From independent sources we obtained 8 parrots that had clinical signs of PDD, were clinically judged to be in the late stages of the disease, and were euthanized for humane reasons. The 8 birds were 1 green-winged macaw (Ara chloroptera), 1 scarlet macaw (A. macao), 2 blue and yellow macaws (A. ararauna), 2 yellow-collared macaws (Primolius auricollis), 1 African gray parrot (Psittacus erithracus), and 1 umbrella cockatoo (Cacatua alba). Four parrots with conditions not related to PDD and euthanized for humane reasons were also included in the study as negative controls.

Immediately after euthanasia, complete necropsies were performed on all birds. Tissue samples from brain, spinal cord, peripheral nerves, lungs, heart, liver, spleen, pancreas, adrenal glands, kidneys, crop, proventriculus, ventriculus, intestine, and cloaca were placed in 10% buffered formalin for histopathologic examination. Tissue sections were stained with hematoxylin and eosin to confirm the clinical diagnosis. Half of each brain was retained for virus isolation, Western blot, and reverse transcription–PCR (RT-PCR).

Specific pathogen–free duck eggs were obtained from the US Department of Agriculture Avian Disease Laboratory (East Lansing, MI, USA). Embryos 9–10 days old were harvested, macerated, and cultured. Primary duck embryonic fibroblasts (DEFs) were used for virus isolation and propagation. DEFs were maintained in Leibowitz L15–McCoy 5A medium (LM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% bovine calf serum (Sigma-Aldrich) and 1% penicillin-streptomycin at 37°C in an atmosphere of 5% CO₂. DEFs were seeded to confluency 24 h before inoculation. DEFs not inoculated with tissue homogenate were maintained in parallel throughout the experiment.

Brain tissue was harvested immediately after euthanasia. Sections of the cerebrum and cerebellum were homogenized, minced, and then passed through an 18-gauge needle in LM complete medium. In instances where immediate culture inoculation was not possible, the brain tissue was frozen at –80°C within minutes of being harvested and was thawed in a 37°C water bath immediately before inoculation. One milliliter of the brain suspension was used to inject previously plated DEF monolayers that were then incubated for 24 h. The injected DEF cultures were then washed once with phosphate-buffered saline (PBS), replaced with fresh LM medium supplemented with 2% fetal calf serum, and incubated for 5–7 days. Infected DEFs were trypsinized and cocultivated with freshly plated DEFs. This procedure was repeated for a minimum of 3 passages.

Infected DEFs passaged a minimum of 3 times were used for Western blot analyses. Samples from infected DEFs were collected by trypsinization and pelleted by centrifugation, and pellets resuspended in PBS were sonicated on ice at 50% intensity for 5 min (Sonifier 250; Branson Ultrasonics Corp, Danbury, CT, USA); 50% intensity). Western blotting was performed as described by Towbin et al. (16) by using 10% polyacrylamide gels and a Mini-Protean II gel electrophoresis apparatus (Bio-Rad, Hercules, CA, USA). The tissue culture preparations were diluted in sample loading buffer containing β-mercaptoethanol at a ratio of 1:1 and heated to 95°C for 5 min before being loaded (30 µg/slot). A prestained sodium dodecyl sulfate–polyacrylamide gel electrophoresis standard covering the 6.5- to 200-kDa range was used for molecular weight estimation (Bio-Rad).

The size-fractionated antigen preparations were transferred to Immobilon polyvinylidene fluoride transfer membranes (Millipore, Bedford, MA, USA) as described by Towbin et al. (16). Transfer efficiency was indicated by the presence of prestained bands on the membranes. After transfer, the membranes were incubated for 2 h in PBS, 0.05% Tween-20, 3% skimmed milk (PBST blocking buffer), then with histopathologically confirmed PDD-positive parrot serum diluted 1:5,000 in PBST blocking buffer for 2 h and with horseradish peroxidase–conjugated goat anti-macaw immunoglobulin Y (Bethyl Inc., Montgomery, TX, USA) diluted 1:10,000 in PBST blocking buffer for 1 h. Membranes were washed with PBST after each step, and all steps were performed at room temperature under constant shaking. Finally, the membranes were incubated for 30 min in Sigma-Fast 3,3′-diaminobenzidine developing substrate (Sigma-Aldrich) and then rinsed in distilled water. The serum from a confirmed PDD-positive parrot used in this experiment has been shown to contain antibodies specific for the 38-kDa ABV N-protein by its reaction with 2 preparations of recombinant protein prepared in Escherichia coli and in mammalian cell systems (11).

Infected DEF were washed 2 times for 5 min each in 0.02M PBS, fixed for 10 min in 2% paraformaldehyde in 0.02 M PBS, and washed 2 times for 5 min each in 0.02 M PBS. Cells were permeabilized in 1% Triton X-100/0.02 M PBS for 10 min and washed 3 times for 5 min each in 0.3% Tween/0.02 M PBS. Blocking was performed for 2 h in 5% dried milk/0.3% Tween/0.02 M PBS. The cells were incubated in a humidified chamber for 30 min at 37°C with the primary antibody (serum from a parrot with histopathologically confirmed PDD) at a 1:500 dilution in 1% dried milk/0.3% Tween/0.02 M PBS. Cells were washed 3 times for 5 min each in 0.03% Tween/0.02 M PBS. The cultures were then incubated in a humidified chamber for 30 min at 37°C with the secondary antibody (horseradish peroxidase– or fluorescein isothiocyanate–conjugated goat anti-macaw immunoglobulin G; Bethyl Inc.) at a 1:500 dilution in 1% dried milk/0.3% Tween/0.02M PBS. Cells were washed 3 times for 5 min each in 0.03% Tween/0.02M PBS and then rinsed in distilled water and mounted with ProLong antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA).

Total RNA was isolated from collected brain tissue and passaged DEF by using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). First-strand cDNA was generated by using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), with 1µg RNA and random primers. PCR for ABV N-protein was performed by using 1–2 µL cDNA and forward (5F: 5′-GCGGTAACAACCAACCAGCAA3-′) and reverse (1212R: 5′-GTTCATTAGTTTGCRAATCCRGTTA3-′) primers, which were developed using GenBank submissions NC_001607.1, FJ169441.1, and FJ169440.1 for reference. Amplification conditions were as follows: 1 cycle at 94°C for 2 min; 35 cycles at 94°C for 30 sec, 50°C for 30 sec, and 72°C for 80 sec; final extension at 72°C for 7 min. PCR products were cloned in TOPO-TA vector (Invitrogen), and individual clones were sequenced after transformation into E. coli. DNA sequencing reactions were performed by using the ABI BigDye Terminator Cycle Sequencing Kit, and sequences were generated with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Sequences were assembled and aligned by using Geneious Pro 4.6.2 software (www.geneious.com). Isolates were assigned to previously defined ABV groups by comparing a 397-nt region to sequences representing avian bornaviruses 1–5 (GenBank accession nos. FJ002329, FJ603688, FJ002328, FJ603687, FJ002335). Evolutionary distances were computed by using a Kimura 2-parameter model with MEGA4 software (www.megasoftware.net).

Experimental infections were performed under Animal Use Protocol no. 2009–033B approved by the Texas A&M University Institutional Animal Care and Use Committee. Three adult Patagonian conures were shown to be seronegative by Western blotting and to be ABV negative by fecal PCR. All 3 birds were known to be chronic carriers of psittacine herpes virus; 1 had a cloacal papilloma, but all were otherwise in good health. Psittacine herpes virus has never been implicated in PDD. Two birds were placed in isolation and inoculated by intramuscular injection with infected DEFs containing 8 × 10⁴ focus-forming units (17) of an ABV4 (M24) originally isolated from a yellow-collared macaw. A large batch of the M24 strain was grown for 5 days on passage 6, and 500-μL aliquots of this batch were frozen at –80°C in freezing medium. Two of the 500-μL aliquots were grown for 5 days, and immunohistostaining (by using the immunofluorescent antibody [IFA] assay described, substituting the fluorescein isothiocyanate–labeled antibodies with horseradish peroxidase–labeled antibodies) was used to visualize and quantify the focus-forming units.

Cytopathic effects were not observed in any of the 12 DEF cultures inoculated with brain tissue harvested from birds with or without clinical signs of PDD. Western blot analyses showed a pronounced ABV N-protein band in extracts of 8 of the 12 cultures. Only DEF cultures inoculated with samples from parrots displaying histopathologically confirmed PDD were positive by Western blotting (Figure 1). ABV N-protein was not detectable in the 4 cultures injected with brain tissue from birds with no histologic evidence of PDD. IFA of infected DEFs also demonstrated ABV N-protein within cells. Foci of antigen-positive cells were apparent 3 days after culture inoculation. Many cells showed both nuclear and diffuse cytoplasmic staining. Other cells showed the characteristic punctate nuclear staining of infected cultures (Figure 2). No positive immunofluorescence was observed in uninfected DEFs or in DEFs inoculated with brain tissue from negative control birds.

RNA was isolated from the brain tissues and infected DEF cultures of 8 parrots with PDD and 4 parrots that were PDD negative. A 397-bp region of the ABV N-gene was amplified from all 8 PDD brain and tissue culture samples but not those from the 4 negative parrots. The amplicons were cloned and their sequences compared with previously described ABV groups (7). One isolate, M25, was most closely related to ABV group 1, whereas the other 7 ABV isolates were most closely related to ABV group 4 (Table). Pairwise comparisons among the group 4 isolates ranged from 94.2% to 99.7% nucleotide identity. When 2–3 complete N-protein gene sequences (1,143 nt) originating from any bird were compared, nucleotide sequence identity ranged from 99.2% to 100% (data not shown).

Two Patagonian conures were challenged with ABV4, strain M24. They were tested by fecal PCR before challenge and at 33, 43, 60, and 62 days postchallenge. Both conures were seronegative by Western blotting before challenge but seropositive for antibodies to the 38-kDa N-protein on day 33 and thereafter. Fecal PCR testing showed that both birds were negative on days 33 and 43. One bird was weakly positive on day 60, but both were strongly positive on day 62. One inoculated bird died on day 66. Necropsy showed a dilated proventriculus and gross lesions characteristic of PDD. Subsequent histopathologic examination confirmed that the bird had a lymphoplasmacytic ganglioneuritis typical of PDD in the crop, proventriculus, gizzard, and intestine (Figure 3). This gangloneuritis included mild to severe infiltration of lymphocytes and a few plasma cells in the serosa, subserosal nerves, and ganglia. The bird also had adrenalitis, encephalitis, and neuritis, as well as a myocarditis. The heart showed a lymphocytic infiltration of the epicardial ganglia as well as in and around Purkinje fibers. Thus, the brain and spinal cord showed multifocal perivascular cuffing and gliosis (Figure 4). The adrenal medulla was infiltrated with lymphocytes and plasma cells.

The second inoculated bird was examined on day 66 and was found to be emaciated and had clinical signs consistent with PDD. It was euthanized for humane reasons. This bird also had gross and histopathologic lesions characteristic of PDD, essentially identical to those described above. The brains of both conures were subjected to PCR for ABV N-protein as described above. Results for both were positive (Figure 5). Sequence analysis of the PCR products confirmed that bird brains contained ABV4 identical to the M24 challenge strain. Brain homogenates from these 2 birds were also cultured on DEFs, and a strong positive PCR signal was obtained at day 16 of culture.

The third conure in this study received uninfected DEFs by both intramuscular and oral routes as described for experimentally infected birds. This bird was housed in an aviary separate from the isolation facilities of the infected birds. It was in apparent good health when euthanized on day 77. Necropsy of the bird conducted, including histopathologic examination of its tissues, and PCR was performed on 4 regions of its brain. No evidence of PDD was seen during necropsy or histopathologic examination, and all 4 brain samples were negative for ABV nucleic acid.