Development of a Dot-Blot Enzyme-Linked Immunosorbent Assay to Detect Avian Bornavirus Antibodies

Abstract

Avian bornavirus (ABV) is the causal agent of a progressive fatal avian neurologic syndrome referred to as Parrot bornavirus syndrome, a complex of clinical problems that can include enteric ganglioneuritis and encephalitis, or neurological deficits. The purpose of this dissertation is to develop a dot-blot enzyme-linked immunosorbent assay (ELISA) with good sensitivity and specificity and a run-time of ≤30 minutes, which could be utilized as a rapid patient-side diagnostic assay. The first objective in optimizing the dot-blot ELISA was to investigate the effect of species-specific secondary antibody in the test performance. Secondly, conditions for the dot-blot ELISA were adjusted to optimize test performance. This included investigating target antigen (recombinant nucleoprotein) concentration, sample dilution and type, and incubation times. Thirdly, the performance of the dot-blot ELISA was evaluated against the western blot, the gold standard for serologic testing of avian bornavirus. Finally, this research generated the first report of gross pathology, histopathological lesions, and viral tissue distribution in Monk parakeets following experimental inoculation with PaBV-2. The research concluded that dot-blot ELISA test characteristics were optimized using an anti-macaw IgY secondary antibody, 0.3 µg of target antigen and two 5-minute incubations and a sample of whole blood or plasma diluted 1:60. The assay had a sensitivity and specificity of 100 and 96% testing a sample population of PaBV-2 infected Monk parakeets at 3 weeks post-inoculation.