Avian Pathology

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Diagnosis of psittacine beak and feather disease

(PBFD) viral infection, avian polyomavirus
infection, adenovirus infection and herpesvirus
infection in psittacine tissues using DNA in situ
hybridization

A. Ramis , K. S. Latimer , F. D. Niagro , R. P. Campagnoli , B. W. Ritchie &

Denise Pesti

To cite this article: A. Ramis , K. S. Latimer , F. D. Niagro , R. P. Campagnoli , B. W. Ritchie &

Denise Pesti (1994) Diagnosis of psittacine beak and feather disease (PBFD) viral infection, avian
polyomavirus infection, adenovirus infection and herpesvirus infection in psittacine tissues using
DNA in�situ hybridization, Avian Pathology, 23:4, 643-657, DOI: 10.1080/03079459408419034

To link to this article: https://doi.org/10.1080/03079459408419034

Published online: 12 Nov 2007.

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Avian Pathology (1994) 23, 643-657

Diagnosis of psittacine beak and feather disease

(PBFD) viral infection, avian polyomavirus
infection, adenovirus infection and herpesvirus
infection in psittacine tissues using DNA in situ
hybridization

A. RAMIS1, K. S. LATIMER2, F. D. NIAGRO 2 , R. P. CAMPAGNOLI2,

B. W. RITCHIE 2 & DENISE PESTI 2
1
School of Veterinary Medicine, Universidad Autónoma de Barcelona,
08193-Bellaterra, Barcelona, Spain, and 2College of Veterinary Medicine,
University of Georgia, Athens, Georgia 30602, USA

SUMMARY
The evaluation of the usefulness of DNA probes in a diagnostic setting to identify
nuclear inclusions in selected viral infections (psittacine beak and feather disease
viral infection, avian polyomavirus infection, adenovirus infection and Pacheco's
parrot disease) is reported. A DNA in situ hybridization method was used to detect
viral nucleic acid in sections of paraffin-embedded tissues coming from birds
naturally and/or experimentally infected. It is concluded that DNA probes used for
polyomavirus (FN-19) and adenovirus (FN-23) are able to identify nucleic acid of
each virus in the cells with nuclear inclusions, and when used for psittacine beak and
feather disease virus (FN-8), and Pacheco's parrot disease virus (FN-49) are able to
detect viral nucleic acid in cells with or without inclusions.

INTRODUCTION
In psittacine birds, infection with psittacine beak and feather disease (PBFD)
virus (Ritchie et al, 1989, 1990; Latimer et al., 1991), polyomavirus (Bernier et
al., 1981; Davis et al., 1981), adenovirus (Scott et al, 1986; Pass, 1987) and
herpesvirus (Simpson et al., 1975; Randall et al., 1979) can produce nuclear
inclusions. Presumptive diagnosis of these viral infections is based on the demon-
stration of eosinophilic, basophilic or amphophilic nuclear inclusions in haema-
toxylin-eosin (HE)-stained tissue sections. Unfortunately, routine histological
stains are not always reliable. Nuclear inclusions in herpesvirus infection may be
eosinophilic or basophilic, resembling adenovirus infection (Gomez-Villamandos
et al, 1992). In both adenovirus and polyomavirus infection, nuclei may contain
amphophilic to basophilic karyomegalic inclusions (Hunter et al., 1979; Jacobson
et al, 1989). However, the lack of karyomegaly in adenovirus and polyomavirus
infections may induce confusion with nuclear inclusions produced by herpesvirus

Received 2 February 1994; Accepted 12 May 1994.

643
644 ARAMIS£7\4L.

or PBFD virus infections (Ritchie et al, 1991b; Scott et al, 1986; Larimer et al,
1991). Finally, nuclear oedema (K. S. Larimer: personal experience) or the
presence of nuclear filaments and granular amorphous material of unknown
significance (Pass and Perry, 1984; Jacobson et al, 1986) in hepatocytes may
mimic eosinophilic viral inclusions.
Definitive diagnosis of viral infection has classically relied upon virus isolation,
determination of virus-neutralizing antibody titre or electron microscopic demon-
stration of characteristic virions (Gaskin, 1989; Ritchie et al, 1991a). Problems
arise because some viruses, like PBFD virus, cannot be grown in routine tissue
cultures so far examined (Ritchie et al, 1989). Furthermore, virus isolation is
expensive, labour intensive and has a long turn-around time. Antibody titre
determination may not reflect active viral infection. For example, virus neutraliz-
ing antibody titres have been shown to be unreliable predictors of active viral
infection and viral shedding in birds with avian polyomavirus (Niagro et al,
1991). A similar situation also may occur in Pacheco's parrot disease (Gaskin,
1989). Electron microscopy is expensive and not universally available in routine
diagnostic laboratories. In addition, only formalin-fixed tissue may be available
for study and tissue fixation may not be optimal for detailed electron microscopic
examination or critical assessment of virion structure.
DNA probes should allow definitive diagnosis of these viral infections because
they are designed to recognize specific or unique nucleic acid sequences for a
given virus (Myerson, 1988). In human viral diseases, DNA in situ hybridization
(ISH) is used routinely to detect viral nucleic acid in biopsy specimens (Herring-
ton et al, 1990). In veterinary medicine, similar procedures have mainly been
used in experimental studies to diagnose PBFD (Greenacre et al, 1992), poly-
omavirus infection (Latimer et al, 1993), adenovirus infection (K. S. Latimer,
unpublished data) and herpesvirus infection (F. D. Niagro et al, unpublished
data).
The purpose of this study was to evaluate the usefulness of DNA probes in a
diagnostic setting to identify nuclear inclusions in selected psittacine viral dis-
eases. These diseases included PBFD, avian polyomavirus infection, adenovirus
infection and herpesvirus infection (Pacheco's parrot disease).

MATERIALS AND METHODS

Birds and specimens
The study population included 18 eclectus parrots (Eclectus roratus) involved in an
adenoviral outbreak; two Pacheco's parrot disease field cases, a blue and gold
macaw (Ara araurand) and a cockatiel (Nymphicus hollandicus); three budgerigars
(Melopsitacus undulatus) with experimentally-induced Pacheco's parrot disease
following intramuscular injection of Parrot Herpesvirus ATCC VR-705 (Ameri-
can Type Culture Collection); four cockatoos (three Cacatua moluccensis and one
Cacatua sulphured) with naturally acquired PBFD; and two field cases of poly-
omavirus infection, a peach-faced lovebird (Agapornis roseicolis) and a yellow-
collared macaw (Ara auricollis).
 DNA PROBES IN PSITTACINE VIRAL DISEASES 645

Tissue specimens examined included two feather biopsy samples from two of
the PBFD suspected cases and a complete set of necropsy tissues from the
remaining birds. All tissue specimens were preserved in 10% neutral-buffered
formalin solution. Tissues were processed routinely, embedded in paraffin wax,
sectioned at 4 /mi, and stained with haematoxylin and eosin (HE). Replicate
tissue sections also were prepared for immunoperoxidase staining as needed.
Lastly, additional portions of some fresh tissues were preserved in glutaraldehyde
for ultrastructural examination.

Standard disease diagnosis

The definitive diagnosis of viral infection (PBFD virus, polyomavirus, adenovirus
and herpesvirus) was based upon a combination of HE-stained sections, im-
munoperoxidase staining, and transmission electron microscopy. Most HE-
stained tissue specimens contained eosinophilic, amphophilic or basophilic
nuclear inclusions in several tissues and organs. Karyomegaly was observed
occasionally in adenovirus and polyomavirus infections.
Immunohistochemistry was performed according to published guidelines
(Stemberger, 1979). Specimens from suspected cases of PBFD virus and avian
polyomavirus infections were stained by the avidin-biotin complex (ABC) im-
munoperoxidase technique. Rabbit polyclonal primary antibody (1:1200 dilution)
was used to detect avian polyomavirus infection. A murine monoclonal primary
antibody (1:50 dilution) was used to diagnose PBFD virus infection (Ritchie et
al., 1991b; Latimer et al., 1992). An indirect immunoperoxidase technique (IIP)
was used to demonstrate herpesvirus infection (Pacheco's parrot disease). This
technique used polyclonal chicken primary antibody (1:1200 dilution), following
a published protocol (Ramis et al, 1992).
Transmission electron microscopy (TEM) was performed on glutaraldehyde
preserved portions of livers containing nuclear inclusions (adenovirus infection,
Pacheco's parrot disease and PBFD). These tissues were postfixed in 2% osmium
tetroxide and embedded in araldite. Ultra-thin sections were cut, stained with
uranyl acetate and lead citrate, and examined ultrastructurally.

DNA in situ hybridization

Viral nucleic acid was demonstrated in paraffin-embedded tissue sections by
DNA in situ hybridization. The four viral-specific DNA probes were designated as
follows: FN-8 (a 30-base oligomer for PBFD virus), FN-19 (a 40-base oligomer
for the avian polyomavirus), FN-23 (a 35-base oligomer for the avian adenovirus)
and FN-49 (a 40-base oligomer for Pacheco's herpesvirus). Each oligonucleotide
was 3' end labelled by the addition of a single digoxigenin-II-dideoxy undine
(Genius 5 system, Boehringer-Manheim). Three-micrometer thick tissue sections
were mounted on 3-aminopropyltriethoxylane-coated slides (Probe-On Plus
slides, Fisher Scientific Inc., USA). A manual capillary-action work station
(MicroProbe system, Fisher Scientific Inc., USA) was used to perform colorimet-
646 A RAMIS ETAL.

ric DNA in situ hybridization, following published guidelines (Unger et ai, 1991;
Greenacre et al., 1992; Chenggis & Unger, 1993; Garcia et al., 1994; Latimer et
al., 1993).
For PBFD, tissue sections were placed in the holder in capillary gap formation,
preheated in the incubation chamber for 10 min at 70°C, and deparaffinized in a
3:1 limolene/xylene solution. The sections were rinsed three times in 100%
ethanol, once in 95% ethanol and twice in Automation buffer (Biomeda Corp.,
USA). Tissue sections were digested by incubating them for 10 min at 37°C in
0.3% pepsin in Autobuffer (pH 2.0). Pepsin activity was arrested by subsequent
incubation for 5 min at 110°C, after which the specimens were rinsed in Auto-
mation buffer. Chemical and thermal denaturation of the target (viral) DNA was
performed by incubating the slides for 5 min at 110°C in 100% formamide. DNA
hybridization of tissue sections was performed by adding a working solution of the
digoxigenin-labelled viral-specific DNA probe for PBFD virus. This solution
comprised 0.5 /A (500 fmol) of probe FN-8 in 100 ^1 of hybridisation mixture.
The hybridisation mixture comprised 22.5% deionized formamide, 7.5% chon-
droitin sulphate, 5 X saline sodium citrate (SSC) and 50 mM phosphate buffer.
The incubation conditions for hybridizing FN-8 probe to its viral DNA target
were 110°C for 5 min, followed by 37°C for 30 min. A negative control was
processed similarly but lacked digoxigenin-labelled DNA probe. After several
washes at room temperature with solutions composed of SSC, Tween 20 and Brij
35 to minimize non-specific hybridisation of DNA probe, the tissue sections were
rinsed twice in buffer 1 (100 mM Tris-HCl pH 7.5, 150 mM NaCl) containing
1% sheep serum and 0.3% Triton X-100. Next, the sections were incubated in an
alkaline phosphatase conjugate of anti-digoxigenin antibody (1:500 dilution) in
Buffer 1, containing 1% Triton and 0.3% sheep serum; incubation conditions
were 37°C for 30 min. Then the tissue sections were rinsed three times in Buffer
1, twice in Automation buffer, and twice in Buffer 2 (100 mM Tris-HCl pH 9.5,
100 mM Na Cl, 50 mM MgCl2) containing 0.4% Tween 20 and 0.25% Brij 35.
A nitroblue tetrazolium (NBT) dye chromagen solution was applied to the tissue
sections at 37°C for 1 h. Lastly, the tissue sections were placed in Buffer 3
(10 mM Tris-HCl, 1 mM EDTA, pH 8.0) until they were counterstained with
fast green, coverslipped and examined.
For avian polyomavirus infection, avian adenovirus infection and Pacheco's
herpesvirus infection, the aforementioned steps were followed modifying only the
concentrations of working solutions [1 ^1 (1 pmol) probe FN-19/100 fû hybridiza-
tion mixture; 2 fû (2 pmol) probe FN-23/100 fû hybridization mixture; and 2 fû
(2 pmol) probe FN-49/lOO/il hybridization mixture]; the incubation conditions
for hybridizing each specific probe (110°C/5min for all probes, followed by
37°C/30 min for FN-19 and FN-23, or 37°C/60 min for FN-49); the incubation
conditions for alkaline phosphatase conjugate of anti-digoxigenin antibody (37°C/
30 min for FN-19 and FN-23 or 37°C/60 min for FN-49); and the time of NBT
dye chromagen solution (2 h for FN-19 and FN-49, or 4 h for FN-23).
Finally, known positive and negative control tissues from birds with previously
confirmed viral infections were used to validate the hybridization procedures. The
 DNA PROBES INPSITTACINE VIRAL DISEASES 647

presence of dark blue-black pigment indicated the presence of target viral nucleic
acid.

RESULTS
Psittacine beak and feather disease (PBFD)
Application of DNA in situ hybridization (ISH) techniques to histological sections
of feathers confirmed the presence of PBFD viral nucleic acid in the nuclear
inclusions of the feather epithelial cells, and also in the cytoplasmic inclusions of
the pulp macrophages, with deposition of the characteristic dark blue-black
pigment in both instances. An intense, diffuse colouration was also seen in
degenerated feather epithelial cells.
In the two birds undergoing a full necropsy (C. sulphurea and C. moluccensis),
viral nucleic acid was detected in non-folliculär epidermis, spleen and liver; the
distribution pattern was similar in the non-follicular epidermis and spleen, whilst
presenting some differences in the liver. In the latter organ, the histopathological
picture in both cases revealed necrotic foci surrounded by a mononuclear
inflammatory infiltrate and markedly oedematous hepatocytes. However, the
appearance of the nuclear inclusions differed between the two cases. In C.
sulphurea they were purple in colour and spherical, with margination of the
nuclear chromatin and of the nucleolus; meanwhile in C. moluccensis they were
eosinophilic, oval in shape and of an increased size, which produced changes in
the structural features of the nucleus. Application of the FN-8 probe only
revealed the presence of viral nucleic acid in the nuclear inclusions in the C.
sulphurea sections, no positive reaction being found in those of the C. moluccensis
specimen. In the latter case, the FN-49 probe was applied to replicate liver tissue
sections in order to eliminate the possibility of concomitant infection with
Pacheco's parrot disease virus. No herpesviral nucleic acid could be detected.
Later, ultrathin liver sections were evaluated with TEM, and it could be shown
that the eosinophilic nuclear inclusions were masses of amorphous material
situated in the central part of the nucleus (Figure 1). In both instances (specimens
of C. sulphurea and C. moluccensis), application of the FN-8 probe revealed the
presence of viral nucleic acid in the nuclei of the endothelial cells lining the
hepatic sinusoids and in the cytoplasm of the Kupffer cells; an isolated positive
reaction was also seen in the cytoplasm of the hepatocytes (Table 1).
With regard to non-follicular epidermis, there was a high density of viral nucleic
acid in the cells of the basal layer, which progressively decreased towards the
corneal layer in the inclusion-free non-follicular areas of the epidermis, as shown
by ISH techniques (Figure 2 a,b). A positive reaction was also present in the
dermis, particularly in the vascular endothelia and in some blood cells. In the
spleen, there were focal aggregations of macrophages associated to the central
arterioles of the white pulp, showing a highly vacuolized cytoplasm and
eosinophilic cytoplasmic inclusions. The positive hybridisation reaction was
648 A RAMIS ETAL.

Table 1. Tissue distribution of psittacine beak and feather disease virus

Number of Species Organ Cell Inclusion FN-S"™ '
birds

2 C. moluccensis Feather Epithelial Nuclear Nucleus

Epithelial b Nucleus and cytoplasm
Macrophages Cytoplasmic Cytoplasm

1 C. moluccensis Feather Epithelial Nuclear Nucleus

Epithelial Nucleus and cytoplasm
Macrophages Cytoplasmic Cytoplasm

Skin epidermis Basal cells Nucleus and cytoplasm

Dennis Endothelia — Nucleus and cytoplasm

Spleen Macrophages Cytoplasmic Cytoplasm

Liver Hepatocytes Nuclear None

Endothelia Nucleus
Kupffer Cytoplasm

1 C. sulphurea Feather Epithelial Nuclear Nucleus

Epithelial Nucleus and cytoplasm
Macrophages Cytoplasmic Cytoplasm
Skin epidermis Basal cells Nucleus and cytoplasm
Dennis Endothelia Nucleus and cytoplasm

Spleen Macrophages Cytoplasmic Cytoplasm

Liver Hepatocytes Nuclear Nucleus

Endothelia Nucleus
Kupffer Cytoplasm

Oesophagus and
crop epithelium Basal cells — Nucleus and cytoplasm
I. propria Endothelia Nucleus and cytoplasm

Gizzard
1. propria Endothelial — Nucleus and cytoplasm

Intestine epithelium Enterocyte Nuclear Nucleus

1. propria Endothelial Nucleus and cytoplasm

Trachea epithelium Epithelial — Cytoplasm

1. propria Endothelial Nucleus and cytoplasm

Lung air capil. Endothelial Nucleus and cytoplasm

atrium Neumocytes — Nucleus and cytoplasm

Kidney Epithelial tubular — Nucleus and cytoplasm

Mesangial Cytoplasm

Myocardium Myocytes — Nucleus and cytoplasm

': positive ISH reaction to FN-8 probe.

b
Absence of inclusion bodies.

confined to the cytoplasm of the majority of the spleen cells, and its intensity was
much greater in the proximity of the PALS (Table 1).
In addition, a positive reaction to the FN-8 probe was seen in other organs of
the digestive, respiratory and urinary tracts and in the myocardium of the C.
 DNA PROBES IN PSITTACINE VIRAL DISEASES 649

Table 2. Tissue distribution of polyomavirus

Number of birds Species Organ Cell Inclusion FN-lotvet

1 A. roseicolis liver Hepatocyte Nuclear Nucleus

Spleen Macrophages of Nuclear Nucleus
white pulp

1 A. auricolis Liver Hepatocyte Nuclear Nucleus

Spleen Macrophages of Nuclear Nucleus
white pulp

"FN-IÇ™: positive ISH reaction to FN-19 probe.

sulphurea specimen. In the digestive tract, the pattern of virus distribution in

tissue was very similar in the oesophagus and crop: an intense dark blue-black
pigment in the cells of the basal epithelial layer, which progressively decreased
when nearing the layers closer to the lumen; positive pigmentation was also found
in the lamina propria (endothelial cells and some blood cells) and in the muscular
layer. Pigmentation was scant and weak in the proventriculus and was only seen
in the epithelial cells of the mucosa and in some endothelial cells of the muscular
layer. In the gizzard a very intense positive reaction was seen in the vascular
endothelium of the lamina propria, but this became weaker in the muscular layer.
In the small intestine, the presence of viral nucleic acid could be demonstrated in
association with the nuclear inclusions of the enterocytes and in the lamina
propria. A positive reaction was also seen in the trachéal epithelium, and in the
endothelium of the blood vessels of the lamina propria and the serosa of the
respiratory tract. In the lung, there was an intense blue-black pigmentation of the
epithelium of the aerial capillaries; this became somewhat weaker in the pneumo-
cytes of the atria. In the kidney, viral nucleic acid was found in the cytoplasm of
the tubular epithelial cells, in the interstitial cells and in the cytoplasm of the
mesangial glomerular cells. Finally, a diffuse positive pigmentation was found in
the nuclei of some myocytes of the myocardium, with fine cytoplasmic spotting in
the majority of them (Table 1).

Table 3. Tissue distribution of adenovirus

Nuclear inclusions

Number of birds Liver Intestine Kidney 1^-23™ *

8 -b - - None
5 +c + — Liver and intestine
4 + - - Liver
1 + — + Liver and kidney

" F N ^ " " : positive ISH reaction to FN-23 probe.

b
—, absence of inclusion bodies.
c
+ , presence of inclusion bodies.
650 A RAMIS ETAL.

Table 4. Tissue distribution of Pacheco's parrot disease virus

Number of Species Organ Cell Inclusion FN-49 we "

of birds

1 A. araucaria liver Hepatocyte Nucleus Nucleus

Endothelial _b Nucleus and cytoplasm
Kupffer Nucleus and cytoplasm

1 N. hollandicus liver Hepatocyte Nucleus Nucleus

Endothelial Nucleus and cytoplasm
Kupffer Nucleus and cytoplasm

Spleen Macrophages Nucleus Nucleus

1 M. undulalus liver Hepatocyte Nucleus Nucleus

Endothelial Nucleus and cytoplasm
Kupffer Nucleus and cytoplasm

Spleen Macrophages Nucleus Nucleus and cytoplasm

2 M. undulatus liver Hepatocytes Nucleus Nucleus

Endothelial Nucleus and cytoplasm
Kupffer Nucleus and cytoplasm

Spleen Macrophages Nucleus Nucleus

Oesophagus Epithelial Nucleus Nucleus

and crop

Cloaca Enterocytes Nucleus Nucleus

*FN-49 m : positive ISH reaction to FN-49 probe.

b
- , indicates absence of inclusion bodies.

Polyomavirus infection
Significant lesions were only observed in the liver and spleen of the two birds
undergoing necropsy. (A. roseicolis and A. auriocottis). In both birds there were a
considerable number of confluent haemorrhagic and necrotic areas in the liver,
with a mainly periportal distribution pattern. Marked karyomegaly was present in
some hepatocytes, with basophilic or neutrophilic inclusions, or both, which in
certain cases had produced margination of the nuclear chromatin and the nucle-
olus. There was little or no inflammatory infiltrate within these necrotic foci.
There were also multiple necrotic foci present in the white pulp of the spleen,
where mononuclear cells containing nuclear inclusions similar in appearance and
staining characteristics to those described in the liver could be seen. The appli-
cation of the FN-19 probe to sections of these organs confirmed the presence of
polyomavirus nucleic acid in the nuclear inclusions (Figure 3 a, b). Neither
significant lesions nor nuclear inclusions were seen in the remaining organs which
were examined (skin and feathers, gastrointestinal tract, pancreas, lung, kidney,
myocardium and encephalon); in no instance was a positive reaction obtained by
application of ISH techniques (Table 2).
 DNA PROBES IN PSITTACINE VIRAL DISEASES 651

Figure 1. Electron micrograph of an hepatocyte eosinophilic nuclear inclusion corresponding to

PBFD in C. moluccensis showing amorphous material situated in the central part of the nucleus
and the absence of viral crystalline arrays in it. Bar = 1 /im.

Adenovirus infection
The most characteristic lesion in all the carcases was hepatitis with abundant
necrotic areas surrounded by a mononuclear inflammatory infiltrate. In 10 of
these cases there were basophilic nuclear inclusions and a marked karyomegaly of
the hepatocytes near the necrotic foci; application of the FN-23 probe revealed a
positive reaction in the nuclear inclusions, whilst there was no sign of a positive
reaction in the sections not containing nuclear inclusions. Congestion and diffuse
inflammatory infiltration of the mucosa was seen in the intestines of the majority
of birds, but basophilic nuclear inclusions in the enterocytes were only detected
in eight of them. A positive reaction to hybridization with the FN-23 probe was
only present in the nuclear inclusions (Figure 4 a,b). Finally, in the majority of
cases, discreet interstitial nephritis with focal haemorrhagic areas was encoun-
tered, and only in one animal were some nuclear inclusions, similar to those
described in the hepatocytes and the enterocytes, seen in the renal tubular
epithelium. On application of the FN-23 probe to renal histological sections, the
characteristic blue-black pigment was seen in the nuclear inclusions of the renal
tubular epithelium of this last case (Table 3).

Pacheco's parrot disease

In all the birds undergoing necropsy the histopathological lesions in the liver were
 652

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Figure 2. PBFD. (a) HE-stained epidermis of unfeathered skin (toe) corresponding to a nuclear
inclusion free area, (b) Replicate skin sectionfollowing DNA in situ hybridization procedure. PBFD
viral nucleic acid (blue-black coloration) is present in the cells of the basal layer and progressively
decrease towards the comeal layer. Bar = 50 fim.

characterized by a focal coagulative necrosis surrounded by a scant inflammatory

lymphoplasmocyte infiltrate; the nuclei of some hepatocytes contained central
eosmophilic nuclear inclusions which displaced nuclear chromatin towards the
periphery. Application of the FN-49 probe evidenced an intense positive reaction
in the nuclei of hepatocytes, with or without nuclear inclusions, to be found on
the periphery of the necrotic foci. Dots of dark blue-black pigment were also to
be seen in the endothelial cells and in the Kupffer cells of the liver sinusoids
(Figure 5 a,b). This positive reaction was present throughout the liver tissue and
showed no particular distribution pattern. In all cases with the exception of the
specimen of A. araucana, there were multiple necrotic foci in the white pulp of the
spleen, surrounded by an inflammatory infiltrate composed mainly of
macrophages, some of which contained eosinophilic nuclear inclusions. Appli-
cation of the FN-49 probe to spleen sections confirmed the presence of her-
pesvirus in the nuclei of the macrophages. Eosinophilic nuclear inclusions were
encountered in cells of the intermediate layer of the epithelium of the oesophagus

Figure 3 . Polyomavirus infection, (a) HE-stained spleen showing basophilic nuclear inclusions close
to a necrotic area in the white pulp, (b) Presence of the viral DNA in the nuclear inclusions is
confirmed using DNA in situ hybridization procedure: polyomavirus nucleic acid (blue-black
coloration) is present in the nuclear inclusions. Bar= 25 urn.
 DNA PROBES IN PSITTACINE VIRAL DISEASES 653

F i g u r e 4 . Adenovirus infection, (a) HE-stained intestinal villi showing basophilic nuclear

inclusions, (b) Presence ofadenoviral DNA in nuclear inclusions is confirmed using DNA in situ
hybridization procedure: adenoviral nucleic acid (blue-black coloration) is present in the nuclear
inclusions. Bar = SO urn.

and crop in the digestive tract of two of the experimentally infected M. undulatus,
as well as in enterocytes of the cloaca. With the FN-49 probe, the dark blue-black
pigment was only present in the nuclear inclusions of the epithelial cells. Neither
significant lesions nor nuclear inclusions were found in the rest of the organs
examined, and application of the FN-49 probe failed to produce a positive
reaction in any instance (Table 4).

DISCUSSION
This study confirms the utility of DNA probes for identification of the causal
agent in the case of four viral infections (PBFD viral infection, polyomavirus
infection, adenovirus infection and herpesvirus infection), in the nuclear or
cytoplasmic inclusions, when the agent has been previously demonstrated by
immunocytochemical and electron microscope techniques. By application of the
FN-8 probe to histological sections of feathers from cases diagnosed as psittacine

F i g u r e 5. Pacheco 's parrot disease, (a) HE-stained liver showing hepatocyte amphophilic nuclear
inclusions, (b) Presence of viral DNA is demonstrated, using DNA in situ hybridization procedure,
in the nuclei of hepatocytes, endothelial cells, and Kupffer cells showing a blue-black coloration.
Bar =25 fim.
654 A RAMIS ETAL.

beak and feather disease (ABC immunoperoxidase technique), the presence of

viral nucleic acid in the nuclear inclusions of the follicular epithelial cells and in
the cytoplasmic inclusions of the pulp macrophages could be confirmed. Its
presence could also be demonstrated in the extracutaneous inclusions in hepato-
cytes and enterocytes (intranuclear), and in spleen macrophages (intracytoplas-
mic). Presence of the viral nucleic acid could also be shown in cells which did not
have inclusion bodies in the cases investigated, but which had been described in
other reports (Latimer et al, 1990), such as cells from the basal epidermal layer,
liver Kuppfer cells, and epithelial cells of the oesophagus and crop. The presence
of PBFD nucleic acid in the endothelial cells of the majority of organs and tissues
(dermis, lamina propria of oesophagus and crop, gizzard, gut and trachea, and in
the endothelium of the pulmonary capillaries) was also significant. Finally, the
positive findings in the kidney (epithelial and interstitial tubular cells, and mesan-
gial glomerular cells) and in the miocardial cells, in the C. sulphured specimen
should be emphasized, as we have been unable to find any published report of
presence of the virus in these cells (Table 1).
The FN-19 and FN-23 probes revealed the presence of polyomavirus and
adenovirus nucleic acids in the nuclear inclusions: a positive ISH reaction was
only seen in the cells with nuclear inclusions. A positive FN-19 reaction only
appeared in association with nuclear inclusions in the hepatocytes and spleen
macrophages of the birds in which a diagnosis of polyomavirus infection had
already been established (ABC immunoperoxidase technique). In the tissue
samples from the outbreak of adenoviral hepatitis in Electus roratus, a positive
FN-23 reaction was only found in the hepatocytes, enterocytes and renal tubular
epithelial cells with obvious nuclear inclusions, and in which the presence of
adenovirus-like particles had already been demonstrated (Tables 2 and 3).
The FN-49 probe, applied to histological sections obtained from experimen-
tally infected birds and from field cases of Pacheco's parrot disease which had
been diagnosed by immunohistological techniques (IIP), and by visualization
(TEM) of herpesvirus-like particles in liver tissue, has demonstrated its effective-
ness in the detection of herpesviral nucleic acid in the nuclear inclusions of the
hepatocytes, spleen macrophages and epithelial cells of the digestive tract
(oesophagus, crop and cloaca), and in other cells not showing the nuclear
inclusions typical of the process (Table 4).
Finally, the presence in one of the birds (C. moluccensis) in the PBFD group, of
hepatocytes with FN-8 and FN-49 negative nuclear inclusions, which corre-
sponded to amorphous intranuclear material (TEM), underlines the specificity of
these techniques and their usefulness in the identification of unknown types of
nuclear inclusions, which have been widely reported in the current literature
(Cardona et al, 1993).
We therefore feel that the application of DNA probes to identify nuclear
inclusions in these four viral infections is an easy to perform diagnostic method
which does not require highly sophisticated equipment and gives reliable results
allowing identification of viral inclusions and their differentiation from other
inclusions of unknown origin or artifacts. In addition, the FN-8 and FN-49
 DNA PROBES IN PSITTACINE VIRAL DISEASES 655

probes are capable of revealing the presence of viral nucleic acid specific for the
PBFD virus or from that of Pacheco's parrot disease virus, even in cells in which
nuclear or cytoplasmic inclusions are absent.

ACKNOWLEDGEMENTS
The authors appreciate the excellent microtomy by Ms Kathy Davis HT(ASCP).
The authors also acknowledge the assistance of Ms M. Steel with the English text.
This project was funded jointly by the Departments of Veterinary Pathology,
College of Veterinary Medicine, University of Georgia (USA) and School of
Veterinary Medicine, Universidad Autönoma de Barcelona (Spain).
This research project was completed during Dr Ramis' sabbatical study at the
Department of Veterinary Pathology, College of Veterinary Medicine, University
of Georgia.

REFERENCES
BERNIER, J., MOWN, M. & MARSOLIS, G. (1981). A generalized inclusion body disease in the budgerigar
(Melopsitacus undulatus). Avian Diseases, 25, 1083-1092.
CARDONA, C.J., TRAMPEL, D.W., BICKFORD, A.A., DAFT, B.M., DROUAL, R. & KINDE, H. (1993). Intranu-
clear inclusions of unknown pathogenic significance from diagnostic cases in three avian species.
Avian Diseases, 37, 224-253.
CHENGGIS, M.L. & UNGER, E. (1993). Application of a manual capillary action work station to colorimet-
ric in situ hibridization. Journal of Histotechnology, 16, 33-38.
DAVIS, R.B., BOZEMAN, L.H., GAUDRY, D., FLETCHER, O.J., LUKERT, P.D. & DYKSTRA, M.J. (1981). A
viral disease of fledgling budgerigars. Avian Diseases, 25, 179-183.
GARCIA, A.P., LATIMER, K.S., NIAGRO, F.D., RITCHIE, B.W. & CAMPAGNOLI, R.P. (1994). Diagnosis of
polyomavirus-induced hepatic necrosis in psittacine birds using DNA probes. Journal of Veterinary
Diagnosis Investigation, 6, 308-314.
GASKIN, J.M. (1989). Psittacine viral diseases: a perspective. Journal of Zoo and Wildlife Mediane, 20,
249-264.
GOMEZ-VILLAMANDOS, J.C., Mozos, E., SIERRA, M.A., PEREZ, J. & MENDEZ, A. (1992). Inclusion bodies
containing adenovirus-like particles in the intestine of a psittacine bird affected by inclusion body
hepatitis. Journal of Wildlife Diseases, 28, 319-322.
GREENACRE, C.B., LATIMER, K.S., NIAGRO, F.D., CAMPAGNOU, R.P., PESTI, D. & RITCHIE, B.W. (1992).
Psittacine beak and feather disease in a scarlet macaw (Ara macad). Journal of the Association of Avian
Veterinarians, 6, 95-98.
HERRINGTON, C.S., BURNS, J. & O'D MCGEE, J. (1990). Non-isotopic in situ hybridisation in human
pathology, In: HARRIS, N. and WILKINSON, D.G. (Eds) In Situ Hybridisation: Application to Develop-
mental Biology and Medicine, pp. 241-269 (Cambridge, Cambridge University Press).
HUNTER, B., GAGNON, A., ONDERKA, D., GOLTZ, J. & HOLMES, B. (1979). Viral hepatitis in budgerigars in
Southern Ontario. Canadian Veterinary Journal, 20, 176.
JACOBSON, E.R., CLUBB, S., SIMPSON, C., WALSH, M., LOTHROP, C , GASKIN, J., BAUER, J., HINES, S.,
KOLLIAS, G.V., POULOS, P. & HARRISON, G. (1986). Feather and beak dystrophy and necrosis in
cockatoos: clinicopathologic evaluations. Journal of American Veterinary Medical Association, 189,
999-1005.
JACOBSON, E.R., GARDINER, C. & CLUBB, S. (1989). Adenovirus-like infection in white-masked lovebirds
(Agapornis personatd). Journal of the Association of Avian Veterinarians, 1, 32-34.
LATIMER, K.S., RAKICH, P.M., KIRCHER, I.M., RITCHIE, B.W., NIAGRO, F.D., STEFFENS, W.L. & LUKERT,
P.D. (1990). Extracutaneous viral inclusions in psittacine beak and feather disease. Journal of
Veterinary Diagnostic Investigation, 2, 204-207.
LATIMER, K.S., RAKICH, P.M., STEFFENS, W.L., KIRCHER, I.M., RITCHIE, B.W., NIAGRO, F.D. & LUKERT,
656 A RAMIS ETAL.

P.D. (1991). A novel DNA virus associated with feather inclusions in psittacine beak and feather
disease. Veterinary Pathology, 28, 300-304.
LATIMER, K.S., NIAGRO, F.D., RAKICH, P.M., CAMPAGNOLI, R.P., RITCHIE, B.W., STEFFENS, W.L., PESTI,
D. & LUKERT, P.D. (1992). Comparison of DNA dot-blot hybridization, immunoperoxidase staining
and routine histopathology in the diagnosis of psittacine beak and feather disease in paraffin-embed-
ded cutaneous tissues. Journal of the Association of Avian Veterinarians, 6, 165-168.
LATIMER, K.S., NIAGRO, F.D., CAMPAGNOU, R.P., RITCHIE, B.W., PESTI, D.A. & STEFFENS, W.L. (1993).
Diagnosis of concurrent avian polyomavirus and psittacine beak and feather disease virus infections
using DNA probes. Journal of the Association of Avian Veterinarians, 7, 141-146.
MYERSON, D. (1988) In situ hybridization, In: COLVIN, R.B., BHAN, A.K. and MCCLUSKEY, R.T. (Eds)
Diagnostic Immunopathology, pp. 475-498 (Raven Press, New York).
NIAGRO, F.D., RITCHIE, B.W., LUKERT, P.D., LATIMER, K.S., STEFFENS, W.L. & PESTI, D. (1991). Avian
polyomavirus: Discordance between neutralizing antibody titers and viral shedding in an aviary.
Proceedings Meeting of the Association of Avian Veterinarians, Chicago, Illinois, pp. 22-26.
PASS, D.A. (1987). Inclusion bodies and hepatopathies in psittacines. Avian Pathology, 16, 581-597.
PASS, D.A. & PERRY, R.A. (1984). The pathology of beak and feather disease. Australian Veterinary Journal,
61, 69-74.
RAMIS, A., FONDEVILA, D., TARRES, J. & FERRER, L. (1992). Immunocytochemical diagnosis of Pacheco's
parrot disease. Avian Pathology, 21, 523-527.
RANDALL, C.J., DAGLESS, M.D., JONES, H.G.R., & MCDONALD, J.W. (1979). Herpesvirus infection
resembling Pacheco's disease in amazon parrots. Avian Pathology, 8, 229-238.
RITCHIE, B.W., NIAGRO, F.D., LUKERT, P.D., STEFFENS, W.L. & LATIMER, K.S. (1989). Characterization
of a new virus from cockatoos with psittacine beak and feather disease. Virology, 171, 83-88.
RITCHIE, B.W., NIAGRO, P.D., LATIMER, K.S., LUKERT, P.D., STEFFENS, W.L. & PESTI, D. (1990).
Advances in understanding the PBFD virus. Proceedings Meeting of the Association of Avian Veterinar-
ians, Phoenix, Arizona, pp. 12-24.
RITCHIE, B.W., NIAGRO, F.D., LATIMER, K.S., DAVIS, R.B., PESTI, D. & LUKERT, P.D. (1991a). Avian
polyomavirus: an overview. Journal of the Association of Avian Veterinarians, 5, 147-153.
RITCHIE, B.W., NIAGRO, F.D., LATIMER, K.S., VERNOT, J., PESTI, D., CAMPAGNOU, R.P. & LUKERT, P.D.
(1991b). Polyomavirus infections in adult psittacine birds. Journal of the Association of Avian
Veterinarians, 5, 202-206.
SCOTT, P.C., CONDRON, R.J. & REECE, R.L. (1986). Inclusion body hepatitis associated with adenovirus-
like particles in a cockatiel (Psittaciformes; Nymphicus hollandicus). Australian Veterinary Journal, 63,
337-338.
SIMPSON, C.F., HANLEY, J.E. & GASKIN, J.M. (1975). Psittacine herpesvirus infection resembling Pacheco's
parrot disease. Journal of Infectious Diseases, 131, 390-396.
STERNBERGER, L.A. (1979). Immunochemistry, 2nd edn (New York, John Willey).
UNGER, E.R., HAMMER, M.L. & CHENGGIS M.L. (1991). Comparison of S35 and biotin as labels for in situ
hibridization: use of an HPV model system. Journal of Histochemistry and Cytochemistry, 39, 145-150.

RESUME
Diagnostic dans les tissus des psittacidés, par hybridation de l'ADN in
situ, de l'infection par le virus de la maladie du bec et des plumes, le
polyomavirus aviaire, l'adénovirus et l'herpès virus
L'utilisation de sondes ADN comme outils de diagnostic pour identifier des inclusions
nucléaires est évaluée pour différentes infections virales: virus de la maladie du bec et des
plumes des psittacidés, polyomavirus aviaire, adenovirus et virus de la maladie de Pacheco du
perroquet. Une méthode d'hybridisation de l'ADN in situ a été appliquée à la détection de
l'acide nucléique virale dans des sections de tissus inclus dans la paraffine, provenant d'oiseaux
naturellement et/ou expérimentalement infectés. Il est conclu que les sondes ADN utilisées
pour le polyomavirus (FN-19) et l'adenovirus (FN-23) sont capables d'identifier
l'acidenucléique de chacun des virus dans les cellules avec inclusions nucléaires; lorsqu'elles
sont utilisées pour les virus de la maladie du bec et des plumes (FN-8) et le virus de la maladie
de Pacheco du perroquet (FN-49), elles sont capables de détecter l'acide nucléique viral dans
les cellules avec/ou sans inclusion.
 DNA PROBES IN PSITTACINE VIRAL DISEASES 657

ZUSAMMENFASSUNG
Diagnose von Infektionen mit Virus der psittacinen Schnabel- und
Federkrankheit (PBFD), aviärem Polyomavirus, Adenovirus und
Herpesvirus in Geweben von Psittaziden mit Hilfe der DNS-in situ-
Hybridisierung
Es wird über die Untersuchung der Brauchbarkeit von DNS-Sonden im Rahmen der Diag-
nostik zur Identifizierung von Kerneinschlüssen bei ausgesuchten Virusinfektionen (psittacine
Schnabel- und Federkrankheit, aviäre Polyomavirusinfektion, Adenovirusinfektion und
Pachecosche Papageienkrankheit) berichtet. Eine DNS-in situ-Hybridisierungsmethode wurde
verwendet, um Virusnukleinsäure in Paraffinschnitten von Geweben natürlich und/oder experi-
mentell infizierter Vögel nachzuweisen. Es wird gefolgert, daß sich DNS-Sonden, die für
Polyomavirus (FN-19) und Adenovirus (FN-23) verwendet wurden, zur Identifizierung der
Nukleinsäure der jeweiligen Virusart in den Zellen mit Kerneinschlüssen eignen, und daß sich
mit DNS-Sonden für Virus der psittacinen Schnabel- und Federkrankheit (FN-8) und Virus
der Pachecoschen Papageienkrankheit (FN-49) Virusnukleinsäure in Zellen mit oder ohne
Einschlüsse nachweisen läßt.

RESUMEN
Diagnóstico de la infección por el virus de la enfermedad de las plumas y
del pico de las psitácidas (PBFD), polioma aviar, adenovirus y her-
pesvirus en tejidos de psitácidas mediante hibridación in situ del ADN
Se describe la utilidad de sondas de ADN en el diagnóstico e identificación de inclusiones
nucleares en diversas infecciones virales (enfermedad vírica de las plumas y del pico de las
psitácidas, infección por el virus del polioma, infección por adenovirus, y enfermedad de
Pacheco del periquito. Se utilizó para ello el método de la hibridación in situ del ADN para
detectar âcido nucleico viral en cortes de tejidos incluidos en parafina procedentes de ayes
infectadas espontâneamente o experimentalmente. De este estudio se concluye que las sondas
de ADN empleadas para poliomavirus (FN-19) y adenovirus (FN-23) son capaces de
identificar el ácido nucleico de dichos virus en células con cuerpos de inclusión; empleando
sondas para el virus de la enfermedad de las plumas y el pico de las psitácidas (FN-8) y de la
enfermedad de Pacheco del periquito (FN-49) se detectó ácido nucleico viral en células con
y sin cuerpos de inclusión.