A sensitive and specific DNA probe for the oyster pathogen Haplosporidium nelsoni

Haplosporidium nelsoni is a pathogen of the eastern oyster, Crassostrea virginica, along the middle Atlantic coast of the U.S. Genomic DNA was extracted from H. nelsoni plasmodia and small subunit (SSU) rDNA was amplified by PCR, cloned and sequenced. The sequence of H. nelsoni SSU rDNA was aligned with that of another haplosporidian, Minchinia teredinis, and with SSU rDNA data of C. virginica and various protists in GenBank. A 21-base oligonucleotide unique to H. nelsoni, designated MSX1347, was commercially synthesized and tested for sensitivity and specificity. In dot blot hybridizations the probe detected 100 pg of cloned H. nelsoni rDNA and the presence of H. nelsoni in 1 microgram of genomic DNA from an infected oyster. It did not hybridize with 1 microgram of genomic DNA from uninfected C. virginica or with cloned SSU rDNA of M. teredinis. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with H. nelsoni plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with oyster tissue, with other common oyster parasites such as P. marinus or Nematopsis sp., or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.), Haplosporidium costale from C. virginica or M. teredinis from shipworms (Teredo spp.).