Conjugative Transfer of the pVA1-Type Plasmid Carrying the pirABvp Genes Results in the Formation of New AHPND-Causing Vibrio

Abstract

Acute hepatopancreatic necrosis disease (AHPND) has caused sharp declines in aquaculture industries of whiteleg shrimp <i>Penaeus vannamei</i> in Asia and the Americas since 2010. <i>Vibrio parahaemolyticus, V. campbellii, V. owensii</i>, and <i>V. punensis</i> have been proved to cause AHPND. However, the mechanisms underlying the burgeoning number of <i>Vibrio</i> species that cause AHPND is not known. All of AHPND-causing <i>Vibrio</i> bacteria (<i>V</i>_AHPND) harbor a highly homologous plasmid (designated as pVA1-type) carrying <i>pirAB</i>^<i>vp</i> toxin genes. In this study, we demonstrate conclusively that the pVA1-type plasmid can be transferred from <i>V</i>_AHPND to non-pathogenic bacteria. We constructed a pVPGX1-<i>Cm</i>^<i>r</i> plasmid (a pVA1-type plasmid) by adding a chloramphenicol resistance gene as a marker in a donor AHPND-causing <i>V. parahaemolyticus</i> 20130629002S01 (<i>Vp</i>2S01). Horizontal transfer of this plasmid was successfully performed from the AHPND-<i>Vp</i>2S01 to a non-pathogenic strain of <i>V. campbellii</i> at the transfer efficiency of 2.6×10^-8 transconjugant/recipient, and DNase I treatment did not eliminate the transfer. The recipient <i>V. campbellii</i> acquired the pVA1-type plasmid and was shown to produce <i>pirAB</i>^<i>vp</i> RNA and proteins. Challenge studies using the transconjugant caused 100% mortality in exposed groups of <i>P. vannamei</i>. The challenged shrimp, infected with the transconjugant bacteria, showed typical gross signs and histological lesions of AHPND. These results demonstrated the conjugative transfer of an AHPND pVA1-type plasmid. It provides timely information for explaining the increased species of AHPND-causing <i>Vibrio</i> bacteria and will be useful in the development of management strategies leading to the prevention and control of AHPND.