Preparation Of Orf Virus Vaccine And Assessment Of Immune Effect

Orf, also known as contagious ecthyma, is a highly contagious disease which oftenoutbreaks on both lambs and adult goats with a mass. The incidence rate can be up to90%and the mortality rate can reach to10%. Adult goats affected with ORFV (Orf virus) will bemalnourished because of ingestion difficulty. Newborn lambs suffering Orf will be growthslowly and even starvation to death because of the difficulty to ingest breast milk. In recentyears, the economic loss of Orf has become increasingly severe with the rapid development ofgoat breeding industry. There is no Orf vaccine available in our country currently. Therefore,Orf virulent isolates (FX0910strain) was the material, this study aimed to prepare and assessthe immune effects Orf inactivated vaccines and FX0910attenuated virulence, safety andimmune effects of test. Research contents and results are as follows.Separation of FX0910virulent strain and immune effects assessment of Orf inactivatedvaccineScabs were isolated from the lips of dairy goats with typical clinical symptoms, then thevirus liquid was filtered and cultured on the bovine testicular primary cells and the virus titerwas determined using the method of Reed-Muench. The10th passage of ORFV wasinactivated about48h with0.4%formaldehyde and then the inactivation effect wasdetermined by inoculating the inactivated virus on cells and lips of lambs.15lambs wereinoculated with inactivated vaccines (109.1TCID50of each) via Groin subcutaneous and thenthe safety of the inactivated vaccine was estimated. The pregnant goats were immunized1mLvia cervical subcutaneous injection42days,28days and14days before delivery respectively.Antibody titer was determined by ELISA while the neutralizing antibody titer was determinedusing the method of fixed virus reacting with different serum dilutions. The protection rate ofOrf inactivated vaccine was also determined. The results were as follows:①ORFV isolatedfrom the scabs was proliferated on the bovine testicular primary cells. There was no cytopathybefore4generations of inoculated cells, while voids and other typical symptoms can beobserved from generation5and the titer of ORFV was determined as108.1TCID50/mL.②Neither cytopathy nor Orf disease was observed on cells or lambs after inoculated withinactivated ORFV, so the vaccine was indeed inactivated. No Orf disease, fever or anorexia appeared on lambs after inoculated with inactivated vaccine, so the safety can be confirmed.③The antibody titers of vaccinated pregnant goats and their lambs were about107,106respectively and ORFV with100-200TCID50can be neutralized by the serum of pregnantgoats and the protection rate of inactivated vaccine of ORFV was60.1%.Virulence attenuation and immune effect assessment of the virulent strain FX0910Isolated and purified ORFV was passaged to85th generation on bovine testicular cells,and then the ORF titter was determined by using the method of Reed-Muench. The85thgeneration ORFV was incubated on oral mucosa of35one-month-old lambs with108.3TCID50for safety testing. As to immune experiment, one-month-old lambs were incubatedtwice by scratching on mouths each other one week with0.2mL ORFV (107.3TCID50/mL) P85of FX0910. Peripheral blood lymphocytes were separated on7days post vaccination and thenantigen-specific lymphocyte proliferation was assessed by MTT assay.18lambs wereselected60d after immunization and divided into FX0910attenuated strain immune group andcontrol group, each of which contain nine lambs, challenged with FX0910virulent strain ofORFV with100TCID50for each lamb. The protective rate of Orf attenuated vaccine wasdetermined according to the morbidity of vaccine lambs. Results are shown below:①ORFVwere passaged to generation85and the titer of the live virus in the vaccine was107.3TCID50/mL.②Lambs were vaccinated by the generation85live ORFV and there was notypical lesion of Orf virus like vesicles, pustules or scabs, make it clear that the generation85live ORFV was safe. It was named FX0910attenuated strain.③ORFV antigen-specificlymphocytes stimulation index was2.88, which was significantly higher than theunvaccinated group.④Typical Orf lesions appeared on lips of all control lambs withinseven days after challenged, and only slight swelling appeared on challenged sites of twoFX0910attenuated strain immune group lambs on the third day but then the swellingdisappeared. There was no Orf lesions on the rest lambs. Only two of the remainingimmunized lambs generated Orf typical lesions in the subsequent30days and recovered after7days.Conclusion1. The inactivated strain of ORFV FX0910were prepared successfully. The ORFVinactivated vaccines were capable to induce the initiation of humoral immune response andthe production of protective neutralizing antibody. The protection rate for lambs was60.1%.2. Obtained an attenuated strain of ORFV FX0910through continuous passage andanimal experiment, and the protection rate for lambs reached98%after immunized with thisattenuated strain.