Avian bornavirus (ABV) is a newly recognized cause of neurological disease and mortality in free-ranging geese and swans in Ontario. To determine the correlation between clinical signs, pathological lesions and presence of ABV in tissues of wild waterfowl, 955 pathology cases from Canada geese (Branta canadensis), trumpeter swans (Cygnus buccinator) and mute swans (Cygnus olor) were reviewed, and 51 cases selected based on the presence of pathology or clinical history suggestive of ABV infection. The presence of virus in brains, assessed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) was highly correlated with the presence of non-suppurative inflammation in the central, peripheral and autonomic nervous systems. Partial sequencing of the ABV-nucleocapsid gene from infected geese indicated a unique waterfowl genotype. To estimate the prevalence of ABV infection in southern Ontario, cloacal swabs and blood samples were collected from 624 asymptomatic free-ranging waterfowl and evaluated using RT-PCR and an enzyme-linked immunosorbent assay, respectively. Thirteen percent of Canada geese caught on the Toronto Zoo site shed ABV in urofeces compared to none of the geese sampled at three other locations. The prevalences of ABV shedding in mute swans, trumpeter swans and mallard ducks (Anas platyrhynchos) were 9.3%, 0% and 0%, respectively. Serum antibodies were present, often at high prevalence, in birds from all four species and at each sampling site. To investigate the possibility of vertical transmission of ABV in wild Canada geese, 53 eggs were collected from an infected flock. ABV was detected in the yolk of one infertile egg. To determine whether poultry species were susceptible to infection with the waterfowl strain of ABV, domestic ducks, chickens and geese were inoculated with brain homogenate from ABV-infected Canada geese. ABV was not detected using immunohistochemistry and RT-PCR in any inoculated or control bird at 45 or 90 days post inoculation. No histological lesions consistent with ABV infection were found in any ducks and chickens; however, non-suppurative inflammation was present in nervous tissues of 5/13 inoculated geese, 5/13 control geese, and 3/8 geese euthanized prior to inoculation, suggesting the presence of a pre-existing, non-ABV neurotropic virus.