Diagnosis, genetic variations, virulence, and toxicity of AHPND-positiveVibrio parahaemolyticusinPenaeus monodon

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp (Penaeus monodon) disease caused byVibrio parahaemolyticus(VP) since 2013 in Bangladesh. The aim of this work was to evaluate a PCR and RT-PCR techniques as rapid methods for detectingV. parahaemolyticusAHPND-positiveP. monodonusing genetic markers. Healthy and diseased shrimp (P. monodon) samples were collected from three monitoring stations. The samples were enriched in TCBS plates and DNA extraction from the cultured bacteria. DNA quantifications, PCR amplification, RT-PCR, and gene sequencing were done for the detection ofV. parahaemolyticusAHPND-positiveP. monodon. The sequence of PCR amplicons showed 100% identity and significant alignment withV. parahaemolyticus. The primers used provided high specificity forV. parahaemolyticusin PCR detection compared with anotherVibriospecies. In the PCR, amplification resulted positive amplicons, whereas, non-AHPND isolates showed no amplicons. Neighbor-joining methods indicated that all genes evolved from a common ancestor and clades have different traits with very low genetic distance and low variability. The pairwise alignment scores ofatpA,tox, blaCARB, 16S rRNA, andpirA genes were 100.0, 98.90, 98.89, 95.53, and 41.42, respectively. The RT-qPCR exposed variable expression levels for all genes in the AHPND-positive strain. Homology analysis and distance matrix exhibited all genes to have the lowest similarity and most divergence, offering the highest specificity. In this study, the expression and variability of target genes confirmed the presence ofV. parahaemolyticusin all sampling sites. The results suggested that PCR amplification, RT-qPCR, and gene sequencing can be used for the rapid detection ofV. parahaemolyticusin AHPND-positiveP. monodonthat may lead to subsequent prevention and treatment research in the future for managing this disease.