Effects On The Mucosal Barrier,antioxidant And Fecal Metabolites Of Small Intestines In Rabbits Infected With E.Magna And E.Intestinalis

Rabbit coccidiosis is a disease caused by a variety of Eimeria species,which has the characteristics of high morbidity and is one of the serious diseases faced by rabbit breeding.Eimeria intestinalis is considered to be the most pathogenic coccidia from the rabbits,Eimeria magna is classified mildly pathogenic.These two Eimeria species parasitize in the small intestines of rabbits.Coccidia invasion can destroy the intestinal epithelial cells,damage the intestinal barrier,disrupt intestinal homeostasis,and increase intestinal colonization of other pathogenic bacteria.In this study,we constructed a model of rabbit infected with E.magna and E.intestinalis to explore the effects of coccidia infection on intestinal mucosal barrier,antioxidant system and fecal metabolites.Thirty 30-day-old conventional rabbits（1054±38 g）were selected in this experiment and randomly divided into three groups.Each rabbit in groupⅠwas infected with 20×10³E.magna oocysts by gavage;each rabbit in groupⅡwas infected with 3×10³E.intestinalis oocysts by gavage;groupⅢwas designated as the control group,and each rabbit was gavaged with 2 m L of normal saline.Six rabbits from each group were dissected for the collection of the duodenum,jejunum and ileum tissues after the 8^thday of infection,a part of which were fixed in 4%paraformaldehyde solution.H.E.and PAS staining were used to study the pathological changes of small intestine after coccidia infection.The other part of small intestine tissue was stored at-80℃,the antioxidant indexes of each group were measured first;and then the effects of coccidia infection on the expression of small intestine injury-related molecules was studied by RT-q PCR and IHC staining.Finally,the feces of groupⅠand groupⅢwere collected to explore the changes of fecal metabolites after Eimeria magna infection based on non-target metabolomic study.The main results are as follows:1.The jejunum and ileum were occupied by a large number of E.magna at different developmental stages in rabbits infected with E.magna.The number of gametophytes and unsporulated oocysts was the largest.The intestinal epithelial cells were shed,accompanied by many bleeding spots.After rabbits infected with E.intestinalis,a small amount of E.intestinalis oocysts was found in the duodenum.The jejunum and ileum tissue,from the intestinal villi to glands,were occupied by a large number of oocysts and other stages of E.intestinalis.Among them,the number of schizonts was the largest.The intestinal structure was completely destroyed.There was much diffuse hemorrhage in the submucosa and intestinal villi.In addition,the jejunal and ileal villi height was significantly lower than that of the control group,the crypt depth significantly deepened,and the VH/CD ratio significantly decreased（P<0.01）.PAS results showed that the number of goblet cells in jejunum and ileum significantly decreased in the infected group.2.The results of RT-q PCR showed that the mRNA expression levels of tight junction proteins（Occludin,Claudin-1 and ZO-1）in the jejunum and ileum of the infected group significantly decreased,and the duodenal changes were not obvious.After infection,the mRNA expression of IL-2 in the duodenum,jejunum and ileum significantly increased,and the mRNA expression of IL-10 significantly decreased（P<0.05）.The mRNA expression of TNF-αonly significantly increased in the ileum after infection（P<0.05）,the changes in the duodenum and jejunum were not obvious.Immunohistochemistry staining results showed that the expression levels of Occludin protein in the duodenum,jejunum and ileum of the infected group were significantly lower than those of the control group,and the expression levels of TNF-αand IL-10 proteins significantly increased（P<0.05）.3.Significant oxidative stress occurred in the intestines from rabbits infected with E.magna and E.intestinalis.The CAT activity and MDA content in duodenum,jejunum and ileum significantly increased in the infection group.The SOD activity,GSH-Px activity and T-AOC significantly decreased（P<0.01）.The changes of antioxidant indexes in jejunum and ileum were significantly greater than those in duodenum.4.A total of thirteen significantly altered metabolites were identified in rabbit feces after E.magna infection,and seven metabolic pathways were disturbed,including tuberculosis,parathyroid hormone synthesis,secretion and action,prolactin signaling pathway,insulin secretion,insulin resistance,thyroid hormone synthesis and thermogenesis.In summary,E.magna and E.intestinalis infection destroy the intestinal epithelial cells of the parasitic sites,damage the intestinal mucosal structure,and cause oxidative stress and inflammation in the small intestines of rabbits.Meanwhile,E.magna and E.intestinalis infection disrupt the intestinal flora homeostasis of the parasitic sites,leading to significant changes in the species and abundance of gut microbiota,which in turn leads to corresponding changes in metabolites.