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 simil ar 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 stain ed tissue sections. Species-specific molecular diagnostics were developed f or H. costale from the small subunit ribosomal DNA (SSU rDNA) sequence. The polymerase chain reaction (PCR) primers amplified a 557 base pair (bp) reg ion of the H. costale SSU rDNA, but did not amplify DNA from oyster (C. vir ginica) or from six other haplosporidans (H. nelsoni, H. louisiana, H. lusi tanicum, Minchinia teredinis, M. chitonis, or M. tapetis). The DNA probe wa s 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 Minchini a teredinis). DNA-based diagnostics for H. costale, in conjunction with mol ecular 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 plas modia 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 oy sters collected in the fall, an unprecedented seasonality for an advanced H . costale infection.