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

The development of diagnostic assays more sensitive and specific than tradi tional histological techniques is important for the management of bonamiasi s 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 g ill and hemolymph. The presence of a 760 bp PCR amplification product corre sponded with B, ostreae infections determined cytologically in 185 oysters from Ireland, Spain, and the USA. All (100%) 'heavily' and 'moderately' inf ected oysters, 86.7% of the 'lightly' infected oysters, and 66.7% of the 's carcely' infected oysters were confirmed to be infected using the PCR. In a ddition, 37.9% of the oysters in which B. ostreae was not detected using cy tology were positive using the PCR. Sampling error and the subjectivity of cytological diagnoses are the likely sources of disagreement between diagno stic methods in oysters with very light infections. The PCR assay developed here is more sensitive and less ambiguous than standard histological and c ytological techniques, Phylogenetic analysis of DNA sequence data confirmed B, ostreae to be a member of the Haplosporidia.