Nondomestic, Exotic, Wild and Zoo Animals–Original Article

Veterinary Pathology
2019, Vol. 56(1) 106-117
Distribution of Viral Antigen and ª The Author(s) 2018
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Inflammatory Lesions in the Central DOI: 10.1177/0300985818798112
journals.sagepub.com/home/vet
Nervous System of Cockatiels (Nymphicus
hollandicus) Experimentally Infected with
Parrot Bornavirus 2

Jeann Leal de Araujo1 , Aline Rodrigues-Hoffmann1 , Paula R. Giaretta1,

Jianhua Guo1, Jill Heatley2, Ian Tizard1, and Raquel R. Rech1

Abstract
Neurotropism is a striking characteristic of bornaviruses, including parrot bornavirus 2 (PaBV-2). Our study evaluated the dis-
tribution of inflammatory foci and viral nucleoprotein (N) antigen in the brain and spinal cord of 27 cockatiels (Nymphicus hol-
landicus) following experimental infection with PaBV-2 by injection into the pectoral muscle. Tissue samples were taken at 12
timepoints between 5 and 114 days post-inoculation (dpi). Each experimental group had approximately 3 cockatiels per group and
usually 1 negative control. Immunolabeling was first observed within the ventral horns of the thoracic spinal cord at 20 dpi and in
the brain (thalamic nuclei and hindbrain) at 25 dpi. Both inflammation and viral antigen were restricted to the central core of the
brain until 40 dpi. The virus then spread quickly at 60 dpi to both gray and white matter of all analyzed sections of the central
nervous system (CNS). Encephalitis was most severe in the thalamus and hindbrain, while myelitis was most prominent in the gray
matter and equally distributed in the cervical, thoracic, and lumbosacral spinal cord. Our results demonstrate a caudal to rostral
spread of virus in the CNS following experimental inoculation of PABV-2 into the pectoral muscle, with the presence of viral
antigen and inflammatory lesions first in the spinal cord and progressing to the brain.

Keywords
Psittaciform 1 orthobornavirus (PaBV), Avian bornavirus, cockatiel, Nymphicus hollandicus, central nervous system, viral distribu-
tion, encephalomyelitis

The family Bornaviridae of the order Mononegavirales
includes mammalian viruses including Borna disease viruses
(BoDV-1 and 2) and a novel zoonotic species transmitted
by variegated squirrels (variegated squirrel bornavirus 1;
VSBV-1)47 as well as viruses of reptiles and aquatic, psit-
tacine, and passerine birds.1 Parrot bornaviruses (PaBVs),
including Parrot bornavirus-2 (PaBV-2), are avian borna-
viruses that belong to the species Psittaciform 1 or 2 orthor-
bornavirus and are the causative agents of proventricular
dilatation disease (PDD), a lethal disease of captive psitta-
cine birds.2,25,31
Several aspects of the pathogenesis of PDD are still not well
understood, including its natural route of infection.26,33,43,53
Experimental inoculations using mixed routes of infection (oral,
oculonasal, subcutaneous, and intramuscular)15,37,41,46 or single
routes (intramuscular, intravenous, or intracerebral)18,42,43 were
able to induce clinical disease and characteristic lesions. How-
ever, histopathology and immunohistochemistry were imple-
mented to analyze late timepoints of infection when the
animals presented clinical signs or at the end of the experiments.
Therefore, crucial information about lesion distribution and viral
progression in the early timepoints was consequently missed.
Nevertheless, recent studies using only oculonasal or oral routes
of infection were not able to establish persistent infection of
PaBV-2 in experimentally inoculated cockatiels.21
Neurotropism is an important feature of mammalian borna-
viruses and has been extensively studied in natural and
1
Department of Veterinary Pathobiology, Texas A&M University, College
Station, TX, USA
2
Department of Small Animal Clinical Sciences, Texas A&M University, College
Station, TX, USA
Supplemental material for this article is available online.
Corresponding Author:
Jeann Leal de Araujo, Department of Veterinary Pathobiology, Texas A&M
University, 307C Manuel Drive, College Station, TX 77840, USA.
Email: lealjeann@gmail.com
de Araujo et al
experimental cases of Borna disease.20,34,48,55 BoDV-1 infec-
tion occurs in horses and sheep in central Europe49 and causes a
lymphoplasmacytic meningoencephalomyelitis with predilec-
tion for the gray matter. It affects primarily the hippocampus,
caudate nucleus, substantia nigra, midbrain, and hypothala-
mus.52,54 Eosinophilic intranuclear inclusion bodies, known
as Joest-Degen bodies, are a sporadic finding in the neurons
of animals affected by Borna disease and particularly promi-
nent in the hippocampus.4 Additionally, retinal degeneration
and lymphoplasmacytic optic neuritis have been reported as a
cause of blindness in horses infected with BoDV-1.5,14 BoDV-
2 was isolated from a horse with severe neurological disease in
Austria, and although it differs from BoDV-1 by more than
15% of nucleotide identity, the clinical and pathological pre-
sentation were identical to BoDV-1 infection.38
The distribution of inflammatory lesions of natural and
experimental bornaviral diseases in other mammals such as
variegated squirrels, humans, and immunocompetent rodents
are similar to those observed in horses and sheep.16,52 In con-
trast, neonatal rodents experimentally inoculated with BoDV-1
lack evidence of encephalitis but suffer significant develop-
mental injury in the central nervous system (CNS), particularly
in the hippocampus and cerebellum, including dentate gyrus
involution and the loss of cerebellar granular cells,
respectively.28,54,55
Neurons, glial, and ependymal cells are the main target cells
of the brains infected by both mammalian and avian borna-
viruses.8–11,58 Immunolabeling is present in the nuclei alone
or nuclei and cytoplasm of these cells, a reflection of the intra-
nuclear replication of bornaviruses,27 a characteristic shared
among only 3 other families of animal RNA viruses: the Ortho-
myxoviridae, Retroviridae, and Nyamiviridae.12,23,54
Although neurotropism is a prominent characteristic of
PaBVs, studies on the precise localization of inflammatory
lesions and viral distribution are limited and lack a systematic
approach.40,44 The aim of this study was to analyze the distri-
bution of inflammatory lesions and PaBV nucleoprotein anti-
gen localization in a chronologic manner to map the CNS areas
affected by this virus.
Material and Methods
Ethics Statement
All procedures in this study were conducted using protocols
approved by the Texas A&M Biosafety and Animal Use Com-
mittees (IACUC 20150-0045) and Institutional Biosafety Com-
mittee (IBC2015-021 and IBC2015-142) and meet all federal
requirements as defined in the Animal Welfare act (AWA),
Public Health Service Policy (PHS), and Humane Care and Use
of Laboratory Animals.
Viral Culture and Inoculum Preparation
Parrot bornavirus 2 (PaBV-2 CK1-34) was inoculated into duck
embryo fibroblasts (DEF, Schubot Exotic Birds health center
107
laboratory cell collection) cultured for 7 passages in minimum
essential medium (MEM) supplemented with 10% fetal bovine
serum (FBS, Gibco, ThermoFisher Scientific, Walthan, MA),
and subsequently maintained in MEM supplemented with 2%
FBS, as previously described.19 The cell debris were removed
by centrifugation at 3000  g for 10 minutes. Serial 10-fold
dilutions of 3 stocks of virus aliquots were analyzed by focus-
forming assays to determine viral titration. Titers above 8 
104 focus forming units per milliliter (FFU/ml) were consid-
ered acceptable for the experimental inoculation as previously
described.18,19
Experimental Animals, Virus Inoculation,
and Infection Timeline
Thirty-four cockatiels (Nymphicus hollandicus) originated
from 2 PaBV-negative aviaries were used for experimental
inoculations of PaBV-2 (CK1-34), as previously described.33
Briefly, cockatiels were divided into 12 groups that corre-
sponded to different euthanasia timepoints (5, 10, 20, 25, 30,
35, 40, 60, 80, 95, 100, and 114 days post-inoculation; dpi).
Each group had 3 experimental cockatiels, except for time-
points 5 dpi (2 cockatiels), 10 dpi (2 cockatiels), 25 dpi
(4 cockatiels), 35 dpi (5 cockatiels), 95 dpi (1 cockatiel), and
100 dpi (2 cockatiels), as described elsewhere.33 Twenty-seven
cockatiels were inoculated with PaBV-2 while 7 cockatiels
were inoculated only with PaBV-free DEF and served as neg-
ative controls. Negative controls were distributed in groups
correspondent to timepoints for 20 dpi, 25 dpi, 30 dpi, 35 dpi,
40 dpi, 60 dpi, and 114 dpi (1 negative control for each of these
timepoints). Cockatiels were inoculated using intramuscular
injection in the right pectoral muscle with 4  104 FFU of
virus. Any clinical signs observed during the experimental
period were documented. At the corresponding timepoints,
cockatiels were anesthetized with isoflurane (IsoThesia, Henry
Schein, Melville, NY) and then euthanized with carbon dioxide
(CO2). The results of the histological and immunohistochem-
ical analyses of other organs from the same experimentally
infected animals are reported elsewhere.33
Histology
Brain and spinal cord of each bird were placed in 4% neutral
formaldehyde for 48 hours. Coronal sections of the brain were
obtained from the cerebrum at the level of the hyperpalium and
nucleus basorostralis (Level I), cerebrum at the level of the
striatum (Level II), and cerebrum at the level of the arcopalium,
thalamus, and midbrain (Level III). Additionally, a sagittal
section was taken encompassing the hindbrain and cerebellum
(Level IV) (Supplemental Figure S1). Three transversal serial
samples from cervical spinal cord between C1 and C13, thor-
acic spinal cord between T1 and T7, lumbosacral spinal cord at
the level of the synsacrum, and the 5 caudal vertebrae (Supple-
mental Figure S2) were collected from all birds. Samples of
spinal cord were collected with intact vertebrae and decalci-
fied, which prevented the destruction of the adjacent spinal
108 Veterinary Pathology 56(1)

Figure 1. Brain, cockatiel. Schematic map of histological landmarks in the 4 selected brain levels (I, II, III, IV). Luxol-fast blue/periodic acid-Schiff
(PAS) stain. Abbreviations: AL, ansa lnticularis; AP, arcopallium; Bas, nucleus basorostralis; Cbl, nucleus cerebellaris intermedius; CbM, nucleus
cerebellaris intermedius; CP, comissura posterior; DBC, nucleus decussationis brachiorum conjunctivorum; DIVA, dorsalis intermedius pars
ventralis anterior; DLP, nucleus dorsolateralis posterior thalami; DMP, nucleus dorsomedialis posterior thalami; GCt, substantia grisea centralis;
HP, hyperpallium; La, nucleus laminaris; Mc, nucleus magnocellularis; NP, nidopallium; OS, nucleus olivaris superior; Ote, optic tectum; Ov,
nucleus ovoidalis; OVH, organum vasculosum of the hypothalamus; P, pallidum; PvT, paraventricular nuclei of the thalamus; Rt, nucleus rotundus;
S, nucleus solitarius; SL, nucleus septalis lateralis; SM, nucleus septalis medialis; SRt, nucleus subrotundus; St, striatum; VeD, nucleus vestibularis
descendens; VeL, nucleus vestibularis lateralis; VMH, ventromedial nucleus of hypothalamus.

ganglia during the trimming process. All samples were pro-
cessed routinely for histologic examination, and when neces-
sary, decalcification was performed using Formical-4
(American master tech, Lodi, CA) as recommended by the
manufacturer. The histological evaluation was done blindly
by a board-certified pathologist (R. R. Rech).
The neuroanatomic location of each inflammatory focus
was mapped in a diagram, and the description of the regions
of the brain was adapted for this purpose and followed what
has been suggested by the avian brain nomenclature consor-
tium,45 avian brain atlases,7,36 and avian medicine researchers
(Fig. 1).13,39,51 A single blood vessel surrounded by layers of
lymphocytes and plasma cells (lymphoplasmacytic perivascu-
lar cuffing) or a focus of inflammation in the neuroparenchyma
(glial nodule) was interpreted as a single inflammatory focus
(Figs. 2, 3) and indicated by a dot in the diagram. The severity
of inflammation was represented by 4 different dot sizes (min-
imal, mild, moderate, or severe), and each bird with
inflammatory lesions was represented by a different color.
Lesions were considered minimal when 1 single layer of peri-
vascular cuffing and no more than 1 glial nodule was in the
analyzed section, mild when vessels had 2 layers of perivascu-
lar cuffing and/or 2 to 3 glial nodules, moderate when vessels
had 3 layers of perivascular cuffing and/or 4 to 7 glial nodules,
and severe when vessels had 4 or more layers of perivascular
cuffing and/or more than 7 glial nodules. All sections were
analyzed separately for each bird and then overlapped to gen-
erate the final diagram using a graphic software (Photoshop
elements, Adobe systems, San Jose, CA). Lymphoplasmacytic
inflammation was scored as follows: – (absent), þ (minimal),
þþ (mild), þþþ (moderate), þþþþ (severe).
Immunohistochemistry
Immunohistochemistry was performed using polyclonal anti-
bodies against a specific region of the PaBV N-protein, as
de Araujo et al
Figures 2–3. Parrot bornavirus 2 infection, brain, cockatiel. Figure 2.
Lymphoplasmacytic perivascular cuffing. HE. Figure 3. Glial focus in the
gray matter. HE.
previously described,33 on serial tissue sections, mounted on
charged slides, and examined by light microscopy. Due to the
intranuclear replication of PaBVs, only cells with intranuclear
or intranuclear and intracytoplasmic immunolabeling were
considered positive. Cells with only intracytoplasmic immuno-
labeling were considered negative. Immunolabeling was scored
as previously described,33 and the progression for each CNS
section was recorded in schematic plates. Briefly, slides were
scored based on the number of N-protein positive cells in the
whole section of brain or spinal cord as follows: – (absent), þ
(1%–3% affected cells), þþ (4%–10% affected cells), þþþ
(11%–25% affected cells), and þþþþ (more than 25%
affected cells). The immunohistochemical evaluation was done
blindly by a board-certified pathologist (A. Rodrigues-
Hoffmann).
Results
Distribution of the Inflammatory Infiltrate
In the spinal cord, lesions were equally distributed among the 3
segments with wide distribution within ventral and dorsal horns
(Fig. 4). For infected birds, inflammation was more prominent
in the central core of the brain, mainly in the thalamus and
hindbrain (Fig. 5).
The progression and severity of the inflammation across
different timepoints in the examined CNS sections are summar-
ized in Table 1. The first spinal cord segment to develop
inflammatory foci was the thoracic spinal cord at 25 dpi.
Inflammation was predominant in the ventral horns accompa-
nied by ganglioneuritis until 35 dpi, when it started to spread to
the dorsal horns. Both cervical and lumbosacral segments of
the spinal cord first developed inflammation at 30 dpi, affecting
both dorsal and ventral horns as well as adjacent spinal ganglia.
All sections presented inflammation, primarily in the gray mat-
ter, throughout all timepoints until 114 dpi. Although less fre-
quent and severe than inflammation in the gray matter,
inflammatory foci in the white matter were first observed close
to nerve insertions of the ventral funiculus at 30 dpi, followed
by the dorsal funiculus at 80 dpi. Then white matter inflamma-
tion spread to ventral, dorsal, and lateral funiculi until 114 dpi.
Ganglioneuritis was observed at all timepoints from 25 to 114
dpi, with the following exceptions: 25 and 114 dpi in the
109
cervical segment, 95 and 100 dpi in the thoracic, and 40 dpi
in the lumbosacral spinal cord. Ganglioneuritis was more
severe and frequent in the lumbosacral spinal segment. Menin-
gitis in the spinal cord was only observed at 80, 95, 100, and
114 dpi, and it was also more frequent and severe in the lum-
bosacral segment. No inflammation was observed in the glyco-
gen body.
At 25 dpi, the first areas of the brain to present inflammation
were the hindbrain and the thalamus. More specifically, ence-
phalitis was more prominent in the gray matter and first spread-
ing through the nucleus magnocellularis, nucleus olivaris
superior, and nucleus laminaris in the hindbrain and through
the paraventricular nucleus of the thalamus. By 30 dpi, inflam-
mation had spread laterally through thalamic nuclei, including
the nucleus ovoidalis and nucleus rotundus, and dorsally to the
commisura posterior, which was the main region of the white
matter affected by inflammation. The nuclei septalis and scat-
tered areas in the cerebrum at the level of the striatum and
nidopallium were also affected at 30 dpi. At 35 dpi, inflamma-
tory foci reached the optic tectum of the midbrain. Inflamma-
tion in the hindbrain was consistently present in the areas
corresponding to the nucleus olivaris superior, nucleus solitar-
ius, nucleus laminaris, and nucleus decussationis. Only 5 had
inflammatory foci in the cerebellum, which only started at 40
dpi and were concentrated at the nucleus cerebellaris interme-
dius and nucleus cerebellaris internus. Inflammation reached
the most dorsal areas of the caudal cerebrum comprising the
mesopallium and hyperpallium at 40 dpi and cranial cerebrum
at 60 dpi affecting both gray and white matter. Hindbrain and
midbrain continuously presented inflammatory foci throughout
60, 80, 95, and 100, until 114 dpi. The cerebrum did not show
inflammation at 95 dpi. At 60, 80, 100, and 114 dpi, inflam-
matory foci were mainly concentrated in the mesopallium and
hyperpallium. Meningitis in the brain was only observed at 60,
100, and 114 dpi, and its severity ranged from minimal to mild.
Distribution of Viral Antigen
Immunolabeling was observed in the nuclei and/or nuclei and
cytoplasm of predominantly neurons, but glial and ependymal
cells were also intermittently positive (Figs. 6–8). Immunola-
beling was observed as early as 20 dpi unilaterally in the neu-
rons and glial cells of the ventral horn and adjacesnt ganglia of
the thoracic spinal cord and at 25 dpi in the cervical and lum-
bosacral segments. At 40 dpi, the immunolabeling spread to the
ependymal cells, adjacent neurons, and glial cells, including
cells in the dorsal horns in all 3 segments of the spinal cord.
Immunolabeling in the glial cells of the white matter was first
observed at 80 dpi. In all following timepoints (80, 95, 100, and
114 dpi), a diffuse labeling involving neurons, glial, and epen-
dymal cells in both gray and white matter as well as neurons of
the adjacent ganglia was observed (Fig. 9).
In the brain, the thalamic nuclei and the hindbrain were
simultaneously the first areas of the brain with PaBV immuno-
labeling at 25 dpi. From 25 to 30 dpi, scattered neurons of the
paraventricular nuclei of the thalamus and glial cells scattered
110
Figure 4. Distribution and severity of inflammation in the cervical
(top), thoracic (middle), and lumbosacral (bottom) spinal cord of
PABV-2-inoculated cockatiels. Animals with inflammatory lesions
are represented by different colored dots and numbers. Each dot
represents an inflammatory focus, and the 4 different dot sizes
represent different degrees of severity (minimal, mild, moderate,
and severe).
Veterinary Pathology 56(1)
throughout the thalamus were positive. At 30 dpi, few neurons
of the nucleus basorostralis and striatum in the cerebrum were
positive. At 35 dpi, the labeling rapidly spread throughout the
central core reaching the nuclei septalis in the subpallium and
neurons of the nuclei cereberallis and also in the granule cell
layer of the cerebellum. Ependymal cells were also positive at
35 dpi, particularly in the organum vasculosum of the hypotha-
lamus, surrounding the third ventricle. At 40 dpi, immunola-
beling was strongly positive in the pallial areas, particularly
prominent in the arcopallium and nidopallium. Also at 40 dpi,
PaBV-positive neurons and glial cells were identified in the
optic tectum and hyperpallium. The most widespread immuno-
labeling for hindbrain was first observed at 60 dpi. A diffuse
pattern affecting virtually all regions of the cerebrum was
observed from 60 to 114 dpi. A widespread pattern affecting
not only the cerebellar nuclei but also the white matter and the
Purkinje cells was first observed at 80 dpi and remained pos-
itive until 114 dpi (Fig. 10).
No inflammatory foci or antigen immunolabeling were
observed in uninfected control birds. Individual data of the
inflammatory infiltrates and viral antigen distribution for the
CNS of each one of the experimental cockatiels are available in
Supplemental Table S1.
Discussion
After 20 dpi, all infected birds experimentally inoculated with
PaBV-2 in the pectoral muscle developed a lymphoplasmacytic
myelitis or lymphoplasmacytic encephalitis or a combination
of both. This was associated with consistent intranuclear immu-
nolabeling of PaBV N protein antigen mainly in neurons, com-
patible with what has been described in natural cases of PDD.40
Although previous studies have been able to identify PaBV
N-protein antigen and inflammatory lesions in the CNS of
natural and experimental cases of PDD,15,18,41,42,43,46 the dis-
tribution of the inflammation and/or immunolabeling were
broadly determined with minimal neuroanatomic mapping.40,44
In this study, immunolabeling for PaBV N-protein preceded the
development of inflammation throughout all timepoints. The
ventral horns of the thoracic spinal cord were the first pos-
itive CNS areas at 20 dpi. The hindbrain and the thalamus
were the first areas of the brain to simultaneously present
viral antigen and inflammation at 25 dpi. The interval
between 25 and 60 dpi was crucial for the viral spread;
during this period, several areas in the gray and white mat-
ter were progressively and consistently affected, and both
inflammation and viral distribution were more concentrated
to the ventral areas of the brain.
PDD has been often described as a gastrointestinal disease,
and signs may include proventricular dilation, regurgitation,
crop impaction, anorexia, weight loss, and undigested seeds
in the feces.53 Experimental studies in cockatiels have shown
gastrointestinal signs prior to the development of neurological
signs,42,43 and ganglioneuritis has been reported as a more
de Araujo et al 111

Figure 5. Distribution and severity of inflammation in 4 selected levels (I, II, III, and IV) of the brain of PABV-2–inoculated cockatiels. Animals
with inflammatory lesions are represented by different colors. Each dot represents an inflammatory focus, and the 4 different dot sizes represent
different degrees of severity (minimal, mild, moderate, and severe).

consistent finding than meningoencephalomyelitis.15,42,43,46
However, it is important to note that PDD is primarily a neu-
rological disease, and gastrointestinal signs are in fact caused
by ganglia and nerve dysfunction.53 As previously demon-
strated,33 lesions in the CNS may develop prior to peripheral
lesions, although this does not necessarily mean that the
affected bird will have clinically evident neurological disease,
especially in the early timepoints of infection. In experimental
studies, the route of inoculation, tissues analyzed, and metho-
dology implemented must be considered to explain the discre-
pancy between the development of neurological and
gastrointestinal signs.
The anatomical localization of lesions in the CNS is a
crucial step for developing differential diagnoses of neuro-
logical diseases13 and consequently, the understanding of
the pathogenesis of neurotropic viruses. Lesions in different
anatomical regions of the CNS may lead to distinct neuro-
logical conditions,34 hence the importance of mapping the
main areas affected in neurological diseases such as PDD.
Although the correlation between affected areas and clinical
presentation of PaBV-infected birds has not been thoroughly
explored, the neuroanatomical localization of lesions in
rodents experimentally infected with BoDV has provided a
good understanding of the clinical presentation. In Lewis
112
Table 1. Progression of the inflammatory lesions and immunolabeling for parrot bornavirus-2 in the central nervous system of infected cockatiels across 12 different timepoints.*

5 dpi 10 dpi 15 dpi 20 dpi 25 dpi 30 dpi 35 dpi 40 dpi 60 dpi 80 dpi 95 dpi 100 dpi 114 dpi

Level IHC/INF IHC/INF IHC/INF IHC/INF IHC/INF IHC/INF IHC/INF IHC/INF IHC/INF IHC/INF IHC/INF IHC/INF IHC/INF

Brain - Level I -/- -/- -/- -/- -/- þ/- þ/- þ/- þþþ/þþþ þþþ/þþ þþ/þ þþþ/þþþþ þþþ/þþþþ
Brain - Level II -/- -/- -/- -/- -/- þ/þþþ þ/þþ þþ/þþþ þþþ/þþþ þþþ/þþþ þ/- þþþ/þþþþ þþþ/þþþþ
Brain - Level III -/- -/- -/- -/- þ/þ þþ/þþþ þþ/þþ þþ/þþþ þþþ/þþþ þþþ/þþþ þþþ/þþþ þþþþ/þþþþ þþþþ/þþþþ
Brain - Level IV -/- -/- -/- -/- þþ/þ þþ/þþþ þþþ/þþ þþ/þþþ þþþ/þþþ þþþ/þþþ þþþ/þþþ þþþþ/þþþþ þþþþ/þþþþ
Cervical spinal cord -/- -/- -/- -/- þþ/- þ/þþ þ/þþþ þþ/þþþ þþþ/þþ þþþ/þþþ þþþ/þþ þþþþ/þþþ þþþþ/þþþþ
Thoracic spinal cord -/- -/- -/- þ/- þþ/þ þ/þþþ þ/þþþ þþþ/þþþ þþþ/þþþ þþþ/þþþþ þþþ/þþþþ þþþþ/þþþþ þþþþ/þþþþ
Lumbosacral spinal -/- -/- -/- -/- þ/- þ/þþ þ/þþ þþ/þ þþ/þ þþþ/þþþ þþþ/þþ þþþ/þþþ þþþþ/þþþþ
cord
Abbreviations: -, absent; þ, minimal; þþ, mild; þþþ, moderate; þþþþ, severe. INF, inflammation score; IHC, immunolabeling score.
*The score of histopathology and immunohistochemistry for each timepoint was based on the average of the results of all birds in the respective timepoint. However, when only one bird presented inflammation or was
positive by IHC, its score was considered representative for the timepoint
de Araujo et al 113

Figures 6–8. Parrot bornavirus 2 infection, cerebrum, cockatiel, immunohistochemistry (IHC) for PaBV-2. Figure 6. Intranuclear and intra-
cytoplasmic immunolabeling for PaBV-2 nucleoprotein in neurons. Figure 7. Intranuclear immunolabeling for PaBV-2 nucleoprotein in glial cells.
Figure 8. Intranuclear immunolabeling for PaBV-2 nucleoprotein in ependymal cells.

Figure 9. Spinal cord, cockatiel. Progression of immunolabeling for PaBV-2 nucleoprotein in the thoracic spinal cord. Immunolabeling was
observed as early as 20 dpi unilaterally in few neurons and glial cells of the ventral horn and adjacent ganglia of the thoracic spinal cord. At 30 dpi,
immunolabeling was more intense and affecting more neurons and glial cells but still focally and unilaterally concentrated in the ventral horns.
Immunolabeling progressively involved all areas of white and grey matter by 80 dpi. IHC for PaBV-2.

rats, experimental infection of BoDV usually causes a wide-
spread lymphoplasmacytic meningoencephalitis with viral
antigen dissemination in all brain regions, which results in
a biphasic neurobehavioral disorder. However, when the rats
are infected with the BoDV-obese variant, inflammation and
viral antigen localization is restricted to the septum, hippo-
campus, amygdala, and ventromedial tuberal hypothalamus,
which results in a neuroendocrine disorder and consequent
obesity.22
The most common neurological signs seen in birds affected
by PDD correspond to both behavioral and motor disorders,
including lethargy, depression, ataxia, seizures, paresis, and
paralysis.26,53 In our study, the concentration of inflammatory
lesions in viscero-limbic components (eg, ventral striatum and
114 Veterinary Pathology 56(1)

Figure 10. Brain, cockatiel. Progression of immunolabeling for PaBV-2 nucleoprotein in 4 brain levels at 4 timepoints. Immunolabeling was
readily visible at 35 dpi in nuclei of the central core, which can be observed at levels III and IV of the brain. The immunolabeling in these areas
progressively spread throughout these sections until 114 dpi. Immunolabeling in sections I and II were more prominent only starting at 80 dpi
and remained widespread until 114 dpi. IHC for PaBV-2.

pallidum), associated nuclei (eg, nucleus dorsolateralis poster-
ior thalami, nucleus dorsomedialis posterior thalami), and
nuclei involved in reward and motivation (eg, paraventricular
nuclei of thalamus)32 are in agreement with the regions that are
usually affected by behavioral disorders that involve the limbic
system in mammals. Lesions in these areas are usually associ-
ated with lethargy, depression, obtundation, and stupor.13
In mammals, the widespread inflammatory lesions in the
cerebrum may explain signs such as seizures, while lesions in
the cerebellum might be associated with ataxia. Although not
well documented in birds, similar circumstances might explain
these clinical manifestations in PDD cases. Purkinje cell
degeneration and necrosis have been reported in some PDD
cases3,40 but were not observed in our study. Glial foci were
sporadically seen in the CNS of our experimental cockatiels,
which might have been associated with neuronal death at some
point of the course of the disease and therefore might play a
role in the pathogenesis of neurological manifestations com-
monly seen in PDD cases. The wide distribution of lesions in
the gray matter of the spinal cord seen in our study explain
clinical signs such as paresis and paralysis, sporadically seen in
PDD cases. Additionally, the high diversity of parrot borna-
viruses can potentially be associated with variable target cells
and lesions and consequently, distinct clinical presentations.42
de Araujo et al
In this study, the inflammatory lesions bear some similari-
ties to what has been described for mammalian bornaviruses.
Lymphoplasmacytic meningoencephalomyelitis with predilec-
tion for gray matter, including areas such as the thalamus and
hypothalamus, and relative sparing of the cerebellum and dor-
sal cerebrum was our main histological finding as reported for
horses, sheep, variegated squirrels, humans, and immunocom-
petent rodents.16,20,21,38,44,55 In contrast, the predilection for
olfactory bulb and hippocampus in mammals, a feature that
fits the olfactory route of infection proposed for BoDV patho-
genesis8 and follows a rostral-caudal progression of BoDV
infection, was not observed in our study.
The natural route of infection for PaBVs remains to be
elucidated. Experimental studies have been able to reproduce
clinical disease using different routes of inoculation, including
single intramuscular, intracerebral, or intravenous
routes,18,42,43 and also by combined intramuscular, oral, ocu-
lonasal, and subcutaneous routes.15,37,41 Nonetheless, experi-
ments using combined routes have nebulous results in regards
to which one of the routes was responsible for causing clinical
disease. Recently, experimental studies using oral and oculo-
nasal routes of infection have failed to induce disease and/or
PDD lesions in cockatiels after 6 months or experiment,21
which may indicate that these routes are unlikely to be associ-
ated with PaBVs’ natural route of transmission. Additionally,
skin injuries due to squirrel bites and scratches have been
reported by family members of the 3 patients who died with
variegated squirrel bornavirus-1 (VSBV-1)–induced encepha-
litis in Germany.24 The successful experiments using intramus-
cular routes of inoculation in psittacine birds and the recent
data about skin injuries in VSBV-1 cases reiterate the impor-
tance of investigating the association of tegument injuries and
intramuscular lesions as a port of entry for PaBVs. However, it
is important to note that although intramuscular inoculation of
PaBV-2 successfully caused PDD in cockatiels of our experi-
ment, there is still not enough data to support that this is the
route of infection in natural PDD cases.
A potential intramuscular route of infection (as was done in
our experiment33) creates the possibility of PaBVs being spread
first to the spinal cord and only reaching the rostral areas of the
late in the course of infection, resulting in a caudal-rostral
progression of infection and explaining the reduced inflamma-
tion and presence of viral N-protein antigen seen in the more
rostral areas during the late timepoints.
Although the hippocampus is associated with spatial mem-
ory in both birds and mammals, its localization and structure
differ enormously between these classes of animals.50 The
mammalian hippocampus is located rostral to the midbrain and
ventral to the thalamus, while the avian hippocampus has a
dorsal location in the pallial region of the cerebrum.29 This
area was not affected in the cockatiels of this study, probably
due the predilection of the virus for the central core of the brain
and consequent inflammation concentrated in the ventral areas
of the brain. Additionally, birds lack a distinguishable dentate
gyrus, which makes the progressive cell death of the granule
115
cells that is reported in the hippocampus of BoDV-infected rats
difficult to assess in birds.30,35
The pathognomonic feature of mammalian BoDV infection,
the eosinophilic intranuclear inclusion bodies known as Joest-
Degen bodies,4 were not observed in this study. Although these
inclusion bodies have been reported in few PDD cases,6,18,57
demonstration of the inclusion bodies in hematoxylin and
eosin–stained sections as observed for mammals56 has not been
consistent, and a careful analysis of the morphological and
staining characteristics of the inclusion bodies must be taken
in consideration to differentiate them from fragmented nucleoli
or artifacts. The immunohistochemical pattern observed in
samples from humans, squirrels, shrews, horses, sheep, and
rodents infected with mammalian bornaviruses17,24,56 has a
similar presentation to the immunolabeling in the nuclei, cyto-
plasm, and neuronal and glial processes noted in the CNS of
birds infected with PaBVs.40,44,58
Our results suggest that the common practice of collecting
and assessing only a single section of the brain could lead to a
false-negative diagnosis in PaBV-infected birds, especially in
the early timepoints of infection. We observed inflammation
in the hindbrain and midbrain from early to late timepoints
(25–114 dpi). On the other hand, the cerebrum at the level of
the hyperpalium remained as the least affected area until 100
dpi. Therefore, sampling limited to rostral areas of cerebrum
would be prone to miss the vast majority of inflammatory
lesions, which might result in the misleading assumption that
gastrointestinal ganglioneuritis was present prior to
meningoencephalomyelitis.
In conclusion, our results suggest that thalamus and hind-
brain were considered the most appropriate sections of the
brain for sampling to determine PaBV-2 infection spread and
inflammation. Little or no difference was observed between the
3 segments of the spinal cord in our study of experimental
PaBV-2 infection, based on distribution of viral antigen and
inflammatory lesions. Further research is needed to understand
whether a similar process happens in naturally infected birds
and elucidate the mode of viral spread from animal to animal as
well as more fully understand the physiologic and behavioral
effects of this temporal progression in the distribution of viral
antigen and inflammation. We advocate sampling the spinal
cord with the vertebral bodies in birds because this results in
outstanding visualization of the spinal ganglia and the decalci-
fication process does not interfere with viral antigen detection.
Acknowledgements
We acknowledge the excellent technical assistance provided by Debra
Turner, Luan Henker, Cintia Queiroz, Caitlin Older, Courtney Smith,
Anna Blick, Mackenzie Branco, Kelly Mallet, Brittany Kerr, Robin
Katchko, Randi Gold, Sierra Guidry, and Kristen Streeter.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
116
Funding
The author(s) disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: We thank
the Avian Health Complex, the Schubot Center, Texas A&M Univer-
sity, and the CAPES-Science without borders program for supporting
this study.
ORCID iD
Jeann Leal de Araujo http://orcid.org/0000-0001-8688-9864
Aline Rodrigues-Hoffmann http://orcid.org/0000-0001-6148-2531
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