Study On The Diagnosis Methods Of ELISA For Antibody Against Corynebacterium Pseudotuberculosis And Serological Epidemiology Of Caseous Lymphadenitis

Caseous lymphadenitis or cheesy gland disease is a chronic infection of sheep and goats caused by Corynebacterium pseudotuberculosis and the disease is distributed worldwide. The main symptoms of CLA include the formation of abscesses in the superficial and visceral lymphnodes, these two mainfestations can occur concomitantly. CLA infection has effects on wool, mutton and milk production, and cause reproductive dysfunction, which in turn leads to heavy economic losses. With the development of the number and scale of the poultry farms, the disease is more serious. So far, it is not clear about the prevalence of the caseous lymphadenitis in major dairy goat breeding of China, such as Shaanxi, Shandong and Yunnan province.One pus samples were collected aseptically from abscessed lymph nodes of naturally infected goat found in Shannxi. The suspected strains were isolated and identified with biochemical test, synergistic lysis test and molecular methods. The somatic and soluble antigen were used to coat the ELISA plate, the blocking buffer was added with different blocking time, the dilution times of serum was determined. The positive criterion was determined. The sensitivity, specificity and repeatability of the assay was detected. The assay was used to test the clinical samples. Also, the assay was used for epidenmiologic survey of CLA in Shannxi, Yunnan and Shandong. The results were as follows:1.A strains of C.p was isolated from abscessed lymph nodes. After incubation for 24 h,small colonies appear, increasing to a diameter of 1-2mm after 48 h.The bacterial colonies were Gram-positive and were pale in colour, dry and friable in consistency, and may be moved freely over the surface of in agar with the point of probe. In sheep blood agar, a β-hemolysis zone can be seen. The cell morphotype was observed to be bacilli or coccoid rod. Physiological and biochemical identification results are consistent with berger bacteria identification manual. The bacterium was highly sensitive to a variety of antibiotics. The synergistic lysis phenomenon was apparent where the soluble factors from each organism come into contact with each other. The 16 s r RNA sequences of the isolated strain was analyzed and compared with the reference strain ATCC19410,the result showed that the hohmolog was more than 99%.2.The somatic antigen was choosed to coat the ELISA plate. The optimal concentration of coating antigen was value 0.080 at OD600,the dilution times of serum was 1∶400,the blocking time was 2h with skimmed milk at 37℃ and the positive criterion was 0.329 at OD450.The sensitivity of the assay was high, The coefficient of variation(CVs) of intro-batch and inter-batch repetition tests were less than 10%, respectively, No cross reaction with the positive serum of orf, goat pox and mastitis was observed in the indirect ELISA. The ELISA results were consistent with the clinical results. The results of epidenmiologic survey indicated that the average relevance ratio of goat with CLA as a whole in China and Shannxi, Shandong, Yunnan was 23.4%,22.3%,6.6%,35.7%.In summary, A strain of C.p was isolated and identified successfully, The established indirect ELISA method can be used for epidenmiologic survey and clinical tests with high sensitivity specificity and repeatability.