Abstract:Bacillus subtilis, B. licheniformis, and B. pumilus, were isolated from healthy Litopenaeus vannamei, and were added to the base feed surface in single and mixed treatments. Probiotic feed was fed to the shrimp daily, and the shrimp were infected by white spot syndrome virus (WSSV) three weeks later. During the virus infection, the cumulative mortality and the number of WSSV copies in the gill tissue were statistically analyzed for each group. The relative expression of cysteinyl aspartate specific proteinase gene (Caspase) and Thioredoxin gene (Trx) in the intestinal tissue of the shrimp was measured by real-time PCR during feeding of probiotic feed and virus infection. The results showed that the cumulative mortality of B. subtilis (Group A), B. licheniformis (Group B), B. pumilus (Group C) and B. subtilis+B. licheniformis+B. pumilus (Group D), were (73.3±7.0)%, (63.3±5.5)%, (75.0±7.9)% and (50.0±5.3)% respectively, significantly lower than that of the control group (Group PBS), where the cumulative mortality of shrimp was 100%. The number of WSSV copies in each experimental group firstly increased, and then declined in the whole infection stage, but the number of WSSV copies of Bacillus groups was significantly higher than that of the control group every time (P<0.05 or P<0.01). The number of WSSV copies in the gill tissue of the mixed Bacillus group was extremely significantly lower than that of the control group, in the 8 d after virus infection. The relative expression of the Caspase gene was not significant in the 21 d of feeding with probiotic feed. After WSSV infection, the relative expression of Caspase in the intestinal tissue of each group firstly increased and then decreased, with time, reaching the maximum at the 18th h. The relative expression of Caspase in the intestinal tissue of the mixed Bacillus group was the highest, with the expression level at the 96th h still significantly higher than that of the control group. Feeding with Bacillus spp. and infection of WSSV, both could stimulate expression of the Trx gene, and the stimulation of feeding with Bacillus spp. was relatively gentle. After WSSV infection, relative expression of the Trx gene in the intestinal tissue of each Bacillus group grew quickly to its maximum at the 18th h, which was extremely significantly higher than that of the control group, and the activation on Trx gene expression from the mixed Bacillus group was the strongest. It can be surmised that the enhancement of the anti WSSV infection ability of shrimp may be related to the reduction of the speed of virus amplification in the target tissue, and the increase of the expression level of the anti-infection genes, such as Caspase and Trx, that were brought by feeding Bacillus spp.
