Isolation And Identification Of Ovine Trueperella Pyogenes And Establishment And Application Of Indirect ELISA Detection Method

Ovine Caseous lymphadenitis（CLA）,also known as Ovine subcutaneous abscess,can lead to the formation of purulent abscesses in goat superficial lymph nodes,visceral lymph nodes and internal organs.The diseased ovine showed weight loss,decreased milk production,reduced fur utilization and carcass meat utilization,and were easily spread among the herds,which seriously restricted the development of the dairy goat industry.At present,there is no effective vaccine prevention and treatment method for the disease,so the early diagnosis and timely isolation of sick animals have become the main measures to control the disease.It was initially thought that CLA was only caused by Corynebacterium pseudotuberculosis,but in recent years,studies have shown that Staphylococcus aureus and Trueperella pyogenes are also important pathogenic bacteria of the disease.Of the three pathogens,T.pyogenes has received the least attention,and there are few serological tests for it.In this study,on the basis of collecting pus samples and isolating T.pyogenes in Guanzhong area,the drug sensitivity and pathogenicity were studied.Using the supernatant protein of the sonicated bacterial solution as the coating antigen,an indirect ELISA of T.pyogenes was established.The detection method and effect evaluation and preliminary application provide a reliable reference for the subsequent detection,prevention and purification of the disease.The following results are obtained in the study:1.24 strains of T.pyogenes were isolated and purified from 272 pus samples,and the isolation rate was about 8.8%.They were Gram-positive bacteria and were rod-shaped or bulb-shaped under the microscope.Adding blood or serum to the medium could promote their growth;cultured on blood agar medium for 24 h,white pinpoint-sized colonies can be seen,and there areβ-type hemolytic rings with diameters 2 to 3 times larger than the colonies.The carrier rate of strain virulence plo、cbp A、nan P、nan H、fim A gene was 100%;the carrier rate of fim C and fim E gene was 83.3%;the minimum inhibitory concentration（MIC）of the drug was detected by the micro-broth dilution method.drug-resistant;isolates were inoculated into mice and goats,and abscesses could be formed at the injection site and ruptured and T.pyogenes were isolated again.2.The established indirect ELISA results show that the optimal coating concentration of antigen is 33.2 ng/m L,the optimal dilution of serum is 1:200,the optimal blocking solution is 10%BSA,and the optimal dilution of enzyme-labeled secondary antibody is 1:10 000;the detected positive critical value was OD₄₅₀=0.366;the sensitivity test showed that the detection result still exceeded the critical value when the positive serum of T.pyogenes was 1:1 280dilution;There was no cross-reaction among disease-positive sera;the intra-assay coefficient of variation was within 7.5%,and the inter-assay coefficient of variation was within 9.1%,with good repeatability.3.The test results of 200 goat serum samples showed that 47 goats with pustules had 5seropositives,accounting for 10.6%;153 goats with no obvious symptoms had 24seropositives,accounting for 15.7%;The number of positives was 29,accounting for 14.5%.In this study,the T.pyogenes isolated from goat pus was identified and some biological characteristics were studied,on this basis,a simple and convenient indirect ELISA method for detecting T.pyogenes was established,the method has strong specificity,high sensitivity and good repeatability,and can be used for clinical serum sample detection,which provides a reference for subsequent detection,prevention and purification of the pathogen.