Investigation Of Gastrointestinal Microbial Flora In Piglets With Different Ages By ERIC-PCR

There are a great variety of species and huge number of microorganisms living in the body of animals. The normal flora is a necessary condition for survival of animals, its diversity and change of structure was important in studying on gastrointestinal microbial community and was closely correlative with health and the development of disease. Enterobacterial Repetitive Intergenic Consensus (ERIC) is the sequence of the bacteria in the intestinal tract newly that finded in these years. The sequence of ERIC is amplificatied by ERIC-PCR based on the reported Enterobcterial Repetitive Intergenic Consensus. Different genome can be distinguished by finger printing of DNA of ERIC-PCR, and this is a very useful and widely used molecular biology way to study the diversity and changes of construction of gastrointestinal microbial community .In order to detect intestinal microbial community diversity and changes of duodenum,jejunum,ileum,caecum,rectum of piglets at 0 day,1 day,3 day,9 day,15 day,21 day,28 day and 35 day,an ERIC-PCR for analyzing the construction of intestinal microbial community in the piglet intestinal tract was developed,and the construction of intestinal microbial flora in every part of intestine of normal piglets at different ages was detected in this study. The result indicated that little number of bacteria was found in intestinal tract even in the newly postnatal piglets , and the number and species of bacteria in every part of intestines were increasing with the growing of piglets. The number and species of of bacteria in piglet intestines at 9 day were very approaching to those at 35 day. Of all parts, the number and species of bacteria in duodenum and jejunum were relatively less , but those of bacteria in caecum and colon were the maximum. It was discovered that : a PCR product about 250 bp was obtained from the appendix of 0-day-old piglets in group A, and none of that obtained from piglets of 0-day-old in group B. And this demonstrated that the intestines were sterile immediately after the piglets was born,and there were some kinds of bacterium lived in the intestines just after two hours. And there was a fragment about 250 bp in the 1 day after postnatalness , the band of jejunum of 1 day piglets in group A was the most, only obtained several weak bands in ileum and appendix. Fragments of 500bp,750bp,1000bp were obtained in appendix ,and only one bands of 500 bp was obtained in jejunum , and none of bands obtained in rectum of days of 1-day-old piglets in group B. The number of bands by ERIC-PCR in every parts of intestines were increasing with the age of piglets.The increasing of number of bands of the piglets was torpidity when the piglets were 15 days old ,and the number reach the apogee at 21 days. Almost all of samples have a bright band of 250 bp by ERIC-PCR.It revealed the changes of intestinal microbial community in group A and B were almost uniform, the number and species of bacteria in every parts of intestines were increasing with the age of piglets. The increasing of number and species of intestinal microbial community of the piglets was torpidity when the piglets were 15 days old ,and the number and species reach the apogee at 21 days. Above all, the number and species of bacteria in duodenum and jejunum were relatively less , the number of bacterium of ileum was increaseing evidently , but those of caecum and colon were the most.Genes of 250bp from one-day-old piglets rectum in group A, 1-day-old piglets jejunum in group B, 21-day-old piglets appendix in group A, 21-day-old piglets rectum in group B, and 35-day-old piglets colon in group B and gene of 500bp from one-day-old piglets jejunum in group B were reclaimed from the agarose gel respectively, cloned into PMD-18 T vector, and then the plasmid transformed DH 5αcompetent cell. The plasmid was extracted and PCR assay was used to identify it. The culture was sent to Shanghai Yingjun corporation for sequencing, and three sequences were obtained: the sequence a was unknown, the sequences of b,d,e,g had higher sequence homology with that of three E.coli strains, which was up to 97%,and the sequence homology between sequence c and bopA gene of Bifidobacterium was up to 99%. It revealed that there are bacillus bifidus living in the intestine of piglets at 1 day, Escherichia coli is the predominance bacteria.