Isolation And Identification Of Bovine Rumen Bacillus And Its Effect On Diarrhea Model In Mice

There are a large number of microbial flora in the gastrointestinal tract of animals.The microorganisms in the flora can participate in various physiological and biochemical activities of the body,and have an impact on the growth,development and immune function of the host.Among them,there are more and more studies on probiotics in the gastrointestinal tract.The important reason is that probiotics can regulate the balance of the gastrointestinal tract and affect the health of animals.The purpose of this experiment is to isolate probiotics by analyzing the diversity of rumen microorganisms in Yanbian yellow calf,and to verify the prevention and treatment function of diarrhea mice,so as to provide a theoretical research basis for the development and application of probiotics in animals in the future.In this experiment,the rumen fluid of three Yanbian yellow calves was collected,and the microbial types were obtained by high-throughput sequencing analysis.In vitro bacteriostatic test,antibiotic susceptibility test,and 16 S r RNA sequencing;BALB/C mice were used in this experiment to successfully establish a mouse model of E.coli diarrhea,and the mice were divided into Bacillus licheniformis treatment group,Bacillus licheniformis group Bacillus prevention group,Bacillus licheniformis and Bacillus subtilis mixed treatment group,Bacillus licheniformis and Bacillus subtilis mixed prevention group,positive model group,The blank control group was used to study the therapeutic and preventive effects of probiotics on diarrhea mice.After 5 days,the mouse serum was collected for ELISA detection of IL-4,IL-6,TNF-α,and IFN-γ.Finally,the mouse small intestine was fixed with formalin,and pathological sections were made to observe the changes of the small intestine.The results of high-throughput sequencing analysis showed that the microbes in the rumen of Yanbian yellow calf were diverse,and the specific distribution was as follows: 17 classes,19 orders,31 families,80 genera,and 89 species.In this experiment,the screening test of probiotic Bacillus in calf rumen juice was carried out.The results of microscopic observation,biochemical identification and 16 S r RNA sequencing showed that the two strains were Bacillus licheniformis and Bacillus subtilis,respectively named bl,bs.Both bl and bs obtained after resistance screening were resistant to acid-base choline salts and high fever;the results of in vitro antibacterial tests showed that both bl and bs had inhibitory effects on E.coli;the results of antibiotic susceptibility tests showed that bl and bs were resistant to chloramphenicol,ceftriaxone,tetracycline,ciprofloxacin,enrofloxacin,vancomycin are all sensitive,are resistant to lincomycin,penicillin,ampicillin,amoxicillin,bl is resistant to erythromycin,clarithromycin It is sensitive to streptomycin and resistant to streptomycin,and bs is sensitive to streptomycin and resistant to erythromycin and clarithromycin.The histological observation results of small intestine showed that: the small intestine section of the blank group showed that the structure of intestinal villi was complete,and the intestinal epithelial cells were intact and tightly arranged;the small intestine section of the treatment group showed that the small intestinal villi were significantly widened,the lamina propria was slightly edema,and the intestinal epithelium was slightly edema.The cell basement membrane was separated from the lamina propria,and inflammatory cells were infiltrated;under the microscope of the small intestine in the prevention group,part of the intestinal villi structure was destroyed,and at the bottom of the lamina propria,chyle duct edema 2 was seen dilated;in the mixed treatment group,the intestinal villi structure was seen under the microscope in the small intestine slice Relatively normal,with slight edema of the villus lamina propria,a small amount of food residues and blue-purple bacterial clumps were seen in the intestinal lumen;in the mixed prevention group,the intestinal villi structure was basically intact,and only part of the intestinal villus epithelial cells were sloughed off into the intestinal lumen,and the lamina propria was light.In the positive model group,the structure of intestinal villi in small intestine slices was obviously destroyed,the lamina propria was severely edema,the basement membrane of intestinal epithelial cells was separated from the lamina propria,the apical epithelial cells were necrotic,and some intestinal villi were broken or dissolved;ELISA results showed that the positive model Compared with the blank control group,the serum levels of IL-6,TNF-α,and IFN-γ in the group mice were significantly higher(p<0.05),and the treatment group,prevention group,mixed treatment group,and mixed prevention group were significantly higher than the positive model group.Decreased(p<0.05);the IL-4 content in the serum of the mice in the positive model group was significantly lower than that in the blank control group(p<0.05).increased(p<0.05).To sum up,based on the species of rumen fluid flora obtained by high-throughput sequencing,two strains of Bacillus were screened out by the experiment,which were identified as Bacillus licheniformis and Bacillus subtilis,and neither strain could be colonized in the gastrointestinal tract.Compound probiotics can effectively protect the intestinal tract,reduce the serum indicators of IL-6,TNF-α,IFN-γ in mice infected with Escherichia coli,and increase the level of IL-4,which has a good preventive effect on bacterial diarrhea in mice.