Isolation Of Lactobacillus From Sheep And Its Protective Effect On Colitis In Mouse

Sheep’s gastrointestinal tract has complex microbes,and gastrointestinal disease is the most harmful to its output.On the one hand,Lactobacillus can replace antibiotics to increase animal output,on the other hand,it has received extensive attention due to its biological activity of inducing intestinal mucosal immunity.However,the isolation and screening of sheep-derived probiotics and their effects on gastrointestinal physiological functions,remains rare.This study intends to isolate a beneficial and safe Lactobacillus from the feces of healthy sheep,and DSS colitis model was used to explore its effect on the intestinal mucosal immune function,enrich the mucosal immunity research on the interaction between Lactobacillus and the intestine,and provide scientific experimental data for promoting the intestinal mucosal immunity of sheep.The results of this study are as follows:1.A total of 13 fresh fecal samples were collected from healthy sheep,and 55 strains of lactic acid bacteria(Number:Y1～Y55)were initially isolated.After Gram staining,5 strains of rod-shaped isolates were selected for probiotic potential test,and the isolate Y15 could tolerate p H=3 and 1%bile salts,sensitive to penicillin G,cefotaxime,ampicillin,clindamycin,erythromycin,and tetracycline,resistant to Streptomycin,kanamycin,gentamicin,ciprofloxacin,sulfisoxazole,norfloxacin,enrofloxacin and vancomycin,could inhibit the growth of Escherichia coli and Staphylococcus aureus,no hemolytic activity.Biochemical tests combined with sequencing results confirmed that it was Lactobacillus curvatus and named it Lactobacillus curvatus Y15.In addition,it grew rapidly at 37°C until 16 h to reach a stable phase and had good acid-producing ability.2.To evaluate the safety of Lactobacillus curvatus Y15 in mouse,twenty SPF male KM mice weighing20±2 g were randomly divided into two groups:control group and experimental group,and were respectively gavaged with sterile PBS and 0.2 m L of Lactobacillus curvatus Y15 bacterial suspension at 10~8CFU/m L for 10 days,the administration of Lactobacillus curvatus Y15 during the period had no effect on the signs,secretions,excreta,body weight gain and feed intake of the mouse.After dissection,there were no obvious pathological changes in the internal organs of the body cavity of all mouse,and no abnormal histopathological changes in the jejunum and colon tissue.Compared with the control group,Y15 did not affect the spleen index,kidney index,blood routine index,serum ALT and serum BUN content,but the liver index in the Y15 group was smaller(P<0.05),and the serum TP content in the Y15 group was higher(P<0.05).3.To explore the effect of Lactobacillus curvatus Y15 on DSS colitis model mouse.Forty-eight SPF male KM mice were selected and given 0.2 m L sterile PBS or Lactobacillus curvatus Y15(10~8 CFU/m L)by gavage every day for 14 days.On the 8th day,5%DSS was added to the drinking water to induce colitis.Lactobacillus curvatus Y15 significantly increased the contents of Ig A and Muc2 in colonic mucosa(P<0.05),and showed a trend of decreasing pro-inflammatory cytokines(IL-1β,IL-6,TNF-α)by ELISA,but the difference was not significant.HE and PAS staining observed that Lactobacillus curvatus Y15 restored the number of colon tissue and goblet cells damaged under DSS treatment,and Q-PCR detected Lactobacillus curvatus Y15 up-regulated the expression of Muc2 m RNA(P<0.05),the expression of Dll 1 m RNA was maintained,and inhibit Notch signaling pathway related genes Notch 1and Hes 1 m RNA expression(P<0.05).In conclusion,Lactobacillus curvatus Y15 meets the safety requirements of probiotics,and can promote intestinal mucosal immunity and alleviate DSS-induced colitis in mouse by regulating immunity and inhibiting the overactivation of Notch signaling pathway.