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Abstracts
Workgroup: Frontiers in Disease Research
Molecular genetic analysis of Perkinsus marinus: Comparisons among laboratory isolates and natural populations
Principal Investigator(s):
David Bushek, University of South Carolina, bushek@sc.edu
Co-Investigator(s):
Kimberly Reece and John Graves, Virginia Institute of Marine Science
Funding Period: Sept. 1997 - May 2000
This project represented a continuation of a previous ODRP project (NA47FL-0162) in which molecular genetic markers were developed to begin investigating the population genetics of P. marinus.
Objective 1 tested ten primer pairs remaining from the previous study and seven new primer pairs were tested, resulting in a total of 23 PCR primer pairs for P. marinus. Thirteen produced a single amplification product and eight were polymorphic, making them useful for discriminating strains. All were made available for use in addressing other ODRP priorities.
Objective 2 significantly expanded the collection of P. marinus isolates available to the research community from twelve to more than 80. Most have been deposited with ATCC for general distribution as well as nearly 100 clonal cultures of many of the isolates.
Objective 3 determined the genetic similarity of P. marinus isolates using the eight polymophic markers from objective 1. No more than two alleles were observed with any marker. Of a potential 6,561 genotypes, only 13 were identified among 162 P. marinus cultures examined. Several clonal cultures indicated that individual oysters were infected with more than one strain of P. marinus. Genetic analyses indicated that three predominant genotypes were relatively genetically distant. Several genotypes were closely related to one of these common genotypes and two were only observed in clonal cultures.
Objective 4: To examine the geographic distribution of P. marinus strains, the US Atlantic coast was divided into two regions based on the historical and current range of the parasite, with the Gulf of Mexico representing a third region. The distribution of genotypes or genetic strains varied within and among regions. Almost 88% of the isolates possessed one of three predominant genotypes (#1, 3 and 4), but these were not equally distributed among regions. These data indicate that while P. marinus is ubiquitously distributed along the Atlantic and Gulf Coasts of the US, different regions possess unique assemblages of genetic strains. Direct isolation of P. marinus DNA from infected oysters was also successfully performed using these molecular tools.
IMPACTS and/or BENEFITS:
- Development of strain-specific molecular markers
- Establishment of numerous geographically and genetically distinct isolates and clonal cultures of P. marinus
- Verification of differential distribution of P. marinus strains indicating potential risks of moving oyster stock between regions despite presence of other strains.
PROJECT PUBLICATIONS AND PRESENTATIONS:
Reece, K.S., D. Bushek, K.L. Hudson and J.E. Graves. 2001. Geographic distribution of Perkinsus marinus genetic strains along the Atlantic and Gulf coasts of the USA, Marine Biology, 139: 1047-1055.
Bushek, D., R. A. Holley and K. S. Reece. 2000. Use of Micromanipulation and "Feeder" Cultures to Clone the Protozoan Oyster Pathogen Perkinsus marinus. J. Eukaryotic Microbiol., 47(2):164-166
Reece, K., J. Graves and D. Bushek. 1997. Molecular markers for population genetic analysis of Perkinsus marinus. Mol. Mar. Biol. Biotech., 6(3):197-206.
Reece, K.S., K.L. Hudson and D. Bushek. Analysis of genetic variation in Perkinsus marinus: Implications for development of DNA-based molecular diagnostics. Oral presentation at the International Conference of the European Association of Fish Pathologists, September 18-23, 1999, Rhodes, Greece
Reece K., D. Bushek and K. Hudson. Molecular genetic studies of Perkinsus: examination of inter- and intra-specific variation. World Aquaculture Society Annual Meeting, May 1-7, 2000. Nice, France.
Reece, K.S. D. Bushek and K. Hudson. 1999. Analysis of the geographic distribution of Perkinsus marinus strains. Nat'l Shellfisheries Assoc. Ann. Mtg., Halifax, Nova Scotia. Apr. 18-22, 1999. J. Shellfish Res. 18(1):320
Reece, K.S., K.L. Hudson and D. Bushek (1999) Analysis of genetic variation in strains of the oyster pathogen Perkinsus marinus. Oral presentation, 24th Annual Eastern Fish Health Workshop. March 9-12, 1999, Atlantic Beach, NC.
Reece, K.S. , J. E. Graves, D. Bushek and K. Hudson. Analysis of genetic variation in the oyster pathogen Perkinsus marinus. 2nd International Conference on Shellfish Restoration. Hilton Head, SC. November 1998., J. Shellfish Res., 17(4):1311-1312.
Reece, K. S., D. Bushek, K. Hudson, and J. Graves. Molecular genetic analysis of Perkinsus marinus, a protozoan pathogen. 3rd International Symposium on Aquatic Animal Health. Baltimore, MD. August 1998
Bushek, D., Reece, K., Graves, J., Holley, R. and Hudson, K. Development of molecular markers for population genetic analysis of Perkinsus marinus. SERRS Spring Meeting. Athens, GA. March 1998
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