Establishment Of PCR Diagnostic Method Of CLA And Comparative Studies

Caseous lymphadenitis in sheep and goat (CLA) caused by Corynebaterium Pseudotuberlosis which could infect multiple animals, such as sheep, goat, camel, horse, bovine, is a kind of zoonotic and chronic infectious. Infected sheep appeared lymph-node swelling, suppurant and caseous necrosis, emerging tubercles in lungs, spleen, and uterus horn. The production of pathogenic sheep was reduced and pathogenic pregnant sheep yielded dead fetus. The disease was unattended for it's slowly development and low lethal rate. Recently, with enlarge after infection, effect of antibiotic treatment to it was not good. So every country was increasing attaching importance to prevention therapy examination of the disease.2 strains bacteria A and B were respectively separated from pre-shoulder lymph-node abscess in 2 sheep, by identification, confirmed as Corynebaterium Pseudotuberlosis. There has some difference between the isolated stains and the reference Corynebaterium Pseudotuberlosis ATCC19410 strain.The ATCC19410 strain was respectively made into exotoxin antigen and inactivated vaccine, and vaccine goat with it, sera was collected as positive serum. Sera collected from cesarean section fetus yielded by somatic agglutination-inhibition test negative and clinical health sheep were used as negative sera. Selecting optimal concentration ratio of enzyme labeling antibody-positive serum and optimal concentration of exotoxin antigen and positive serum, by ELISA test, setting blank, positive, negative contrast, positive determining valure was obtained. We set it as standard for examining 46 samples, detected out 2 positive sheep, and positive rate 4.35%, which was agreed with the result of somatic agglutination-inhibition test.According to the known Corynebaterium pseudotuberlosis strain 16S rRNA sequence, one pair of primers were designed, applied to PCR, and perspective fragments were obtained. By purposive fragments sequence compared, it showed the homology of strain A and strain ATCC19410, strain B and strain ATCC19410 were respectively 96.8% and97.1%. The result shows CLA PCR test was build successfully. Streptococcus, Br. melitensis, M. tuberculosis and Corynebaterium pseudotuberlosis were amplified with the pair of primers, except one special fragment was amplified in Corynebaterium pseudotuberlosis, no fragment in other bacteria, which displayed CLA PCR test had strong specificity. 17 samples were tested, 12 samples were positive, positive rate 70.6%, consistent with the result of isolation and identification for pathogenic bacterium.