Objectives: Falconry, the use of birds of prey to hunt wild quarry, has a long tradition in the Arabian region. During the last decades the Arabian falconers have shifted from species that have naturally occurred in the region to falcons that are adapted to colder climates as well as hybrid birds, of which the immunity can be impaired. As a result, aspergillosis, most commonly caused by Aspergillus fumigatus, has emerged as one of the main health problems in this group of birds. This disease in falcons may be associated with high mortality and difficulties in clinical and laboratory diagnosis. We previously cloned an immunogenic protein, Afmp1p, in A. fumigatus and showed that anti-Afmp1p antibodies were present in human patients with A. fumigatus infections, making Afmp1p potentially useful for serodiagnosis of human aspergillosis. In this study, we hypothesise that an Afmp1p-based enzyme-liked immunosorbent assay (ELISA) could also be applied for serodiagnosis of aspergillosis in falcons. The aim of this study was to develop an Afmp1p-based ELISA system for the serodiagnosis of falcon aspergillosis.

Methods: Total IgY was first purified from falcon sera and used to raise a polyclonal anti-falcon IgY antibody using a guinea pig model. This polyclonal antibody was then conjugated with horseradish peroxidase for use in subsequent ELISA. Next, laboratory falcons were experimentally infected with A. fumigatus intratracheally and had their blood drawn at different timepoints post-infection for the characterisation of any anti-Afmp1p antibody response developed. Western blotting was carried out to examine the presence of any immunoreaction between Afmp1p and the sera of the experimental falcons before and after infection. An Afmp1p-based ELISA was then developed to characterise the change in anti-Afmp1p antibody profiles of the experimentally infected falcons throughout the infection course. The performance of this Afmp1p-based ELISA was evaluated using serum samples from 129 apparently healthy falcons and 12 falcons diagnosed with aspergillosis by endoscopy taken during routine annual health checks.

Results: A guinea pig polyclonal antibody against falcon IgY was generated, and its titre (as half maximal effective concentration [EC50]) and detection limit were 1:7,332 and 2,538 pg/mL, respectively. This polyclonal antibody against falcon IgY possessed a high specificity to falcons and did not cross-react with the IgYs of other bird species, including chicken, duck and goose. All four laboratory falcons experimentally infected with A. fumigatus through the intratracheal route developed culture- and histology-proven invasive aspergillosis. Anti-Afmp1p antibodies were first detected in their sera from day 12–50 post-infection. For the Afmp1p-based ELISA, the mean±SD absorbance at 450 nm using sera from 129 apparently healthy falcons was 0.186±0.073. Receiver operating characteristics curve analysis showed an absorbance cut-off value of 0.407. Using this cut-off, one negative serum gave an absorbance outside the normal range, giving a specificity of 99.2%. For the 12 field sera from falcons with confirmed aspergillosis, nine gave absorbance values equal to or above the cut-off, giving a sensitivity of 75%. 

Conclusion: The Afmp1p-based ELISA is useful for serodiagnosis of falcons with aspergillosis in veterinary microbiology laboratories, complementing endoscopy, fungal culture and histology.

Serodiagnosis of aspergillosis in falcons (Falco spp.) by Afmp1p-based enzyme-linked immunosorbent assay