, the emerging microbial pathogens share certain characteristics including: poor or no response to therapy, existence in asymptomatic carrier states, ability to establish themselves in wild fish populations, they result in significant losses (economic and ecologic) and they can be transmitted via horizontal means and by vertical modes despite topical egg disinfection. Controlling these agents therefore often relies on better regulatory approaches and an improved understanding of their biology. Understanding the biology of agents allows much more prudent decisions on where to exercise control, short of completely preventing the movements of the host fish species. Research directed at understanding where the principal risks for transmission or movement of these agents occurs is therefore a critical step in the process.

Important Developments ­ Past
Perhaps the most important development in our hope to contain or control emerging disease problems is the movement towards international standards for fish health inspections/certification. The Office of International Epizootics or OIE is gaining greater stature as the international standard for the conduct of fish health certification/inspection. Through the Code and the Manual, standardized lists of pathogens and methods for the detection are now available. Participation in these international programs and at a regional level when such organizations exist are keys to improving the climate associated with an increasing international trade in aquatic animals.

Important Development/Most Interesting Aspects for the Future
Diseases, both new and known, will continue to be part of lives of both captive and wild fish populations. Minimizing their effects through better management will be a consequence of the developing science of fish health. Encouraging the participation of member countries in existing international forums, particularly those benefiting from the trade in aquatic species must occur. Additionally, agencies both national and international will have to commit more resources to accommodate an ever-growing list of aquatic species in international trade. The movement of pathogens between continents are occurring in species which current codes and manuals do not address. A thorough evaluation of the principal species moving in international trade should be followed by the development of procedures and guidelines for their inspection and certification. Once established, we must coordinate the surveillance and certification programs at regional, national and international levels. Lastly, our ability to create fair and effective control programs (that will not be contested between member countries) will rely on a vastly increased database on the biology, ecology and distribution of these agents.

References
Bloch, B., and Larsen, J.L. 1993. An iridovirus-like agent associated with systemic infection in cultured turbot Scophthalmus maximus fry in Denmark. Dis. Aquat. Org. 15:235-240.

Byrne, P.J., MacPhee, D.D., Ostland, V.E., Johnson, G., and Ferguson, H.W. 1998. Haemorrhagic kidney syndrome of Atlantic salmon Salmo salar L. J. Fish Dis. 21:81-91.

Castic, J. 1998. Viral diseases in fish mariculture. Bull. Eur. Assoc. Fish Pathol. 17(6):220-228.

Fryer, J.L., and Maule, M.J. 1997. The rickettsia: an emerging group of fish pathogens. Emerging Infectious Diseases 3(2):137-144.

Ghittino, C., Eldar, A., and Hedrick, R. 1998. Recent knowledge on the taxononmy of Gram-positve cocci pathogenic for fish. Third International Symposium of Aquatic Animal Health, August 30 ­ September 3, 1998, Baltimore, Maryland (see abstract).

Hedrick, R.P., and McDowell, T.S. 1995. Properties of iridoviruses form ornamental fish. Veterinary Research 26:423-427.

Hyatt, A.D., Hine, P.M., Jones, J.B, Whittington, R.J., Kearns, C., Wise, T.G., Crane, M.S.,and Williams, L.M. 1997. Epizootic mortality in the pilchard (Sardinops sagax neopilchardus) in Australia and New Zealand in 1995. II. Identification of a herpesvirus within gill epithelium. Dis. Aquat. Org. 28I 17-29.

Grotmol, S., Totland, G.k., and Kryvi, H. 1997 Detection of a nodavirus-like agent in heart tissue from reared Atlantic salmon Salmo salar suffering from cardiac myopathy syndrome (CMS). Dis. Aquat. Org. 29:79-84.

Muroga, K. 1997. Viral diseases of vultured marine fish in Japan. Proceedings NRIA International Workshop on "New Approaches to Viral Diseases of Aquatic Animals", January 21-24, 1997, Kyoto, Japan, pages 12-121.

Nakajima, K. 1997. Red sea bream iridovirus in Japan: Antigen analusis and comparison with other fish iridoviruses. Proceedings NRIA International Workshop on "New Approaches to Viral Diseases of Aquatic Animals", January 21-24, 1997, Kyoto, Japan, pages 108-118.

Nakajima, K., Maeno, Y., Kurita, J., and Inui, Y. 1997. Vaccination against red sea bream iridoviral disease in red sea bream. Fish Pathology 32(4):205-209.

Ono, S., Nagai, A., and Sugai, N. 1986. A histopathological study on juvenile colorcarp, Cyprinus carpio, showing edema. Fish Pathology 21:167-175.

Vagsholm, I., and Djupvik, H.O. 1998. Risk factors for spinal deformities in Atlantic salmon Salmo salar L. J. Fish Dis. 21:47-54.

Whittington, R.J. and Hyatt, A.D. 1997. Diagnosis and prevention of epizootic hematopoietic necrosis virus (EHNV) infection. Proceedings NRIA International Workshop on "New Approaches to Viral Diseases of Aquatic Animals", January 21-24, 1997, Kyoto, Japan, pages 80-91.

Update on Shellfish Pathogen Listed in the OIE Aquatic Animal Health Code

[TOP]

H. Grizel (1)* and F. Berthe (2)

1.	IFREMER, B.P.171,1 rue Jean Vilar, 34203 Sète France hgrizel@ifremer.fr
2.	IFREMER, B P 133, 17390 La Tremblade France fberthe@ifremer.fr

The International Aquatic Animal Health Code prepared by the "Office International des Epizooties" (O.I.E.) has been realized after the agreement on the application of Sanitary and Phytosanitary Measures (S.P.S. agreement) which is under the General Agreement on Tariffs and Trade (G.A.T.T.). The aim of the S.P.S. agreement is to minimize the negative effects of health restriction on international trade. Until 1994, several diseases, mainly of vertebrates, has been listed as a notifiable disease to the O.I.E. The choice of diseases to establish the list of notifiable diseases of bivalves has been made following the recommendations of a special O.I.E. working group which prepared a decisional diagram (O.I.E. report, 1994). The main criteria are: (I) geographical distribution of the diseases (one or several areas), (II) losses due to the diseases (economical impact), (III) infectiosity of the pathogen and level of spread. The possibility to eradicate the disease is also one of the parameters for vertebrates, but in our case field experiences showed the difficulties to do that (Grizel, 1985; Van Banning, 1982; 1987).
Following these basic recommendations, the O.I.E. Fish Disease Commission has selected in a first time diseases of bivalves which: (I) are in one or more areas, or in one or more countries, (II) induce important mortalities (up than 40% in the affected areas and during several years), (III) spread quickly from the initial centre of infection, (IV) represent, with the lack of scientific data (mainly epidemiological data), a real risk for the most important reared species (e.g. the world-wide species, Crassostrea gigas). Between the two hundred pathogenic agents of bivalves reported in the literature, few of them correspond to the proposed criteria. In most of the cases, agents have been described during routine examinations or during a short period of mortalities. More over, except for the first description of the pathogenic agent, very little information is available, for long period impacts of a disease: importance, epidemiological situation, potential hosts and etc. So, a first list of diseases has been submitted to the member countries for discussion.
The main remarks on this first proposal concerned: (a) the number of listed diseases, some countries, such as Canada wish to add more diseases. The result of the examination of the comments, through the O.I.E. screen, was the non-eligibility of the proposed diseases; (b) the choice of the diseases retained. After exchanges with several pathologists of bivalves, the O.I.E. Fish Disease Commission eliminated the Iridovirus (O.V.V.D. disease and Iridovirosis of C. angulata). The main reason was the actual absence of epidemic situation, in the world, related with these viruses; (c) the question of the specificity of the pathogen agent. Considering the G.A.T.T. recommendations it is not possible to assume, without evidence, that in an infected area all the bivalves are potential carriers. For this reason, the list of notifiable diseases specified clearly the name, genus and species of the pathogens and of the susceptible hosts for each pathogen; (d) the procedure to establish the status of a country, a zone or an establishment was adapted for each disease. The general principle is coming from the valuable experience of the National laboratories in Europe, applied for the European zoning. For bivalves an official mollusc health surveillance scheme is required for two years prior to establishing their status; (e) the certification for live molluscs is based on the results coming from the official health surveillance scheme. The certificate includes all the pathogens listed as notifiable diseases (Table1).

Table 1.- Diseases Notifiables to the O.I.E..

Diseases	Pathogens
Bonamiosis	Bonamia ostreae and B. sp. (Australie, Nouvelle-Zélande)
Haplosporidiosis	Haplosporidium nelsoni and H. costale
Marteiliosis	Marteilia refringens and M. sydneyi
Mikrocytosis	Mikrocytos mackini and M. rougheyi
Perkinsiosis	Perkinsus marinus and P. olseni

Others serious diseases can also be mentioned on the certificate. Finally a bilateral agreement is necessary between the two countries which want to trade in live molluscs. For trade, all different shipments must be accompanied by an international certificate. In the case where the shipment is composed of batches from different zones or establishments, each batch needs a certification; (f) the general information on diagnostic techniques, sampling rules and examination of stocks where abnormal mortalities occurred, based on the valuable experience of National reference laboratories.

For bivalves, in spite of the recent development of new diagnostic tools, such as DNA probes, monoclonal or polyclonal antibodies, smears or histological technique remain the best tools to diagnose at protozoon diseases or causes of abnormal mortalities. The major consequences of these recommendations for culture and trade is the need to obtain historical data on epidemiology of bivalves. To accomplish this, a national mollusc health surveillance scheme, for a period of at least two years, is necessary to establish the status of country, zone or establishment. However, the absence of this information allow importing countries to refuse all introductions of live animals on its own territory.

For the future the research should be focused more on epidemiological data (health surveillance scheme, experimental and cross infections, the relationship between the environment, rearing techniques and diseases) and on developing the diagnostic tools for protozoons and viruses (Grizel, 1997). More over, it will be necessary to validate new diagnostic methods using the appropriate experimental plan and tests. Currently, such work is occurring in Europe between national reference laboratories.

Literature cited
Grizel, H. 1985. Etude des récentes épizooties de l'huître plate Ostrea edulis Linné et de leur impact sur l'ostréiculture bretonne. Thèse Do ctorat d'Etat en Sciences Naturelles, Université de Montpellier, 145 pp.

Grizel, H. 1997. Les maladies des mollusques bivalves: risques et prévention. Rev. sci. tech. Off. Int. Epiz., 16 (1), 161-171.

O.I.E., 1994. Rapport sur la classification des maladies. Paris, 15-17 septembre,9 pp.

Van Banning, P. 1982. Some aspects of the occurrence, importance and control of the oyster pathogen Bonamia ostreae in the Dutch oyster culture. In Proc. International Colloquium on Invertebrate Pathology, 6-10 septembre, Brighton (Royaume-Uni), 261-263.

Van Banning, P. 1987. Further results of the Bonamia ostreae challenge tests in Dutch oyster culture. Aquaculture, 67, 191-194.

Update on Fish Pathogens Listed in the OIE International Aquatic Animal Health Code

[TOP]

BJ Hill

The Centre for Environment, Fisheries & Aquaculture Science, Weymouth Laboratory, The Nothe, Weymouth, Dorset DT4 8UB, UK

The Office International des Epizooties (OIE) is the world organization for animal health. One of its principle aims is to facilitate international trade in animals and animal products (including aquatic species) whilst reducing the risk of transfer of serious diseases from one country to another, by international standardization of health controls and preventative measures. The general principles and standards for minimum health guarantees for trade in fish between countries are laid down in the OIE International Aquatic Animal Health Code. These guarantees must be based on inspection by the competent authorities, epidemiological surveillance and standard methods for laboratory examinations and disease diagnosis as described in the OIE Diagnostic Manual for Aquatic Animal Diseases. Since January 1, 1995, the Sanitary and Phytosanitary (SPS) Agreement of the World Trade Organization (WTO) has required Member Countries not to introduce or maintain animal health measures which give a higher level of protection than that recommended by international standards, unless it can be scientifically justified and based on risk assessment analysis. The OIE Code and Manual are the only international standards and recommendations currently recognized by the WTO for this purpose (Chillaud, 1996).

The Code and Manual have been prepared by the Fish Diseases Commission (FDC), a specialist Commission of the OIE, with input from the OIE International Animal Health Code Commission and the Standards Commission and taking account of the opinions of leading experts in the field of fish disease in different OIE Member Countries. The first editions were published in 1995 and the second, amended versions were published in 1997 (OIE, 1997 a, b). Because of the rapid changes in aquaculture world-wide and the epizootiological situation of serious diseases, together with advances in diagnostic methods and increasing scientific knowledge, the Code and Manual will require frequent updating. OIE Member Countries are encouraged to send to the OIE via their national Delegates suggestions for amendments, including the rationale for such changes. These suggestions are considered by the FDC, along with any amendments its own members propose, at one or both of its bi-annual meetings and if accepted, are proposed to the OIE International Committee at the OIE General Session in May each year. If agreed by the International Committee, the amendments are included in the next edition of the Code and Manual. It is envisaged that updated versions of the Code and Manual will be printed approximately every 4 years.

The contents of the Code and Manual are based on the same principles and definitions applied to terrestrial animals, but adapted to be more appropriate for aquatic species and include detailed information on definitions, ethics of certification, import risk analysis and import/export procedures. For international trade in aquatic animals, health certification under the Code is only generally required for those diseases classified as notifiable to the OIE, but other significant diseases are identified for which health guarantees on imports of aquatic animals may be justified for some countries. Thus, at present, the Code lists two separate categories of disease: "notifiable diseases" and "other significant diseases". "Notifiable diseases" are defined by OIE as those that are considered to be of socio-economic and/or public health importance within countries and that are significant in the international trade of aquatic animals and aquatic animal products. The notifiable aquatic animal diseases are generally regarded as having potential for serious damage to national aquaculture industries or wild populations of fish, molluscs and crustaceans. For these diseases, reports on occurrence in each OIE Member Country are normally submitted to OIE once a year, but more frequent reporting may be necessary, particularly in the event of the first occurrence of a disease in a particular country or zone of a country previously considered to be free of from that disease. "Other significant diseases" are defined as those that are of current or potential international significance in aquaculture but that have not been included in the list of notifiable diseases because they are less important or because their geographic distribution is either limited or is too wide for notification to be meaningful, or is not yet sufficiently defined, or the aetiology of the disease is not well enough understood or approved diagnostic methods are not available.

The fish diseases currently included in these categories are:

Notifiable diseases:

Epizootic haematopoietic necrosis Infectious haematopoietic necrosis Oncorhynchus masou virus disease Spring viraemia of carp Viral haemorrhagic septicaemia

Other significant diseases:

Channel catfish virus disease Viral encephalopathy and retinopathy Infectious pancreatic necrosis Infectious salmon anaemia Epizootic ulcerative syndrome Bacterial kidney disease (Renibacterium salmoninarum) Enteric septicaemia of catfish (Edwardsiellosis) Piscirickettsiosis (Piscirickettsia salmonis) Gyrodactylosis (Gyrodactylus salaris)

Whilst the Code deals mostly with the principles and procedures of issuing health certificates and conducting veterinary checks on animals, the Diagnostic Manual covers the scientific aspects of the sampling and testing required for health certification. The purpose of the Diagnostic Manual is to provide a uniform approach to the diagnosis of the OIE-listed diseases so that the requirements for health certification in connection with trade in aquatic animals and their products, can be met. Although many scientific publications exist describing the diagnosis and control of aquatic animal diseases, the Diagnostic Manual is intended to be a key document describing the methods that can be applied in aquatic animal health laboratories all over the world, thus increasing efficiency and promoting improvements in aquatic animal health world-wide. The methods described in the Manual are all directly diagnostic methods. Due to insufficient development of serological methodology, the detection of fish antibodies to pathogens has not so far been accepted as a routine screening method for assessing the health status of fish populations. However, the validation of some serological techniques for diagnosis of certain infections could arise in the near future, making the use of serology more widely accepted for diagnostic and health screening purposes.

At present, the methods in the Manual are based either on isolation of the pathogen followed by its specific identification, the demonstration of pathogen-specific antigens using an immunological detection method, or demonstration of the presence of the genome of the pathogen through use of PCR or DNA probes. In recent years, scientific advances in the application of PCR technology is not only allowing the identification of pathogens after their isolation, but also the direct detection of low numbers of the pathogens in fish tissues. Furthermore, such techniques are allowing differentiation between strains of specific viruses arising from different geographical origins. Although more generally being used for detection and identification of viruses, PCR methods have also been developed for detection and differentiation of parasites such as Gyrodactylus salaris and bacteria such as Renibacterium salmoninarum. However, as good as PCR methods can be for such purposes, a "PCR-positive" test on a sample of fish in the isolation of the pathogen or other methods used to demonstrate its presence, is not yet globally accepted as evidence of infection. Similarly, the PCR method has not yet become a fully accepted alternative to the pathogen isolation methods described in the Diagnostic Manual for use in screening fish populations for health certification purposes. It is likely, though, that in time it will.

By its very nature, the Manual is much more likely to need frequent updating than is the Code. The Code mostly deals with the principles and procedures that are widely accepted and are not likely to change much over time. However, the steady advances in scientific knowledge of the diseases and the development of more sensitive/specific tests for the pathogens, new additions of the Manual can be seen as "out of date" in some aspects within a relatively short period of time.

The increasing activity in research into fish diseases and the development of expertise in countries where previously there was little activity in the fish disease area, will inevitably lead to a gradual increase in the known host and/or geographical ranges of the OIE-listed fish diseases. One or more of the listed diseases can be expected to discovered for the first time in some countries and fish species not previously known to be susceptible to a particular pathogen will be found, afterall, to be so. Furthermore, the international trade in live fish (and their products) will continue to provide a possible route for transfer of the listed diseases to countries (or regions of the world) and host species previously not affected, particularly where robust health certification standards are not applied. Conventional wisdom on some diseases will inevitably have to change, sometimes profoundly so.

Just some examples in recent years include:

• detection of viral haemorrhagic septicaemia (VHS) infection for the first time outside Europe (west coast of North America) (Meyers and Winton, 1995), for the first time in UK and Ireland (in turbot farms on coastal islands) (Ross, 1994; McArdle, 1997), and in Norway and Sweden after many years of being believed to be absent (Håstein, 1998);
  
• the discovery of VHS virus in a wide variety of wild marine fish populations along the Pacific coast of North America (Meyers and Winton, 1995) and in the North Sea (Smail, 1995), English Channel (Dixon et al, 1997) and Baltic Sea (Olesen, 1997), indicating a natural reservoir of the virus in the marine environment;
  
• the occurrence of infectious salmon anaemia (ISA) for the first time outside Norway, both in Europe (Scotland) (Rodger et al 1998) and North America (Atlantic coast of Canada) (Mullins et al,1998);
  
• the detection (in the UK) of spring viraemia of carp (SVC) virus in koi carp and goldfish imported from China, suggesting the first existence of SVC disease outside Europe (author's laboratory - unpublished results);
  
• the further spread of epizootic ulcerative syndrome (EUS) through India and Nepal, and recently to Pakistan, affecting a wide range of freshwater fish species (Das and Das, 1993; Lilley et al, 1997);
  
• the detection of bacterial kidney disease in Denmark for the first time (Lorenzen et al, 1997).

These and other changes in the epizootiological situation for the OIE-listed diseases are considered by the OIE Fish Diseases Commission each year and a synopsis of collated new developments for the listed diseases and any new diseases is published in the Commission's annual report. However, there is not global awareness of the FDC's annual report, nor do all fish disease specialists have ready access to it following its presentation to and acceptance by the OIE International Committee at the General Session each year.

Reliable, up-to-date information on the occurrence of fish diseases in other countries is needed by the veterinary services (or other competent authority) of a country in order to make assessments of the risk of introducing serious exotic diseases into its territory through importation of live fish (or their products). Official data on the occurrence of the OIE-listed diseases of fish in OIE Member Countries is published in the annual World Animal Health report from OIE, but is dispersed throughout in summary form or in statistical tables. It can be time-consuming to determine which countries are affected by a particular fish disease and no information is given on host species affected, or on the epidemiological situation regarding the "other serious diseases" listed by OIE. Such information is available in the Diagnostic Manual or is otherwise scattered throughout the scientific literature from which it can be a major effort to retrieve. It was therefore decided at the author's laboratory (formerly the MAFF Fish Diseases Laboratory) that a computer database on the prevalence of all the OIE listed diseases by country and host species was required to provide a simpler and more rapid means of information retrieval. OIE published data on the geographical and host ranges (natural and experimental) of all 10 notifiable diseases and 16 "other significant" diseases (of fish, molluscs and crustacea) published in the World Animal Health reports for all OIE Member Countries for 1994, 1995 and 1996 have been entered, together with additional information taken from the 1997 edition of the OIE Diagnostic Manual for Aquatic Animal Diseases. Further information on the geographical and host ranges of the diseases published in the scientific literature has been added as "non-OIE" data to ensure that the epidemiological picture is as complete as possible. It is intended to enter subsequent data from future OIE World Animal Health reports as soon as they become available each year and, if possible, the existing archive data for earlier years will also be entered. An automatic computer-based search of the current scientific literature is being carried out at the author's laboratory and all relevant new information is entered on a monthly basis into the "non-OIE data" section, together with details of the reference and an abstract.

The main switchboard for the database contains a number of interactive buttons for either accessing sub-switchboards or for rapid data retrieval. Essentially, the information is grouped into OIE data, Non-OIE data and All data. Each specific data area is accessed by a simple button press by clicking on the mouse. The database is divided into tables designed to cover each disease, host species, disease location and information source (reference). Within these general areas, more specific items include information on the natural or experimental occurrence of the disease and the taxonomic position of the host species. For each disease, lists of individual and host species affected are provided and for each country or species the source of the data (and in most cases an abstract of the publication) for the occurrence of a particular disease in a particular country, and further details of the host species can be called up instantly by a click on the mouse button. Alternatively, a report of all the OIE listed diseases known to occur in a particular country, or a particular host species, can be displayed. The database will be invaluable to those wishing to know the official OIE published data on the occurrence of listed fish diseases in its 150 Member Countries and/or the additional data in the published scientific literature.

The UK's Ministry of Agriculture, Fisheries and Food has offered to make the information available internationally through the official channels of OIE and at the OIE General Session in May 1997, the International Committee confirmed the designation of the CEFAS Weymouth Laboratory as the OIE Collaborating Centre for Information on Aquatic Animal Diseases with a mandate to provide Member Countries with information from the database. At its General Session in May 1998, the International Committee agreed to the proposal that the database be made available initially to Member Countries only via the reserved websites restricted to the national Delegates so that the information presented for individual countries can be checked and any inaccuracies notified to the Collaborating Centre. Since the information in the database would be valuable to a much wider audience in Member Countries than just the OIE Delegates, the Collaborating Centre has suggested that in future it should be included in the OIE website on the internet for public use. However, the database contains much epidemiological information additional to that officially report to the OIE, so placing the database on the Delegates' reserved websites gives the opportunity for it to be checked by the official services of each country who can then notify the Centre of any errors or omissions. All being well, the Fish Diseases Commission will propose to the International Committee in May 1999 that the database is included in the OIE website for public use.

REFERENCES

Chillaud, T. (1996). The World Trade Organisation agreement on the application of sanitary and phytosanitary measures. In: Preventing the spread of aquatic animal diseases, Rev. sci. tech. Off. int. Epiz., 15(2), 733-741.

Das, M.K. and Das, R.K., (1993). A review of the fish disease epizootic ulcerative syndrome in India. Environ. Ecol., 11(1), 134-145.

Dixon, P.F., Feist, S., Kehoe, E., Parry, L., Stone, D.M. and Way, K., (1997). Isolation of viral haemorrhagic septicaemia virus from Atlantic herring Clupea harengus from the English Channel. Dis. Aquat. Org., 30 (2), 81-89.

Håstein, T. (1998). Personal communication.

Lilley, J.H., Hart, D., Richards, R.H., Roberts, R.J., Cerenius, L. and Soderhall, K., (1997) Pan-Asian spread of single fungal clone results in large scale fish kills. Vet. Rec. 140, 653-654.

Lorenzen, E., Olesen, N.J., Korsholm, H., Hever, O.E. and Evensen, A., (1997). First demonstration of Renibacterium salmoninarum/BKD in Denmark. Bull. Eur. Ass. Fish Pathol., 17(3/4), 140-144.

McArdle, J. (1997). Personal communication.

Meyers, T.R. and Winton, J.R., (1995). Viral haemorrhagic septicaemia virus in North America. Ann. Review Fish Dis., 5, 3-24.

Mullins, J.E., Groman, D. and Wadowska, D., (1998). Infectious salmon anaemia in saltwater Atlantic salmon (Salmo salar L.) in New Brunswick, Canada. Bull. Eur. Ass. Fish Pathol., 18(4), 110-114.

Office International des Epizooties (OIE), (1997). Diagnostic manual for aquatic animal diseases. OIE, Paris 267 pp.

Office International des Epizooties (OIE), (1997). International aquatic animal health code. OIE, Paris 203 pp.

Olesen, N. (1997). Personal communication.

Rodger, H.D., Turnbull, T., Muir, F., Millar, S. and Richards, R.H., (1998). Infectious salmon anaemia (ISA) in the United Kingdom. Bull, Eur. Ass. Fish Pathol., 18(4), 115-116.

Ross, K., McCarthy, U., Huntly, P.J., Wood, B.P., Stuart, D., Rough, E.I., Smail, D.A. and Bruno, D.W., (1994). An outbreak of viral haemorrhagic septicaemia (VHS) in turbot (Scophthalmus maximus) in Scotland. Bull. Eur. Ass. Fish Pathol., 14(6), 213-214.

Smail, D.A., (1995). Isolation and identification of viral haemorrhagic septicaemia (VHS) virus from North Sea cod (Gadus morhua L.). Int. Counc. Explor. Sea (ICES). CM 1995/F: 15 (mimeo).

Toxicology and Carcinogenesis

[TOP]

David E. Hinton

Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine & UC Systemwide Lead Campus Program in Ecotoxicology, University of California-Davis, Davis California 95616 USA

INTRODUCTION
The liver is the most common organ site of experimentally induced tumors in fish and this organ is also the site of tumors linked to pollutant exposure in wild species. In 1964, following detection of liver neoplasms in white sucker (Catostomus commersonii) and brown bullhead (Ictalurus nebulosus) from a reservoir in Deep Creek, Maryland, USA, a contaminant etiology was proposed (1). Since then hepatic neoplasms have been found with high or repeated prevalences in populations of additional species from North America, as reviewed (2), from the North Sea and central European rivers (3,4). Comparisons of tumor prevalences in age-matched groups form multiple sampling sites and comprehensive analyses of sediment and fish tissue have clearly established this link for two species, English sole (Pleuronectus vetulus) (5) and brown bullhead (6). Controlled laboratory administration of contaminated sediment or sediment extracts from various sources produced liver tumors in rainbow trout (Oncorhynchus mykiss) and medaka (Oryzias latipes) (7-9). In a conference of this nature, it is appropriate to consider various aspects of hepatic carcinogenesis in fishes. Except for rare direct-acting chemical carcinogens, most potentially carcinogenic substances must be metabolized to ultimate carcinogenic forms. The ultimate carcinogenic form of the genotoxic carcinogen binds to target cellular macromolecules (DNA) and an adduct is formed. Once DNA adducts are formed, they may be excised and repaired correctly; or, the resultant toxicity may cause death of the affected cell. In these instances the process may be halted. If the DNA is misrepaired and/or if the cell divides prior to repair, daughter cells with DNA mutations will result. In a clonal expansion type of way, the initial morphologic stage associated with the neoplastic process, a focus of cellular alteration, occurs. The further promotion and progression of foci to bridging lesions such as adenomas and to the endstage, hepatocellular carcinoma, occurs over a chronic time frame and is regarded as a multistep process (10) with a striking similarity exemplified between the process as seen in certain fishes and that of the more conventional and better characterized rodent models (11,12). How these altered fish cells survive and preferentially grow, in a toxic milieu, provides us with valuable information on the cell biology of survival and growth.

CHARACTERIZATION OF FOCI OF CELLULAR ALTERATION
Because foci of cellular alteration occur following exposure to known carcinogens and prior to development of adenoma and carcinoma, the lesions have received appreciable attention. Three basic phenotypes are seen with conventional hematoxylin and eosin procedures. These are basophilic, eosinophilic and clear cell. In addition to histologic phenotypes, enzyme histochemical procedures may be used to mark foci. Teh and Hinton (13) have shown that the same battery of enzyme histochemical procedures which mark foci of rodent liver marks these lesions in medaka liver. Moore and Myers (14) reviewed enzyme characteristics of foci and included a review of immunohistochemical and biochemical observations on cytochrome P450 1A and glutathione S transferase (GST) of foci and neoplasms. Foci generally are deficient in phase I but enriched in certain phase II enzymes, quinone oxido-reductase and UDPGdH. However, they concluded that the pattern associated with GST needs additional work. Fohler and Van Noorden (15) established metabolic changes and cell proliferation indices for extrafocal liver, foci and neoplasms of European flounder. Small foci with increased activity of G6PDH and elevated expression of proliferating cell nuclear antigen (PCNA) appeared prior to development of tumors. High initial velocities of G6PDH and high PCNA labelling index of early foci persisted in hepatocellular adenomas and carcinomas (15).

GENDER AS A HOST FACTOR IN CARCINOGENESIS OF FISHES
For those studies in which results were separated by sex of fish, females consistently showed more susceptibility to tumor development. Masahito et al. (16) studied "spontaneous" liver neoplasms in medaka and found that between 3 and 5 years of age, the numbers of males with tumors rose from 0 to 3.8% while that of females rose from 1.3 to 7.1%. Similar effects were reported in sexually mature salmonids (17,18). DDT proved carcinogenic in the rainbow trout model and a 3X greater prevalence was seen in females versus males (see discussion Nunez et al.(19)). After exposure of zebrafish (Brachydanio rerio) to diethynitrosamine, an increased prevalence of 1.4 to 3X was seen in females (20). Field studies have also indicated gender-related susceptibility to hepatocarcinogenesis. Common dab (Limanda limanda) were exensively surveyed in the North Sea and more foci and neoplasms were seen in females than in males (21). Similarly, female European flounder (Platichthys flesus) of the North Sea showed prevalences of hepatic neoplasia 2X that of males (4). The extensive and carefully-designed surveys of coastal US waters by National Oceanographic & Atmospheric Administration and National Marine Fisheries Service scientists (22) are noteworthy since they consistently report no gender differences with respect to hepatic neoplasia prevalence. Cooke and Hinton (23) used an initiation-promotion assay to investigate, for the first time, the effects of tumor modulators, estradiol (E2) and hexachlorocyclohexane (HCH), on hepatic foci in fish (medaka). Their results showed that post-initiation (brief diethylnitrosamine exposure) treatment with either E2 or HCH increased the numbers of basophilic foci but had an opposite effect on eosinophilic foci. E2 and HCH positively modulated (promoted) tumor development in the medaka diethylnitrosamine model (23). Aspects of gender-specific growth in hatchling, immature and sexually mature control medaka were studied and compared to gender-specific growth medaka receiving a single exposure as hatchlings to the complete carcinogen, diethylnitrosamine (DEN) (24). Body weights of control females were significantly greater than that of males at weeks 8, 20, 32, and 44. Liver weights and hepatosomatic indices were significantly greater in females versus males at all ages. In the DEN-exposed fish, liver weights were greater in females for all weeks except 4 and 6. Female hepatosomatic indices proved significantly greater than their male counterparts at all times. Because sampling was extended through 44 weeks, it was possible to determine whether the gender specific growth enhancement of the females was associated with tumor promotion. Females reached tumor first and at higher prevalence than their male counterparts. Apparently, gender specific growth of medaka livers can operate as a positive modulator of DEN-induced hepatic carcinogenesis. Physiological and morphological studies with rainbow trout have described a strong linkage between gonadal development and liver growth. This has been associated with the normal role of the female liver in vitellogenesis (25-27); and, more recently, also with zona radiata protein or choriogenin (28) production.

CONCLUDING REMARKS
Carcinogenesis and especially hepatocarcinogenesis of fishes affords an excellent opportunity to investigate metabolic activation and deactivation schema. The enhanced growth of specific populations of cells with phenotypic and genotypic alteration enable linkage of molecular to cellular and tissue phenomena. Work is needed in immune system control of neoplastic growth, and we need a better understanding of signal transduction, oncogene activation and phosphorylation states and their role in growth of foci and neoplasms in fishes. By necessity, much of this work has been descriptive and for continued expansion of the field, tools to address mechanisms of normal and abnormal growth in fishes are essential.

ACKNOWLEDGMENTS
This work was suported by: the U.S. Department of Health and Human Services grant CA45131-11 from the National Cancer Institute, the U.S. Environmental Protection Agency (EPA) grant R 825298, the EPA Center for Ecological Health Research at UC Davis grant CR 819658, by grant 5 P42 ESO4699 from the National Institute of Environmental Health Sciences, NIH with funding provided by EPA, and by funds from the Toxic Substances Research and Teaching Program, University of California. Contents of this publication are solely the responsibility of the author and do not necessarily represent the official views of the NIEHS, NIH or EPA.

REFERENCES

1.)	Dawe, C.J., M.F. Stanton, and F.J. Schwartz. 1964. Hepatic neoplasms in native bottom-feeding fish of Deep Creek Lake, Maryland. Cancer Research. 24:1194-1201.
2.)	Harshbarger, J.C. and J.B. Clark. 1990. Epizootiology of neoplasms in bony fish of North America. Science Total Environment 94:1-32.
3.)	Peters, N., A. Kohler, and H. Kranz. 1987. Liver pathology in fishes from the Lower Elbe as a consequence of pollution. Diseases Aquatic Organisms 2:87-97.
4.)	Vethaak, A.D. and P.W. Wester. 1996. Diseases of flounder Platichthys flesus in Dutch coastal and estuarine waters, with particular reference to environmental stress factors. II. Liver histopathology. Diseases Aquatic Organisms 26:99-116.
5.)	Malins, D.C., B.B. McCain, D.W. Brown et al., 1987. Toxic chemicals, including aromatic and chlorinated hydrocarbons and their derivatives, and liver lesions in white croaker (Genyonemus lineatus) from the vicinity of Los Angeles. Environmental Science and Technology 21:765-770.
6.)	Baumann, P.C. and J.C. Harshbarger. 1995. Decline in liver neoplasms in wild brown bullhead catfish after coking plant closes and environmental PAHs plummet. Environmental Health Perspectives 103:168-170.
7.)	Maccubbin, A.E., N. Ersing, J. Weinar, and J.J. Black. 1987. In vivo bioassays using rainbow trout and medaka fish embryos. In: Short-term Bioassays in the Analysis of Complex Environmental Mixtures. S.S. Sandhu, D.M. Demarini, M.J. Mass, M.M. Moore, and J.L. Mumford, eds. Plenum Press, New York, pp.209-223.
8.)	Metcalfe, C.D., V.M. Cairns, and J.D. Fitzsimons. 1988. Experimental induction of liver tumours in rainbow trout (Salmo gairdneri) by contaminated sediment from Hamilton Harbour, Ontario. Canadian Journal Fisheries and Aquatic Science 45:2161-2167.
9.)	Fabacher, D.L., J.M. Besser, C.J. Schmitt et al. 1991. Contaminated sediments from tributaries of the Great Lakes: chemical characterization and carcinogenic effects in medaka (Oryzias latipes). Archives Environmental Contamination and Toxicology 20:17-34.
10.)	Farber, E. 1980. The sequential analysis of liver cancer induction. Biochimica Biophysica Acta 605:149-166.
11.)	Bannasch, P., H. Zerban, and H.J. Hacker. 1997. Foci of altered hepatocytes, rat. In: Digestive System. Second Edition, J.A. Popp, U. Mohr, and T.C. Jones (eds.)3-37. Springer-Verlag, Berlin.
12.)	Boorman, G.A., S. Botts, T.E. Bunton, et al. 1997. Diagnostic criteria for degenerative, inflammatory, proliferative non-neoplastic and neoplastic liver lesions in medaka (Oryzias latipes): Consensus of a National Toxicology Program Pathology Working Group. Toxicologic Pathology 25:202-210.
13.)	Teh, S.J. and D.E. Hinton.1993. Detection of enzyme histochemical markers of hepatic preneoplasia and neoplasia in medaka (Oryzias latipes). Aquatic Toxicology 24: 163-182.
14.)	Moore, M.J. and M.S. Myers. 1993. Pathobiology of Chemical - Associated Neoplasia in Fish. Chapter 8. In: Aquatic Toxicology: Molecular, Biochemical and Cellular Perspectives. D.C. Malins and G.K. Ostrander (eds.) Lewis Publishers, Boca Raton. pp. 327-386.
15.)	K. Fohler, A. and C.J.F. Van Noorden. 1998. Initial velocities in situ of G6PDH and PGDH and expression of proliferating cell nuclear antigen (PCNA): sensitive diagnostic markers of environmentally induced hepatocellular carcinogenesis in a marine flatfish (Platicthyl flesus L.). Aquatic Toxicology 40:233-252.
16.)	Masahito, H.R.H. Prince, K. Aoki, N. Egami et al. 1989. Life-span studies on spontaneous tumor development in the medaka (Oryzias latipes). Japanese Journal Cancer Research 80:1058-1065.
17.)	Takashima, F. and T. Hibiya. 1972. Hepatic tumors in yamame, Oncorhynchus masou ishikawae: a preliminary report. Bulletin Japanese Society Scientific Fisheries 38:955-964.
18.)	Takashima, F. 1976. Hepatoma and cutaneous fibrosarcoma in hatchery-reared trout and salmon related to gonadal maturation. Progress Experimental Tumor Research 20: 351-366.
19.)	NuF1ez, O., J.D. Hendricks, and G.S. Bailey. 1988. Enhancement of aflatoxin B1 and N-methyl-N'-nitro-N-nitrosoguanidine hepatocarcinogenesis in rainbow trout Salmo gairdneri by 17-estradiol and other organic chemicals. Diseases Aquatic Organisms 5:185-196.
20.)	Stanton, M.F. 1965. Diethylnitrosamine-induced hepatic degeneration and neoplasia in the aquarium fish, Brachydanio rerio. Journal National Cancer Institute 34:117-130.
21.)	Vethaak, D. and J. van der Meer. 1991. Fish disease monitoring in the Dutch part of the North Sea in relation to the dumping of waste from titanium dioxide production. Chemistry and Ecology 5:149-170.
22.)	Myers, M.S., C.M. Stehr, O.P. Olson et al. 1994. Relationships between toxicopathic hepatic lesions and exposure to chemical contaminants in English sole (Pleuronectes vetulus), starry flounder (Platichthys stellatus), and white croaker (Genyonemus lineatus) from selected marine sites on the Pacific Coast, USA. Environmental Health Perspectives 102:200-215.
23.)	Cooke, J.B. and D.E. Hinton. 1998. Promotion by 17-estradiol and -hexachlorocyclohexane of Hepatocellular Tumors in Medaka (Oryzias latipes). Aquatic Toxicology. In press.
24.)	Teh, S.J. and D.E. Hinton. 1998. Gender-specific growth and hepatic neoplasia in medaka (Oryzias latipes). Aquatic Toxicology 41:141-159.
25.)	Olivereau, M. and J. Olivereau. 1979. Effect of estradiol-17 on the cytology of the liver, gonads and pituitary, and on plasma electrolytes in the female freshwater eel. Cell and Tissue Research 199:431-454. 26.) Ng, T.B., N. Y. S. Woo, P.P.L. Tam, et al., 1984. Changes in metabolism and hepatic ultrastructure induced by estradiol and testosterone in immature female Epinephelus akaara (Teleostei, Serranidae). Cell and Tissue Research 236: 651-659.
26.)	van Bohemen, C.G., J.G.D. Lambert, and J. Peute. 1981. Annual changes in plasma and liver in relation to vitellogenesis in the female rainbow trout, Salmo gairdneri. General and Comparative Endocrinology 44:94-107.
27.)	Murata, K., H. Sugiyama, S. Yasumasu et al., 1997. Cloning of cDNA and estrogen-induced hepatic gene expression for choriogenin H, a precursor protein of the fish egg envelope (chorion). Proceedings National Academy of Science USA 94:2050-2055.