Detection of Bonamia ostreae in fixed Ostrea edulis tissues by use of specific PCR assays

Infection by the parasite Bonamia ostreae has infected and caused major mortality of the flat oyster, Ostrea edulis, over the last 25 years throughout the coasts of Europe and the United States of America. The conventional techniques for the diagnosis of infection with Bonamia ostreae are typically by histology and cytology. Both have a low sensitivity and Bonamia ostreae in weekly infected oysters can remain undetected when analyzed by such techniques. Molecular methods like the Polymerase Chain Reaction have recently been applied for a more reliable and sensitive detection of Bonamia ostreae. The aim of this project was to optimize a PCR for the specific detection of the 18S Small Ribosomal subunit rDNA gene of Bonamia ostreae in formalin fixed Ostrea edulis tissues. While the PCR was successfully optimized for purified oyster DNA from fresh tissue it was difficult to apply on formalin fixed oyster tissues due to poor quality DNA from the fixed tissues. Ethanol fixed tissues were also tested for Bonamia ostreae, however, the primers were not specific for Bonamia ostreae and uninfected oysters also tested positive which led to the conclusion that the PCR could not be used as a reliable detection method for Bonamia ostreae in oysters. Despite using alternative primers which were designed to amplify other components of the Bonamia ostreae genome no consistent results were achieved to reliably use the PCR method for the accurate detection of Bonamia ostreae in oysters. The conclusion of this project is that other genomic sites in Bonamia ostreae must be identified as a target for PCR for this test to be specific