Cold temperature stress and damaged skin induced high mortality in barramundi (Lates calcarifer) challenged with Vibrio harveyi.

Most diseases in aquaculture are caused by opportunistic pathogens. One of them, Vibrio harveyi, is a widespread Gram-negative bacterium that has become an important pathogen of aquatic species in marine environments. Here, we propose the use of the causal pie model as a framework to conceptualize the causation of vibriosis in juvenile barramundi (Lates calcarifer) and to establish an effective challenge model. In the model, a sufficient cause, or the causal pie, is a constellation of component causes that lead to an outcome (e.g. vibriosis). In the pilot study, a high cumulative mortality (63.3% ± 10.0%, mean ± SE) was observed when V. harveyi was administered by intraperitoneal injection using a high challenge dose [107 colony-forming units (CFU) fish-1 ], but low or no mortality was observed in fish subject to cold stress or fish with intact skin when challenged by immersion. We, therefore, tested the use of a skin lesion (induced with a 4-mm biopsy punch) combined with cold temperature stress to induce vibriosis following the causal pie model. After challenge, fish were immediately subject to cold stress (22°C) or placed at an optimal temperature of 30°C. All groups were challenged with 108  CFU mL-1 for 60 min. A considerably higher mortality level (72.7% ± 13.9%) was observed in fish challenged with both a skin lesion and cold stress compared with mortality in fish only having a skin lesion (14.6% ± 2.8%). V. harveyi was re-isolated from all moribund fish and was detected by species-specific real-time PCR in gills, head kidney and liver, regardless of challenge treatment confirming vibriosis as the cause of disease. Parenchymal tissues had histopathological changes consistent with vibriosis. Whole-genome sequence (WGS) is provided for the Vibrio harveyi isolate examined in this study. Overall, the causal pie model was a useful framework to conceptualize the design of the experimental challenge model, in which both cold stress and skin damage were identified as component causes of vibriosis with high mortality. This conceptual framework can be applied to other opportunistic pathogens in aquaculture or to the study of co-infections in fish.