Fig 1.
Distribution of infected (green) and control (blue) cockatiels throughout timepoints.
Cockatiels that naturally died during the experiment are represented in orange.
Fig 2.
Clinical signs of cockatiels (Nymphicus hollandicus) infected with PaBV-2.
Lethargy and depression of CK19 at 35 dpi (A) and ingesta-stained feathers in the foreneck and breast of CK24 (60 dpi) after regurgitation (B).
Fig 3.
Comparison of proventriculus size (arrowheads) in a control (A) and infected (B) cockatiels of the same timepoint (35 dpi). Note the distention of the proventricular wall in CK19 when compared to a control cockatiel (CK15).
Table 1.
Summary of inflammatory lesions, PCR and IHC results observed in 12 sequential timepoints following days post infection.
Fig 4.
Sequence of histological findings and immunohistochemical labelling in tissues from infected cockatiels during early and late infection.
The first tissue to present lymphoplasmacytic inflammation and positive labelling was the site of inoculation, which showed marked lymphoplasmacytic myositis (A) and positive immunolabeling in the nuclei of inflammatory cells as well as in Schwann cells of intercostal and pectoral nerves (B) in early infection (CK2, 5 dpi) and reduced inflammatory infiltrates (C) and no positive immunolabeling (D) at late infection (CK34, 114 dpi). PaBV then reached the spinal cord through adjacent ganglia and nerves, which provoked minimal myelitis (E) and infection of few neurons (F) in the early timepoints (CK9, 25 dpi) and severe inflammation (G) and widespread viral distribution (H) in late infection (CK26, 80 dpi). When PaBV reached the brain (CK10, 25 dpi), minimal inflammation (I) and positive immunolabeling of few neurons (J) was observed, whereas severe inflammation (K) accompanied by widespread immunolabeling (L) was observed in chronically infected cockatiels (CK30, 100 dpi). Only after invasion of the CNS, PaBV was identified at 30 dpi (CK19) in the GI tract (N), initially with mild inflammation (M) that progressed to moderate to severe ganglioneuritis (O) and diffuse immunolabeling in ganglia, smooth muscle and few scattered epithelial cells (P) in the late infection timepoints (CK28, 80 dpi). Adrenalitis was only observed in small areas of the adrenal medulla (Q) accompanied by scattered PaBV positive cells (R) in the early infection (CK14, 30 dpi), however, adrenalitis and ganglioneuritis (S) were severe in late infection (CK25, 60 dpi) and followed by wide spread immunoimmunolabeling (T) affecting the adrenal medulla and adjacent ganglia. Finally, tissues such as skin had no inflammation (U) or immunolabeling (V) in the early infection timepoints (CK7, 20 dpi) but presented multifocal positive immunolabeling pattern (X) in late infection (CK34, 114 dpi), accompanied by mild to moderate inflammation (W).
Fig 5.
Chronologic infection pathway based on PaBV N-protein detection by IHC.
Positive immunolabeling after intramuscular inoculation (1) was first observed at 20 dpi in the spinal cord (2), followed by brain (3) at 25 dpi. Ganglia in the GI tract (4) and adrenal gland (5) were first positive at 30 dpi. Positive immunolabeling in the epicardial ganglia (6) was first observed at 35 dpi. Extra-neural tissues such as skin (7) only had positive immunolabeling at late infection timepoints, as early as 80 dpi.