ABSTRACT
An indirect immunofluorescence serologic assay, PCR assay, and histopathology were used to screen for psittaciform orthobornaviruses (PaBV) in wild Cacatuidae in Victoria, Australia. Anti-PaBV antibodies were detected, but PCR and histopathology did not detect PaBV. This study presents the first evidence of PaBV in wild birds in Australia.
Psittaciform orthobornaviruses (PaBV) are enveloped RNA viruses widespread in captive psittacine birds. To date, eight genotypes have been detected in psittacine birds (Psittaciformes), with PaBV-2 and PaBV-4 the most common (Honkavuori et al. 2008; Kistler et al. 2008; Weissenböck et al. 2009; Rubbenstroth et al. 2012; Philadelpho et al. 2014). The mode of transmission of PaBV remains unclear; the virus is shed intermittently in both urine and feces, and has been detected in feathers and feather dander, but the fecal-oral route of infection requires cofactors such as epithelial damage or immunosuppression (Piepenbring et al. 2012; Heckmann et al. 2020). Vertical transmission is possible, but uncommon (Lierz et al. 2011). While many infections are subclinical, PaBVs are the etiologic agent of proventricular dilatation disease (PDD), a fatal neurologic disease. As a result, PaBVs have had major impacts on pet bird owners, aviculture, and captive breeding projects for avian conservation (Lierz et al. 2009; Heffels-Redmann et al. 2011; Piepenbring et al. 2012; Encinas-Nagel et al. 2014).
There is limited information regarding the prevalence of PaBV in wild birds, although it has been documented in free-ranging psittacines in Brazil (Encinas-Nagel et al. 2014). Reports of PDD and PaBV in Australia are rare, and all relate to imported, nonnative pet psittacines (Sullivan et al. 1997; Doneley et al. 2007). Previously there have been no reports of PDD or bornavirus infections in wild birds in Australia.
We aimed to determine if PaBV is present in wild cacatuids (Cacatuidae) in Victoria, Australia. Samples were collected from 55 wild Sulphur-crested Cockatoos (Cacatua galerita), Long-billed Corellas (Cacatua tenuirostris), Little Corellas (Cacatua sanguinea), and Galahs (Eolophus roseicapillus). The birds originated from four regions in rural Victoria (Ararat, 37°17′0″S, 142°55′0″E; Moriac, 38°14′0″S, 144°10′0″E; Kaniva, 36°22′0″S, 141°14′0″E; and Edenhope, 37°03′0″S, 141°18′0″E districts; n=44) or were wild birds originating from rural areas of greater Melbourne. The birds were sampled on admission to the Australian Wildlife Health Centre, Healesville Sanctuary, Healesville, and the Burwood Bird and Animal Hospital, Burwood, Victoria, Australia (n=11; Table 1).
Samples opportunistically collected from free-ranging Cacatuidae; Sulphur-crested Cockatoo (Cacatua galerita), Little Corella (C. sanguinea), Long-billed Corella (C. tenuirostris), and Galah (Eolophus roseicapillus) in Victoria, Australia. Sera were collected from live birds, choanal and cloacal swabs were collected from all birds, and brain samples from a limited number of dead birds. Samples were tested for Psittaciform orthobornavirus antibodies (sera) and antigen (swabs and brain tissue).

From each live bird (n=24), 0.5–1.0 mL of blood was obtained by jugular venipuncture and placed into a lithium heparin tube. The whole blood was spun down, serum extracted, and stored at –80 C. An indirect immunofluorescence assay (IIFA) was performed to detect antibodies against PaBV (Herzog et al. 2010).
A swab moistened in sterile phosphate-buffered saline was taken from the choana and cloaca of each bird, placed into RNA-later (Qiagen, Hilden, Germany), and stored at –80 C. Real-time PCR was used to detect PaBV RNA in the swabs (Kistler et al. 2008).
Brain tissue was obtained opportunistically from six birds. In each case, one section of brain was placed into RNA-later and stored at –80 C, with a second piece fixed in 10% buffered formalin. The brain was tested by PCR as it was for the swabs. The formalin-fixed brain tissues were embedded in paraffin, sectioned at 4 µm, and stained with H&E.
One of 24 birds was serologically positive (prevalence 4%; 95% confidence interval 3.36–4.96). This Little Corella, originating from suburban Melbourne (Nunawading, 37°49′1.2″S, 145°10′37.2″E), which presented with nonspecific signs of illness, had a positive specific antibody titer of 1:640; it also tested positive for Chlamydia psittaci, an adenovirus, an alphaherpesvirus, and beak and feather disease virus (Sutherland et al. 2019). Psittaciform bornavirus RNA was not detected in any of the choanal/cloacal swabs (0/55) or brain samples (0/6). Histologic lesions characteristic of PaBV infections were not detected in the brain sections (0/6).
Detecting birds with subclinical PaBV infection is challenging because infected birds may or may not be seropositive or shedding virus, and in some instances are neither seropositive nor shedding virus (Lierz et al. 2009; Herzog et al. 2010; Villanueva et al. 2010; Heffels-Redmann et al. 2011). Additionally, available PCR assays do not detect the sequences of all PaBVs. To increase the probability of detecting evidence of known or novel PaBV infections, we used both PCR and serologic assays. Serology is considered an important tool, as the IIFA uses whole cells infected with the virus, therefore providing an array of antigens that detect all known PaBVs that would potentially cross-react with antibodies of novel bornaviruses (Herzog et al. 2010). While it is possible that the serologic assay detected a serologically cross-reactive virus, not PaBV, this is unlikely as the IIFA used is highly specific (Herzog et al. 2010). Our prevalence of infection was low compared to previous studies. In a study of captive birds in Europe, overall seroprevalence was 17%; the prevalence of infection increased to 20.7% when birds were screened using both serology and a PCR assay (Heffels-Redmann et al. 2011). Infection prevalence varied from 4.5–40%, depending on species, but Cacatua spp. and Eolophus roseicapilla had infection rates of 40% and 20%, respectively (Heffels-Redmann et al. 2011). A study of free-ranging Brazilian parrots detected PaBV RNA in 30.2% of birds tested by PCR assays of cloacal swabs or tissue, although only 5% were seropositive (Encinas-Nagel et al. 2014). Failure to detect PaBV in choanal and cloacal swabs in our study could result from a low prevalence in the sampled bird population or a failure of the PCR assay to detect a novel PaBV variant or PaBV-like virus.
Our study has detected the first evidence of PaBV in a wild psittacine bird in Australia. Whether this infection represents a spill-over event from PaBV-infected captive birds in Australia infected with a known and probably introduced PaBV genotype (Vall-Llosera and Cassey 2017), or a novel bornavirus or borna-like virus that is enzootic in native Australian birds, is unknown. Captive birds commonly escape into the wild (Vall-Llosera and Cassey 2017) and are therefore potential sources of PaBV infection to wild birds. Also, large numbers of wild birds are rehabilitated in Australia every year and released back to the wild; many wildlife rehabilitators have pet birds, which provide another potential opportunity for PaBV spread to wild bird populations. Whatever the source of the PaBV infection in this Little Corella, given the potential significance of the introduction of PaBV into native psittacines, additional screening is indicated.
Samples tested in this study were collected under the Department of Economic Development Jobs Transport and Resources (DEDJTR) Victoria Scientific Procedures Fieldwork Licence 01180. This study was approved by the Wildlife and Small Institutions Ethics Committee (DEDJTR, Victoria) under application 01.15. The authors thank the Association of Avian Veterinarians Australasian Committee Research Fund for providing financial assistance in support of this project and the staff at the Australian Wildlife Health Centre, Healesville Sanctuary for their assistance with sample collection.