Isolation And Identification Of Mastitis Pathogens In Large-Scale Pasture Dairy Cows And Study On Drug Resistance And Pathogenicity Of Klebsiella Pneumoniae

Cow mastitis is one of the important diseases that cause economic losses in dairy farms.Some pathogenic bacteria can cause zoonotic diseases or food poisoning,threatening to human health.Klebsiella pneumoniae can cause cow mastitis under some conditions and it is also a common zoonotic pathogen.This study investigated the pathogenic bacteria of cow mastitis in large-scale pastures,analyzed the pathogenicity and drug resistance of isolated Klebsiella pneumoniae,and established a rat model of Klebsiella pneumoniae mastitis,which provide theoretical basis and practical guidance for the drug resistance,prevention and control of cow mastitis.The results are as follows.(1)Epidemiological investigation of pathogens of cow mastitis in large-scale pastures856 milk samples of mastitis were collected from 11 large-scale dairy farms in Fujian,Jiangsu,Guangzhou,Hebei,Anhui,Shanxi,Inner Mongolia,and Zhejiang provinces,then the bacteria were cultured andidentified.The results showed that the detectable rate of bacteria in mastitis samples was 84.46%.Pathogenic bacteria leading to mastitis are mainly environmental pathogenic bacteria,including Escherichia coli(22.74%),Klebsiella pneumoniae(14.55%),and Enterobacter cloacae(9.05%),etc.Infectious pathogenic bacteria were also isolated,such as Staphylococcus aureus(4.25%)and Streptococcus agalactiae(1.71%).There were differences in the separation of mastitis pathogens in different regions.The conclusions show that conditional pathogenic bacteria are more dangerous and increase with the standardization of modern breeding.(2)Virulence genes and drug resistance analysis,biofilm formation ability of 119 Klebsiella pneumoniae isolated from mastitisFor 119 strains of Klebsiella pneumonia.PCR was used to detect the virulence genes.Biofilm formation capacity were also tested.Drug sensitivity tests were carried out,and 13 types of beta-lactamase gene carrying were tested in ESBL-positive strains.The results showed that magA,wcaG,rmpA,wabG,fimH,uge,Aerobactin,ybtA,and ureA genes were found in 119 strains of Klebsiella pneumonia,but IucB,kfuBC and allS were not got.Biofilm of 103 strains were positive;119 strains of Klebsiella pneumoniae have the highest resistance ability to ampicillin,with a resistance rate of 94.96%,and resistance to some cephalosporins and aminoglycosides.But it was most sensitive to kanamycin with a sensitivity rate of 97.48%.28 strains of Klebsiella pneumoniae yielding ESBLs were detected.TEM,SHV,OXA-1 and CTX-M of β-lactamase genes were detected as 19,22,2 and 19 strains,others were not found.The resistance rate of strains yielding ESBLs was higher than that of strains non-yielding ESBLs.This results indicate that carrying rate of the main virulence genes in Klebsiella pneumoniae isolated from large-scale pasture is high,and some isolates are highly pathogenic.The high positive rate of biofilms is more likely to produce multi-drug resistance,and some strains carry multiple β-lactamase genes,so the multi-drug resistance of Klebsiella pneumoniae should be concerned and guarded against by pastures.(3)Establishment of rat mastitis model induced with Klebsiella pneumoniae and pathogenicity studyFemale mice with lactation for 4 days were used as experimental animals.100 μL of Klebsiella pneumoniae(1 × 109 cfu/mL)was injected into the fourth pair of mammary.Blood and mammary gland samples were collected at 6 h,12 h,and 24 h after injection.Clinical symptoms and changes of the mammary glands were observed.CBC,pathological changes of mammary glands,and expression of related inflammatory genes were detected.The results showed that the female rats in the experimental group were depressed,the mammary areas were swollen,the mammary glands was edema and severely congested,and there was milk clot.The count of WBC was significantly reduced,compared with the control group(p<0.01).Histopathological observation showed that a large number of inflammatory cells appeared in the acinus in the experimental group,and some epithelial cells were shed.Fluorescence quantitative PCR detection found that the gene expression levels of IL-1β,IL-6,IL-8,TNF-αand NOD2 of the mammary tissues in the experimental group were significantly increased(p<0.01)compared with the control group.Therefore,the rat mastitis model was successfully established with Klebsiella pneumoniae.Klebsiella pneumoniae promoted the release of related inflammatory factors and lead to breast mammary damageIn summary,the main pathogenic bacteria causing mastitis in large-scale pastures are mainly conditional pathogens,such as Escherichia coli and Klebsiella pneumoniae,and some Klebsiella pneumoniae have multi-drug resistance,which should be focused in pastures.A large number of neutrophils were found in the acinus and some epithelial cells were damaged when rat mammary glands were infected with Klebsiella pneumoniae.And the bacteria promote the release of related inflammatory factors,causing mastitis.This study provides investigation reports and basic theoretical research,which can guide to the prevention and control of mastitis in China.