Detection of antibodies against serotypes 1 and 2 infectious bursal disease virus by commercial ELISA kits

Two distinct serotypes of infectious bursal disease virus (IBDV) are recognized in chicken and turkey flocks in the United States. Serologic testing of chicken flocks for serotype 1 viruses is routinely performed to monitor disease status and vaccination. Earlier studies indicated that enzyme-linked immunosorbent assay (ELISA) test detects antibodies to both serotypes of the virus, while the virus neutralization (VN) test is serotype specific. It is useful to evaluate currently available commercial ELISA kits for their ability to differentiate between antibodies elicited by the two serotypes. Three trials were performed in which chickens were orally inoculated with either a high or a low dose of serotype 1 STC or serotype 2 OH strains of IBDV. Sera collected at 0, 7, 14, and 21 days from these chickens and antisera procured from naturally infected broiler (n=20) and layer (n=30) flocks were tested with five different commercial ELISA kits and by VN. All ELISA kits detected different levels of antibodies elicited against serotype 1 of the virus and moderate and high levels of antibodies against serotype 2 virus. A correlation existed between the ELISA and the VN titers of experimentally infected chickens. All serum samples tested from the commercial layer flocks and 65% of the broiler flocks had antibodies against the OH strain. However, no correlation between the VN titers and ELISA titers was observed for the commercial broilers and layers sera by the majority of the kits. The results indicated that currently available commercial ELISA kits detect antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses.     

Heteroduplex mobility assay for genotyping infectious bursal disease virus

A heteroduplex mobility assay (HMA) was developed to genotype infectious bursal disease virus (IBDV). This method analyzed 390-base pair (bp) polymerase chain reaction (PCR) products, encompassing the hypervariable region of the VP2 gene. IBDV strains from the United States and other countries were analyzed. The HMA was able to differentiate standard, antigenic variants and very virulent strains of IBDV. Minor differences between different strains from the same subtype were also detected. Close relationships between field IBDV with vaccines prepared with Delaware E strain were determined by HMA. The results obtained by HMA were confirmed by restriction fragment length polymorphism (RFLP) and phylogenetic analysis of nucleotide sequences. The HMA proved to be a useful technique to rapidly genotype different field strains of IBDV and should prove to be a useful tool in epidemiologic studies.     

Isolation of infectious bursal disease virus from turkeys with arthritic and respiratory symptoms in commercial farms in Quebec.

Infectious bursal disease viruses (IBDVs) were isolated from turkeys showing symptoms of arthritis and respiratory disease in commercial poultry farms in the province of Quebec, Canada. Synovial fluids collected from hock joints of arthritic birds and peripheral blood leukocytes obtained from the birds with respiratory problems were used for virus isolation in embryonated chicken eggs, and Vero and BGM-70 cell cultures. The infected cells were evaluated for the presence of IBDV by indirect immunofluorescence assay using monoclonal antibodies. The viruses were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of viral genome and by electron microscopy. Although one of these turkey isolates tested was neutralized by serotype 1-specific commercial chicken antisera, preliminary results indicated that there are antigenic differences between the Quebec isolate, IBDV QT-1, and the existing strains of IBDV belonging to serotype 1.     

Lack of pathogenicity of five serotype 2 infectious bursal disease viruses in chickens

The pathogenic potential of five strains of serotype 2 infectious bursal disease virus (IBDV) for specific-pathogen-free chickens was examined. There were no gross or microscopic lesions in the inoculated chickens. Bursa-to-body-weight ratios of IBDV-infected chickens were not significantly different from those of uninfected controls. Virus-neutralizing antibodies to IBDV of serotype 2, but not serotype 1, were detected in infected chickens. This study indicated that the serotype 2 viruses examined were infectious but not pathogenic in chickens.     

In ovo vaccination of specific-pathogen-free chickens with vaccines containing multiple agents.

We used in ovo technology to protect chickens against multiple diseases by inoculating vaccines containing mixtures of live viral agents. A single in ovo injection of a vaccine containing serotypes 1, 2, and 3 of Marek's disease virus (MDV), a vaccine strain of serotype 1 infectious bursal disease virus (IBDV), and recombinant fowl pox vaccine with HN and F genes of Newcastle disease virus (rFP-NDV) induced protection against virulent MDV, IBDV, Newcastle disease virus, and fowl poxvirus. The multiple-agent vaccine induced specific antibodies against the viral agents present in the mixture and did not adversely affect the survival of hatched chickens. Inoculation of a vaccine containing serotypes 1, 2, and 3 of MDV and IBDV did not affect hatchability of eggs, although the addition of rFP-NDV to the mixture reduced hatchability by 23%-26%. In ovo vaccination with a vaccine containing MDV and IBDV vaccine viruses did not exacerbate the inhibitory effect of individual viral agents on humoral and cellular immune competence.     

Differentiation of infectious bursal disease viruses directly from infected tissues with neutralizing monoclonal antibodies: evidence of a major antigenic shift in recent field isolates

Two neutralizing monoclonal antibodies (MCAs), R63 and B69, were used in antigen-capture enzyme immunoassays to verify the presence of infectious bursal disease virus (IBDV) in infected bursal tissues. The intra-serotype-common neutralization site defined by the R63 MCA was present on all IBDV isolates and laboratory strains tested. However, the neutralization site defined by the B69 MCA was found on only classic or older IBDV strains; it was not found on recently isolated variants of IBDV or on a majority of recent field viruses examined. The data suggest that a major antigenic shift in IBDV has occurred in the field and that this shift involves, at a minimum, the deletion or alteration of one of two neutralization sites previously found on classic IBDV strains.     

Differentiation of infectious bursal disease viruses by restriction enzyme analysis of RT-PCR amplified VP1 gene sequence

In order to differentiate infectious bursal disease virus (IBDV) isolates/strains, a quick method of RT-PCR followed by restriction enzyme analysis of VP1 gene sequence is being reported for the first time. A 480 bp fragment, comprising one of the RNA dependent RNA polymerase motifs of VP1 gene sequence of an Indian classical virus, an attenuated vaccine strain, Georgia and two Indian field isolates, genetically similar to reported very virulent strains of IBDV, was amplified by RT-PCR. Restriction enzyme digestion of PCR products with Taq1 enzyme generated distinct profile for field isolates, different from the classical and attenuated viruses, whereas restriction profile with BstNI restriction enzyme was similar in all the viruses, irrespective of the pathotype. Therefore, the present results suggest that Taq1 digestion can be taken up for the differentiation of field isolates from the classical and vaccine strains. The sequence analysis of VPI gene of reported very virulent IBD viruses from Europe and Japan, using 'MapDraw' programme of Lasergene software, revealed similar restriction enzyme profile as in Indian field isolates.     

Group and strain-specific neutralization sites of infectious bursal disease virus defined with monoclonal antibodies

Two somatic cell hybridizations were performed utilizing splenocytes from mice immunized with one or more strains of infectious bursal disease virus (IBDV). Supernatants from hybridoma cell lines were initially screened by the enzyme-linked immunosorbent assay (ELISA) against multiple strains of IBDV. Cell lines that secreted antibodies with ELISA reactivity patterns of interest were cloned, and their monoclonal antibodies (MCAs) were subsequently tested in cross-virus-neutralization tests. Two of the nine MCAs selected exhibited strong neutralizing activity and precipitated IBDV antigens in agar gel precipitin tests as well. MCA B69 significantly neutralized only the cloned D78 strain of IBDV, whereas MCA R63 neutralized all IBDV strains (representing both serotype I and II viruses) against which it was tested. Results of competitive ELISAs that used the R63 and B69 MCAs showed that the two neutralization sites on the D78 strain were not overlapping.