Antigen tissue distribution of Avian bornavirus (ABV) in psittacine birds with natural spontaneous proventricular dilatation disease and ABV genotype 1 infection

Abstract

Tissues of 10 psittacines from aviary 1 ("case birds") and 5 psittacines from different aviaries were investigated for the presence of Avian bornavirus (ABV) antigen by immunohistochemistry using a polyclonal serum specific for the viral nucleocapsid (N) protein. Seven of 10 case birds had clinical signs, and necropsy findings consistent with proventricular dilatation disease (PDD) while 3 case birds and the 5 birds from other aviaries did not exhibit signs and lesions of this disease. In birds with clinical signs of PDD, ABV antigen was largely limited to neuroectodermal cells including neurons, astroglia, and ependymal cells of the central nervous system, neurons of the peripheral nervous system, and adrenal cells. ABV antigen was present in the nuclei and cytoplasm of infected cells. In 2 case birds that lacked signs and lesions of PDD, viral antigen had a more widespread distribution and was present in nuclei and cytoplasm of epithelial cells of the alimentary and urogenital tract, retina, heart, skeletal muscle, and skin in addition to the mentioned neuroectodermal cells. ABV RNA was identified by reverse transcription polymerase chain reaction (RT-PCR) in tissues of all 7 case birds available for testing from aviary 1, including 4 birds with PDD lesions and the 3 birds without PDD lesions. Sequencing and phylogenetic analysis indicated the presence of ABV genotype 1 in all cases. Findings further substantiate a role of ABV in PDD of psittacine bird species.

Figure 1

Phylogenetic tree showing the relationship of the Avian bornavirus (ABV) strain identified in the current study (bird 8) to previously published ABV sequences (with their respective GenBank accession numbers). Partial nucleoprotein sequence was aligned using ClustalW, and phylogenetic relationships were deduced by neighbor joining analysis using MEGA version 4.0.2, applying a Jukes-Cantor model, and performing 1,000 pseudo-replicate analyses. Bootstrap values are given at the respective nodes; scale bar indicates number of nucleotide substitutions per side.

Figure 2

Photomicrographs of the immunoreactivity for Avian bornavirus (ABV) antigen in psittacine birds with clinical signs and lesions of proventricular dilatation disease. Envision system horseradish peroxidase: nucleocapsid protein of ABV; polyclonal antiserum. A, cerebrum, cockatiel, bird 3: bilateral symmetrical immunoreactivity in the nuclei septalis. Bar = 1.0 mm. B, brainstem, cockatiel, bird 1: immunoreactive neurons. Note that the nucleus of some neurons is strongly immunoreactive (white arrow) while in another neuron the cytoplasm is immunoreactive (black arrow). In addition, the nuclei of astrocytes are immunoreactive (arrowheads). Bar = 50.0 μm. C, cerebellum, Nanday parakeet, bird 7: immunoreactive Bergmann glia. Note that the nuclei and processes of Bergman glia are immunoreactive (black arrows) but the Purkinje cells are negative for ABV antigen in this case (white arrow). Bar = 50.0 μm. D, cerebrum, Nanday parakeet, bird 7: immunoreactive glial cells. The processes and nuclei of glial cells in the neuropil are immunoreactive (black arrows). The cell process of 1 glial cell extends to a capillary (white arrow). Bar = 50.0 μm. E, cerebrum, cockatiel, bird 2: ependymal cells (black arrows) and subependymal glial cells (white arrows) are immunoreactive. Bar = 50.0 μm. F, crop, blue-crowned parakeet, bird 6: nuclei and cytoplasm of neurons of a subserosal ganglion (black arrows) and smooth muscle cells of the crop muscularis layer (m, white arrows) are strongly immunoreactive. The epithelial lining (e) of the crop is on the top of the picture. Bar = 100 μm.

Figure 3

Photomicrographs of the immunoreactivity for Avian bornavirus (ABV) antigen in 2 psittacine birds without clinical signs and lesions of proventricular dilatation disease. Envision system horseradish peroxidase: nucleocapsid protein of ABV; polyclonal antiserum. A, mesentericoceliac ganglion, salmon-crested cockatoo, bird 10: nuclei of numerous neurons (black arrow), cytoplasm of occasional neurons (white arrow), and cytoplasm and nuclei of numerous satellite cells are immunoreactive. Bar = 100 μm. B, heart, cockatiel, bird 5: cytoplasm and nuclei of cardiomyocytes is immunoreactive. Bar = 200 μm. C, kidney, cockatiel, bird 5: cytoplasm and nuclei of epithelial cells of proximal tubuli is immunoreactive (black arrow). Note an inflammatory “lymphofollicular” aggregate in the center of the pictures (arrowheads). Multiple cells within the aggregate (presumably macrophages) are immunoreactive (white arrow). Bar = 100 μm. D, pancreas, salmon-crested cockatoo, bird 10: nuclei of numerous acinar pancreatic cells (black arrows) and cytoplasm and nuclei of islet cells are immunoreactive (white arrows). Bar = 50 μm. E, retina, salmon-crested cockatoo, bird 10: photoreceptors (black arrow), nuclei of outer nuclear layer neurons (white arrow), inner nuclei layer neurons, neurons of the ganglion cell layer (white arrowhead), and nuclei of Müller cells are immunoreactive (black arrowhead). In addition, multiple photoreceptors are immunoreactive (black arrow). Bar = 100 μm. F, skin, salmon-crested cockatoo, bird 10: numerous epithelial cells of a feather follicle are immunoreactive. Bar = 200 μm.

Figure 4

Photomicrographs of the immunoreactivity for Avian bornavirus (ABV) antigen in an ABV RNA–negative eclectus parrot (case 11) without clinical signs and lesions of proventricular dilatation disease. Envision system horseradish peroxidase: nucleocapsid protein of ABV; polyclonal antiserum. A, pancreas: the cytoplasm of islet cells has a fine granular immunoreactivity (black arrows). Bar = 20.0 μm. B, proventriculus, the cytoplasm of few individual epithelial cells has immunoreactivity (black arrows). Bar = 50.0 μm.

Figure 5

A, photomicrographs of the immunoreactivity for Avian bornavirus (ABV) antigen in the mesencephalon (colliculus) of a cockatiel (bird 5) that was positive for ABV by polymerase chain reaction. Cytoplasm and nuclei of virtually all cells (neurons and astrocytes) in the photomicrograph are strongly positive. Bar = 50 μm. Inset: low magnification photomicrograph of the immunoreactivity for ABV antigen in the brain of the cockatiel depicting the location of the colliculus (*). C = cerebellum; BS = brainstem. Bar = 2 mm. B, transmission electron microscope micrograph, cockatiel, bird 5 (tissue from the colliculus was postfixed and processed): spherical 84–104 nm virus-like particles (arrowheads) were present in the cytoplasm of a neuronal cell process. mA = myelinated axon; M = mitochondrion. Bar = 500 nm.