Fatal Proventricular Dilatation Disease in Captive Native Psittacines in Brazil

Author(s): Rogério Venâncio Donatti , Maurício Resende , Francisco Carlos Ferreira Junior , Marcus
Vinícius Romero Marques , Roselene Ecco , H. L. Shivaprasad , José Sérgio de Resende , and Nelson
Rodrigo da Silva Martins
Source: Avian Diseases, 58(1):187-193. 2014.
Published By: American Association of Avian Pathologists
DOI: http://dx.doi.org/10.1637/10588-061013-Case.1
URL: http://www.bioone.org/doi/full/10.1637/10588-061013-Case.1

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AVIAN DISEASES 58:187–193, 2014

Case Report—
Fatal Proventricular Dilatation Disease in Captive Native Psittacines in Brazil
Rogério Venâncio Donatti,A Maurı́cio Resende,A Francisco Carlos Ferreira Junior,A Marcus Vinı́cius Romero Marques,A
Roselene Ecco,B H. L. Shivaprasad,C José Sérgio de Resende,A and Nelson Rodrigo da Silva MartinsAD
A
Departamento de Medicina Veterinária Preventiva, and
B
Departamento de Clı́nica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antônio Carlos,
6627, 30.123-970, Belo Horizonte, MG, Brasil
C
California Animal Health and Food Safety Laboratory System–Tulare, University of California, Davis, 18830 Road 112, Tulare, CA 93274
Received 19 June 2013; Accepted 16 October 2013; Published ahead of print 16 October 2013

SUMMARY. An outbreak of proventricular dilatation disease (PDD), a fatal inflammatory disease of psittacines (Aves:
Psittaciformes), is described in native Brazilian psittacines. Twenty captive psittacines that died of suspected PDD were necropsied
and 10 were submitted to histopathology, reverse transcriptase PCR (RT-PCR), and immunohistochemistry (IHC) for avian
bornavirus (ABV). Examined species were one pileated parrot (Pionopsitta pileata), three vinaceous-breasted parrots (Amazona
vinacea), two blue-winged macaws (Primolius maracana), one scarlet macaw (Ara macao), one chestnut-fronted macaw (Ara severa),
one scaly-headed parrot (Pionus maximiliani), and one red-browed Amazon parrot (Amazona rhodocorytha). Gross examination and
histopathology revealed typical PDD lesions in all birds. The presence of ABV was confirmed in four psittacines including one red-
browed Amazon parrot, one blue-winged macaw, one scarlet macaw, and one chestnut-fronted macaw. In the red-browed Amazon
parrot and in one blue-winged macaw, IHC demonstrated ABV antigens in the nucleus and cytoplasm of cells in various organs.
This is the first description of PDD by ABV in Brazilian psittacines and indicates the necessity for adopting a strategic control plan
for reducing its impact in native birds.
RESUMEN. Reporte de Caso—Enfermedad de dilatación proventricular fatal en psitácidos nativos en cautiverio en Brasil.
Un brote de la enfermedad de dilatación proventricular (con las siglas en inglés PDD), que es una enfermedad inflamatoria fatal
de los psitácidos (Aves : Psittaciformes ), se describe en psitácidos nativos de Brasil. Se practicó la necropsia de veinte psitácidos
cautivos que se sospechó murieron de la enfermedad de dilatación proventricular y diez fueron sometidos a estudio histopatológico,
por transcripción reversa y PCR, inmunohistoquı́mica (IHC) para bornavirus aviar (ABV). Las especies examinadas fueron uno
lorito carirrojo (Pionopsitta pileata), tres loros vinosos (Amazona vinacea), dos guacamayos maracaná (Primolius maracana), un
guacamayo macao (Ara macao), un guacamayo severo (Ara severa), un loro de Maximilian (Pionus maximiliani), y una amazona de
frente roja (Amazona rhodocorytha). El examen macroscópico y la histopatologı́a revelaron lesiones tı́picas la enfermedad de la
dilatación proventricular en todas las aves. La presencia del bornavirus aviar se confirmó en cuatro psitácidos como en la amazona
de frente roja, un guacamayo maracaná, el guacamayo macao y el guacamayo severo. En la amazona de frente roja y en un
guacamayo maracaná, la inmunohistoquı́mica demostró antı́genos del bornavirus aviar en el núcleo y el citoplasma de las células en
diversos órganos. Esta es la primera descripción de la enfermedad de dilatación proventricular por bornavirus aviar en psitácidos
brasileños y también indica la necesidad de adoptar un plan de control estratégico para la reducción de su impacto en las aves
nativas.
Key words: Amazona rhodocorytha, Ara macao, Ara severa, avian bornavirus, Brazil, immunohistochemistry, macaw,
proventricular dilatation disease, Primolius maracana, parrot, RT-PCR
Abbreviations: ABV 5 avian Bornavirus; BDV 5 bornavirus disease virus; DEPC 5 diethylpyrocarbonate; H&E 5 hematoxylin
and eosin; IHC 5 immunohistochemistry; M 5 matrix; N protein 5 nucleoprotein; PDD 5 proventricular dilatation disease; RT-
PCR 5 reverse transcriptase PCR; SPF 5 specific-pathogen-free

Proventricular dilatation disease (PDD) was first recognized in
psittacines in the late 1970s (16) and its occurrence has been
reported in various parts of the world including North America,
Europe, Australia, and South Africa (11,14,16,24). PDD is one of
the most common and often fatal diseases of psittacines. It has been
observed in more than 80 species of birds, including psittacine and
nonpsittacine species such as canary (Serinus canaria), Canada goose
(Branta canadensis), trumpeter swan (Cygnus buccinator), toucan
(Ramphastos toco), finches (Carduelis chloris), peregrine falcon (Falco
peregrinus), red-tailed hawk (Buteo jamaicensis), golden eagle (Aquila
chrysaetos), mouse bird (Colius spp.), honey creeper (Cyanerpes spp.),
D
Corresponding author. E-mail: nrsmart@gmail.com
long-wattled umbrellabird (Cephalopterus penduliger), bearded barbet
(Lybius dubius), and roseate spoonbill (Platalea ajaja) (11,14,16,24).
Clinical signs of PDD include anorexia, regurgitation, passing of
undigested seeds in the feces, diarrhea, lethargy, loss of weight, and
neurologic signs as well as sudden death (2,11,12,14). PDD is
characterized by dilation of the proventriculus, in most cases, with
associated microscopic changes such as lymphoplasmacytic gang-
lioneuritis involving the gastrointestinal tract, nonsuppurative
encephalomyelitis, peripheral neuritis, and ganglionitis, myocarditis,
adrenalitis, and lesions in the eye and skin (2,12,27,30,31,40). PDD
involves mainly the autonomic nervous system of the digestive tract
including the esophagus, crop, proventriculus, ventriculus, and
duodenum. The clinical signs are a result of gastrointestinal
dysfunction such as dysphagia, regurgitation, weight loss, and the
presence of undigested food in stools (12,27). Intracytoplasmic and

187
188 R. V. Donatti et al.

Table 1. PDD-affected psittacine species included in the avian bornavirus detection study.A
Scientific name (common name) No. sampled Conservation status Geographic range Estimated population Reference
Amazona rhodocorytha (red- 1 Endangered Atlantic forest fragments of 2295 (2004–2006) (3)
browed Amazon parrot) eastern Brazil
Amazona vinacea 3 Endangered Brazil (South and Southeast), 1500–2000 (Brazil) (4)
(vinaceous-breasted parrots) Paraguay Argentina
Ara macao (scarlet macaw) 1 Least concern From Mexico to the Amazon Brazil (Amazon Forest): (5)
forest 20,000–50,000
Ara severa (chestnut-fronted 1 Not assessed by IUCN From Panama to Peru Unknown; (6)
macaw) (Pacific coast), Brazil (Amazon
basin, Atlantic forest)
Pionopsitta pileata 1 Least concern South and Southeast Brazil Unknown (uncommon) (7)
(pileated parrot) (Atlantic forest), Paraguay,
Argentina
Pionus maximiliani 1 Least concern Brazil Northeast, Midwest, Unknown; fairly (8)
(scaly-headed parrot) Southeast, and South common
Primolius maracana 2 Near-threatened Brazil, Paraguay, Argentina Unknown; abrupt (9)
(blue-winged macaws) decline
A
Source: International Union for Conservation of Nature and Natural Resources (IUCN) Red List. (www.iucnredlist.org).

intranuclear eosinophilic inclusion bodies in nerve cells in the celiac
ganglion and myenteric plexus were described in parrots with
neuropathic gastric dilatation (2,25,30,31,40).
The etiology of PDD remained unknown until 2008, although
virus was suggested (13). Two independent studies described a novel
bornavirus from birds with PDD (18,23), which consists of an RNA
virus member of Bornaviridae. Avian bornavirus (ABV) shared only
65% nucleotide sequence with the known bornavirus disease virus
(BDV) of mammals (18,19,20,23,36). Based on nucleotide sequence
analysis of numerous ABV isolates from psittacines, five distinct
genotypes, designated ABV1, ABV2, ABV3, ABV4, and ABV5, have
been identified (23). A sixth ABV genotype has been described from
a canary with typical PDD lesions (39). ABV has also been detected
in clinically healthy parrots (25).
The diagnosis of PDD has relied on suggestive signs and lesions in
tissues by histopathology and immunohistochemistry, the confir-
matory tests for demonstrating ABV (11,24,25,30,38,39,40), and by
PCR (18,23,38). The objective of this report is to describe
pathologic and molecular findings of natural PDD cases in native
captive psittacines at a conservation center in Brazil.
CASE REPORT
Birds and history. The psittacine species included in this study
(Table 1) were one pileated parrot (Pionopsitta pileata), three
vinaceous-breasted parrots (Amazona vinacea), two blue-winged
macaws (Primolius maracana), one scarlet macaw (Ara macao), one
chestnut-fronted macaw (Ara severa), one scaly-headed parrot
(Pionus maximiliani), and one red-browed Amazon parrot (Amazona
rhodocorytha) (10). These birds represented the most-typical PDD
lesions from a total of 20 birds evaluated in a triage. Birds were from
one collection of an authorized conservation breeder in Minas Gerais
state, Brazil, originally confiscated by the environmental police and
after triage for rehabilitation in the Center for Triage of Wild
Animals–Belo Horizonte, Brazilian Institute of Environment and
Natural Renewable Resources (CETAS–BH, IBAMA).
All psittacines of the present report were maintained in captivity
in two large aviaries and managed with adequate food (balanced
parrot ration, fruits), potable water (chlorinated), and other needs
accordingly. The aviaries are well adapted for housing most common
species, such as the scaly-headed parrot, or when birds are not
intended for reproduction or are species known to reproduce
collectively, such as the red-browed Amazon. The conservation
facility housed 34 psittacine species with more than 400 individuals
in total. Dead birds were submitted to necropsy and preliminary
laboratory analysis at the Avian Diseases Laboratory of the
Veterinary College of the Universidade Federal de Minas Gerais
(UFMG).
Prostration, weight loss, and poor body condition were observed
in all birds. The birds in the study were submitted to the clinic for
special care such as heating and force feeding. The fatalities most
suggestive of PDD were necropsied and sampled for histopathology
and molecular analysis. Although all necropsied birds had a history
of prostration, weight loss, and food regurgitation, neurologic signs
were rare. Occurrences extended for 15 mo (February 2010 to May
2011) and did not appear to peak; but in contrast, appeared to cease
for a period of time, from April to November 2010, before resuming
the long outbreak. Subsequent clinical and pathologic follow-up
informed the re-emergence of PDD-like disease in the conservation
facility in 2013, including a new host species, golden parakeet
(Guaruba guarouba), and in blue and yellow macaw (Ara ararauna)
of another conservation unit, neither studied here.
Differential diagnosis. In order to investigate Newcastle disease
and influenza viruses, chicken embryo inoculation was performed in
9–11-day-old specific-pathogen-free (SPF) embryos via the allantoic
cavity with brain tissue from affected birds treated with antibiotics
and antimycotic (1000 units/ml penicillin G, 1 mg/ml streptomycin
sulfate and 2.5 mg/mL amphotericin B). Routine bacteriology was
performed on the liver for aerobic bacteria using McConkey and
blood agar.
Pathology. Necropsies were carried out in 20 birds to determine
cause of death and 10 birds with lesions typical for PDD were
selected for further studies. Portions of brain, brachial nerve, trachea,
lung, liver, spleen, crop, ventriculus, proventriculus, intestine,
kidney, ovary, testis, and adrenal gland were collected and conserved
frozen (280 C) or fixed in 10% neutral buffered formalin. Fixed
tissues were processed for histopathology by routine methods,
embedded in paraffin, sectioned at 5 mm, and stained with
hematoxylin and eosin.
Immunohistochemistry. Selected sections from paraffin-embedded
tissues were cut at three micrometers of thickness and deparaffinized.
Immunohistochemistry (IHC) using monoclonal antibodies against
the nucleoprotein (N protein) of ABV was performed on brain,
proventriculus, peripheral nerves, adrenal gland, liver, skeletal muscles,
 Proventricular dilatation disease in Brazilian psittacines
and testes based on a previously published protocol (38). Tissues were
selected from parrots (n 5 5) submitted to necropsy showing the most
typical gross lesions.
Molecular techniques. Total RNA of the PDD-affected
psittacines was extracted from frozen (280 C) brain tissue. Samples
were homogenized and RNA was isolated from 400 ml of lysed
sample using phenol-chloroform (TrizolH, Invitrogen, Carlsbad,
CA). Total RNA was subjected to reverse transcription (M-MLV
Reverse Transcriptase, Promega, Madison, WI) into cDNA. Primers
and reaction protocols were previously described (23,38) for RT-
PCR of ABV. Frozen brain tissue was evaluated according to a
protocol previously described (23) using primers ABV-F, GGR-
CAAGGTAATYGTYCCTGGATGGCC with genomic location
1905–1931, and ABV-R, CCAACACCAATGTTCCGAAGMGC
with genomic location 2242–2263, for amplifying a 360-bp sequence
within positions 1905–2263 of the matrix protein. The paraffin-
embedded tissue block of multiple 10-mm recuts from a red-browed
parrot were subjected to RNA extraction followed by ABV RT-PCR
using primers ABV F Weissenböck, CAAGGTAATYGTYCCTG-
GATGG with genomic location 1908–1929, and ABV-R Weissen-
böck, ACCAATGTTCCGAAGMCGAWAY with genomic location
2237–2259, with a 352-bp product (38). Sequencing of the putative
matrix (M) gene region was performed as previously described (35).
This primer pair would amplify a sequence which corresponds to
nucleotide positions 1908–2259 of the complete genome of ABV
strain ‘‘bil’’ (GenBank accession no. EU781967).
One microliter of diethylpyrocarbonate (DEPC) water was added
to the 2-ml samples, which were heated to 95 C for 2 min and then
1.5 ml of each primer was added. An additional 6 ml of DEPC water
were added for a total of 12 ml. Cycles for cDNA preparation were of
70 C for 5 min with an additional 5 ml of buffer, 1 ml of DNTP mix,
and 1.5 ml of M-MLV, and a final extension at 42 C for 5 min. For
each PCR reaction, 8.15 ml of DEPC water, 1.6 ml of MgCl2
(25mM), 2.0 ml DNTP mix (20 mM), 1.0 ml of forward and 1.0 ml
of reverse primer oligonucleotide, 0.25 ml GOTAC-FLEX (Pro-
mega), 4 ml 103 buffer, and 2.0 ml of cDNA were used. After
denaturation at 95 C for 2 min, the 30 cycles consisted of 95 C for
20 sec, 55 C for 30 sec, 72 C for 1 min, and a final extension at 72 C
for 5 min.
Following the routine laboratory diagnosis, further PCR investi-
gations on total DNA were performed according to previously
described protocols for beak and feather disease virus-BFDV (41)
and Chlamydophila psittaci (34).
Sequencing by the dideoxynucleotide method (35) was performed
in an automated capillary sequencer (ABI 310, Applied Biosystems,
Foster City, CA). PCR reactions with a BigDyeH Terminator v3.1
Cycle Sequencing Kit (Applied Biosystems) were performed
according to the manufacturer recommendation using 1 ml of PCR
product in a thermocycler (PTC-100, MJ Research Inc., Waltham,
MA). Products were purified with isopropanol and ethanol,
homogenized in formamide, denatured at 95 C for 2 min, and the
microtube was placed in flaked ice. The software employed was
Sequencing Analyses version 5.2 (Applied Biosystems).
Analyses of sequences. Electropherogram analyses of sense and
anti-sense sequences were performed with BioEdit software (15)
version 7.0.9 for evaluation of nucleotide bases quality. Sequences
were obtained at the National Center for Biotechnology Information
database (NCBI, http://www.ncbi.nlm.nih.gov) (28) and aligned
with GenBank sequences using Clustal W or X (37). The algorithms
for similarity of nucleotides or deduced amino acids were evaluated,
respectively, using BLAST 2.0 (Basic Local Alignment Search
Tool, BLASTn) or BLASTx (http://www.ncbi.nlm.nih.gov/BLAST/)
189
(1,28). Clustal W version 1.6 implemented in Molecular Evolutionary
Genetics Analysis (MEGA 5.0/ www.megasoftware.net) (36) version
5.0 for Windows was used for alignment of nucleotide or deduced
amino acid sequences.
The phylogenetic analyses of nucleotide and deduced amino acids
sequences were performed by neighbor-joining (MEGA 5.0 software).
Topology confidence tests (bootstrap) with 1000 oversampling were
performed using nucleotide substitution by Kimura 2 (22) or amino
acid JTT substitution (21).
RESULTS
All birds presented severe proventricular dilatation, with the
proventriculus filled with undigested food, compatible with PDD.
The first two birds (pileated parrot and two vinaceous-breasted
parrots) were studied from February 2010 to May 2011. Gross
changes were characterized by cachexia indicated by loss of pectoral
muscle mass (Fig. 1), crop distended with food, hepatomegaly,
splenomegaly, a proventriculus and ventriculus distended and with
undigested food (Fig. 2), lungs congested and edematous, intestinal
and brain congestion, enlarged adrenal glands, and air sac opacity. In
March 2010, one blue-winged macaw was received with similar
lesions except for a ventriculus with erosions and ulcers. By April
(2010), a similarly affected scarlet macaw (not shown) was received,
presenting all previously described lesions and hepatomegaly with
white-yellowish foci (1 cm), enlarged adrenal gland, and presenting a
high infection by adult nematodes of Trichuridae (possibly genus
Capillaria) and eggs in the small intestine. For a period of 6 mo
afterwards, no new cases were received from this breeder, who
informed us of no further deaths. However, disease and deaths re-
emerged by November 2010, with one vinaceous-breasted parrot
presenting all previously described lesions and opacity of air sacs. In
December 2010, one blue-winged macaw was received with similar
lesions and, after 11 days, an Amazon parrot was necropsied, also
with similar poor body condition and lesions. By May 2011, one
chestnut-fronted macaw and one scaly-headed parrot were examined,
both with the previously described lesions, with the proventriculus
distended and filled with a large quantity of undigested food,
hepatomegaly, splenomegaly, focal ulcerative areas on ventriculus
koilin, and small intestines with coccidian oocysts revealed by fresh
direct intestinal mucosa smears microscopy.
Attempts of virus isolation of the central nervous tissues were
negative for Newcastle disease virus and avian influenza, after three
passages with 7 days of incubation each, in 8–11-day-old SPF
chicken embryos inoculated via the allantoic membrane. Except for
one coliform isolate, no other relevant bacterium or fungi was
isolated from the sampled livers.
On histopathology, ganglion neuritis, neuritis, or both were
observed in most organs of the digestive tract and also in the brachial
and digestive peripheral nerves. These lesions are summarized in
Table 2. All birds presented mild or moderate infiltration of
lymphocytes and plasma cells in different organs. Three birds
showed thickened ventriculus serosa and moderate multifocal to
coalescing infiltration of lymphocytes and plasma cells in the nerves
and ganglia, with loss of neurons and axons (Fig. 3). In two birds the
inflammation was mild. The lymphocyte and plasma cell infiltra-
tions were also observed in the ventriculus muscular layer and
submucosa, associated with the nerves and perivascular space.
Similar lesions associated with nerves and ganglia were observed in
the crop and proventriculus (Fig. 4) of all birds. In one bird the
esophagus was also involved. Another three birds showed multifocal
lesions similar to those observed in the ventriculus. Focal or
190 R. V. Donatti et al.

Fig. 1. Amazona rhodocorytha in poor body condition, as evidenced by the pectoral muscle atrophy (insert). This is a large parrot (about 40 cm
total length), almost entirely green except for the forehead, ranging from red to orange and yellow between the beak and eyes.
Fig. 2. Amazona rhodocorytha. Proventriculus and ventriculus markedly distended. Insert: the lumen of both viscera can be seen filled with a
large quantity of undigested food.

multifocal erosions and ulcerations were occasionally observed on
the esophageal, crop, and proventricular mucosae. In the adrenal
gland (Fig. 5), there was mild to intense multifocal to coalescing
lymphocyte infiltration in the medullary regions, obscuring its
normal architecture. Three birds showed testes with mild multifocal
infiltration of lymphocytes and lack of spermatogenesis. In these
birds, an intense differentiation of lymphocytes to plasma cells was
observed in the spleen. In one bird, an infiltration of lymphocytes
and plasma cells was observed in the brachial nerve; this bird also
had several elongated cysts suggestive of Sarcocystis observed within
 Proventricular dilatation disease in Brazilian psittacines
Table 2. Histologic lesions of selected PDD-affected psittacines.
Bird BR PN ADR HRT
Primolius maracana ++ 2 + ++
Ara macao + 2 NE 2 ++
Amazona vinacea ++ ++ ++ NE
Primolius maracana + ++ ++ NE
Amazona rhodocorytha ++ + +++ NE
BR 5 brain; PN 5 peripheral nerves; ADR 5 adrenal gland; HRT 5 heart; PROV 5 proventriculus; VENTR 5 ventriculus; INT 5 intestine;
CR/ESO 5 crop/esophagus. Other organs: Lung, liver, spleen, kidney, gonads, or skeletal muscle. (+) mild; (++) moderate; (+++) marked; (2) no
lesions; (NE) not examined.
the skeletal muscle fibers. Lesions in the brain were absent or
minimal in most birds.
IHC revealed ABV antigen in various organs in two out of five
birds tested. The N protein-specific monoclonal antibody labeled the
nucleus and cytoplasm of neurons and glial cells throughout the
medullary cells of the adrenal gland (Fig. 6), in the axis cylinders of
the peripheral nerves, in the brain (Fig. 7), and in the myenteric
ganglion and glial cells of the proventriculus. The liver, skeletal
muscles, testis, and a few peripheral nerves were negative.
RT-PCR confirmed ABV-specific RNA in four out ten of
psittacines tested including one red-browed Amazon parrot, one
blue-winged macaw, one scarlet macaw, and one chestnut-fronted
macaw. In the red-browed Amazon parrot and in one blue-winged
macaw, IHC revealed ABV antigens in the nucleus and cytoplasm of
cells in various organs.
The ABV sequence (accession no. KF704677) recovered from a
red-browed Amazon parrot was submitted to BLAST search against
GenBank sequences, and confirmed as ABV genotype 4 (not shown),
being up to 91% similar to sequences described in psittacine
species from Israel and Canada, respectively (GenBank accession
numbers FJ002341, GQ496351, FJ0023331, FJ002330, FJ002340,
and FJ002342). Further studies are under way for obtaining ABV
sequences for phylogenetic analysis of local compared to worldwide
strains.
DISCUSSION
Clinical history and gross and histologic lesions were indicative of
PDD in all psittacines investigated. ABV-specific RNA was detected
by RT-PCR and antigens by IHC, indicating ABV’s involvement
with this condition. The extended outbreak was in agreement with
reports previously described (26,36). Similarly long outbreaks, in
crowded aviaries with birds affected during several weeks, have been
previously described (2,18,24,25,27,30,38).
PDD was preliminarily diagnosed based on clinical, pathologic,
and histopathologic findings in all 20 birds examined. In 10 birds,
additional IHC and PCR tests were implemented, confirming ABV
in four. ABV infection is possibly disseminated in the investigated
conservation facility, considering the proximity of aviaries. The
infection has been previously considered widespread in aviaries
(30,38), and possibly maintained by apparently healthy psittacines,
as detected in fecal swabs. Transmission was previously described to
occur by fecal-oral route in mallards (Anas platyrhynchos) and
determined that ABV PCR-positive, apparently healthy birds ranged
from 20% to 80% of flocks (24).
The apparent lack of detection of ABV by IHC in three out of five
birds, possibly false-negatives, may have resulted from denatured
ABV key antigens for IHC due to excessive cross-linking of proteins
(prolonged fixation in formaldehyde) or other etiologies for PDD-
like disease in our region (14,16). Other diseases which would cause
191
PROV VENTR INT CR/ESO Other organs
++ ++ + + 2
++ ++ ++ 2
++ + ++ + Testis +
++ ++ ++ NE 2
++ +++ + ++ 2
central and peripheral nervous system signs were discarded,
considering that central nervous system-inoculated embryos and
the bacteriology-mycology of livers did not result in relevant
isolation. None of the birds presented DNA sequences of beak and
feather disease virus or Chlamydophila psittaci, although a few birds
of the aviaries have previously shown to be positive for sequences of
each DNA separately or in coinfection.
The dysfunction of the digestive tract was associated with
inflammation, necrosis, and loss of neurons in muscular and serosal
ganglia, as observed in PDD cases reported previously (2,12,18,
23,24,27,29,30,31,38,40), and affected the crop, proventriculus,
ventriculus, and intestinal functions. Peripheral neuritis has been
previously reported in sciatic, brachial, and vagal nerves
(2,11,27,29,30,31,33,40) with local nervous tissue damage resulting
in crop and gastrointestinal tract stasis, lack of glandular secretions,
regurgitation, inappetence, undigested food in feces, and functional
malnutrition, leading to starvation and death.
The definitive diagnosis of PDD can be made based on gross and
histologic lesions (14). Molecular biological techniques, however,
represent an option for ante mortem diagnosis and allow the
implementation of a monitoring program for adopting preventive
measures aiming to reducing transmission to susceptible birds (11).
The IHC is a useful tool for the direct demonstration of virus antigens
associated with lesions in tissues affected by ABV, supporting the
concept of a viral etiology for PDD and allowing a better
understanding of the pathogenesis (12,14,18,23,24,27,30,31,38). In
the PDD tissues the cellular response observed histologically, along
with the IHC response, possibly indicates the role of autoimmunity in
the pathogenesis of this disease. Autoimmunity, as previously
suggested on the pathogenesis of Borna disease (32), represents a
novel perspective for understanding the disease in birds.
From lesions here described, PDD must be considered a
condition for concern in native Brazilian psittacines in captivity.
Our findings have indicated avian bornavirus as the potential
etiology. Samples here studied were originally from a large psittacine
collection which contained more than 400 individuals of 34
different species.
Although the epidemiology of ABV, as suggested for mammals,
describes virus shed by shrews (17) or taken from infected insects in
pastures (33), birds here examined did not have contact with shrews,
a species not present in Brazil, nor had they access to consumption of
pasture. Also, due to hygiene, aviaries did not enable the important
development of insect populations, as premises were routinely
cleaned and birds were maintained on wire-net floor cages-aviaries
suspended above the ground. However, regarding potential risks of
introducing the infection into the facility here studied, the proximity
of aviaries, enabling airborne and mechanical transmissions of
feathers, feces, and secretions, and the absence of biosecurity or
quarantine were here considered determinants for risk of infection by
ABV as well as other etiologies. A preliminary control strategy was
proposed for the conservation facility, including pretesting birds in
192 R. V. Donatti et al.

Fig. 3. Ventriculus subserosal ganglia. Amazona rhodocorytha. Moderate lymphocyte and plasma cell infiltration associated with loss of neurons
and axons. H&E.
Fig. 4. Proventricular subserosal ganglion. Amazona rhodocorytha. Mild lymphoplasmacitic infiltration associated with loss of neurons. H&E.
Fig. 5. Adrenal gland. Amazona rhodocorytha. There are many lymphocytes and a few plasma cells infiltrating the medullary chords. H&E.
Fig. 6. Adrenal gland. Amazona rhodocorytha. Nucleus and cytoplasm of the cells of the medullary chords cells positive for avian bornavirus
antigen. IHC.
Fig. 7. Brain, cerebrum. Amazona rhodocorytha. Nucleus and cytoplasm of neurons and a few glial cells positive for avian bornavirus
antigen. IHC.

quarantine for absence of ABV (by PCR) and development of
negative flocks in isolation. A concern has arisen from the
information, indicating a history of purchasing a macaw (Ara severa)
that was housed with potentially infected exotic psittacines, enabling
fecal-oral transmission (14,16) and suggesting an exotic origin for
the described ABV outbreak. It remains of ultimate importance to
perform ABV testing for separation according to ABV health status
of all psittacines in conservation facilities. Of particular concern is
the lack of biosecurity represented by maintaining exotic and native
psittacines in the same premises. The description of PDD and
detection of ABV in Brazilian psittacines indicates the necessity of
adopting a strategic health plan for avoiding its spread and reducing
its impact in native birds.
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ACKNOWLEDGMENTS
We are indebted to CAPES, CNPq, FAPEMIG, and FEP-MVZ for
funding and to the Animal Science Post Graduate Board (Colegiado de
Pós-Graduação em Ciência Animal) for support. This study is part of
the National Institute of Science and Technology-Brazilian Livestock
Genetic-Sanitary Information (Instituto Nacional de Ciência e Tecno-
logia [INCT] Informação Genético-Sanitária da Pecuária Brasileira
IGSPB]). We would like to thank Dr. H. Weissenböck for performing
immunohistochemistry for avian bornavirus. We express our gratitude
to Dr. Kirsi, S. Honkavuori, and Dr. Thomas Briese for sharing
positive-control samples. We are grateful to Dr. Vasco Azevedo of the
Laboratory of Cellular and Molecular Genetics LGCM-ICB (Labor-
atório de Genética Celular e Molecular do Instituto de Ciências
Biológicas [UFMG]) for the additional laboratory support for RNA
extractions.