| Bovine mastitis have a high incidence in dairy farming industry,which has poor treatment effect and is easy to recur,causing huge economic losses to dairy farming industry and causing serious food safety probles.The invasion of pathogenic microorganisms are the main cause of dairy cow mastitis.There are many pathogenic microorganisms that cause the disease of dairy cow mastitis,and the infection situation and pathogenesis are extremely complicated.There are few studies on lactococcus acidus causing dairy cow mastitis at home and abroad.This paper aims to provide theoretical basis and reference for the prevention and treatment of dairy cow mastitis by studying the isolation,identification,genotyping and pathogenic characteristics of Lactococcus lactis causing clinical mastitis.Research methods:Lactococcus lactis was isolated from milk samples of dairy cows with clinical mastitis by colony characteristics,microscopic examination,catalase test,16 SrRNA sequencing and sequence alignment.Phenotypic analysis were carried out by biochemical test,antibiotic sensitivity of the isolates were detected by broth microdilution method,and genotypig of the isolates was performed.According to the results of typing,strains were selected for lethal test of zebrafish to select virulent strains.Different concentrations of lactococcus lactis virulent strains were injected into the mammary gland of BALB/ C mice,and the optimum concentration of attack bacteria was screened by autopsy and pathological observation.Fifty mice after delivery were divided into two groups: blank group(control)and challenged group(LL-1).The degree of mammary gland tissue damage was observed by autopsy and HE staining at different modeling time;Observe the change of bacterial quantity in mammary tissue at different time;The expression levels of IL-6,IL-1β and TNF-α in breast tissues at different time were detected by ELISA;qRT-PCR was used to detect the expression levels of ASC,P65 and phosphorylated P65 in breast tissue 48 h after bacterial attack.Twenty mice were divided into 4 groups for antibiotic treatment experiment,with 5 mice in each group:control,LL-1,LL-H and LL-L.The modeling method was the same as above.24 h after bacterial attack,LL-H and LL-L were intramuscularly injected with 30 mg/Kg and 10 mg/Kg marbofloxacin,while LL-1 and control were injected with the same dose of physiological saline at the same position.After 48 hours of modeling,the mice were sacrificed for tissue dissection and pathological observation;the amount of bacteria in breast tissue was observed;The expression of TNF-α in breast tissue was detected by immunohistoch emistry;the expression levels of IL-6,IL1-β and TNF-α in tissues were also detected by ELISA;TUNEL was used to observe the apoptosis of tissues.Bacterial cells were co-cultured according to MOI 5.The effect of Lactococcus lactis on the release of MAC-T lactate dehydrogenase was determined at different bacterial attack times.Bacterial cells were co-cultured according to MOI 50.The invasion rate and adhesion rate of bacteria to cells were observed at different bacterial attack times.The bacterial cells were co-cultured according to MOI 5,and the damage of bacterial cells under at different bacterial attack times were observed by scanning electron microscopy and Gram staining.Results:8 strains of Lactococcus lactis were successfully isolated from the milk of 457 dairy cows with clinical mastitis,the clinical isolation rate was 1.75%.Eight isolates were genotyped by RAPD,and three subtypes were identified.All isolates were sensitive to marbofloxacin and vancomycin.The highest fatality rate was LL-1,which was selected as a virulent strain.The expression of IL-6,IL-1β and TNF-α in breast tissue was significantly increased when LL-1 was injected into mouse breast tissue,and the acinar structure was destroyed and a large number of inflammatory cells appeared.The contents of P65,phosphorylated P65 and ASC in tissues were significantly increased by qRT-PCR.Mastitis mice were treated with marbofloxacin at high and low doses,and significantly reduced the levels of three inflammatory factors and bacteria amount in the tissues of the two treatment groups.TUNEL observation showed that antibiotic treatment could significantly reduce the rate of apoptosis.Lactococcus lactis can damage cells.With the increase of co-culture time of bacterial cells,the adhesion rate and invasion rate of Lactococcus lactis to MAC-T also increased gradually.Lactococcus lactis was observed to damage cells by scanning electron microscopy and Gram staining.Conclusion:Eight strains of Lactococcus lactis were successfully isolated from milk of dairy cows suffering from clinical mastitis.Phenotype has certain differences,according to the genotyping of a total of three subtypes.All isolates were sensitive to marbofloxacin and vancomycin.LL-1 can induce murine mastitis,activate NF-κB/ P65 signaling pathway,make mammary gland epithelial cell denaturation necrotic,destroy acinar structure,cause tissue damage,leading to cell apoptosis.Marbofloxacin can alleviate the inflammatory response and reduce the number of apoptosis,and the optimal dosage is 30 mg/Kg.Lactococcus lactis has strong cytotoxicity to cells,and has adhesion and invasion ability to MAC-T,which can damage cells.Marbofloxacin can be used in clinical treatment of dairy cow mastitis induced by Lactococcus lactis,providing theoretical basis and reference for the prevention and treatment of dairy cow mastitis. |
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