Study On The Changes Of Intestinal Flora And Its Effects On Immune Function During Orf And Mastitis In Goats

Goat breeding industry has become the important industry of animal husbandry in China.The economic benefits brought by the development greatly promote the development of animal husbandry.Epidemiological investigation showed that Orf and mastitis of goats had a high outbreak rate in goat cultured areas,which not only affected the healthy development of goat farming,but also greatly reduced the economic benefits of dairy industry.Studies have shown that the immune system plays an important role in resisting the outbreak of Orf and mastitis.Therefore,it is particularly important to clarify the pathogenesis of these two diseases for the development of goat breeding industry.In recent years,many studies suggest that the intestinal flora plays an important role in the body's barrier,nutrition metabolism and immunoregulation.The diversity of the intestinal flora and the balance of intestinal microecological are important to animal body's immune function,and gut-breast pathway also has a regulatory role in the process of the body's immune.However,the role of intestinal flora in Orf and the regulation of intestinal flora and milk microorganisms in mastitis are not very clear.Based on this,the experiment research the influence of intestinal flora on Orf,changes of intestinal flora and milk microorganism in goat mastitis,and change of the body's immune function in the process.By analyzing the correlation between microorganism and immune function indexes,the bacteria that were correlated with the immune function indexes were screened out.Finally,the effects of beneficial Enterococcus faecium and Bacillus subtilis on the differentiation of peripheral blood lymphocytes of goats were studied in vitro.The results not only help to clarify the role of intestinal flora in the pathogenesis of Orf and mastitis,but also provide a new idea for the prevention and treatment of the disease.In this study,the 16 S r RNA sequencing method was used to analyze the fecal microorganisms of ORFV carrier goats,and the fecal and milk microorganisms of dairy goats with mastitis were sequenced and analyzed to analyze the significant difference of microbial composition between diseases and healthy conditions;we used fluorescent quantitative PCR,flow cytometry,immunofluorescence staining,histopathological staining and other methods to detect the expression of IL-17 A and related cytokines in peripheral blood and inflammatory tissues,the number and proportion of ??T cells,and the pathological changes in inflammatory sites during the process of goat Orf and goat mastitis.Through the correlation analysis,the beneficial bacteria genera with significant correlation were screened.We further analyzed the effects of Enterococcus faecium and Bacillus subtilis on the differentiation of peripheral blood lymphocytes of goats by fluorescence quantitative PCR and flow cytometry.The results obtained are as follows:1.In the Orf group,the treatment of ORFV carrier goats with antibiotics significantly promoted the outbreak of Orf,and the proportion of animals with clinical symptoms of ORFV increased.2.In the Orf group,16 S r RNA sequencing results showed that the richness of ORFV-carrying faecal microorganisms decreased significantly during the treatment of antibiotics,and the relative abundance of Firmicutes increased significantly after the outbreak of Orf at the phylum level,and the relative abundance of Spirochaetes,Bacteroidetes and Cyanobacteria decreased significantly.There was no significant difference in fecal microbial richness between healthy goats and ORFV-carrying goats without antibiotic treatment.3.The expression level of IL-17 A in lip tissue and peripheral blood mononuclear cells(PBMCs)of goats treated with antibiotics was significantly increased in the experimental group.The expression level of IL-17A-related cytokines in PBMCs was also significantly increased,both the number and percentage of ??T cells were significantly increased,and the expression levels of neutrophil chemokines IL-8 and CCL4 were also significantly increased.4.In the goat mastitis group,it was found that compared with the milk of healthy goats,the number of total microbial groups in the milk of goats with mastitis was significantly reduced,and the relative abundance of Cyanobacteria and Proteobacteria were significantly decreased at the phylum level,and the relative abundance of Firmicutes was significantly increased.However,the fecal microorganisms of the goats with mastitis increased significantly in Cyanobacteria and Bacteroidetes,while the relative abundance of Firmicutes decreased significantly.5.In goat mastitis group,immunofluorescence staining results showed that IL-17 A was significantly increased in breast tissue during mastitis.IL-17 A and its related cytokines were significantly increased in goat peripheral blood and milk,and the expression of neutrophil chemokines was also significantly increased.Immunofluorescence staining of ??T cells in breast tissue showed a significant increase in the number of ??T cells.Flow cytometry showed a significant increase in the number and percentage of ??T cells in breast milk.6.In vitro,we stimulate PBMCs by Enterococcus faecium or Bacillus subtilis and their metabolites: the expression of IL-12 and T-bet were significantly decreased;The expression of IL-4 and IL-13 in Enterococcus faecium supernatant group was increased significantly;The expression of IL-17 A and IL-6 in Enterococcus faecium supernatant group were significantly decreased,the expression of IL-6 and TGF-? in Bacillus subtilis supernatant group were significantly decreased.The expression level of IL-10 in Enterococcus faecium supernatant group was significantly increased.ELISA results showed that expression of IL-17 A decreased significantly in Enterococcus faecium and Bacillus subtilis groups.Following conclusions can be drawn:1.Antibiotic treatment of ORFV carrier goats led to changes in intestinal flora that promoted the outbreak of Orf and pathological damage.2.Goat intestinal flora of ORFV carrier goats plays a key role in controlling the inflammatory pathological process of Orf,possibly by inhibiting the pro-inflammatory immune response mediated by IL-17 A.3.The milk microorganisms and fecal microorganisms of goats with mastitis were significantly different from those of healthy goats.4.IL-17 A and its related cytokines may drive systemic and mammary gland inflammatory responses in goat mastitis.5.Enterococcus faecium can inhibit the differentiation of PBMCs toward Th1 and Th17 in vitro,and promote the differentiation toward Th2.Bacillus subtilis also could inhibit the differentiation toward Th1 and Th17.