Analysis Of Intestinal Flora And Identification Of Potential Probiotics In Preweaned Goats

In recent years,the mutton sheep breeding industry in Jiangsu Province has developed steadily.However,lamb diarrhea remains one of the problems that troubles farmers.It not only affects fattening,but also causes dehydration and death in severe cases,resulting in economic losses.In this study,we collected fecal samples of preweaned goats from a sheep farm in Jiangsu for metagenomic sequencing,and analyzed their gut microbiota composition and dynamic changes at different ages.Besides,we analyzed the differences in intestinal flora between preweaned diarrhea goats and healthy goats.In addition,traditional culture,MALDITOF and PCR were used to isolate and identify potential probiotics in the intestines of preweaned goats,and then analyzed their biological characteristics,in order to provide new ideas for the intestinal health care and diarrhea diagnosis and treatment.1.Analysis of intestinal flora in healthy goats before weaningTwo clinical health goats were random selected from a farm in Jiangsu Province,China.Fecal specimens of each goat were sampled at the age of 1,3,5,10,20,30 and 45 days old respectively.The DNA in fecal samples was extracted by high-temperature pyrolysis method.The genomic DNA was randomly fragmented.The fragments were treated for end repairing and adding adaptors to both ends.Size selection of adaptor-ligated DNA was then performed by DNA cleanup beads.Then,each sample was amplified by PCR using P5 and P7 primers.The qualified PCR products were sequenced by pair end PE150 on the Illumina Hiseq System.After being optimized and integrated,the sequencing data were blasted against the constructed microbial database.Combined with the species annotation results of genes and the gene abundance table,we calculated the abundance of each species under each classification level for each sample.Following data analysis,a total of 3444701 valid sequences were obtained in all samples.The total sequence length is 3590843589 bp,in which the shortest sequence length is 500 bp,the longest sequence length is 980213 bp,the average sequence length is 1429.32 bp and the GC content is 46.38%.All samples were annotated to 53 phyla,77 classes,176 orders,303 families,625 genera and 2439 species.As the age increased,the intestinal flora of preweaned goats continuously changed,and the species composition,community diversity,and richness fluctuated.The 1st,3rd,and 20th days after birth of preweaned goats were important points for intestinal flora succession.Bacteria such as Bacteroides fragilis,Ruminococcus gnavus,Lactococcus garvieae,Butyricicoccus pullicaecorum,Akkermansia muciniphila were closely related to gut health.2.Analysis of intestinal flora in diarrhea goats before weaningA total of 8 fecal samples of preweaned diarrhea goats from the above sheep farms were randomly collected.The sample processing and sequencing methods were the same as above.The results showed that a total of 2745732 valid sequences were obtained in all samples.The total sequence length is 2737598234 bp,in which the shortest sequence length is 500 bp,the longest sequence length is 871956 bp,the average sequence length is 1656.87 bp and the GC content is 47.56%.All samples were annotated to 56 phyla,79 classes,179 orders,316 families,697 genera,and 2597 species.Compared with preweaned healthy goats of the same or similar age,the proportion,species,and diversity of intestinal microflora in diarrhea lambs had changed,mainly manifested by a significant decrease in the relative abundance of Bacteroidetes and a significant increase in the relative abundance of Firmicutes and Proteobacteria,including a decrease in the relative abundance of 13 bacteria(Bacteroides fragilis,Butyricoccus pullicaecorum,Akkermansia muciniphila,Lactobacillus reuteri,etc.)and an increase in the relative abundance of 12 bacteria(Staphylococcus aureus,Escherichia coli,Lactococcus garvieae,Helicobacter pylori,Streptococcus pneumoniae,Klebsiella pneumoniae,etc.).3.Isolation and identification of probiotics in the intestinesA total of 27 strains of intestinal probiotics were isolated and identified from 232 lamb fecal samples,including 10 strains of Bacteroides fragilis,7 strains of Bacteroides ovatus,5 strains of Bacteroides uniformis,3 strains of Parabacteroides distasonis,2 strains of Lactobacillus reuteri.One strain from each of the above five strains with good growth and stable passage was selected for biological characteristics.B.fragilis,B.ovatus,B.uniformis,P.distasonis and L.reuteri entered a logarithmic growth phase at 6 h,4 h,2 h,6 h and 6 h,respectively,entered a plateau phase at 14 h,14 h,10 h,16 h and 12 h,respectively,and entered a recession phase at 22～24 h,20～22 h,18 h,20 h,and 22～24 h,respectively.L.reuteri and B.ovatus had certain resistance to the acidic environment of pH 2.0 and pH 4.0,while the remaining three strains had poor acid resistance.Under the condition of 0.1%bile salt concentration,B.fragilis was well tolerated,followed by B.ovatus and L.reuteri.Under the condition of 0.2%and 0.3%bile salt concentrations,L.reuteri was well tolerated.Microdilution method was used to test drug sensitivity on all isolates.The results showed that among the 27 isolates,8(4 strains of B.fragilis,2 strains of B.ovatus,2 strains of B.uniformis)were sensitive to metronidazole,7(6 strains of B.fragilis,1 strain of B.ovatus)were sensitive to cefoxitin,3(B.ovatus)were sensitive to ceftriaxone sodium,1(B.ovatus)was sensitive to clindamycin hydrochloride,and 2(B.ovatus)were sensitive to tetracycline hydrochloride.All isolates were not sensitive to ciprofloxacin hydrochloride and ampicillin sodium.A strain of B.fragilis was selected for animal toxicity research.The results showed that BF1 had no significant impact on body weight,diet,and mental state of mice,and there was no visible pathological changes in anatomy.