Chicken recombinant antibodies specific for very virulent infectious bursal disease virus

Summary. A phage-displayed single chain variable fragment (scFv) antibody library was constructed from the immune spleen cells of chickens immunized with very virulent infectious bursal disease virus (vvIBDV) strain CS89. A library consisting of around 9.2 × 10⁷ clones was subjected to 3 rounds of panning against captured CS89 virus. Analysis of individual clones by nucleotide sequencing revealed at least 22 unique scFv antibodies binding to vvIBDV in ELISA. Testing of the scFv antibody panel in ELISA against classical, variant or vaccine strains and a wide variety of vvIBDV isolates from the UK, China, France, Belgium, Africa, Brazil, Indonesia and the Netherlands identified one antibody, termed chicken recombinant antibody 88 (CRAb 88) that was specific for vvIBDV. CRAb 88 was capable of recognizing all vvIBDV strains tested regardless of their country of origin and showed no reactivity with classical, variant or vaccine strains, lending support to the use of this scFv as a powerful diagnostic tool for the differentiation of vvIBDV strains. Immunoprecipitation studies revealed that CRAb 88 was directed towards a highly conformational epitope located within the major neutralizing protein VP2. Sequence analysis of the hypervariable region of VP2 of the IBDV strains tested indicate that Ile(256) and Ile(294) may play roles in binding of CRAb 88. This is the first reagent of its type capable of positively distinguishing vvIBDV from other IBDV strains.     

Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease

Summary.  The right hand end Nde I fragment 3 (90.8–100 map units) of the fowl adenovirus serotype 10 (FAV-10) was characterised so as to allow the location of an insertion site for recombinant vector construction. Infectious bursal disease virus (IBDV) VP2 gene from the Australian classical strain 002/73, under the control of the FAV-10 major late promoter/leader sequence (MLP/LS) was inserted into a unique Not I site that was generated at 99.5 map units. This recombinant virus was produced without deletion of any portion of the FAV-10 genome. When administered to specific pathogen free (SPF) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the FAV-10/VP2 recombinant induced a serum VP2 antibody response and protected chickens against challenge with IBDV V877, an intermediate virulent classical strain. Birds were not protected when the recombinant was delivered via the conjunctival sac. Accepted December 18, 1997 Received August 26, 1997     

The working mechanism of an immune complex vaccine that protects chickens against infectious bursal disease.

The role of immune complexes (Icx) in B-cell memory formation and affinity maturation allow for their potential use as vaccines. Recently, a new immune complex vaccine has been developed that is currently under field trials conducted in commercial poultry. This immune complex vaccine is developed by mixing live intermediate plus infectious bursal disease virus (IBDV) with hyperimmune IBDV chicken serum (IBDV-Icx vaccine). Here we have investigated the infectivity of this vaccine as well as the native IBDV (uncomplexed) vaccine in terms of differences in target organs, in target cells and speed of virus replication. At various days after inoculation on day 18 of incubation (in ovo) with either one dose of virus alone or the IBDV-Icx vaccine, the replication of IBDV and the frequency of B cells and other leucocyte populations were examined in the bursa of Fabricius, spleen, and thymus using immunocytochemistry. With both vaccines, IBDV was detected associated with B cells, macrophages and follicular dendritic cells (FDC) in bursa and spleen, although complexing IBDV with specific antibodies caused a delay in virus detection of about 5 days. Most remarkable was the low level of depletion of bursal and splenic B cells in IBDV-Icx vaccinated chickens. Furthermore, in ovo inoculation with the IBDV-Icx vaccine induced more germinal centres in the spleen and larger amounts of IBDV were localized on both splenic and bursal FDC. From these results we hypothesize that the working mechanism of the IBDV-Icx vaccine is related to its specific cellular interaction with FDC in spleen and bursa.     

Immunosuppressive effect of infectious bursal disease virus on vaccination against infectious bronchitis

Groups of broiler chicks hatched with parental antibodies to infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV) were vaccinated against IBV at 1 day of age via the oculonasal routes and inoculated with virulent IBDV at 1, 5, 10, 15 or 20 days of age. While the non-IBDV inoculated birds were solidly immune against IBV challenge at an age of 29 days, immunity in the IBDV infected birds was depressed. This depression, which was most serious in the birds IBDV inoculated at 1 or 5 days of age, coincided with a delayed infiltration of the Harderian gland by lymphocytes and immunoglobulin-bearing cells. In the groups inoculated at older ages infiltration did not seem to be delayed but moderate destruction of plasma cells was observed 7 to 14 days later. The neutralisation index to IBV, which was significantly higher in the non-IBDV infected than in the infected birds at the day of challenge, increased sharply after challenge in the IBDV infected but not in the non-infected birds. All IBDV-inoculated birds developed typical lesions when 17 to 26 days old whereas precipitins reappeared when birds were 29 to 33 days old.     

Comparison of vaccines against infectious bursal disease.

A comparative study was undertaken of 9 products from 7 different sources intended for use as vaccines against infectious bursal disease of chickens (IBD). A range of properties was found in laboratory tests for safety, efficacy and immunosuppressive effect. No vaccine caused clinical disease after administration to chicks at 7 days of age, but one caused a significant impairment of weight gain, and when given to day-old chicks caused some morbidity and deaths.Most vaccines affected the bursa of Fabricius and histological examination of this organ revealed varying degrees of tissue damage which correlated with the reduction in size of this organ. The effects with the different products ranged from no damage to damage almost as rapid and severe as that produced by a fully virulent field strain of the agent.Selected products which differed in their effect on the bursa were tested for their immunosuppressive properties by assessing the response to live Newcastle disease vaccine administered after the IBD vaccine. The effect on the response to Newcastle disease vaccine was found to be correlated with the degree of tissue damage.Variations in the ability of the vaccines to protect against IBD challenge were also found, but these did not depend on the degree of damage to the bursa.These studies enable proposals for standard tests for IBD vaccines to be formulated in respect of safety, potency and lack of immunosuppressive effect. Only 2 of the 9 vaccines tested satisfied these standards.     

A monoclonal antibody capture enzyme-linked immunosorbent assay for detecting antibodies to infectious bursal disease virus.

A monoclonal antibody capture enzyme-linked immunosorbent assay (mAb-ELISA) for antibodies to infectious bursal disease virus (IBDV) in chicken sera was developed and compared with conventional ELISA. When sera from farm chickens were tested by the two ELISAs and serum neutralization (SN), the correlation rate between SN and mAb-ELISA was 100% (49/49), and that between SN and conventional ELISA was 81.6% (40/49). In mAb-ELISA, all of the sera that were antibody-negative by SN had low absorbance values (below 0.05), and the absorbance values correlated closely with the SN titers. In the conventional ELISA, however, the sera antibody-negative by SN had various absorbance values ranging from 0.06 to 0.32. mAb-ELISA had much lower non-specific reactions than the conventional ELISA against sera from IBD-negative chickens.     

Heat inactivation of serotype 1 infectious bursal disease virus.

Heat inactivation curves for infectious bursal disease virus serotype 1 (IBDV) strain 52/70 in bursal homogenate supernatant were constructed for 70 degrees C, 75 degrees C and 80 degrees C. Biphasic, multiple kinetics curves were produced with an initial rapid drop in infectivity followed by a more gradual decline. In this second phase the approximate times taken to reduce the infectivity by 1 log(10) at each temperature were: 70 degrees C (18.8 min), 75 degrees C (11.4 min) and 80 degrees C (3.0 min), confirming the heat resistance of this virus.     

Isolates of infectious bursal disease virus from India are of very virulent phenotype

Four Indian field isolates, a classical virulent and an attenuated vaccine strains of Infectious bursal disease virus (IBDV) have been characterized by sequence analysis of part of the VP1 gene (from nucleotide 1538-1979) comprising one of viral RNA dependent RNA polymerase motifs. Sequence alignment of these viruses with reported viruses of other countries revealed Indian IBDV field isolates to be 100% similar to very virulent Japanese (OKYM), European (UK661) and Bangladesh (BD3/99) IBD viruses at amino acid level, whereas they had 0.2-0.9% divergence at nucleotide level. Out of the total 24 nucleotide changes found in the Indian field isolates, as well as reported very virulent viruses, only one resulted in amino acid change S-P at 562 position. The Indian field isolates displayed nucleotide divergence of 10.6-11.6% and amino acid divergence of 2.8-3.5% from the classical virulent and attenuated vaccine strains. The RNA dependent RNA polymerase motif from amino acid 528-541, present in the sequence analyzed, was conserved among all the viruses, irrespective of pathotype and serotype. In the phylogenetic tree, based on nucleotide sequence, Indian field viruses were grouped with reported very virulent viruses in one lineage whereas, classical virulent, attenuated vaccine and serotype 2 strains formed part of the second lineage. But in the phylogenetic tree based on amino acid sequence alignment, the serotype 2 strain OH grouped with Indian field isolates and reported very virulent viruses in one lineage and classical virulent and attenuated vaccine strains formed the second lineage.     

Rapid detection of infectious bursal disease antibodies by counterimmunoelectrophoresis

Counterimmunoelectrophoresis was used to detect infectious bursal disease virus-specific precipitating antibodies in chicken sera. The method was found to be sensitive, simple, reproducible and rapid - detecting precipitins after 45 min. Results could be read visually, without staining, and they compared favourably with results obtained with the agar-gel precipitation test.     

Current status of vaccines against infectious bursal disease

Infectious bursal disease virus (IBDV) is the aetiological agent of the acute and highly contagious infectious bursal disease (IBD) or “Gumboro disease”. IBD is one of the economically most important diseases that affects commercially produced chickens worldwide. Along with strict hygiene management of poultry farms, vaccination programmes with inactivated and live attenuated viruses have been used to prevent IBD. Live vaccines show a different degree of attenuation; many of them may cause bursal atrophy and thus immunosuppression with poor immune response to vaccination against other pathogens and an increase in vulnerability to various types of infections as possible consequences. Depending on their intrinsic characteristics or on the vaccination procedures, some of the vaccines may not induce full protection against the very virulent IBDV strains and antigenic variants observed in the last three decades. As chickens are most susceptible to IBDV in their first weeks of life, active immunity to the virus has to be induced early after hatching. However, maternally derived IBDV-specific antibodies may interfere with early vaccination with live vaccines. Thus new technologies and second-generation vaccines including rationally designed and subunit vaccines have been developed. Recently, live viral vector vaccines have been licensed in several countries and are reaching the market. Here, the current status of IBD vaccines is discussed.     

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.     

Reassortant infectious bursal disease virus isolated in China

Infectious bursal disease virus (IBDV) is a bi-segmented, double-stranded RNA virus which belongs to the genus Avibirnavirus of the family Birnavirideae. In this study, we determined the complete nucleotide sequences of a reassortment IBDV strain TL2004 with segments A and B derived from attenuated and very virulent strains of IBDV. This strain is pathogenic to SPF-embryonated eggs and chickens, although it is not as virulent as very virulent strain. Genomic sequence in GenBank analysis showed that both types of natural genetic reassortment of infectious bursal disease virus emerged in China. Our findings, which strongly suggest genetic exchange between attenuated and very virulent strains of IBDV, emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines.     

Isolation of infectious bursal disease virus from turkeys

A virus, which is morphologically identical and antigenically related to two previously known isolates of infectious bursal disease (IBD) virus, was isolated from pooled faeces of 6-week-old turkeys with diarrhoea. It is concluded that this virus, designated TY89, is an isolate of IBD virus. The isolation of TY89 was heavily dependent upon the use of electron microscopy and the immunofluorescence technique. Antibody to TY89 virus was detected by both indirect immunofluorescence and serum neutralisation tests in 28 of 95 (29%) sera collected from 20-week-old turkeys. The available evidence suggests that this virus has only recently been introduced into turkeys in Northern Ireland. It is believed that this is the first report of natural infection of turkeys with IBD virus.     

Validation of five commercially available ELISAs for the detection of antibodies against infectious bursal disease virus (serotype 1)

In this study, the results are reported from a validation study of five commercially available enzymelinked immunosorbent assays (ELISAs) for the detection of antibodies against infectious bursal disease virus (IBDV), serotype 1. The specificity of the ELISAs varied from 63.8 to 100%. All ELISAs reached a sensitivity of 100% on sera between 14 and 21 days post-vaccination (d.p.v.) with two classical vaccines and a Delaware variant-E virus. Overall, most birds became positive between 8 and 11 d.p.v. As expected, the ELISA with the lowest specificity showed the highest sensitivity at 5 d.p.v. When the decrease in maternally derived antibodies against IBDV was measured, a highly significant correlation (P < 0.001) was found for all ELISAs and the virus neutralization test (VNT). R ² varied from 0.44 to 0.76, whereas the slope varied from 0.33 to 0.57. This indicates that there is a certain correlation between VNT and ELISA titres, but that the correlation differs from ELISA to ELISA. For all ELISAs, no significant (P < 0.05) differences in level of antibodies were detected between antibody titres in breeder serum, egg yolk and progeny at 1 and 4 days of age. This validation of five commercially available ELISAs for the detection of antibodies against IBDV (serotype 1) shows that there are significant differences in their performance. Therefore, choosing an ELISA or interpreting ELISA results without knowing the technical performance should be avoided.     

Susceptibility of chicken mesenchymal stem cells to infectious bursal disease virus

Infectious bursal disease virus (IBDV) is the causative agent of one of the most important viral diseases affecting the poultry industry worldwide. The virus causes an acute, highly contagious and immunosuppressive disease in chickens. Previous studies have demonstrated that in addition to B cells, macrophages can support the replication of IBDV. Since mesenchymal stem cells in bone marrow regulate the differentiation and proliferation of hematopoietic precursors, the interaction between IBDV and mesenchymal stem cells was investigated. Mesenchymal stem cells were isolated from chicken bone marrow. The classical IM strain and the variant strain-E of IBDV, both adapted to grow in a chicken macrophage cell line, were used to infect mesenchymal stem cells. Primary chicken mesenchymal stem cells were highly susceptible to replication of IBDV. Both viruses induced cytopathic effects and replicated to high titers in mesenchymal stem cells. The finding that IBDV can replicate in mesenchymal stem cells provides new information on the susceptible target cell population within the host and contributes to the understanding of the pathogenic potential of the virus.