饲料中添加复合芽孢杆菌对凡纳滨对虾抗病毒感染能力及抗病基因表达的影响

自健康凡纳滨对虾(Litopenaeus vannamei)分离到枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(B. licheniformis)和短小芽孢杆菌(B. pumilus)，将上述芽孢杆菌以单一和3株复合的方式包裹在基础饲料表面，制成益生菌饲料；每日投喂对虾，3周后进行白斑综合征病毒(WSSV)人工感染。统计实验组和对照组的累积死亡率，测定对虾鳃组织内WSSV拷贝数，分析对虾肠道组织含半胱氨酸的天冬氨酸蛋白酶基因(Caspase)和硫氧还原蛋白基因(Trx)的相对表达量。结果显示，感染实验结束时，A组(枯草芽孢杆菌)、B组(地衣芽孢杆菌)、C组(短小芽孢杆菌)和D组(枯草芽孢杆菌+地衣芽孢杆菌+短小芽孢杆菌复合益生菌)的对虾累积死亡率分别为(73.3±7.0)%、(63.3±5.5)%、(75.0±7.9)%和(50.0±5.3)%，显著低于对照组(PBS组)(100%)；在整个感染阶段，各实验组的病毒拷贝数呈先上升后下降的趋势，但对照组呈现一直上升趋势，且显著高于实验组。抗病基因表达结果显示，WSSV感染后，各组对虾肠道Caspase相对表达量随感染时间的延长呈先上调再下调的趋势，且在18 h各组对虾肠道Caspase表达量达到最大值；益生菌摄取和WSSV感染都能刺激Trx的表达，益生菌的刺激相对平缓，且各实验组对虾肠道Trx相对表达量在WSSV感染后的18 h时陡升到最大值，极显著高于对照组，且以D组的激活能力最强。研究证实，枯草芽孢杆菌、地衣芽孢杆菌和短小芽孢杆菌均可提高对虾抗WSSV感染能力，复合芽孢杆菌抗病毒能力最突出。对虾抗病力的提高可能与芽孢杆菌减缓了病毒在靶组织的增殖速率、提高了Caspase和Trx基因表达水平相关。

Effects of single or mixed Bacillus on WSSV infection and immune-related gene expression in Litopenaeus vannamei

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.