| The infectious laryngotracheitis (ILT) is an acute and highly contagious disease of chickens characterized by latent infection. The attenuated live vaccines of ILT remained a variety of side effects including spreading vaccine virus to non-vaccinated animals, increasing virulence during in vivo passages, and occurrence of long term "carrier" birds. Therefore, a new safe and efficient ILTV vaccine is needed. In this study, we devoted to construct a stable polyvalent vaccine against both MD and ILT. Marek’s disease virus (MDV) CVI988/Rispens vaccine strain was used as the vector to construct the recombinant virus which expressed the glycoprotein B of infectious laryngotracheitis virus (ILTV), and immune effect of the recombinant virus against MD and ILT was evaluated.The transfer vector pMDV-gB-GFP which contained the gB of ILTV used the sorf1-sorf2of IRS-US region from MDV CVI988/Rispens genome as the insertion site for foreign genes, long terminate repeat (LTR) of the reticuloendotheliosis virus (REV) as the promoter and the green fluorescent protein (GFP) as the report gene. The transfer plasmid pMDV-gB-GFP and MDV CVI988/Rispens strain gene were co-transfected into chicken embryo fibroblast (CEF) cells to obtain recombinant virus rMDV-gB-GFP. Then we got the purified rMDV-gB-GFP by limiting dilution method and obtained rMDV-gB by using Cre recombinase to remove the EGFP gene. Results of PCR, IFA and western-blot showed that ILTV gB gene was cloned into the genome of CVI988/Rispens and expressed stably.We tested the protective efficacy of rMDV-gB against ILTV challenges in SPF chickens. SPF chickens were divided into four groups:rMDV-gB group, rMDV-gB and ILTV attenuated vaccine combined immunization group, ILTV attenuated vaccine group, challenges control group. The first two groups were immunized with5000plaque forming unit (PFU) of the rMDV-gB virus; rMDV-gB and ILTV attenuated vaccine combined immunization group and ILTV attenuated vaccine group were inoculated with ILTV attenuated vaccine when chichens were21days old. All the chichens were challenged with ILTV virulent strain when chickens were42days old by the intratracheal route,105EID50per chicken. We observed the symptoms of challenged chickens and conducted the necropsy and histopathologic examination10days after challenge. The results showed that, the protection rate of rMDV-gB group was65%which was significantly higher than ILTV attenuated vaccine group (protection rate was41%). rMDV-gB and ILTV attenuated vaccine combined immunization did not improve the protective effect against ILTV which indicated1-day-old chicks inoculated with rMDV-gB could estabilish effective immunity against ILTV.SPF chickens were immunized with rMDV-gB to test the protective efficacy against MDV. Chickens were randomly divided into CVI988/Rispens group, rMDV-gB group and challenges control group, each group contains20SPF chickens. The rMDV-gB group was subcutaneously inoculated with5000PFU of rMDV-gB virus, while the CVI988/Rispens group inoculated with5000PFU of MDV CVI988/Rispens vaccine strains. All of the SPF chickens were challenged with500PFU of vvMDV RBIB strain at7days post-immunization. After challenged with MDV, we observe the chickens for90days. The result showed that protection rate of rMDV-gB group was100%just as the CVI988/Rispens strain group. All chickens of control group showed clinical signs. The protective efficacy of rMDV-gB experiment demonstrated that the immunization of rMDV-gB virus could provide complete protection against vvMDV... |
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