【作者】 刘彤；

【导师】 徐春生； 甘尚权；

【作者基本信息】 石河子大学 ， 兽医（专业学位）， 2021， 硕士

【摘要】 目的:本研究以中国荷斯坦公犊牛为试验动物,研究复合益生菌对大肠杆菌K99致犊牛腹泻的小肠组织结构的影响以及肠胶质细胞(EGCs)分泌因子GSNO、Sox10的分布表达及含量的影响。腹泻是导致犊牛死亡的主要原因,严重影响了养牛业的发展,而抗生素被禁用,因此,其替代品复合益生菌被应用于防治动物腹泻,可以抑制犊牛腹泻的发生。EGCs在肠黏膜保护中具有重要作用。本试验探究复合益生菌对犊牛腹泻小肠黏膜形态和EGCs分泌GSNO、Sox10的影响,以期为犊牛腹泻的防控提供参考依据。方法:选择体况相似的健康5日龄荷斯坦公犊牛24头,体重约为40kg。自犊牛出生后饲喂足量的初乳及鲜乳。将犊牛随机分为3组,常规饲养管理,对照组每头每日灌服10m L生理盐水;益生菌组在试验第一天饲喂含菌量为1×10~9CFU/m L的大肠杆菌K99 10m L,之后每天饲喂复合益生菌3g,复合益生菌由嗜酸乳杆菌(S1)、枯草芽孢杆菌(K1)、布拉迪酵母菌(B1)组成,比例为1:1:3,含菌量为1×10~9CFU/m L;感染组每头每天灌服大肠杆菌K99 10m L,直至犊牛发生腹泻。于7d、14d、21d分别随机挑选2头犊牛宰杀并取组织样,在试验期到之时,于次日清晨晨饲前宰杀。取小肠组织进行HE染色及免疫组化,并将小肠均浆取上清进行ELISA检测。结果:1.利用石蜡切片HE染色,观察犊牛小肠各段组织病理学变化。对照组犊牛十二指肠、空肠及回肠肠绒毛完整;而益生菌组小肠各段组织结构也较完整,无绒毛断裂、脱落现象,并且肠绒毛高度增加,空肠的益生菌组肠绒毛显著高于对照组(P<0.05);感染组小肠绒毛有明显的绒毛断裂,脱落现象,十二指肠与空肠肠绒毛高度低于对照组(P<0.05),十二指肠、空肠、回肠肠绒毛高度均低于益生菌组(P<0.05)。2.利用ELISA方法检测犊牛EGC分泌因子GSNO、Sox10的含量,GSNO结果为,三组的十二指肠中的GSNO的含量无显著差异(P>0.05);空肠中益生菌组含量高于对照组(P>0.05),感染组含量低于对照组(P<0.05),益生菌组含量高于感染组(P<0.05);在回肠中,益生菌组含量高于对照组(P>0.05),感染组含量低于对照组(P<0.01),益生菌组含量高于感染组(P<0.01)。益生菌组Sox10含量高于对照组(P<0.05);在空肠,益生菌组Sox10含量高于感染组(P<0.05),对照组含量高于感染组(P>0.05);在回肠益生菌组含量高于对照组(P<0.05),感染组含量低于对照组(P<0.05)。3.利用免疫组化的方法观察犊牛EGC分泌因子GSNO、Sox10的分布情况,其中GSNO及Sox10在对照组犊牛小肠中主要表达于肠绒毛固有层、小肠腺、黏膜上皮;益生菌组主要表达于肠绒毛固有层、小肠腺、黏膜上皮、黏膜下层及血管周围;感染组主要表达于黏膜上皮、肠绒毛固有层。并且益生菌组强表达,对照组表达,感染组弱表达。结论:大肠杆菌K99可以对小肠结构造成损伤,从而造成哺乳期犊牛发生腹泻,复合益生菌可以在一定程度上保护肠道粘膜,进而增强肠道屏障功能。大肠杆菌K99可导致小肠胶质细胞分泌因子GSNO、Sox10的分泌降低;复合益生菌可对肠道屏障起到一定的保护作用,提高GSNO、Sox10的分泌。更多还原

【Abstract】 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.更多还原