Isolation and identification of haemolytic Vibrio parahaemolyticus from haemolymph of cultured shrimp Litopenaeus vannamei

Abstract

The pandemic pathogen Vibrio parahaemolyticus is normal inhabitant of the marine environment which causes pathogenesis in both the human beings as well as farmed aquatic animals including shrimps. In the present study, V. parahaemolyticus was isolated from the haemolymph of the widely cultured shrimp, Litopenaeus vannamei. The cultured shrimps were brought from local shrimp farm in Ernakulam district to the laboratory and haemolymph was drawn aseptically. The haemolymph was enriched overnight in 3 per cent alkaline peptone water, streaked on TCBS agar and incubated at 30 degree celsius for 24 hours. Ten well isolated colonies were picked, purified and haemolytic studies were carried out on human blood agar. Among ten isolates, four isolates that produced distinct zone of beta-haemolysis on blood agar plate were selected and biochemical identification was carried out using API 20 NE identification system. The results revealed that the four isolates were V. parahaemolyticus. PCR detection was carried out using the species specific ToxR gene primers and all the four isolates produced the 368-bp amplicon that is specific to V. parahaemolyticus. Thermostable direct hemolysin and TDH-related hemolysin are considered to be major virulence factors of V. parahaemolyticus. Though the four isolates were haemolytic on human blood, none of the strains were positive for TDH and TRH genes which suggest that some unknown factors might have been responsible for haemolysis. It was concluded that the haemolymph of shrimp harbours pathogenic bacteria such as V. parahaemolyticus which invade when the animal is under stress and produce disease mainly by the production of haemolytic enzymes. Therefore, proper management of the farms has to be carried out regularly to prevent the outbreak of disease.