Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp

Abstract

The 69 kb plasmid pVPA3-1 was identified in Vibrio parahaemolyticus strain 13‑028/A3 that can cause acute hepatopancreatic necrosis disease (AHPND). This disease is responsible for mass mortalities in farmed penaeid shrimp and is referred to as early mortality syndrome (EMS). The plasmid has a GC content of 45.9% with a copy number of 37 per bacterial cell as determined by comparative quantitative PCR analyses. It consists of 92 open reading frames that encode mobilization proteins, replication enzymes, transposases, virulence-associated proteins, and proteins similar to Photorhabdus insect-related (Pir) toxins. In V. parahaemolyticus, these Pir toxin-like proteins are encoded by 2 genes (pirA- and pirB-like) located within a 3.5 kb fragment flanked with inverted repeats of a transposase-coding sequence (1 kb). The GC content of these 2 genes is only 38.2%, substantially lower than that of the rest of the plasmid, which suggests that these genes were recently acquired. Based on a proteomic analysis, the pirA-like (336 bp) and pirB-like (1317 bp) genes encode for 13 and 50 kDa proteins, respectively. In laboratory cultures of V. parahaemolyticus 13-028/A3, both proteins were secreted into the culture medium. We developed a duplex PCR diagnostic method, with a detection limit of 10(5) CFU ml(-1) and targeting pirA- and pirB-like genes in this strain of V. parahaemolyticus. This PCR protocol can reliably detect AHPND-causing strains of V. parahaemolyticus and does not cross react with non-pathogenic strains or with other species of Vibrio isolated from shrimp ponds.

Fig. 1

Plasmid pVPA3-1. All predicted open reading frames (ORFs) are shown as arrows in different colors. Direction of arrowhead represents the transcriptional orientation

Fig. 2

Schematic representation of the comparative analysis of the pirA- and pirB-like genes in Vibrio parahaemolyticus with the pirA and pirB genes of Photorhabdus luminescens. Regions for the pirA and pirB genes are shown with a symmetrical gene order. Translated BLAST (tblastx, score cut-off: 30) was used to align translated genome sequences. The color intensity is proportional to the sequence homology. Nucleotide base pairs are indicated between grey lines and GC contents (%) are shown

Fig. 3

PCR detection of pirA- and pirB-like genes. (A) Amplifications with pathogenic acute hepatopancreatic necrosis disease (AHPND)-Vibrio parahaemolyticus strains and non-pathogenic strains. Pathogenic strains collected from Mexico (Lane M: 1 kb plus DNA ladder; Lane 1: strain 13-511/A1; Lane 3: strain 13-306/D4, see Table 2) and Vietnam (Lane 2: strain 13-028/A3; Lane 4: strain 12-194G); non-pathogenic strains from shrimp farms in Vietnam (Lane 5: strain 13-028/A2), India (Lane 6: strain 13-488L), and USA (Lane 7: strain 13-431). (B) Determination of sensitivity by serially diluting V. parahaemolyticus 13-028/A3 DNA; bacterial concentrations were as follows: 1 × 10⁷ (Lane 1), 1 × 10⁶ (Lane 2), 1 × 10⁵ (Lane 3), 1 × 10⁴ (Lane 4), and 1 × 10³ CFU ml⁻¹ (Lane 5), and a non-template control (Lane 6). Lane M: 1 kb plus DNA ladder