Effect Of Probiotics On K99 Induced Diarrhea In Calves And The Effect Of EGC Secreting Factors GSNO And Sox10

Objective:To study the effect of compound probiotics on the structure of small intestine and the distribution,expression and content of GSNO and Sox10 secreted by glial cells(EGCs)in calves with diarrhea induced by Escherichia coli K99.Diarrhea is the main cause of calf death,which seriously affects the development of cattle industry,and antibiotics are banned.Therefore,its alternative compound probiotics are applied to prevent and treat animal diarrhea,which can inhibit the occurrence of calf diarrhea.EGCs have an important role in intestinal mucosal protection.In this study,we investigated the effects of compound probiotics on the morphology of small intestinal mucosa and the secretion of GSNO and Sox10by EGCs in calf diarrhea in order to provide a reference basis for the prevention and control of calf diarrhea.Methods:Twenty-four healthy 5 Dutch male calves with similar parity and gestational age,weighing about 40 kg,were selected.The calves were fed adequate amounts of colostrum and fresh milk since birth.Calves were randomly divided into 3 groups for routine feeding and management.Each calf in the control group was given 10 m L of normal saline daily;each calf in the probiotic test was fed with 3 g of compound probiotics at a bacterial content of 1×10~9CFU/m L of E.coli K99 10 m L on the first day,followed by 3 g of compound probiotics daily.The compound probiotics consisted of Lactobacillus acidophilus(S1),Bacillus subtilis(K1),and Saccharomyces cerevisiae boulardii(B1)at a ratio of 1:1:3 and a bacterial content of 1×10~9CFU/m L;each calf in the infection group received 10 m L of E.coli K99 every day until diarrhea occurred in calves.Two calves were randomly selected and slaughtered and tissue samples were taken at 7 days,14 days,and 21 days,respectively,and slaughtered before feeding the next morning when the experimental period arrived.Small intestinal tissues were taken for HE staining and immunohistochemistry,and the supernatant of small intestinal homogenates was taken for ELISA.Results:1.The histopathological changes of each segment of the small intestine of calves were observed by HE staining of paraffin sections.The intestinal villi of duodenum,jejunum and ileum in the control group were intact;while the tissue structure of each segment of small intestine in the probiotic group was also intact,without villus breakage or shedding phenomenon,and the height of intestinal villi increased.The intestinal villi in the probiotic group of jejunum were significantly higher than those in the control group(P<0.05);the intestinal villi in the infected group had significant villus breakage and shedding phenomenon,the height of intestinal villi in the duodenum and jejunum was lower than that in the control group(P<0.05),The height of intestinal villi in the duodenum,jejunum,and ileum was lower than that in the probiotic group(P<0.05).2.The contents of GSNO and Sox10 in EGC secretory factors of calves were detected by ELISA.The results of GSNO showed that there was no significant difference in the contents of GSNO in the duodenum of the three groups(P>0.05);the contents of probiotic group in the jejunum were higher than those in the control group(P>0.05),the contents in the infected group were lower than those in the control group(P<0.05),and the contents in the probiotic group were higher than those in the infected group(P<0.05);in the ileum,the contents in the probiotic group were higher than those in the control group(P>0.05),the contents in the infected group were lower than those in the infected group(P<0.01).The Sox10 content in the probiotic group was higher than that in the control group(P<0.05);in the jejunum,the Sox10 content in the probiotic group was higher than that in the infection group(P<0.05),and the content in the control group was higher than that in the infection group(P>0.05);in the ileum,the content in the probiotic group was higher than that in the control group(P<0.05),and the content in the infection group was lower than that in the control group(P<0.05).3.The distribution of GSNO and Sox10 secreted by EGC in calves was observed by immunohistochemical method,in which GSNO and Sox10 were mainly expressed in the intestinal villus lamina propria,small intestinal glands,and mucosal epithelium in the small intestine of calves in the control group;the probiotic group was mainly expressed in the intestinal villus lamina propria,small intestinal glands,mucosal epithelium,submucosa,and perivascular;and the infection group was mainly expressed in the mucosal epithelium and intestinal villus lamina propria.And it was strongly expressed in the probiotic group,expressed in the control group,and weakly expressed in the infected group.Conclusion:Escherichia coli K99 can damage the structure of the small intestine,resulting in diarrhea in lactating calves,and compound probiotics can protect the intestinal mucosa to a certain extent,which in turn enhances intestinal barrier function.Escherichia coli K99 could decrease the secretion of GSNO and Sox10 secreted by small intestinal glial cells;compound probiotics could play a protective role in the intestinal barrier and increase the secretion of GSNO and Sox10.