The Effect Of Lactobacillus Buchneri On The Intestinal Microflora, TLR2 And TLR4 Gene Expression Of Weaning Rex Rabbit

Diarrhea disease, which is one of the major disease of Rex rabbit with large-scaled culture, makes a huge economic loss during rabbit breeding. After diarrhea occurred, the intestinal microflora disturbed rapidly, the gut lost the physical and biological barriers which was important for immune defence, and the function of Toll-like receptor ?TLR? to recognize and eliminate invadeed microbe was also destroyed. However, probiotics, as a biologicals, has a great potential and advantage to prevent diarrhea of Rex rabbit. In these research, thus, we compared the difference of caecum microflora between healthy and diarrheal Rex rabbit, and the effect of Lactobacillus buchneri?L.buchneri? on the structure of intestinal microflora, TLR2 and TLR4 gene expression of weaning Rex rabbit. The results as followed:1. Application of PCR-DGGE and Quantitative PCR ?qPCR? to analyze the difference of caecum microflora between healthy and diarrheal Rex rabbit. The result showed that, the PCR-DGGE bands were not significant different between healthy and diarrheal Rex rabbit ?P>0.05?, while the diversity index of caecum microflora of diarrheal rabbit was higher than healthy rabbit. The clustering analysis and principal component analysis ?PCA? could distinguish diarrhea group from health group. The population of Clostridium leptum group. Bifidobacterium spp., Clostridium coccoides, Ruminococcus albus, Butyrivibrio fibrisolvens and Fusobacterium rausnitziiwere sharply decreased?P<0.01?, other detected bacteria was not changed ?P>0.05?,however, the number of Escherichia coli,which belongs to the Enterobacteriacae,was greatly increased ?P<0.05?, other detected species had no changed.The results prove that, after suffering from diarrhea, the population of benefical bacteria decreased, some harmful bacteria increased in the caecum of diarrheal Rex Rabbit, the caecum flora structure is difference but not significant ?P>0.05?, indeed the structure of caecum flora has a tendency to complicate in the diarrheal rabbit.2. The effect of L.buchneri on the intestinal microflora. TLR2 and TLR4 gene expression with different viable count (1.0×10⁴ and 1.0×105CFU·g^-1) of L. buchneri in weaning Rex rabbit's feed.60 weaning Rex rabbits ?including CT Group, LC Group, HC Group. each group has 20 rabbits? after 4 weeks feeding, the total and daily body weight gain of LC group was higher than HC group and CT group ?P<0.01?. however, the HC group had no change compared with CT group?P>0.05?. The PCR-DGGE and qPCR were performed to detected the microbial diversity of intestinal microfiora: qPCR was used for TLRs gene expression testing in the intestinal segments; The cytokines in the peripheral blood were tested by ELIS A kits. We noticed that the H', Ea and S had a "V-sharp" change tendency, from duodenum to caecum, in the same group, the ileum at the lowest-point; the H'?duodenum P<0.01. jejunum P<0.01, caecum P<0.05?, Ea ?duodenum P<0.01, jejunum P<0.01. caecum P<0.05? and S ?duodenum P<0.01, jejunum P<0.01, caecum P<0.05? of two L.buchneri group increased sharply, while, the H'. Ea and S of ileum were just increasd in LC group ?P<0.01?, HC group had no changed. The quantity of detected bacteria in the intestinal tract were increased in both L.buchneri adding group, especially, the abundance of totall bacteria ?PO.01?, Firmicutes ?P<0.01?, Bacteroidetes ?P<0.05?, Enterobacteriaceae?P<0.01?. Clostridium leptum group ?P<0.05? and Butyrivibrio fibrisolvens ?P<0.01? were increased in jejunum; totall bacteria ?P<0.01?, Firmicutes ?P<0.05?, Bacteroidetes ?P<0.01?. Clostridium leptum group ?P<0.01?, Butyrivibrio fibrisolvens ?P<0.05? and Clostridium cluster I ?P<0.01? were increased and Enterobacteriaceae ?P<0.01? decreased in the ileum. however, other specieses, Lactobacillus spp ?P<0.05?. Bifidobacterium spp. ?P<0.05? and Fibrobacter succinogenes ?P<0.05? were increased in the HC group. LC group had no change:In the caecum. juest few species were influenced by using L. buchneri., including Enterobacteriaceae decreased significantly ?P<0.01?, Lactobacillus spp ?P<0.05?. Bifidobacterium spp. ?P<0.05? and Fibrobacter succinogenes ?P<0.05? were increased in HC group, others had no change. Forfurther analysis, the population of E. coil in the ileum and caecum were decreaseed?P<0.05?. actually, in the jejunum the abundance of E.coil aslo decreased but there was no statistical significance?P>0.05?. Thus. L.buchneri could increase the abundance of intestinal microflora of Rex rabbit. improve the population of beneficial bacteria and inhibit some harmful bacteria.When compared the qPCR reslut of TLRs gene expression in the intestinal tissue between L.buchneri adding groups and CT group, it indicated that TLR2 and TLR4 genes were down-regulated in all L. buchneri adding groups. The TLR2 and TLR4 gene expression, in the jejunum and caecum of L.buchneri adding groups, were down-regulated ?P<0.01?; in the ileum,, the TLR2 and TLR4 gene expression of LC group were down-regulated ?P<0.01?, However, in the HC group, only TLR4 gene expression was down-regulated ?P<0.05?, TLR2 had no change. It indicated that the L.buchneri could down-regulated the TLR2 and TLR4 gene expression.The concentration ofTNF-? in the peripheral blood was increased significantly ?P<0.01?, when L.buchneri adding groups compared with CT group, and the concentration of HC group higher than LC group ?P<0.05?. On the contrary, the IL-4 was decreased in both L.buchneri adding groups?P<0.05?, and HC group lower than LC group?P<0.05?. The concentration of IL-1, IL-6 and INF-ywere decreased in the HC group?P<0.05?, but not in the LC group ?P>0.05?. IL-10 and IL-12 had some changes, but there was no no statistical significance.Conclusion:1) The population of beneficial bacteria decreased, some harmful bacteria increased in the caecum of diarrhea Rex Rabbit;the caecum flora structure is difference but not significant, indeed the structure of flora has a tendency to complicate in the diarrhea rabbit.2) The L.buchneri could improve the abundance of gut microbiota, improve the abundance of benefical bacteria and inhibit some harmful bacteria; the L.buchneri could affect increase the rate of B/F, and suitable does of L.buchneri benefits to feed conversion ratio; the L.buchneri chould down-regulate the expression of TLR2 and TLR4, promote the the tolerance of commensalmicrobiota and improve the production of TNF-? at some degree.