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Contents lists available at SciVerse ScienceDirect

Veterinary Parasitology

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hort communication

nfection with a Hepatozoon sp. closely related to Hepatozoon felis in aild Pampas gray fox (Lycalopex – Pseudalopex – gymnocercus)

o-infected with canine distemper virus

ederico Giannitti a,∗, Santiago S. Diabb, Francisco A. Uzalb, Karina Fresnedac,aniel Rossid, Dalit Talmi-Franke, Gad Banethe

California Animal Health and Food Safety (CAHFS) Laboratory System, University of California Davis, West Health Sciences Drive, Davis 95616, CA, USACalifornia Animal Health and Food Safety (CAHFS) Laboratory System, University of California Davis, San Bernardino Branch, 105 West Central Avenue, Sanernardino 92408, CA, USACentro de Diagnóstico Veterinario (CEDEVET), Chile 962 (7000), Tandil, Buenos Aires, ArgentinaPrivate Practice, Urquiza 351 (8360), Choele Choel, Rio Negro, ArgentinaSchool of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

r t i c l e i n f o

rticle history:eceived 16 August 2011eceived in revised form 24 October 2011ccepted 1 November 2011

eywords:ampas gray foxepatozoonanine distemper virus (Morbillivirus)rotozoa8S rRNA gene

a b s t r a c t

A species of Hepatozoon closely related to Hepatozoon felis found in the skeletal and cardiacmuscle of a wild Pampas gray fox (Lycalopex gymnocercus) is described. The fox was euth-anized after showing severe incoordination. On necropsy and histopathology there wasbilateral, diffuse, severe, sub-acute, necrotizing bronchointerstitial pneumonia, with intra-cytoplasmic and intranuclear eosinophilic inclusion bodies. Canine distemper virus wasdetected by immunohistochemistry in the bronchiolar epithelium, syncytial cells, alveolarmacrophages and pneumocytes. The skeletal muscle and myocardium contained multipleround to oval protozoan cysts ranging from 64 �m × 75 �m to 98 �m × 122 �m, with acentral eosinophilic meront-like core surrounded by concentric rings of mucinous mate-rial resembling Hepatozoon americanum cysts but smaller in size. Macrophages within rarepyogranulomas and monocytes/macrophages in adjacent sinusoidal blood vessels in theskeletal muscle contained intracytoplasmic round protozoa consistent with merozoites ordeveloping gamonts of Hepatozoon. Hepatozoon sp. infection was confirmed by PCR of skele-tal muscle and the sequenced 18S rRNA PCR product was found to be 99% identical to H. felis

by BLAST analysis and deposited in GenBank as accession number HQ020489. It clusteredtogether in the phylogenetic analysis with published H. felis sequences and separately fromH. canis, H. americanum and other Hepatozoon species. However, the close relatedness of thefox Hepatozoon to H. felis does not rule out infection with a different and possibly unknownHepatozoon species.

. Introduction

Please cite this article in press as: Giannitti, F., et al., Infection wa wild Pampas gray fox (Lycalopex – Pseudalopex – gymnocercus(2011), doi:10.1016/j.vetpar.2011.11.006

Hepatozoon is a genus of protozoan parasites thatelongs to the phylum Apicomplexa which includes more

∗ Corresponding author.E-mail addresses: fgiannitti@yahoo.com, fgiannitti@cahfs.ucdavis.edu

F. Giannitti).

304-4017/$ – see front matter. Published by Elsevier B.V.oi:10.1016/j.vetpar.2011.11.006

Published by Elsevier B.V.

than 300 species that infect mammals, reptiles, birds, andanurans (Smith, 1996). Vertebrates are the intermediatehosts that harbor the merogonic and the gametogoniclife stages of the parasite, which undergoes its sexualreproduction and sporogony in a bloodsucking inverte-

ith a Hepatozoon sp. closely related to Hepatozoon felis in) co-infected with canine distemper virus. Vet. Parasitol.

brate, its definitive host. Ticks, mites, sand flies, tsetseflies, mosquitoes, fleas, lice, reduviid bugs, and leechesare the vectors identified so far (Baneth et al., 2007). Todate, 49 species belonging to the genus Hepatozoon have

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been described in domestic and wild mammals. Infectiontakes place by ingesting the arthropod containing infec-tive sporozoites. No salivary transfer during the courseof the arthropod vector’s blood meal has been demon-strated for Hepatozoon sp. The gamont stage of the parasiteusually develops in mammalian leukocytes (Smith, 1996),whereas the meront stages develop in different visceralorgans which vary according to the infecting species andmay include hematopoietic, cardiac and musculoskeletaltissues (Panciera et al., 2001).

Free-ranging red foxes (Vulpes vulpes) have beenreported to be seropositive for Hepatozoon canis in Israel(Fishman et al., 2004) and H. canis infection was confirmedin free-ranging red foxes in Italy (Gabrielli et al., 2010), Slo-vakia (Majláthová et al., 2007) and Croatia (Dezdek et al.,2010) by PCR. H. canis infection has also been reportedin gray foxes (Pseudalopex gymnocercus) in Brazil (Criado-Fornelio et al., 2006). The objective of this study is to reportinfection with a Hepatozoon sp. closely related to Hepa-tozoon felis in a free ranging Pampas gray fox (Lycalopexgymnocercus) with concomitant pneumonia caused bycanine distemper virus (CDV, Morbillivirus).

2. Materials and methods

A wild adult female Pampas gray fox was found ona farm close to the city of Choele Choel in the provinceof Río Negro, in the north-central Patagonia region ofArgentina (Latitude: 39◦16′0′′S, Longitude: 65◦41′0′′W) onAugust, 2008 (winter in the Southern hemisphere). Theanimal showed severe incoordination and apparent blind-ness when found and was easily hand-caught. The foxwas humanely euthanized due to poor prognosis and thecarcass was submitted to the “Laboratorio de Diagnós-tico Veterinario” in the city of Bahía Blanca, where a fullnecropsy was performed approximately 12 h after death.Samples of brain (cerebral cortex, brainstem, cerebel-lum and medulla oblongata), lung, heart, skeletal muscle,skin, kidney, liver, small and large intestine and stom-ach were formalin-fixed, paraffin-embedded, sectionedat 5 �m and stained with hematoxylin and eosin (HE)for routine histopathology. CDV immunohistochemistrywas performed as previously reported (Stanton et al.,2003) on sections of lung that were placed on positivelycharged slides. Antigen retrieval was performed by auto-claving the slides for 10 min in citrate buffer (AntigenUnmasking Solution, Citrate-Based, Vector LaboratoriesInc., Burlingame, CA). Non-specific binding sites wereblocked by the application of 0.5% casein in tris-bufferedsaline-tween-20 buffer for 10 min at room temperature.The slides were then incubated with a commercially avail-able mouse monoclonal anti-CDV nucleoprotein antibody(CDV-NP, VMRD Inc., Pullman, WA) at a dilution of 1:3000.A secondary biotinylated goat anti-mouse IgG antibody(BA-9200, Vector Laboratories Inc., Burlingame, CA) wasapplied to samples at a dilution of 1:200, followed bysignal amplification with horseradish peroxidase-labeled

Please cite this article in press as: Giannitti, F., et al., Infection wa wild Pampas gray fox (Lycalopex – Pseudalopex – gymnocercus(2011), doi:10.1016/j.vetpar.2011.11.006

streptavidin (SP-2011, Jackson ImmunoResearch Laborato-ries, Inc., West Grove, PA). Finally, the antigen–antibodycomplex was demonstrated following reaction with 3-amino-9-ethylcarbazole (AEC K4001, Dako, Carpinteria,

PRESSitology xxx (2011) xxx– xxx

CA). Slides were counterstained with hematoxylin and cov-erslipped. Brain from a raccoon infected with CDV was usedas a positive control. The anti-CDV nucleoprotein primaryantibody was replaced with pre-immune serum for nega-tive control slides.

PCR for Hepatozoon sp. was performed at the KoretSchool of Veterinary Medicine in Israel. Formalin-fixed,paraffin-embedded skeletal muscle samples were deparaf-finized and DNA was extracted using the QIAampDNA FFPE tissue kit (QIAgen, CA, USA). PCR was per-formed using the Piroplasm F (CCAGCAGCCGCGGTAATTC)and Piroplasm R (CTTTCGCAGTAGTTYGTCTTTAACAAATCT)primers targeting the 18S rRNA region as previouslydescribed (Tabar et al., 2008). The amplified DNA seg-ment was purified using ExoSAP-IT (USB, Cleveland, OH)and sequenced at the Center for Genomic Technolo-gies, Hebrew University of Jerusalem. The DNA sequenceobtained was compared for similarity to sequences in Gen-Bank using the BLAST program hosted by NCBI, NationalInstitutes of Health, USA (http://www.ncbi.nlm.nih.gov).Sequences were further analyzed using the MEGA ver-sion 3.0 (www.megasoftware.net) and a phylogenetictree was constructed by the Maximum-Likelihood algo-rithm in agreement with the Minimum-Evolution and theNeighbor-Joining algorithms using the Kimura2-parametermodel. Bootstrap replicates were performed to estimatethe node reliability, and values were obtained from 1000randomly selected samples of the aligned sequence data.

3. Results

On necropsy, the carcass was found to be in goodnutritional condition and moderate state of postmortempreservation. The right cranial, middle, caudal and the leftcranial and caudal lung lobes had multifocal to coalescingirregular areas of bright red and tan to white discolorationand mottling, and rubbery to firm texture of the pulmonaryparenchyma. No ticks or other ectoparasites were notedgrossly and no other significant gross abnormalities wereobserved.

Histologically, there was diffuse, severe, sub-acute,necrotizing bronchointerstitial pneumonia with type IIpneumocyte hyperplasia and syncytial cell formation in thealveoli. Multifocally, bronchiolar epithelial and syncytialcells contained multiple round to oval and homogeneousvariable sized (3–5 �m) deeply eosinophilic intracyto-plasmic and rare intranuclear inclusion bodies (Fig. 1). Nosignificant histological abnormalities were observed in anyof the other tissues examined histologically. CDV antigenwas detected immunohistochemically in the bronchiolarepithelial cells, syncytial cells, alveolar macrophages andtype I and II pneumocytes; all these cells showed pre-dominantly intracytoplasmic granular immunostaining(Fig. 2). The skeletal muscle and myocardium containedseveral round to oval protozoan cysts in different stagesof development that ranged from 64 �m × 75 �m to98 �m × 122 �m (minor and major axis, respectively), and

ith a Hepatozoon sp. closely related to Hepatozoon felis in) co-infected with canine distemper virus. Vet. Parasitol.

were characterized by a round to oval, central, eosinophilicmeront-like core or developing merozoites surrounded byconcentric rings of mucinous material. These structuresmorphologically resembled Hepatozoon sp. meronts, in

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F g pneu3 ristic of4

pa2at

Fs

ig. 1. Histopathology of the lung showing bronchointerstitial necrotizin–5 �m diameter intracytoplasmic eosinophilic inclusion bodies characte00×.

articular the so-called “onion skin” cysts of Hepatozoon

Please cite this article in press as: Giannitti, F., et al., Infection wa wild Pampas gray fox (Lycalopex – Pseudalopex – gymnocercus(2011), doi:10.1016/j.vetpar.2011.11.006

mericanum (Vincent-Johnson et al., 1997; Panciera et al.,001; Cummings et al., 2005). An infiltrate of macrophagesnd neutrophils was evident in the interstitial connectiveissue of the skeletal muscle adjacent to the Hepatozoon

ig. 2. Immunostaining for canine distemper virus in lung tissue showing positivyncytial cell (center), 400×.

monia with type II pneumocyte hyperplasia and multiple round to oval, CDV within syncytial cells and pneumocytes. Hematoxylin & Eosin stain,

cysts, often forming discrete pyogranulomas presum-

ith a Hepatozoon sp. closely related to Hepatozoon felis in) co-infected with canine distemper virus. Vet. Parasitol.

ably where mature collapsed cysts had freed merozoitesinto the surrounding interstitium. Macrophages withinthe pyogranulomas and monocytes/macrophages in thelumen of adjacent sinusoidal blood vessels contained

e reaction in pneumocytes lining the alveoli, alveolar macrophages and a

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on skin”aterial,

Fig. 3. Hepatozoon sp. cysts in skeletal muscle. Several oval protozoan “onicore surrounded by concentric rings of mucinous mucopolysaccharide m& Eosin stain, 200×.

intracytoplasmic round protozoa consistent with mero-

Please cite this article in press as: Giannitti, F., et al., Infection wa wild Pampas gray fox (Lycalopex – Pseudalopex – gymnocercus(2011), doi:10.1016/j.vetpar.2011.11.006

zoites or developing gamonts (Figs. 3 and 4) (Pancieraet al., 2001; Cummings et al., 2005).

Hepatozoon sp. infection was confirmed by PCR of skele-tal muscle tissue. The PCR product was sequenced and

Fig. 4. Hepatozoon sp. “onion skin” shaped cyst in skeletal muscle showing a singlof multiple individual merozoites. Leukocytes infected with parasites (arrows) pror developing gamonts are present within large sinusoidal blood vessel at the edgestain, 600×.

shaped cysts up to 122 �m in diameter, with a round, central, eosinophilicmorphologically resembling Hepatozoon americanum cysts. Hematoxylin

found to be 337 base pairs long. By BLAST analysis, it

ith a Hepatozoon sp. closely related to Hepatozoon felis in) co-infected with canine distemper virus. Vet. Parasitol.

was found to be 99% identical to H. felis with the clos-est match being H. felis isolate Spain 2 18S ribosomal RNAgene (GenBank accession AY628681) from a Spanish cat(Criado-Fornelio et al., 2006). The new sequence from the

e large nucleus consistent with an early meront prior to the developmentesumably monocytes or macrophages containing Hepatozoon merozoites

of a pyogranuloma adjacent to the cystic structure. Hematoxylin & Eosin

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Fig. 5. A Neighbor Joining (NJ) tree phylogram comparing 18S rRNA DNA sequences from the Argentinean fox Hepatozoon, Hepatozoon felis, Hepatozooncanis, and Hepatozoon americanum and with Babesia vogeli and Babesia canis as outgroups. The GenBank accession numbers, country of origin and specieso for each

AnfoHH

4

p(btdcwttdaieaa

ihi

idm(HoBtt(df

f infected animals from which the sequences were derived are included

rgentinean fox was deposited in GenBank as accessionumber HQ020489. Phylogenetic analyses using three dif-

erent algorithms were similar and showed that sequencesbtained from the Hepatozoon infected fox clustered with. felis sequences from Spain and differed from H. canis and. americanum (Fig. 5).

. Discussion

The clinical signs and the gross and histopathologicalulmonary findings are highly suggestive of CDV infectionCaswell & Williams, 2007); this diagnosis was confirmedy immunohistochemistry. Clinical disease was consideredo be most likely caused by CDV infection. Although theetection of Hepatozoon sp. meronts in the skeletal mus-le and myocardium was probably an incidental finding,e cannot rule out a possible contribution of the infec-

ion by this parasite to the clinical condition observed inhis fox. Co-infection of CDV and Hepatozoon sp. has beenescribed before in wild carnivores (Goller et al., 2010),lthough never in foxes. In this case, it is probable thatmmunosuppression due to the viral infection may havexacerbated a pre-existing Hepatozoon sp. infection andllowed increased merogony and dissemination of the par-site in the host’s tissues (Baneth, 2011).

Subclinical H. canis infection has been reported in dogsn Buenos Aires City, Argentina (Eiras et al., 2007) before;owever this is the first report of Hepatozoon sp. infection

n a wild animal in this country.Although H. felis and H. canis are closely related genet-

cally (Criado-Fornelio et al., 2006), they appear to targetifferent host tissues. While H. canis has been reportedostly in peripheral blood, spleen and bone marrow

Baneth et al., 1998), but never in muscular tissues of dogs,. felis was reported mostly in skeletal and cardiac musclesf domestic cats and wild felids (Klopfer et al., 1973;eaufils et al., 1998; Kubo et al., 2006, 2010). In contrasto H. canis, the target tissues of H. americanum include

Please cite this article in press as: Giannitti, F., et al., Infection wa wild Pampas gray fox (Lycalopex – Pseudalopex – gymnocercus(2011), doi:10.1016/j.vetpar.2011.11.006

he canine striated skeletal muscles and myocardiumVincent-Johnson et al., 1997), a location similar to thatescribed in the fox of this report. However, the cystsound in our fox measured up to 122 �m in diameter

sequence.

and were considerably smaller than H. americanum cystsin dog tissues described to be 186 ± 52 �m in diameter(Vincent-Johnson et al., 1997). Furthermore, the Hepato-zoon DNA amplified and sequenced from the fox tissues inthis study clustered together in the phylogenetic analysiswith published H. felis sequences and separately from H.canis, H. americanum and other Hepatozoon species.

An intrasarcoplasmic (intra-myocyte) location of the“onion skin” cysts in the muscles of this fox was not clearlydemonstrated by routine histologic sections stained withHE and it seems more likely that the cysts were present inthe interstitium or within unidentified host cells locatedbetween myocytes. This tissue distribution has been pre-viously proposed for species of Hepatozoon infecting wildfelids, including a species related to H. felis (Kubo et al.,2006, 2010). However, the morphology and size of the car-diac cysts in these felids do not resemble the ones describedin this fox.

To the best of our knowledge no H. felis infectionshave been reported in members of the family Canidae. AHepatozoon sp. similar to H. felis was detected in ticks col-lected from wild Tasmanian devils, a marsupial carnivore.Although the parasite was not actually found in the marsu-pial’s tissues, this finding suggests that this infection mightbe possible in non-felid animal species (Vilcins et al., 2009).

Not much is known about the arthropod vectors of H.felis, its transmission in general, and whether it exclusivelyinfects domestic cats or can also infect other mammalianhosts. The fact that the Hepatozoon sp. detected in this fox ismost closely related to H. felis does not rule out a differentand perhaps unknown Hepatozoon species. To the best ofour knowledge, the only Hepatozoon species reported toinfect Pampas gray foxes (L. gymnocercus) so far is H. canis(Criado-Fornelio et al., 2006).

The ticks Rhipicephalus sanguineus, a vector of H. canis,and Amblyomma tigrinum, have been reported to infestfoxes in the region where this fox originated (Guglielmoneand Nava, 2005, 2006) and their role as possible vectors of

ith a Hepatozoon sp. closely related to Hepatozoon felis in) co-infected with canine distemper virus. Vet. Parasitol.

this Hepatozoon sp. should be further investigated.More investigation is needed to identify the Hepato-

zoon species which infect domestic and wild carnivores,to understand the epidemiology of these infections, to

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establish their actual clinical, biological and pathologi-cal behavior and to clarify the phylogenetic relationshipbetween isolates and the vectors of these parasites.

Conflict of interest statement

The authors of this paper have no financial or personalrelationship with other people or organizations that couldinappropriately influence or bias the content of the paper.

Acknowledgements

The authors wish to thank Dr. Bradd Barr for criticalrevision of this article and Karen Sverlow for technicalassistance with CDV immunohistochemistry.

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