| OBJECTIVES:Bovine mastitis is a common infectious disease that affects the daily management of dairy farms.Its occurrence and development are affected by both pathogens and host genetic factors.Among them,Streptococcus agalactiae as an important environmental pathogen,plays a role in mastitis in dairy cows,host genetic factors affect the susceptibility to mastitis in dairy cows as well.Although previous studies have accumulated many research results in the pathogenic and epidemiology of bovine mastitis,the pathogenic characteristics of bovine mastitis,the genetic relationship with human strains and their association with host immune gene polymorphism are still needed to further be uncovered.This study intends to comprehensively and systematically analyze the pathogenic characteristics and the genome sequence of Streptococcus agalactiae,the genetic evolution relationship with human strains,and association with host immune genes(BOLA and CD14)polymorphism,which would provide basis for comprehensive control and treatment of Streptococcus agalactiae-causing bovine mastitis in dairy cows.METHODS:(1)Twenty-one dairy farms of 10 provinces in China were selected to carry out Streptococcus agalactiae isolation and culture from subclinical bovine mastitis,Streptococcus agalactiae,screening by differential medium,isolation and culture of pathogens,capsular serotyping,genotyping,major virulence genes(hemolysin III,C-β protein,C-α protein,surface protein rib,hyaluronate lyase,CAMP factor,β-hemolysin/cytolysin,C5 a peptidase)detection and anti-drug susceptibility(Erythromycin,clindamycin,ampicillin,cefepime,vancomycin,levofloxacin,chloramphenicol)test,which would comprehensively and systematically elucidate the epidemiological characteristics of Streptococcus agalactiae in bovine subclinical mastitis in China.(2)Eight Streptococcus agalactiae isolates from bovine subclinical mastitis were carried out whole genomic sequencing by Illumina Novaseq and PacBio Sequel platforms.After assembling and splicing,the Streptococcus agalactiae genome sequences were annotated and analyzed by bioinformatic tools,and elucidated the genomic characteristics.(3)Using Xinjiang hospitals as the research site,the human Streptococcus agalactiae were isolated and identified from human samples,and then were detected the capsular serotype,genotype,virulence gene and anti-drug resistance.Furthermore,the dominant isolates were conducted whole genome sequencing and phylogenetic analysis,and compared the difference with bovine isolates.(4)The milk and blood samples were collected and done subclinical mastitis test.Then,the positive samples were used to isolate Streptococcus agalactiae and detect BOLA and CD14 gene polymorphism detection.The effects of subclinical mastitis on daily milk yield and the association of polymorphisms of BOLA and CD14 genes with Streptococcus agalactiae susceptibility were also revealed.RESULTS:(1)A total of 2,225 milk samples were collected from 21 dairy farms in 10 provinces,including Hainan,Henan,Liaoning,Inner Mongolia,Shaanxi,Shaanxi,Shandong,Sichuan,Xinjiang and Zhejiang,in China,and 946 samples were tested as subclinical mastitis by LMT,the detection rate was 42.52%(95%CI 40.46%-44.57%).From the above subclinical mastitis milk samples,133 bovine mastitis Streptococcus agalactiae isolates were isolated,the isolation rate was 14.06%(95%CI 11.94%-16.37%),the highest isolation rate of Streptococcus agalactiae was 21.68%(95%CI 16.90%-26.45%)in Xinjiang,and the lowest was in Zhejiang(6.90%,95%CI 0.03%-13.41%).The serotype of bovine mastitis Streptococcus agalactiae was serotype Ia;the genotypes were mainly distributed in ST103,ST67 and ST4,and ST103 was the dominant genotype.In addition to the conserved virulence genes,the dominant virulence genes were C-α protein(16.54%,22/133),surface protein(13.53%,18/133),the detection rate of hemolysin III and C5 a peptidase were 2.26% and 3.76%,no C-β protein gene was detected.Furthermore,1 vancomycin-resistance(0.75%,1/133),2 ampicillin/cefotaxime/levofloxacin multi-resistance(1.50%,2/133)and 1 ampicillin/cefepime/levofloxacin/vancomycin multiresistance(0.75%,1/133)bovine mastitis Streptococcus agalactiae were found in this study,all these isolates were ST103 and isolated from Xinjiang and Liaoning.(2)Eight bovine mastitis Streptococcus agalactiae isolates genomic sequences were obtained.These isolates encoded 2170-2237 genes,carried 24-29 virulence genes,and carried tetracycline-resistant ribosomal protection protein 、 defensin resistant mprF 、 ANT(6)、chloramphenicol acetyltransferase(CAT)、major facilitator superfamily(MFS)antibiotic efflux pump antimicrobial resistance gene families.(3)A total of 9 human Streptococcus agalactiae were collected from Xinjiang hospitals,and their capsular serotypes were mainly Ia,Ib and III strains.The genotypes were mainly ST10,ST485,ST314,ST19 and ST1168.Seven,3,3 and 1 isolates were resistant to clindamycin(77.78%),erythromycin(33.33%),levofloxacin(33.33%)and cefepime(11.11%).After whole genome sequencing,4 human Streptococcus agalactiae genome draft map obtained,which encoded 2105-2225 genes,carried 24 or 37 virulence genes and 7 antimicrobial resistance gene families,such as APH(3’)、ABC-F ATP-binding cassette ribosomal protection protein、defensin resistant mprF、Erm 23 S ribosomal RNA methyltransferase、major facilitator superfamily(MFS)antibiotic efflux pump、ANT(6)、tetracycline-resistant ribosomal protection protein.(4)There are different ST types in bovine and human strains.The proportion of isolates carrying hymolysin III and C-α protein in human strains is significantly higher than that in bovine strains,while hyaluronate lyase in human is significantly lower than the latter(p< 0.001);the ratio of human isolates resistant to erythromycin and clindamycin was higher than that of bovine strains(p<0.001).Human isolates SA007 is in the same evolutionary branch as bovine strains SA002,SA009,SA003,SA080,SA090,SA091 and SA098.When compared to human strains,bovine isolates did not carry fbsA,lmb,pilA,pilB,pilC,srtC3 and srtC4,and did not carry ADR(3’),ABC-F ATP-binding cassette ribosomal protection protein and Erm 23 S ribosomal RNA methyltransferase as well.(5)Bovine subclinical mastitis could cause milk production to decrease by 3.65±1.09kg/day,accounting for 12.15%±3.36% of daily milk production;Bovine subclinical mastitis caused by Streptococcus agalactiae could cause to decrease by 5.62 ± 1.56kg/day of daily milk production,accounting for 18.71%±5.19 of daily milk yield.Tweenty-five 25 mutation loci,including BOLA(80C→G),BOLA(93T→C),BOLA(188C→G),BOLA(191G→T)were found in the polymorphism of BOLA and CD14 genes.),BOLA(196A→G),BOLA(218C→G),BOLA(222A→G),BOLA(233G→T),BOLA(209T→A),CD14(g528 A→C),CD14(g612 A→ G),CD14(g1022 A→G),CD14-3(g285A→C),CD14-6(g161 A→G),CD14-6(g173 A→G),BOLA(219A→G),BOLA(241C →G),BOLA(243 T→G),BOLA(244 A→T),BOLA(245 C→G),BOLA(266 T→G),BOLA(267 T→C),BOLA(268 A→G)25,BOLA(269 T→G),BOLA(270 T→G)were found,among them,BOLA(218C→G)allele was associated with Streptococcus agalactiae infection in dairy cows(p<0.05),and could Increased risk of infection with Streptococcus agalactiae in dairy cows(OR=2.343;95% CI 1.148-4.781).CONCLUSIONS:(1)The dominant serotype of Streptococcus agalactiae in subclinical mastitis in major provinces of China was type Ia,and the dominant genotype was ST103.Except conserved virulence genes,the dominant virulence genes was C-α protein and surface protein rib.These isolates of bovine carried a higher anti-drug-resistance to levofloxacin,1 vancomycinresistance isolate,2 ampicillin/cefotaxime/levofloxacin and 1 ampicillin/cefotaxime multi-drug resistant isolates were found.(2)Twelve bovine mastitis and human Streptococcus agalactiae were determined.(3)Nine strains of human Streptococcus agalactiae were collected were mainly serotypes Ia,Ib and III,and genotypes ST10,ST485,ST314,ST19 and ST1168,and carried a higher antidrug-resistance(non-susceptible)to Clindamycin,erythromycin and levofloxacin.(4)(3)The genomic sequences of 8 bovine and 4 human Streptococcus agalactiae were obtained,of which showed differences in capsular serotype,virulence genes and anti-drug resistance genes.(4)The BOLA(218C→G)allele was associated with Streptococcus agalactiae infection,which increased the potential risk of infection with Streptococcus agalactiae in dairy cows(OR=2.343;95% CI 1.148-4.781). |
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