Establishment And Application Of Method For Ornithobacterium Rhinotracheale Detection

Ornithobacterium rhinotracheale disease (ORT) is caused by Ornithobacterium rhinotracheale, which primarily an acute, highly exposed to respiratory diseases infect chickens and turkeys. The clinical features are respiratory tract symptoms, growth retardation and increased mortality. The pathological features are with severe unilateral or bilateral cellulose suppurative pneumonia and airbags inflammation. At present, the disease is popular and forms an upward trend in many developed countries, for example Europe, the America, South Africa, and so on. Further more it brings about serious harm in poultry industry. China has reported the isolation of the bacteria. Therefore, it should cause people's attention. The topic has made a detailed study of the Ornithobacterium rhinotracheale disease diagnostic methods, and successfully established PCR, digoxigenin-labeled probe, indirect ELISA detection methods. It laid the foundation for diagnosis and epidemiological investigation of the ORT, and it is also have a great significance for the prevention and the control of this disease.Three parts were included in the studies:1.PCR method for Ornithobacterium rhinotracheale detectionBased on the 16S rRNA sequence in GenBank for Ornithobacterium rhinotracheale, a pair of primers was selected for amplification the 671bp fragment to establish a PCR method for ORT detection. This method could amplify the specific fragment in the reference strains of ORT, but no fragment in chicken E.coli, Haemophilus paragallinarum, and Salmonella Pullorum and Avian pasteurella multocida. A sensitivity test indicated that the assay's lowest detection was 90pg DNA. The PCR methodology appeared to be highly sensitive and specific for the purpose. Positive results were obtained on two isolated strains by using this newly developed PCR method. Therefore, it could be applicable for ORT diagnosis.2.Study on Digoxigenin-labeled Probe for detection of Ornithobacterium rhinotrachealeThe 671bp specificity fragment was amplified by PCR from 16S rRNA in ORT and through reclamation and clean, this specificity fragment was labeled by Digoxigenin. A novel DIG-Labeled DNA probe method was established for detecting the ORT. From specificity test, it showed that this probe can developed specificity nucleate hybridization with different serotype of ORT reference strains, but no in chicken E.coli, Haemophilus paragallinarum, Salmonella Pullorum and Avian pasteurella multocida. It indicated that this probe could detect the lowest of 100pg/μL by sensitivity test. The isolation strains were detected on the basis of this probe and 2 were positive, and on total of 211 doubtful samples were 4 positive, which was identical in PCR.3.Establishment and application of indirect ELISA method for detection of Ornithobacterium rhinotrachealeOuter membrane protein (OMP) was prepared and purified from the standard strain ORT of type- A, and through the optimization for concentration of coating antigen, dilution of serum and dilution of HRP IgG by chessboard titration test to develop an indirect ELISA for detection of serum antibodies against ORT. Result of chessboard titration test showed that the optimal concentration of coating antigen was 11.85μg/mL. The optimal dilution of serum was 1∶20. The optimal dilution of HRP IgG was 1:1000. The optimal time of combination of antigen with antibody was one hour, and the optimal combination time of serum with HRP IgG was 45min. The criteria for evaluation were as follows: the serum was positive if OD490nm≥0.639, the serum was negative if OD490nm≤0.350. The serum was suspicious if OD490nm was between 0.350 and 0.639. The method showed stability, repeatability and specificity. The indirect ELISA detected a total of 205 serum sample originated from chickens which were doubted ORT and the positive efficacy was 11.7% (24/205). This research could provide a high sensitive, powerful specificity convenient and easy to walk method for diagnosing ORT.