Detection of V. harveyi in shrimp postlarvae and hatchery tank water by the Most Probable Number technique with PCR

Abstract

V. harveyi is the cause of serious disease in the shrimp industry in Thailand during cultivation. In this study, the gyrB gene of V. harveyi NICA, isolated from shrimp in Thailand, was sequenced. A pair of specific primers (A2B3) was designed that allowed amplification of a 363 bp gene fragment of V. harveyi. No cross reaction was detected in 17 other Vibrio species tested except for V. carchariae which is a synonym for V. harveyi. The possibility of using A2B3 for confirmation and enumeration of V. harveyi by PCR was demonstrated. Of 40 possible V. harveyi strains isolated from seafood on the basis of their growth on TCBS plates and biochemical reactions, 36 gave a reaction with the specific primers. The primers could detect V. harveyi at a level of as few as 15 cells/ml. The Most Probable Number (MPN) technique was applied to enumerate V. harveyi. We have demonstrated that when PCR was applied directly to the enrichment broth of shrimp artificially inoculated with V. harveyi, the MPN value was no different from the MPN value obtained using the standard technique with selective agar. This technique was employed to enumerate V. harveyi in postlarvae and hatchery tank water. V. harveyi were detected in 18 out of 21 postlarval samples and in 14 out of 21 tank water samples. The numbers of V. harveyi detected in postlarvae and water were 150–1.1 × 10 8/g postlarvae and 7–4.6 × 10 4/ml of water samples, respectively. Screening of postlarvae to reduce the high risk of V. harveyi contamination in cultivation ponds is suggested as a measure to prevent the catastrophic losses caused by V. harveyi disease.