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Journal of Veterinary Diagnostic
http://vdi.sagepub.com/content/13/4/365
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DOI: 10.1177/104063870101300418
2001 13: 365 J VET Diagn Invest
Susan Clubb and Maria Crane
Laura E. Leigh Perkins, Raymond P. Campagnoli, Barry G. Harmon, Christopher R. Gregory, W. L. Steffens, Ken Latimer,
Detection and Confirmation of Reptilian Adenovirus Infection by in Situ Hybridization

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365
J Vet Diagn Invest 13:365368 (2001)
Detection and conrmation of reptilian adenovirus infection by
in situ hybridization
Laura E. Leigh Perkins, Raymond P. Campagnoli, Barry G. Harmon, Christopher R. Gregory,
W. L. Steffens, Ken Latimer, Susan Clubb, Maria Crane
Abstract. Adenovirus infections are documented in at least 12 different species of reptiles. In contrast to
their mammalian and avian counterparts reptilian adenoviruses are not well characterized as to their pathogenic
potential and their ability to cause primary disease. In the diagnostic setting, fresh tissues are often not available
for virus isolation, and the conrmation of reptilian adenovirus infections is dependent largely upon electron
microscopy for the identication of intranuclear viral inclusions associated with histopathologic changes. The
diagnosis of adenovirus infection in 2 different species of snake was conrmed by the application of DNA in
situ hybridization. Using an aviadenovirus specic oligoprobe, adenoviral DNA was observed in the nuclei of
hepatocytes, Kupffer cells, endothelial cells, and enterocytes. Electron microscopy of the liver conrmed the
presence of intranuclear viral particles morphologically consistent with an adenovirus. DNA in situ hybridization
on formalin-xed tissues can serve as a suitable alternative to electron microscopy in the diagnosis of reptilian
adenovirus infections. Both affected snakes had other concurrent diseases, suggesting that the adenovirus may
not have been the primary pathogen.
Adenoviruses have been reported in at least 12 different
species of reptiles of the orders Crocodylia (crocodiles, al-
ligators, caimans) and Squamata, including both the Serpen-
tes (snakes) and Sauria (lizards) suborders. The reptiles in
which adenoviruses have been identied include those be-
longing to zoologic collections and commercial breeders. In
these species, adenoviruses have been incriminated as the
cause of gastroenteritis, hepatitis, nephritis, pneumonia, and
encephalitis.
1,59,11,12,15,16
Adenovirus infection has been re-
ported in reptiles with
5,6,12
and without
5,79,15,16
concurrent dis-
ease, and in the majority of communications, only individual
animals have been affected.
15
Therefore, the pathogenic po-
tential of the adenoviruses and their ability to cause primary
disease in reptiles remains uncertain.
Conrmation of adenovirus infection in the majority of
the cases is based upon morphologic features of the virus or
viral inclusions as determined by electron microscopy. In
these accounts, tissues suitable for virus isolation were not
available. In 3 reports, isolation and propagation of a reptil-
ian adenovirus in cell culture was successful, with a virus-
induced cytopathogenic effect.
7,11,15
However, in only 1 in-
vestigation were Kochs postulates fullled by the reproduc-
tion of disease, specically identifying the isolated adeno-
virus as the primary etiologic agent of hepatitis and
subsequent death in a common boa.
7
In contrast to reptilian adenoviruses, the pathogenesis and
disease-causing potential of human adenoviruses and those
of mammalian (mastadenovirus) and avian (aviadenovirus)
origin are well characterized. In the diagnostic setting, DNA
in situ hybridization (ISH) is a useful adjunct to microscopic
and ultrastructural identication of mastadenoviruses and
From the Departments of Veterinary Pathology (Perkins, Cam-
pagnoli, Harmon, Steffens, Latimer) and Small Animal Medicine
(Gregory), College of Veterinary Medicine, University of Georgia,
Athens, GA 30602, Hurricane Aviaries, 3319 East Road, Loxahatch-
ee, FL 33470 (Clubb), and Zoo Atlanta, 800 Cherokee Avenue S.E.,
Atlanta, GA 30315 (Crane).
Received for publication May 22, 2000.
aviadenoviruses.
2,3,14,19
In the present study, adenovirus in-
fections in a common boa (Boa constrictor imperator) and
a Mojave rattlesnake (Crotalus scutulatus scutulatus) were
diagnosed using ISH.
An adult male common boa from a zoologic collection
presented with general malaise, listlessness, dehydration, an-
orexia, and dermatitis of unknown duration. Treatment with
ceftiofur was ineffectual, and the snake was found dead
shortly after treatment. An adult male Mojave rattlesnake
from a zoologic collection apparently was in good health but
was found dead the day after an abdominal mass was iden-
tied.
Except for the abdominal mass in the rattlesnake, post-
mortem lesions were not reported. Formalin-xed tissues
from both snakes were independently submitted to the De-
partment of Pathology of the University of Georgia College
of Veterinary Medicine.
Fixed tissues from both snakes were routinely processed
and embedded in parafn. Three-micrometer sections were
stained with hematoxylin and eosin (HE) and examined by
light microscopy. Replicate unstained sections of the liver
and gastrointestinal tracts were placed on silicon-coated
slides for DNA ISH tests. The digoxigenin-labeled, adeno-
virus-specic oligoprobes, designated FN-23 and FN-96, had
the following nucleic acid sequences: 5-TCGGA-
CATCGGGGTCAAGTTCGACACGCGCAACTTC-3 (FN-
23) and 5-CGCCTTCAACCGCTTTCCCGAAAACGA-
GATTCTGAAGCAA-3 (FN-96). These probes are based
upon nucleotide sequences that code for a structural capsid
protein of a chicken adenovirus (type 10) (GenBank acces-
sion M87008).
18
The probes were designed to provide max-
imum cross-reactivity for the detection of adenovirus infec-
tions among numerous avian species. Fortunately, the probes
also cross-reacted with adenoviruses from these snakes. The
technique for ISH using either of the 2 oligoprobes, detec-
tion, and counterstaining was described previously.
13
Nitrob-
lue tetrazolium dye and fast green were used as the substrate
chromagen and counterstain, respectively. Two avian poly-
omavirus (APV) oligoprobes were used as negative probe
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366 Brief Communications
Figure 1. Liver, common boa. Intranuclear inclusion bodies in hepatocytes (arrows). HE.
Figure 2. Liver, common boa. Adenoviral DNA in nuclei of hepatocytes (arrows). ISH with FN-96 aviadenovirus oligoprobe and fast
green counterstain.
Figure 3. Transmission electron micrograph. Liver, common
boa. Adenovirus particles in the nucleus of a hepatocyte. Bar 200
nm.
controls. Sections of chicken liver containing conrmed ad-
enoviral inclusions were used as positive controls, with in-
tranuclear deposition of a blue-black chromagen indicating
the detection of adenoviral nucleic acid. Negative controls
using the APV oligoprobes lacked chromagen deposition.
Formalin-xed liver from the common boa was submitted
for examination by transmission electron microscopy. The
tissue was rexed in 2% paraformaldehyde, 2% glutaralde-
hyde, and 0.2% picric acid in 0.1 M cacodylate HCl buffer
(pH 7.2), rinsed, and postxed in 1% osmium tetroxide in
0.1 M cacodylate HCl. The tissue was then inltrated and
embedded in an eponaraldite mixture and allowed to po-
lymerize in a 75 C oven overnight. Blocks were trimmed,
and 1-m sections were stained with 1% toluidine blue in
1% sodium borate and evaluated for the presence of inclu-
sions. Tissues containing viral inclusions were sectioned at
approximately 65 nm and placed on 200-mesh copper hex
grids. The grids were then poststained with 5% methanolic
uranyl acetate and Reynolds lead citrate and viewed with a
transmission electron microscope.
a
Multifocal to conuent hepatic necrosis and hemosiderin
deposition in hepatocytes and Kupffer cells were observed
in the liver sections from the boa. Anisokaryosis of hepa-
tocytes and marked increases in sinusoidal inammatory
cells, which were predominantly macrophages, also were ob-
served. Large basophilic intranuclear inclusion bodies were
present in hepatocytes, Kupffer cells, and occasionally in
endothelial cells (Fig. 1). The boa also had concurrent en-
terohepatic amebiasis with corresponding ulcerative enteri-
tis. Small eosinophilic intracytoplasmic inclusion bodies
were observed in the epithelial cells of renal tubules, tracheal
mucosa, lung, and gastric mucosa. These inclusions were
consistent in morphology with those identied in previously
described cases of retrovirus-induced wasting disease of boa
constrictors.
17
In the liver sections, adenoviral DNA was de-
tected by ISH in hepatocytes, Kupffer cells, and sinusoidal
endothelial cells (Fig. 2). The chromagen deposition was
granular and conned to the nucleus. Intranuclear round to
hexagonal virus particles, with both electron-dense and elec-
tron-lucent cores, were observed with transmission electron
microscopy. These viral particles ranged in size from 65 to
70 nm and were typical of adenoviral particles (Fig. 3). In-
tranuclear adenovirus particles in reptiles typically form a
paracrystalline array
1,11,15,16
; however, this structural organi-
zation of the virus particles was not observed ultrastructur-
ally in tissues from this boa.
In the Mojave rattlesnake, the most striking lesions were
in the gastrointestinal tract. Severe multifocal hemorrhage
and ulceration with intralesional ameobae and basophilic in-
tranuclear inclusion bodies in enterocytes were observed
(Fig. 4). Additional lesions included bacterial nephritis and
cellulitis, splenic brosis, myocardial necrosis with pericar-
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367 Brief Communications
Figure 4. Intestinal tract, Mojave rattlesnake. Intranuclear inclusion body in an enterocyte (arrow). HE.
Figure 5. Intestinal tract, Mojave rattlesnake. Adenviral DNA in the nucleus of an enterocyte (arrow). ISH with FN-23 aviadenovirus
oligoprobe and fast green counterstain.
dial edema, and a renal carcinoma in the abdomen. In the
intestinal tract, adenoviral DNA was detected by ISH within
scattered enterocytes (Fig. 5).
Replicating mastadenoviruses and aviadenoviruses charac-
teristically form large basophilic intranuclear inclusions in in-
fected cells that can be detected microscopically. Similar in-
tranuclear inclusions have been consistently identied in rep-
tilian tissues infected with adenoviruses. Tissues in which ad-
enovirus inclusions have been identied include liver (present
report),
1,6,7,9,12,16
biliary ductular epithelium,
12
pancreatic acinar
epithelium,
5
renal tubular epithelium,
1,9,16
gastrointestinal mu-
cosa (present report),
5,8
respiratory epithelium,
1,8
Kupffer cells
of the liver (present report), splenic macrophages,
5
and en-
dothelium (present report).
9,12
However, other reptilian viruses
that replicate in the nucleus, such as herpesviruses, also are
capable of forming similar intranuclear inclusions.
4,20
In ad-
dition, nonviral intranuclear inclusions have been reported in
reptilian tissues.
10
Currently, ultrastructural evaluation and/or
virus isolation are the main diagnostic tools for the positive
identication of the causative agents of the inclusions. In the
present study, ISH was used to detect adenoviral DNA in
formalin-xed, parafn-embedded tissues to obtain a deni-
tive diagnosis and to the origin of the inclusion bodies ob-
served in the tissues of 2 captive reptiles.
In this and previous studies, adenovirus inclusions have
been associated with epithelial necrosis regardless of the
specic anatomic location. In addition, an adenovirus isolate,
originally obtained from a moribund boa, was shown to
cause similar disease and death when experimentally inoc-
ulated into a neonatal boa.
7
Thus, adenoviruses appear to be
capable of causing severe disease in reptiles. Several reptiles
in previous reports also were undergoing stress, such as re-
production and recent importation, or were experiencing co-
existing infections, such as enteric amebiasis. Likewise, con-
current amebiasis and retroviral infection (common boa) and
amebiasis and neoplasia (Mojave rattlesnake) were present
in the infected snake of this report. Thus, as with the path-
ogenesis of some mammalian and avian adenoviruses, im-
munosuppression may play a role in adenovirus-induced dis-
ease in reptiles. However, there are incidences of adenoviral
infections in reptiles without other concurrent diseases.
6,8
10,15,16
Therefore, the disease-causing potential of adenovirus-
es in reptiles cannot be determined based on the rather lim-
ited number of cases in which presence of this virus has been
conrmed. Further research is required to dene the epide-
miology and virulence of adenoviruses in captive reptiles.
Because of the high sensitivity of ISH,
3
this technique may
be useful for determining the prevalence and pathogenic po-
tential of adenovirus infections.
Acknowledgements. We thank Mary Ard for technical assis-
tance and Dr. Frank Niagro for part in developing the probes.
Sources and manufacturers
a. JEM-1210, IOEL USA Peabody, MA.
References
1. Frye FL, Munn RJ, Gardner M, et al.: 1994, Adenovirus like
hepatitis in a group of related Rankins dragon lizards (Pogona
henrylawsoni). J Zoo Wildl Med 25:167171.
2. Gomes SA, Nascimento JP, Siqueira MM, et al.: 1985, In situ
hybridization with biotinylated DNA probes: a rapid diagnostic
test for adenovirus upper respiratory infections. J Virol Methods
12:105110.
3. Goodwin MA, Latimer KS, Resurreccion RS, et al.: 1996, DNA
in situ hybridization for the rapid diagnosis of massive necro-
tizing avian adenovirus hepatitis and pancreatitis in chicks. Avi-
an Dis 40:828831.
4. Hauser B, Mettler F, Rubel A: 1983, Herpesvirus-like infection
in two young boas. J Comp Pathol 93:515519.
5. Helstab A, Bestetti G: 1984, Virus associated gastrointestinal
diseases in snakes. J Zoo Anim Med 15:118128.
6. Jacobson ER: 1984, Adenovirus-like infection in two Nile croc-
odiles. J Am Vet Med Assoc 185:14211422.
7. Jacobson ER: 1985, Adenovirus-like infection in a boa constric-
tor. J Am Vet Med Assoc 187:12261227.
by guest on May 14, 2014 vdi.sagepub.com Downloaded from
368 Brief Communications
8. Jacobson ER, Gardiner CH: 1990, Adeno-like virus in esopha-
geal and tracheal mucosa of a Jacksons chameleon (Chamaeleo
jacksoni). Vet Pathol 27:210212.
9. Jacobson ER, Kollias GV: 1986, Adenovirus-like infection in a
Savannah monitor. J Zoo Anim Med 17:149151.
10. Jacobson ER, Seely JC, Novilla MN, Davidson JP: 1979, Heart
failure associated with unusual hepatic inclusions in a Deckerts
rat snake. J Wildl Dis 15:7581.
11. Juhasz A, Ahne W: 1992, Physiochemical properties and cyto-
pathogencity of an adenovirus-like agent isolated from a corn
snake (Elaphe guttata). Arch Virol 130:429439.
12. Julian AF, Durham PJK: 1985, Adenviral hepatitis in a female
bearded dragon (Amphibolurus barbatus). NZ Vet J 30:5960.
13. Latimer KS, Niagro FD, Williams OC, et al.: 1997, Diagnosis
of avian adenovirus infections using DNA in situ hybridization.
Avian Dis 41:773782.
14. Montone KT, Furth EE, Pietra GG, Gupta PK: 1995, Neonatal
adenovirus infection: a case report with in situ hybridization
conrmation of ascending intrauterine infection. Diagn Cyto-
pathol 12:341344.
15. Ogawa M, Ahne W, Essbauer S: 1992, Reptilian viruses: ade-
novirus-like agent isolated from royal python (Python regius).
J Vet Med Ser B 39:732736.
16. Schumacher J, Jacobson ER, Burns R, Tramontin RR: 1999,
Adenovirus-like infection in two rosy boas (Lichanura trivir-
gata). J Zoo Wildl Med 25:461465.
17. Schumacher J, Jacobson ER, Homer BL, Gaskin JM: 1994, In-
clusion body disease in boid snakes. J Wildl Dis 25:511524.
18. Sheppard M, Trist H: 1992, Characterization of the avian ade-
novirus penton base. Virology 188:881886.
19. Smyth JA, Benko M, Moffett DA, Harrach B: 1996, Bovine
adenovirus type 10 identied in fatal cases of adenovirus-asso-
ciated enteric disease in cattle by in situ hybridization. J Clin
Microbiol 34:12701274.
20. Watson GL: 1993, Herpesvirus in red-headed (common) agamas
(Agama agama). J Vet Diagn Invest 5:444445.
by guest on May 14, 2014 vdi.sagepub.com Downloaded from