A Natural Vibrio parahaemolyticus ΔpirA(Vp) pirB(Vp+) Mutant Kills Shrimp but Produces neither Pir(Vp) Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VP(AHPND) isolates) that harbor a pVA plasmid encoding toxins PirA(Vp) and PirB(Vp). These are released from VP(AHPND) isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA(Vp) but carried pirB(Vp). Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB(Vp) was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VP(AHPND) isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA(Vp) gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA(Vp) gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA(Vp) gene sequence and in the downstream pirB(Vp) gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB(Vp) mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir(Vp) toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions. IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VP(AHPND) isolates) that harbor the pVA1 plasmid encoding toxins PirA(Vp) and PirB(Vp). The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus 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 ponds experiencing an outbreak of EMS that is not ascribed to VP(AHPND). Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VP(AHPND). Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.