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Abstracts
Workgroup: Frontiers in Disease Research
Life Cycle Studies of Haplosporidium nelsoni (MSX) Using PCR Technology.
Principal Investigator(s):
Eugene M. Burreson, Virginia Institute of Marine Science, School of Marine Science, College of William and Mary, gene@vims.edu
Co-Investigator(s):
Susan E. Ford, Haskin Shellfish Research Laboratory, Rutgers University, Port Norris, NJ 08349 and Nancy A. Stokes, Virginia Institute of Marine Science, School of Marine Science, College of William and Mary
Funding Period: 12/1/95-11/30/98
The oyster pathogen Haplosporidium nelsoni, also known as MSX, has been the causative agent for oyster mortalities in many areas of the east coast of the U.S. for the past 45 years. Although the distribution and intensity of H. nelsoni seem to respond to salinity changes, with increased intensity during drought conditions and decreased intensity during very wet years, the overall virulence and intensity of the pathogen have not decreased since the original epizotics in 1957 and 1959. The presence of this disease is a major obstacle to aquaculture and oyster restoration efforts.
The complete life cycle of H. nelsoni, and of all other haplosporidians, remains a mystery. The life cycle stage infective to oysters and the source of that stage have not been identified. Previous attempts to infect oysters directly with H. nelsoni spores have been unsuccessful, thus leading to speculation that parasite transmission between oysters occurs via an obligate intermediate host.
The overall objective of this project was to elucidate the complete life cycle of H. nelsoni using newly-developed PCR primers specific for this organism. Field samples were collected from both Chesapeake and Delaware Bay and processed for DNA extraction and PCR amplification. PCR-positive samples were subjected to in situ hybridization using a H. nelsoni-specific DNA probe and/or DNA sequencing to confirm identity of the amplicon.
Field samples consisted of size-fractionated water (250:m, 75:m, 35:m and 10:m) samples and sediment (aerobic top layer and 250:m) and macroinvertebrates from both locations. Sampling was conducted weekly in 1996, every other week in 1997 and 1998. Total genomic DNA was extracted from each sample and subjected to PCR using H. nelsoni-specific primers. Initial difficulties with removal of Taq DNA polymerase inhibitors in the environmental samples, especially in sediment, were overcome with ethidium bromide/high salt purification of the DNA and addition of bovine serum albumin to the PCR reactions.
There were very few Delaware Bay samples that yielded H. nelsoni PCR product during the course of the project. None of the sediment samples and only four water samples, all in spring/summer 1997, were PCR-positive. Eight samples of macroinvertebrates tested positive for H. nelsoni. These included a variety of organisms, such as nereid and spionid worms, dog whelk, gammarid amphipod, and pea crab.
Samples that yielded H. nelsoni PCR product were more frequent from the lower Chesapeake than from the Delaware Bay, corresponding to the higher MSX disease prevalence in oysters from this location. PCR-positive results were obtained in many of the Chesapeake water, sediment, and macroinvertebrate samples. Most of the PCR-positive macroinvertebrates were polychaetes, including spionid, nereid, capitelid, and orbiniid worms; however, a variety of other organisms such as gammarid amphipod, isopod, and gastropods also yielded H. nelsoni PCR product. Each size fraction of water or sediment was PCR-positive at some point during the study. The only obvious pattern observed was the absence of positive PCR in all samples between late August 1997 and late May 1998 and between late October 1996 and early July 1998 in sediment samples.
Sequencing of the amplicons yielded the H. nelsoni sequence in every case, demonstrating that positive samples were not the result of cross-reactivity. In situ hybridizations with the H. nelsoni-specific DNA probe were conducted on PCR-positive macroinvertebrate samples to discriminate between true infections and those where H. nelsoni simply adhered to the external surface or passed through the gut. Unfortunately, none of the samples used for in situ hybridization revealed H. nelsoni cells. We suspect that H. nelsoni spores were widely distributed, especially in the Chesapeake sampling location, and that many of the PCR amplicons were from spores either free in the water or sediment or within the guts of organisms. If there were samples with MSX disease, they were probably masked by the abundance of PCR-positive samples from H. nelsoni not representing true infections.
IMPACTS and/or BENEFITS:
This project allowed us to optimize and validate the H. nelsoni-specific PCR assay for environmental samples. This optimized assay has been used in other laboratories to confirm the presence of H. nelsoni. Application of molecular diagnostics to environmental samples was still new at the time of this project and we learned much about the abilities and limitations of PCR assays for large-scale surveys such as this one, and used that information to improve our approach for subsequent life cycle studies.
PROJECT PUBLICATIONS:
Stokes, N. A., B. S. Flores, E. M. Burreson, K. A. Alcox, X. Guo, and S. E. Ford. 1997. Life cycle studies of Haplosporidium nelsoni (MSX) using PCR technology. Journal of Shellfish Research 16:336.
Stokes, N. A., B. S. Flores, K. A. Ashton-Alcox, J. R. Pharo, S. E. Ford, and E. M. Burreson. DNA-based molecular diagnostics for Haplosporidium nelsoni (MSX) life cycle studies. Abstract for Third International Symposium on Aquatic Animal Health, Baltimore, MD, 30 August-3 September, 1998.
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