Using bacterial whole genome sequencing to identify toxin genes associated with disease outbreaks in black tiger shrimp (Penaeus monodon) aquaculture production

Abstract

Vibrio-related diseases cause significant economic impact on global shrimp production. Although toxin expression is a well-established mechanism of Vibrio-related disease in humans, the role of toxin expression in Vibrio disease in aquaculture generally is not well defined. The current study investigated a sustained, rapid onset, mass mortality at a commercial tiger prawn, Penaeus monodon, hatchery. Affected shrimp were between mysis-2 and post-larvae-15. Disease outbreaks were prevented through prophylactic treatment with oxytetracycline, suggesting a bacterial-related causative agent. Conversely, mortality was not reduced with erythromycin treatment. Whole genome sequencing (WGS) using the Illumina Nextera XT protocol performed on mixed microbial cultures from erythromycin and non-erythromycin infused thiosulphate-citrate-bile salts-sucrose (TCBS) TCBS agar on a MiSeq, and postlarval samples on a HiSeq. The WGS revealed several Vibrio-related sequences dominated by reads with highest homology to Vibrio harveyi and Vibrio alginolyticus. In addition, a range of putative virulence genes encoding for antibiotic resistance and toxin production were identified. Taqman qPCR assays were designed to specifically quantify four of the putative toxin-genes found, namely the Zonula occludens toxin (ZOT), hemolysin d toxin (HylD), YaFO toxin-antitoxin and Repeats–in-Toxin (RTX) genes. The qPCR assays confirmed the presence of the toxin target genes with low cycle threshold value (Ct) in the bacterial cultures and postlarval samples. Specifically, the ZOT and HylD genes were detected in high copy number in the non-erythromycin and erythromycin-treated cultures. Although the detection of toxin-genes does not demonstrate causation of mortality, the application of WGS combined with qPCR analysis has revealed the presence of a range of virulence factors within the hatchery system and allowed a rapid implementation of management protocols to prevent entry of genetically toxin-armed Vibrio spp. until more conclusive studies into disease mechanisms could be conducted.