Study On Microorganism Diversity In Fresh Milk Of Dairy Cows And Breast Tissue Transcriptome With Mastitis

Dairy cow mastitis is the most common disease in dairy farms,which causes serious economic losses to dairy farming and has been troubling the healthy development of dairy industry for a long time.The etiology of cow mastitis is generally believed to be the infection of mammary gland vesicles caused by exogenous pathogenic bacteria invading the mammary tissue,and it is also believed that the occurrence of cow mastitis is related to the imbalance of endogenous microorganisms.This study through the analysis of the incidence of mastitis in dairy farms,separation of pathogenic bacteria,drug sensitive test,microbial diversity in milk sample testing and transcriptome analysis of mastitis cows breast tissue,the occurrence of mastitis and the relationship between microbial diversity in breast,breast tissue in the molecular immune mechanism,so as to provide new perspectives for the mechanism of dairy cow mastitis.In order to explore the relationship between the microbial diversity in milk,the molecular immune mechanism in breast tissue and cow mastitis.This study through the analysis of the incidence of mastitis in dairy,separation of pathogenic bacteria,drug sensitive test,microbial diversity in milk sample testing and transcriptome analysis of mastitis cows breast tissue.The relationship between the occurrence of mastitis and the diversity of microorganisms in milk and the molecular immune mechanism of breast tissue provide a new perspective for the study of the pathogenesis of cow mastitis.Section one:A total of 1,827 dairy cows were selected to study the incidence of mastitis in some large dairy farms in Jiangsu province.Milk samples were collected continuously for 7 months,once a month at the same time.According to the results of somatic cell detection,20 milk samples with the number of somatic cells more than 1 million/ml were randomly selected from the cattle farm for bacterial isolation and identification and drug susceptibility test.The results showed that the incidence of mastitis in dairy cows was seasonally related,and the incidence of mastitis in winter was generally lower than that in summer.A total of 6 kinds of bacteria were detected in 20 milk samples from a cattle farm in Nantong,and the highest detection rates were Escherichia coli,Citrobacter,Klebsiella pneumoniae and Acinetobacter.The results of drug sensitivity test showed that ampicillin sodium,penicillin potassium and streptomycin sulfate had the best bacteriostatic effect.Section two:In this experiment,45 milk samples from a cattle farm in the area of Nantong in Jiangsu province were randomly selected to study the microbial diversity in normal and mastitis milk samples,and divided into three groups according to the results of somatic cell detection:the normal group(less than 200,000/ml),the subclinical group(50-1 million/ml)and the clinical group(over 1 million/ml).Bacterial isolation and identification were performed on milk samples with a concentration of more than 1 million/ml of somatic cells.The results showed that Streptococcus agalactiae was detected in milk samples of mastitis.Meanwhile,16SrRNA high-throughput sequencing technology was used to analyze bacterial DNA in three groups of milk samples.After cluster analysis of sequencing results,a total of 394 OTU species were obtained,covering 383 phyla,375 classes and 160 genera.Species abundance analysis showed that the phylum classification level was mainly Firmicutes,Cyanobacteria and Proteobacteria,while the dominant genera were mainly Bacillus,Lactobacillus and Oceanobacillus.The microbial diversity of fresh milk increased with the level of somatic cells.Fresh milk from cows with clinical mastitis had the highest microbial diversity.Serratia and Streptococcus are the main pathogenic bacteria of clinical mastitis in dairy farm.Section three:The purpose of this study was to investigate the molecular mechanism and the changes of gene expression during the immune defense process in the breast tissues of Streptococcus pyogenes dairy cow mastitis,two of which were dairy cow mastitis individuals caused by Streptococcus pyogenes infection,and the other two were normal controls.Transcriptome sequencing using RNA-seq technology in the mammary tissue of two groups of cows found that there were a total of 2481 differentially expressed genes between healthy mammary glands and mammary glands of mastitis,among which 2061 were up-regulated and 420 differentially expressed genes were down-regulated.Comparison with GO database showed that the significantly enriched GO term was the extracellular exosome,extracellular space,inflammatory response,defense response to bacterium and Heparin binding.The KEGG pathway enrichment analysis found 48 significantly enriched biological pathways,the most significant of which included the PI3k-Akt signaling pathway,the cytokine-cytokine receptor interaction pathway,the HTLV-I infection pathway,the tumor pathway,and the chemokine signaling pathway.In addition,59933,59770,61140 and 61222 selective shear events were identified in four samples.2,831 and 2,502 new transcripts were found in the mastitis group and the normal group,respectively.These new findings will provide data for the study of the molecular mechanism of the mammary tissue response to different pathogenic bacteria infections in dairy cows.In this experiment,it was found that there were many pathogenic bacteria of dairy cow mastitis in some dairy farms in Jiangsu province,and the combination of ampicillin sodium,penicillin potassium and streptomycin sulfate had the best bacteriostasis effect.Compared with normal milk,fresh milk of dairy cows with mastitis showed no significant difference in microbial diversity,but some species abundance showed significant difference,indicating that breast health of dairy cows was closely related to microbial homeostasis in the udder.The new discovery of transcriptome sequencing results of mammary tissue of dairy cows with Streptococcus pyogenes mastitis can provide data for the study of the molecular mechanism of the mammary tissue of dairy cows in response to different pathogenic bacteria infections.