| A probiotic Bacillus PAS38 was identified by the combination of traditional methods and modern molecular biology techniques methods.It was made as microbial additives suspension and the chickens were oral administered by gavge.Then the dynamic change of Bacillus PAS38 was detected in vivo and the ecological balance of intestinal flora by using modern molecular biology.The results were as follows:The probiotic PAS38 was identified by using the 16S rDNA sequence analysis method.The general primers were designed according to conservative sequence of bacteria 16S rDNA.The genomic DNA of Bacillus PAS38 was extracted and amplified the target 16S rDNA sequence.At the same time,this sequence was analysised by using the NCBI with BLAST database.Furthermore,according to the morphological,physiological and biochemical characteristics,and based on its position in phylogenetic tree,the Bacillus strain of PAS38 was finally identified as Bacillus cereus.In order to observe Bacillus growth and decline changes in the digestive tract of chicken by TaqMan Real-Time Fluorescence Quantitative PCR,28-day-old chickens were administered orally with the PAS38 suspension twice a day at the dose of 2mL/1kg(10~9CFU/mL)for three consecutive days by gavage.The samples of intestinal microbial communities were collected everyday until the end of the ninth day.The results showed that strain PAS38 group:from the first day to the fourth day,the diversification of Bacillus sp in all the digestive tracts had significantly increased;on the fifth day,there was no significant increase of the diversification in the duodenum,while in the rest digestive tracts the diversification increased significantly;on the sixth day,there was a significant increase in the cecum and rectum,however,in the others there was no significant increase.Finally,from the seventh day to the ninth day,in all the digestive tracts there was no significant increase.The results showed that the chickens were oral administered the PAS38 strains for three days,the number of Bacillus sp in the intestinal tract can be increased rapidly;this tendency could be maintained into the fourth day followed by returning to the normal level.During the third day after stopping being fed with PAS38,the samples were collected and identified. The diversity structure of intestinal microbial communities was analyzed by ERIC-PCR agarose electrophoresis pattern and dendrogram.The results showed that both the diversity and the brightness of the bands had been markedly improved.The location of the bands which represented the same digestive tract within a certain period of time was relatively stable and appeared some regularity,two main bright bands were observed in the location of about 250bp and 500bp respectively.In the PAS38 group except the cecum,the differences of PAS38 in the ileum were very significant,jejunum and duodenum were significant;the total number of amplified bands from the jejunum sample was more than others;the total number from the ileum was lower,while cecum and duodenum were the last.Similarity coefficient (Sc) increased most greatly in the jejunum,comparison with 81%in the control group,it increased to 91%;comparison with 88%in the control group,it increased to 94%in the ileum;comparison with 83% in the control group,it increased three percentage to 86%in the cecum;the least influence was in the duodenum,it only increased two percentage to 95%.All of theses phenomenon indicated PAS38 played a more obvious regulatory role in ileum microorganisms,while it played weakly regulatory role in the duodenum.PCR-DGGE analysis showed that after stopping being oral administered PAS38 for three days,the number of feature bands and total bands was significantly more than that in the control group.Apart from the crop,there were significant differences in the cecum,jejunum,ileum,rectum and duodenum. Compared with the control group,the total similarity coefficient increased by 12 percentage to 91%in the crop;the total similarity coefficient increased by 6 percentage to 77%in the duodenum;while the total similarity coefficient increased by 23 percentage to 75%in the jejunum;similarity coefficient in the ileum had also improved 6 percentage to 84%and the similarity coefficient in the cecum improved 11 percentage to 91%.As for the rectum,the coefficient of the total fell by 2 percentage to 89%.On the PCR-DGGE fingerprint,the specific sequences of those recovery bands were identified.The results showed that there were many different kinds of bacteria,such as Lactobacillus johnsonii,Lactobacillus acidophilus,Streptococcus sp.,Acinetobacter sp.,Lactobacillus salivarius,Lactobacillus reuteri, Acinetobacter lwoffii,Clostridium sp and so on,most of which were Lactobacillus.It was worth noting that Lactobacillus reuteri was the dominant microflora in the jejunum after being oral administered PAS38.This phenomenon indicated that Bacillus PAS38 had played an important role in establishing dominant microorganisms in intestinal bacteria and regulating the intestinal microbial balance to promote animals to be healthy. |
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