Chicken recombinant antibodies specific for very virulent infectious bursal disease virus.

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 x 10(7) 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.

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.

Characterization of infectious bursal disease virus isolates from Indonesia indicates the existence of very virulent strains with unique genetic changes.

Sequencing of the hypervariable region of viral protein VP2 of infectious bursal disease virus (IBDV) isolates obtained from non-vaccinated chickens in Indonesia showed that the majority (16/17) were closely related to published very virulent (vv)IBDV strains. Four isolates contained identical amino acid sequences to Asian and European vvIBDVs, sharing vv-specific amino acid residues 222(Ala), 256(Ile), and 294(Ile). Eight isolates differed by one amino acid at position 222(Ala-->Ser); however, this change did not alter the pathogenicity or antigenicity of these strains. Two isolates, with amino acid substitutions at positions 272(Ile-->Thr) and 279(Asp-->Asn), did not cause clinical disease or mortality, and were therefore considered to be naturally occurring, attenuated mutants of vvIBDV. The results illustrate variability that might occur among vvIBDV strains.

Characterisation of an Indonesian very virulent strain of infectious bursal disease virus.

An Indonesian very virulent (vv) strain of infectious bursal disease virus (IBDV), designated Tasik94, was characterised both in vivo and at the molecular level. Inoculation of Tasik94 into 5-week-old specific-pathogen-free (SPF) chickens resulted in 100% morbidity and 45% mortality. The complete nucleotide and predicted amino acid sequences of genomic segments A and B were determined. Across each of the three deduced open reading frames (ORFs), Tasik94 shared the greatest nucleotide homology to Dutch vv strain D6948. Phylogenetic analyses were performed using 15 full-length polyprotein sequences and a total of 105 VP2 hypervariable region sequences from geographically and pathogenically diverse strains. In each case, Tasik94 grouped closely with vv strains, particularly those from Europe. The deduced VP1, VP2, VP3, VP4 and VP5 protein sequences of Tasik94 were aligned with those from published strains and putative virulence determinants were identified in VP2, VP3 and VP4. Alignment of additional protein sequences across the VP2 hypervariable region confirmed that residues Ile[242], Ile[256] and Ile[294] were highly-conserved amongst vv strains, and may account for their enhanced virulence.

Studies of the pathology of velogenic Newcastle disease: virus infection in non-immune and immune birds.

The pathology of velogenic viscerotropic Newcastle disease virus infection was compared in 7-and 20-week-old groups of non-immune birds and birds with two levels of immunity as determined by the haemagglutinin inhibition test. In non-immune birds the bursa at 7 and 20 weeks was the only lymphoreticular organ to show sustained reticular and lymphoid cell reactions until death took place. Caecal tonsil and spleen were extensively necrotized on day 4 after contact exposure, and similar changes occurred in lung and proventriculus. There was evidence of lymphoid recovery in birds which survived for 18 days. In immune birds the spleen showed two main responses. The first, acute reticular cell response around the ellipsoids indicated that renewed exposure to antigen was often associated with localized cell degeneration. The second, immunological, reaction was rapid formation of germinal centres which occurred somewhat earlier in 20-week-old birds (4-5 days). Especially from the second week, reticular (dendritic) cell and lymphoid hyperplasia occurred diffusely in the bursal medulla of both age groups although marked atrophy and cellular depletion, probably of physiological origin, was a feature of 20-week-old birds with high antibody levels. In the gastro-intestinal tract, germinal centre formation was most marked in the caecal tonsils at 20 weeks. With the Indonesian ITA strain of ND virus, degenerative and inflammatory changes in the brain were mild in all groups up to day 18 irrespective of immune status.

The pathogenesis of velogenic Newcastle disease virus infection of chickens of different ages and different levels of immunity.

Chickens of 7 weeks or 20 weeks of age were divided into three groups according to their antibody status (high, low, absent) and were infected with a velogenic viscerotropic Newcastle disease virus. To follow patterns of viral replication, birds were necropsied at regular intervals up to 22 days and organs were sampled from each bird. In non-immune birds, virus could be isolated from all organs examined. In birds with antibody, virus was most frequently isolated from the proventriculus, cecal tonsil, bursa, and brain. However, because no one organ could be recommended for all situations, all four should be sampled for field diagnosis. In immune birds, although clinical signs were either mild or absent, widespread virus replication occurred up to 19 days post-challenge.