Incidence and Molecular Typing of Vibrio parahaemolyticus from Tiger Shrimp Culture Environments along the Southwest Coast of India

Abstract

Vibrio parahaemolyticus is one of the most prevalent food-borne pathogens along the southwest coast of India, where marine foods are frequently consumed. Shrimp (Penaeus monodon) and environmental samples were collected from aquaculture farms located in and around Cochin. Confirmation of the biochemically identified strains with species-specific toxR gene and detection of virulent genes viz., tdh and trh was performed by polymerase chain reaction (PCR). The phenotypic markers for the presence of tdh and trh genes were assayed by Kanagawa phenomenon and urease activity, respectively. Protease activity was examined to identify other potential virulence factors. After phenotypic characterization of bacterial strains fingerprinting of genomic DNA was carried by various typing methods, viz., random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence (ERIC), repetitive extragenic palindromic sequence (REP), and ribosomal gene spacer sequence (RS) PCR methods to assess the genetic diversity within the isolates. Eighteen percent of the samples were found positive for the incidence of V. parahaemolyticus by biochemical protocols and toxR (368 bp) targeted PCR. PCR analyses revealed 1% of the samples positive for tdh (269 bp) and trh (500 bp) gene. RAPD analysis revealed clustering of toxigenic strains into a single group. Cluster analysis revealed the conglomeration of isolates into two, five, and seven major groups using RS, ERIC, and REP PCR methods, respectively. RS PCR generated fewer amplified bands compared to REP and ERIC PCR methods, thus giving scope for higher discrimination. Moreover, RS PCR patterns were more discernible visually from other patterns, suggesting RS PCR as a considerably practical method for routine use.

Key Words:

• PCR
• Penaeus monodon
• tdh
• trh
• typing
• Vibrio parahaemolyticus

ACKNOWLEDGMENTS

The authors are grateful to Dr. K. Devadasan, director, Central Institute of Fisheries Technology, Cochin, and Prof. (Dr.) Mohan Joseph Modayil, director, Central Marine Fisheries Research Institute for providing necessary facilities and encouragement for carrying out the study. The guidance and support rendered by Dr. Nirmala Thampuran, head of the Microbiology Fermentation Biotechnology Division, Central Institute of Fisheries Technology, Cochin, to carry out this work is gratefully acknowledged.