| Antibiotic resistance of the animal intestinal microbiome usually associated with excessive use of antibiotics,and it would lead to the failure of medical treatment.And Antibiotic resistance genes in bacteria may be transferred from animal to human,which may threaten public health.In China,since January,2020,it was forbidden to use antibiotic-supplemented feed for farming animals,this ban may contribute to animal derived food safety and security of public health.In addition,the nationwide reduction of the use of antibiotics had made it becomes an important task to find alternative products.At present,Bacillus has been proposed as an alternative to the use of antibiotics.It can not only produce the spore structure(conducive to long-term storage)under non-biological conditions,but also produce metabolites with antibacterial effects,improve intestinal immune function,reduce diarrhea rate,and promote growth.However,there are few studies on the isolation,identification and probiotic properties of sheep-origin Bacillus.Therefore,the purpose of this study was to isolate Bacillus with good probiotic properties,non-toxic to animals and improving intestinal immune function from the fecal of Xinjiang fine wool sheep,and to explore its mechanism of action on intestinal immune barrier.1.Isolation,identification and selection of Bacillus spp..We tried to isolate Bacillus spp.from Xinjiang fine-wool sheep faecal,and they were identified by Gram staining,biochemical characteristics and molecular biology tests.Then,the dominant probiotic Bacillus spp.was screened by hemolysis,p H and bile salt tolerance tests.The results show that,a total of 14 Bacillus spp.strains were isolated(Bacillus licheniformis,Bacillus subtilis,etc.).Among them,only Bacillus licheniformis B63 had non-hemolysis and was well tolerated to low p H and 0.3%bile salt concentration.Therefore,B.licheniformis B63 was selected for subsequent research.2.Safety of B.licheniformis B63 and its effect on intestinal immune barrier.Firstly,the growth curve of the strain was determined as well as the antibacterial and antibiotic sensitivity tests in vitro were carried out.After that,24 Kunming mice(24.85±1.92 g,half male and half female)were selected for acute toxicity test.The mice were randomly divided into control group(Control)and B.licheniformis B63 group(B63).The control group and B63 group were intraperitoneally injected on the 1th day,with saline and 4.0×10~8 CFU/m L B.licheniformis B63 0.2 m L,respectively.The status of the mice was observed and the body weight was recorded every day.On the 8th day,the mice were dissected,the spleen and liver were removed,weighed and fixed for preparation of tissue sections.Then 32 male SD rats(301.55±21.15 g)were selected for subacute toxicity test.They were randomly divided into control group,B.licheniformis B63 low dose group(B63-L),medium dose group(B63-M)and high dose group(B63-H).The control group and B.licheniformis B63 different dose groups were given PBS and 4.0×10~6 CFU/m L,4.0×10~7 CFU/m L,4.0×10~8 CFU/m L B.licheniformis B63 2 m L,respectively,for 28 days.Every day,the mice were observed and the body weight was recorded.On the 29 th day,the spleen and liver were dissected,weighed and were used to prepare tissue sections,and blood was collected for blood routine test.The serum was collected to detect the levels of immunoglobulins(Ig A,Ig G,Ig M)and cytokines(IL-2,IL-6,IL-10),the jejunum and colon tissues were collected for histopathological observation,and the gene expression levels of colon cytokines(IL-4,IL-5,IL-10)and Muc2 were detected.The results showed that the B63 strain entered the logarithmic growth phase after 2 h of culture,and entered the platform phase after 8 h of culture.B63 strain has a high inhibitory effect on Escherichia coli,Salmonella typhi and Staphylococcus aureus,and was sensitive to most commonly used antibiotics.The results of acute and subacute toxicity tests showed that B63 strain had no significant effect on organs and blood indexes.After intragastric administration of B63 strain,the content of IL-6 in serum of SD rats decreased significantly(P<0.05).When the gavage dose was 4.0×10~7 CFU/m L,the villus height of jejunum increased significantly(P<0.05),and the gene expression of IL-4 and Muc2 increased significantly(P<0.05).3.The whole genome sequencing of B.licheniformis B63.The genome information of B.licheniformis B63 was characterized by whole genome sequencing technology.After quality control and evaluation of the sequencing data,the genome sequence assembly and prediction were carried out.The gene function annotation information and drug resistance and virulence genes,metabolic systems and pathogen-host interaction were analyzed jointly.The results showed that the genome sequence of B63 strain was 4 329 555bp in length,which was composed of only one circular chromosome and did not contain plasmid structure.The average GC content of the genome was 45.82%.There are 7 genomic islands,including 1 virulence island and 1 drug resistance island.B63 can produce secondary metabolites with bacteriostatic action,such as surfactin,cyclic lipopeptide and bacitracin.The number of co-coding genes was 4 402,and the COG function was annotated to genes related to antibiotic sensitivity,virulence factors and immunity.GO function annotation to flagellar composition and spore structure genes;k EGG metabolic function was annotated to antigen processing and presentation,immune response,anti-Salmonella infection,HSP90 and other related pathways.4.The effect of B.licheniformis B63 on intestinal barrier in mice infected with Salmonella.40 Kunming mice(25.48±3.27 g,half male and half female)were randomly divided into control group(C),model group(M),prevention group(P),and treatment group(T).The control group was stimulated by intragastric.The model group was fed with Salmonella for 7 d.The prevention group was fed with B.licheniformis B63 for 7d and then fed with Salmonella for 7 d.The treatment group was fed with Salmonella for 7 d and then fed with B.licheniformis B63 for 7 d.The intragastric volume of B.licheniformis B63 was 0.2 m L for each mouse.The intragastric volume of Salmonella was 0.5 m L for each mouse.The body weight and signs were recorded.After the feeding,the blood was collected and the mice were dissected.The organ was weighed to calculate the organ coefficient and used to prepare the tissue section.Serum was collected to detect Ig A,Ig G,IL-4,IL-6,IL-10 and Alanine Transaminase(ALT);the colon was collected to prepare paraffin sections for HE staining,PAS staining and immunohistochemical test to perform the colon structure observation,goblet cell count and tight junction protein(ZO-1,OCLN,CLDN)histological score(H-score),respectively.Colon RNA was extracted to detect the gene expression of ZO-1,OCLN,CLDN,IL-4,IL-5,IL-6,Muc2 and Signal Transducers and Activators of Transcription 6(STAT6)transcription factors,and the secretion of Secretory Immunoglobulin A(SIg A)in colon was detected by ELISA.The results showed that compared with the control group,the daily gain of mice in model group was significantly decreased(P<0.05),the coefficients of liver and spleen were significantly increased(P<0.05),the morphological changes of organs were observed,the content of ALT in serum was significantly increased(P<0.05),the muscular layer of colon was thickened and the depth of colon crypt was significantly increased(P<0.05).The expressions of IL-5and Muc2 in colon were significantly decreased(P<0.05),and the secretion of SIg A was significantly decreased(P<0.05).Compared with the model group,the daily weight gain of mice in the treatment group was significantly increased(P<0.05),the liver coefficient was significantly decreased(P<0.05),the contents of Ig A and Ig G in serum were significantly increased(P<0.05),there was no significant change in colon structure while the number of goblet cells was significantly increased(P<0.05),and the H-score values of ZO-1,OCLN and CLDN proteins were increased.The expression of ZO-1,OCLN,CLDN,IL-4,IL-5,STAT6,Muc2 and the secretion of SIg A in colon were significantly increased(P<0.05),and the expression of IL-6 was significantly decreased(P<0.05).Compared with the model group,the daily weight gain of the mice in the prevention group did not change significantly and showed negative growth,the liver coefficient decreased significantly(P<0.05),the contents of Ig A and Ig G in serum increased significantly(P<0.05),the H-score values of ZO-1,OCLN and CLDN proteins increased,the expression of OCLN,CLDN,IL-4,IL-5 and Muc2 in the colon increased significantly(P<0.05),the expression of IL-6 decreased significantly(P<0.05).Conclusion:In this study,a strain of B.licheniformis B63 with good safety and probiotics was isolated from Xinjiang fine-wool sheep fecal,which has the ability to colonise the intestine and produce antibacterial substances.At the concentration of 4.0×10~7 CFU/m L,it can promote the growth performance of salmonella typhi infected mice,and improve the intestinal mucosal immune function by increasing SIg A secretion and Muc2 gene expression. |
