| Infectious Laryngotracheitis (ILT) and Newcastle Disease (ND) are two major respiratory infectious diseases of chickens and cause great economic losses to poultry industry. Although the vaccines made by traditional methods have played important roles in control of infectious diseases, new generation of vaccines, which are safer and can be easily differentiated from wild-type viruses are in urgent needed. For those advantages, fowipox virus has been used as a vector to generate a variety of recombinant vaccines, some of them have already been commercialized. To develop a fowipox virus based multi-valent vaccine is especially significant to poultry industry since coinfections of different pathogens are often seen in many modern poultry farms.In the study, a transfer vector containing F gene of NDV primed with LP2E?2 promoter was constructed and designated as pSY681+NDV F. The transfer vector was used to co-transfect chicken embryo filprpblast, (CEF) cells with rFPV-ILTVgB (a recombinant fowpox virus expressing gB protein of ILTV and Lac Z gene of E coli.) following lipofectin transfection procedure. A recombinant FPV (designated as rFPV-NDV F) was obtained by identifying white plaques in background of blue coloration and purified through several passages of single plaque. PCR results showed that F gene was in the genome of rFPV-NDV F and indirect immuno-fluorescence test showed F protein expressed on rFPV-NDV F infected cells.In order to generate a multi-valent recombinant fowipox virus, a transfer vector containing NDV F gene primed with P7.5 promoter and ILTV gB gene primed with LPaEP2 promoter was constructed and designated as pSY-gB-F. The vector was than used to transfect S-FPV-017 infected CEF cells, and a recombinant FPV (designated as rFPV-gB-F) was obtained by identifying blue coloration of plagues and purified through several passages of single plaque. PCR amplification showed that both F gene and gB genewere in the genome of rFPV-gB-F. Expression of gB was identified by Western blotting, and expression of F protein was detected by indirect immuno-fluorescence test and Dot-ELISA.Immune potency of rFPV-gB-F was tested on SPF chickens. 90 four-week-old SPF chickens were divided into six groups: group 1 inoculated with rFPV-gB-F(20), group 2 with La Sota vaccine(10), group 3 with ILT vaccine(10), group 4 with rFPV-ILTVgB(10), group 5 with S-FPV-017(20), and group 6 as control with immunization(20). After immunization, antibodies against ILTV, NDV and FPV were detected respectively. The results showed that rFPV-gB-F could induce antibody to NDV, but titer was significantly lower than that of La Sota vaccine group. Antibody against ILTV induced by rFPV-gB-F and rFPV-ILTVgB were in similar level, which is relatively lower than that of ILT vaccine group. Four weeks after inoculation, ten chickens from each group inoculated with La Sota, S-FPV-017, rFPV-gB-F and control respectively, were challenged with 103 ELD50NDV F48E9 strain. All chickens from La Sota vaccine group were survived, but no chickens from S-FPV-017 and control groups were survived within a week after challenge. Only 2/10 of chickens inoculated with rFPV-gB-F survived lethal challenge, but time for developing clinical signs and death were delayed in contrast to S-FPV-017 and control groups. Five weeks after vaccination, ten chickens from each group inoculated with rFPV-gB-F, ILT vaccine, rFPV-ILTVgB, S-FPV-017 and control respectively, werechallenged by larynx injection of 10 EIDso ILTV WG strain. Chickens inoculated with. rFPV-gB-F, rFPV-ILTVgB or ILT vaccine were all protected from death, but 50%(5/10) of chickens from S-FPV-017 and control groups were protected. Measures from virus isolation and PCR detection showed that rFPV-gB-F and rFPV-ILTVgB were as efficient as commercial ILT vaccine in protection'of chickens against ILTV infection.Taking all results together, rFPV-gB-F protected SPF chickens against lethal challenge by virulent ILTV, and offered partial protection to NDV challenge. Further investigation is... |
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