复合噬菌体与枯草芽孢杆菌对肠道产气荚膜梭菌的抑制效果及免疫代谢功能

Compound and Bacillus subtilis inhibit intestinal Clostridium perfringens and improve immunometabolism of microbiota

摘要:

【背景】产气荚膜梭菌感染一直以来困扰着家禽养殖业，限抗以来感染率呈上升趋势。生物源噬菌体以其独特的抑菌作用、安全无残留等优点，已成为潜在的减抗关键技术之一。【目的】明确产气荚膜梭菌噬菌体、沙门氏菌噬菌体及枯草芽孢杆菌联用对蛋鸡养殖的增效作用，通过对肠道组织结构形态变化、产气荚膜梭菌消减和菌群多样性的分析，为家禽减抗养殖中细菌性疾病防控提供有效的减抗技术方案。【方法】选取105只260日龄海兰褐蛋鸡，随机分为5组：A：产气荚膜梭菌噬菌体SD72组；B：产气荚膜梭菌噬菌体SD72&枯草芽孢杆菌联用组；C：沙门氏菌噬菌体JDF1-6组；D：SD72/JDF1-6复合噬菌体&枯草芽孢杆菌联用组；E：空白对照组。连续饮水饲喂42 d后取盲肠内容物测定产气荚膜梭菌数量，观察肠道组织结构，通过16S rRNA基因扩增子测序分析明确肠道微生物的群落分布、多样性及代谢功能。【结果】与对照组相比，D组SD72/JDF1-6复合噬菌体&枯草芽孢杆菌联用组肠道中产气荚膜梭菌的数量减少1.48 log₁₀ (CFU/mL)，空肠隐窝深度显著降低(P<0.05)，绒毛高度/隐窝深度显著增大(P<0.05)。微生物多样性分析结果显示，各组盲肠菌群中均以厚壁菌门和变形菌门为主，在属水平上乳杆菌属(11.26%)相对丰度较高。血清免疫指标及代谢功能分析显示，IgA、IgG和IgM的含量显著提高(P<0.05)，肠道微生物基因主要富集于Kyoto Encyclopedia of Genes and Genomes (KEGG)的遗传信息处理通路，其中D组在排泄系统和免疫疾病代谢相关通路中富集基因相对丰度高于对照组755.95%。【结论】复合噬菌体和枯草芽孢杆菌联用能够有效改善蛋鸡肠道健康，提高机体免疫水平，促进微生物组免疫疾病代谢相关基因表达，提高肠道菌群代谢功能，为家禽减抗健康养殖中细菌性疾病的防控提供重要的理论依据和生物资源。

Abstract:

[Background] Clostridium perfringens has been a major pathogen affecting the poultry industry, and its infection rate has been on the rise since the use of antibiotics was restricted. Bacteriophages with unique antibacterial effects and safety without residues have become a primary approach to reduce the use of antibiotics. [Objective] To evaluate the synergistic effects of Clostridium perfringens phage, Salmonella phage, and Bacillus subtilis on laying hens by analyzing the histological changes of the intestinal structure, the reduction of C. perfringens, and microbial diversity, so as to provide a scheme for the prevention and control of bacterial diseases in poultry with antibiotic reduction. [Methods] A total of 105 260-day-old Hyline Brown hens were selected and randomized into 5 groups of A: Clostridium perfringens phage SD72, B: SD72+Bacillus subtilis, C: Salmonella phage JDF1-6, D: SD72+JDF1-6+ Bacillus subtilis, and E: blank control. After the hens were treated for 42 days, the cecum contents were collected to determine the number of C. perfringens, and the intestinal structure was observed. The 16S rRNA gene amplicon was sequenced to reveal the intestinal microbial community structure, diversity, and metabolism. [Results] Compared with the blank control group, D group showed decreased number of C. perfringens (by 1.48 log₁₀ (CFU/mL)), decreased depth of jejunal crypts (P<0.05), and increased villus height/crypt depth (P<0.05). Firmicutes and Proteobacteria were dominant in cecum contents of laying hens in all the treatment groups after drinking water, and Lactobacillus(11.26%) had higher relative abundance at the genus level. The serum levels of IgA, IgG, and IgM were significantly increased (P<0.05), and intestinal microbial genes were mainly enriched in the genetic information processing pathways. The abundance of enriched genes in excretory system and immune disease-related pathways in group D was 755.95% higher than that in the blank control group. [Conclusion] The combination of mixed phages and B. subtilis could improve the intestinal health and immunity of laying hens by promoting the expression of microbial genes related to immune disease and improving the immunometabolism of intestinal flora. The findings provide a theoretical basis and biological resources for the prevention and control of bacterial diseases in poultry with reduced antibiotics.