A natural Vibrio parahaemolyticus ΔpirA^VppirB^Vp+mutant kills shrimp but produces neither Pir^Vptoxins nor acute hepatopancreatic necrosis disease lesions

Abstract

© 2017 American Society for Microbiology. Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPNDisolates) that harbor a pVA plasmid encoding toxins PirAVpand PirBVp. These are released from VPAHPNDisolates 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 pirAVpbut carried pirBVp. Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirBVpwas 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 VPAHPNDisolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirAVpgene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirAVpgene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirAVpgene sequence and in the downstream pirBVpgene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirABVpmRNA 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 PirVptoxins 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.