Isolation And Identification Of Corynebacterium Pseudotuberculosis And Establishment And Application Of Indirect ELISA Detection Method

Corynebacterium pseudotuberculosis(C.p)is an important pathogen that causes pathological changes such as swollen lymph nodes and internal organs,and caseous necrosis in various animals such as goats,sheep,cattle and camels.The disease caused by infection of the pathogen in sheep and goats is commol/Lonly referred to as caseous lymphadenitis(CLA).There are currently no effective treatments and preventive measures,and it can lead to a decline in the performance of the diseased sheep and an obstacle to reproductive function.As a progressive and consumptive chronic zoonosis,it severely constrains the development of the sheep industry and causes great economic losses.In the past,the disease did not cause sufficient attention from farmers because of its slow duration and low mortality.At this stage,with the continuous expansion of the breeding scale of dairy goats in China,there have been reports on the isolation of the pathogens in the major dairy goat breeding areas in China.At present,there is no vaccine to prevent and control the disease in China,and the therapeutic effect of antibiotics is very limited.Therefore,once a sheep is infected,it is easy to spread to the whole group and it is difficult to eradicate.Therefore,it is possible to establish an effective and accurate detection method.It is an effective means to prevent and control the pathogen.n this study,a large number of pus samples with typical clinical symptoms of Corynebacterium pseudotuberculosis were collected from some areas of Shaanxi,including Yaoxian,Longxian and Fuping,the traditional isolation and identification methods,biochemical tests and 16 S rRNA PCR sequencing were used to identify the strains and the strains were isolated and purified for drug sensitivity test and animal curative test.The virulence factor phospholipase D(PLD)of Corynebacterium pseudotuberculosis was selected as a candidate gene,and the recombinant plasmid pET-28a-sumo-PLD was constructed for prokaryotic expression and protein purification.The protein concentration was determined by gray-scale analysis software Image-Pro Plus and verified by Western blot,indicating that the recombinant PLD fusion protein can specifically react with its positive serum;Authenticating.Using the purified PLD fusion protein as a coating antigen,screening for optimal antigen coating concentration and serum dilution,selection of blocking solution,optimization of rabbit anti-goatase secondary antibody dilution ratio,and positive critical value It was determined that an indirect ELISA assay was established and the sensitivity,specificity,and reproducibility of the method were evaluated.After the preliminary application of the established indirect ELISA method,a preliminary epidemiological survey was conducted on 186 serum samples from Yaoxian,Longxian and Fuping in Shaanxi.The main results of this pilot study are as follows:1.The isolated strain was cultured for 48 h at 37 ?,and the growth was poor on nutrient agar.The blood agar grew well and formed a colony with a size of about 1 mm~2mm,white or light yellow dry and easy to push;Gram stain was positive,the shape of the bacteria under the microscope is small spherical,rod-shaped Combined with physical morphology,biochemical test and 16 S rRNA PCR sequencing,the results were identified as Corynebacterium pseudotuberculosis,and infected mice caused cheese-like abscess lesions.The minimum inhibitory concentration of 32 isolates of Corynebacterium pseudotuberculosis was detected.The results showed that the resistance rate of penicillin was 21.9%(7/23)and the resistance rate of gentamicin was 12.5%(4/32).Both clindamycin and linezolid are sensitive.2.The phospholipase D(PLD)was expressed by the recombinant plasmid pET-28a-sumo-PLD.The results showed that the main form was inclusion body.The concentration after purification was 2.63mg/ml.The results of Western blot showed the recombinant PLD fusion protein can specifically react with its positive serum3.The purified PLD was used as the coating antigen,in order to establish an indirect ELISA assay.The results showed that the optimal antigen coating concentration was 1.64?g/mL,the optimal serum dilution ratio was 1:200,and the dilution ratio of the enzyme-labeled secondary antibody was 1:5000,the positive cut-off value was determined to be 0.368 by testing the cord blood of 48 healthy newborn lambs.When the positive serum dilution ratio is 1:1600,the OD450 detection value is still higher than the positive critical value,and there is no cross-reaction with sheep's mouth sore,sheep mastitis and M.hemolyticus positive serum.The intra-assay repeat coefficient of variation ranges from 3%.~10.5%,the inter-assay repeat coefficient of variation ranged from 5% to 15%,indicating that the indirect ELISA assay has good sensitivity,specificity and repeatability.Using the established indirect ELISA method,the serum of 186 dairy goats in Guanzhong area of Shaanxi Province was detected,including 42 positive samples,the positive rate was 22.6%(42/186).In conclusion:Combined with the above test results,32 strains of Corynebacterium pseudotuberculosis were successfully isolated;the isolated strains of Corynebacterium pseudotuberculosis were sensitive to various antibiotics,and the drug resistance level waslow;the Pseudotuberculosis phospholipase D protein was used as the coating antigen.The established indirect ELISA diagnostic method has the characteristics of strong specificity,high accuracy and excellent repeatability,and can be used for serological diagnosis of coryneform bacteria of the flock.