Development of a co-agglutination method for detection of Aeromonas hydrophila as causative agent of motile Aeromonas septicemia (MAS) disease in gourami (Osphronemus goramy)

Aeromonas hydrophila is an opportunistic pathogen causing high mortality and economic burden in freshwater fish farming. This study aims to develop a coagglutination method for detecting and creating Aeromonas hydrophila diagnostic rapidly. In this study, we injected rabbits (±2kg weight) with 1mL of A. hydrophila antigen suspension 1.2 x 109cfu mL–1 at one week intervals (three times, intra vena) respectively. The gouramis (15.48±0.55g-1 weight) were infected by Aeromonas hydrophila, Aeromonas sobria, Aeromonas salmonicida, Streptococcus agalactiae, and Pseudomonas aeruginosa separately with 0.1 mL fish–1 and 108 cfu mL–1 bacterial cell suspensions. The antiserum was purified to couple with the Staphylococcus aureus suspension protein A, in a 1:1 (v/v) ratio and used by the co-agglutination reagent. We compared this method with standard polymerase chain reaction (PCR) for A. hydrophila detection. The rabbit antibody reaction occurred only against A. hydrophila antigen showing specificity of the gourami tissue supernatant within 10-30 seconds. The sensitivity test had a detection limit of 106 cfu mL–1. Comparison detection method with PCR showed that positive result of A. hydrophila was located in 209 bp. Coagglutination method could detect A. hydrophila in the internal organ of fish at 12h after injection, but the PCR method could detect at one hour after injection. This research concluded that co-agglutination method could detect A. hydrophila specifically, sensitively, rapidly and practically in laboratory and field examination.