| Ornithobacterium rhinotracheale(ORT) is a relatively recently discovered bacterium associate with respiratory tract infections in poultry. Respiratory diseases of turkeys and chickens with Ornithobacterium rhinotracheale infection are accompanied by heavy economic loss due to increased mortality, drops in egg production, reduction of egg shell quality, decreased hatchability, growth retardation, increased medication costs and condemnation rates. Though cases of ORT infection are reported from around the world, there have few reports in China. The objectives of the present paper were to study the isolation and identification of ORT from commercial poultry in Guangxi province and to develop a rapid .diagnosis technique of ORT.20 ORT strains were isolated from 466 chicken samples collected from Guangxi different commercial flocks with respiratory clinic signs by culturing on blood plates. By comparing the isolation rate of the different breeds, the age and the organs of chickens, the results show that the isolation rate of the indigenous breed chickens and fast-growth chickens were higher than others with the value of 45.4% and 5.6% respectively, chickens from 4 to 10 weeks have the highest isolation rate with a value of 31.4%. Biochemical identification of all of the isolates were tested by using nine kinds of biochemical reagents, results show that all of isolates were consistent with the ORT reference strain. Serotyping all of isolates were examined by SPAT and AGP, result show that all of isolates have positive reaction to the antiserum which were made by ORT reference strains ORT2. All of isolates were also tested by PCR assay, a 784bp fragment of 16S rRNA gene were amplified from all of 20 isolates, but not from other closely related bacteria. Furthermore, we sequenced the PCR products nucleotides of 5 isolates and 2 ORT reference strains directly, The results of homologous comparisons of the nucleotide sequence among 5 isolates and 2 ORT reference strains indicated that Guangxi ORT isolates were from 99.2% to 100% identical with those of other oversea isolates. To investigate pathogenicity of chickenswith the ORT isolates, we challenged fast-growth chickens with 3 ORT isolates and 1 ORT reference strain. Clinic signs were observed in chickens of infection test. The morbidity rate of each group ranges from 42.5% to 87.5%. By comparing the average daily weight gain and the pathologic lesions, the results shows that the average daily weight gain and the pathologic lesions are significant difference between the ORT challenge groups and the control group(p≤0.05), the average daily weight gain of challenged groups with ORT isolates was lease than that of the control groups, and the pathologic lesions in the tracheal and air sac are more serious than that of the control group. 20 isolates were proved to ORT from the result of those researches, and it also demonstrate that chickens in Guangxi flocks were infected ORT with some proportion. ORT were isolated first from chickens in Guangxi province in this study, and it is also the first time to study the isolation and identification of ORT isolates in detail in China.A set of specific PCR primers were designed and synthesized according to the published sequence of ORT 16S rRNA gene. A PCR assays were developed to detect of ORT in chickens samples. The PCR assays can amplify a 784bp specific fragments from ORT strains, but not from other closely-related bacteria. The primary application with PCR system in ,28 experiment challenged chickens samples with ORT and 8 chickens sample from field infection have show that, the positive rate of 28 challenge chickens samples and 8 field infection chicken samples is 71.4% and 87.5% respectively; The positive rate of 7 health chickens samples detected by the PCR assay was 0%. The results demonstrated that the developed PCR assay is a specific, sensitive and rapid diagnostic technique for ORT infections in chicken samples. The PCR assay is useful in the epidemiological investigations of ORT. |
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