| Infectious laryngotracheitis（ILT）and fowlpox are two important infectious diseases affecting chicken husbandry,which cause great economic loss.Fowlpox virus（FPV）is suitable to be used as a viral vector,which has characteristics that strict host specificity,a large genome,and the existence of a large number of non-essential regions for replication.Our laboratory has constructed the recombinant FPV genetically engineered vaccine which expresses the major protective antigen gB of the epidemic strain of ILTV（rFPV-ILTV gB）for the prevention and control of these two kinds of diseases.Then the immune efficacy,physical and chemical properties,and biological safety characteristics have been preliminarily investigated.In this study,we systematically evaluated the safety and immune efficacy of rFPV-ILTV gB in SPF and commercial chickens under different inoculated conditions,which provides basic information for the subsequent clinical trial and production of this candidate vaccine.1 Safety evaluation of rFPV-ILTV gBTo evaluate the safety of the candidate vaccine.In this study,the growth curve of rFPV-ILTV gB in chicken embryo fibroblast with different multiplicity of infection（MOI）was measured by the plaque counting method.The batch of basic seed of rFPV-ILTV gB was prepared according to the best MOI and time point of collection.After vacuum freeze-drying,its properties,mycoplasma,exogenous virus,bacteria,vacuum degree,and residual water were tested.21-dayold SPF chickens and Hy-Line brown laying hens were immunized with rFPV-ILTV gB,wtFPV282E4（wild-type FPV vaccine）,Cr-FPV（commercial recombinant FPV genetic engineering vaccine）at the doses of 2×104 PFU/3 μL and PBS,respectively.The safety of the vaccine was evaluated by observing clinical symptoms,pathological changes,and record of body weight gain.The same method was used to evaluate the safety of rFPV-ILTV gB in SPF chickens after repeated inoculation,over-dose inoculation and non-clinical usage age inoculation.The results showed that the indexes of lyophilized basic seed batch met with the standard of Chinese Veterinary Pharmacopoeia.Slight local scabs were observed SPF chickens and Hy-Line brown laying hens inoculated with rFPV-ILTV gB,which disappeared quickly without systemic symptoms,and the growth and weight gain of chickens were not affected.The rFPV-ILTV gB vaccination with different usage conditions and dosages have good safety which did not affect diet desire,growth,and weight gain of inoculated SPF chickens.2 Establishment of FPV and ILTV challenge modelsTo establish the challenge models of FPV 102 and 1370 strains,49-days-old SPF chickens were randomly divided into 9 groups,6 groups were challenged by FPV 102 and FPV 1370 at the doses of 102,103 and 104 EID50/0.1 mL via smearing feather bursa on the back,respectively;2 groups were challenged by FPV 102 and FPV 1370 at the dose of 103 EID50/3 μL via stabinoculation on the wing,respectively;the last group was inoculated with PBS as a control.To establish the challenge models of ILTV 119 and WG strains,49-days-old SPF chickens were randomly divided into 7 groups,6 groups were challenged by ILTV 119 and ILTV WG at the doses of 102,103 and 104 EID50/0.1 mL,respectively;the last group was inoculated with PBS as a control.The minimum infection dose of ILTVs and FPVs were determined by combining clinical symptoms and pathological changes.The results showed that the minimum infection dose of FPV 102 and FPV 1370 were 103 EID50/0.1 mL,and the incubation period was 6 days and 9 days,respectively.The minimum infection dose of ILTV 119 and ILTV WG were 104 EID50/0.1 mL,and the incubation period was 3 days and 4 days,respectively.In this study,reliable FPV and ILTV challenge models were established,which laid a foundation for evaluating the immune efficacy of rFPV-ILTV gB.3 Immune efficacy evaluation of rFPV-ILTV gB3.1 The minimum immune dosage of rFPV-ILTV gBTo further evaluate the immune efficacy and determine the minimum immune dose of rFPVILTV gB,21-day-old SPF chickens were immunized with different doses of rFPV-ILTV gB,then ILTV 119 and FPV 102 were used for the challenge viruses.The results showed that the minimum immune dose of rFPV-ILTV gB was 104 PFU/3 μL.3.2 Immune efficacy of rFPV-ILTV gB in SPF chickens21-day-old SPF chickens were immunized with 2×104 PFU/3μL rFPV-ILTV gB,Cr-FPV,wt-FPV282E4,respectively,and PBS was set as a control.ILTV 119 and FPV 102 were used to challenge the chickens on 28 days after immunization to evaluate the immune efficacy.Serum samples were collected every week since the day of immunization,and the antibody levels against ILTV and FPV were detected by indirect ELISA.The results showed that the antibody levels against ILTV and FPV in the rFPV-ILTV gB group were both the highest among the four groups.Antibody levels against ILTV（0.583±0.449 OD450）and FPV（0.639±0.106 OD450）peaked at 28 and 21 days after immunization,respectively.After the challenge,the ILTV and FPV antibody levels in each group increased rapidly.The challenge protection rates of rFPV-ILTV gB,Cr-FPV,wt-FPV282E4 and PBS group against ILTV 119 were 90%,80%,0%,0%,respectively,and the challenge protection rates against FPV 102 were 90%,70%,80%,0%,respectively.3.3 Immune duration of rFPV-ILTV gB in SPF chickensTo explore the effective immune duration of rFPV-ILTV gB,the challenge protection experiment was conducted at different time points after immunization.The results showed that the protective rates of rFPV-ILTV gB against ILTV and FPV 102 were 60%and 70%,respectively on 21 days after immunization,then the protective rates increased to 100%and 90%,respectively on 28 days after immunization.After 50 days of primary immunization,the chicken was boosted with the same dose.The protective rates of ILTV 119 and FPV 102 were 90%and 100%on 56 days after the second vaccination.3.4 Immune efficacy of rFPV-ILTV gB in commercial laying hensSerum samples of Hy-Line brown laying hens were collected weekly to detect ILTV and FPV antibody levels from 7 days of age.At 21 days of age,they were divided into 4 groups and vaccinated with rFPV-ILTV gB,Cr-FPV,wt-FPV282E4 and PBS,respectively.Spleen samples were collected on 7 and 14 days after immunization.The mRNA relative transcription levels of IL-2,IL-18 and IFN-γ were detected by qRT-PCR.The immune efficacy of the vaccine was evaluated in Hy-Line brown laying hens on 28 days after immunization challenged by ILTV 119 and FPV 102.Indirect ELISA results showed that the maternal antibody levels against ILTV and FPV were 0.699±0.182 OD450 and 0.328±0.103 OD450,respectively on 7 days of age,and then decreased gradually to 0.312±0.093 OD450 and 0.151±0.033 OD450,respectively on 21 days of age.The antibody levels against ILTV（0.670±0.088 OD450）and FPV（0.868±0.119 OD450）peaked at 28 days and 21 days post vaccination by rFPV-ILTV gB.The mRNA transcription level of rFPV-ILTV gB induced cytokines was the highest in all immune groups at 14 days post vaccination.The challenge protection rates of rFPV-ILTV gB,Cr-FPV,wt-FPV282E4 and PBS against ILTV 119 were 100%,90%,60%and 70%,respectively,and those against FPV 102 were 100%,50%,100%and 0%,respectively.3.5 Immune efficacy of rFPV-ILTV gB in commercial laying hens at non-clinical used ageFifteen-day-old Hy-Line brown laying hens were immunized with rFPV-ILTV gB,ILTV 119 or FPV 102 were challenged at 28 days post vaccination.The results showed that the protection rate of rFPV-ILTV gB against ILTV 119 was 80%,while the control group had no symptom,indicating that there was interference of ILTV maternal antibody for rFPV-ILTV gB vaccination.The protective rate of rFPV-ILTV gB against FPV 102 was 100%,and all hens of the control group were infected.The chicken was boosted with rFPV-ILTV gB at 50 days after primary immunization,and the protection rate of rFPV-ILTV gB against ILTV 119 and FPV 102 was both 100%at 28 days after the boost.In conclusion,rFPV-ILTV gB shows good safety and immune efficacy for SPF chickens and commercial laying hens and can be used as a very potential candidate vaccine against ILTV and FPV. |
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