Analysis Of Intestinal Flora Of Healthy Captive Rhinopithecusroxellana At Different Ages

Rhinopithecusroxellana was I key animal protected in our country,because some factors changed in captive conditions such as environmental factors and food,Rhinopithecusroxellana were vulnerable to illness,especially the elderly and young.This study combined DGGE,Real-time PCR and 16S rDNA high throughput technologies to analysis the intestinal bacteria of the young,adult and old Rhinopithecusroxellana.Colecting feces of Rhinopithecusroxellana of all ages,12 for the young,13 for the adult,12 for the old.Randomly selected 4 samples in each age,amplified by PCR with 16S rDNA V3 universal primer,which had the GC clip,products were analyzed by DGGE,picked the universality and specificity dominant bands on the fingerprint,send them to company for detection after purified.The results showed that,clustering analysis and PCA analysis made obvious distinction among the young,adult,old Rhinopithecusroxellana,the Shannon index and richness index of the young were significantly lower than the adult and old(P<0.05),no significant difference between adult and old(P>0.05),no significant difference in the evenness index of all ages(P>0.05),the universality and specificity dominant bands was a total of 20,dominant bacteria were mainly concentrated in the Firmicutes(12 dominant bands)and Bacteroidetes(5 dominant bands).The Clostridium of adult and young of Rhinopithecusroxellana-was rich in the microflora,the Prevotella of the adult was rich,and the Ruminococcus for the old.Randomly selected 3 samples in each age,amplified by PCR with the specific primer of Eubacteria?Lactobacillus spp.?Enterobacteriaceae family,Bifidobacterium spp.,and products were imported into DH5a after connected with pMD 19-T Vector,and the positive clones were screened by plate culture,extracted the plasmid and computed the copy number,diluted them whih 10 fold serial dilution,detected the value of Ct,established the standard curve.The samples were detected,and conversed the number of Eubacteria?Lactobacillus spp?Enterobacteriaceae family?Bifidobacterium spp..The results showed that,the standard curve had a high correlation(R2>0.99),successfully constructed standard curve.The number of Eubacteria in the young samples was significantly less than the adult(P<0.05),significantly more than the old(P<0.05),the number of Eubacteria in the adult samples was significantly more than the old(P<0.01),but the number of the three were in the same level(1011).The number of Enterobacteriaceae family in the young samples was significantly less than the adult and old(P<0.05),but there was no significant difference between the adult and old(P>0.05).The number of Lactobacillus spp.in the adult samples was significantly more than the young and old(P<0.01),but there was no significant difference between the young and old(P>0.05).The number of Bifidobacterium spp.in the old samples was significantly less than the adult and young(P<0.01),and the young was significantly less than the adult(P<0.01).12 samples of the young,13 samples of the adult and 12 samples of the old were send to the Shenzhen Huada Gene Science and Technology Limited Company for high throughput 16S rDNA sequencing.After removing primers,impurity,chimerism,with the software of Qiime 1.8 analysised the pecies composition of intestinal microflora,as well as beta analysis and alpha analysis.The results showed that,in 37 samples there were 652004 sequences,the average was 17621.730±6067.612,the average length was 252 bp,according to similarity of 97%,got 20531 OUT,the average was 2376.162±722.155.In alpha analysis,there was a significant difference in the old and adult shannon index(P<0.05),no significant difference between the adult and young(P>0.05),the difference was not significant in chao 1 and observed species(P>0.05).Rhinopithecusroxellana in different age were divided according to analysis of PCoA and clustering.The dominant bacteria were mainly Firmicutes and Bacteroidetes.The ratio of Firrmicutes in young,adult,old were 62%,70%,83%,the old was significantly higher than the young and adult(P<0.01),the adult was significantly higher than the young(P<0.05).The ratio of Bacteroidetes in young,adult,old were 32%?20%?7%,the young was significantly higher than the old and adult(P<0.01),and the adult was significantly higher than the old(P<0.01).In class level,the Clostridia of the old samples was significantly more than the young and adult(P<0.01),the adult was significantly higher than the young(P<0.05).But the Bacteroidia of the young samples was significantly more than the old and adult(P<0.01),the adult was significantly higher than the old(P<0.01).In order level,the Clostridiales of the old samples was significantly more than the young and adult(P<0.01),the adult was significantly higher than the young(P<0.05).But the Bacteroidales of the young samples was significantly more than the old and adult(P<0.01),the adult was significantly higher than the old(P<0.01).In family level,the Ruminococcaceae of the old samples was significantly more than the young(P<0.01),no difference between the adult and the old(P>0.05).In conclusion,the structure of intestinal flora in young,adult,old Rhinopithecusroxellana had a significant difference with age increasing.As the age increased,the intestinal bacteria diversity of Rhinopithecusroxellana showed a trend of first increased and then decreased.In intestinal microflora of Rhinopithecusroxellana,Firmicutes and Bacteroidetes dominated,Firmicutes and the dominant bacteria in low level of Firmicutes increased with age increasing,but Bacteroidetes and the dominant bacteria in low level of Bateroidetes decreased.