Diagnosis, genetic variations, virulence, and toxicity of AHPND-positive Vibrio parahaemolyticus in Penaeus monodon
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Diagnosis, genetic variations, virulence, and toxicity of AHPND-positive Vibrio parahaemolyticus in Penaeus monodon Md. Mer Mosharraf Hossain, et al. [full author details at the end of the article] Received: 25 May 2020 / Accepted: 18 September 2020 / Published online: 28 September 2020 # Springer Nature Switzerland AG 2020
Abstract
Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp (Penaeus monodon) disease caused by Vibrio parahaemolyticus (VP) since 2013 in Bangladesh. The aim of this work was to evaluate a PCR and RT-PCR techniques as rapid methods for detecting V. parahaemolyticus AHPND-positive P. monodon using 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 of V. parahaemolyticus AHPND-positive P. monodon. The sequence of PCR amplicons showed 100% identity and significant alignment with V. parahaemolyticus. The primers used provided high specificity for V. parahaemolyticus in PCR detection compared with another Vibrio species. 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 of atpA, tox, blaCARB, 16S rRNA, and pirA 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 of V. parahaemolyticus in all sampling sites. The results suggested that PCR amplification, RT-qPCR, and gene sequencing can be used for the rapid detection of V. parahaemolyticus in AHPND-positive P. monodon that may lead to subsequent prevention and treatment research in the future for managing this disease. Keywords Diagnosis . Penaeus monodon . Vibrio parahaemolyticus . Acute hepatopancreatic necrosis disease (AHPND genetic variations, virulence, and toxic gene)
Introduction Vibriosis is one of the major disease problems in shellfish and finfish aquaculture responsible for the mortality of cultured shrimp worldwide (Mohney et al. 1994). Opportunistic pathogens
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with highly virulent strains (Ishimaru et al. 2009) include Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Vibrio parahaemolyticus (Kannapiran et al. 2009). In an initial outbreak in the southwest of China in 2009, acute hepatopancreatic necrosis disease (AHPND) had its rapid, lethal effect in early stages of shrimp and had spread all over the world. Over the past 40 years, Asia’s fa
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