A Review of Methods for Detection of Hepatozoon Infection in Carnivores and Arthropod Vectors

Abstract

Vector-borne protists of the genus Hepatozoon belong to the apicomplexan suborder Adeleorina. The taxonomy of Hepatozoon is unsettled and different phylogenetic clades probably represent evolutionary units deserving the status of separate genera. Throughout our review, we focus on the monophyletic assemblage of Hepatozoon spp. from carnivores, classified as Hepatozoon sensu stricto that includes important pathogens of domestic and free-ranging canine and feline hosts. We provide an overview of diagnostic methods and approaches from classical detection in biological materials, through serological tests to nucleic acid amplification tests (NAATs). Critical review of used primers for the 18S rDNA is provided, together with information on individual primer pairs. Extension of used NAATs target to cover also mitochondrial genes is suggested as a key step in understanding the diversity and molecular epidemiology of Hepatozoon infections in mammals.

Introduction

The apicomplexan genus Hepatozoon (apicomplexan suborder Adeleorina, family Hepatozooidae Wenyon, 1926) includes more than 300 species infecting a wide range of vertebrates from amphibians to mammals (Smith 1996, Baneth 2011). In recent phylogenetic studies, the genus Hepatozoon is described as paraphyletic and its split into a number of distinct genera has been suggested (Barta et al. 2012, Karadjian et al. 2015).

The life cycle of Hepatozoon sensu lato is heteroxenous, and a range of invertebrates (argasid and ixodid ticks, mosquitoes, fleas, triatomines, tsetse flies, and biting lice) serve as vectors for individual species. The vertebrate host is usually infected by ingestion of a hematophagous arthropod, although infection can be acquired also by intrauterine transmission or by predation. Infection through ingestion of an infected invertebrate vector is unlikely for many Hepatozoon s.l. from reptiles, where development can be alternatively completed through a three-host life cycle along an arthropod—paratenic host—reptile axis (Landau 1973; Sloboda et al. 2007).

In cases of infection via ingesting an infected invertebrate host, the sporozoites released from sporocysts within oocysts enter the circulation and migrate to various organs (liver, bone marrow, spleen, kidneys, lungs, intestines, and lymph nodes) where they undergo merogony. When merozoites are released from mature meronts into the bloodstream, they enter the blood cells and transform into gamonts, which are later ingested with the blood meal by a competent vector. Subsequent development includes the production of microgametes and macrogametes (in different parts of vector organs) that fuse and develop to polysporocystic oocysts (Smith 1996, Kim et al. 1998, O'Dwyer 2011).

The taxonomy of Hepatozoon s.l. is unsettled and different phylogenetic clades probably represent evolutionary units deserving the status of separate genera. Throughout our review, we focus on the monophyletic assemblage of Hepatozoon spp. from carnivores, classified as Hepatozoon sensu stricto by Karadjian et al. (2015).

Species of Importance

Hepatozoon spp. has been described in a range of mammalian hosts, mainly carnivores and rodents. While the infections in most mammalian hosts are typically subclinical, species that infect canid and felid carnivores (Hepatozoon americanus, Hepatozoon canis, Hepatozoon felis) are of veterinary importance. Currently, the ubiquitous presence of H. canis and closely related species in European foxes is attracting considerable attention (Najm et al. 2014, Hodžić et al. 2015) inasmuch as the main tick vector of this species, Rhipicephalus sanguineus, is not endemic in some areas where infection is abundant in local foxes.

H. canis infections in domestic dogs can vary from subclinical to severe, with animals manifesting extreme lethargy, cachexia, and anemia (Baneth and Weigler 1997). Infection with this species or its variants in wildlife canids has not been clearly associated with clinical disease to date. In contrast, Hepatozoon americanum infection of domestic dogs is associated with severe disease characterized by muscular pain induced by myositis, severe lameness, and subsequent muscle atrophy. Infections with H. felis primarily target striated muscle in domestic cats, but these also are mainly subclinical (Baneth et al. 2013). Nevertheless, infection with this species or a closely related variant has resulted in severe myositis in a wild Pampas gray fox (Giannitti et al. 2012).

In carnivores, vectors have been identified only for two Hepatozoon species: R. sanguineus and Amblyomma ovale for H. canis (Baneth et al. 2001, 2007, Forlano et al. 2005) and Amblyomma maculatum for H. americanum (Mathew et al. 1998). The diversity of Hepatozoon species in carnivores is probably more complex than is presently known because considerable DNA heterogeneity in conserved gene sequences of Hepatozoon from carnivore hosts is frequently seen (Starkey et al. 2013). However, a lack of data on different manifestations of infection, life cycle variations, antigenic variability, and vector competence constitute a serious drawback for further taxonomic work.

Hepatozoon Detection in Blood, Tissues, and Invertebrate Vectors

Due to their complex life cycle, stages of Hepatozoon can be detected in a variety of tissues. In vertebrate hosts, blood is the most commonly used material for intravital diagnostics. Inasmuch as the gamonts are mostly present in white blood cells within mammalian hosts, however, using the buffy coat layer for microscopic and molecular diagnostics increases the sensitivity of detection (Sasanelli et al. 2010, Otranto et al. 2011). Depending on the given Hepatozoon species, life stages can be further detected by cytology or biopsy histopathology in the bone marrow, spleen, lymph nodes, or striated muscle. At necropsy, almost any parenchymal tissue can be used for parasite detection by nucleic acid amplification tests (NAATs). The spleen is nevertheless the primary target organ for the parasite's detection (Baneth et al. 2007). However, the ability of some Hepatozoon spp. to survive in tissues of hosts accidentally infected by ingestion of other infected hosts (e.g., in bats after presumed ingestion of infected arthropods) complicates the interpretation of PCR positivity in tissues (Pinto et al. 2013, Karadjian et al. 2015). Polysporocystic oocysts (or DNA) of Hepatozoon species can also be detected in bloodsucking arthropod vectors. Detection of DNA in questing ticks is highly suggestive for the vectorial role. On the contrary, the presence of Hepatozoon DNA in blood-engorged arthropods should not be considered proof of their vectorial capacity unless mature oocysts are demonstrated microscopically (Giannelli et al. 2013).

Diagnosis by Microscopy

The traditional direct diagnostic methods for rapid detection of Hepatozoon include thick and thin blood smears and buffy coat smear (Fig. 1). These methods are effective only in animals with high parasitemia. Sensitivity is noticeably diminished for subclinical infections and for those Hepatozoon species (such as H. americanum) that typically produce very low parasitemia levels during clinical disease (Vincent-Johnson et al. 1997). Romanowsky stains, including Giemsa stains and such commercial rapid stains as Diff-Quick, are suitable for detecting Hepatozoon spp. in blood. Hepatozoon life stages can be detected also in histopathology sections of tissues demonstrating the architecture of the parasite's tissue stages in a clear manner. Histopathology is also helpful in characterizing inflammatory response to infection with different Hepatozoon species in distinctive hosts (Klopfer et al. 1973, Baneth and Weigler 1997, Vincent-Johnson et al. 1997, Baneth et al. 2013).

FIG. 1.

Hepatozoon gamonts in white blood cells manifested in blood smears remain the keystone for clinical diagnosis of infected mammalian hosts. (a) Hepatozoon canis in a dog from Romania, (b) Hepatozoon felis in the blood of a Kenyan cat, (c) Hepatozoon sp. in buffy coat smear from a Romanian red fox. Stained by Giemsa stain (a, b) and Diff-Quick (c). Scale bar = 20 μm, all images is same scale. Color images available online at www.liebertpub.com/vbz

Serological Tests

Serological assays have been developed for detecting antibodies for Hepatozoon spp. in dogs. An indirect fluorescent antibody (IFA) test for anti-H. canis antibodies using gamont antigen has been used in epidemiological studies in Israel, Japan, and Turkey (Shkap et al. 1994, Baneth et al. 1996, Inokuma et al. 1999, Karagenc et al. 2006), and an IFA assay using sporozoite antigens derived from ticks has been developed for detecting anti-H. americanum antibodies. The latter assay was found to be as sensitive as a muscle biopsy in diagnosing this infection (Mathew et al. 2000). Furthermore, an enzyme-linked immunofluorescence assay (ELISA) for anti-H. canis antibodies based on gamont antigen has also been developed (Gonen et al. 2004). A low degree of cross-reactivity was found when using the H. canis ELISA with sera of dogs infected with H. americanum (Gonen et al. 2004). In the past decade, serological tests have largely been replaced by NAATs.

NAATs for Hepatozoon species

Similar to other vector-borne protists, molecular diagnostic approaches relying mainly on the amplification of 18S rRNA gene fragments have prevailed during the past two decades in Hepatozoon detection and genetic characterization in mammals, while studies on reptilian Hepatozoon s.l. use ITS-1 as an additional or alternative target for both diagnostic PCR assays and phylogenetic analyses (Kim et al. 1998, Boulianne et al. 2007). To date, sequences of the 18S rRNA gene comprise the only available genetic data from Hepatozoon species infecting carnivores (Table 1, Fig. 2). The major bulk of sequences originating from different host species are ca 250–700 bp in length, and suggests a monophyletic character of Hepatozoon species from carnivores (Criado-Fornelio et al. 2006, Barta et al. 2012, Najm et al. 2014). However, the variability of the 18S rDNA within Hepatozoon species combined with the short length of the sequences used in some phylogenetic analyses complicate definitions of species status among Hepatozoon spp. (see e.g., Karadijan et al. 2015). Neighbor-joining analysis of partial 18S RNA sequences has revealed five groups of H. canis isolates from dogs based on individual point mutations, thereby demonstrating the variability of H. canis within the microenvironment in Croatia (Vojta et al. 2009). Foxes from the same country showed less variability (Dežđek et al. 2010).

FIG. 2.

Length and position of 18S rDNA fragments amplified by various primer pairs. The nucleotide positions are extrapolated according to the reference sequence of H. canis AY461378 (shown in gray). Color images available online at www.liebertpub.com/vbz

Table 1.

Examples of Primers for PCR Amplification of 18S Sequences of Hepatozoon spp.

Primer name	Sequence (5′ → 3′)	Product size (bp) ^a	Nested PCR ^b	Specific ^c	Reference ^d
BTHL1	CCA GCA GCC GCG GTA ATT C	380	No	No	Tabar et al. (2008)
BTHL2	CTT TCG CAG TAG TTY GTC TTT AAC AAA TCT
HepF	ATA CAT GAG CAA AAT CTC AAC	670	No	Yes	Inokuma et al. (2002)
HepR	CTT ATT ATT CCA TGC TGC AG
HAM-1	GCC AGT AGT CAT ATG CTT GTC	1760	No	No	Criado-Fornelio et al. (2006)
HPF-2	GAC TTC TCC TTC GTC TAA G
HEP-1	GCG CAA ATT ACC CAA TT	660	No	Yes	Criado-Fornelio et al. (2006)^*
HEP-4	TAA GGT GCT GAA GGA GTC GTT TAT
BT1-F	GGT TGA TCC TGC CAG TAG T	430	No	No	Criado-Fornelio et al. (2003)
BT1-R	GCC TGC TGC CTT CCT TA
BTH-1F	CCT GMG ARA CGG CTA CCA CAT CT	750	Yes	No	Criado- Fornelio et al. (2003)
BTH-1R	TTG CGA CCA TAC TCC CCC CA
RLB-F	GAG GTA GTG ACA AGA AAT AAC AAT A	550	No	No	Majláthová et al. (2007) and Gubbels et al. (1999)
RLB-R	TCT TCG ATC CCC TAA CTT TC
H14Hepa18SFw	GAA ATA ACA ATA CAA GGC AGT TAA AAT GCT	620	Yes	Yes	Hodžic et al. (2015)
H14Hepa18SRv	GTG CTG AAG GAG TCG TTT ATA AAG
GF2	GTC TTG TAA TTG GAA TGA TGG	610	Yes	No	Zintl et al. (2011)
GR2	CCA AAG ACT TTG ATT TCT CTC
4558F	GCT AAT ACA TGA GCA AAA TCT CAA	1180	No	Yes	Mathew et al. (2000) and Wozniak et al. (1994)
2773R	CGG AAT TAA CCA GAC AAA T
Piroplasmid F	CCA GCA GCC GCG GTA ATT	390	No	No	Baneth at al. 2013
Piroplasmid R	CTT TCG CAG TAG TTY GTC TTT AAC AAA TCT
KIM18SF	GAA ATT AGA GTG TTT C	600	No	No	Williams et al. (2014)
KIMR2	ACC CTA TTT AGC AGG TTA AG
Piro A1	AGG GAG CCT GAG AGA CGG CTA CC	520	No	No	O'Dwyer et al. (2009) and Jefferies et al. (2003)
Piro B	TTA AAT ACG AAT GCC CCC AAC
BmF1	GCG ATG TAT CAT TCA AGT TTC TG	1160	Yes	No	Simpson et al. (2005)
BmR1	TGT TAT TGC CTT ACA CTT CCT TGC
BmF2	ACGGCT ACCACATCTAAGGAAGGC	700	Yes	No	Simpson et al. (2005)
BmR2	TCTCTCAAGGTGCTGAAG GA
Babesia-F	GTG AAA CTG CGA ATG GCT CA	740	No	No	Oyamada et al. (2005)
Babesia-R	CCA TGC TGA AGT ATT CAA GAC

Expected approximate size of PCR product for Hepatozoon spp. sequence.

Primers used in cited literature in nested PCR protocol.

Yes = primers specific only for Hepatozoon spp. sequences; no = primers with broader range of amplified species.

Reference to first use of primers for Hepatozoon spp. sequence amplification, original annotation of primers in brackets.

Sequence of primer HEP-1 was originally published as 5′-CGC GAA ATT ACC CAA TT-3′. As this very probably contains a mistyping error, we suggest here the correct sequence to anneal to Hepatozoon 18S.

Molecular detection of Hepatozoon spp. is highly influenced by the specificity of the primer pair and the PCR cycle conditions that are used. Most of the primers used are universal enough to amplify either a broad range of apicomplexan protists (e.g., BTHL 1 and 2, BTH-1F and −1R, BmF and R) or piroplasmids (e.g., Piroplasmid F and R, Piro A1 and B, BT1F and R). It should be recognized that several primer pairs amplify Hepatozoon 18S despite mismatches in target sequence (GF2 and GR2, BmF and R, Babesia-F and -R) (Table 1). For this reason, it is always recommended to confirm the identity of PCR products by sequencing a representative amount of samples from each particular set. The difference of ca 40 bp in length of the 18S PCR products for piroplasmids and Hepatozoon species can be helpful in distinguishing them when universal primers are used (Fig. 3).

FIG. 3.

Demonstration of H. canis and Babesia canis 18S PCR products amplified by nested PCR using nonspecific primers (BTH-1F, BTH-1R, and GF2, GR2), showing ca 40 bp length difference between the amplicons; 100 bp DNA Ladder (New England BioLabs).

The choice of primers should reflect the purpose and design of the particular study. For diagnostic PCR, specific primers amplifying short PCR products are recommended (e.g., H14Hepa18SFw and Rv, Hep-1 and -4, HepF and R). High sensitivity can be achieved using a nested PCR protocol (e.g., a combination of universal BTH-1F and −1R with more specific Hep-1 and -4 or H14Hepa18SFw and Rv; Fig. 2), but this may come at the cost of increased contamination risk. For phylogenetic analyses and species determination, amplification of longer fragments (using, e.g., HAM1-HPFR, 4558F −2773) is advisable (Table 1).

Quantitative PCR

In recent years, quantitative PCR (qPCR) has been frequently used in studies because it enables quantification and detection of mixed infections from various biological sources, without laborious evaluation of results by gel electrophoresis. This notably has not been the case, however, for studies of Hepatozoon species. Only two qPCRs assays targeting the conserved 18S rRNA gene have been developed to estimate the prevalence and intensity of Hepatozoon infection in carnivore species (Criado-Fornelio et al. 2007, Li et al. 2008). Using the primers HEP1 and HEP2, Criado-Fornelio et al. (2007) failed to separate species infecting canids and felids by melting peak temperature analysis, while qPCR combined with FRET revealed single target nucleic copies with 100% specificity and enabled differentiation of dog species (H. canis and H. americanum) from canine blood (Li et al. 2008, Kelly et al. 2013). The first and to date only qPCR protocol based on melting curve analysis that enables differentiation between mixed infection of adeleorinid (Hepatozoon s.l.) and eimeriorinid (Schellackia and Lankesterella) protists was developed to study their presence in reptiles (Maia et al. 2014).

Relevance of Various Detection Techniques for Clinical Diagnosis in Dogs and Cats

The assays used to diagnose Hepatozoon infections in dogs and cats include detection of gamonts in blood smears, cytology of tissues, serology, and PCR. While detection by microscopy of blood smears is easy and rapid when parasitemia is sufficient, as in most clinical infections with H. canis, it is not sensitive enough to detect H. americanum and H. felis infections. Furthermore, follow-up treatment for H. canis should be carried out using PCR and buffy coat microscopy because detection by microscopy is considerably less sensitive than is PCR (Sasanelli et al. 2010, De Tommasi et al. 2014). A study from Turkey demonstrated that detection of H. canis by PCR is far more sensitive than is examination of blood by light microscopy. In that study, the prevalence of infection among 349 dogs was 10.6% according to blood smear evaluation and 25.8% according to blood PCR (Karagenc et al. 2006). Additionally, a comparative study on the bone marrow, blood, and buffy coat of dogs infected with H. canis has found that buffy coat and blood are the best tissues for detecting H. canis infection in dogs using PCR (Otranto et al. 2011). Sensitive and specific real-time qPCR assays are able to detect both H. americanum and H. canis while distinguishing between the two species (Criado-Fornelio et al. 2007, Li et al. 2008). Serological assays for canine hepatozoonosis are indicative of exposure to the parasite and can be used for epidemiological surveys, but they lack the sensitivity and specificity of real-time PCR.

Conclusions and Future Perspectives

A plethora of studies employing molecular tools for detecting Hepatozoon s.l. in domestic and free ranging mammals have been published in the past decade. To date, both molecular diagnosis and phylogenetic analyses rely solely on nuclear 18S rDNA data. Extending the range of available molecular markers to plastid or mitochondrial genes will greatly improve the understanding of diversity within the genus Hepatozoon and intergeneric relationships between individual clades of hemogregarines. The recently published mitochondrial genome of Hepatozoon (Bartazoon) catesbianae (Leveille et al. 2014) opened a range of opportunities in the search for alternative phylogenetic markers and diagnostic primers also for mammalian Hepatozoon spp. Inasmuch as the prevalence assessed by PCR in clinically healthy dogs and cats can reach more than 25% in areas of endemic occurrence, and because extremely sensitive diagnostic PCR also detects subclinical carriers, direct demonstration of gamont presence in circulating white blood cells (in blood smears or buffy coat smears) remains an important clinical diagnostic tool.

Footnotes

Acknowledgments

The international collaboration of all authors was facilitated by COST Action TD1303. The University of Veterinary and Pharmaceutical Sciences team was supported by grant COST CZ LD14048 and the project CEITEC 2020 (LQ1601) with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Programme II. The microphotographs of Hepatozoon were kindly provided by Martina Gallusová (Fig. 1b) and Andrei Mihalca (Fig. 1c). Barbora Mitková kindly contributed to the review of primers used in the literature ().

Author Disclosure Statement

No competing financial interests exist.

References

Barta

, Ogedengbe

, Martin

, Smith

. Phylogenetic position of the adeleorinid coccidia (Myzozoa, Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) inferred using 18S rDNA sequences. J Eukaryotic Microbiol, 2012; 59:171–180.

Baneth

. Perspectives on canine and feline hepatozoonosis. Vet Parasitol, 2011; 181:3–11.

Baneth

, Samish

, Alekseev

, Aroch

, et al. Transmission of Hepatozoon canis to dogs by naturally-fed or percutaneously injected Rhipicephalus sanguineus ticks. J Parasitol, 2001; 87:606–611.

Baneth

, Samish

, Shkap

. Life cycle of Hepatozoon canis (Apicomplexa: Adeleorina: Hepatozoidae) in the tick Rhipicephalus sanguineus and domestic dog (Canis familiaris). J Parasitol, 2007; 93:283–299.

Baneth

, Sheiner

, Eyal

, Hahn

, et al. Redescription of Hepatozoon felis (Apicomplexa: Hepatozoidae) based on phylogenetic analysis, tissue and blood form morphology, and possible transplacental transmission. Parasit Vectors, 2013; 6:102.

Baneth

, Shkap

, Presentey

, Pipano

. Hepatozoon canis: The prevalence of antibodies and gamonts in dogs in Israel. Vet Res Commun, 1996; 20:41–46.

Baneth

, Weigler

. Retrospective case-control study of hepatozoonosis in dogs in Israel. J Vet Intern Med, 1997; 11:365–370.

Boulianne

, Evans

, Smith

. Phylogenetic analysis of Hepatozoon species (Apicomplexa: Adeleorina) infecting frogs of Nova Scotia, Canada, determined by ITS-1 sequences. J Parasitol, 2007; 93:1435–1441.

Criado-Fornelio

, Buling

, Cunha-Filho

, Ruas

, et al. Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp. Vet Parasitol, 2007; 150:352–356.

10.

Criado-Fornelio

, Martinez-Marcos

, Buling-Saraña

, Barba-Carretero

. Molecular studies on Babesia, Theileria and Hepatozoon in southern Europe: Part I. Epizootiological aspects. Vet Parasitol, 2003; 113:189–201.

11.

Criado-Fornelio

, Ruas

, Casado

, Farias

NAR

, et al. New molecular data on mammalian Hepatozoon species (Apicomplexa: Adeleorina) from Brazil and Spain. J Parasitol, 2006; 92:93–99.

12.

De Tommasi

, Giannelli

, de Caprariis

, Ramos

RAN

, et al. Failure of imidocarb dipropionate and toltrazuril/emodepside plus clindamycin in treating Hepatozoon canis infection. Vet Parasitol, 2014; 200:242–245.

13.

Dežđek

, Vojta

, Ćurković

, Lipej

, et al. Molecular detection of Theileria annae and Hepatozoon canis in foxes (Vulpes vulpes) in Croatia. Vet Parasitol, 2010; 172:333–336.

14.

Forlano

, Scofield

, Elisei

, Fernandes

, et al. Diagnosis of Hepatozoon spp. in Amblyomma ovale and its experimental transmission in domestic dogs in Brazil. Vet Parasitol, 2005; 134:1–7.

15.

Giannelli

, Ramos

RAN

, Dantas-Torres

, Mencke

, et al. Experimental evidence against transmission of Hepatozoon canis by Ixodes ricinus . Ticks Tick Borne Dis, 2013; 4:391–394.

16.

Giannitti

, Diab

, Uzal

, Fresneda

, et al. Infection with a Hepatozoon sp. closely related to Hepatozoon felis in a wild Pampas gray fox (Lycalopex–Pseudalopex–gymnocercus) co-infected with canine distemper virus. Vet Parasitol, 2012; 186:497–502.

17.

Gonen

, Strauss-Ayali

, Shkap

, Vincent-Johnson

, et al. An enzyme-linked immunosorbent assay for antibodies to Hepatozoon canis . Vet Parasitol, 2004; 122:131–139.

18.

Gubbels

, de Vos

, van der Weide

, Viseras

, et al. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J Clin Microbiol, 1999; 37:1782–1789.

19.

Hodžić

, Alić

, Fuehrer

, Harl

, et al. A molecular survey of vector-borne pathogens in red foxes (Vulpes vulpes) from Bosnia and Herzegovina. Parasit Vectors, 2015; 8:88.

20.

Inokuma

, Ohno

, Yamamoto

. Serosurvey of Ehrlichia canis and Hepatozoon canis infection in dogs in Yamaguchi Prefecture, Japan. J Vet Med Sci, 1999; 61:1153–1155.

21.

Inokuma

, Okuda

, Ohno

, Shimoda

, et al. Analysis of the 18S rRNA gene sequence of a Hepatozoon detected in two Japanese dogs. Vet Parasitol, 2002; 106:265–271.

22.

Jefferies

, Ryan

, Muhlnickel

, Irwin

. Two species of canine Babesia in Australia: Detection and characterization by PCR. J Parasitol, 2003; 89:409–412.

23.

Karadjian

, Chavatte

, Landau

. Systematic revision of the adeleid haemogregarines, with creation of Bartazoon n. g., reassignment of Hepatozoon argantis Garnham, 1954 to Hemolivia, and molecular data on Hemolivia stellata . Parasite, 2015; 22:31.

24.

Karagenc

, Pasa

, Kirli

, Hosgor

, et al. A parasitological, molecular and serological survey of Hepatozoon canis infection in dogs around the Aegean coast of Turkey. Vet Parasitol, 2006; 135:113–119.

25.

Kelly

, Xu

, Lucas

, Loftis

, et al. Ehrlichiosis, babesiosis, anaplasmosis and hepatozoonosis in dogs from St. Kitts, West Indies. PLoS One, 2013; 8:e53450.

26.

Kim

, Smith

, Desser

. The life history and host specificity of Hepatozoon clamatae (Apicomplexa: Adeleorina) and ITS-1 nucleotide sequence variation of Hepatozoon species of frogs and mosquitoes from Ontario. J Parasitol, 1998; 84:789–797.

27.

Klopfer

, Nobel

, Neumann

. Hepatozoon-like parasite (schizonts) in the myocardium of the domestic cat. Vet Pathol, 1973; 10:185–190.

28.

Landau

. A comparison of the life cycles of Toxoplasma and Hepatozoon, with reference to the general phenomenon and the role of cyst formation in the Coccidia. Ann Trop Med Parasitol, 1973; 67:403–407.

29.

Leveille

, Ogedengbe

, Hafeez

, Tu

, et al. The complete mitochondrial genome sequence of Hepatozoon catesbianae (Apicomplexa: Coccidia: Adeleorina), a blood parasite of the green frog, Lithobates (formerly Rana) clamitans . J Parasitol, 2014; 100:651–656.

30.

, Wang

, Allen

, Little

, et al. Diagnosis of canine Hepatozoon spp. infection by quantitative PCR. Vet Parasitol, 2008; 157:50–58.

31.

Maia

, Harris

, Carranza

, Gómez-Díaz

. A comparison of multiple methods for estimating parasitemia of hemogregarine hemoparasites (Apicomplexa: Adeleorina) and its application for studying infection in natural populations. PLoS One, 2014; 9:e95010.

32.

Majláthová

, Hurníková

, Majláth

, Petko

. Hepatozoon canis infection in Slovakia: Imported or autochthonous?. Vector Borne Zoonotic Dis, 2007; 72:199–202.

33.

Mathew

, Ewing

, Panciera

, Woods

. Experimental transmission of Hepatozoon americanum Vincent-Johnson et al., 1997 to dogs by the Gulf Coast tick, Amblyomma maculatum Koch. Vet Parasitol, 1998; 80:1–14.

34.

Mathew

, Van Den Bussche

, Ewing

, Malayer

, et al. Phylogenetic relationships of Hepatozoon (Apicomplexa: Adeleorina) based on molecular, morphologic, and life-cycle characters. J Parasitol, 2000; 86:366–372.

35.

Najm

, Meyer-Kayser

, Hoffmann

, Pfister

, et al. Hepatozoon canis in German red foxes (Vulpes vulpes) and their ticks: Molecular characterization and the phylogenetic relationship to other Hepatozoon spp. Parasitol Res, 2014; 113:2679–285.

36.

O'Dwyer

. Brazilian canine hepatozoonosis. Rev Bras Parasitol Vet, 2011; 20:181–193.

37.

O'Dwyer

, Lopes

VVA

, Rubini

, Paduan

KDS

, et al. Babesia spp. infection in dogs from rural areas of São Paulo State, Brazil. Rev Bras Parasitol Vet, 2009; 18:23–26.

38.

Otranto

, Dantas-Torres

, Weigl

, Latrofa

, et al. Diagnosis of Hepatozoon canis in young dogs by cytology and PCR. Parasit Vectors, 2011; 4:55.

39.

Oyamada

, Davoust

, Boni

, Dereure

, et al. Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies. Clin Diagn Lab Immunol, 2005; 12:1343–1346.

40.

Pinto

, Helgen

, Fleischer

, Perkins

. Hepatozoon parasites (Apicomplexa: Adeleorina) in bats. J Parasitol, 2013; 99:722–724.

41.

Sasanelli

, Paradies

, Greco

, Eyal

, et al. Failure of imidocarb dipropionate to eliminate Hepatozoon canis in naturally infected dogs based on parasitological and molecular evaluation methods. Vet Parasitol, 2010; 171:194–199.

42.

Shkap

, Baneth

, Pipano

. Circulating antibodies to Hepatozoon canis demonstrated by immunofluorescence. J Vet Diagn Invest, 1994; 6:121–123.

43.

Simpson

, Panciera

, Hargreaves

, McGarry

, et al. Myocarditis and myositis due to infection with Hepatozoon species in pine martens (Martes martes) in Scotland. Vet Rec, 2005; 156:442–446.

44.

Sloboda

, Kamler

, Bulantová

, Votýpka

, et al. A new species of Hepatozoon (Apicomplexa: Adeleorina) from Python regius (Serpentes: Pythonidae) and its experimental transmission by a mosquito vector. J Parasitol, 2007; 93:1189–1198.

45.

Smith

. The genus Hepatozoon (Apicomplexa: Adeleina). J Parasitol, 1996; 82:565–585.

46.

Starkey

, Panciera

, Paras

, Allen

, et al. Genetic diversity of Hepatozoon spp. in coyotes from the south-central United States. J Parasitol, 2013; 99:375–378.

47.

Tabar

, Altet

, Francino

, Sánchez

, Ferrer

, Roura

. Vector-borne infections in cats: Molecular study in Barcelona area (Spain). Vet Parasitol, 2008; 151:332–336.

48.

Vincent-Johnson

, Macintire

, Lindsay

, Lenz

, et al. A new Hepatozoon species from dogs: Description of the causative agent of canine hepatozoonosis in North America. J Parasitol, 1997; 83:1165–1172.

49.

Vojta

, Mrljak

, Ćurković

, Živičnjak

, et al. Molecular epizootiology of canine hepatozoonosis in Croatia. Int J Parasitol, 2009; 39:1129–1136.

50.

Williams

, Berentsen

, Shock

, Teixiera

, et al. Prevalence and diversity of Babesia, Hepatozoon, Ehrlichia, and Bartonella in wild and domestic carnivores from Zambia, Africa. Parasitol Res, 2014; 113:911–918.

51.

Wozniak

, Telford

Jr , Mclaughlin

. Employment of polymerase chain reaction in the molecular differentiation of reptilian haemogregarines and its applications to preventative zoological medicine. J Zoo Wildl Med, 1994; 25:538–547.

52.

Zintl

, Finnerty

, Murphy

, de Waal

, et al. Babesias of red deer (Cervus elaphus) in Ireland. Vet Res, 2011; 42:7.