ABSTRACT
Newcastle disease is a serious infectious disease in the poultry industry. The commercial vaccines can only offer limited protection and some of them are expensive and need adjuvants. At present, DNA vaccines are widely used. However, the immune responses induced by DNA vaccines are too slow and low. Here, we constructed the transfer vectors with a different number of C3d as molecular adjuvants (n = 1, 2, 4, or 6), and the vectors were cloned into the optimal eukaryotic expression plasmid (pVAXI-optiF) that expressed the F gene of Newcastle disease virus (NDV), and named pVAXI-F(o)-C3d1, pVAXI -F(o)-C3d2, pVAXI-F(o)-C3d4, and pVAXI-F(o)-C3d6, respectively. Cell transfection test indicated that pVAXI-F(o)-C3d6 showed the highest expression. In vivo immunization showed that the chickens immunized with pVAXI-F(o)-C3d6 intramuscularly induced better immune responses than the chickens immunized with the other plasmids. The protective efficacy of pVAXI-F(o)-C3d6 was 80% after challenge with the highly virulent NDV strain F48E9. The results in this study showed that C3d6 could be used as a molecular adjuvant to quickly induce an effective immune response to control NDV.
Subject(s)
Adjuvants, Immunologic , Complement C3d/immunology , Newcastle Disease/immunology , Newcastle disease virus/immunology , Recombinant Fusion Proteins/immunology , Vaccines, DNA/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cell Proliferation/drug effects , Chickens/genetics , Chickens/immunology , Chickens/virology , Cloning, Molecular , Complement C3d/genetics , DNA, Viral , Disease Models, Animal , Genetic Vectors , HEK293 Cells , Humans , Immunization , Immunoglobulin A/blood , Immunoglobulin G/blood , Lymphocytes/immunology , Newcastle Disease/genetics , Newcastle Disease/prevention & control , Newcastle disease virus/genetics , Plasmids , Poultry Diseases/immunology , Poultry Diseases/prevention & control , Poultry Diseases/virology , Recombinant Fusion Proteins/genetics , Vaccination , Vaccines, DNA/genetics , Vaccines, DNA/pharmacology , Viral Fusion Proteins/genetics , Viral Fusion Proteins/immunology , Viral Vaccines/genetics , Viral Vaccines/pharmacologyABSTRACT
Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek's disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens.
