In early 2013, Vibrio parahaemolyticus colonizing the shrimp stomach was discovered as the causative agent of acute hepatopancreatic necrosis disease (AHPND). Causative isolates were negative for two human-pathogen markers, thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh). Using isolates of Vibrio previously isolated from disease outbreak ponds in Thailand exhibiting histopathology of AHPND, we found that most were positive for the V. parahaemolyticus species-specific marker gene lecithin-dependent hemolysin (ldh)(non-pathogenic for humans) but negative for the two human-pathogen markers given above.

Subjecting 4 arbitrarily selected V. parahaemolyticus isolates obtained from a single farm in November 2012 to the previously reported immersion challenge protocol, we confirmed that 3 caused high mortality at different rates accompanied by characteristic lesions of AHPND (similar to a positive control isolate from China) while 1 caused high mortality without such lesions. A negative control isolate obtained from shrimp pond sediment in March 2008 caused no mortality and no hepatopancreatic lesions. However, all 4 of the selected Thai isolates gave positive test results with an AP2-AHPND detection method released in December 2013. Thus, the lethal isolates of V. parahaemolyticus obtained from a single disease outbreak differed somewhat in details of pathogenicity, supporting a previous proposal that virulence of AHPND isolates might be determined by mobile genetic elements such as plasmids or bacteriophages. This is being verified by total DNA sequencing of several isolates.

We are also trying to identify the toxin(s) responsible for AHPND virulence using proteomic techniques in order to develop more specific AHPND detection methods and perhaps therapeutic measures. In addition, we are testing V. parahaemolyticus AHPND isolates in dual immersion challenges with bacterial isolates from the genera Delftia, Rhodococcus and Leifsonia (identified as associated with AHPND in our previous metagenomic study) and Shewanella to determine whether these isolates can act synergistically or in an additive manner to increase virulence of AHPND bacteria. We have shown that these isolates on their own may cause high mortality using the same immersion challenge protocol used with V. parahaemolyticus AHPND isolates, but with different histopathology. In addition to AHPND we are investigating three concurrent HP pathologies [high prevalence of the microsporidian Enterocytozoon hepatopenaei, of vermiform, aggregated transformed microvilli (ATM) (sometimes mistaken for gregarines) and of distorted hepatopancreatic tubules.