Molecular identification and characterization of Vibrio parahaemolyticus causing Acute Hepatopancreatic Necrosis Disease in cultured Shrimp

Abstract

Acute Hepatopancreatic Necrosis Disease (AHPND), also called Early Mortality Syndrome (EMS), is a recently emergent shrimp bacterial disease caused by strains of Vibrio parahaemolyticus that contain a unique virulent plasmid, resulted in substantial economic losses since 2009.AHPND has caused severe mortalities up to100% in farmed populations of marine shrimp Penaeus monodon. The purpose of this thesis work was to identify and characterize the pathogenic strain of V. parahaemolyticus causing EMS in cultured shrimp using classical and molecular techniques. To conduct this work, shrimp samples were collected from three different locations of south-west shrimp farming region of Bangladesh viz.Sadar Upazilla of Satkhira district, Mongla and Morrelganj Upazilla under Bagerhat district. The shrimp samples were processed for microbial load count and to isolateV. parahaemolyticus strains. Four types of bacteriological culture media were used to enumerate the Total Bacterial count (TBC), Total Vibrio count (TVC). Besides Classical microbiology, molecular approaches (16S rRNAgene sequencing, pathogenic gene PCR using AP3 and AP4 primers) wereperformed to identify the pathogenic strains of V. parahaemolyticus causing AHPND in cultured shrimp. Shrimp suffering from AHPND showed significantly atrophy of hepatopancreas (HP), pale to white hepatopancreas due to pigment loss in connective tissue capsules and guts with discontinuous or no contents. In this study, TBC, TVC on TCBS and on HiChrome showed little variation of AHPND affected shrimp. TBC was found highest(6.37×108cfu/g) in shrimp of Satkhira Sadar Upazilla, whereas highest number of Total Vibrio on TCBS agar (2.40×107 cfu/g) was found in shrimp of Mongla Upazilla. In this study, among 46 isolates, representative eighteen isolates were checked for the species-specificdetection of V. parahaemolyticus using ldh primers; and tdhprimerswere used for the detection of human pathogenicity of V. parahaemolyticus. Detection of ldh gene fragment in the isolates showed positive result for ldhbut isolates were negative for human toxigenic gene tdh. The representative isolates were also subjected to 16S rRNA gene sequencing and were identified as V. parahaemolyticus. Multiple sequence alignment was performed to find out the polymorphic sites among the sequenced strains with considering 1386 bp nucleotides where 1.15 % dissimilarities were observed.Six strains (Vp2, Vp4, Vp21, Vp27, Vp35 and Vp39) were highly similar to each other but dissimilar to only two sites (34, 35 bp) on their polymorphic region. Phylogenetic analysis also confirmed the taxonomic positionof the isolates as V. parahaemolyticus. On the other hand, 18 V. parahaemolyticus isolates were further characterized to check the AHPND positivity using AP3 and AP4 primer. Twelve isolates showed positive result for AP3 and fourteen isolates for AP4 primer that indicate those isolates were AHPND positive which caused EMS in cultured shrimp.This study also reported that all the representative strainsof AHPND positive V. parahaemolyticuswere resistant to Gentamycin whereas all the strain showed sensitivity to Chloramphenicol, Nalidixic Acid,Nitrofurantion and Tetracycline. This report also agreed with the mortalities of shrimp that occurred within 30 days after stocking in shrimp farms of three different south-west regions of Bangladesh which caused by AHPND positive V. parahaemolyticus;and to the best of my knowledge, this is the first report of this shrimp pathogen in Bangladesh.