Co-expression, purification and bioassay of three avian viral antigens / 生物工程学报

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.

Immune response elicited by the oral administration of an intermediate strain of IBDV in chickens

The immune response elicited by the oral inoculation of an intermediate strain of infectious bursal disease virus was studied in chickens. A strong over expression of IL-6, IL-8, IFNα and IFNγ was observed in bursa at 3 days post inoculation together with an increase in splenic NO2 release. An influx of T-lymphocytes was also detected.

Effects of Dangguibuxue Tang, a Chinese herbal medicine, on growth performance and immune responses in broiler chicks

The effects of Dangguibuxue Tang (DBT) on growth performance and immunity response in immunosuppressed broiler chicks were investigated in this study. 240 one-d-old broiler chicks (DaHeng S01) were randomly divided into 4 groups, 2.0% DBT-treatment (A), 0.5% DBT-treatment (B), cyclophosphamide-control (C), and control group (D). From 4 d to 7 d of age, chicks in group A, B and C were given cyclophosphamide (CY) at a dosage of 100mg/kg body weight (BW) daily by intraperitoneal injection to induce immunosuppression. Chicks in group D were given an equal volume of physiological saline daily by intraperitoneal injection and considered normal chicks. Groups A and B were supplemented with 2.0% or 0.5% of DBT in the drinking water from 8 d to 42 d of age. Groups C and D did not receive any additional medication. The results revealed that chicks from group B had lower feed:gain rate (FGR), lower total mortality, higher immunity organ indexes, higher levels of Newcastle disease (ND) antibody and infectious bursal disease (IBD) antibody, higher interleukin-2 and interleukin-6 levels, and greater lymphocyte proliferative responses to concanavalin A (ConA) during the experiment than those from group C. However, no significant difference in the immunity status in the two levels of DBT-treatment was observed. These results indicate that supplementation of 0.5% of DBT can improve both cellular immunity and humoral immunity in immunosuppressed broiler chicks.

Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens

A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.

Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-gamma on protective immunity by a DNA vaccine against IBDV in chickens

This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-gamma (ChIFN-gamma) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-atilde was constructed. Twice at 2-week intervals, twoweek-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-gamma were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-gamma was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-gamma groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-gamma had no significant effect.

Passive immunization using purified IgYs against infectious bursal disease of chickens in Pakistan

Infectious bursal disease (IBD) is an acute and highly contagious disease of young chickens caused by Birnavirus. Mortality of infected birds can be best prevented if injected with antibodies. The present study was an attempt to raise specific hyper-immune polyclonal antibodies against IBD virus in Pakistan. Commercial layers divided into four groups were injected with IBD vaccine subcutaneously according to four different treatment regimens. Eggs were collected daily and antibodies were purified from yolk with dextran sulphate. Titers of antibodies in serum and yolk were evaluated with enzyme linked immunosorbant assay and agar gel precipitation test. Antibody titers were significantly higher in yolk than serum. Eggs collected at 28 days post-vaccination had maximum antibody titers. Of treatment regimens, T3 was found to be most effective for hyperimmunization. Lyophilized antibodies stored at 4oC did not lose their activity till the end of experiment. IBD virus infected birds were injected with purified antibodies which induced 92% recovery as compared to control birds. The study implicates that the purified antibodies may be useful as a therapeutic agent to cure IBD infected birds.

Early and transient induction of nitric oxide (NO) in infectious bursal disease virus infection is T-cell dependent: a study in cyclosporin-A treated chicken-model.

The level of nitric oxide (NO) in the supernatants of mitogen (PHA) stimulated lymphocyte cultures from infectious bursal disease (IBD) virus infected T-cell suppressed and immune competent chickens was monitored. The immune competent chickens when infected with IBD virus showed 4-6 folds increased levels of NO as compared to uninfected chickens. The levels of NO in T-cell suppressed chickens were comparable to uninfected control chickens, in spite of markedly increased hemorrhage suggesting that the muscular hemorrhage observed in IBD in not solely and directly related with NO production. The immune suppressed chickens that did not induce NO production after IBD virus infection showed more severe lesions and supported enhanced virus replication. Taken together it may be suggested that NO production after IBD virus infection, may exert antiviral effect since the immune-suppressed chickens that failed to induce NO showed more severe disease and higher magnitude of virus replication, but does not seem to correlate with the hemorrhagic lesions which in fact may be as a result of the net outcome of various host-factors and the determinants responsible for virus virulence and virus clearance.

Virus enhancement following infection with antibody-coated infectious bursal disease virus (IBDV) in chickens.

A novel concept of vaccination, employing virus-antibody complex has been reported for the control of infectious bursal disease in chickens. A comparison of virus replication, serum neutralizing antibody response and pathogenicity in chickens inoculated with the antibody coated virus, prepared by mixing virus and antibody in different ratios (1:1, 1:0.1, 1: 0.01) and virus alone without antibody, has been made. Antibody coated virus (when mixed in certain crucial ratios) replicated to a higher magnitude in the target organ, caused more severe pathogenesis but induced a primary serum neutralizing antibody response almost comparable. The results may have important implications in understanding of pathogenesis and development of control strategies against infectious bursal disease virus, specially employing immune complex vaccine.

Western blot detection of infectious bursal disease virus infection

In order to evaluate the use of a Western blot methodology for the diagnosis of infectious bursal disease virus (IBDV) infection, chickens were experimentally infected with IBDV strains and tested for the presence of viral antigens and antibodies by a blocking Western blot test (bWB). The viral proteins obtained from the bursa of Fabricius (BF) were transferred to a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the chicken sera obtained by heart puncture were used for the detection of these proteins. In order to eliminate nonspecific reactions, we used a rabbit anti-chicken serum (blocking tool). By the use of the bWB test, two distinct viral proteins of 43-kDa (VP2) and 32-kDa (VP3) were detected. We suggest the use of this methodology for the detection of IBDV infection in animals suspected of having IBDV reinfection and a chronic subclinical form of the disease. With the use of the rabbit anti-chicken sera for blocking, this method is practical, sensitive and less time consuming.

Evaluation of two vaccination programs to control infectious bursal disease in commercial broilers

An experimental study was carried out to compare protection induced by two infectious bursal disease [IBD] vaccination programs in commercial broilers. Chickens inoculated with live vaccine at 7 and 21 days old were protected against mortality but clinical signs of IBD were noted after challenge at 28 and 35 days old. Chickens inoculated with both live vaccine [at 7 and 21 days old] and inactivated vaccine [at 8 days old] were better protected against challenge at 28 and 35 days of age. This vaccination program protected challenged chickens against both mortality and clinical IBD signs. However, the bursa: body weight ratios were lower than those of the control birds [P < 0.05]

electrophoretic pattern of serum proteins in chickens vaccinated with newcastle [different strains and gumboro vaccines

The present work was planned to study the effect of vaccination with different strains of Newcastle disease virus vaccine [NDVV] as well as vaccination with Gumboro vaccine on the electrophoretic pattern of serum proteins in our local breed [Baladi]. One hundred and fifty three one day old chicks were used. Our results revealed that there was a significant increase in the levels of total serum proteins, albumin, total globulins and gamma-globulin in the sera of chickens vaccinated with NDVV whereas there was no significant difference between Gumboro vaccinated chickens and control group in the total serum proteins and its fractions. The chickens vaccinated with komarov strain of NDVV showed the highest mean values of total serum proteins, albumin, total globulins and gamma-globulin whereas the lowest values were observed in the chickens vaccinated with Gumboro and Lasota strain of NDVV. Concerning the type of vaccine and weeks post-vaccination. The mean values of total serum proteins, albumin, total globulins and beta-globulins were significantly increased in chickens vaccinated with NDVV either with komarov or Lasota strains at the 1st week post-vaccination. In the 2nd week post-vaccination, the mean values of beta-globulin fraction in group vaccinated with NDVV and chickens vaccinated with NDVV [without last dose] and Gumboro vaccine was significantly decreased. Furthermore, the total serum proteins were significantly decreased in chickens vaccinated with Gumboro and NDVV [K]. In the 3rd week post-vaccinated with Gumboro [L]. It was concluded that the vaccination of chicks with Gumboro vaccine at 14 day of age delay the response of immune system for vaccination with Newcastle disease virus vaccine [NDVV]