利用PCR-DGGE方法分析不同鸡群的盲肠微生物菌群结构变化

Structural change analysis of cecal bacterial flora in different poultry breeds using PCR-DGGE

研究不同品种、饲养阶段的健康和不良鸡群对盲肠细菌种群结构和多样性的影响。使用基于16S rDNA的PCR-DGGE技术,结合割胶回收DNA进行克隆和测序,分别以4、6、10、16、20、40周龄蛋鸡及1、2、4、6、7、8周龄肉鸡健康、不良鸡群盲肠内容物为样本,研究其中特定细菌类群的16S rDNA序列片段指纹图谱,并进行聚类分析,鉴定特异性和共性种群。在两品种健康鸡群盲肠内容物的细菌群落中,Lactobacillus属菌株的相似性均高于不良鸡群,并且在不同饲养阶段的健康、不良鸡群间指纹图谱平均条带数差异显著(P<0.05);而Bacteroides属菌株在健康鸡群盲肠内容物细菌的相似性与不良鸡群较为相近,健康、不良鸡群间平均条带数差异显著(P<0.05);Clostridium属菌株在蛋鸡20、40周龄的平均条带数差异不显著(P>0.05),但肉鸡各周龄健康、不良鸡群间的平均条带数差异显著(P<0.05)。序列测序结果,在蛋鸡产蛋期健康、不良鸡群样本中均检测到能动乳杆菌(Lactobacillus agilis),而育雏和育成期中均检测到鸟乳杆菌(Lactobacillus aviaries)和不可培养细菌;两品种的健康、不良鸡群样本中均检测到Bacteroides属的生酸拟杆菌(Bacteroides acidifaciens)、不可培养物细菌(Uncultured bacterium);而健康、不良蛋鸡群样本中均检测到Clostridium属不可培养的变形菌(Uncultured proteobacterium),健康肉鸡群中检测到索氏志贺菌(Shigellasonnei),而两品种不良鸡群中均缺乏此类菌种。结果显示,不同品种、饲养阶段的鸡群,其盲肠细菌群落的组成差异显著,并且细菌种群结构对鸡群的生长发育影响较大。

This study investigated the effects of normal and slow poultry development on the structural diversity of cecal bacterial flora using different chicken breeds and feeding stages.Cecal samples were collected from normal and slow developing,4,6 10,16,20,and 40-week-old commercial layers,and 1,2,4,6,7,and 8-week-old broilers chickens.Polymerase chain reaction and denaturing gradient gel electrophoresis（PCR-DGGE） of bacterial 16S ribosomal deoxyribonucleic acid（rDNA） was used,in combination with cloning and sequencing of amplified fragments to produce bacterial flora DNA fingerprints.Cluster analysis was further used to identify the specific and common community members.Lactobacillus species were more similar in normal chickens of both breeds than in slow developing chickens.A significant difference（P0.05） in the average 16S rDNA PCR-DGGE fingerprint band number was observed between normal and slow developed chickens of the same breed at different ages.Bacteroides fingerprints indicated similar cecal bacteria in the two breeds of normal and slow developing chickens.Average band numbers for the 16S rDNA PCR-DGGE fingerprints of the two breeds at different ages were not significantly different（P0.05）,while band numbers between normal and slow developed chickens was significant（P0.05）.Clostridium specific fingerprints were not significantly different between 20-week and 40-week-old commercial layers（P0.05）,and significantly different between healthy and poor broiler chickens of different ages（P0.05）.Lactobacillus agilis was most abundant in both normal and slow developing commercial layers during the laying period,while Lactobacillus aviaries and an unculturable bacteria were abundant during the brooding and rearing periods.Lactobacillus animalis,Bacteroides acidifaciens and an unculturable Bacteroides sp.,along with unculturable Clostridium were common in both chicken breeds undergoing normal and slow development.Unculturable proteobacteria were also abundant in commercial layers.Shigella sonnei was prevalent in normal broiler chickens,but absent in the slow developing chickens.Substantial differences in the cecal bacterial community composition exist between different chicken breeds at different developmental stages.Furthermore,bacterial population structure potentially has a major influence on chicken growth.