Infectious Bursal Disease Virus Antigen Research Articles

Factors Affecting The Indirect Immunoperoxidase Staining Of Infectious Bursal Disease Virus Antigens In Cells

THE application of the Immunoperoxidase (IP) antibody technique for the definitive diagnosis of Infectious Bursal Disease (IBD) in chickens has earlier been described (Okeke and Lang, 1982). During that study it was noted that the Indirect method of IP was more sensitive in the demonstration of IBD viral antigens than the direct method. Consequently, more formation was sought on the specific technical parameter and the consequences that resulted by deviations from the codified procedures employed in the Indirect technique with the aim of obtaining an even better result. Studies of pH, serum dilutions, serum incubation time, conjugate dilutions, conjugate incubation time and developing time were conducted to find out what Influence such factors could have on the final staining product.

Naturally occurring cell-adapted classic strain of infectious bursal disease virus

Infectious bursal disease virus (IBDV), the etiological agent of infectious bursal disease (IBD), is a variable RNA virus of Avibirnavirus. Some artificially attenuated vaccine strains of IBDV can adapt to cell culture of chicken embryo fibroblast (CEF) cell or its immortalized cell line DF1 in vitro while wild-type IBDV cannot. In this study, for the first time, a naturally occurring cell-adapted classic strain (genogroup 1) of IBDV named IBD17JL01 was identified in China. Animal experiments showed that IBD17JL01 could severely damage the central immune organ of infected chickens. Sequence analysis of the full-length genome revealed the peculiar molecular characteristics of IBD17JL01 with a few amino acid substitutions that might be involved in cell-tropism, antigenicity, and virulence of IBDV. Identification of this novel strain is beneficial to our understanding of the complexity of the epidemiology of IBDV. And the expansion of viral cell-tropism might increase the potential risk of the reassortment of different IBDVs including the live vaccines.

Novel variants of infectious bursal disease virus can severely damage the bursa of fabricius of immunized chickens

In recent years, atypical infectious bursal disease (IBD) with severe immunosuppression has brought new threats to the poultry industry and has caused considerable economic losses. Novel variant infectious bursal disease virus (IBDV) has been identified as the etiological pathogen and for unknown reasons is widespread in poultry on many chicken farms in China that have been immunized with vaccines against very virulent IBDV (vvIBDV). Using immunoprotection experiments in specific-pathogen-free chickens, we first verified that novel variant IBDV could severely damage the bursa of Fabricius of the important immune organ of immunized chicken in the presence of antibodies induced by three types of vvIBDV vaccines, which is a primary reason for the current epidemic of atypical IBD. Monoclonal antibody reactivity patterns and cross-neutralization assays further confirmed the obvious antigenic mismatch between novel variant IBDV and vvIBDV. Sequence analysis of the genome of novel variant IBDV (SHG19 strain) was performed and the key amino acid residues that might be involved in antigenicity and virulence differences of novel variant IBDV compared to vvIBDV were further analyzed. This study not only determined the primary reason for the atypical IBD epidemic, but also remind us of the urgency for developing new vaccines against novel variant IBDV.

Functional characterization of a plant-produced infectious bursal disease virus antigen fused to the constant region of avian IgY immunoglobulins.

Infectious bursal disease virus (IBDV) is the cause of an economically important highly contagious disease of poultry, and vaccines are regarded as the most beneficial interventions for its prevention. In this study, plants were used to produce a recombinant chimeric IBDV antigen for the formulation of an innovative subunit vaccine. The fusion protein (PD-FcY) was designed to combine the immunodominant projection domain (PD) of the viral structural protein VP2 with the constant region of avian IgY (FcY), which was selected to enhance antigen uptake by avian immune cells. The gene construct encoding the fusion protein was transiently expressed in Nicotiana benthamiana plants and an extraction/purification protocol was set up, allowing to reduce the contamination by undesired plant compounds/proteins. Mass spectrometry analysis of the purified protein revealed that the glycosylation pattern of the FcY portion was similar to that observed in native IgY, while in vitro assays demonstrated the ability of PD-FcY to bind to the avian immunoglobulin receptor CHIR-AB1. Preliminary immunization studies proved that PD-FcY was able to induce the production of protective anti-IBDV-VP2 antibodies in chickens. In conclusion, the proposed fusion strategy holds promises for the development of innovative low-cost subunit vaccines for the prevention of avian viral diseases.

Isolation of Infectious Bursal Disease Virus Using Indigenous Chicken Embryos in Kenya

Infectious bursal disease virus (IBDV) isolates were recovered from outbreaks to initiate activities towards developing a local vaccine strain. Use of indigenous chicken embryos was exploited to determine their potential, promote utilization of local resources for research, and enhance household economic activities. Bursa of Fabricius (BFs) samples from outbreaks shown to be IBDV positive was homogenized and inoculated in 4-week-old specific pathogen-free (SPF) IBDV seronegative white leghorn chicks. The harvested virus was inoculated into 11-day-old indigenous chicken embryos that were IBDV seronegative and passaged serially three times after which they were inoculated into 4-week-old indigenous chicks to test for presence and virulence of propagated virus. Out of 153 BFs collected from outbreaks, 43.8% (67/153) were positive for IBDV antigen and 65.7% (44/67) caused disease in SPF chicks. The embryo mean mortalities were 88% on primary inoculation, 94% in 1st passage, 91% in 2nd passage, and 67% in 3rd passage. After the third passage in embryos all the 44 isolates were virulent in 4-week-old indigenous chicks. The results show that indigenous chicken embryos support growth of IBDV and can be used to propagate the virus as an alternative viral propagating tool for respective vaccine preparation.

In vitro characterization of chicken bone marrow-derived dendritic cells following infection with very virulent infectious bursal disease virus

Infectious bursal disease is caused by infectious bursal disease virus (IBDV), an immunosuppressive virus that targets immune cells such as B cells and macrophages. However, the involvement of dendritic cells (DCs) during IBDV infection is not well understood. In this study the in vitro effects of live and inactivated very virulent IBDV (vvIBDV) UPM0081 on bone marrow-derived DCs (BM-DC) were characterized and compared with BM-DC treated with lipopolysaccharide (LPS). Morphologically, BM-DC treated with LPS and vvIBDV showed stellate shape when compared to immature BM-DC. In addition, LPS-treated and both live and inactivated vvIBDV-infected BM-DC expressed high levels of double positive CD86 and major histocompatibility complex class II antigens (>20%). vvIBDV-infected BM-DC showed significantly higher numbers of apoptotic cells compared to LPS. Replication of vvIBDV was detected in the infected BM-DC as evidenced by the increased expression of VP3 and VP4 IBDV antigens based on flow cytometry, real-time polymerase chain reaction and immunofluorescence tests. Levels of different immune-related genes such as interleukin-1β (IL-1β), CXCLi2 (IL-8), IL-18, interferon gamma (IFN-γ, IL-12α, CCR7 and Toll-like receptor-3 (TLR3) were measured after LPS and vvIBDV treatments. However, marked differences were noticed in the onset and intensity of the gene expression between these two treatment groups. LPS was far more potent than live and inactivated vvIBDV in inducing the expression of IL-1β, IL-18 and CCR7 while expression of Th1-like cytokines, IFN-γ and IL-12α were significantly increased in the live vvIBDV treatment group. Meanwhile, the expression of TLR3 was increased in live vvIBDV-infected BM-DC as compared to control. Inactivated vvIBDV-treated BM-DC failed to stimulate IFN-γ, IL-12α and TLR3 expressions. This study suggested that BM-DC may serve as another target cells during IBDV infection which require further confirmation via in vivo studies.

Histopathological and immunohistochemical diagnosis of infectious bursal disease in poultry birds.

Aim:The aim of the present study was to diagnose infectious bursal disease (IBD) using gross, histopathological, and immunopathological approaches and to compare efficacy of immunohistochemical techniques with conventional diagnostic techniques.Materials and Methods:A total of 33 samples were collected from the six different poultry farms from Ludhiana and the nearby districts. Upon gross analysis of the necropsied birds, the relevant tissue samples such as bursa, kidney, junction of proventriculus and gizzard, heart, and muscles were then processed for histopathological and immunohistochemical studies.Results:Varied macroscopic changes were noted in bursa, characterized as swollen, hemorrhages to atrophy in size. Nonetheless, hemorrhages over thigh muscles were rarely seen. Histologically, the bursa showed prominent fibrotic and atrophic changes. Rarefaction of bursal follicles with intermittent infiltration of lympho-mononuclear cells with chronic cystic changes was additional changes, considered to be paramount for IBD. Expression and localization of IBD specific viral antigens were noticed mainly intracellular to the rarefied areas of bursal follicle section(s), in conjunction to inner lining of the cystic cavities of affected follicles. In addition, the junction of proventriculus and gizzard, the heart muscle, respiratory ciliated epithelium, and proventriculus also revealed positive expression to IBD virus (IBDV) antigen. Advanced immunopathological techniques, i.e., immunofluorescence further testified the evidence of antigen as positive green signal within affected follicles. Further consideration to the reliability of various techniques employed, positive correlation (r=0.64623) was emerged out with conventional pathological scoring.Conclusion:It is concluded that the bursa acts as an organ of choice for demonstrating IBDV antigen for specific diagnosis of disease using immunohistochemistry (IHC), and IHC staining is a precise, specific, rapid, and reliable method to demonstrate the IBDV antigen in the altered tissues due to IBDV infection.

Protective efficacy of a DNA vaccine construct encoding the VP2 gene of infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens

Infectious bursal disease (IBD) is an acute, infectious, immunosuppressive disease affecting young chicken worldwide. The etiological agent IBD virus (IBDV) is a double stranded RNA virus with outer capsid protein VP2 of IBDV is the major antigenic determinant capable of inducing neutralizing antibody. DNA vaccines encoding VP2 has been extensively studied achieving only partial protection. However, the efficacy of DNA vaccines against IBDV can be augmented by choosing a potential molecular adjuvant. The goal of the present study is to evaluate the immune response and protective efficacy of a DNA vaccine encoding the C-terminal domain of the heat shock protein 70 (cHSP70) of Mycobacterium tuberculosis gene genetically fused with the full length VP2 gene of IBDV (pCIVP2-cHSP70) in comparison to a ‘DNA prime-protein boost’ approach and a DNA vaccine encoding the VP2 gene (pCIVP2) alone. The results indicate that both pCIVP2-cHSP70 and ‘DNA prime-protein boost’ elicited humoral as well as cellular immune responses. Chickens in the pCIVP2-cHSP70 and ‘DNA prime-protein boost’ groups developed significantly higher levels of ELISA titer to IBDV antigen compared to the group immunized with pCIVP2 alone (p<0.01). However, significantly higher levels of lymphocyte proliferative response, IL-12 and IFN-γ production were found in the pCIVP2-cHSP70 group compared to ‘DNA prime-protein boost’ group. Additionally, chickens immunized with pCIVP2-cHSP70 and ‘DNA prime-protein boost’ vaccines were completely protected against the vvIBDV whereas pCIVP2 DNA vaccine alone was able to protect only 70%. These findings suggest that the truncated C-terminal HSP70 mediated DNA vaccine genetically fused with the VP2 gene construct stimulated both humoral and cell mediated immune responses and conferred complete protection against IBDV. This novel strategy is perhaps a seminal concept in utilizing HSP70 as an adjuvant molecule to elicit an immune response against IBD affecting chickens.

English

AIM: The study involved the evaluation of the bursa of fabricius in healthy and experimentally infected quails with infectious bursal disease virus (IBDV). METHODS: One hundred and fifty days old quail chicks were obtained and divided into two groups. The Group B birds were inoculated with two drops of IBDV antigen while Group A were inoculated with two drops of phosphate buffered saline per os and kept for 14 weeks. Birds were weighed, sacrificed and dissected to remove the bursae on which morphometric and histological procedures were done. RESULTS: Infected bursae showed an initial increase in size which later decreased for a while before attaining a second peak. Histologically, the normal bursae showed the general plan of gastrointestinal tract structures with the lamina propria containing non capsulated lymphoid follicles, which varied in arrangement and number. The infected bursae revealed interfollicular edema, lymphocytolysis, haemorrhages, fibroplasia and keratinization of the bursal substances. CONCLUSION: This study could serve as a guide to poultry clinicians and pathologists in prompt diagnosis of infectious bursal disease in both clinical and subclinical states as well as advancing the knowledge of the bursa of Fabricius in normal and in abnormal states. KEY WORDS: Quail, Bursa of Fabricius, Gastrointestinal tract.

Mucosal application of cationic poly(d,l-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease

Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens. The virus protein (VP) 2 induces neutralizing antibodies, which protect chickens against the disease. The aim of this study was to develop a cationic poly(d,l-lactide-co-glycolide) (PLGA) microparticle (MP) based IBDV-VP2 DNA vaccine (MP-IBDV-DNA) for chickens to be delivered orally and by eye drop route. The tested IBDV-VP2 DNA vaccines were immunogenic for specific-pathogen-free chickens and induced an antibody response after intramuscular application. Co-inoculation with a plasmid encoding chicken IL-2 (chIL-2) or CpG-ODN did not significantly improve protection against IBDV challenge. However, the application of a MP-IBDV-DNA vaccine alone or in combination with a delayed oral and eye drop application of cationic MP loaded with CpG-ODN or chIL-2 improved protection against challenge. The MP-IBDV-DNA-vaccinated chickens showed less pathological and histopathological bursal lesions, a reduced IBDV antigen load as well as T-cell influx into the bursa of Fabricius (BF) compared to the other groups (p<0.05). The addition of chIL-2 loaded MP improved challenge virus clearance from the BF as demonstrated by lower neutralizing antibody titers and reduced IL-4 and IFN-α mRNA expression in the bursa at 7 days postchallenge compared to the other challenged groups. Overall, the efficacy of the IBDV-DNA vaccine was improved by adsorption of the DNA vaccine onto cationic PLGA-MP, which also allowed mucosal application of the DNA vaccine.

An in vivo experimental model to determine antigenic variations among infectious bursal disease viruses

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus causing infectious bursal disease in chickens. IBDV undergoes antigenic drift, so characterizing the antigenicity of IBDV plays an important role for identification and selection of vaccine candidates. In this study, an in vivo experimental model was developed to differentiate a new antigenic variant of IBDV. To this end, a hyper-immune serum to IBDV E/Del-type virus was generated in specific pathogen-free chickens and a standard volume of the hyper-immune serum was serially diluted and injected in specific pathogen-free birds via intravenous, subcutaneous, or intramuscular routes. The chickens were bled at different time points in order to evaluate the dynamics of virus neutralization titres. Based on the results, chickens were injected with different serum dilutions by the subcutaneous route. Twenty-four hours later, chickens were bled and then challenged with 100 median chicken infectious doses of the E/Del virus and a new IBDV variant. Chickens were euthanized at 7 days post infection and the bursa of Fabricius was removed for microscopic evaluation to determine the bursal lesion score. The determined virus neutralization titre along with the bursal lesion score was used to determine the breakthrough titre in the in vivo chicken model. Based on the data obtained, an antigenic subtype of IBDV was identified and determined to be different from E/Del. This model is a sensitive model for determination of IBDV antigenicity of non-tissue culture adapted IBDV.

Transition from acute to persistent infection: Antigen persistence and cytotoxic T cells response in infectious bursal disease virus infected SPF chickens (P6129)

Abstract Described by Cosgrove about a half century ago, infectious bursal disease (IBD) is as an acute, highly contagious viral infection of young chickens, which last for 7 to 10 days and leads to immunosuppression. Here, we are reporting for the first time that in IBD virus (IBDV) -infected, specific pathogen free (SPF) chickens bursa, the virus antigen, macrophages, CD4+, CD8+ cells, Fas, Fas ligand (FasL), and caspase-3 were consistently detected by immunohistochemistry up to 56 days post inoculation (DPI). Two-week-old SPF chickens were inoculated intraocularly with standard challenge strain (STC) using a 104embryo infectious dose. Histopathology of bursal tissues revealed severe lymphoid follicular depletion at DPI 5-28. A distinctive pattern of bursal follicles in the form of small and large follicles was detected from DPI 35 to 56. At 42 and 56 DPI a characteristic clustered pattern of IBDV antigen, macrophages, CD4+, CD8+ cells, Fas, FasL, and caspase-3 were detected in or around the small bursal follicles as compared to large bursal follicles. This is the first report on the persistence of IBDV antigen causing a latent infection, in bursal tissues of SPF chicken. Detection of persistent IBDV infection is highly significant in the elucidation of virus pathogenesis, immunology and epidemiology and it raises many questions that need to be answered in future studies.

The host genotype influences infectious bursal disease virus pathogenesis in chickens by modulation of T cells responses and cytokine gene expression

The immunopathogenesis of infectious bursal disease virus (IBDV) was investigated in different layer and broiler type chickens in comparison to highly susceptible specific-pathogen-free layers (SPF-Wh-LT) often used for experimental studies. Layer-type chickens (LT) of all genetic backgrounds showed significantly higher IBDV antigen loads in the bursa of Fabricius (BF) compared to broiler type birds (BT) (P<0.05). The variation between IBDV-infected and virus-free birds in the percentage of splenic and intrabursal B cells, T cells and macrophages differed between genetic backgrounds as well as the expression levels of cytokines. The most susceptible SPF-Wh-LT showed high levels of circulating type I IFN starting at 2days post infection (dpi) up to 7dpi coinciding with clinical IBD, while less susceptible birds showed a delayed response. Circulating cytokine levels were poorly associated neither with intrabursal nor with splenic mRNA expression of these cytokines. Detected cytokines varied in expression levels and timing between infected groups of different genetic background. These data suggest that variations in the activity of immune cell populations contribute to differences in infectious bursal disease between birds of various genetic backgrounds.

DETERMINATION OF SENSITIVITY AND SPECIFICITY OF IN-HOUSE SANDWICH ELISA FOR THE DETECTION OF INFECTIOUS BURSAL DISEASE VIRUSES

The study was designed for the development of an In-House sandwich ELISA as a suitable serological method for the rapid detection of infectious bursal disease virus (IBDV). The test was also designed to compare and evaluate its sensitivity and specificity with other traditional methods used for the detection of IBDV from field outbreak cases prevalent among the poultry population of Bangladesh. To develop the In-House sandwich ELISA, hyper-immune serum was raised against live IBDV vaccine in rabbit which was used to coat each of the 96-well flat bottomed polystyrene microtitre plate whereas, hyper-immune sera raised in chickens against IBDV used as secondary antibody. The newly developed In-House sandwich ELISA was standardized by dispensing different dilutions (10-1 up to 10-4) of rabbit serum. Among them, the 10-2 dilution of serum showed most suitable reading for the detection of IBD virus and used to coat the plate to evaluate its sensitivity and specificity. Sensitivity test was done by different dilutions (10-0 to 10-4) of reference IBD virus. The virus dilution, 10-3 was the highest dilution having lowest capacity to bind with coated antibody of the ELISA plate which indicated that IBD viruses was absent in the dilutions of above 10-3. The cut-off value of negative control samples was determined as 0.937 which indicated titer of tested samples &gt;0.937 was positive and &lt;0.937 was negative. Specificity test was performed using different known viruses (IBDV and NDV) using different dilutions (10-1 up to 10-4). Only the IBDV showed positive result which indicated high specificity of newly developed ELISA plate. A total of 26 samples (feces, cloacal swab, spleen and bursa) from control group, experimental and natural IBDV outbreaks were used as field viral antigen for the evaluation of sensitivity and specificity of the newly developed In-House sandwich ELISA. In case of experimental infection, 5 (62.5%) of 8 feces sample but none of cloacal swab were positive for IBDV whereas, all bursa and spleen samples were positive by both In–House sandwich ELISA and AGIDT. In case of natural outbreak cases, 6 of 6 bursal samples and 4 of 6 spleen samples were positive by In-House sandwich ELISA whereas, AGIDT detected all bursal and 3 spleen samples. No virus was detected from the samples of control group. The result showed 92.85% specificity of the developed sandwich ELISA for detection of IBDV with AGIDT which indicated that the developed ELISA is a sensitive, specific, cost effective and reliable tool for the detection of IBDV antigen from a large number of field samples.DOI = http://dx.doi.org/10.3329/bjvm.v8i2.11185 Bangl. J. Vet. Med. (2010). 8 (2) : 97-106

Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen

Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV.

DNA-mediated vaccination conferring protection against infectious bursal disease in broiler chickens in the presence of maternal antibody

The objective of the present study was to determine if a DNA vaccine carrying large segment gene of infectious bursal disease virus (IBDV) could confer protection against infectious bursal disease (IBD) in broiler chickens in the presence of maternal antibody. Broiler chickens with maternal antibody titers to IBDV were intramuscularly injected with a DNA plasmid coding for VP2, VP3, and VP4 genes of IBDV strain variant E (VE) (P/VP243/E) at 1-day, 1-week, and/or 2 weeks old. The dose of P/VP243/E used ranging from 400 μg to 10 mg. Broiler chickens at 3 weeks old were orally challenged with IBDV strain (VE) and observed for 10 days. Only broiler chickens vaccinated with 7.5 or 10 mg of P/VP243/E 3 times had 90 or 100% protection against challenge by IBDV strain VE and protected broiler chickens had significantly higher ( P < 0.05) bursa weight/body weight ( B/ B) ratios, significantly lower ( P < 0.05) bursal lesion scores, and the absence of IBDV antigens in bursae determined by immunofluorescent antibody assay (IFA). Antibody titers to IBDV as determined by enzyme-linked immunosorbent assay (ELISA) or virus neutralization (VN) assay in chickens of each group in each trial were gradually decreased prior to challenge. There was no significant difference ( P > 0.05) in ELISA or VN titers to IBDV among all groups of broiler chickens or among the groups of broiler chickens vaccinated with various dose of P/VP243/E before challenge. Broiler chickens in the groups receiving 7.5 or 10 mg of P/VP243/E had significantly lower ( P < 0.05) ELISA or VN titers to IBDV than those in the challenge control (CC) groups or the other groups vaccinated with various dose of P/VP243/E after challenge. Broiler chickens in the groups vaccinated with 10 mg of P/VP243/E 3 times had significantly higher ( P < 0.05) stimulation indices for IBDV-stimulated lymphocyte proliferation response than those in the vector control (VC) or CC group at 14, 21, 24, or 31 days after first DNA vaccination. The results indicated that DNA vaccination with DNA encoding large segment gene of IBDV confers protection against challenge by IBDV in broiler chickens with maternal antibody to IBDV.

Detection of Antibodies to Infectious Bursal Disease Virus (IBDV) by Agar Gel Immunodiffusion using Recombinant VP2 Protein

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease of chicken. Agar gel immunodiffusion using IBDV antigen extracted from bursa of Fabricius of infected chicken has been used officially for diagnosis of IBDV in Korea. In this study, in order to replace the IBDV whole virus antigen with non-infectious antigen, recombinant VP2 protein (rVP2) of IBDV was produced using recombinant baculovirus expression system. Purified baculovirus-expressed rVP2 was used as an antigen in an agar gel immunodiffusion (AGID). rVP2 antigen precipitated specifically IBDV antibodies. AGID using rVP2 antigen detected anti-IBDV antibodies from 6 dpi to 28 dpi (termination of the experiment) when specific pathogen free chickens were experimentally infected with IBDV 52/70 strain. This was consistent with result by AGID using IBDV antigen, virus neutralization test (VNT) and a commercial ELISA kit (except for one serum). The sensitivity of rVP2 was the same with that of IBDV antigen when field sera (n=324) were tested by AGID. However, AGID using rVP2 antigen detected maternal antibodies from broiler chickens (n=20) on a broiler farm up to 15 days old, although the detection rate of the AGID was relatively low compared to a commercial ELISA kit. Our results indicate that IBDV whole virus antigen from IBDV infected chickens would be replaced with recombinant VP2 protein as an antigen for AGID.

Dose and timing requirements for immunogenicity of viral poultry vaccine antigen: investigations of emulsion-based depot function

The release requirements for vaccine antigens delivered by adjuvants with presumed depot function are poorly understood. Water-in-oil (W/O) emulsions are routinely used in many poultry vaccines. They strongly activate antibody production, and are regarded as a depot from which antigens are slowly released, resulting in prolonged antigen residence. However, from earlier studies we concluded that W/O adjuvant activity is partly based on the immunostimulatory activity of the oil phase. Here we assess the dose and regimen requirements for viral antigen in immunization experiments in chickens. Three-week-old to 4-week-old White Leghorn chickens were repeatedly injected with inactivated infectious bursal disease virus antigen over 48 days. Our aim was to compare the antibody responses in repeatedly injected animals, receiving fractioned doses of antigen, with the responses in animals receiving only one injection of the full dose of antigen formulated in either a W/O emulsion or in saline. We observed that repeated administration of small amounts of antigen results in a gradual increase of specific humoral immune responses during the immunization regimen. Immunization with a higher first dose evoked an early higher antibody response, which, however, reached a similar plateau level at the end of the regimen. When compared with lower first-dose regimens, a slow decline of serum antibody titre 2 weeks after the end of antigen injections indicated that repeated injection of small doses of antigen indeed mimics the efficacy of depot-forming adjuvants. All regimens of fractioned antigen in saline, however, proved less effective, when compared with a single-dose vaccination of the cumulative amount of antigen formulated in a W/O emulsion. From our data we confirm that W/O emulsions are very effective vaccine vehicles for improving antigen-specific humoral responses in chickens, owing to a combination of antigen residence-prolonging activity and direct immune stimulation.

An ELISA for detection of infectious bursal disease virus and differentiation of very virulent strains based on single chain recombinant chicken antibodies

Two chicken single-chain variable antibody fragments (scFv) designated scFv154 and scFv88, previously shown to react with either all or very virulent (vv) infectious bursal disease virus (IBDV) strains, respectively, were evaluated for use in an enzyme-linked immunosorbent assay (ELISA) for differentiation of vvIBDV. Specificity and sensitivity of the vvIBDV ELISA was assessed when scFv154 and scFv88 were expressed as soluble antibodies (sAb), phage antibodies (pAb) or hyper-phage antibodies (hpAb). The highest test sensitivity and specificity was obtained using hpAb154 to detect all IBDV and pAb88 to differentiate vvIBDV strains. Such an ELISA was eight to 16 times more sensitive for IBDV antigen detection than the mouse monoclonal antibody ELISA. Using field samples, the scFv ELISA was able to differentiate between flocks infected with vvIBDV and those infected with classical or variant IBDV. In one instance IBDV was detected in a flock found to be negative by the monoclonal antibody ELISA. The results showed that scFv can be utilized as highly specific and sensitive ELISA reagents for the detection and discrimination of avian pathogens.

L-Arginine stimulates immune response in chickens immunized with intermediate plus strain of infectious bursal disease vaccine

Infectious bursal disease continues to pose an important threat to the commercial poultry industry, especially after the emergence of virulent infectious bursal disease virus (IBDV). l-Arginine, a ubiquitous, basic amino acid is used as an immunostimulant in variety of human and animal studies with great success in immunosuppressed hosts. In the present study, we evaluated the immunomodulatory effects of l-arginine on humoral and cellular immune response in chickens immunized with live intermediate plus (IP) strain of IBDV vaccine. Chickens vaccinated with IP strain and supplemented with 2% l-arginine showed 100% protection after challenge with virulent IBDV compared to 80% protection induced by IP strain alone. IP strain vaccine elicited good antibody titres in both the groups, IP + l-arginine and IP alone, however, the antibody titres in IP + l-arginine group were significantly higher ( P < 0.05) than IP vaccinated chickens. Mitogenic response of peripheral blood lymphocytes to specific IBDV antigen stimulation was significantly higher in IP + l-arginine group ( P < 0.05). Peripheral blood lymphocytes were able to rapidly clear the suppressive effects of IP strain and virulent challenge virus in IP + l-arginine group. These results strongly suggest that l-arginine plays a vital role in modulation of protective immune response against IBDV.