| Aims:In order to screening the virulence of Mycoplasma ovipneumoniae strains,using Mycoplasma ovipneumoniae infected Goat trachea epithelial cells and SPF chicken embryos.To evaluate the virulence of Mo isolates using experimental challenge.To provide effective tools for Mo etiological and pathogenic research and provide datas for vaccine.Methods:(1)Indirect immunofluorescence assay for detection of Mo adhesion to GTEC:Mo adhesion goat tracheal epithelial cells,Primary antibody is rabbit serum injected with Mycoplasma ovipneumoniae,with FITC-labeled goat anti-rabbit IgG was the secondary antibody.By optimizing the Mo titer,adhesion time,working concentration of the primary antibody and secondary antibody.FL3 strain MoGH3-3 strain and A3 strain were carried on indirect immunofluorescence assay according to the optimal reaction conditions.qPCR was used to determine the adhesion rate of Mo.(2)Experimental challenge of Mo in chicken embryos:The 7-days old SPF chicken embryos were infected with different concentrations of Mo by injection of yolk sac and allantoic cavity.The mortality and Mo detection rate(PCR detection and MTB isolation)of infected chicken embryos were employed to evaluate the optimal inoculation route,dose and sampling site.Three different isolates of Mo were submitted to injection of chicken embryos for pathogenicity comparison.(3)Experimental challenge of Mo in goats:Six goats were infected with FL3 stain by injection of nasal cavity and trachea.Observed and recorded the change of Clinical symptoms and autopsy.The antibody of Mo was measured by indirect ELISA in serum.The histopathological changes of tissue were observed by HE staining and immunohistochemical reaction.qpcr detection the payload of Mo in different tissus,including lungs?lives?kidneys and spleens.Results:(1)Indirect immunofluorescence assay optimal conditions:adhesion titer was 1×10~8 CCU/ml,adhesion time was 2h,optimal working concentration of the primary antibody was 1:200,and the optimal working concentration of the secondary antibody was 1:250.All three strains could infected GTEC and showed different fluorescence and adhesion rate:FL3 strain(6.35%)>MoGH3-3 strain(5.12%)>A3strain(3.12%).(2)The results showed that the infection optimal of ways was the 7-days old SPF chicken embryos were infected with 10~9 CCU/mLof Mo 0.2mL/per by injection of yolk sac.Mo cells per embryo and the sampling site for detection is also yolk sac.Three Mo isolates revealed good infectivity and lethality to the chicken embryo.The chicken embryos mortality caused by FL3 strain(45%)and MoGH3-3(40%)are both higher than that by A3 strain(25%).The detection rate of Mo from the chicken embryos in FL3 group(100%)is higher than that of MoGH3-3 stain(85%)and that of A3 strain(90%).(3)FL3strain-infected goats developed clinic symptoms including high temperature?cough?depression and nasal discharge.The antibody of Mo significantly increased to be positive after infection 7 days(OD?0.3).one goat showed necropsies in lungs and one goat showed lung adhesions to heart and chest wall in the infection group.Mo were isolated from the lungs,nasal swabs and paratracheal lymph nodes.HE staining observed inflammatory cell infiltration in bronchioles and alveolar and enlarged alveolar septum.In the infection group,Immunohistochemical results showed that Mo was mostly distributed in cytoplasm and intercellular substance.The content of Mo is different in lungs?livers?spleens and kidneys,lungs was higher than kidneys?livers and spleens.(P<0.05).Conclusions:(1)Fl3 strain has stronger adhesion to GTEC than A3 strain and MoGH3-3stain.(2)The chicken embryo infection model of Mycoplasma ovipneumoniae was preliminarily established,which proved that Fl3 strain had the highest lethality to chicken embryos.(3)The virulence of strain FL3 is confirmed with goat challenge test,which agrees with the results of GTEC adhesion assay and SPF-CE infection.It shows that Mo strain FL3 would be a candidate for development of Mo vaccine. |
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