Detection of Bonamia ostreae Based on Small Subunit Ribosomal Probe

Abstract

Bonamia ostreae is a protozoan parasite of the flat oyster, Ostrea edulis, which has caused significant loss of oysters in Europe over the last decade. B. ostreae was purified from infected flat oysters and DNA was extracted. The nearly complete small subunit rDNA gene of B. ostreae was amplified using universal oligonucleotides and the PCR product was cloned and sequenced. BLAST research with this sequence revealed similarities to Haplosporidium nelsoni, Haplosporidium costale, and Minchinia teredinis. These data suggest that B. ostreae may be included in the genus Haplosporidium. Specific B. ostreae primers were designed for labeling, by PCR, a probe. This probe was successfully used by in situ hybridization to detect B. ostreae in infected flat oysters, thus confirming the accuracy of this SSU rDNA sequence. The probe lead also to the detection of Bonamia sp. in infected Tiostrea chilensis and H. nelsoni in infected Crassostrea virginica but not Mikrocytos mackini infected Crassostrea gigas. These primers were also used to detect B. ostreae from infected oyster tissues by PCR. This B. ostreae SSU rDNA gene sequence provides genetic information as a first step toward elucidation of the taxonomic boundaries among the microcell organisms. Moreover, the development of DNA detection assays will be valuable specific diagnostic tools.