Identification Of The Main Pathogen In Clinical Endometritis Of Dairy Cows And Establishment Of Multiplex PCR Detection Method

Cow endometritis is the main disease of postpartum cows,which seriously affects the conception rate of cows and reduces economic benefits.The main cause of the disease is postpartum bacterial infection,because it has a wide variety of pathogenic bacteria,all of which are difficult to control.In recent years,due to the abuse of antibiotics,the resistance of bacteria has increased,making the disease more difficult to control.In response to the above situation,we conducted surveys and sample collections on four dairy farms in Baoji City,Weinan City,Xi'an City and Yangyang District of Xianyang City in Shaanxi Province.And The samples were isolated and identified for pathogenic bacteria and tested drug sensitivity.Based on the above experimental results,we establish a multiplex PCR assay for major pathogens.The results as follow:1.74 samples of uterine pus were collected from 4 areas in Guanzhong,Shaanxi Province.And thirteen kinds of bacteria were isolated from 66 effective samples,with a total of 113 bacteria,including 38 strains of Escherichia coli,29 strains of Trueperella pyogenes,19 strains of Streptococci?Including 13 strains of Streptococcus uberi,3 strains of Streptococcus dysgalactiae and 3 strains of Streptococcus pluranimalium?,11 strains Staphylococcus?Including 1 strain of Staphylococcus aureus,4 strains of Staphylococcus sciuri,3 strains of Staphylococcus xylosus and 3 strains of Staphylococcus haemolyticus?,6strains of Bacillus,5 strains of Aerococcus viridans,4 strains of Proteus mirabilis,and 1strain of Pseudomonas aeruginosa.2.We used Kunming mice for animal pathogenicity tests on the isolated bacteria.Among them,Escherichia coli,Staphylococcus aureus,Proteus mirabilis,Pseudomonas aeruginosa and Trueperella pyogenes are more pathogenic,followed by Streptococcus uberis and Streptococcus dysgalactiae,and other bacteria are less pathogenic.3.The results of drug susceptibility test showed that the isolates were more resistant to ceftazidime,penicillin G,ampicillin,kanamycin and clindamycin,and the antibacterial effects of cefquinome,piperacillin and amoxicillin/clavonic acid were good.4.We have established a multiplex PCR assay that can simultaneously detect six pathogens.We based on the ycjM gene of pathogenic Escherichia coli,the plo gene of Trueperella pyogenes,the nuc gene of Staphylococcus aureus,the pauA gene of Streptococcus uberis,the O antigen acetyltransferase gene of Pseudomonas aeruginosa,and the positive regulatory factor R gene of urease synthesis of Proteus mirabilis to design Specific Primers.The amplified product bands were 548 bp,338 bp,424 bp,1135 bp,708bp,and 951 bp.The results showed that the optimal reaction system was:5.6?L of reaction primer?Including 0.75?L of Escherichia coli,0.7?L of Trueperella pyogenes,0.5?L of Staphylococcus aureus,1.5?L of Streptococcus uberis,0.95?L of Pseudomonas aeruginosa and 1.2?L of Proteus mirabilis?,ddH₂O 3.9?L,r Taq?RR901A?12.5?L,total reaction system 25?L;The optimal annealing temperature was 58?;The optimal cycle number was 30 times.5.The minimum sensitivity of the template for multiplex PCR detection was 40 pg/?L for Escherichia coli,40 pg/?L for Trueperella pyogenes,40 pg/?L for Staphylococcus aureus,4 pg/?L for Staphylococcus aureus,4 pg/?L for Pseudomonas aeruginosa and 4pg/?L for Proteus mirabilis.The clinically collected samples were tested by this method,and the results were good.