热应激环境下蛋鸡肠道微生物菌群多样性

Diversity analysis of the intestinal microbial flora of laying hens under heat stress

通过分析热应激环境下蛋鸡肠道菌群结构的变化,旨在揭示热应激对肠道微生态环境的影响机理,为探索炎热环境下家禽肠道菌群定植规律提供理论依据。试验选择16周龄济宁百日鸡96只,随机分成对照组[(24±1)℃,Ⅰ]和热应激[(38±1)℃]组,分别在2个人工环境气候舱中饲养,各组设6个重复,每个重复8只,试验持续14 d。采用16S rDNA的变性梯度凝胶电泳(Denatured gradient gel electrophoresis,PCR-DGGE)技术和实时荧光定量(Real-time quantitative RT-PCR)以及多变量统计(Principal Components Analysis,PCA)分析等手段,分析热应激2d(Ⅱ)、7d(Ⅲ)和14 d(Ⅳ)时,对十二指肠、空肠及回肠内容物菌群多样性以及菌群数量变化。PCA分析结果显示,热应激暴露过程中十二指肠部位菌群组成与对照组保持相似的趋势,但热应激2 d时空肠部位菌群组成有分开趋势;到7 d时空肠与回肠部位菌群组成分开明显,而14 d时菌群组成具有明显差异;热应激7、14 d时空肠和回肠部位末检测到敏感乳杆菌(Lactobacillus agilis),回肠部位也末检测到约氏乳杆菌(Lactobacillus johnsonii)、不可培养细菌(Uncultured bacterium)等芽孢杆菌纲,而热应激不同时间段空肠和回肠部位可检测到不可培养细菌(Uncultured Escherichia sp)、溃疡拟杆菌(Bacteroides helcogenes)、卵形拟杆菌(Bacteroides ovatus)和索氏志贺氏菌(Shigella sonnei)等拟杆菌纲和γ-变形菌纲;其中约氏乳杆菌、敏感乳杆菌数量变化在空肠部位减少最明显(P<0.05),空肠和回肠卵形拟杆菌、不可培养的拟杆菌数量明显上升(P<0.05)。热应激环境下蛋鸡空肠、回肠部位菌群多样性较为丰富,其抑制乳杆菌属、不可培养细菌的增殖,促进卵形拟杆菌的繁殖,而促进卵形拟杆菌的繁殖,导致消化道菌群平衡的破坏。

Structural changes in the intestinal microbial flora of laying hens were analyzed to determine the influence of heat stress on the intestinal microbial environment and explore microbial colonization of the poultry intestinal tract in the hot environment. Ninety-six 16-week-old Jining Bairi chickens were randomly allotted into control group Ⅰ（ 24 ± 1） ℃ or the heat stress（ 38±1） ℃ group. Each group was divided into six replicate subgroups,each consisting of eight individuals. The duration of the experiment was 14 days. Denaturing gradient gel electrophoresis combined with multivariate statistical analysis and real-time quantitative analysis of the 16 S rDNA were used to analyze the diversity of the microflora in the duodenum,jejunum,and ileum. Changes in the abundance of the microflora were also studied. The effect was examined at 2 days（ group Ⅱ）,7 days（ group Ⅲ）,and 14 days（ group Ⅳ） of heat exposure. PCA was carried out and the results showed that the composition of the microflora in parts of the duodenum remained similar between the heat-stressed and control groups,but that the microbial composition of the jejunum area varied between the heat-stressed and control animals after 2 days. At 7 days,significant differences were found in the composition of the microflora in the jejunum and ileum compared with that at 2 days and at 14 days. The influence of heat stress on the bacterial composition in each part of the intestinal tract was most pronounced at the 7-day time point. Bacilli,Lactobacillus agilis in the jejunum and ileum,and Lactobacillus johnsonii and other uncultured bacteria in the ileum,were not detected at the 7- or 14-day time points,while Bacteroidetes and γ-Proteobacteria,uncultured Escherichia sp,Bacteroides helcogenes,Bacteroides ovatus,and Shigella sonnei,were detected in the jejunum and ileum at different time points. Decreases in the overall abundance of L. johnsonii and L. agilis were most obvious in the jejunum（ P0.05）,while the abundance of B. ovatus and uncultured Escherichia sp.in the jejunum and ileum was obviously increased（ P 0.05）. The number of bacterial cells of each of the various strains ranged between 1.76—6.58 lg CFU / g. The results indicated that the proliferation of lactobacilli and uncultured bacteria in the jejunum and ileum of laying hens was inhibited under heat stress,while the growth of B. ovatus in the jejunum and ileum was promoted,resulting in disruption of the digestive tract microflora balance. The intestinal microflora of laying hens under heat stress is relatively abundant,and the diversity of the flora in the jejunum and ileum showed distinct patterns with increasing heat stress exposure time. The effect of heat stress on jejunal microbial flora was most pronounced,followed by the ileum and the duodenum. The types of lactobacilli in the jejunum and ileum were significantly different at the different time points. The effects of thermal stress were detected in the duodenum and jejunum,particularly in reference to four kinds of pathogenic bacteria. The results suggest that heat stress can inhibit Lactobacillus species and promote the proliferation of oval Bacteroidetes,thereby disrupting the balance of the digestive tract microflora.