A new EMS-associated bacterium of the genus Roseateles found in Shrimp

Abstract

Early Mortality Syndrome (EMS), which is an unusually high mortality observed within approximately 35 days of rearing post larvae of shrimp in a culture pond, is a serious threat to the shrimp farming industry especially in China, Malaysia, Vietnam and Thailand. Studies have demonstrated that Vibrio parahaemolyticus harbouring a plasmid containing toxin genes (VPAHPND) is a major causative agent of EMS in the form of acute hepatopancreatic necrosis disease (AHPND). However, shrimp specimens in a number of shrimp ponds exhibiting EMS did not show the pathological characteristics associated with AHPND and the cause of mortality in those ponds remains unexplained. We hypothesized that there are other pathogens involved in EMS or associated with VPAHPND that cause EMS. In this study, we reported a discovery of a bacterium from the genus Roseateles that could kill shrimp in its own right but also work synergistically in mixed challenges to increase the virulence of VPAHPND. In vivo bioassays indicated that the 4-day LC50 of Roseateles alone for shrimp was 105 CFU/ml while that at 102 CFU/ml was 45%. However, mortality of shrimp challenged with VPAHPND at 103 CFU/ml was 65% on day 3 post immersion challenge, while addition of Roseateles at a concentration of 102 CFU/ml (i.e., an additional 100 cells) in a co-challenge test with VPAHPND at 103 CFU/ml (i.e., 1100 cells total) resulted in 75% cumulative mortality on the same day 3 post challenge. A mixture of both at 103 CFU/ml each (2000 cells total) gave 100% mortality in 3 days while 100% mortality from VPAHPND alone required 105 CFU/ml (100,000 cells). These results indicate that a combination of Roseateles and VPAHPND increases shrimp mortality in a synergistic manner. A specific, nested PCR detection method for shrimp Roseateles was developed based on its 16S-23S rRNA intergenic spacer region (ITS region).