To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.
Animals ; Antigens, Viral/genetics* ; Biological Assay ; Chickens/immunology* ; Escherichia coli/genetics* ; Infectious bursal disease virus/immunology* ; Poultry Diseases ; Vaccines, Synthetic/isolation & purification* ; Viral Structural Proteins/immunology* ; Viral Vaccines/immunology*

In order to observe the effect of the immune and weight of chickens after use the attenuated vaccine with low dose of chicken infectious anemia virus (CIAV). In this study, the effects of low dose of CIAV on the weight of SPF chickens and NDV antibody production were observed by simulated experiments. The results showed that 10 EID50 and 5 EID50 CIAV per plume attenuated NDV vaccines were used to cause the weight loss of SPF chickens. Compared with the use of the non contaminated vaccine group, it has significant difference. And NDV antibody levels compared with the use of the non contaminated groups also decreased after use the vaccine with two doses of CIAV contaminated. It has significant difference. A certain proportion of CIAV antibody positive was detected at the beginning of the second week after use the NDV vaccine with two doses of CIAV contaminated. The detection of a high proportion of CIAV nucleic acid was detected in the first week after the use of a contaminated vaccine. The results of the study demonstrate the effects of CIAV pollution on the production and immune function of SPF chickens, and it is suggested that increasing the detection of viral nucleic acid can help save time and improve the detection rate in the detection of exogenous virus contamination by SPF chicken test method.
Animals ; Antibodies, Viral ; immunology ; Chicken anemia virus ; genetics ; immunology ; physiology ; Chickens ; Circoviridae Infections ; immunology ; veterinary ; virology ; Poultry Diseases ; immunology ; virology ; Specific Pathogen-Free Organisms ; Vaccines, Attenuated ; administration & dosage ; genetics ; immunology

We wished to ascertain the prevalence as well as the genetic and antigenic variation of infectious bronchitis viruses (IBVs) circulating in the Guangxi Province of China in recent years. The S1 gene of 15 IBV field isolates during 2012-2013 underwent analyses in terms of the similarity of amino-acid sequences, creation of phylogenetic trees, recombination, and serologic identification. Similarities in amino-acid sequences among the 15 isolates of the S1 gene were 54.3%-99.6%, and 43.3%-99.3% among 15 isolates and reference strains. Compared with the vaccine strain H120, except for GX-YL130025, the other 14 isolates showed a lower similarity of amino-acid sequences of the S1 gene (65.1-81.4%). Phylogenetic analyses of the S1 gene suggested that 15 IBV isolates were classified into eight genotypes, with the predominant genotype being new-type II. Recombination analyses demonstrated that the S1 gene of the GX-NN130048 isolate originated from recombination events between vaccine strain 4/91 and a LX4-like isolate. Serotyping results suggested that seven serotypes prevailed during 2012-2013 in Guangxi Province, and that only one isolate was consistent with the vaccine strain H120 in serotype (which has been used widely in recent years). The serotype of recombinant isolate GX-NN130048 was different from those of its parent strains. These results suggested that not only the genotype, but also the serotype of IBV field isolates in Guangxi Province had distinct variations, and that increasing numbers of genotypes and serotypes are in circulation. We showed that recombination events can lead to the emergence of new serotypes. Our study provides new evidence for understanding of the molecular mechanisms of IBV variations, and the development of new vaccines against IBVs.
Animals ; Antibodies, Viral ; blood ; Chickens ; China ; Coronavirus Infections ; blood ; veterinary ; virology ; Genetic Variation ; Genotype ; Infectious bronchitis virus ; classification ; genetics ; immunology ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; blood ; virology ; Sequence Homology, Amino Acid ; Spike Glycoprotein, Coronavirus ; chemistry ; genetics ; immunology

In Korea, several outbreaks of low pathogenic AI (H9N2) viral infections leading to decreased egg production and increased mortality have been reported on commercial farms since 1996, resulting in severe economic losses. To control the H9N2 LPAI endemic, the Korea Veterinary Authority has permitted the use of the inactivated H9N2 LPAI vaccine since 2007. In this study, we developed a killed vaccine using a low pathogenic H9N2 AI virus (A/chicken/Korea/ADL0401) and conducted safety and efficacy tests in commercial layer farms while focusing on analysis of factors that cause losses to farms, including egg production rate, egg abnormality, and feed efficiency. The egg production rate of the control group declined dramatically 5 days after the challenge. There were no changes in feed consumption of all three groups before the challenge, but rates of the control declined afterward. Clinical signs in the vaccinated groups were similar, and a slight decline in feed consumption was observed after challenge; however, this returned to normal more rapidly than the control group and commercial layers. Overall, the results of this study indicate that the safety and efficacy of the vaccine are adequate to provide protection against the AI field infection (H9N2) epidemic in Korea.
Animals ; Chickens ; Emulsions ; Female ; Influenza A Virus, H9N2 Subtype/*immunology ; Influenza Vaccines/*immunology/*standards ; Influenza in Birds/immunology/prevention & control ; Oviparity ; Specific Pathogen-Free Organisms ; Vaccines, Inactivated/immunology

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.
Animals ; Antibodies, Viral/blood ; Cercopithecus aethiops ; Chickens ; *HN Protein/genetics/immunology ; Immunogenicity, Vaccine/*immunology ; Newcastle Disease/immunology ; Newcastle disease virus/enzymology/*genetics/immunology ; Specific Pathogen-Free Organisms ; Vaccines, DNA/genetics/*immunology ; Vaccines, Inactivated/immunology ; Vero Cells ; *Viral Fusion Proteins/genetics/immunology ; Viral Vaccines/genetics/*immunology/*standards

OBJECTIVEChlamydia psittaci is an avian respiratory pathogen and zoonotic agent. The wide prevalence of C. psittaci poses a threat to the poultry industry and its employees. However, few commercial kits are available for detecting avian antibodies excluding the in-house ELISA kit. In this study, we developed a novel ELISA kit for detecting antibodies against C. psittaci based on the N-terminal fragment of polymorphic outer membrane protein D (PmpD-N) as the coating antigen.

METHODSThe antigen concentrations, primary antibody, and cut-off value were determined and optimized. The ELISA, designated PmpD-N ELISA, was assessed for sensitivity, specificity, and concordance using sera samples from 48 experimentally infected and 168 uninfected SPF chickens.

RESULTSThe sensitivity and specificity of PmpD-N ELISA were 97.9%, 100%, respectively, while the concordance was 98.1% as compared to that of MOMP-ELISA. No cross-reaction with positive sera for other avian pathogens was found. Using PmpD-N ELISA, 799/836 clinical samples were positive, including 93.0% and 98.1% positivity in layers and broilers, respectively.

CONCLUSIONThese data indicate that indirect ELISA with PmpD-N as the antigen candidate is a promising approach for the surveillance of C. psittaci infection.

Animals ; Bacterial Proteins ; analysis ; Chickens ; Chlamydophila psittaci ; genetics ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; veterinary ; Membrane Proteins ; analysis ; Poultry Diseases ; diagnosis ; microbiology ; Psittacosis ; diagnosis ; microbiology ; veterinary ; Sensitivity and Specificity

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H2O2, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGDelta3 (SGDeltarpoSDeltahmpDeltassrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGDelta3 mutant did not cause any mortality after inoculation with either 1 x 10(6) or 1 x 10(8) colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGDelta3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGDelta3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGDelta3 could be a promising candidate for a live Salmonella vaccine against FT.
Administration, Oral ; Animals ; Bacterial Proteins/*genetics/immunology ; *Chickens ; Female ; Poultry Diseases/*immunology/microbiology ; Salmonella Infections, Animal/*immunology/microbiology ; Salmonella Vaccines/administration & dosage/genetics/*immunology ; Salmonella enterica/immunology/*physiology ; Vaccines, Attenuated/administration & dosage/genetics/immunology ; Virulence

Hazardous biochemical agents in animal husbandry indoor environments are known to promote the occurrence of various illnesses among workers and animals. The relationship between endotoxin levels in dust collected from chicken farms and various immunological markers was investigated. Peripheral blood was obtained from 20 broiler chickens and 20 laying hens from four different chicken farms in Korea. Concentrations of total or respirable dust in the inside the chicken farm buildings were measured using a polyvinyl chloride membrane filter and mini volume sampler. Endotoxin levels in the dust were determined by the Limulus Amebocyte Lysate Kinetic method. Interferon-gamma production by peripheral blood mononuclear cells stimulated with concanavalin A was significantly lower in broilers or layers from the farms with higher endotoxin concentrations than the chickens from the farms with lower endotoxin levels. An opposite pattern was observed for plasma cortisol concentrations with higher cortisol levels found in chickens from the farms with higher endotoxin levels. When peripheral lymphocytes were examined, the percentage of CD3-Ia+ B cells was lower in layers from farms with higher endotoxin levels than those from locations with lower endotoxin levels. Overall, these results suggest a probable negative association between dust endotoxin levels and cell-mediated immunity in chickens.
Animal Husbandry ; Animals ; Biomarkers/blood ; Chickens/*immunology ; Dust/*analysis ; Endotoxins/*analysis ; *Housing, Animal ; *Immunity, Cellular

The bursa of Fabricius (BF) is the acknowledged central humoral immune organ in birds. Bursal septpeptide II (BSP-II) is an immunomodulatory bioactive peptide isolated from BF. To understand the effects of BSP-II on immune induction, gene expression profiles of hybridoma cells treated with BSP-II were evaluated. Pathway analysis showed that regulated genes were involved in cytokine-cytokine receptor interactions, T cell receptor signaling pathway, and pathway in cancer. It was observed that BSP-II reduced tumor cells proliferation and stimulated p53 expression. These results indicate potential mechanisms underlying the effects of the humoral immune system on immune induction, including antitumor activities. Our study has provided a novel insight into immunotherapeutic strategies for treating human tumors.
Animals ; Antineoplastic Agents/*pharmacology ; Avian Proteins/*pharmacology ; Bursa of Fabricius/immunology ; Cell Proliferation/drug effects ; Chickens/*immunology ; Hybridomas/drug effects ; Immunologic Factors/*pharmacology ; Oligonucleotide Array Sequence Analysis/veterinary ; Signal Transduction/*drug effects ; *Transcriptome

The recent human infection with avian influenza virus revealed that H9N2 influenza virus is the gene donor for H7N9 and H10N8 viruses infecting humans. The crucial role of H9N2 viruses at the animal-human interface might be due to the wide host range, adaptation in both poultry and mammalian, and extensive gene reassortment. As the most prevalent subtype of influenza viruses in chickens in China, H9N2 also causes a great economic loss for the poultry industry, even under the long-term vaccination programs. The history, epidemiology, biological characteristics, and molecular determinants of H9N2 influenza virus are reviewed in this paper. The contribution of H9N2 genes, especially RNP genes, to the infection of humans needs to be investigated in the future.
Animals ; Chickens ; virology ; China ; epidemiology ; Humans ; Influenza A Virus, H7N9 Subtype ; genetics ; Influenza A Virus, H9N2 Subtype ; genetics ; immunology ; physiology ; Influenza in Birds ; epidemiology ; transmission ; virology ; Influenza, Human ; epidemiology ; transmission ; virology ; Vaccination ; Viral Proteins ; classification ; metabolism