No.

6 December 2009

www.cvbd.org
®
CVBD DIGEST
Canine hepatozoonosis –
a summary for the practitioner
Cutting-edge information brought to you by the CVBD® World Forum
 ® No.6 December 2009
CVBD DIGEST Canine hepatozoonosis – a summary for the practitioner

Introduction

When Bayer HealthCare, Animal Health Division, called for the 1st International
CVBD® Symposium in 2006, this was the first step to address the global threat
of canine vector-borne diseases (CVBD®). This was based on the belief that
vector-borne diseases of the dog should be treated as one topic and dealt with
on a global level in an interdisciplinary way. During the past years, four sym-
posia have taken place and CVBD® have become a global issue, even sparking
public interest. Many of the parasite-transmitted diseases affect humans as
well as animals. The dog as man’s best friend plays an important role – both
being affected by, and serving as a host for some of the zoonotic pathogens.

At the first symposium, the participants agreed to form the CVBD® World
Forum. Besides gathering knowledge, the main task for this group of inter-
national experts has been to raise awareness for the specific regional risks
of CVBD® and to foster preventive measures. For this reason, the CVBD® World
Forum created a website (www.cvbd.org) to provide the veterinary practitioner
with cutting-edge and clinically relevant scientific information on CVBD®.

In CVBD® Digest, relevant findings from CVBD® symposia are presented

periodically to veterinary practitioners. During the symposia, hepatozoonosis
has become a major topic of interest, as it is now regarded as a global
problem: meanwhile, Hepatozoon canis has been identified in most if not all
continents using molecular diagnostics. The recent discovery of a related
species in the US, Hepatozoon americanum, with severe clinical signs and a
poorer prognosis, has accelerated research into the Hepatozoon life cycle and
associated pathophysiology. Nevertheless, there are still a lot of uncertainties
in this research area. One reason might be that these infectious agents have
an atypical route of transmission, mostly via ingestion of the transmitting ticks.
However, when it comes to prevention, the use of an ectoparasiticide that
repels and kills the transmitting ticks, as is recommended to reduce the risk
of other CVBD®, is a promising way to minimize the risk of Hepatozoon trans-
mission in the dog.
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No.6 December 2009
Canine hepatozoonosis – a summary for the practitioner CVBD DIGEST 3

Cutting-edge information brought to you by the CVBD World Forum

Canine hepatozoonosis –
a summary for the practitioner
Author: Friederike Krämer differences in definitive host, pathology and life
Institute for Parasitology, University of Veterinary Medicine cycle12 led to the description of a separate species
Hannover, Germany H. americanum in 1997.13 Comparative genetic and
antigenic studies further substantiated that, although
Canine hepatozoonosis is caused by the two apicom-
related to H. canis, H. americanum is a distinct spe-
plexan parasites Hepatozoon canis and Hepatozoon
cies.12,14,15 A brief comparison of these pathogens is
americanum. Although phylogenetically related, the
provided at the end of this text in Tab. 2.
two species differ in a variety of aspects, including
clinical signs, life cycle, and host spectrum. In con-
trast to other canine vector-borne diseases (CVBD),
the main transmission route for both of these para-
Life cycle
sites is via ingestion of the infected tick vectors. This
All Hepatozoon spp. share a basic life cycle that
article gives an overview of H. canis infection with
includes sexual development and sporogony in a
a comparison to H. americanum infection. When it
hematophagous invertebrate definitive host, and
comes to prevention, tick control using a repellent
merogony followed by gamontogony in a vertebrate
ectoparasiticide is beneficial for the dog.
intermediate host. Definitive hosts for Hepatozoon
spp. are blood-sucking invertebrates, including ticks,
lice, reduviid bugs, and leeches. The gamont stage is
Historical background and taxonomy
found in blood cells of the vertebrate host, typically
in leukocytes (see Fig. 1).
The two canine pathogens Hepatozoon canis (see
Fig. 1) and H. americanum are hepatozoid apicom-
plexan protozoa. They belong to a diverse group of
parasites that includes more than 300 Hepatozoon
spp., of which 46 have been described in mammals.1
The genus Hepatozoon now belongs to the family
Hepatozoidae of the suborder Adeleorina.1,2,3
Canine hepatozoonosis was first diagnosed in India
at the beginning of the last century, and the causa-
tive agent was classified as Leukocytozoon canis.4,5
In 1908, Miller6 described the genus Hepatozoon,
into which this canine parasite was subsequently
transferred.7 During this time, the Brown Dog tick
Rhipicephalus sanguineus was established as the
main invertebrate host for the protozoan.8
Prior to 1978 when the first cases with severe clinical
signs were detected in the Gulf Coast region of Fig. 1 Blood-smear showing Hepatozoon canis gamonts
Texas, USA, canine hepatozoonosis was only known from the blood of a dog. Two protozoa can
in the Old World.9,10,11 At first, it was assumed that be observed as oval structures within leukocytes.
(With kind permission of Gad Baneth, Rehovot, Israel)
the pathogen was a virulent strain of H. canis, but
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4 CVBD DIGEST No.6 December 2009
Canine hepatozoonosis – a summary for the practitioner

Transmission The bone marrow has also been shown to be a

Transmission of Hepatozoon spp. to vertebrates is major site for merogony.7,21,22
by ingestion of all or part of the definitive inverte- Meronts of H. canis are found rarely in muscle and
brate host. In the case of canine hepatozoonosis, have their own characteristic morphologic “wheel
a dog may ingest an infected tick, either during spoke” arrangement of merozoites within the
grooming or when eating tick infested prey such as meront (see Fig. 2).23 It is likely that the invasion of
small rodents. Direct transmission from infected ro- leukocytes by (micro)merozoites and transformation
dents to dogs is currently being investigated: Inges- to gamonts takes place in the visceral tissues prior
tion of H. americanum sporozoites by cotton rats led to returning to the circulation20, where these stages
to the development of cystozoites in the rats’ mus- can be taken up again by blood-feeding ixodid ticks.
cle tissue.16 Muscle from infected rats was infectious
to a dog that subsequently developed the character-
istic signs of American canine hepatozoonosis
(ACH).17 However, muscle from H. americanum in-
fected dogs did not result in transmission.13,18 There
are no feeding studies evaluating the infectivity of
H. canis cysts.19

Merogony – Development in vertebrates

The exact dispersion route of H. canis sporozoites
after oral ingestion of H. canis oocysts containing
sporozoites within sporocysts is unknown. It is not
clear whether the sporozoites penetrate the gut and
disseminate hematogenously to their target organs
or whether they are first engulfed by a phagocytic
host cell prior to migration via the lymph or blood to Fig. 2 Hepatozoon canis meront in the spleen. Note the
other tissues.20 Initial merogony may take place in “wheel spoke” arrangement of the merozoites within
the meront. (With kind permission of Gad Baneth, Rehovot, Israel)
the liver or alternatively the gut lymph tissue or
mesenteric lymph nodes.

INFO BOX 1
VERTEBRATE LIFE CYCLE OF H. AMERICANUM
After the ingestion of oocysts by the vertebrate resulting in the so-called “onion skin cysts”.
host, sporocysts are freed, releasing sporozoites. This seems to shield the parasite from the dog’s
It is assumed that the sporozoites cross the gut immune system until merogony is completed and
wall and are carried via the lymphatic system or the cystic structure is ruptured.24 Mature meronts
the bloodstream to tissues throughout the release merozoites, causing local inflammation
body.24 Parasitized host cells have been demon- with an associated systemic reaction and overt
strated lodged between myofibres in a variety illness. Highly vascular granulomas evolve with
of skeletal muscles soon after experimental expo- parasites in macrophages, where presumably
25,26
sure to infective oocysts. The trophozoite gamogony commences.27 Parasites enter leuko-
found in macrophage-like cells (mainly in striated cytes, which subsequently circulate in the blood-
muscle) apparently transforms the host cell stream as gamonts and may be consumed by
into a mucopolysaccharide-producing entity, blood-feeding ixodid ticks.
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No.6 December 2009
Canine hepatozoonosis – a summary for the practitioner CVBD DIGEST 5

Merozoites released
from mature meronts

Merogony Dog Parasite enters

(liver, gut lymph tissue, leukocytes
bone marrow)

Sporozoites enter Gamontogony in

host’s tissue visceral tissue

Intermediate host
Dog ingests Tick ingests
oocysts-harbouring gamont-harbouring
tick leukocytes
Brown Dog tick
Rhipicephalus sanguineus

Mature oocysts Gametogenesis and

in hemocoel fertilization

Definitive host

Sporogony

Fig. 3 Life cycle of Hepatozoon canis (modified after reference 24). After transmission to the vertebrate host (left side), mero-
gony and gamonotogony both take place in the host’s (i.e. dog) tissues. The transmission of gamonts to ticks via blood
feeding initiates sporogony inside the tick and development continues until the sporozoites mature and become infective.

Sporogony – Development in invertebrates zygote gives rise to one oocyst containing hundreds
After ingestion by the tick, gamonts are released of sporocysts and thousands of sporozoites.
from the canine leukocytes. As shown for H. ameri- For H. canis, it has been suggested that sexual devel-
canum, some time later zygotes can be observed opment can take place outside the tick gut lumen.20
within cells of the tick gut’s wall. Sporogony then
follows within the tick gut cells, eventually giving
rise to oocysts packed with hundreds of sporocysts, Vertical transmission
each containing sporozoites.28 The host cell is dis-
torted; some oocysts become dislodged and remain As well as ingestion of sporocyst-containing oocysts
in the tick’s body cavity (hemocoel). From there, they within ticks and cystozoites in muscle tissue of rats
are discharged mechanically when the tick’s body is (as in the case of H. americanum), transplacental trans-
ruptured on ingestion by a vertebrate host. A single fer of H. canis has been reported in Japanese dogs.29
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6 CVBD DIGEST No.6 December 2009
Canine hepatozoonosis – a summary for the practitioner

Host range
(invertebrate and vertebrate) Distribution

Although the invertebrate host for H. canis has been For H. canis the distribution of the definitive host,
known for a century to be the Brown Dog tick, the Brown Dog tick, is decisive. This tick is found
Rhipicephalus sanguineus8, several attempts to in temperate and tropical regions worldwide, and
transmit H. americanum with R. sanguineus have the infection with H. canis has been identified in
not been successful. Other ixodid ticks were also many parts of the Old and some regions in the New
studied (Dermacentor variabilis, Amblyomma amer- World (see Fig. 4). More recently, infections with
icanum), until the nymphs of Amblyomma mac- H. canis have also been detected in the USA in dogs
ulatum, the Gulf Coast tick, were found to be (see Tab. 1) either as single or as co-infections with
consistently susceptible to infection with H. ameri- H. americanum.40 This refutes the general assump-
canum.30 The invertebrate host range of H. ameri- tion that H. canis is not present in North America.
canum seems to be narrower than that of the
vertebrate host. The distribution of the definitive host and tick
Reports from South America suggest that the vector of H. americanum, the Gulf Coast tick
Cayenne tick, Amblyomma cajennense, is a possible (A. maculatum), was historically limited to areas
vector for Hepatozoon spp.24 Japanese scientists along the Gulf Coast and southern Atlantic coast41
have found oocysts in Haemaphysalis spp. taken of North America. Recently, the range of the Gulf
from dogs with hepatozoonosis31, but it is unclear Coast tick has expanded northwards42,43 and the
whether they are those of H. canis or a different pathogen has also been reported from states outside
Hepatozoon spp. In dogs, the only Hepatozoon the recognized range of A. maculatum40, presumably
species described to date are H. canis and H. amer- due to relocation of infected dogs from endemic
icanum.19 In other vertebrate hosts, various Hepato- areas.19
zoon species have been detected in grey squirrels32,
raccoons33, bobcats, ocelots34 and in a crab-eating
fox from Brazil.35

INFO BOX 2
CURRENT ASSUMPTIONS ON H. AMERICANUM EPIDEMIOLOGY
Naturally occurring infection with Hepatozoon ticks and a vertebrate host such as rodents.
americanum in wild rodents, rabbits or verte- One hypothesis is that H. americanum is a newly
brates other than canids has not yet been de- emerged species and that its appearance in co-
10,19,36
monstrated, despite testing in endemic areas. yotes and dogs is a recent event.39 Dogs are not
Coyotes (Canis latrans) have been reported to be among the favored hosts for A. maculatum, but
naturally infected with H. americanum10,37,38 and all three life stages will feed to repletion on this
may be an important component of the emerg- host under experimental conditions. There is evi-
ing problem in domestic dogs.38 It remains to be dence that birds are hosts for larvae and nymphs,
determined whether both coyotes and domestic and that small mammals such as mice, wood rats,
dogs are simply being inserted into an already voles, lagomorphs, and shrews are also hosts for
existing enzootic cycle involving Gulf Coast these immature stages.39
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No.6 December 2009
Canine hepatozoonosis – a summary for the practitioner CVBD DIGEST 7

No occurrence
Endemic occurrence

Fig. 4 Geographical distribution of canine hepatozoonosis in different parts of the world. Countries where the endemic occurence
has been reported are highlighted in red. The data was gathered by Bayer HealthCare Animal Health from recent scientific
publications to provide a comprehensive picture of the endemic situation of several CVBDs including hepatozoonosis in Asia-
Pacific (Fig. 4a), Europe (Fig. 4b) and Latin America (Fig. 4c). More specific regional information can be obtained from
www.cvbd.org.

Total H. americanum Total %

State specimens H. americanum H. canis and H. canis positive

Alabama 268 83 6 9 36.6%

Georgia 63 18 2 0 31.8%

Mississippi 56 23 3 4 53.6%

Texas 50 12 0 0 24.0%

Louisiana 42 10 0 1 26.2%

Oklahoma 17 9 0 0 52.9%

North Carolina 16 1 0 0 6.3%

Virginia 10 1 1 0 20.0%

Othersa 92 10 2 0 13.0%

Total 614 167 14 14 31.8%

a) Includes specimens from the remaining 20 states from which less than 10 specimens were submitted, and specimens with unidentifiable sources.

Table 1 Geographical distribution of Hepatozoon spp.-positive blood samples in the continental United States. Hepatozoon spp.
have been amplified from the specimens using 18 S rDNA Fluorescence Resonance Energy Transfer-PCR. In California,
Kentucky, Nebraska, North Carolina, Oklahoma, Texas, Vermont, and Washington, H. americanum, but not H. canis, was
detected (after reference 40).

infection with other tick-borne pathogens (such as

Clinical presentation Ehrlichia, Babesia etc.). Leukocyte counts are usu-
ally normal or slightly elevated and numerous
H. canis infection ranges from a subclinical state in gamonts can be observed, infecting up to 100% of
dogs with low level parasitemia, to a severe life- neutrophils.44 Only rarely do H. canis-infected dogs
threatening illness with fever, lethargy, anemia, and display osteoproliferative lesions,45 in contrast to
emaciation in dogs with high parasitemia22 or co- H. americanum infected animals.
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Canine hepatozoonosis – a summary for the practitioner

INFO BOX 3 In addition, muscle biopsy might reveal the existence

of the characteristic morphologic feature referred
CLINICAL PRESENTATION to as a “wheel spoke” arrangement of merozoites
within the meront.23 In contrast, H. americanum
OF AMERICAN CANINE
meronts form “onion-skin cysts”.
HEPATOZOONOSIS (ACH)
Hepatozoon americanum infected dogs usually Treatment
present with fever, myalgia, myasthenia, and
wasting. They suffer from severe pain and are There are no substances reported to eliminate all the
different developmental stages of Hepatozoon sp.
often reluctant to move. Dogs may also exhibit
making the aim of chemotherapy the alleviation of
mucopurulent ocular discharge46 and usually clinical signs. For H. canis, combination therapy of
there is generalized periosteal exostosis of imidocarb diproprionate and tetracyclines or tetra-
cycline hydrochloride has been shown to achieve clin-
bones on radiograph, and myositis of striated
ical cure. However, because of very slow elimination
muscles.47,48 Hematological examination typi- of gamonts in the peripheral blood, in certain cases
cally reveals leucocytosis (15,000 to more than imidocarb diproprionate had to be administered
200,000/µl), with a mature neutrophilia over eight weeks.51
Treating dogs with ACH is often frustrating, due to
(greater than 95%)39, but gamonts are only
frequent relapses that may result in exacerbating
rarely found in blood smears. episodes of disease. Additionally with each relapse,
the chances of developing complications of glomeru-
lonephropathy, amyloidosis, vasculitis, and cachexia
increase.49 A treatment protocol has been developed
that is effective in alleviating overt disease. It con-
Diagnosis sists of a combination of trimethoprim-sulfadiazine,
clindamycin, and pyrimethamine (TCP), administered
The following diagnostic tests apply mainly to daily for 14 days. This is followed by the long term
H. canis. For H. americanum, some information is use of decoquinate, an effective anticoccidial drug. If
provided in the final table. For more information, the the protocol is not strictly adhered to, relapse is likely
corresponding literature should be referred to.24,40,49 to occur within weeks to months after decoquinate
treatment is discontinued.52
• Blood smears: Parasitemia is usually distinct so Apart from this antiprotozoal regimen, supportive
that diagnosis of infection can readily be con- care with nonsteroidal anti-inflammatory (NSAIDs)
firmed by examination of blood smears. drugs is very important. Some dogs with undiag-
• Serology: Baneth and colleagues have reported nosed ACH may recover as a result of good care by
the use of a serological test for the diagnosis of owners.24
H. canis hepatozoonosis in Israel.50
• PCR: Recently a real-time PCR has been devel-
oped using EDTA-whole blood samples and Prevention
subsequently fluorescence resonance energy
transfer (FRET) probes to detect a signature poly- As in all canine vector-borne diseases (CVBD), control
morphism in the amplified DNA. This combined of the vector is of major importance in prevention of
test system differentiates between H. canis and disease by minimizing the risk of disease transmis-
H. americanum and reveals single target nucleic sion. This is also the case for Hepatozoon spp. infec-
acid copies in a PCR sample derived from an tion, even though the way of transmission is not via
aliquot of ~140 µl canine blood with essentially the tick’s saliva, but mainly via ingestion of infected
100% specificity.40 ticks. Since dogs may ingest ticks while grooming
their fur, the use of an ectoparasiticide that repels
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No.6 December 2009
Canine hepatozoonosis – a summary for the practitioner CVBD DIGEST 9

and kills ticks is advantageous. A broad spectrum ec- roaming outdoors. With respect to H. americanum,
toparasiticide will also minimise the risk for other dogs in endemic areas should be restricted from
CVBDs transmitted by sandflies, fleas or mosquitoes. eating raw meat or organs from wildlife, that could
Additionally, owners should frequently examine their possibly be infected.
dogs to remove ticks, particularly after hunting or

Hepatozoon canis Hepatozoon americanum

Tick vector Rhipicephalus sanguineus Amblyomma maculatum
(Brown Dog tick) (Gulf Coast tick)

Primary clinical signs frequently asymptomatic; can cause fever, pain, lameness, mucopurulent
lethargy, fever, weight loss ocular discharge; may wax and wane

Common laboratory anemia; extreme leukocytosis is rare extreme leukocytosis (20,000 –

abnormalities but may be seen in dogs with very 200,000 leukocytes/µm3), anemia,
high parasitemia elevated alkaline phosphatase,
low glucose

Concurrent infection very common occasional

or immunosuppression

Geographic distribution Africa, Middle East, Asia, Southern United States, Central and South
Europe, South America recently America
United States40

Tissue stages “wheel-spoked” meronts found meronts exhibit blastophore

primarily in spleen, bone marrow, formation muscle lesions consisting
lymph nodes of “onion-skin” cysts, meronts,
pyogranulomas, myositis

Radiographic lesions none (except one case reported periosteal proliferation

in Japan)

Severity of disease subclinical to severe, usually mild; severe; guarded prognosis

good prognosis

Frequency of gamonts common parasitemia 1–100% rare; parasitemia usually <0.1%

in peripheral blood (usually ~1%)

Gamont characteristics size: 11.0 x 4.3 µm; ultrastructure: size: 8.8 x 3.9 µm; ultrastructure: tail-
fine fibril-like structure surrounding like appendage; lacks fine fibril-like
parasitophorous vacuole structure seen in H. canis

Antibodies H. canis IFA shows high frequency of H. americanum IFA shows good corre-
antibodies in general dog population lation with muscle biopsy and low
(Israel) cross-reactivity on H. canis IFA

PCR real-time PCR on EDTA-whole blood real-time PCR on EDTA-whole blood

samples, with subsequent use of FRET samples, with subsequent use of FRET
probes, differentiating between probes, differentiating between
H. canis and H. americanum; 100% H. canis and H. americanum; 100%
specifity40 specifity40

Treatment imidocarb diproprionate, doxycycline trimethoprim/sulfadiazine, pyrime-

thamine, clindamycin, decoquinate

Table 2 Comparison of Hepatozoon americanum and Hepatozoon canis (modified after reference 49)

IFA: immunofluorescent antibody; FRET: fluorescence resonance energy transfer

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Canine hepatozoonosis – a summary for the practitioner

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* Members of the CVBD World Forum
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CVBD DIGEST No.6 December 2009
Canine hepatozoonosis – a summary for the practitioner

www.cvbd.org