Differential Diagnosis Of Mixed Haplosporidium Costale And Haplosporidium Nelsoni Infections In The Eastern Oyster, Crassostrea Virginica, Using Dna Probes
Haplosporidium costale and Haplosporidium nelsoni are morphologically similar pathogens of the eastern oyster Crassostrea virginica. In the absence of the spore stage, infections of the two species are extremely difficult, if not impossible, to distinguish using traditional light microscopy of stained tissue sections. Species-specific molecular diagnostics were developed for H. costale from the small subunit ribosomal DNA (SSU rDNA) sequence. The polymerase chain reaction (PCR) primers amplified a 557 base pair (bp) region of the H. costale SSU rDNA, but did not amplify DNA from oyster (C. virginica) or from six other haplosporidans (H. nelsoni, H. louisiana, H. lusitanicum, Minchinia teredinis, M. chitonis, or M. tapetis). The DNA probe was used with in sim hybridizations of oyster tissue sections to visualize H. costale plasmodia and prespore stages; it did not hybridize with oyster (C. virginica) or other haplosporidans (H. nelsoni, H. louisiana, or Minchinia teredinis). DNA-based diagnostics for H. costale, in conjunction with molecular tools previously developed for H. nelsoni, have overcome limitations of histological examination. From in situ hybridizations using both probes, some Virginia oysters previously diagnosed with H. costale were found to have mixed infections consisting of approximately 80 to 90% H. costale plasmodia and 10 to 20% H. nelsoni plasmodia, Plasmodia of H. costale were not found in epithelial tissue, only in connective tissue. In addition, use of the DNA probe confirmed the presence of H. costale plasmodia in Virginia oysters collected in the fall, an unprecedented seasonality for an advanced H. costale infection
