Detection of PirA/B Toxin Genes for Acute Hepatopancreatic Necrosis Disease (AHPND) and Vibrio parahaemolyticus in Penaeus vannamei Culture from Major White Shrimp Producing Farms in Malaysia

Abstract

The acute hepatopancreatic necrosis disease (AHPND) epidemic from 2010 to 2013 significantly affected white shrimp (Penaeus vannamei) production in Malaysia. This study aims to determine the status of AHPND in P. vannamei culture from detecting PirA/B toxin genes in hepatopancreas tissues and isolation of Vibrio parahaemolyticus for identification of pathogenic strain from major white shrimp producing farms in Malaysia. Bacteria from the hepatopancreas organ were cultured on tryptic soy agar and identified using API® 20 NE (bioMérieux, France) for Vibrio species. Confirmation of PirA/B toxin genes in hepatopancreas and V. parahaemolyticus isolates were determined by polymerase chain reaction (PCR). Twenty-three V. parahaemolyticus isolates were identified from 7.7% of the analysed samples. Fourteen (14) (4.7%) were detected with PirA/B toxin genes from districts of Johor such as Batu Pahat (1) and Kota Tinggi (8), Alor Setar, Kedah (3), Tawau, Sabah (1), and Kuching, Sarawak (1). In contrast, the other nine isolates (3%) contained only the plasmid. Genomic and phylogenetic tree analysis of four V. parahaemolyticus isolates carrying PirA/B toxin genes from this study showed that only one strain (Vp14) harbours the PirA/B complete genes in addition to displaying full sequence homology and coverage to the pVA1 plasmid. In contrast, other strains (AAT22, IKK3, and PK3) displayed partial sequence homology of plasmid harbouring key genes associated with conjugative transfer function but not the plasmid segments containing PirA/B toxin genes. Hence, this study showed that six farms were negative from AHPND. In contrast, four farms were positive with PirA/B toxin genes in juveniles from Pekan, Pahang (26.7%), Kuching and Sarikei, Sarawak (10% respectively), and Alor Setar, Kedah (3.3%).