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Background: Beak and feather disease virus (BFDV) is a circovirus that infects captive and wild psittacine birds, and is of conservation concern. The haemagglutination inhibition (HI) assay is used to determine antibody titres against BFDV, and the use of dried blood spots (DBS) on filter paper stored at room temperature has been suggested to be an equally valid technique to the use of frozen serum. However, research on other pathogens has found variable results when investigating the longevity of antibodies stored on DBS at room temperature. Consequently, we aimed to test the temporal stability of antibodies to BFDV in DBS samples stored long-term at room temperature. A further goal was to add to the current knowledge of antibody response to naturally acquired BFDV infection in crimson rosellas (Platycercus elegans). Methods: Blood was collected from wild P. elegans in Victoria, Australia, that had been live-trapped (n = 9) or necropsied (n = 11). BFDV virus load data were obtained from blood stored in ethanol by real-time quantitative PCR (qPCR); antibody titres were obtained by HI assay from either DBS or serum samples, which had been collected concurrently. All HI assays were performed commercially by the Veterinary Diagnostic Laboratory (VDL) in Charles Sturt University, Australia, who were blind to BFDV blood status. Results: HI titres from DBS stored at room temperature declined significantly over time (~80 weeks). By contrast, frozen serum samples assayed after 80 weeks in storage all had high HI titres, only varying up to one dilution step from the initial HI titres obtained from DBS at 3-6 weeks after sampling. Weak HI titres from DBS samples all came back negative when the test was repeated only nine weeks later. Novel high HI titres were reported in P. elegans, and while most birds with high antibody titres had corresponding negative qPCR results, a single subadult presented with high HI titres and virus load simultaneously. Conclusion: Detection of antibodies on filter paper stored at room temperature decreases over time, increasing the chances of false negatives in these samples, and in repeated testing of samples with weak HI titres. Consequently, serum should be the preferred sample type to use for seroepidemiological studies on BFDV in parrots and other bird species. When not possible, it may help to store DBS on filter paper at -20 °C or lower. However, prompt testing of DBS samples (e.g., <6 weeks in storage) is recommended pending further research on antibody temporal stability. We also show that P. elegans, especially adults, can produce high antibody titres against BFDV, which may help them resist infection. [ABSTRACT FROM AUTHOR]

Background Beak and feather disease virus (BFDV) is a circovirus that infects captive and wild psittacine birds, and is of conservation concern. The haemagglutination inhibition (HI) assay is used to determine antibody titres against BFDV, and the use of dried blood spots (DBS) on filter paper stored at room temperature has been suggested to be an equally valid technique to the use of frozen serum. However, research on other pathogens has found variable results when investigating the longevity of antibodies stored on DBS at room temperature. Consequently, we aimed to test the temporal stability of antibodies to BFDV in DBS samples stored long-term at room temperature. A further goal was to add to the current knowledge of antibody response to naturally acquired BFDV infection in crimson rosellas (Platycercus elegans). Methods Blood was collected from wild P. elegans in Victoria, Australia, that had been live-trapped (n = 9) or necropsied (n = 11). BFDV virus load data were obtained from blood stored in ethanol by real-time quantitative PCR (qPCR); antibody titres were obtained by HI assay from either DBS or serum samples, which had been collected concurrently. All HI assays were performed commercially by the Veterinary Diagnostic Laboratory (VDL) in Charles Sturt University, Australia, who were blind to BFDV blood status. Results HI titres from DBS stored at room temperature declined significantly over time (~80 weeks). By contrast, frozen serum samples assayed after 80 weeks in storage all had high HI titres, only varying up to one dilution step from the initial HI titres obtained from DBS at 3–6 weeks after sampling. Weak HI titres from DBS samples all came back negative when the test was repeated only nine weeks later. Novel high HI titres were reported in P. elegans, and while most birds with high antibody titres had corresponding negative qPCR results, a single subadult presented with high HI titres and virus load simultaneously. Conclusion Detection of antibodies on filter paper stored at room temperature decreases over time, increasing the chances of false negatives in these samples, and in repeated testing of samples with weak HI titres. Consequently, serum should be the preferred sample type to use for seroepidemiological studies on BFDV in parrots and other bird species. When not possible, it may help to store DBS on filter paper at −20 °C or lower. However, prompt testing of DBS samples (e.g.,

Indirect transmission of pathogens can pose major risks to wildlife, yet the presence and persistence of wildlife pathogens in the environment has been little studied. Beak and feather disease virus (BFDV) is of global conservation concern: it can infect all members of the Psittaciformes, one of the most threatened bird orders, with infection often being lethal. Indirect transmission of BFDV through contaminated nest hollows has been proposed as a major infection source. However, data on whether and for how long nest sites in the wild remain contaminated have been absent. We determined the BFDV status of birds (parents and nestlings) for 82 nests of Crimson Rosellas, Platycercus elegans and Eastern Rosellas, Platycercus eximius. In 11 of these nests (13.4%, 95% confidence interval 6.9–22.7), we found an infected parent or nestling. Using nest swabs, we then compared BFDV presence at three points in time (before, during and after breeding) in three groups of nest boxes. These were nest boxes occupied by infected birds, and two control groups (nest boxes occupied by uninfected birds, and unoccupied nest boxes). Detection of BFDV on nest swabs was strongly associated with the infection status of parents in each nest box and with the timing of breeding. During breeding, boxes occupied by BFDV-positive birds were significantly more likely to have BFDV-positive nest swabs than boxes occupied by BFDV-negative birds; nest swabs tested BFDV-positive in 80% (28.4–99.5) of nests with parental antigen excretion, 66.7% (9.4–99.2) of nests occupied by parents with BFDV-positive cloacal swabs and 66.7% (22.3–95.7) of nests occupied by parents with BFDV–positive blood. 0% (0–52.2) of nests with BFDV–positive nestlings had BFDV–positive nest swabs. Across all boxes occupied by BFDV-positive birds (parents or nestlings), no nest swabs were BFDV–positive before breeding, 36.4% (95% CI 10.9–69.2) were positive during breeding and 9.1% (0.2–41.3) remained positive after breeding. BFDV was present on nest swabs for up to 3.7 months. Our study provides novel insights into the potential role of nest cavities and other fomites in indirect transmission of BFDV, and possibly other pathogens, and offers a non-invasive method for surveillance of pathogens in wild bird populations.

Indirect transmission of pathogens can pose major risks to wildlife, yet the presence and persistence of wildlife pathogens in the environment has been little studied. Beak and feather disease virus (BFDV) is of global conservation concern: it can infect all members of the Psittaciformes, one of the most threatened bird orders, with infection often being lethal. Indirect transmission of BFDV through contaminated nest hollows has been proposed as a major infection source. However, data on whether and for how long nest sites in the wild remain contaminated have been absent. We determined the BFDV status of birds (parents and nestlings) for 82 nests of Crimson Rosellas, Platycercus elegans and Eastern Rosellas, Platycercus eximius. In 11 of these nests (13.4%, 95% confidence interval 6.9-22.7), we found an infected parent or nestling. Using nest swabs, we then compared BFDV presence at three points in time (before, during and after breeding) in three groups of nest boxes. These were nest boxes occupied by infected birds, and two control groups (nest boxes occupied by uninfected birds, and unoccupied nest boxes). Detection of BFDV on nest swabs was strongly associated with the infection status of parents in each nest box and with the timing of breeding. During breeding, boxes occupied by BFDV-positive birds were significantly more likely to have BFDV- positive nest swabs than boxes occupied by BFDV-negative birds; nest swabs tested BFDV-positive in 80% (28.4-99.5) of nests with parental antigen excretion, 66.7% (9.4-99.2) of nests occupied by parents with BFDV-positive cloacal swabs and 66.7% (22.3-95.7) of nests occupied by parents with BFDV-positive blood. 0% (0-52.2) of nests with BFDV-positive nestlings had BFDV-positive nest swabs. Across all boxes occupied by BFDV-positive birds (parents or nestlings), no nest swabs were BFDV- positive before breeding, 36.4% (95% CI 10.9-69.2) were positive during breeding and 9.1% (0.2-41.3) remained positive after breeding. BFDV was present on nest swabs for up to 3.7 months. Our study provides novel insights into the potential role of nest cavities and other fomites in indirect transmission of BFDV, and possibly other pathogens, and offers a non-invasive method for surveillance of pathogens in wild bird populations. [ABSTRACT FROM AUTHOR]