THE NEED FOR MOLECULAR TOOLS IN THE STUDY OF MOLLUSC PATHOGENS - P.M. HINE

National Institute of Water and Atmospheric Research, PO Box 14-901,

Wellington, New Zealand.

Introduction

The OIE list of notifiable diseases of molluscs and the pathogens causing them comprises marteiliosis (Marteilia refringens, Marteilia sydneyi), bonamiosis (Bonamia ostreae, Bonamia sp.), mikrocytosis (Mikrocytos mackini, Mikrocytos roughleyi), haplosporidiosis (Haplosporidium nelsoni, Haplosporidium costale) and perkinsosis (Perkinsus marinus, Perkinsus olseni). This paper gives a brief overview of the areas in which molecular tools are needed to overcome problems associated with these diseases, and considers the needs of the Asian region.

Infection levels

Moderate to heavy infections with Marteilia refringens, Marteilia sydneyi, both Bonamia spp., and both Haplosporidium spp. are relatively easy to detect by routine histology. Both Perkinsus spp. can be cultured using Ray's Fluid Thioglycollate Medium (RTFM) (Bushek et al., 1994), allowing light infections to be amplified, and consequently detected. Mikrocytos spp. are much harder to detect. Mikrocytos roughleyi infects the haemocytes of Sydney rock oysters (Saccostrea commercialis) in eastern (Georges River) and western (Carnarvon, Albany) Australia, and ultrastructurally it has a single mitochondrion. Mikrocytos mackini infects connective tissue cells of Pacific oysters (Crassostrea gigas) off the coast of British Columbia, and ultrastructurally it lacks a mitochondrion. Currently it is thought that these two pathogens are not closely related, and M. roughleyi may be more closely related to Bonamia spp. Macroscopically both Mikrocytos spp. produce macroscopic pustular or abcess-like lesions in cases of heavy infection, and if occurring in the known range of these two pathogens, may allow presumptive diagnosis. However, microscopically both Mikrocytos spp. are only ~2?m in diameter, do not stain well, consequently they are difficult to detect in moderate to light infections (Hervio et al., 1996). Therefore probes are needed to detect light infections with all OIE listed pathogens except (Perkinsus spp.), and light to moderate infections with Mikrocytos spp.

The identification of species

The inter-relationships of all the OIE listed notifiable pathogens are currently uncertain. Marteilia sydneyi was initially distinguished from the previously described Marteilia refringens, on the grounds that the latter possessed refringent granules, whereas the former did not. However, M. sydneyi does possess refringent granules (Roubal et al., 1989). Also it is unclear how the Marteilia sp. that caused a massive epizootic in calico scallops off the coast of Florida (Moyer et al., 1993) relates to described species. Although distinction on the basis of cleavage patterns during development seems to overcome these uncertainties, the distinction of Marteilia spp. is still being questioned (Bower et al., 1994).

The two species of Bonamia have also not yet been clearly distinguished, although B. ostreae has dense forms that are seldom seen in Bonamia sp, and the latter has a vacuolated stage (Hine, 1991) that has not been reported from B. ostreae. The two species of Haplosporidium can be distinguished by spore size, and H. nelsoni can be distinguished from all other Haplosporidium spp. as it sporulates in epithelial cells of the digestive diverticulae, and the other species sporulate in connective tissue. Currently, H. costale cannot be distinguished reliably from other Haplosporidium spp. except H. nelsoni. Mikrocytos spp. do not resemble each other closely and M. roughleyi may be more closely related to Bonamia spp. (see above). Perkinsus olseni shows similarities to Perkinsus atlanticus (Hamaguchi et al., 1998), but Perkinsus marinus also shows similarities to P. atlanticus (Robledo et al., 1997). Specific probes are currently available, or are being developed for, Marteilia refringens, Marteilia sydneyi, Bonamia spp., Haplosporidium nelsoni, and H. costale.

Life cycles

Bonamia spp., Mikrocytos mackini, and Perkinsus spp., transmit directly from one host to another. Haplosporidium spp. and Marteilia spp. cannot be transmitted directly from one to another, and probably require an intermediate host (Roubal et al., 1989; Berthe et al., 1998). Probes currently developed or being developed for Haplosporidium spp. and Marteilia spp. will be used to identify the DNA of these pathogens in likely alternative hosts, such as filter feeding or detrivorous invertebrates.

The Asian region

The OIE listed diseases of molluscs mainly infect bivalves in temperate regions. This is true of Bonamia spp. in temperate oysters (Ostrea, Tiostrea, Crassostrea), Mikrocytos spp. in temperate oysters (Crassostrea, Saccostrea), and Haplosporidium spp. of temperate oysters (Crassostrea). Marteilia refringens is also a parasite of temperate bivalves (Ostrea, Tiostrea, Crassostrea, Mytilus, Cerastoderma), and although Marteilia sydneyi occurs in the subtropics of southern Queensland, it is primarily a parasite of temperate oysters (Saccostrea). Marteilia refringens, Bonamia ostreae, Mikrocytos mackini, Haplosporidium costale and Perkinsus marinus have not been reported from the Asian region.

Marteilia sydneyi, Bonamia sp., Mikrocytos roughleyi and Perkinsus olseni have been reported from Australia, and a Perkinsus sp. from clams (Tapes philippinarum) in Japan (Hamaguchi et al., 1998). The Japanese isolate had sequences intermediate between P. olseni in Australia, and P. atlanticus in Manila clams (Tapes philippinarum) from Spain. Perkinsus atlanticus from Ruditapes philippinarum and Ruditapes decussatus around Spain and Portugal, are closely related to P. olseni (Robledo et al., 1997). It may be therefore that P. olseni was moved from Asia in Manila clams to Europe, where it was described as P. atlanticus. If so, P. olseni/atlanticus may be widely distributed throughout Southeast Asia.

Although Haplosporidium nelsoni has not been formally reported from Asia, a Haplosporidium sp., similar in size and pathology to H. nelsoni, occurs in Pacific oysters (Crassostrea gigas) in California and in Matsushima Bay, Japan, from which the Californian stocks derived (Friedman et al., 1991; Friedman, 1996). A sensitive and specific probe for H. nelsoni (Stokes and Burreson, 1995) labels the Californian and Japanese Haplosporidium, suggesting that it is also H. nelsoni, and that H. nelsoni was originally introduced into California in Japanese Pacific oysters.

Therefore, some of the temperate OIE listed diseases occur in Australia and Japan, and it is likely that Haplosporidium nelsoni and Perkinsus olseni/atlanticus occur more widely in Asia than is currently realized. As bivalve health expertise becomes more widespread in Asia, other serious diseases of tropical, as well as temperate bivalves, are likely to emerge. One such pathogen may be a Haplosporidium sp. pathogenic in silverlip pearl oysters (Pinctada maxima) in northwestern Australia (Hine and Thorne, 1998). Also, an apparently infectious disease that has caused massive mortalities among akoya pearl oysters (Pinctada fucata) in Japan since 1994 (Miyazaki et al., 1998) may well prove to be a serious disease in Asia. Currently a parasite related to Marteilia, called Marteilioides chungmuensis, which parasitizes the ova of Pacific oysters (Crassostrea gigas) is having a serious impact on oyster production, and may also prove to be a problem in Asia. Once such problems have been identified, molecular tools will need to be developed to detect low infection levels, distinguish species and study life cycles, as for the currently listed diseases.

The OIE protocols are designed to control spread of aquatic animal diseases, using a certification and reporting system that requires a national infrastructure, based in law, and a network of skilled and experienced aquatic animal health specialists, including technicians, inspectors and pathologists. Development of such systems occurs as industries to be serviced develop, and therefore are found where aquaculture industries are well established. Such established industries use hatchery production or natural spat settlement as their source of stock. Certification allows stock to be traded between farms, and the identification and establishment of disease free zones minimizes risk of disease spread.

In developing bivalve farms, it is often necessary to initially acquire brood stock from the wild, or to move bivalves into an area to enhance natural spat settlement. Such movements are already taking place throughout Asia. This process must be undertaken with extreme care to minimize the risk of introducing disease onto the farm site. Studies on the parasites and diseases of wild bivalves in northwestern Australia have shown that the prevalence of potentially serious diseases in wild stocks may be extremely low (Table 1). Prevalences of ~0.1% are common. To detect such infection with 95% confidence, it is necessary to sample 2,994 animals, and even then a light infection may well be missed. Molecular tools are needed to detect such low levels of infection before stocks are moved.

References

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Bower, S.M., McGladdery, S.E. and Price, I.M. (1994). Synopsis of infectious diseases and parasites of commercially exploited shellfish. Annual Review of Fish Diseases 4, 1-199.

Bushek, D., Ford, S.E. and Allen, S.K. (1994). Evaluation of methods using Ray's fluid thioglycollate medium for diagnosis of Perkinsus marinus infections in the eastern oyster, Crassostrea virginica. Annual Review of Fish Diseases 4, 201-217.

Friedman, C.S. (1996). Haplosporidian infections of the Pacific oyster, Crassostrea gigas (Thunberg), in California and Japan. Journal of Shellfish Research 15, 597-600.

Friedman, C.S., Cloney, D.F., Manzer, D. and Hedrick, R.P. (1991). Haplosporidiosis of the Pacific oyster, Crassostrea gigas. Journal of Invertebrate Pathology 58, 367-372.

Hamaguchi, M., Suzuki, N., Usuki, H. and Ishioka, H. (1998). Perkinsus protozoan infection in short-necked clam Tapes (=Ruditapes) philippinarum in Japan. Fish Pathology 33, 473-480.

Hervio, D., Bower, S.M. and Meyer, G.R. (1996). Detection, isolation, and experimental transmission of Mikrocytos mackini, a microcell parasite of Pacific oysters Crassostrea gigas (Thunberg). Journal of Invertebrate Pathology 67, 72-79.

Hine, P.M. (1991). Ultrastructural observations on the annual infection pattern of Bonamia sp. in flat oysters Tiostrea chilensis. Diseases of Aquatic Organisms 11, 163-171.

Hine, P.M. and Thorne, T. (1998). Haplosporidium sp. (Haplosporidia) in hatchery-reared pearl oysters, Pinctada maxima (Jameson, 1901), in north Western Australia. Journal of Invertebrate Pathology 71, 48-52.

Miyazaki, T., Goto, K., Kobayashi, T. and Miyata, M. (1998). An emergent virus disease associated with mass mortalities in Japanese pearl oysters Pinctada fukata martensii. In: Proceedings of the VIIth International Colloquium on Invertebrate Pathology and Microbial Control. Sapporo, Japan, August 23-28^th 1998, pp. 154-159.

Moyer, M.A., Blake, N.J. and Arnold, W.S. (1993). An ascetosporan disease causing mass mortality in the Atlantic calico scallop Argopecten gibbus (Linnaeus, 1758). Journal of Shellfish Research 12, 305-310.

Robledo, J.A.F., Wright, A.C., Coss, C.A., Vasta, G.R. and Goggin, C.L. (1997). Further studies of conserved genes from Perkinsus isolates. Journal of Shellfish Research 16, 342.

Roubal, F.R., Masel, J. and Lester, R.J.G. (1989). Studies on Marteilia sydneyi, agent of QX disease in the Sydney rock oyster, Saccostrea commercialis, with implications for its life cycle. Australian J.ournal of Marine and Freshwater Research 40, 155-167.

Stokes, N.A. and Burreson, E.M. (1995). A sensitive and specific DNA probe for the oyster pathogen Haplosporidium nelsoni. Journal of Eukaryote Microbiology 42, 350-357.

Table 1. Prevalence of parasites in Saccostrea spp. IVI = intranuclear virus-like inclusions. RLOs = Rickettsiales-like organisms.