Analysis Of Intestinal Microbiota Of Newborn Piglets In Jiangxi And Screening Of Lactobacillus Inhibiting CpA

Intestinal microbies is important for the piglet health.Imbalance of microbiota is closely related to diarrhea which causes significant economic losses,but the difference and potential pathogens remain little known in intestinal microbiota between healthy and diarrhea piglets.Clostridium perfringens（Cp）has been reported to be associated with diarrhea in piglets.Researches,including the toxin genes,health and diarrhea isolate clonal cluster and the population of Cp,are beneficial to prevent and cure Cp-induced diseases.As a well-known probiotic,Lactobacillus has various probiotic functions and participates in the regulation of bacterial microbiota balance.Screening of Lactobacillus with good inhibition against Cp has a great application prospect.Piglet feces were collected from various areas in Jiangxi.Some piglet feces were used to extract total DNA,and the V3-V4 region of 16 S rDNA was amplified and sequenced.The optimized sequences were used for bioinformational analysis.The result of comparison between health group and diarrhea group at OTU level revealed that diarrhea-causing bacteria may be opportunistic pathogens.The difference in α diversity index between the two groups was not significant,and the functional prediction showed that there was only a difference in the relative abundance of each function.Random forest and LEfSe analysis of intestinal microbiota in newborn piglets showed that Fusobacterium mortiferum,unclassified Proteus,unclassified Tyzzerella,Prevorellaceae_UCG₀03 and Alispes shahill were top 5 key microorganisms that led to a transition from health to diarrhea.Among them,Fusobacterium mortiferum,which had significant abundance differences in the healthy and diarrhea groups,may be closely related to piglet diarrhea.The relative abundances of the two major groups belonging to Clostridium were quite different.The abundance of Clostridium sensu stricto₂ in diarrhea group is 4.5 times than that in healthy group.Based on the results of intestinal microflora analysis of healthy/diarrhea newborn piglets,Cp in pig feces samples were further isolated and cultured,and the main Cp toxin gene of fecal DNA was detected,and the number of Cp in feces was quantified by real-time PCR.24 Cp type A isolates from healthy/diarrhea newborn piglets from different regions were typed via multilocus sequence typing analysis（MLST）.The isolates were extracted with DNA,and 7 housekeeping genes and α-toxin genes were amplified and sequenced.The obtained ST number was used to construct the phylogenetic tree.By comparing the polymorphisms of the allele sequences of the strains,the potential mutations in the strain genome were known.The test results of Cp toxin gene in piglet feces indicated that both were β2+ type A.The detection rates of alpha toxin genes in healthy and diarrhea piglets were 85.4%（41/48）and 81.3%（39/48）,respectively,and the β2 toxin genes were79.2%（38/48）and 77.1%（37/48）,respectively.The results of phylogenetic tree indicatedthat the β2 toxin gene was highly conserved,with only mutations of no more than 3 bases,and the highest homology with the corresponding sequence of GenBank accession number AJ537530.1.The difference in Ct value of RT-PCR results was not significant,indicating that the number of Cp bacteria may not differ significantly in the healthy and diarrhea groups.The results of 24 CpA MLST indicated that the strain had genetic diversity and the alpha toxin gene had little effect on typing.The overall genetic difference of CpA from healthy/diarrhea piglets was not significant,but the CpA clustering of diarrhea piglets in the same area was significantly higher than that of healthy piglets.Screening CpA antagonistic bacteria against Clostridium enteritis in piglets based on microecological balance theory.Lactobacillus were isolated from the sow fecal samples and tested for acid production capacity and bile salt tolerance.The MRS medium was modifed to grow both Cp and Lactobacillus,and the growth curve and the expression time of α and β2 toxin were determined.Lactobacillus with good and stable Cp inhibition were screened,and the co-culture method was used to determine the effect on toxin expression.Lactobacillus amylovorus and Lactobacillus reuteri were successfully isolated,and the latter still had good activity in the culture medium containing bile salts for 15 h.The bacteriostatic test results showed that Lactobacillus amylovorus was less bacteriostatic than Lactobacillus reuteri.The results indicated that the growth phase was reached after 6 hours culture,and only β2 toxin gene expression was detected.Co-culture results showed that Lactobacillus reuteri screened had a good inhibitory effect on β2 expression.In this study,the high-throughput sequencing of bacterial 16 SrDNA was used to analyze the difference of healthy/diarrhea intestinal microbies in newborn piglets,and further analysis was carried out on the bacteria causing diarrhea in newborn piglets.MLST of CpA from healthy/diarrhea newborn piglets was performed to further determined the differences in health and diarrhea CpA isolates and genetic diversity.Using CpA and its β2toxin gene as the research object,Lactobacillus reuteri was screened,which could produce CpA proliferation and β2 gene expression.It has great potential to be developed as microecological agents for the prevention and treatment of Clostridium enteritis in piglets.