DOI: 10.2478/s11686-010-0030-6 © W. Stefan´ski Institute of Parasitology, PAS Acta Parasitologica, 2010, 55(3), 210–214; ISSN 1230-2821 Apicomplexan parasites of red foxes (Vulpes vulpes) in northeastern Poland

Grzegorz Karbowiak1*, Viktória Majláthová2, Joanna Hapunik1, Branislav Pet’ko2,3 and Irena Wita1 1W. Stefański Institute of Parasitology of the Polish Academy of Sciences, Twarda 51/55, 00-818 Warsaw, Poland; 2Parasitological Institute of Slovak Academy of Sciences, Hlinková 3, Košice, 040 01, Slovakia; 3Faculty of Health Care, Catholic University in Ružomberok, Miloša Vesela 21, Ružomberok 034 01, Slovakia

Abstract Molecular detection of apicomplexan parasites in splenic samples of red foxes collected from northeastern Poland was conducted by PCR amplification of a fragment of the 18S rRNA spanning the V4 gene region of . Positive PCR products were further analysed by restriction fragment length polymorphism (RFLP) and sequencing to identify species. One hundred and eleven red foxes (Vulpes vulpes) were acquired from 15 localities in the Mazovian Province and 27 foxes were acquired from the Mazurian Lakeland. Apicomplexan 18S rDNA was detected in 15.9% of 138 fox spleens examined. Three apicomplexan species were identified: canis was detected in 11.6% of the spleen samples, was detected in 3.6% of the spleen samples and a sp. was sequenced from 1 sample (0.7%). This data represent the first record of H. canis, T. gondii and a B. sp. from naturally infected red foxes in Poland. Infected foxes may act as sylvatic reservoirs of these api- complexan parasites as well as serving as a source of infection for definitive hosts and vectors.

Keywords Babesia sp., Hepatozoon canis, Toxoplasma gondii, molecular analysis, Vulpes vulpes

Introduction related to B. microti, B. rodhaini, and equi. Zahler et al. (2000) suggest that the fauna of small piroplasms in The phylum Apicomplexa contains unicellular obligate para- is more complex than previously assumed and that it includes sites with great importance in terms of human and veterinary at least two different species. They proposed therefore to name medicine. Species differ in their life cycles, modes of trans- the new species described by them as Theileria annae which mission, geographic distributions and disease potential. Red is used by some authors only. foxes are recognized as an important sylvatic reservoir for Hepatozoonosis, a less known but wide spread disease of zoonotic helminthes in the Slovak Republic and Poland (An- dogs caused by H. canis and H. americanum, is prevalent in tolová et al. 2004, Gawor et al. 2004, Dubinský et al. 2006). dogs from Europe, Northern and Southern America, and Asia However, very little is known about the prevalence of blood (Ewing et al. 2000; Ewing and Panciera 2003; Criado-Forne- and tissue apicomplexan parasites, some of which are lio et al. 2003, 2007; Jittapalapong et al. 2006; Forlano et al. zoonotic, in foxes. 2007). In dogs in Poland this parasite was not recorded so far. Babesia canis, B. vogeli and B. gibsoni, apicomplexan par- Competent reservoirs of canine and hepatozoono- asites of red blood cells, are tick-borne agents of disease in sis in Poland are unknown; however, it is known that these dogs from Europe and North America (Cacciò et al. 2002, pathogens are able to infect wild carnivores. Both natural and Birkenheuer et al. 2003, Adaszek and Winiarczyk 2008). An- experimental infections have been reported (Davis et al. 1978, other pathogenic species morphologically similar to Babesia Maede et al. 1982, Conceicão-Silva et al. 1988, Heerden et microti, a parasite of rodents and humans, was isolated from al. 1995, Alencar et al. 1997, Kocan et al. 1999, Garrett et al. dogs in Spain and Germany (Camacho et al. 2001). Pairwise 2005). identities as well as analyses of the 18S rDNA demonstrated Our study focused on molecular detection of apicomplexan that this Babesia, referred to as a B. microti-like agent, is only parasites in blood and tissue of red foxes from northeastern distantly related to the other canine Babesia: it is more closely Poland. Fox carcasses had been frozen then held before ship-

*Corresponding author: [email protected] Apicomplexa of red foxes in Poland 211

ment to our laboratory until rabies testing was completed. each positive sample, 13 µl amplified DNA was digested at Splenic tissue represented a suitable organ for molecular 65°C overnight in a solution containing 5U of Tru1I (300 analysis of haemotagenous parasites since the spleen functions U/ml) and 1× buffer R (Fermentas). Electrophoresis was con- as a reservoir for blood components and removes damaged or ducted in 16% polyacrylamide gel at 150 V for 3 h. The gels parasitized cells. were stained with SYBR gold nucleic acid gel stain (Molecu- lar Probes, Leiden, the Netherlands) for 20 min, and bands were visualized with a UV transilluminator. The profile of Materials and methods H. canis, T. gondii and the Babesia sp. contain products in size: 250 bp, 175 bp, 50 bp, 40 bp, 35 bp; 250 bp, 175 bp, 50 Animals bp, 45 bp, 40 bp, 35 bp, 30 bp; 250 bp, 200 bp, 125 bp, 100 bp, 75 bp, 65 bp, 50 bp, 45 bp, 40 bp, 35 bp, 30 bp, respectively. Frozen spleen samples (total 138) from 111 red foxes were ac- Specimen of different RFLP profiles were further analysed by quired from the Sanitary and Epidemiological Station in sequence analysis (Fig. 1). Ostrołęka located in the Mazovian Province, Poland and 27 spleen samples were acquired from hunters in the Mazurian DNA sequencing of PCR products Lakeland. Habitats of the Mazovian Province (51°00΄– 53°30΄N; 19°00΄–22°30΄E) consist of marshy floodplains bor- Sequencing was performed at the Department of Molecular dered by dry terraces of river Vistula. The foxes were collected Biology (Faculty of Natural Science Commenius University, from 15 localities in the Mazovian Province. All animals were Bratislava, Slovak Republic). PCR products were purified by shot during routine huntings and delivered to Epidemiological using a QIAquick PCR purification kit (Qiagen). The com- Stations by the hunters. The Mazurian Lakeland (53°52΄– plementary strands of each sequenced product were manually 53°45΄N; 21°35΄–21°19΄E) includes more than 2,000 lakes assembled. Sequences were compared with GenBank entries originally formed by meltwaters from the Vistula ice sweet. by Blast N2.2.13. Sequence similarity was calculated by This region contains numerous marshes, forest mosaics, EMBOSS Align, a pairwise alignment algorithm (http:// meadows, pastures and sand dunes. www.ebi.ac.uk/emboss/align). The following sequences were sent to GenBank: H. canis (EU165370), T. gondii (EU165368), PCR amplification and a Babesia sp. (FJ480420).

DNA from fox spleen samples were isolated and purified using Genomic Mini AX tissue kit (A&A Biotechnology), ac- cording to the manufacturer’s protocol. PCR amplification was performed in a 25-µl reaction mixture from the MasterTaq DNA polymerase kit (Eppendorf AG, Hamburg, Germany) containing 10.4 µl deionized water, 5 µl 5 × TaqMaster PCR Enhancer, 2.5 µl 10 × Taq buffer (with 15 mmol/l Mg2+), 1.5

µl 25-mmol/l solution of Mg (OAc)2, 0.1 µl Taq DNA poly- merase (5 U/µl), 0.5 µl deoxynucleoside triphosphate (dNTP) mix (10 mmol/l) (Fermentas, Vilnius, Lithuania), 1.25 µl of each primer (10 pmol/µl) (Invitrogen, Paisley, Scotland), and 2.5 µl DNA template. For detection of apicomplexan para- sites, primers amplifying a fragment of the 18S rRNA span- ning the V4 region of apicomplexans were used, RLB-F (GAGGTAGTGACAAGAAATAACAATA) and RLB-R (TCTTCGATCCCCTAACTTTC) were originally designed for use in reverse line blot (Gubbels et al. 1999). B. canis iso- late (EU165369) was used as a positive control. PCR prod- ucts were electrophoresed on 1% agarose gels stained with GoldView Nucleic Acid Stain (Bejing SBS Genetech Co. Ltd.). The size of amplified fragments was ~ 530 bp.

RFLP analysis

The PCR products of the 18S rDNA were further analysed by Fig.1. RFLP profiles of apicomplexan parasites identified in red restriction fragment length polymorphism (RFLP), according foxes. Abbreviations: a – ladder; b and c – Hepatozoon canis; to previously described protocol (Majláthová et al. 2006). For d – Toxoplasma gondii; e – Babesia sp. 212 Grzegorz Karbowiak et al.

Results is non-pathogenic in red foxes. Conceicão-Silva et al. (1988) arrived at that conclusion based on light-microscopic and his- The DNA of apicomplexan parasites was detected in 22 tological studies of foxes in Portugal. However, Maede et al. (15.9%) spleen samples from red foxes, all originating from (1982) described the pathological symptoms of hepatozoono- the Mazovian Province. There was no evidence of parasites in sis in young foxes as similar to that observed in ill dogs. spleen samples of foxes from the Mazurian Lakeland. Three Toxoplasma gondii is a widespread apicomplexan parasite species of apicomplexan parasites were identified: H. canis, that infects a broad range of animals and man. The definitive T. gondii and a Babesia sp. H. canis, detected in 16 samples, host is the cat while other animals and man act as intermedi- was the most prevalent (11.6%). T. gondii was present in 5 ate hosts. Transmission can occur through several routes of samples (3.6%) and a Babesia sp. was found in 1 sample exposure. Ingestion of sporulated oocysts passed in the faeces (0.7%). Infection rates for H. canis and T. gondii were variable of an infected cat through contamination of drinking water, among localities within the Province, ranging from 0 to 50% food or objects, ingestion of cysts contained in the tissues of for H. canis and 0 to 12.5% for T. gondii. infected meat animals, or transplacental transmission from an Hepatozoon sequence obtained from foxes in Poland was infected mother to the offspring are the main methods of trans- identical to H. canis sequence from Canis familiaris in Brazil mission. The presence of T. gondii in ticks has been shown (EU 165370). A comparison of the sequence from red foxes in (Siuda 1993); however, the significance of this finding is not Poland (EU165370) with the sequence of H. canis 5LIMI clear. T. gondii has previously been detected in wild carnivores strain (DQ869309) obtained from red foxes in Slovakia re- (Hůrková and Modrý 2006, Prestrud et al. 2008); however, vealed 99% identity. Despite the high degree of sequence sim- this is the first report of T. gondii in red foxes from Poland. ilarity the restriction profiles for the 2 fox isolates were Consumption of infected rodents or infected meat would be different (Fig. 1), indicating different strains of H. canis in red the most likely source of exposure for red foxes to this para- foxes from the 2 locations. site. However, T. gondii infections in red foxes does not rep- Sequence (EU165368) obtained from one of 5 PCR prod- resent a direct health risk to people in Poland. ucts revealed 99% identity with T. gondii (TGU03070, Evidence of Babesia sp. in red foxes from Poland should M97703, L37415). Sequence (FJ480420) from the Babesia alert veterinarians to the presence of this parasite, a newly rec- sp. was different from B. canis sequences, and revealed 100% ognized pathogen of dogs associated with a high risk of identity with a sequence of B. microti (AY144702) found in azotemia and mortality (Camacho et al. 2001). Based on the foxes in the USA as well as with a sequence (AY144700) de- observation of Camacho et al. (2001), Ixodes hexagonus is the tected in dogs from Spain. main candidate as a vector for this protozoan parasite. I. hexa- gonus is a widespread nidicolous tick which is present in Poland (Siuda et al. 1993). Discussion The decision to use spleen samples was motivated by dif- ficulties in obtaining fresh blood from foxes and the necessity Canine hepatozoonosis is an emerging tick transmitted dis- to test foxes for rabies. Fox carcasses were frozen before trans- ease. The brown tick is the port to the Sanitary and Epidemiological Station. Moreover, main vector of the causative agent, H. canis. The autochtho- for the safety of the investigators, rabies testing had to be com- nous occurrence of this parasite is restricted to regions where pleted before transporting materials to the laboratory. How- the tick vector occurs. However, H. canis has been detected in ever, spleen samples should be adequate for diagnosis of blood naturally infected red foxes in Slovakia where R. sanguineus and tissue parasites through molecular techniques since the is not known to be present (Majláthová et al. 2007). R. san- spleen is part of the reticuloendothelial system and a reservoir guineus is rarely found in Poland (Siuda 1993). Finding evi- for blood components. This was verified previously by Cria- dence of H. canis in Poland might imply that the range of the do-Fornelio et al. (2003) who used this material to diagnose tick vector has expanded. Alternatively, another tick species H. canis in foxes. Identical materials have also been used suc- may act as a suitable definitive host and vector of H. canis in cessfully to detect B. microti in Microtus oeconomus (Kar- Poland and Slovakia. bowiak, unpubl.). Our results confirm the usefulness of spleen A wide range of carnivorous hosts including domestic samples for molecular diagnosis of blood parasites when fresh dogs, jackals (Shamir et al. 2001), coyotes (Davis et al. 1978), blood cannot be obtained, as well as for apicomplexan para- foxes (Conceicão-Silva et al. 1988) and wild dogs (Heerden et sites located in internal organs. Some parasites, such as He- al. 1995) are susceptible to H. canis, with high infection rates patozoon and Toxoplasma, are present in almost every internal detected in red foxes from France and Portugal (48%) (Con- organ (Conceicão-Silva et al. 1988, Criado-Fornelio et al. ceicão-Silva et al. 1988), Israel (24%) (Fishman et al. 2004), 2003). Japan (Maede et al. 1982) and Spain (Criado-Fornelio et al. This data represents the first record of H. canis, T. gondii, 2003). A high prevalence (15.3%) in red foxes from the Ma- and a Babesia sp. in red foxes from Poland. Currently, fox zovian Province was also observed. A high infection rate with- populations in Poland have been increasing (Panek and Bre- out pathology and signs of illness might suggest that H. canis siński 2002, Goszczyński et al. 2008). Foxes frequently en- Apicomplexa of red foxes in Poland 213

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(Accepted April 9, 2010)