饲料添加益生菌对凡纳滨对虾肠道菌群、Toll受体及溶菌酶基因表达及抗感染的影响

以初体重为(7.20±1.38)g的凡纳滨对虾(Litopenaeus vannamei)为研究对象,在室内养殖箱进行3周的养殖实验和2周的哈维氏弧菌(Vibrio harveyi)人工感染实验,其中对照组每日投喂普通商品饲料,实验组每日投喂在普通商品饲料中添加地衣芽孢杆菌(Bacillus licheniformis)1.0×10~8 CFU/m L配制成的实验饲料。目的是研究饲料中添加益生菌对凡纳滨对虾肠道菌群、Toll受体及溶菌酶基因表达量和抗哈维氏弧菌能力的影响。实验结果表明,在饲料中添加地衣芽孢杆菌可显著提高对虾抗哈维氏弧菌感染的能力,其相对免疫保护率为22.22%。与对照组相比,实验组可显著降低对虾肠道内弧菌数量(P0.05)。感染哈维氏弧菌后,实验组溶菌酶(lysozyme,LZM)m RNA的相对表达量在12 h、18 h、24 h、36 h、48 h、72 h均显著高于对照组(P0.05)。实验组感染哈维氏弧菌后在6 h、12 h、18 h、24 h、36 h、48 h、72 h、7 d的Toll受体(Toll receptor)m RNA的相对表达量均显著高于对照组(P0.05)。实验结果提示:饲料中添加地衣芽孢杆菌可有效提高对虾抗哈维氏弧菌感染的能力,这种能力的提高可能是通过降低对虾肠道内的弧菌量,并提高抗病相关基因的表达量实现的。

Effects of adding probiotics on promoting intestinal bacteria, Toll receptors, and lysozyme immune gene expression and resistance to Vibrio harveyi in Litopenaeus vannamei

The white shrimp,Litopenaeus vannamei(Decapoda, Penaeidae), isomnivorous, grows quickly, and has a low food nutrition demand, which has made it an economically important crustacean species in China. However, the single breed type and high-density culture can result in bacterial and viral disease outbreaks.Vibrio is the dominant bacteria in shrimp and causes disease when shrimp are immunocompromised. In this study,L. vannamei (initial body length, 7.90 cm±1.15 cm; initial body weight, 7.20 g±1.38 g) were purchased from Qingdao Baorong Aquaculture Co., Ltd. A 3-week feeding experiment and a 2-weekVibrio harveyi infection experiment were per-formed to evaluate the effects of adding the probioticBacillus licheniformisto feed on promoting intestinal bacte-ria, non-specific immune gene expression, and resistance toV. harveyi. The experimental shrimp were divided into control and experimental groups. Control group shrimp were fed commercial feed throughout the experiment, and the experimental group shrimp were fed the same feed supplemented with 1.0×108CFU/mLB. licheniformis.The numbers ofVibrioand total intestinal bacteria inL. vannameiwere checked every 7 days using 22l6E and TCBS media. After 3 weeks of feeding, all shrimp were injected with 1.0×107CFU/mLV. harveyi(50μL; dose deter-mined in preliminary experiment).Then, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, and 72 h, as well as 7 d later, three shrimp from each group were captured randomly to extract RNA from the gill. The RNA was reversed transcribed into cDNA and tested for lysozyme and Toll receptor expression levels. The cumulative mortality rate of the con-trol group was 100%, and the cumulative mortality rate of the experimental group was 22.78% after 14 days. The relative protection ratio was 22.22%. The experimental feed significantly reduced the number ofVibrioin the shrimp intestine compared with that in the control group (P<0.05). Relative lysozyme mRNA expression levels in the experimental group 12 h, 18 h, 24 h, 36 h, 48 h, and 72 h after theV. harveyichallenge were significantly higher than thosein the control group(P<0.05). Relative Toll receptor mRNA expression levels in the experimental group 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, and 72 h, as well as 7 d after theV. Harveyichallenge were significantly higher than those in the control group (P<0.05). These results indicate that addingB. licheniformisto shrimp feed effectively improved the ability ofL.vannamei to resist aV. harveyi infection, possibly by reducing the number of Vibrio in the intestine and improving immune-related gene expression.