DAO 42:199-206 (2000)  -  doi:10.3354/dao042199

Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia

Ryan B. Carnegie1,*, Bruce J. Barber1, Sarah C. Culloty2, Antonio J. Figueras3, Daniel L. Distel1,4

1School of Marine Sciences and
4Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono, Maine 04469, USA
2Dept of Zoology and Animal Ecology, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland
3Instituto de Investigaciones Marinas, CSIC, Eduardo Cabello, 6, 36208 Vigo, Pontevedra, Spain

ABSTRACT: The development of diagnostic assays more sensitive and specific than traditional histological techniques is important for the management of bonamiasis in flat oysters Ostrea edulis. A specific polymerase chain reaction (PCR) protocol was developed for the detection of very small amounts of Bonamia ostreae (Pichot et al. 1980) ribosomal DNA (rDNA) in bulk DNA from oyster gill and hemolymph. The presence of a 760 bp PCR amplification product corresponded with B. ostreae infections determined cytologically in 185 oysters from Ireland, Spain, and the USA. All (100%) Œheavily¹ and Œmoderately¹ infected oysters, 86.7% of the Œlightly¹ infected oysters, and 66.7% of the Œscarcely¹ infected oysters were confirmed to be infected using the PCR. In addition, 37.9% of the oysters in which B. ostreae was not detected using cytology were positive using the PCR. Sampling error and the subjectivity of cytological diagnoses are the likely sources of disagreement between diagnostic methods in oysters with very light infections. The PCR assay developed here is more sensitive and less ambiguous than standard histological and cytological techniques. Phylogenetic analysis of DNA sequence data confirmed B. ostreae to be a member of the Haplosporidia.

KEY WORDS: Bonamia ostreae · PCR · Haplosporidia · Ostrea edulis

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