Correlation Between Infection Patterns Of Dairy Cow Mastitis Pathogens And Milk Quality Traits And Analysis Of Drug Resistance Genes

Mastitis is one of the most serious constraints affecting the healthy development of the dairy industry,causing serious harm to the entire industry chain.In this study,we collected the mastitis milk samples and routine milk samples from several dairy farms in Jiangsu and Shandong,and conducted an epidemiological investigation of mastitis in dairy cows.The pathogens were isolated and cultured by bacteriological methods,and 16S rDNA sequencing was used to identify the prevalence of sampled pasture mastitis.The situation,the establishment of a small pathogen strain library,combined with the DHI determination of dairy farms,analysis of the correlation between bacterial infection patterns and milk quality traits,based on the drug sensitivity test,the detection of the drug resistance genes of the main epidemic strain Klebsiella pneumoniae,to reveal The infection law and drug resistance mechanism of cow mastitis pathogens provide experimental basis for breeding mastitis resistance in dairy cows.This study mainly includes the following aspects:1.Isolation and 16S rDNA gene sequence analysis of pathogenic bacteria of cow mastitis:Epidemiological investigation was caried out on pastures in Jiangsu and Shandong provinces to isolate and identify pathogenic bacteria of mastitis.A total of 882 routine milk samples(n1=223,N2=220,N3=193,N4=246)were randomly collected from pasture 1,pasture 2,pasture 3 and pasture 4.The pathogenic bacteria were identified by bacteriology and 16S rDNA sequencing.The results showed that 39 strains of 11 pathogenic bacteria were isolated from the mastitis samples of Ranch 1,38 strains of 15 pathogenic bacteria were isolated from the mastitis samples of Ranch 2,12 strains of 6 pathogenic bacteria were isolated from the mastitis samples of Ranch 1 and 4 strains of 3 pathogenic bacteria were isolated from the mastitis samples of Ranch 4.228 strains of 36 pathogenic bacteria were isolated from routine milk samples of Ranch 1,275 strains of 32 pathogenic bacteria were isolated from routine milk samples of Ranch 2,226 strains of 42 pathogenic bacteria were isolated from routine milk samples of Ranch 3 and 322 strains of 67 pathogenic bacteria were isolated from routine milk samples of Ranch 4.2.To explore the relationship between the bacterial infection pattern of cow mastitis and the composition of milk and the number of somatic cells.The infection modes of milk isolates in four areas were classified as single bacterial infection,two bacterial infection and three bacterial infection.The corresponding DHI detection data were collated and analyzed by single factor variance analysis and correlation analysis.The results showed that:1.There were no significant differences in true protein rate,fat rate,crude protein rate,lactose rate and total solids rate among different bacterial infection modes(P>0.05),and no significant differences in SCC among different bacterial infection modes(P>0.05).2.With the increase of SCC range,the true protein rate,fat rate,crude protein rate and total solids rate in milk components were significantly different between SCC less than 300,000/ml and other three groups(P<0.05).There was no significant difference in true protein rate,fat rate,crude protein rate,lactose rate and total solids rate between groups when SCC was larger than 300 and less than 1000 kg/ml(P>0.05).There was no significant difference in true protein rate,fat rate,crude protein rate,lactose rate and total solids rate between groups when the milk composition was larger than 500,000/ml(P>0.05).When SCC was less than 300,000/ml and larger than 1000,000/ml,there were no significant differences in true protein rate,fat rate,crude protein rate and total solid content(P>0.05)and lactose rate(P<0.05).It was revealed that there was no correlation between bacterial infection patterns and milk components,and the changes of SCC in milk affected the changes of other milk properties.3.The drug sensitivity of pathogenic bacteria isolated from mastitis:using NCLLS susceptibility test standard and disc diffusion method to detect mastitis isolates on six drugs(ampicillin,ciprofloxacin,lincomycin,gentamicin,Sensitivity of streptomycin,penicillin),The results showed that the isolates from the four pastures showed full resistance to lincomycin,and the inhibition zone of the experiment was zero.Almost all of the isolates from cow mastitis samples from four pastures showed resistance to ampicillin,lincomycin and penicillin.Among them,Bacillus licheniformis isolated from Jielong farm,Enterococcus glycolyticus and Proteus mirabilis isolated from xuyi farm were sensitive,while Pseudomonas putidaf isolated from Jielong farm was moderately sensitive.The isolated strain was sensitive to ciprofloxacin,gentamicin and streptomycin.Among them,B.alkalophilus and Pseudomonas aeruginosa isolated from the Honghongweigang dairy industry showed resistance,and Bacillus subtilis showed moderate sensitivity.4.Detection of resistance genes of 38 strains of Klebsiella pneumoniae isolated from Pasture 1:Cases of cow mastitis caused by Klebsiella pneumoniae increased year by year,with long course of disease and easy re-infection.The drug resistance of 38 strains of Klebsiella pneumoniae was determined by drug sensitivity test,and the positive rate of drug resistance genes was determined by PCR gel electrophoresis,?-lactamase gene TEM,SHV,LEN,OKP,CTX-M-1 group,CTX-M-2group,CTX-M-9group,PER,GES,VEB,aminoglycoside gene aac(6)-Ib,aadA4/5,aadA6/16,aph(3)-I,aph(3')-IIb,efflux pump mdfA,zygote plasmid genetic marker traA,trbC,insert sequence genetic marker IS 1133,quinolone gyrA,parC,16s rRNA methylase gene armA,rmtA,rmtB,rmtC,rmtD a total of 28 genes,Results The detection rates of 28 genes in 38 strains of Klebsiella pneumoniae were as follows:OPK(100%),TEM(42.11%),SHV(68.42%),LEN(97.37%)of ?-lactamases.The detection rates of other CTX-M-1 group,CTX-M-2 group,CTX-M-9 group,PER,GES,and VEB are zero.The detection rates of aminoglycosides aac(6)-Ib(31.68%),aph(3)-I(78.95%),aph(3')-IIb(13.16%),aadA4/5 and aadA6/16 Zero.The efflux pump mdfA(15.79%),the zygosity plasmid genetic marker trbC(76.32%),the detection rate of trbA was zero.Insert sequence genetic marker IS1133(21.05%),transposon genetic marker IntI2(21.05%),merA detection rate is zero,quinolones parC(100%),gyrA(81.58%),16S rRNA methylase rmtA(100%),rmtB(86.84%),rmtD(7.89%),armA and rmtC have a detection rate of zero.Most strains contain multiple combinations of resistance genes.2,analysis of drug susceptibility test results:in 38 strains of Klebsiella pneumoniae,89.47%were resistant to ampicillin,34.21%were resistant to cefotaxime,7.89%were resistant to cefotaxime,and 39.47%were resistant to ceftazidime.50.00%were resistant to amoxicillin,73.68%were resistant to penicillin,18.42%were resistant to piperacillin,and closely related to?-lactamase resistance gene;7.89%were resistant to gentamicin,7.89%Tobramycin resistance,associated with aminoglycoside resistance genes;34.21%resistance to chloramphenicol,related to quinolone resistance genes;31.58%resistance to tetracycline;to cefotaxime,ciprofloxacin Meropenem is sensitive.Some mobile genetic elements have an inseparable relationship to the transfer of drug resistance genes.The complexity of the experimental results also suggests the complexity of the bacterial resistance mechanism.