Detection of ctx gene positive non-O1/non-O139 V. cholerae in shrimp aquaculture environments

Abstract

Water and post-larvae samples from black tiger (Penaeus monodon) shrimp hatcheries; pond water, pond sediment and shrimp from aquaculture farms were screened for the presence of V. cholerae. A V. cholerae-duplex PCR method was developed by utilizing V. cholerae species specific sodB primers and ctxAB genes specific primers. Incidence of V. cholerae was not observed in shrimp hatchery samples but was noticed in aquaculture samples. The incidence of V. cholerae was higher in pond water (7.6%) than in pond sediment (5.2%). Shrimp head (3.6%) portion had relatively higher incidence than shrimp muscle (1.6%). All the V. cholerae isolates (n = 42) belonged to non-O1/non-O139 serogroup, of which 7% of the V. cholerae isolates were potentially cholera-toxigenic (ctx positive). All the ctx positive V. cholerae (n = 3) were isolated from the pond water. Since, cholera toxin (CT) is the major contributing factor for cholera gravis, it is proposed that the mere presence of non-O1/non-O139 V. cholerae need not be the biohazard criterion in cultured black tiger shrimp but only the presence of ctx carrying non-O1/non-O139 V. cholerae may be considered as potential public health risk.

Keywords: Black tiger shrimp; Choleratoxigenic; Duplex PCR; Non-O1 and non-O139; Penaeus monodon; Vibrio cholerae; ctx.

Fig. 1

PCR for the detection of V. cholerae using species specific primers. Lane 1–3, V. cholerae isolates from shrimp pond water; Lane 4–5, V. cholerae isolates from shrimp pond sediment; Lane 6, V. cholerae isolates from shrimp muscle; Lane 7, V. cholerae (MTCC 3906); All V. cholerae yielded the species specific amplicon (248 bp) with sodB primers; Lane8, 100 bp DNA ladder (GeNei)

Fig. 2

Duplex PCR for the simultaneous detection of V. cholerae and differentiation of cholera toxin producing V. cholerae isolates. Lane 1, Negative control; Lane 2–3, Cholera toxin negative V. cholerae isolates from shrimp pond water; Lane 4–6, Cholera toxin positive V. cholerae isolates from shrimp pond water; Lane 7, V. cholerae (MTCC 3906); Lane 8, 100 bp DNA ladder (Gene Ruler™, Fermentas)

Fig. 3

PCR for testing the O1 and O139 status of ctx bearing V. cholerae isolates from shrimp pond water. Lanes 1–3, ctx positive V. cholerae isolates from shrimp aquaculture system failed to yield O1 specific or O139 specific amplicon indicating that the isolates were V. cholerae non-O1/non-O139; lane 4, Known V. cholerae O1 isolate from laboratory culture collection; lane 5, V. cholerae (MTCC 3906) – V. cholerae O139; lane 6, Known V. cholerae non-O1/non-O139 from laboratory culture collection; lane 7, 100 bp DNA ladder (GeNei)

Fig. 4

Performance of V. cholerae duplex-PCR in different food matrices. Lane 1, Tiger shrimp, (Penaeus monodon); Lane 2, 3 spot crab (Portunus sangulnolentus); Lane 3, Freshwater fish (carp, Labeo rohita); Lane 4, marine fish (tuna, Euthynnus affinis) meats spiked with V. cholerae (ctx positive) cells yielded positive PCR result for the presence of Vibrio cholerae species specific amplicon (248 bp) and cholera toxin specific amplicon (777 bp amplicon); Lane 8, 100 bp DNA ladder (GeNei)