Development of an immersion challenge model for Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Streptococcus agalactiae, the causative agent of streptococcosis in fish, induces significant mortality in the tilapia aquaculture. To elucidate the pathogenesis of S. agalactiae in tilapia, a simple and feasible challenge model was established in this study. Here, we report the development of an immersion challenge model that mimics a natural outbreak of S. agalactiae. In this model, tilapia were allowed to stress under high temperature, and hypoxia (33.5 +/- 0.5 degrees C, 2.0 +/- 0.5 mg L-1 dissolved oxygen), and the bacterial exposure increased the mortality. The infection concentration assay showed the cumulative mortality to be 6.67 +/- 3.34%, 32.22 +/- 1.92%, and 53.3 +/- 1.92% for the tilapia in the stressed group (under high temperature and hypoxia after immersed in 6.24x10(7), 3.12x10(8) and 1.56x10(9) CFU mL(-1) for 20 min), which were higher than the cumulative mortality (0, 0, and 4.42 +/- 1.95%) of the unstressed control group. The infection time assay showed the cumulative mortality to be 6.67 +/- 3.34%, 26.67 +/- 3.34%, and 35.56 +/- 1.93% for the tilapia in the stressed group (under high temperature and hypoxia after immersed with 3.06x10(8) CFU mL(-1) for 5, 20 and 60 min), which were higher than the cumulative mortality (0, 0, and 0%) of the unstressed control group. No mortality was observed in tilapia under stressed water conditions. The high mortality may attributed to the increased bacterial loads at 24 h post infection in the liver (6.81 x 10(4) vs. 2.63 x 10(3) CFU mg(-1)), spleen (1.85 x 10(6) vs. 8.69 x 10(4) CFU mg(-1)), and kidney (3.84 x 10(5) vs. 2.14 x 10(4) CFU mg(-1)) in the stressed fish compared to the unstressed fish. The serum non-specific immune parameters (adenosine deaminase, acetylcholinesterase, catalase, and superoxide dismutase) were also found to be decreased in the stressed fish. In addition, the transcripts of inflammatory-related genes (TNF-alpha, IL-1 beta, and IL10) exhibited up-regulation, whereas IgM and IgT exhibited down-regulation in the head kidney of the stressed fish, which could also increase the probability of disease. In conclusion, these findings indicate that the mortality of tilapia increased upon exposure to high temperature and hypoxia with S. agalactiae. The immersion challenge model may provide an insight into the evaluation of pathogenesis and vaccine efficacy.