CHARACTERIZATION OF PIR A/B TOXIN-CARRYING Vibrio STRAINS FROM MEXICAN NORTHWESTERN SHRIMP FARMS

Since the first outbreaks in 2013, Vibrio parahaemolyticus was identified as responsible for causing Acute Hepatopancreatic Necrosis Disease (AHPND), due to the presence of the binary toxins Pir A/B; pathogenic for shrimp.  However, other species of Vibrio have been reported containing these toxins: V. punensis, V. harveyi, V. campbellii, and V. owensii. The aim of this study was to characterize Pir A/B toxin-carrying Vibrio strains present in shrimp hepatopancreas (HP) from ponds with or without symptoms of AHPND, and isolated from sediments and pond water from shrimp farms in the northwestern coast of Mexico.

Samples were collected and immediately enriched in TSB with 1.5% NaCl at 30°C for 18-24 h. After incubation, they were inoculated onto TCBS agar plates and further purified in CHROMagar Vibrio (CV), incubation at 30°C for 18-24 h. Isolates were tested by nested PCR for the presence of the pir A/B gene. Strain identification was carried out by sequencing a 1,487 bp fragment of the 16S rRNA gene of positive strains; and by multiplex PCR with specific genes for V. parahaermolyticus (flaE), V. alginolytiucs (vppC), and V. harveyi (hly). Nested PCR showed to be highly specific and sensitive for the detection of the pir A/B-carrying Vibrio strains (Fig. 1), detecting a total of 20 isolates from shrimp HP (15), sediment (3), and water (2). All isolates formed green colonies in TCBS; but only 16 strains formed mauve colonies in CV, which is expected for V. parahaemolyticus, while 4 strains formed blue colonies with a clear halo, expected for V. cholerae and V. vulnificus, according to the manufacturer specifications.

The multiplex PCR amplified only for the flaE gene of V. parahaemolyticus in all the isolates. No amplification was obtained for the other genes (Fig. 2). The sequencing of the 16S rRNA gene showed high identity values for all the species of the Harveyi clade of the Vibrio species. A phylogenetic tree showed a poor resolution of the sequences with very low bootstrap values (Fig. 3).