Establishment And Application Of Indirect ELISA Assay For Detection Of Antibodies Against Corynebacterium Pseudotuberculosis In Goats

Goat pseudotuberculosis,also known as Caseous lymphadenitis(CLA),can lead to the formation of pyogenic abscesses in superficial lymph nodes,visceral lymph nodes and internal organs of goats.The sick sheep lose weight so rapidly that the milk production and the utilization of fur and carcass meat are decreased.It is also easy to spread among herds,which has seriously restricted the development of dairy goat industry.At present,there is no effective treatment and no feasible immunization plan for this disease.Therefore,rapid detection of diseased animals and isolation them from large groups become the main measure to prevent the disease.So that,it is necessary to establish a diagnostic method for detecting goat pseudo-tuberculosis antibody.In this experiment,the contents of pustules in lymph nodes of suspected diseased sheep were sampled for isolation and identification of pathogenic bacteria.An indirect ELISA method for the detection of antibodies against Corynebacterium pseudotuberculosis was established using heat inactivated bacterial suspension as the coating antigen.This method was applied to serological investigation of goat pseudotuberculosis in Guanzhong area of Shaanxi province.The study yielded the following results:1.After separation and purification,a Gram-positive bacterium was obtained,which was club-shaped under the microscope.The bacterium grew well on serum broth solid medium at 37?,and circular,dry,easily-pushed,opaque and grayish-white colonies with a diameter of about 1?2 mm could be observed.Cultured in serum broth liquid medium,grew into granules with a surface film and deposited at the bottom of the conical flask.The results of 16S r RNA PCR sequencing and PCR identification of specific genes confirmed that the strain was Corynebacterium pseudotuberculosis.The isolation was inoculated with milk goats and the abscess ulceration was formed at the injection site.The results of drug resistance test showed that the strain was sensitive to tetracycline,neomycin,ciprofloxacin,compound sulfamethoxazole and other antibiotics,while it was low sensitive to streptomycin and only resistant to amikacin.2.The indirect ELISA results showed that the optimal antigen coating amount was 100?L of bacterial suspension with OD_600nm=0.084,the blocking time of 5%BSA was 2 h,the dilution ratio of primary antiserum was 1:400,that of enzyme-labeled secondary antibody was 1:5 000,and the critical OD_450nm value of serum was 0.352.This method only showed specific reaction to the positive serum of goat pseudo-tuberculosis while there was no cross-reaction or false positive in the positive serum of the other six common goat diseases,so the specificity was strong.The coefficient of variation of intra-assay and inter-assay was less than 9.5%,demonstrating good repeatability.The sensitivity test showed that the test result still exceeded the threshold when the sheep pseudo-tuberculosis positive serum was diluted at 1:1 280,indicating a strong sensitivity.3.In Guanzhong area of Shaanxi Province:Fuping,Pucheng,Longxian,Jingyang and Lantian,the positive rates of goat anti-Pseudo-Mycobacterium tuberculosis antibodies were36.9%,45.6%,33.3%,30.9%and 35.2%,respectively.There was no regional difference in the positive rate of goat anti-Pseudo-Mycobacterium tuberculosis antibodies in Guanzhong area.The positive rate of antibody against Corynebacterium pseudotuberculosis in goats was significantly different among diseased animals at different ages and sexes(P<0.01)and was significantly different among different breeding scales(P<0.05).The positive rate of antibody in adult sheep was higher than that in juvenile sheep and also higher in female animals than that in male animals.With the increase in the scale of aquaculture,the prevalence of disease showed an increasing trend and the prevalence rate was the highest in large and medium-sized farms.