Disseminated Acanthamoeba sp. infection in a dog.

Abstract

Several species of free-living amoebae can cause encephalomyelitis in animals and humans. Disseminated acanthamoebiasis was diagnosed in pyogranulomatous lesions in brain, thyroid, pancreas, heart, lymph nodes, and kidney of a one-year-old dog. Acanthamoeba sp. was identified in canine tissues by conventional histology, by immunofluorescence, by cultivation of the parasite from the brain of the dog that had been stored at -70 degrees C for two months, and by PCR. The sequence obtained from the PCR product from the amoeba from the dog was compared to other sequences in the Acanthamoeba sp. ribosomal DNA database and was determined to be genotype T1, associated with other isolates of Acanthamoeba obtained from granulomatous amebic encephalitis infections in humans.

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Disseminated Acanthamoeba sp. infection in a dog
J.P. Dubeya,*, J.E. Bensonb, K.T. Blakeleyc, G.C. Bootond, G.S. Visvesvarae
aUnited States Department of Agriculture, Agricultural Research Service,
Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Beltsville, MD 20705-2350, USA
bIllinois Department of Agriculture, Animal Disease Laboratory, Galesburg, IL 61402-2100, USA
cAll Pets Veterinary Clinic, 722 W Jackson, Macomb, IL 61455, USA
dDepartment of Molecular Genetics and Department of Evolution, Ecology, and Organismal Biology,
The Ohio State University, Columbus, OH 43210-1092, USA
eDivision of Parasitic Diseases, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, GA 30341, USA
Accepted 10 November 2004
Abstract
Several species offree-living amoebae can cause encephalomyelitis in animals and humans. Disseminated acanthamoebiasis
was diagnosed in pyogranulomatous lesions in brain, thyroid, pancreas, heart, lymph nodes, and kidney of a one-year-old dog.
Acanthamoeba sp. was identified in canine tissues by conventional histology, by immunofluorescence, by cultivation of the
parasite from the brain of the dog that had been stored at ?70 8C for two months, and by PCR. The sequence obtained from the
PCRproductfromtheamoebafromthedogwascomparedtoothersequencesintheAcanthamoebasp.ribosomalDNAdatabase
and was determined to be genotype T1, associated with other isolates of Acanthamoeba obtained from granulomatous amebic
encephalitis infections in humans.
# 2004 Elsevier B.V. All rights reserved.
Keywords: Acanthamoeba; Dog; Encephalitis; In vitro cultivation; PCR
1. Introduction
Several species of the genera Acanthamoeba, Bala-
muthia, and Naegleria are free-living amoebae that can
cause encephalomyelitis in animals and humans (Mart-
inezandVisvesvara,1997;Visvesvara,1999).Acantha-
moebaspp.infectionshavebeenreportedtocauseacute
to chronic central nervous system (CNS) infections in
threedogs(Ayersetal.,1972;Pearceetal.,1985;Bauer
etal.,1993;Brofmanetal.,2003).WereportAcanthamo-
eba sp. infection ina dog and documentisolation ofthe
parasitefromitsbrainstoredat?70 8Cfortwomonths.
2. Materials and methods
2.1. Naturally-infected dog
An approximately one-year-old Labrador-type
male dog was brought to All Pets Veterinary Clinic,
www.elsevier.com/locate/vetpar
Veterinary Parasitology 128 (2005) 183–187
* Corresponding author. Tel.: +1 301 504 8128;
fax: +1 301 504 9222.
E-mail address: jdubey@anri.barc.usda.gov (J.P. Dubey).
0304-4017/$ – see front matter # 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2004.11.022

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Macomb, Illinois, USA with a complaint of muscle
stiffness and apparent discomfort with anorexia. The
animal had been obtained as a stray about five months
previously, was allowed to run at large, and had no
history of vaccination. Subjective signs of discomfort
were noted on flexion of the extremities and cervical
spine and on palpation of the abdomen, primarily the
dorsal right quadrant. Rectal temperature, hematolo-
gicandserumenzymevalueswere allwithinreference
normal ranges. Abdominal and cervical radiographs
were unremarkable. Dexamethasone, enrofloxacin,
and intravenous lactated ringer’s solution were
administered. After 24 h the animal was subjectively
less stiff, but had developed nystagmus. Oxytetracy-
cline was administered as an intravenous bolus. The
following day the dog was in lateral recumbancy and
unable to rise, had multiple seizures, and repeatedly
pawed at the face. Due to the deteriorating condition
the owners elected euthanasia.
2.2. Postmortem examination
A complete necropsy examination was made.
Brain, lung, heart, liver, kidney, spleen, mesenteric
lymph node, thyroid, thymus, pancreas, multiple
sections of intestine, stomach, urinary bladder, and
skeletal muscle were collected, fixed in neutral
buffered 10% formalin, and routinely processed for
histologic examination. Sections were examined after
staining with hematoxylin and eosin (H and E).
Unfixed tissues were retained at ?70 8C. Brain was
examined by flourescent antibody test for rabies virus
antigen.
2.3. Immunofluorescence for amoeba species
Immunofluorescence tests were conducted by
reacting sections of brain with polyclonal anti-
Acanthamoeba castellanii, anti-Naegleria fowleri,
and anti-Balamuthia mandrillaris antibodies (Visves-
vara, 1999).
2.4. Isolation and cultivation of amoebae
Attempts were made to isolate the organism from
frozen-thawed unfixed brain tissue. The brain had
been stored at ?70 8C for 60 days and was
transferred on dry ice from the laboratory in
Galesburg, Illinois to the Centers for Disease
Control (CDC), Atlanta, Georgia, where cultivation
was attempted as previously described (Visvesvara,
1999). Approximately 3 g of brain tissue was
triturated in about 0.5 ml of amoeba-saline and
one-half of the mixture was inoculated onto a
monkey kidney cell (E6) monolayer and the other
half onto nonnutrient agar coated with Escheria coli.
Amoebae grew out of both cultures within a week
and were identified as Acanthamoeba sp. based on
the characteristic cyst morphology (Visvesvara,
1999). Amoebae growing
subsequently washed off of the agar plate and
inoculated intoAcanthamoeba
containing 100 mg/ml gentamicin and 100 mg/ml
imipenem and incubated at 35 8C. After 24 h
of incubation the culture medium was removed
and replaced with fresh medium. Three such
transfers were sufficient to obtain a culture free of
bacteria.
withbacteriawere
growthmedium
2.5. Isolation of DNA and PCR
A centrifuged pellet of amoebae was suspended in
200 ml UNSET buffer and DNA extracted (Schroeder
et al., 2001; Booton et al., 2002). Subsequently,
nuclear 18S rDNA Acanthamoeba genus specific
amplicon ASA.S1 was amplified by PCR using genus-
specific primers JDP1 (50-GGCCCAGATCGTTTA-
CCGTGAA-30) and JDP2 (50-TCTCACAAGCTGC-
TAGGGAGTCA-30) (Schroeder et al., 2001; Booton
et al., 2002). Electrophoresis on a 1% agarose gel
produced a product of an expected size of ?450 bp
(Fig. 1). The PCR product was then sequenced using
an ABI 310 automated fluorescent sequencing system
(Applied Biosystems, Foster City, CA) (Schroeder
et al., 2001; Booton et al., 2002). The PCR analyses
were performed on amoebae cultivated from the brain
tissue because all the fresh brain tissuewas usedup for
initiating the culture.
Additionally, mitochondrial 16S rDNA multiplex
PCR also was performed to test for the presence of
either Acanthamoeba spp. or the closely related
pathogenic amoeba Balamuthia mandrillaris (Booton
et al., 2003a). The Acanthamoeba-specific PCR
amplimer was directly sequenced using primers
previously used in our laboratory for sequence
analysis of this gene (Ledee et al., 2003).
J.P. Dubey et al./Veterinary Parasitology 128 (2005) 183–187 184

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J.P. Dubey et al./Veterinary Parasitology 128 (2005) 183–187185
Fig. 1. Acanthamoeba sp. (arrows) in sections of the brain of the dog. A–D, H and E; E and F, immunofluorescence pattern of amoebae in the
brain tissue after reaction with anti-Acanthamoeba antibodies. Bar in A applies to parts A–D. Note trophozoites (A–C) with prominent eccentric
nucleus, and a cyst (D) with irregular outline of the cyst walls.

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3. Results and discussion
The carcass was in good condition and body fat
stores were normal. There was abundant 4 mm pale
foci scattered within the myocardium. The kidneys
had multifocal to coalescing, 2–5 mm reddish brown,
slightly raised foci primarily in the cortex.
Histologically in the brain stem, cerebellar pedicle,
and spinal cord, the meninges and superficial neuropil
were infiltrated with macrophages, lymphocytes, and
neutrophils. Locally extensive necrosis and spongiosis
accompanied by gitter cells were present in the
superficial neuropil. Occasional blood vessels were
characterized by fibrinoid necrosis and thrombosis.
Scatteredwithintheaffectedareaswereroundamoeba-
like protozoa. In H and E-stained sections these
protozoaresembledenlargedmacrophages.Underhigh
illumination they could be distinguished from macro-
phagesby theireccentricnucleus anda large nucleolus
based on optimally stained sections (Fig. 1A–D). A
vacuole around the amoebae was also helpful in
locating amoebae. Multiple sections of spinal cord
demonstrated decreasingly severe lesions distally to
about the thoracolumbar junction. The kidney had
multifocal to coalescing infiltrates of macrophages,
lymphocytes, and neutrophils, with occasional intrale-
sional amoebae. Severe, locally extensive pyogranu-
lomatous infiltrates were also identified in the
myocardium, thyroid, lymph node, and pancreas, also
containing occasional organisms, occasionally within
macrophages. Minimal widely scattered primarily
perivascular infiltrates of mononuclear cells were in
the pulmonary alveolar walls, but no organisms were
detected in H and E stained sections.
Rabies testing was negative. The amoebae in the
section treated with anti-A. castellanii antibodies
reacted brightly (Fig. 1E and F), but showed no
reactivity with the other antisera. The polyclonal
antibody is genus but not species specific, and
identified the isolate as Acanthamoeba sp.
The source of infection in this case was not
identified. The route of entry has been speculated tobe
oral, nasal, or ocular. In this case, extensive systemic
spread was identified, indicative of hematogenous
spread. The histological appearance of the lesions
suggests concurrent development, and does not point
to a specific route of infection. The multicentricity of
the infection was striking and consistent with
previously reported cases, although lesions in thyroid
have not been reported previously in dogs. Unlike
earlier reports, pulmonary lesions were not a
prominent feature. Clinical disease progressed rapidly
once manifested, a common feature of granulomatous
amebic encephalitis in nonhuman species.
Clinical pathology data in this case, however, were
not revealing. Fortunately, brain tissue had been
frozen and in vitro cultivation and subsequent PCR
(Fig. 2) helped to reach a definitive diagnosis. The
nuclear 18S rDNA sequence obtained was compared
to other sequences in the Acanthamoeba ribosomal
DNA database and was determined to be genotype T1,
which is associated with other isolates of Acantha-
moeba obtained from granulomatous amebic ence-
phalitis infections(Stothardetal.,1998).Genotype T1
was originally associated with a strain (CDC:V006)
that was isolated in 1982 from the brain tissue of a
granulomatous encephalitis case.
Next, multiplex PCR targeting the mitochondrial
16S rDNA revealed (Fig. 3) that the culture contained
only Acanthamoeba as it produced only the Acantha-
moeba-specific PCR amplimer of ?600 bp (Ledee
et al., 2003). Direct sequencing of this product
confirmed that it was Acanthamoeba sp. Furthermore,
the 16S rDNA sequence was identical to other
Acanthamoeba. sp. genotype T1 isolates, the same
J.P. Dubey et al./Veterinary Parasitology 128 (2005) 183–187186
Fig. 2. Acanthamoeba genus-specific nuclear 18S rDNA PCR.
Legend: arrow indicates position of Acanthamoeba specific PCR
amplimer; C, canine isolate PCR; Ac+, Acanthamoeba positive
control; M = 1 kbp marker; (- - -) negative control is template-free
master mix.

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genotypic conclusion provided by the nuclear 18S
rDNA analysis. If Balamuthia were present a second
PCRproductof1075 bpwouldhavebeenproducedby
the Balamuthia-specific primer set used in the
multiplex PCR, however, no Balamuthia-specific
band was observed (Booton et al., 2003a, 2003b).
Therefore, the presence of Acanthamoeba-specific
amplimers in both nuclear and mitochondrial rDNA
analyses lead to the conclusion that the infection of
this dog was due to Acanthamoeba, specifically
genotype T1. Lastly, multiplex analysis indicates that
no Balamuthia infection was present in this canine.
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