| Yersinia enterocolitica is a kind of enteric pathogenic bacteria which can infect both people and animals and caused widespread concern from 1980 s. Y.enterocolitica is also a common foodborne pathogen of worldwide diarrhea. Currently clinical treatment of Y.enterocolitica infection is using antibiotics. However,a long-term irrational using of antibiotics treatment will not only cause the functional disturbance of gastrointestinal tract and digestive tract microecological imbalance and other adverse reactions,but also lead to antibiotic residue and induce pathogen resistance. Researches have indicated that probiotics are effective in the treatment of intestinal bacterial infections,but the mechanism is not completely clear. Additionally,there are few reports about probiotics in the prevention and treatment of human and animal infections caused by Y. enterocolitica. Lactobacillus and Bifidobacteria are common probiotics among intestinal normal flora of animals in the natural state,which have the advantage of being adapted to the environment of the gastrointestinal tract of animals and the long-term colonization ability in the intestine. Therefore,The aim of this study is to screen excellent Lactobacillus and Bifidobacteria that has acid-resistance activity,bile salt-resistance ability,trypsin-resistance ability,antibacterial activity and antioxidant activity from healthy animals and infant intestinal contents or feces. And to validate and better understand the mechanism of the anti-Y.enterocolitica ability of these probiotics based on establishing BALB/c mice infection model by gavaging Y.enterocolitica.The main research process of this study is described as follows:First,the Y.enterocolitica isolates were isolated from 58 diarrhea stool samples collected from weaned piglets in Heilongjiang Province. After biochemical identification and molecular identification,18 Y.enterocolitica were isolated and named as Y1 ~ Y18. The detection result of six virulence genes(ail and virF,yadA,ystA,ystB,and rfbC) by polymerase chain reaction shows that among the 18 Y.enterocolitica isolates,10 isolates carries ystB genes,3 isolates carried the ail gene,and the rest of 5 isolates carries no virulence genes. The result of animal pathogenicity test shows that there exists different pathogenicity of these isolates when they were orally infected BALB/c mice and weaning piglets. The isolates that carry ail gene used to be described as typical pathogenic strains such as Y7 and Y17 show different pathogenicity to mice and weaning piglets. Meanwhile those typical non pathogenic isolates without virulence genes,such as Y9 and Y16 were actually pathogenic to mice and weaning piglets. In addition,these isolates that show pathogenicity can cause different levels of diarrhea. Besides,the diarrhea and strain carrier duration are also different,as well as the colonization site of isolates in the weaned piglets,such as mesenteric lymph nodes,intestinal contents and arthrosis fluid.At the same time,to screen lactic acid bacteria and Bifidobacteria that natural state exists in intestinal respectively from stool of healthy animals and healthy breast feeding infants by Isolation and culture on the modified MRS selective medium and BLM selective medium. Based on colony morphology,gram staining and microscopic examination,catalase test,biochemical test and 16 SrRNA gene sequence analysis,lactic acid bacteria and Bifidobacteria were isolated. Then gradually screening were performed through acid-resistance test,trypsin-resistance test,bile salt-resistance test,detection of antioxidant ability and inhibitory effect on pathogen,such as Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and Salmonella typhimurium. Finally,14 Lactic acid bacteria and 7 Bifidobacteriawere selected and respectively named as L1~L14 and B1~B7. These isolates including 11 Lactobacillus brevis, 2 Lactobacillus johnsoii, 1 Lactobacillus plumtarum, 3 Bifidobacterium infantisand 4 Bifidobacterium breve.At last,establish model of BALB/c mice respectively orally infected by isolate Y16(group T1) and standard strain ATCC9610(group T2),model of BALB/c mice respectively orally infected by isolate Y16(group L1) and standard strain ATCC9610(group L2) after treated with Lactobacillus brevis L5,and model of BALB/c mice respectively orally infected by isolate Y16(group L1) and standard strain ATCC9610(group L2) after treated with Bifidobacterium breve B3. Through the detection of weight change,of cecal contents and feces of bacterial count,detection of the content of IgG in serum and intestinal mucous SIgA,observation of pathological changes occur in vivo,oxidative stress related substances and enzyme levels of detection,intestinal mucosal barrier function based on detecting of mRNA levels and associating with inflammatory signaling pathway protein expression level of detection,the Protective effect of probiotics Lactobacillus brevis L5 and Bifidobacterium breve B3 against intestinal infection with Y.enterocolitica Y16 and ATCC9610 was studied and its mechanism was researched.The results of this study showed that L5 and B3 can not only reduce pathogen colonization; promote the expression of tight junction proteins,protect the integrity of intestinal mucosal; inhibit inflammatory reaction in mice by inducing inflammation inhibitor and inhibiting production of pro-inflammatory cytokines showing a protective effect on the intestinal of mice infected by Y.enterocolitica. This study also found that Lactobacillus brevis L5 and Bifidobacterium breve B3 showed a protective effect on mice by inducing more s IgA while being infeced by Y.enterocolitica and help mice to resist oxidation harm which was induced by Y. enterocolitica infection. This study also found that the protective effect of L5 and B3 on these mice may be achieved through the regulation of MAPK p38 and NF-κB signaling pathway. This study provides further evidence that probiotics are potential therapy on Y.enterocolitica intestinal infection,to provide reference data for the mechanism study of probiotics in the treatment or prevention of intestinal bacterial infection.This study further confirms that the probiotics are potential treatment modalities to Y.enterocolitica infection. It also found the probiotic’s functions of in vivo antioxiding and stimulating specific sIgA secretion in intestinal tract. In consideration of the expression changes of inflammatory signal pathway proteins,we speculated that the functions of probiotics,such as regulating immunity and inhibiting pathogen infection may achieved by inducing the signaling pathway protein expression. This study also found that Lactobacillus brevis L5 and Bifidobacterium breve B3 showed a protective effect on mice by inducing more sIgA while being infeced by Y.enterocolitica and help mice to resist oxidation harm which was induced by Y.enterocolitica infection. And it may be new mechanism of probiotics to inhibit the intestinal tract infection caused by pathogenic bacteria. This study provides further evidence that probiotics are potential therapy on Y.enterocolitica intestinal infection,to provide reference data for the mechanism study of probiotics in the treatment or prevention of intestinal bacterial infection. This study can also provide high quality probiotics for the development of new probiotics preparation. |
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