||Morga Benjamin, Arzul Isabelle, Faury Nicole, Gagnaire Beatrice, Chollet Bruno, Renault Tristan
||13th EAFP Internation Conference "Diseases of Fish and Shellfish" European Association of Fish Pathologists
||SSH, Suppression subtractive hybridization, Cytometry, Bonamia ostreae, Parasite, Ostrea edulis, Oyster
||Bonamiosis due to the intrahaemocytic protistan parasite Bonamia ostreae is a European endemic disease affecting flat oysters Ostrea edulis. Control of bonamiosis requires a good knowledge of the parasite life cycle including host-parasite interactions. In this study, we investigated interactions between parasites and haemocytes by flow cytometry and Suppression Subtractive Hybridization (SSH). In both approaches, analyses were performed after 2 hours of contact between haemocytes and parasites purified from highly infected flat oysters. Flow cytometry analyses consisted in testing haemocyte activities including esterase activities, reactive oxygen species (ROS) production and phagocytosis after contact with live parasites and parasites inactivated by heating at 100°C for 5 minutes. Two amounts of parasites per haemocytes (5 per 1 and 10 per 1) were tested and haemocytes alone were used as controls. Contact experiments were performed three times. Flow cytometry revealed a decrease of esterase activities and an inhibition of ROS production after contact with live parasites, while phagocytosis did not present variation in comparison with haemocytes alone. Inactive parasites induced same modifications of haemocyte activities than live parasites but to a lesser extent. SSH performed between haemocytes alone and haemocytes in contact with parasites for 2 hours allowed obtaining 1104 clones among which 391 presented a differential expression between both tested conditions. These clones were sequenced. Sequence analysis allowed the identification of genes expressed by haemocytes and parasites during in vitro infection including genes potentially involved in oyster defence mechanisms and in parasite survival within haemocyte. These genes of interest will be selected for development of real time PCR in order to follow their expression in the context of experimental infections of flat oysters by injection of purified parasites. These results also contribute to improve knowledge of both Ostrea edulis and Bonamia ostreae genomes for which very few data are available. Flow cytometry as well as SSH are two interesting techniques for a better understanding of host pathogen interactions but these in vitro approaches need to be validated in vivo.