| Fuping County in Shaanxi Province has the title of "China’s Goat Milk Capital".The dairy goat industry has become one of the most characteristic leading industries in the region and an important part of the local economic development.With the expansion of its breeding scale,the prevalence of mastitis in dairy goats has caused serious economic losses to the development of the industry.The purpose of this study was to explore the diversity of microbial communities in the milk of dairy goat mastitis,the prevalence and some biological characteristics of the main pathogens,and to provide a scientific basis for the prevention and treatment of dairy goat mastitis in this region.In this study,clinical mastitis goat milk samples and healthy goat milk samples were collected from three dairy goat farms in Fuping County,and the bacterial community composition and abundance changes in the samples were analyzed by metagenomic sequencing..The clinical mastitis milk samples and healthy milk samples were analyzed for bacterial community composition and abundance changes in samples.Laboratory bacterial isolation and culture and 16 S r RNA gene sequencing were used to identify the main pathogenic bacteria in the 84 clinical mastitis milk samples collected.The minimum inhibitory concentration(MIC),drug resistance genes and virulence genes were detected for the four major pathogenic bacteria isolated and purified,including coagulase-negative Staphylococcus,Staphylococcus aureus,Pseudomonas aeruginosa,and Cryptobacterium pyogenes;In addition,the cell plate culture method was used to detect the biofilm forming ability of the strains.The test results are as follows.1.According to the metagenomic high-throughput sequencing data of the 12 samples that were successfully sequenced,a total of 1 kingdom,35 phyla,87 classes,211 orders,377 families,708 genera,and 1107 species were obtained by cluster analysis.The results of data analysis showed that both mastitis milk and healthy breast milk have rich bacterial diversity,but there are great differences in the bacterial community structure and abundance,and there are also certain differences between different samples in the mastitis group.The main dominant phyla in the samples were Proteobacteria,Firmicutes,Actinobacteria,Bacteroidetes and Fusobacterium,but no Fusobacterium was detected in healthy milk.And both mastitis samples and healthy samples have their own dominant bacterial phyla.The dominant bacterial genus in the overall samples were Enterobacter,Cryptobacter,Clostridium,Staphylococcus and Bacteroides,but Cryptobacter and Fusobacterium were not detected in healthy goat milk.2.84 mastitis milk samples were isolated and cultured,purified,stained and identified by microscopy and 16 S r RNA gene sequencing.A total of 74 strains of bacteria were obtained(88%),including 26 strains of coagulase-negative staphylococci(31%),18 strains of Staphylococcus aureus(21%),and 13 strains of Pseudomonas aeruginosa(15%),6 strains of Cryptobacterium pyogenes(7%),3 strains of Streptococcus(4%),2 strains of Escherichia coli(3%),2 strains of Corynebacterium pseudotuberculosis(3%),2 strains of Mansoni hemolytica(3%),1 strain of Bacillus(1%),and 1 strain of Enterococcus(1%).3.Antibacterial drug susceptibility testing of 4 major pathogens found that all tested isolates had the highest resistance rates to penicillin,followed by tetracyclines.Nearly all tested isolates showed varying degrees of sensitivity to vancomycin,clindamycin and chloramphenicol.The detection results of antibiotic resistance genes of four pathogens showed that the detection rate of β-lactam drug resistance genes was the highest,including CNS 88%,Staphylococcus aureus 83%,Pseudomonas aeruginosa 100%,Cryptobacterium pyogenes 100%,then Resistance genes tet(M)(CNS 80%,Staphylococcus aureus 61%,Pseudomonas aeruginosa 84%)and tet(K)(CNS 65%,Staphylococcus aureus 55%,Pseudomonas aeruginosa 53 %,cryptobacillus pyogenes 33%),Quinolones glr B(CNS 34%,Staphylococcus aureus 38%)and glr A(Pseudomonas aeruginosa 23%)and aminoglycoside resistance gene aac A-aph D(CNS 42%)While no chloramphenicol resistance gene was detected in all tested isolates.4.The detection results of virulence genes of main pathogens showed that the virulence genes of Staphylococcus aureus with higher detection rate were hlb(100%),hla(88%),sea(77%),ica A(66%)and clf A(61%),While the virulence genes of coagulase-negative staphylococci with higher detection rate were sea(80%),seb(73%),ica C(65%)and hla(50%).The highest virulence gene carrier rate of Pseudomonas aeruginosa was T2 SS secretion system tox A(84%)and phospholipase gene plc H(76%).The carrier rate of C.pyogenes hemolysin gene plo and neuraminidase gene nan H were both 100%.5.Biofilm test results show.,Staphylococcus aureus can form biofilms(18/18),9strains of biofilm formation ability are strongly positive(+++);20(20/26)strains of coagulase-negative staphylococci can form biofilms 4 strains were strongly positive(+++);10(10/13)strains of Pseudomonas aeruginosa could form biofilm,and 6 strains were strongly positive(+++);There are 4 strains(4/6)of Cryptobacter pyogenes which can form biofilm,and 4 strains are all moderately positive(++).In conclusion,this study conducted a comparative analysis of the changes in the structure and abundance of microflora in goat milk with mastitis by means of metagenomic sequencing technology,proving that there is a close relationship between dysbiosis and the pathogenesis of mastitis.In addition,the investigation showed that the main pathogenic bacteria of dairy goat mastitis in this area were coagulase-negative Staphylococcus,Staphylococcus aureus,Pseudomonas aeruginosa and Cryptobacterium pyogenes,while Streptococcus and Escherichia coli were less.And for the main pathogenic bacteria,sensitive drugs were screened and some of their biological characteristics were obtained.All the above results enrich the relevant experimental data of dairy goat mastitis disease in this area,and hope to provide reference for the prevention and treatment of mastitis in this area. |
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