DETECTION OF HAPLOSPORIDIUM-NELSONI (HAPLOSPORIDIA, HAPLOSPORIDIIDAE) IN OYSTERS BY PCR AMPLIFICATION

Haplosporidium nelsoni is a protistan pathogen of the eastern oyster Crassostrea virginica, and has contributed to the decline of the oyster population in the Chesapeake Bay. From comparison of the sequence data of the 16S-like rDNA of H. nelsoni with those of Minchinia teredinis and other related organisms, 2 oligonucleotides which were specific to H. nelsoni and suitable for use as PCR primers were identified. These primers amplified a 564 base pair fragment of the small subunit (SSU) rRNA gene of H. nelsoni, but did not amplify genomic oyster DNA or the SSU rRNA genes of the haplosporidians Haplosporidium costale, Haplosporidium louisiana, or M. teredinis. The PCR primers were able to detect the H. nelsoni SSU rDNA from 50 ng of infected oyster genomic DNA or from 10 fg of cloned H. nelsoni SSU rDNA. The ability of the PCR primers to diagnose H. nelsoni-infected oysters was compared to the established techniques of hemolymph settlement analysis in Farley chambers and histological examination from a sample of 20 oysters. Hemolymph settlement analysis detected infection in 10 oysters and histology revealed infections in 11 oysters. PCR amplification of DNA from hemolymph initially detected infections in 15 oysters and reamplification of the PCR products detected an additional 4 infections. PCR amplification is a more sensitive diagnostic assay for H. nelsoni than traditional techniques.