Isolation And Identification And Study On Pathogenicity Of Ornithobacterium Rhinotracheale

Ornithobacterium rhinotracheale (ORT) is a Gram-negative pleomorphic, rod-shaped bacterium. It grows slowly, producing only pinpoint colonies on blood agar at 37℃in the presence of 5% CO2 at 24h. Optimal growth occurs when plates have been incubated for 48h .Colonies then appear gray to grayish white, opaque, convex and circular with a diameter of 1-3mm. The clinical symptoms associated with O. rhinotracheale infection have included lacrimation, nasal exudates, sinusitis, dyspnoea, coughing, oedema in facial subcutis, growth retardation and a drop in feed consumption. Postmortem lesions associated with O. rhinotracheale infection have been identified as fibrinopurulent pneumonia, air sacculitis, peritonitis with foamy exudates and arthritis. Outbreaks of ORT infection have subsequently occurred in ORT, a causative agent of respiratory disease in fowl, was isolated from different commercial flocks showing respiratory symptoms in Shandong province. A slide agglutination test was used to detect ORT. A PCR was performed to detect ORT. In order to determine the serotype of ORT, an agar gel precipitation test was performed Antimicrobial susceptibility was also tested. Five strains were isolated. All of the isolates gave a positive reaction to the ORT antiserum. A 671 bp fragment of the 16S rRNA gene was amplified from all of the five isolates, but not from closely related bacteria used as a control. Three serotypes were identified: three of the isolates were serotype A, one isolate was serotype B and one was serotype D. All of the isolates were found to be susceptible to Amoxicillin, Ceftriaxone, Amikacin, ofloxacin, enrofloxacin and ciprofloxacin.The broiler and SPF chicks were artificially infected through the trachea challenge, digestive tract challenge and intramuscular challenge. The incidence and clinical symptoms of chicks were observed. And the body weight gain of chicks of each test group and negative control group were weighed. Dynamical observation of detoxification conditions of the experimental group and negative control group were done using the established PCR detection methods. The chicks of experimental group and the negative control group were killed randomly after infection at different times. The heart, liver, lungs and trachea were collected to be fixed and stained by conventional H.E. staining. Then the pathological changes of these organs were observed. The distribution of the ORT in chicks issue was studied with IFA (indirect fluorescent antibody test; Their bloods were collected to be carried out the routine blood test and the detection of blood parameters. The chicks were caused dyspnea, coughing and other symptoms by being inoculated ORT. And the weight gain of these chicks significantly decreased. Histopathological changes were characterized by liver, heart necrosis extensive hemorrhage in the trachea and lungs and inflammatory cell infiltration. IFA positive signals were mainly distributed in the lungs and trachea. The exhaust bacteria stage continued from the second day post challenge to the eighth day. The HGB, WBC, RBC, BUN, and AST significantly changed.Two clean NewZealandrabbits were vaccinated with oil-emulsion vaccine of ORT. The rabbit anti-ORT specific IgG was extracted by caprylic-ammonium sulphate method, purified by Sephadex G200 chromatography after dialyzing. An IFA test was developed to the rapid diagnosis of ORT by using the anti-ORT IgG as the primary antibody and the fluoresceinisothiocyanated(FITC) conjugate goat anti-rabbit IgG as the second antibody. The research suggested that the dilution of primary antibody was 1:25 and Sections were incubated overnight at 4℃. Then sections incubated at 37℃for 30min with diluted FITC-labelled-secondary antibody (1:200). The IFA was applied to detect the ORT antigen in different organs of the artificially infected chicks. The results showed that the heart, lung, liver and trachea have the most posive rate. It suggested that the target organs that ORT mainly attacked were lung and trachea.The IFA established in this study had been demonstrated to be a rapid, sensitive, specific and economical test for the diagnosis of ORT.