| Dairy mastitis is one of the most common and highest morbidity diseases in dairy industry,and Bacillus cereus is an important conditional pathogen in dairy mastitis.With the use of antibiotics,more selectivity and possibilities are provided for the evolution of drug-resistant bacteria.The emergence of drug-resistant strains,especially multi-drug-resistant strains,leads to the increasingly serious problem of the drug resistance of Bacillus cereus.In addition,the toxic plasmid of Bacillus cereus can carry horizontal transfer,and many Bacillus cereus themselves carry toxin genes,which makes the harm of Bacillus cereus gradually increasing,especially antibiotic-resistant strains and multi-drug-resistant strains are becoming potential threats to veterinary and public health.In this study,clinical mastitis milk samples of dairy cows from three large-scale ranches in Inner Mongolia were collected for epidemiological investigation of mastitis source pathogens,and the main pathogens were isolated and identified.The resistance phenotype,virulence gene and pathogenicity of the isolated Bacillus cereus were detected.The objective was to understand the dominant pathogenic bacteria of mastitis infection in dairy cows in Inner Mongolia,the distribution of drug resistance and virulence genes of isolated Bacillus cereus,and to determine its pathogenicity in mice,so as to provide experimental basis for the prevention and control of the epidemic spread of Bacillus cereus from mastitis in Inner Mongolia.In this study,the bacterial isolation culture method was used for morphological identification,16SrDNA was used for molecular biological identification,and 54 samples of clinical mastitis milk samples were collected for bacterial isolation and identification.The results showed that 12 kinds of bacteria were isolated in this experiment,a total of 170 strains,including 1 sterile milk sample,the total detection rate of bacteria samples was 98.14%.The detection rate of Bacillus cereus was the highest(30.58%,52/170),followed by Staphylococcus(21.76%,37/170).The detection rates of Enterococcus faecalis,Arthrobacter and actinobacillus were 0.59%(1/170).Of the 53 milk samples with bacteria detected,10(19%)had one bacteria detected,6(11%)had two bacteria detected,16(30%)had three bacteria detected,11(21%)had four bacteria detected,5(9%)had five bacteria detected,4(8%)had six bacteria detected,and 1(2%)had seven bacteria detected.The mixed infection rate was 81%.The results of bacterial isolation and identification showed that the pathogens of mastitis in dairy cows in Inner Mongolia were mainly Bacillus and staphylococcus,and there were mixed infections of various pathogens,and mixed infections accounted for a large proportion,which revealed that the mixed infections in this area were relatively serious.The drug tolerance of 52 strains of Bacillus cereus to 8 kinds of non-β-lactam antibiotics was analyzed.The results were as follows:52 strains of Bacillus cereus were sensitive to gentamicin,amicacin and roxithromycin.26 plants are waxBacillus is sensitive to tetracycline;Eleven strains of Bacillus cereus were sensitive to tetracycline.Six strains of Bacillus cereus mediated tetracycline;Three strains of Bacillus cereus mediated erythromycin;Five strains of Bacillus cereus mediated clindamycin;Two strains of Bacillus cereus mediated compound neosumine;One strain of Bacillus cereus mediated roxithromycin;52 strains of Bacillus cereus were sensitive to gentamicin,amicacin and roxithromycin,but insensitive to erythromycin,tetracycline,ciprofloxacin,clindamycin and cotrimoxazole,and tetracycline sensitive strains were the least.Among 52 isolates of Bacillus cereus,30 strains were found to be drug-resistant,and the proportion of drug-resistant was 57.6%.The proportion of drug resistance detected was 42.3%,among which 12 strains of Bacillus cereus were single drug resistant,10 strains of Bacillus cereus were double drug resistant,and no multiple drug resistant strains were found.There were 5 drug resistance types in total,which were:tetracycline,ciprofloxacin,tetracyclineCotrimoxazole,tetracycline-ciprofloxacin,cotrimoxazole-ciprofloxacin.The prevalence of drug resistance spectrum in this survey is shown in the table:tetracycline and tetracycline and cotrimoxazole,accounting for 50%and 31.9%of drug-resistant strains,respectively;tetracycline-ciprofloxacin resistant strains and cotrimoxazole-ciprofloxacin resistant strains accounted for 9.1%and 4.5%,respectively;ciprofloxacin resistant strains accounted for 4.5%;However,it showed high sensitivity to gentamicin,roxithromycin,amikacin and other drugs.The main virulence factors carried by 52 strains of Bacillus cereus from mastitis were detected by PCR.It was found that all 52 strains of Bacillus cereus carried nheA,nheB and entFM genes.99.9%of Bacillus cereus carried 4 or more virulence factors.98.1%of Bacillus cereus carried nheC gene;bceT gene was found in 36.5%of Bacillus cereus.32.7%of Bacillus cereus carried hblA gene;30.8%of Bacillus cereus carried cytK gene;26.9%of Bacillus cereus carried hblC gene;25%of Bacillus cereus carried ces gene;25%of Bacillus cereus carried the EM1 gene;21.2%of Bacillus cereus carried the hblD gene.Two strains of Bacillus cereus with different number of virulence genes were used to challenge mice.The results showed that the two strains of Bacillus cereus had different effects on ALT,AST,LDH,ALP,SOD,GSH-Px and MDA in mouse plasma:ALT,AST,LDH,ALP,SOD,GSH-Px and MDA were significantly increased in groups Ⅰ and Ⅱ.The above indexes suggested that different degrees of liver function injury occurred in groupsⅠ and Ⅱ after challenge.In addition,the two strains of Bacillus cereus with different number of virulence genes had significantly enhanced the expression of inflammatory genes in mice liver,and the expression of inflammatory genes in mice liver in group Ⅱwas stronger than that in group Ⅰ,indicating that Bacillus cereus carrying virulence genes in group Ⅱ had greater damage to liver.The liver and kidney of challenged mice werecollected,and HE staining showed that the two strains of Bacillus cereus with different number of virulence genes were very harmful to the liver and kidney of mice,and both could cause liver and kidney inflammation.Tetracycline-ciprofloxacin combined treatment decreased the expressions of IL-6,IL-1β,and TNF-α genes in mice,suggesting that the combination of antibiotics inhibited the expression of liver inflammatory genes.The results of this study revealed the widespread spread of Bacillus cereus in clinical mammary infection of dairy cows and its potential pathological harm to the host,and provided experimental basis for the prevention and control of Bacillus cereus mastitis of dairy cows. |
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