The Study On Detection And Identification Of Bonamia Ostreae From Oysters

Bonamiasis is a parasitic disease of flat oysters that has resulted in serious mortalities and destroyed the oyster industries in several countries. It is listed by OIE as a Notifiable Disease. There is no Bonamiasis occurrence in our country. However, lately, we have identified Bonamia-infected flat oysters in aquatic nursery at the east bays of our country. Thus, it is of economic importance to develop the powerful diagnostic methods of Bonamiasis, especially for the international trade and native aquaculture industry.A series of examination and detection methods were established in this study. First of all, we collected oyster samples from east China bays. By making wet smears of oyster's heart (ventricle preferably), blood smears and paraffin section of the gill and mantle, the histological examination methods were developed. Results showed that Bonamia-like plasmodia were found in Hemolymph by microscope examination. Bonamia was recognized as ring-shaped and 2-5μm in diameter, with purple nucleus and pale blue cytoplasm when dyed with Giemsa's stain. Using the above identified positive samples as materials, the specific polymerase chain reaction (PCR) method was developed for the sensitive detection of Bonamiosis. When combined with PCR-RFLP method, the Bonamia species isolated in this study could be identified to be Bonamia ostreae. To develop an in situ hybridization detection method, the Bonamia Specific PCR products of 300bp in size, was chosen to be labeled with Digoxigenin, Results indicated that the DIG-labeled probe was specific for the detection of Bonamia infection.In the end, the real time quantitative PCR method was developed to detect Bonamia contamination in oysters. The primers and TaqMan probe were designed to target the Internal Transcribed Spacer 1 (ITS-1) region of rRNA gene, a relatively conserved genomic region among Bonamia species. Reaction solutions and conditions were optimized to obtain better sensitivity and specificity for the genus Bonamia. Results indicated that the sensitivity was 10 copies of plasmid DNA inserted with targeted DNA fragment. And specific enough to distinguish Bonamia from other protistan parasites.111 samples of oyster, collected from east China bays, were subjected to real time PCR detection. As result, the Bonamia infection rate was 60.36%.In conclusion, the examination and identification methods of Bonamia infection from Oysters, developed in this experiment, will supply powerful tools for the entry-exit inspection and quarantine of Bonamia, and can meet the demands of epidemiological investigation of Bonamia in Oyster farms in our country.