Isolation And Identification Of Pathogenic Bacteria Of Subclinical Mastitis In Dairy Cattle And Establishment Of Lamp Detecting Method

The bovine mastitis is one of the most common diseases in dairy industry,which seriously inhibit the safety and health development purpose of dairy industry.The bovine mastitis cause serious economic losses for dairy industry and dairy farming,and even harm to human health.The incidence and harm of subclinical mastitis is higher than that of clinical mastitis.The main cause of infection of bovine mastitis is pathogenic microorganisms.In order to investigate the pathogens distribution of subclinical mastitis around Xianyang City,two large-scale dairy farms in this areas were selected,and then mastitis pathogens were isolated and identificated,drug sensitivity were further tested.The results will provide references for the prevention and treatment of subclinical mastitis in this area.To lay a foundation for the rapid and accurate diagnosis of subclinical mastitis in dairy cattle,loop-mediated isothermal amplification(LAMP)detection method for the above main pathogens was establish.The main results of this test are as follows:1.The mastitis pathogens from total of 48 milk samples,which were collected from two dairy farms in Xianyang city of Shaanxi province were detected by pathogens isolated and identificated and biochemical reaction.Total of 9 strains of 92 pathogens were identified,including Escherichia coli,Staphylococcus aureus,Salmonella,Klebsiella,Enterobacteria,fungi,Proteus,Staphylococcus epidermidis and Shigella sp.The main pathogens of subclinical mastitis in dairy cattle were mainly staphylococcus aureus and Escherichia coli,which accounting for more than 60% of the total identified pathogens.The rate of mixed infection was 83.33 %.The results of antimicrobial susceptibility test showed that gram-negative bacilli in two dairy farms were both sensitive to ceftazidime;the gram-negative bacilli isolated from the two dairy farms produced severe resistance to aminosamine antibiotics and macrolide antibiotics;two dairy farms of Gram-positive cocci were sensitive to tetracycline and levofloxacin,but had a serious drug resistance to penicillin G,oxacillin and clindamycin.2.A set of LAMP primers was designed for the specific gene alkaline phosphatase(phoA)of Escherichia coli,and the specificity and sensitivity experiments were carried out afteroptimizing the reaction conditions.LAMP reaction system can be completed within 50~60minutes,and the positive result was observed by green or green fluorescence.The minimum detection limit of this method for Escherichia coli was 241 fg/μl,which was sensitive 100 times to PCR method.There was no cross reaction with the common bovine mastitis pathogens.This study successfully established a rapid,specific,sensitive,convenient operation and easy judgement of LAMP visual inspection method used in Escherichia coli mastitis.