A Natural Vibrio parahaemolyticus ΔpirAVp pirBVp+ Mutant Kills Shrimp but Produces neither PirVp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus (VP) isolates that harbor a pVA plasmid encoding toxins PirvpA and PirvpB (VPAHPND). These are released from VPAHPND that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that VP isolate XN87 lacked PirvpA but carried PirvpB Unexpectedly, western blot analysis of proteins from culture broth of XN87 revealed absence of both toxins and the lack of PirvpB was further confirmed by ELISA assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. By contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that pVA plasmid of XN87 contained a mutated PirvpA gene interrupted by out-of-frame insertion of a transposon gene fragment. The upstream region and beginning of the original PirvpA gene remained intact, but the insertion caused a 2-base reading-frame shift in the remainder of the PirvpA gene sequence and in the downstream PirvpB gene sequence. RT-PCR and sequencing of 5HP revealed a bi-cystronic PirvpAB mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed some VP isolates carrying mutant pVA plasmids that produce no Pirvp toxins can cause mortality in EMS outbreak ponds but may not have been previously recognized as AHPND-related because they do not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates that harbor pVA1 plasmid encoding toxins PirvpA and PirvpB (VPAHPND). The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). VP isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in EMS ponds that is not ascribed to VPAHPND Thus, they pose an additional threat to shrimp farmers that would be missed by current testing for VPAHPND Moribund shrimp from EMS ponds that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates that can then be confirmed by additional PCR tests for presence of a pVA plasmid.