Abstract
A newly recognized, emerging baculovirus infection has caused high mortalities in several populations of penaeid shrimp in the United States and several Asian countries, and most recently in a population of feeder crayfish at the National Zoological Park (NZP). The National Zoo’s Department of Invertebrates (DOI) receives weekly shipments of feeder crayfish, which are fed live to cephalopods (mainly common cuttlefish exhibited in the collection). In early December 1995, the DOI reported a high mortality rate in their feeder crayfish for at least 1 month prior to submitting the dead crayfish to the Zoo’s Department of Pathology. At the onset of this epidemic, two species of crayfish (Orconectes punctimanus and Procambarus sp.) were supplied by two different local distributors. Between December 1995 and mid-March 1996, approximately equal numbers of the crayfish species were received and combined in large plastic holding tanks in the DOI until fed out. At the peak of the epidemic, greater than 90% of Orconectes either arrived dead or died within the first 24 hours after arrival. There were no mortalities of Procambarus until mid-March 1996, at which time 11 out of ∼100 died within 4 days after arrival. At the end of March, Orconectes sp. were no longer purchased due to the high losses. Shortly thereafter, Procambarus mortalities increased to 20–30% through the summer of 1996.
Crayfish were submitted for postmortem examination at seven timepoints between December 1995 and May 1996. Gross lesions were limited to exoskeleton discoloration and mottling, especially prominent on the cephalothorax, with occasional extension down the chelipeds. Histologically, within the basal epithelial cell layer of the integument, there were large, deeply basophilic intranuclear inclusion bodies, often with attendant necrosis and separation of the overlying chitinous layer of the integument. Additionally, there were similar intranuclear inclusions within skeletal muscle and epithelial cells of the gill, stomach, intestine, hepatopancreas, compound eye, and antennal gland, with multifocal necrosis. Transmission electron microscopic examination of the cuticular epithelium and antennal gland revealed large, rod-shaped to elliptical intranuclear viral particles that measured ∼100–350 nm, with morphological characteristics of the Baculoviridae family.
No further baculovirus-associated mortalities occurred in DOI until early February 1997, when approximately 20% of a shipment of crayfish (Orconectes punctimanus) died within 24 hours after arrival to the DOI. Mortalities attributed to the virus have continued to occur at a low rate as of April 1997.
In 1992, a baculovirus was determined to be the causative agent of a fatal shrimp disease that resulted in high mortalities and economic loss to the shrimp culture industry of Taiwan and other Asian countries.2,4,7 Until then, baculoviruses had been isolated from and caused disease only in arthropods, mainly insects. In 1995, the cultured shrimp industry in the southern United States experienced outbreaks of a baculovirus disease, which resulted in high mortalities.5,6,9 The source was believed to be from imported Asian shrimp. Clinical signs in the acutely affected shrimp included lethargy, anorexia, and loose cuticles with white target-like spots on the exoskeleton measuring 0.5–2.0 mm in diameter. These spots, composed of abnormal deposits of calcium salts, were most prominent on the inside surface of the carapace and led to the name white spot disease or white spot syndrome. The etiologic agent was called white spot syndrome virus (WSSV). WSSV is extremely virulent, and the natural host range includes several species of penaeid shrimp (P. japonicus, P. monodon, others).2-4,7,8 Similar to the crayfish at the National Zoo, some populations of shrimp may have cumulative mortalities up to 100% within 3–10 days of onset of clinical signs.5 Experimental susceptibility to WSSV has been demonstrated in many cultured and wild shrimp, crabs, lobster, and the red swamp crayfish, Procambarus clarkii.1,11
Using in-situ hybridization probes developed from the Thailand WSSV isolate and an isolate from the Yellow Sea region of China by co-author DVL5,8 at the University of Arizona Aquaculture Pathology Laboratory, a strong positive signal was demonstrated in the intranuclear inclusions of affected NZP crayfish. Additionally, infectivity trials utilizing disease-free shrimp that were injected with or fed our WSSV probe-positive crayfish yielded a syndrome identical to WSSV reported in penaeid shrimp. These results strongly indicate the virus present in crayfish from the National Zoo is an Asian-derived WSSV.
The source of the baculovirus has so far been undetermined. At this time, because of multiple vendor involvement, it has not been clear when or if any of these crayfish originated from wild populations. Significant natural crayfish mortalities have not been reported to our knowledge, but the possibility remains that crayfish are becoming infected at their point of origin (either natural habitats or crayfish farms) in the southeast United States. Other possible sources of infection include the introduction of baculovirus-contaminated or infected food fed to the crayfish, or cross-contamination during handling by vendors or staff at the zoo. Since the early mortalities occurred right after delivery of Orconectes to the National Zoo, it is likely they arrived infected. Contamination of holding tanks may have contributed to perpetuation of the virus following the earlier outbreaks.
The significance of this newly recognized, fatal baculovirus in decapods is threefold. First, the American Fisheries Society Endangered Species Committee has determined 112 out of 338 native crayfish species are either endangered or threatened.10 The introduction of nonindigenous crayfish species alters the local habitat and now represents a potential source of baculovirus transmission to wild, endangered crayfish. Second, baculovirus from infected crayfish from any source may be transmitted to other wild decapods. Third, economic losses to the farmed crayfish and shrimp industries are likely. Therefore, it is of utmost importance to eliminate exposure of farmed and wild populations of decapods to baculovirus-infected arthropods.
Acknowledgments
The authors thank the following individuals for their contributions to our study: Dr. Mary Allen, Andrew Keech, Christopher James, and Earl Pinkney. This research was supported in part by a Friends of the National Zoo Senior Fellowship, FONZ 96-3545A.
Literature Cited
1. Chang, P.S., C.F. Lo, Y.C. Wang, and G.H. Kou. (in press). Detection of white spot syndrome associated virus (WSSV) in experimentally infected wild shrimp, crabs and lobsters by in-situ hybridization. Second International Conference on the Culture of Penaeid Prawns and Shrimps, May, 1996.
2. Chou, H.Y., C.Y. Huang, C.H. Wang, H.C. Chiang, and C.F. Lo. 1995. Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Diseases of Aquatic Organisms. 23: 165–173.
3. Durand, S., D.V. Lightner, L.M. Nunan, R.M. Redman, J. Mari, and J.R. Bonami. 1996. Application of gene probes as diagnostic tool for the white spot baculovirus (WSSV) of penaeid shrimps. Diseases of Aquatic Organisms. 27: 59–66.
4. Inouye, K., S. Miwa, N. Oseko, et al. 1994. Mass mortalities of cultured Kuruma shrimp Penaeus japonicus in Japan in 1993: electron microscopic evidence of the causative virus. Fish Pathology. 29: 149–158.
5. Lightner, D.V. 1996. A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA. 304 p.
6. Lightner, D.V., R.M. Redman, B.T. Poulos, L.M. Nunan, J.L. Mari, and K.W. Hasson. (in press). Risk of spread of penaeid shrimp viruses in the Americas by the international movement of live shrimp for aquaculture and frozen shrimp commodity markets. Revue Scientifique et Technique Office International des Epizooties.
7. Lo, C.F., C.H. Ho, S.E. Peng, et al. 1997. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Diseases of Aquatic Organisms. 27: 215–225.
8. Nunan, L.N., and D.V. Lightner. 1997. Development of a non-radioactive gene probe by PCR for detection of white spot syndrome virus (WSSV). Journal of Virological Methods. 63: 193–201.
9. Rosenberry, B. (ed.). 1996. Whitespot and Yellowhead in the United States. In: World Shrimp Farming 1996. Aquaculture Digest, San Diego, CA. 164 p.
10. Taylor, C.A., M.L. Warren, J.F. Fitzpatrick, et al. 1996. Conservation status of crayfishes of the United States and Canada. Fisheries. 21: 25–38.
11. Wang, Y.C., C.F. Lo, P.S. Chang, and G.H. Kou. (in press). White spot syndrome associated virus (WSSV) infection in cultured and wild decapods in Taiwan. Second International Conference on the Culture of Penaeid Prawns and Shrimps, May, 1996.