养殖水体中添加蜡样芽孢杆菌PC465对凡纳滨对虾抗病力的影响 [中文引用][英文引用]

将蜡样芽胞杆菌(Bacillus cereus)PC465添加到凡纳滨对虾(Litopenaeus vannamei)养殖水体中至终浓度分别为10⁴、10⁵和10⁶ CFU/mL,以无益生菌添加的养殖组为对照组,定期检测实验对虾肠道和养殖水体中的细菌总数、弧菌总数以及水体中氨氮含量和亚硝酸氮含量。实验结果表明,水体中添加益生菌能降低凡纳滨对虾肠道内细菌数量,且与对照组相比差异显著(P < 0.05);高浓度的益生菌处理组可以明显降低养殖水体内弧菌数量(P < 0.05),但是益生菌并没有显著影响水体中的氨氮含量和亚硝酸氮含量。养殖4周后进行WSSV投喂感染实验,感染实验表明,实验组H组(益生菌浓度为10⁶ CFU/mL)和M组(益生菌浓度为10⁵ CFU/mL)凡纳滨对虾的累计死亡率分别为63.9%和74.6%,显著低于对照组100%的累计死亡率(P < 0.05)。感染实验期间采用荧光定量PCR方法测定了凡纳滨对虾3种免疫相关基因的表达情况,统计数据显示,益生菌处理组的脂多糖-β-1, 3-葡聚糖结合蛋白(lipopolysaccharide-β-1, 3-glucan-binding protein, LGBP)、β-1, 3-葡聚糖结合蛋白-脂蛋白(beta-1, 3-glucan-bindingprotein-lipoprotein, βGBP-HDL)、热激蛋白70(heat shock proteins, Hsp70) mRNA的表达量在WSSV感染后呈显著上调趋势。实验结果提示,水体中添加蜡样芽孢杆菌PC465可以提高凡纳滨对虾抗WSSV感染能力,其作用机制可能是降低对虾肠道和水体中的细菌和弧菌数量,或调节免疫相关基因的表达水平。

We evaluated the effect of three different doses of Bacillus cereus as a probiotic added to rearing water on the number of bacteria, water quality, and disease resistant ability of Litopenaeus vannamei compared with those in a control group. Shrimp were divided into four groups treated with different probiotic doses: group H (10⁶ CFU/mL), group M (10⁵ CFU/mL), group L (10⁴ CFU/mL), and the control group (0 CFU/mL). The intestinal tracts of the shrimp were sampled every 10 days to count total bacteria and Vibrio spp.. A water sample was collected at 8: 00 every 5 days. Ammonia-nitrogen and nitrite levels were measured by spectrophotometry. The shrimp were challenged with white spot syndrome virus (WSSV) after 4 weeks, and dead shrimp were removed and recorded every day until the end of the experiment. Quantitative real-time polymerase chain reaction (PCR) analysis was performed to evaluate the expression of three immune-related genes after 0, 12, 24, 48, 96, and 192 h of exposure. The results showed that adding the probiotic significantly reduced the number of bacteria in the L. vannamei intestinal tract compared with that in the control group. It also reduced the number of Vibrio spp. in culture water (P < 0.05). However, no differences in ammonia-nitrogen or nitrite levels were detected, which contrasts with most previous studies. Cumulative shrimp mortality rates in group H (10⁶ CFU/mL) and group M (10⁵ CFU/mL) were 63.9% and 74.6%, respectively, after WSSV challenge, which were significantly lower than that in the control group with 100% mortality (P < 0.05). Lipopolysaccharide, β-1, 3-glucanbinding protein, and beta-1, 3-glucan-binding protein-lipoprotein mRNA levels were significantly upregulated 48 h after the WSSV infection, compared with those in the control group (P < 0.05). Heat shock protein 70 mRNA expression was significantly upregulated 96 h after the WSSV infection compared with that in the control group but the difference between the three experimental groups was not significant. We conclude that B. cereus as a probiotic added to rearing water reduced the number of bacteria in the intestinal tract of L. vannamei and Vibrio spp. in culture water (P < 0.05) and did not increase ammonia-nitrogen or nitrite level. Adding B. cereus to the water improved the survival rate of WSSV-infected L. vannamei. Moreover, B. cereus stimulated the expression of immune-related genes, which could improve disease resistance.