Isolation And Identification Of Main Pathogenic Bacteria Of Bactrian Camel Mastitis In Xinjiang And Establishment And Application Of Multiplex PCR Detection Method

In recent years,the dairy camel industry in Xinjiang has developed rapidly and has become a powerful engine for the strategy of ’ rural revitalization ’ in some areas.However,the prevalence of mastitis has seriously threatened the healthy development and public health of camel industry.At present,the research on Bactrian camel mastitis in Xinjiang is not deep enough,the types of main pathogenic bacteria and their drug resistance are not clear,and convenient and reliable detection methods have not been established,which seriously restricts the prevention and control of this disease.It is an urgent production problem to be solved.In this study,in order to find out the prevalence of mastitis and the main pathogenic bacteria in Bactrian camels in Altay,Xinjiang,and establish a multiplex PCR method suitable for the detection of camel mastitis,the following studies were carried out : 1.388 milk-producing Bactrian camels in Altay were selected as the research objects.The clinical examination method and CMT method were used to detect the whole group of mastitis,and then the positive milk samples were collected aseptically for bacterial isolation and identification.A total of 154 strains were isolated and identified,including 11 strains of Escherichia coli,4 strains of Staphylococcus aureus,7 strains of Acinetobacter,82 strains of Enterococcus(33 strains of Enterococcus faecalis),9 strains of Klebsiella,7 strains of Laurella,17 strains of Citrobacter freundii,7 strains of Citrobacter morinii,6 strains of Proteus,3 strains of Lactococcus,and 1 strain of Lactobacillus.The results of epidemiological survey showed that the average infection rates of clinical and occult mastitis were 4.40 % and 21.30 %.The infection rates of intensive farms were 20.00 % and 17.19 % respectively.The results of drug sensitivity showed that all the isolates tested by Staphylococcus aureus were resistant to penicillin G,and Enterococcus faecalis had obvious resistance to chloramphenicol and tetracycline.Three virulence genes ast A,Fyu A and irp2 were detected in Escherichia coli,five virulence genes clfa,hla,hlb,sea and fnb B were detected in Staphylococcus aureus,five virulence genes gel E,efa A,ace,agg and esp were detected in Enterococcus faecalis,and seven virulence genes were detected in Mycoplasma.The results of virulence genes showed that all the tested strains carried multiple virulence genes and had strong pathogenicity.2.According to the bacterial isolation of camel mastitis in different regions and in this experiment,a total of five bacteria,Staphylococcus aureus,Enterococcus faecalis,Escherichia coli,Streptococcus agalactiae and Mycoplasma,were selected to construct the multiplex PCR method.After the exploration and optimization of the single PCR and multiplex PCR reaction conditions,50μL system was used for the multiplex PCR reaction,and the annealing temperature was 58 °C.The primers of the five-fold PCR reaction had good specificity,and the bands were generated by Mycoplasma at the template concentration of 1 ng / μL,and by Staphylococcus aureus at the template concentration of 10 ng / μL.The bands were produced by Enterococcus faecalis at the template concentration of 10 ng / u L,Escherichia coli at the template concentration of 10 ng / u L,Streptococcus agalactiae at the template concentration of 1 ng / u L.The clinical detection of the collected samples was consistent with the previous isolation and identification.