Isolation Of Main Bacteria Of Bovine Mastitis In Hunan Province And Establishment Of Rapid Detection By LAMP Assay

Bovine Mastitis is a disease with high incidence rate and infection rate in breast milk culture.Pathogenic microorganism infection is the main cause.Hunan Province is located in the Central and South China,High humidity and hot climate limit the scale of dairy cattle breeding.Therefore,there is lack of researches about the prevention and control of cow diseases.Information regarding the prevention and control of the related dairy cattle epidemic.In order to investigate the main pathogenic bacteria of cow mastitis in large-scale dairy farms in Hunan Province,in the present study,based on the data obtained from the Dairy Production Performance Testing Center（DHI）of Hunan Animal Husbandry and Veterinary Research Institute,we collected milk with mastitis from four large-scale farms and isolated and identified the pathogens followed by drug resistance test on the main pathogens.Meanwhile,in order to timely,quickly and accurately monitor the occurrence of cow mastitis,we further established visual loop-mediated isothermal amplification of DNA（LAMP）assays,which combined with the use of a small animal disease detector.The current study can provide scientific basis for the prevention and control of cow mastitis in Hunan Province.Through analysis of the DHI data,77 milk samples with more than 5×10⁵somatic cells/m L were collected from four cow farms.The bacteria were isolated from the mastitic milk samples,and identified basing on the biochemical character,gram’s staining,PCR amplification and the sequences of 16S r DNA.The results showed a total of 37 bacteria with162 strains were identified,among which 24,29,48 and 61 pathogens were isolated from A,B,C and D farms respectively.The main pathogens of cow mastitis in Hunan Province were Staphylococcus aureus（25.31%,41 strains）,Staphylococcus squirrel（20.37%,33 strains）,Bacillus licheniformis（5.56%,9 strains）,Escherichia coli（5.56%,9 strains）,Klebsiella pneumoniae（4.32%,7 strains）.In addition,there were 4 strains（2.47%）of rigovirus,Bacillus cereus,Staphylococcus saprophyticus,Bacillus pumilus,Corynebacterium and Staphylococcus haemolyticus,respectively.Analysis of the infection found that Staphylococcus aureus and Escherichia coli were detected in the all four dairy farms,while Staphylococcus squirrel and Klebsiella pneumoniae were only detected in the farm D,and the other bacterial infection were different in the four farms.The antibiotic sensitive tests against the three main pathogens including staphylococcus aureus,Escherichia coli and Klebsiella pneumonia were performed.The results showed that all the three pathogens were resistant to amoxicillin,and sensitive to gentamicin and ciprofloxacin.The LAMP rapid detection assays for Staphylococcus aureus,Escherichia coli and Klebsiella pneumoniae were established.The established visual LAMP assays were more sensitive,and the minimum nucleic acid detection quantity is 5×10^-5ng for Staphylococcus aureus,5×10^-6ng for Escherichia coli and 5×10^-7ng for Klebsiella pneumonia,which is lower with 1×10³～1×10⁴folds when compared to those detected by common PCR assay.Moreover,the results can be determined by observation of the amplification curve within 30min.The established LAMP assays are simple and suitable for rapid detection of large quantities of samples in farms.Collectviely,the present study indicates that Staphylococcus aureus,Staphylococcus squirrel,Bacillus licheniformis,Escherichia coli and Klebsiella pneumoniae are the main pathogens of mastitic milk samples in large-scale dairy farms in Hunan Province.The established LAMP assay combining use of the small animal disease detector can rapidly detected the pathogens causing cow mastitis.