Effects Of Pueraria Polysaccharide On Blood Related Cytokines And Intestinal Flora In Healthy And Diarrhea Calves

The etiology of calf diarrhea is complex,which is a frequent and multiple disease,and seriously affects the healthy development of dairy industry.At present,antibiotics are mainly used for prevention and treatment.However,long-term,large-scale and even excessive use of antibiotics not only leads to the decline of curative effect,antibiotic residues and drug-resistant strains production,but also seriously affects human health.Pueraria polysaccharide（PPL）has anti-bacterial,anti-inflammatory,antioxidant and immune regulating functions,which is highly consistent with the treatment needs of calf diarrhea.Therefore,this study explored the effects of PPL on inflammatory,antioxidant,intestinal immune cytokines and intestinal microflora of healthy and diarrhea calves,and provided theoretical basis for the study of PPL mechanism and clinical application.Experiment 1 The effect of PPL on blood related cytokines of newborn healthy and diarrhea calves.The semi closed unified feeding house was selected in a large-scale dairy farm,the body weight was 45.34±5.52 kg,and the age was 7.58±1.61 days twenty four healthy calves were randomly divided into group C and group H（12 calves/group）and 12Chinese Holstein calves with typical diarrhea symptoms（group T）.Group H and group T were given PPL at the dose of 0.4 g/kg,while group C was given the same amount of normal saline once a day for 5 consecutive days.The plasma cytokines contents of the inflammation,oxidative stress,intestinal damage biomarker,immune were measured by ELISA on the 1^st,3^rd,5^th and 7^th day.The results showed that:（1）the levels of IL-1β,TNF-α,MDA,DAO all extremely raised（P<0.01）,the concentration of IL-2 significantly increased（P<0.05）;TGF-β,GSH-Px,SOD,Ig A both extremely decreased（P<0.01）;CAT,CD4 both significantly declined（P<0.05）;and IL-4,IL-10,NOS,ET,s Ig A,Ig G all were not significant respectively（P>0.05）.（2）After oral administration of PPL to diarrhea calves,GSH-Px,Ig A,Ig G significantly improved（P<0.05）;IL-1β,IL-2,IL-4,IL-10,TNF-α,TGF-β,NOS,CAT,MDA,SOD,DAO,ET,CD4,s Ig A both had not significant discrepancy（P>0.05）.All indexes had no significance compared with group C on 7^th day of experiment.（3）After oral administration of PPL to healthy calves,IL-1β,MDA showed a extremely declining trendency（P<0.01）,IL-2,TNF-α,DAO were significantly decreased（P<0.05）;GSH-Px,Ig A were extremely increased（P<0.01）,TGF-β,SOD,CD4,Ig G were significantly increased（P<0.05）;IL-4,IL-10,NOS,CAT,ET,s Ig A all were not significantly various（P>0.05）.The results showed that diarrhea caused severe inflammatory reaction,oxidative damage and immune suppression in calves.PPL could significantly or extremely significantly improve the anti-inflammatory,antioxidant and immune functions of healthy calves and diarrhea calves,reduce the inflammatory reaction of diarrhea calves,alleviate oxidative damage,and improve the body immunity,so as to treat calf diarrhea.Experiment 2 The effect of PPL on intestinal microflora of newborn healthy and diarrhea calveson blood related cytokines of newborn healthy and diarrhea calves.The results were based on 16S r DNA gene high-throughput sequencing showed that two samples of D1 and D5 feces were collected from calves in groups C and T during the experiment,denoted as groups Cf1 and Cf2,Tf1 and Tf2 in D1 and D5,respectively.Based on 16S r DNA high-throughput sequencing,we found that:（1）Firmicutes and Proteobacteria are the two major dominant bacteria in the feces of experimental calves,were respectively 42.21%,22.51%in Cf1 group and 35.80%,19.87%in Cf2 group as well as respectively 42.24%,21.03%in Tf1 group and 42.51%,13.35%in Tf2 group;Bacteroides（18.69%）was the major dominant genus of feces in Cf group,Escherichia-Shigella（16.92%）was the major advantageous genus of feces in Tf group.（2）The number of operational taxonomic units（OTUs）,Shannon index and ace index were not different among the groups（P>0.05）,Simpson index of Tf2 group was extremely significantly higher than that of Cf1,Cf2 and Tf1 groups（P<0.01）,and Chao1 index of Cf2 group was significantly higher than that of Tf1 group（P<0.01）.βdiversity of Cf2 group was significantly higer than Cf1 group（P<0.05）,Cf1 group has no significance compared with Tf1（P>0.05）,Tf2 group was significant higer than Tf1 group（P<0.05）,Tf1 group had no significance compared with Cf1group（P>0.05）.（3）Lef Se analysis of the influence of intestinal microbial species abundance on the differential effect showed that two phyla from Cf1/Cf2,Patescibacteria and Cyanobacteria,were mainly enriched in Cf2 group.In Cf1/Tf1,Verrucomicrobia,Actinobacteria and Bacteroides were mainly concentrated in Cf1 group,while Fusobacteria was mainly concentrated in Tf1 group.In Tf1/Tf2,one Proteobacteria was mainly enriched in Tf1 group,while three Bacteroidetes,Actinobacteria and Epsilonbacteraeota were mainly enriched in Tf2 group.A phylum of Verrucomicrobia in Cf2/Tf2 was mainly enriched in Cf2group.（4）The results of the random forest algorithm model which was used to regression forecast the intestinal flora of healthy and diarrhea calves showed that Bifidobacteria,Brucella,Paracella,Desulfovibrio and Lachnospiraceae＿UCG＿006 were the five most important species;while the correlation analysis showed that Coprobacter was negatively correlated with Clostridium sensu stricto 2（r=-0.697,P<0.05）;ace index was positively correlated with SOD（r=0.29,P<0.05）;Clostridium sensu stricto 1 was extremely positively correlated with IL-1β（r=0.38,P<0.01）;Bifidobacterium was significantly negatively correlated with Glycan biosynthesis and metabolism（r=-0.30,P<0.05）and so on.Conclusion:（1）PPL can significantly or extremely significantly improve the anti-inflammatory,antioxidant and immune contents of healthy calves and diarrhea calves,reduce the inflammatory reaction of diarrhea calves,alleviate oxidative damage,and improve the body immunity.（2）Compare sicked calves with healthy calves,diarrhea leads to dramatic changes in intestinal flora diversity and abundance and then oral administration of PPL affects intestinal structure and function by exerting a probiotic role to promote diarrheic calves restore to health.