| Calf diarrhea is the foremost cause of calf death and it is very serious especially if it occurs before weaning.It not only affects the health of calves,but also significantly diminishes their later production performance,thereby restricting the development of the cattle industry.Previous studies have shown that the gut microbiota of the calves in early life plays a critical role in their long-term health.A good early gut health can minimize the calf susceptibility to gut infections before weaning.However,the mechanism of perturbation of the gut microbiota in calf diarrhea is unclear,and the improvement in their health after probiotic supplementation remains to be understood.Therefore,improving the survival rate of calves by effectively preventing and treating calf diarrhea,promoting the repair of the damage caused to the calf gastrointestinal tract and formation of healthy microflora are crucial issues to be addressed in current dairy farming.This can be great practical significance to improve the economic benefits of the calves.Objective: In the present study,high-throughput sequencing technology was used to explore the changes of host intestinal function and microbiota in Ecoli K99-infected newborn calves at different time periods,and the main differential microbial genera associated with calf diarrhea was explored.Subsequently,the compound microbial preparations were developed according to the different microbial genera,and the effect of the compound microbial preparations on the incidence of diarrhea,growth performance,immune function and intestinal microflora in pre-weaning calves was evaluated.The regulation mechanism of the intestinal damage in neonatal calves infected with Ecoli K99 can provide a scientific basis of the improvement in the intestinal health of calves by probiotics and assist the production guidance.Methodology and Results: In this study,six experiments were performed,and the specific research methodology and results are presented as follows:Experiment 1: Effects of Ecoli K99 infection on the intestinal microbiota,intestinal epithelial development and mucosal barrier function in newborn calvesSixty Holstein bull calves were randomly divided into 2 groups.They were given sterile saline(group C)and Escherichia coli suspension(antibiotics were administered on the next day,group D),respectively.The experimental period was of 21 days.Blood samples,intestinal contents and tissue mucosa were collected on days 7,14 and 21.Furthermore,the feed intake,diarrhea rate,serum immunity,SIg A,intestinal epithelial structure and the microflora of different intestinal segments were analyzed and determined.Pearson’s rank correlation coefficient was used to analyze the correlation between the microorganisms and intestinal epithelial development,and the immunoglobulin index.It was found that the concentration of SIg A in the jejunum and ileum,the height of villi,and the relative expression of GHR and INSR genes in the mucosa of group D was significantly lower than that of group C on days 7,14 and 21.The relative abundance of Bifidobacterium,Lactobacillus,Prevotella＿7 and Saccharomyces cerevisiae was significantly lower,while the relative abundance of Bacteroides and Apiotrichum of calf jejunum was significantly higher in group D than that of group C.The results indicate that the gut microbial disturbances were associated with the gut mucosal function.Experiment 2: Study on the optimization of the ratio of 3 strains of the compound microecological preparation for preventing calf diarrheaOn the basis of Experiment 1,the tolerance of L.acidophilus,S.cerevisiae,B.subtilis of different concentrations in the artificial gastric juice,intestinal juice,bovine bile salts and their resistance to Ecoli K99,Salmonella,Staphylococcus aureus were studied.Using Ecoli K99 as the indicator bacteria,the bacteriostatic effect of three probiotics in different proportions was studied to determine their optimal combination.The results showed that the three probiotics exhibited a good tolerance in different concentrations in the artificial gastric juice,intestinal juice and bovine bile salts,and the optimal ratio of L.acidophilus,B.subtilis and S.cerevisiae was 3:3:1.The results indicated a theoretical basis of the microecological preparations in preventing the diseases of the digestive tract in young calves.Experiment 3: Effects of compound probiotics on the incidence of diarrhea,immune function and fecal microflora in pre-weaning calvesThe effects of different doses of a multispecies probiotic(MSP)mixture on growth performance,the incidence of diarrhea rate and immune function,and fecal microbial diversity and structure were evaluated in pre-weaning Holstein dairy calves at WK2,WK4,WK6,and WK8.Forty Holstein newborn calves were selected and randomly divided into 4 treatment groups,namely: C(control group)and the probiotic administered groups T1(0.5 g/head/day),T2(1 g/head/day)and T3(2 g/head/day)group;the experimental period was of 56 days.Calf feed intake and fecal scores were recorded daily during the experiment.In 2nd,4th,6th,and 8th week of the experiment,the calf body size,serum immunity,antioxidant indexes and the fecal microbiota was measured and analyzed.Pearson rank correlation coefficient was used to analyze the main fecal microbial genera and growth performance or serum immunoglobulin in week 2.The results showed that the incidence of diarrhea 4 weeks before birth in T3 group was significantly lower than the control,T1 and T2 groups.Increase in the body height and concentration of T-AOC,Ig A,Ig G and Ig M in the serum was noted in the T3 group between 2 to 8 weeks.Compared with group C,MSP supplementation reduced the relative abundance of Bacteroides in T1 and T2 groups and increased the relative abundance of Bifidobacterium,Lactobacillus,Collinsella and Saccharomyces at week 2 in the T3 group.The results indicate that supplementing MSP 2g/head/day early in calf life could be an effective way to potentially prevent the calf diarrhea and improve the growth performance.Experiment 4: Effect of compound probiotics on resistance to E.coli K99 infection in calvesForty newborn Stan calves were randomly divided into 2 groups with 20 calves in each group.Group I was the control group(group C)and group II were fed MSP 2 g/head/day(group P).The production performance,diarrhea rate and SIg A concentration in the serum and rectal stool samples was noted during the period of the study.The results showed that the addition of compound probiotics to the diet reduced the diarrhea rate in the newborn calves and improved the immune performance and resistance to E.coli K99 infection in the newborn calves.Experiment 5: Effects of compound probiotics on the growth performance and intestinal epithelial development in the E.coli K99-infected newborn calvesThirty six newborn Holstein bull calves were randomly assigned into three treatment groups,including the control group(group C)that was given sterile normal saline,E.coli K99 suspension administered group(group D,antibiotics were given on the next day)and P group(E.coli K99 suspension was administered and MSP 2 g/day was fed on the next day).The experimental period was of 21 days.The growth performance,blood samples,serum and intestinal immunity(Ig A,Ig G,etc.)and intestinal tissue were assessed to analyze the incidence of diarrhea.The results showed that MSP supplementation increased the ADG of E.coli K99-infected newborn calves,improved the intestinal nutrient digestion and absorption,regulated the intestinal energy metabolism,and helped to improve and repair the intestinal tract structure of the E.coli K99-infected calves.Experiment 6: Effect of compound probiotics on the intestinal mucosal barrier function and microbiota in the E.coli K99-infected calvesThe design of this experiment is the same as the Experiment 5.A combination of high-throughput sequencing and metaproteomics was used to analyze the interaction between the changes in the gut microbiota and the host intestinal mucosal barrier function.The results showed that MSP administration significantly changed the fungal Chao1(abundance),Shannon(diversity)and post-intestinal microbiota diversity in the small intestine on the 21 st day after infection compared with the D group.Addition of MSP significantly increased the relative abundance of Firmicutes and Actinobacteria in the duodenum and jejunum and significantly decreased the relative abundance of Bacteroidetes in the jejunum compared to group D(P = 0.03).At the same time,the relative abundances of Bifidobacterium,Prevotella＿7,Ascomycota and Saccharomyces in the jejunum of the P group were significantly increased(P < 0.01).The concentration of SIg A in duodenum,jejunum and ileum mucosa of group P was significantly higher than that of group D(P < 0.01).The relative expression of IL-1β,TLR4,ZO-1 and occludin m RNA in the jejunum mucosa and the concentration of total SCFA in jejunum of group P was significantly higher than that of groups C and D(P < 0.01).Thus,MSP supplementation significantly enhanced the intestinal energy metabolism and defense mechanism in the E.coli K99-infected calves by reducing the expression of HSP90,thereby reducing the inflammation and promoting the recovery of the mucosal function.Conclusion:(1)The diversity and structure of the gut microbiome changed dramatically,when the calves were infected with E.coli K99.On day 21 post-infection,the structure and diversity of the bacterial and fungal flora in the gut of calves changed.The relative abundances of Bifidobacterium,Lactobacillus,Prevotella＿7and Saccharomyces cerevisiae decreased,and the relative abundances of Bacteroides and Apiotrichum exhibited an increase.(2)The three probiotic strains of L.acidophilus,B.subtilis,S.cerevisiae could tolerate the gastric juice,intestinal juice and bovine bile salts,and exhibited inhibitory effects on the bovine E.coli K99,Salmonella,and Staphylococcus aureus.The compound probiotics exhibited an obvious inhibitory effect on E.coli K99,and the optimal ratio of L.acidophilus: B.subtilis: S.cerevisiae was 3:3:1.(3)The optimal addition amount of MSP was found to be 2g/head,and the optimal addition time was four weeks before the life of the calf.The lower incidence of diarrhea in MSP-supplemented calves was a result of the modulation of the fecal microbial composition and diversity.(4)MSP supply regulated the growth of the damaged intestinal epithelial cells in E.coli K99-infected calves,enhanced the host intestinal energy metabolism and defense mechanisms,reduced the inflammation,and promoted the recovery of the mucosal function. |
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