404156

le, BanethCutaneous Hepatozoon canis infection

XXXXXX10.1177/1040638711404156Litt

Journal of Veterinary Diagnostic Investigation

Cutaneous Hepatozoon canis infection 23(3) 585­–588

© 2011 The Author(s)
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DOI: 10.1177/1040638711404156
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Liz Little,1 Gad Baneth

Abstract. A 7-month-old mixed-breed intact female dog was presented to a private veterinarian with a 2 cm in diameter
raised, pruritic, alopecic, subcutaneous, fluctuant swelling over the right eye. Cytology of the mass revealed many degenerate
neutrophils, moderate numbers of eosinophils, moderate numbers of macrophages, rare mast cells, and few erythrocytes.
Rare neutrophils contained a protozoal agent compatible with a Hepatozoon gamont. Real-time polymerase chain reaction
of peripheral blood was positive for Hepatozoon canis. The complete sequence identity of the amplified 18S ribosomal RNA
fragment from the dog’s blood confirmed H. canis and proved it was relatively distant from the corresponding fragment
sequence of Hepatozoon americanum. This case is important in documenting an unusual presentation of infection with H.
canis outside of the southern United States.

Key words: Cutaneous; dogs; Hepatozoon; protozoa.

Hepatozoonosis is a tick-borne infection caused by an api-

complexan protozoon from the family Hepatozoidae. All
species of genus Hepatozoon share a basic life cycle that
includes asexual development and sporogony in a hematoph-
agous invertebrate definitive host, and merogony followed
by gamontogony in a vertebrate intermediate host.4 Unlike
most tick-borne protozoal pathogens that are transmitted via
tick bites, Hepatozoon transmission takes place by ingestion
of a tick containing Hepatozoon oocysts. Hepatozoon gam-
onts from dog isolates originating in different geographical
areas are morphologically similar, and therefore it was
believed, until 1997 that canine hepatozoonosis was caused
by a single species.16 Since then, research has led to the rec-
ognition of 2 distinct Hepatozoon species in dogs, H. ameri-
canum and H. canis, with very distinct clinical syndromes.2 Figure 1. Fine-needle aspirate sample of the lesion over the
Polymerase chain reaction (PCR) can now differentiate eye; dog. Note neutrophilic and eosinophilic inflammation with
between the 2 distinct species based on the unique 18S ribo- many intracellular and extracellular cocci and a single intracellular
somal RNA (rRNA) gene sequence.15 Although not always gamont. Wright–Giemsa. Bar = 20 µm.
necessary to make a diagnosis, PCR can aid in cases that
have an atypical presentation for Hepatozoon.
A 7-month-old mixed-breed intact female dog was pre-
sented to a private veterinarian with a 2 cm in diameter mg/dl), and mild increased activity of alkaline phosphatase
raised, pruritic, alopecic, subcutaneous, fluctuant swelling (191 U/l, ref. interval 5–131 U/l).
over the right eye. The mass had been present since adoption Aspirate smears made from the lesion over the eye were
from a shelter in New Jersey, 3 weeks prior. No other abnor- stained with Wright–Giemsa.a The direct smears were highly
malities were found on clinical examination. Surgical cellular and mildly hemodilute. Smears contained many
removal was declined. Results of a complete blood cell count degenerate neutrophils, moderate numbers of eosinophils,
and serum chemistry panel were unremarkable with the
exception of mild normocytic, normochromic anemia (hema- From IDEXX Laboratories, Westbrook, ME (Little), and the School of
tocrit value: 33.7%, reference [ref.] interval: 36–60%), mild Veterinary Medicine, Hebrew University, Israel (Baneth).
thrombocytosis (592 × 103/ml, ref. interval: 170–400 × 103/ 1
Corresponding Author: Liz Little, IDEXX Laboratories, 2010 Cabot Blvd
ml), hyperphosphatemia (10.4 mg/dl, ref. interval: 2.5–6.0 West, Suite D-1, Langhorne, PA 19047. lizklitte@gmail.com

Downloaded from vdi.sagepub.com at UNIV OF CALGARY on May 24, 2015

586 Little, Baneth
moderate numbers of macrophages, rare mast cells, and few
erythrocytes. Many intracellular and extracellular bacterial
cocci were identified. Rare neutrophils contained an ellipsoi-
dal structure measuring 11 µm × 4 µm that either displaced
or overlaid the nucleus (Fig. 1). This structure was light blue
with a variably distinct 3–4-µm round magenta-stained
nucleus-like structure. Morphology was most compatible
with a Hepatozoon gamont. Based on the geographical loca-
tion (New Jersey) and the presence of gamonts in a tissue
aspirate, a tentative diagnosis of H. americanum was made.
Lesion cultures were positive for moderate growth of
Staphylococcus intermedius. Based on sensitivity testing, the
initially prescribed 12.5 mg/kg of amoxicillin and clavulanic
acidb was discontinued, and trimethoprim and sulfamethoxa-
zole (15 mg/kg) and clindamycin hydrochloride dropsc (10
mg/kg) were prescribed. The lesion had decreased in size by
approximately 50% on amoxicillin and clavulanic acidb
alone.
Upon receiving the cytology results, a peripheral blood
sample was submitted to the Molecular Diagnostics
Laboratory (Auburn University, Auburn, Alabama) for real-
time PCR for the 18S rRNA gene of Hepatozoon. Copy num-
bers of the Hepatozoon spp. 18S rRNA gene were quantified,
and the amplified sample was speciated via melting curve
analysis.15 Test results were a high positive, with 600 copies
per µl of blood of H. canis.
An additional sample was then submitted to the Hebrew
University School of Veterinary Medicine in Israel for addi-
tional genetic characterization. Polymerase chain reaction
for the amplification of a fragment of the Hepatozoon 18S
rRNA gene was carried out as previously described,20 and
the 352 base pairs amplicon was sequenced at the Alexander
Silberman Institute of Life Sciences at the Hebrew University
of Jerusalem using a commercial sequencing kitd and genetic
analyzer.e Sequencing was performed from products of both
the reverse and forward primers. Obtained sequences were
evaluated with the ChromasPro software version 1.33f and
compared to sequence data available from GenBank using
the BLAST 2.2.9 program (http://www.ncbi.nlm.nih.gov/
blast/Blast.cgi). Analysis of the partial 18S rRNA gene
sequence determined that the dog was infected with H. canis
with 100% similarity to a H. canis sequence from Spain
(GenBank AY150067).8 The closest H. americanum
GenBank accession (no. AF176836) had only a 90% similar-
ity to the amplified sequence.18
Hepatozoon canis was first described in the blood of dogs
in India in 1905 (Christophers S: 1906, Leucocytozoon canis.
Scientific Memoirs by the Officers of the Medical and
Sanitary Departments of the Government of India. Office of
the Superintendent of Government Printing, Calcutta, India.
Available at: http://books.google.com/books?id=3jTlAAAA
MAAJ&pg=PA23&dq=Scientific+Memoirs+by+the+Officers+
of+the+Medical+and+Sanitary+Departments+of+the+Gove
rnment+of+India+1907&hl=en&ei=OAAmTbuQDoT78
Aak7_WwAQ&sa=X&oi=book_result&ct=result&resnum=
Downloaded from vdi.sagepub.com at UNIV OF CALGARY on May 24, 2015
1&ved=0CCMQ6AEwAA#v=onepage&q&f=false.
Accessed on January 6, 2011) and is well documented in
tropical, subtropical, and temperate regions all over the
world.10,11,13 The main vector of H. canis is the brown dog
tick (Rhipicephalus sanguineus).3 Although this tick is com-
monly found throughout the United States, H. canis was only
recently identified in dogs in the southern United States.1 In
addition to ingestion of infected ticks, horizontal transmis-
sion through the uterus from the dam to its offspring has also
been demonstrated in H. canis.19 The majority of dogs
infected with H. canis develop subclinical infections (less
than 5% neutrophils infected) and only rarely does infection
cause severe disease. The dogs that develop high parasitemia
(90–100% of neutrophils infected) can exhibit pyrexia, leth-
argy, anemia, and emaciation.6 High parasitemia can result in
extreme neutrophilia with greater than 50,000 gamonts per
µl of blood. Other clinical laboratory findings associated
with H. canis include mild anemia, thrombocytopenia,
hyperglobulinemia, and increased creatine kinase and alka-
line phosphatase activities.3
Hepatozoon americanum, a well-documented pathogen that
causes American canine hepatozoonosis (ACH) in the southern
United States, was first recognized in Texas, but the disease has
since been reported in dogs from several other states, including
Louisiana, Alabama, Georgia, Florida, Tennessee, and
Oklahoma.9 Dogs become infected with H. americanum when
they ingest the vector tick, Amblyomma maculatum, containing
oocysts. The infection is proposed to occur during grooming,
ingestion of tick-infested prey,10 or ingestion of cystozoites in
rodent paratenic hosts.14 Amblyomma maculatum is primarily
found along the Gulf Coast, but has spread to Oklahoma and
Kansas.7 Hepatozoon americanum causes severe clinical signs
such as high pyrexia, cachexia, depression, muscle atrophy,
anemia, generalized pain, and weakness. These clinical signs
have been attributed to merogonous cysts in skeletal muscle and
bony proliferative lesions.16 A marked leukocytosis ranging
from 20,000 to 200,000 leukocytes/µl of blood is typically
found with parasitemia not usually exceeding 0.1% of circulat-
ing leukocytes.3 Unlike the subclinical course common to H.
canis, ACH is often fatal if left untreated. Immunodeficient
dogs, such as those with immature immune systems, congenital
immunodeficiencies, and/or concurrent infections (toxoplasmo-
sis, leishmaniasis, babesiosis, or ehrlichiosis), are more suscep-
tible to infection with H. canis, whereas H. americanum can
cause severe clinical signs in immunocompetent dogs.3
The present case is unusual in that the diagnosis of
H. canis was made on a tissue lesion from an otherwise
asymptomatic dog, and thus emphasizes the importance of a
patient’s travel history, since H. canis is not endemic to New
Jersey. The complete sequence identity of the amplified 18S
rRNA fragment from the dog’s blood with H. canis (GenBank
accession no. AY150067) and the fact that it was distant
from the corresponding fragment sequence of H. america-
num (GenBank accession no. AF176836) confirmed the
diagnosis of H. canis infection. Hepatozoon canis is found
 Cutaneous Hepatozoon canis infection 587
primarily in hemolymphatic tissues, whereas H. americanum
infects mainly muscular tissues.3 Before the advent of a species-
specific PCR, the most common method of diagnosing H.
canis infection was demonstration of gamonts in a blood
smear. Less frequently, H. canis meronts can also be detected
in histopathologic specimens or in cytologic preparations
made from aspirates of impression smears of hemolymphatic
tissues. Since H. americanum infects mainly muscular tis-
sue, diagnosis typically relies on unique “onion skin” cyst
lesions in muscle biopsies.3 An enzyme-linked immunosor-
bent assay is available for detection of antibodies reactive
with gamont (H. canis) or sporozoite antigens (H. america-
num) in sera; however, it is mainly utilized for epidemiologi-
cal studies.12,17 In more recent studies in the United States,
diagnosis of H. canis has been based on positive PCR results
in asymptomatic dogs or those suspected of having H. amer-
icanum. In a study published in 2008 of Hepatozoon species
in the United States,1 2 out of 200 randomly sampled asymp-
tomatic shelter dogs in Payne County, Oklahoma were posi-
tive for H. canis. In addition, a review of PCR results on 274
blood samples submitted to the Molecular Diagnostic
Laboratory at Auburn University revealed H. americanum,
H. canis, and a mixture of H. americanum and H. canis in 68
(24.8%), 2 (0.7%), and 7 (2.6%), respectively, of 77 canine
blood samples found positive for Hepatozoon spp.1 In
another study,15 examination of 614 ethylenediamine tetra-
acetic acid blood samples from dogs with suspected hepato-
zoonosis identified H. americanum in 167 samples (27.2%),
H. canis in 14 (2.3%), and a coinfection of the 2 species in 14
(2.3%) cases. Hepatozoon was not identified in the remain-
ing 68.2% of cases. Although hepatozoonosis was thought to
be only endemic in the southeastern United States, the afore-
mentioned study also documented Hepatozoon spp. DNA in
samples from California, Nebraska, Vermont, Virginia, and
Washington State. These studies confirmed H. canis is pres-
ent in North America and that coinfections with H. america-
num do occur, despite different tick vectors.
Since the dog in the current report was otherwise healthy,
it is presumed that gamonts were identified in the skin lesion
due to extravasation of infected circulating neutrophils to the
site of the bacterial infection. No gamonts were reported by
a technician on routine review of the blood smear.
Unfortunately, the peripheral blood smear was no longer
available for pathologist review when requested. Since H.
canis has not been documented to preferentially infect sub-
cutaneous tissue, it seems unlikely that H. canis was the ini-
tiating cause of the lesion. Additionally, the lesion resolved
with routine antibiotic treatment. Additional diagnostics
(tick-borne disease titers or PCR, biopsy of the lesion) and
treatment with imidocarb dipropionate were declined by the
owners, due to the otherwise healthy status of the patient. At
the time of publication, the patient was reported as healthy
with no clinical signs of disease.
Based on the geographic location of this patient (New
Jersey), further inquiries revealed that the puppy had been
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whelped in a Texas shelter and subsequently transported to a
shelter in New Jersey for adoption services. The dam or litter-
mates could not be located for additional diagnostics. Infection
in this dog could have occurred via vertical transmission from
the dam or ingestion of a tick, likely in the Texas shelter envi-
ronment. The transportation timeline for this animal was not
available, but it is unlikely infection occurred by ingestion of a
tick in New Jersey since naturally occurring infections have not
yet been confirmed in this state. In an experimental transmis-
sion study, H. canis gamonts appeared in the peripheral blood
28 days postinoculation.5
The hyperphosphatemia and increased alkaline phospha-
tase were attributed to bone growth in the young dog in the
present study, particularly in the absence of skeletal lesions.
The mild normocytic–normochromic anemia was attributed
to the age of the patient and perhaps anemia of inflammatory
disease, despite the lack of an inflammatory leukogram.
Thrombocytosis could be attributed to epinephrine-mediated
splenic contraction, underlying inflammatory disease, and/or
erythropoietin-induced thrombopoiesis due to a decreased
erythropoietic state.
The current case is important in documenting an unusual
presentation of infection with H. canis outside of the southern
United States. Veterinarians and diagnosticians all over the
United States should be aware of the unique differences between
H. canis and H. americanum. The diagnosis of canine hepatozo-
onosis is typically accomplished by the identification of gam-
onts in blood smears or of “onion skin” cysts in muscle
biopsies.11 Although the clinical presentation and the aforemen-
tioned diagnostic techniques are usually adequate in differenti-
ating between the 2 infections, PCR should be pursued if the
clinical presentation is unusual. Dogs with a low H. canis para-
sitemia generally have a good prognosis if treated with the rec-
ommended protocols, but the prognosis for dogs with high
parasitemia is guarded.3,6 Treatment for H. americanum is more
difficult since infected dogs are more debilitated at the time of
diagnosis and because there is no effective therapy to eliminate
the tissue stages of this organism.4
Acknowledgements
The authors thank Dr. Onesios at the Hoboken Animal Hospital
in Hoboken, NJ, for her clinical assistance and Dr. Susan Little,
Department of Veterinary Pathobiology, Center for Veterinary
Health Sciences, Oklahoma State University, Stillwater, OK, for
her molecular expertise and advice.
Sources and manufacturers
a. Caligor Inc., Greenville, PA.
b. Clavamox®, Pfizer Inc., New York, NY.
c. Antirobe Aquadrops®, Pfizer, New York, NY.
d. BigDye® Terminator v3.1 Cycle Sequencing Kit, Applied
Biosystems, Foster City, CA.
e. ABI PRISM 3100 Genetic Analyzer, Applied Biosystems,
Foster City, CA.
f. © Technelysium Pty Ltd., Digital River Inc., Eden Prairie, MN.
588 Little, Baneth
Declaration of conflicting interests
The authors declared that they had no conflicts of interest with
respect to their authorship or the publication of this article.
Funding
The authors declared that they received no financial support for
their research and/or authorship of this article.
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