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Abstracts
Workgroup: Frontiers in Disease Research
Infectivity , pathogenicity, and epizootiology of the clam parasites Perkinsus chesapeaki and Perkinsus andrewsi in Chesapeake Bay oysters: have we been misinterpreting Perkinsus marinus epizootiology?
Principal Investigator(s):
Eugene M. Burreson, Virginia Institute of Marine Science, College of William and Mary, gene@vims.edu
Co-Investigator(s):
Kimberly S. Reece, Virginia Institute of Marine Science, College of William and Mary
Christopher F. Dungan, Maryland Department of Natural Resources, Oxford Cooperative Lab
Nancy A. Stokes, Virginia Institute of Marine Science, College of William and Mary
Christopher F. Dungan, Maryland Department of Natural Resources, Oxford Cooperative Lab
Nancy A. Stokes, Virginia Institute of Marine Science, College of William and Mary
Funding Period: 10/01/01 - 09/30/03
PROJECT OBJECTIVES AND RESULTS:
The overall objectives are to determine the infectivity and pathology of P. chesapeaki and P. andrewsi to oysters and to use that information to reevaluate our understanding of P. marinus epizootiology in oysters.
Year 1: Develop specific DNA probes and PCR primers for P. marinus, P. chesapeaki and P. andrewsi based on ITS or LSU sequences of the ribosomal RNA gene complex.
Year 2: Determine infectivity and pathogenicity of P. chesapeaki and P. andrewsi to oysters with laboratory challenge experiments. Determine prevalence and intensity of P. chesapeaki and P. andrewsi, as compared to P. marinus, in selected oyster and clam populations in Chesapeake Bay.
PROJECT RESULTS YEAR 1 and YEAR 2 (current year): The internal transcribed spacer region (ITS) of the ribosomal RNA gene complex was chosen for the design of species-specific PCR primers for Chesapeake Bay Perkinsus species. Uncloned and clonal cultures of three Perkinsus isolates were sequenced at the ITS region. One isolate was from Mya arenaria, the type host for P. chesapeaki, and two were from the razor clam, Tagelus plebeius. Our ITS sequence data showed two clades within a single monophyletic group, one clade containing the GenBank-deposited P. chesapeaki sequence, the other clade containing the GenBanked sequence for P. andrewsi. Sequences obtained from individual clonal cultures from both hosts were found in both clades suggesting that P. chesapeaki and P. andrewsi are the same species. Large subunit rRNA sequences (LSU) were also determined to confirm the findings based on ITS sequences. Clonal cultures isolated from M. arenaria and T. plebeius were used and once again the Perkinsus sequences from both hosts grouped into a monophyletic clade. To further test whether these two species were the same, the previously developed P. andrewsi-specific primers (Coss et al. 2001b) were tested with these same clonal isolates and they amplified the DNA from all of the cultures. Due to the evidence suggesting that P. chesapeaki and P. andrewsi are the same species, one PCR primer set was developed to specifically amplify these ITS sequences, and one in situ hybridization probe was designed to target the LSU region. An in situ hybridization probe specific to P. marinus was also designed based on the LSU region sequences. The PCR primers were optimized with cultures and are currently being optimized for use with infected host materials. Real-time/quantitative PCR was optimized with Perkinsus genus-specific primers in water samples during year one and will continue to be optimized with infected host material during the second year.
Oyster beds located adjacent to a variety of clam species were sampled during the first year throughout the Chesapeake Bay in order to determine prevalence and intensity of P. marinus and P. chesapeaki/andrewsi in oyster populations. Specifically, 7 sites were sampled in the Maryland portion of the Chesapeake Bay for Crassostrea virginica and M. arenaria. T. plebeius was also sampled at 5 of the 7 sites in MD. Macoma balthica was found at only one site in MD with 7 individuals. In the Virginia portion of the Chesapeake Bay, there were 3 sampling sites; C. virginica and M. mercenaria were collected from 2 sites and C. virginica, M. balthica and T. plebeius were collected from 1 site. All samples were assessed for Perkinsus prevalence by Ray's fluid thioglycollate medium (RFTM) assay, processed for histology, and the gill was processed for use in DNA analysis. The RFTM assay showed greater than 80 percent prevalence of Perkinsus in all C. virginica samples except one MD sample with 52 percent prevalence. Prevalence of Perkinsus in the clam species by RFTM was usually very high prevalence, with a few exceptions. High prevalence of Perkinsus infections was found in all of the M. arenaria samples, except for one sample from MD. In addition, Perkinsus was found at a high prevalence in all but one of the T. plebeius samples. Perkinsus was found in Macoma balthica in both states, however, only one M. mercenaria sample was found to have Perkinsus infections and only at a 3 percent prevalence. The DNA samples are currently being extracted for screening by PCR. The histology sections are paraffin embedded and awaiting results of the PCR analysis before the final processing for in situ hybridization analysis.
IMPACTS and/or BENEFITS: The thorough DNA sequencing clonal cultures of Perkinsus species isolated in Chesapeake Bay in order to develop PCR primers has elucidated significant intra-specific variation within clonal cultures at the ITS locus. ITS sequences previously thought to be either P. chesapeaki or P. andrewsi- specific were found simultaneously within the genome of several clonal cultures. This observation suggests that P. chesapeaki and P. andrewsi should be synonymized. Sequence data from several clonal Perkinsus cultures isolated from various hosts, including M. balthica, and from several different geographic locations in the Chesapeake Bay, as well as sequences of other genes were collected for this study and further support synonymization. These observations suggest that intra-, as well as inter-specific, sequence variation needs to adequately characterized before designing molecular diagnostics and to use molecular data to support descriptions of new species of Perkinsus, and other pathogens. The field studies in this project are beneficial to further understand the epizootiology of P. marinus on oysters. It will help to indicate whether the assumption that positive results from RFTM assays as only P. marinus infections has been accurate, or whether some infections are actually caused by other Perkinsus species. Furthermore, this field study will be useful in comparing the RFTM diagnosis with PCR analysis and quantification.
PROJECT PUBLICATIONS:
Dungan, C.F., R.M. Hamilton, K.L. Hudson, C.B. McCollough and K.S. Reece. (2002) Two Epizootic Infectious Diseases in Chesapeake Bay Commercial Clams Mya arenaria and Tagelus plebius. Dis. Aquat. Org. 50:67-78.
Reece, K.S. (2002) Utilization of Molecular Genetic Data for Detection, Identification and Description of Perkinsus Species. .Oral presentation at the National Shellfisheries Association Conference April 14-18, 2002, Mystic, CT. Abstract in J. Shellfish. Res. 21(1):376.
Dungan, C.F., R.M. Hamilton, C.B. McCollough, K.S. Reece, and K.L. Hudson. (2002) Epizootic diseases in Chesapeake Bay clams. Oral presentation at the National Shellfisheries Association Conference April 14-18, 2002, Mystic, CT. Abstract in J. Shellfish. Res. 21(1):372.
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