亚剂量硫酸新霉素对黄羽肉鸡回肠黏膜菌群组成的影响

Impact of Subclinical Doses of Neomycin Sulfate on the Ileal Mucosa-Associated Microbiota Composition of Broiler Chickens

以1日龄岭南黄肉鸡（♂）为试验动物,研究在日粮中长期添加亚剂量硫酸新霉素对黄羽肉鸡回肠黏膜菌群动态变化与组成的影响。于1、7、14、21、28、35及42日龄分别从A组（饲喂基础日粮）和D组（饲喂基础日粮＋50mg·kg-1硫酸新霉素）采集鸡回肠肠段并提取黏膜细菌基因组DNA,采用PCR-DGGE技术分析鸡回肠黏膜菌群结构,同时应用16SrDNA基因序列技术,建立42日龄鸡回肠黏膜细菌16SrDNA的随机克隆文库。DGGE图谱分析发现,A、D两组1-28日龄回肠黏膜细菌结构存在明显差异,其相似性为40%-60%;A、D两组肉鸡1-35日龄菌群结构发生变化的程度不同,A组的动态变化值（%change）为9%-33%,D组的动态变化值为17%-45%。分析回肠文库发现,A组文库有94个序列,共产生9个OTUs（Operational Taxonomic Units）,其中未知菌属（微杆菌属）所占克隆总数比例最大为48.94%（46/94）,其次是乳杆菌属占38.30%（36/94）;D组文库有93个序列,共产生6个OTUs,其中乳杆菌属所占克隆总数比例最大为49.46%（46/93）,其次是微杆菌属占45.16%（42/93）;两组回肠文库中其他菌属的组成种类与数量存在差异,且未发现与大肠杆菌相关的序列。A组与D组42日龄肉鸡回肠黏膜菌群的组成及多样性存在差异,尤其是乳杆菌属的组成比例,这种差异可能是抗生素持续压力的影响所致。

The experiment was conducted to study on the effects of subclinical doses of neomycin sulfate on the ileal mucosa-associated microbiota（MAM）composition and dynamic change of broiler chickens at 1to 42 days old.Chicken ileal segments were collected in group A（basal diet）and B（basal diet with 50mg·kg-1 Neomycin Sulfate）,and MAM DNA were extracted at day 1,7,14,21,28,35 and 42,respectively.The community of the ileal MAM between two groups were analyzed by Polymerase chain reaction-denaturing gradient gel electrophoresis（PCR-DGGE）at various ages,and the random clone library of 16 SrDNA V3region of the MAM in ileum at 42 days old between Group A and D was established by analysis of 16 SrDNA gene sequences.DGGE profiles showed that the structure of the MAM in ileum between the two groups were of remarkable difference during day 1-28,and the similarity of the MAM in ileum was 40%-60%.The alterations of MAM of ileum were remarkably different between the two groups during day 1-35,with9%-33% change in group A and 17%-45% change in group D.Analysis of the library in ileum indicated that nine operational taxonomic units（OTUs）（form 94 gene sequences or clones）were detected in the group A,as compared with 6OTUs（form 93clones）in the group D.Exiguobacteriumand Lactobacillus were predominant in the group A and D,counting for 48.94%,38.30%and 45.16%,49.46%,respectively.The amount of other species found in ileum in group A were different from that in group D.In addition,sequence known being related to Colibacillus were not detected in group A and D.These results indicated that the composition and diversity of the MAM in ileum at day 42 does exist difference between group A and D,especially the proportion of Lactobacillus,these difference might be caused by the continuous antibiotic pressure selection.