The Epidemiological Investigation Of Contagious Ecthyma In Jilin Province And Development Of Its Inactivated Vaccine

Contagious ecthyma, also known as contagious pustular stomatitis, contagiouspustular dermatitis and Sore mouth, is an acute and high contagious disease of sheep,goat and humans which caused by Orf virus. The disease is characterized by theformation of erythema, papule, pustules, ulcers and crusts in the skin or mucosa of thelips, hooves, breasts and vulva. Currently, the disease is widespread worldwide. Thesheep flocks in the northern and northwestern areas is continuing to have theoccurrence of the disease. The occurrence of the disease could cause severe economiclosses to sheep industry. Because ORFV has strong resistance, the disease is hard toclear from the flocks once infection and may continue for several years. In addition,the human also could infect by direct contact, especially in Pastoralists, veterinariansand shearing, slaughtering, breeding and fur processing staff. So, the disease causes aserious threat to human health and becomes an important hidden danger of the worldpublic health security.In recent years, the occurrence of the disease showed the tendency of increase andhad became a main loemia which caused severe harm to sheep industry. To understandthe prevalence condition of the disease in the Jilin Province, we carried out thedetection of Orf virus nucleic acid by PCR for628scab samples suspected Orf virusinfection which collected from85sheep farmers during2006to2010. Andanti-ORFV serum antibodies were detected by indirect ELISA method for2160serumsamples collected from the same sheep farms at the same period. The prevalenceregularity was initially clarified by for the investigation of Orf virus infection inflocks in Jilin Province. This not only established foundation for the research ofmolecular pathogenesis of Orf virus, but will also provided important theoretical basisfor the formulation of scientific prevention and control measures against Orf. Theinvestigation result of molecular epidemiology showed that ORFV nucleic acid couldbe detected from the scabs collected from sheep of different ages, areas and breeds.The average rate of ORFV infection in flocks was56.47%(48/85) and the average rate of ORFV infection in scabs was24.36%(153/628). In different areas, theORFV-positive rate ranged from16.42%to32.60%. The ORFV-positive rates in theflocks of Songyuan and Baicheng were higher. The investigation result of serosurveyshowed that the ORFV serum antibody positive rate ranged from18.95%to53.49%,and ORFV antibodies positive rate in Nongan, Songyuan and Baicheng wererelatively high. The above results indicated that ORFV infection were very commonin the Jilin Province. In addition, the investigation and analysis of ORFV infection inthe sheep of different ages, sexes and breeds were performed in the study. The aboveresults of epidemiological investigation would provide important theory basis for theprevention and control of ORFV in the Jilin Province.In order to understand the type and the molecular characteristics of ORFVepidemic isolate and provide scientific basis and material foundation for theprevention and control of ORFV, the virus isolation and identification forORFV-positive materials were performed by virus isolation, microscopy, animalregression experiments, PCR and so on. In order to confirm the reason for the diseasein the flock from the pathogen, the systemic virus isolation and identification forORFV-suspected materials collected from some flock in Baicheng area in2010wereperformed. First, the primary ovine fetal turbinate cells (OFTu) and Madin-Darbybovine kidney cells (MDBK) were respectively used for the virus isolation. The OFTucells appeared the typical CPE after blind passage2, and MDBK cells for virusisolation, the fifth generation appeared typical CPE. The cell fluid showed CPE werecollected and purified by sucrose density gradient (W/V) centrifugation. The typicalparapoxvirus virions appeared ovoid shape were observed by electron microscopynegative staining. The result showed that virus isolate was obtained successfully. Inorder to further identify the virus isolate, the specific primers aimed to ORFV, CPV,BTV, FMDV were respectively synthesized in order to perform on the diagnosis ofsimlar symptoms. The results of PCR and RT-PCR showed that the amplificationusing ORFV primers was positive, thus excluded the possibility of of CPV, BTV orFMDV infection. So, the disease was finally diagnosed as ORFV infection. In order tofurther understand the proliferation ability and dynamic invasion process of the ORFVisolate on OFTu cells, the morphology development process of ORFV isolate and theultrastructural changes of infected cells were observed by the manufacture of ultrathinsections in different infection peroids. In the early stage, the changes of mitochondrion which were most sensitive to injury were more significant. Themitochondrion showed swelling, severe deformation and the rupture and disappear. Atthe later stage of infection, rough endoplasmic ER also occurred great expansion. Theperinuclear gap increased. In addition, a large of oval mature viral particles wereobserved in the cytoplasm of infected cells. The indicated that ORFV particles weresuccessfully copied in the cytoplasm of infected cells. In addition, the virulence of theORFV isolate was determined using OFTu cells. The result showed that the titre ofORFV isolate on OFTu cells was10-8.33/0.1mL. This indicated the ORFV isolate hadmore stronger virulence. In order to further identify its pathogenicity, animalregression experiments were performed on rabbits and sheeps. We successfullyduplicated the similar clinical symptoms with the natural cases. The disease was finaldiagnosed as ORFV wild virus infection by the systemic virus isolation andidentification, and the analysis of pathogenicity.Because ORFV infection were still very common in flock in Jilin Province, andthere is no effective method for the prevention and treatment of the disease, so thedevelopment of a safe and efficient vaccine using ORFV local epidemic isolate for theprevention and treatment of the disease had practical importance. In the study, theobtained ORFV local epidemic isolate was used as a vaccine candidate strains. TheOFTu cells were used for ORFV proliferation by optimizing the conditions of theproliferation in vitro. On the basis of determining the the ORFV optimal growthconditions, the ORFV Jilin isolate was determined as F0generation. The isolate wasinocubated on OFTu cells to perform passage. The original seed bank (F3generation)and foundation seed bank (F6generation) were successfully prepared, and the culturecharacteristics, purity and virulence of their3rd generation of culture (original seed:F6; basic seed: F9) were measured. The results showed that established ORFVoriginal seed and foundation seed bank corresponded with "the new veterinarybiological products quality standards". The vaccine antigens were prepared using thevirus of the foundation seed bank. The virus fluid of the titer (TCID50) of10-7.0/0.1mLat least was collected. After inactivated by0.4%formalin, ORFV oil emulsioninactivated vaccines were prepared. The physical properties, bacteria and mycoplasmatesting were performed on the random collected samples. The above detection resultswere consistent with "China Veterinary Pharmacopoeia" and " People’s Republic ofveterinary biological products quality standard". The vaccines of three batches were safe by the safety test. The ORFV oil emulsion inactivated vaccines mainly inducedthe Th1type immune response, however, the level of antibody was more lower. Inaddition, the vaccines could pruduce effective immunoprotection. The above obtainedresults would provide important theory basis and technique support for the preventionand control as well as the clear of the disease in the flocks in the Jilin Province.