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Contents lists available at ScienceDirect

Veterinary Parasitology

jo u r nal homep age: www.elsev ier .com/ locate /vetpar

hort communication

irofilaria immitis: An emerging parasite in dogs, red foxesnd golden jackals in Hungary

. Tolnaia, Z. Széll a, Á. Sprochb, L. Szeredib, T. Srétera,∗

Laboratory of Parasitology, Fish and Bee Diseases, Veterinary Diagnostic Directorate, National Food Chain Safety Office, Tábornok utca, Budapest H-1143, HungaryLaboratory of Mammalian, Poultry and Wildlife Pathology, Veterinary Diagnostic Directorate, National Food Chain Safety Office,ábornok utca 2, Budapest H-1143, Hungary

r t i c l e i n f o

rticle history:eceived 11 February 2014eceived in revised form 2 April 2014ccepted 4 April 2014

eywords:eartwormirofilaria immitis

a b s t r a c t

Hungary was not considered to be a heartworm (Dirofilaria immitis) endemic country until2007, when the first autochthonous canine infection was described. Herein we report addi-tional autochthonous heartworm infections in two dogs (Canis familiaris), twenty red foxes(Vulpes vulpes) (n = 534; prevalence: 3.7%; 95% CI = 2.4–5.7%) and two golden jackals (Canisaureus) (n = 27; prevalence: 7.4%; 95% CI = 2.1–23.4%) coming from eight counties. The iden-tification of the parasite was based on morphology, morphometrics and amplification of 12SrDNA followed by sequencing in all cases. Our results indicate that Hungary became a D.immitis endemic country in the past decade. The prevalence and intensity of heartworm

revalenceistributionoged foxolden jackal

infection in wild canids is similar to or lower than that observed in the Mediterraneancountries of Europe (3.7–7.4% vs. 0.4–12.7% and 1.5 vs. 2.9–4.4 worms/animal). These find-ings are in line with the results of the recently developed climate based forecast model topredict the establishment of D. immitis in Hungary.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Heartworm disease is caused by the nematode Dirofi-aria immitis. Patent infections are possible in numerous

ild and companion animal species. Wild animal reservoirsnclude wild canids and probably some other carnivorepecies in Europe (McCall et al., 2008; Simón et al., 2012).n companion animals, heartworm disease is diagnosed

ainly in dogs and less commonly in cats and ferrets, dueo the differences of diagnostic techniques and the life-

Please cite this article in press as: Tolnai, Z., et al., Dirofilaria immjackals in Hungary. Vet. Parasitol. (2014), http://dx.doi.org/10.1

pan of the parasites in these animals (Genchi et al., 2007).eartworm infection may lead to serious and potentially

atal cardiopulmonary disease caused primarily by adult

∗ Corresponding author. Tel.: +36 1 460 6322; fax: +36 1 252 5177.E-mail address: SreterT@nebih.gov.hu (T. Sréter).

http://dx.doi.org/10.1016/j.vetpar.2014.04.004304-4017/© 2014 Elsevier B.V. All rights reserved.

heartworms and their antigenic products (Genchi et al.,2007; Simón et al., 2012). Several species of mosquitoes canserve as intermediate hosts worldwide (McCall et al., 2008).Infected mosquitoes can transmit the parasite to humans,but the infection does not become patent. The infective lar-vae reach the lungs, become encapsulated and die causinggranulomatous reactions called “coin lesions” in humans.The lesions are medically significant because radiographi-cally they appear similar to metastatic lung cancer (Genchiet al., 2007; Simón et al., 2012). Heartworm disease hasbeen reported worldwide in many countries with tem-perate, semitropical or tropical climates (McCall et al.,2008; Simón et al., 2012). In Europe, D. immitis infections

itis: An emerging parasite in dogs, red foxes and golden016/j.vetpar.2014.04.004

occur mainly in the southern countries including Spain,Portugal, France, Italy, Greece and Turkey (Genchi et al.,2009). These countries are considered to be the historicallyendemic region of the continent. Recent reports from other

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European countries indicate an expansion of the dis-tribution of the parasite (Genchi et al., 2005, 2009,2011). In Eastern Europe, Bulgaria, Croatia, Romania andSerbia are currently known to be endemic, and spo-radic autochthonous cases were also reported from CzechRepublic, Slovakia and Hungary (Genchi et al., 2007).Herein we provide evidence that Hungary should also beconsidered as a D. immitis endemic country.

2. Materials and methods

The carcass of a 7-year-old male shepherd Mudi dog(Dog 1) and a 12-year-old female mongrel dog (Dog 2) wassent to the National Food Chain Safety Office of Budapestwith suspicion of poisoning in February 2013 and January2014. During necropsy, the heart and vena cava were slitopen and visually inspected. Although the blood was par-tially clotted, blood samples could be collected from theheart of dogs and examined by the modified Knott test(Genchi et al., 2007).

In order to reveal the spatial distribution of D. immitis inthe warmest region of Hungary, heart and lungs of red foxes(Vulpes vulpes) killed by hunters and sent in individual plas-tic bags at +4 ◦C to the National Food Chain Safety Officewere screened for heartworm infections from November2013 to January 2014. Red fox carcasses, representing morethan 1% of the total fox population (n = 534), were randomlyselected out of all the foxes from the southern countiesas described (Széll et al., 2008) (Fig. 1). All golden jackal(Canis aureus) carcasses (n = 27) sent to the laboratory fromNovember 2007 to January 2014 were included in thepresent study (Fig. 1). Carcasses were individually labelledby the hunters with an identification number reporting theinformation on the nearest place to killing on the topo-graphic map and the date of collection. If the nearest placeto hunting was a human settlement, the animal positionwithin a municipality (illustrated with a symbol in Fig. 1)was randomly chosen. The heart, pulmonary artery andlungs of the animals were slit open and examined visuallyfor the presence of parasites. In Dirofilaria positive cases,various amount of non-clotted blood samples were col-lected from the heart or the body cavities and examined bymodified Knott test (Genchi et al., 2007). Parasite speciesidentification was based on morphology and morphomet-rics.

Species identification was also confirmed by 12S rDNA-based PCR in all cases (Casiraghi et al., 2001). DNA wasextracted from worms and purified as described (Sréter-Lancz et al., 2007). The fragments of 12S rDNA wereamplified (Casiraghi et al., 2001), and amplicons werefurther characterised by sequence analysis (Sréter-Lanczet al., 2007). Sequencing data were visually inspected forreading errors and combined using Chromas (Technely-sium, Tewantin) and MultAlin (Corpet, 1988) programmes.Sequences were identified by comparison with GenBankentries using the BLAST software (National Center forBiotechnology Information, 2014).

Please cite this article in press as: Tolnai, Z., et al., Dirofilaria immjackals in Hungary. Vet. Parasitol. (2014), http://dx.doi.org/10.1

The locality of origin of the dogs, foxes and jackals andthe number of D. immitis adults were marked on the map(vector point layer) by the Quantum GIS 1.8.0 software(QGIS Team, 2012) (Fig. 1). Statistical calculations were

PRESSology xxx (2014) xxx–xxx

carried out with MedCalc 12.7 (MedCalc Software, Ostend,Belgium) and EpiTools (Sergenat, 2014) programmes.

3. Results and discussion

During necropsy, four male and thirteen female adultworms were detected in the right ventricle of the heartof Dog 1, and a male adult worm was found in the venacava of Dog 2 (Table 1). The worms collected from theheart of Dog 1 and vena cava of Dog 2 and the microfilariaedetected in the blood of Dog 1 were identified as D. immi-tis on the basis of the characteristic morphological featuresand morphometrics of the parasites (Genchi et al., 2007).The identification was also confirmed by 12S rDNA-basedPCR. The results of sequencing showed 100% agreementwith D. immitis (GenBank accession no. AJ544831). Dog1 was born in a sheep farm near to Hajdúbagos, countyHajdú-Bihar (Fig. 1) and has never been outside of thefarm and the surrounding grazing area. Dog 2 was bornin Újlorincfalva, county Heves and has never left the vil-lage (Fig. 1). Therefore, both cases should be consideredautochthonous. In 2007 and 2009, similar autochthonousheartworm infections were detected in a dog and a ferretin counties Jász-Nagykun-Szolnok and Baranya (Jacsó et al.,2009; Molnár et al., 2010).

Twenty foxes (3.7%; 95% CI = 2.4–5.7%) and two jack-als (7.4%; 95% CI = 2.1–23.4%) coming from eight counties(Baranya, Békés, Borsod-Abaúj-Zemplén, Csongrád, Fejér,Heves, Jász-Nagykun-Szolnok, Pest) in 2013 and 2014were infected with low number of D. immitis adults(m ± SE = 1.5 ± 0.2; range: 1–5 worms/animal) (Fig. 1;Table 1). All worms except one isolated from the pulmonaryartery were detected in the right ventricle of the heart.Microfilariae were not found in the uterus of the femaleworms or in the blood samples of the infected wild canids(Table 1). Species identification was based on morphol-ogy, morphometrics and amplification of 12S rDNA genefollowed by sequencing in all cases. The presence of lownumber of adult heartworms in foxes and jackals with-out microfilaraemia might indicate that these species arenot good hosts for D. immitis (Marconcini et al., 1996).Therefore, the true impact of foxes and jackals on thetransmission dynamics of dirofilariosis should be assessed(Simón et al., 2012).

Our results indicate that the Hungarian lowland is a D.immitis endemic region. The prevalence and intensity ofheartworm infection in wild canids seems to be similar toor lower than that observed in the Mediterranean countriesof Europe (3.7–7.4% vs. 0.4–12.7% and 1.5 vs. 2.9–4.4worms/animal) (Gortázar et al., 1994, 1998; Manas et al.,2005; Genchi et al., 2007; Magi et al., 2008). These findingsare in line with the results of the recently developed cli-mate based forecast model to predict the occurrence of theparasite in Europe (Genchi et al., 2005, 2007). The yearlyaverage predicted heartworm generations are between 1and 5 in Hungary. Although it is lower than that predicted

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in the majority of the territory of the Mediterranean Basin(1–10 generations), the climate of Hungary was thought tobe suitable for the establishment of D. immitis in Hungary(Genchi et al., 2005, 2007).

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Fig. 1. Map of Hungary showing uninfected (open symbols) and Dirofilaria immitis infected red foxes (Vulpes vulpes) (filled circles), golden jackals (Canisaureus) (filled triangles) and dogs (Canis familiaris) (filled squares).

Table 1Number of heartworms (Dirofilaria immitis) detected in dogs (Canis familiaris), red foxes (Vulpes vulpes) and golden jackals (Canis aureus) in Hungary andthe results of the Knott test.

Host species Nearest human settlement Number of heartworms Result of the Knott test

Dog Hajdúbagos 13 gravid females + 4 males PositiveÚjlorincfalva 1 male Negative

Red fox Tószeg 1 male NegativeValkó 1 male NegativeNagyköru 1 non-gravid female NegativeKiskunlacháza 2 males NegativeSzeghalom 1 non-gravid female NegativeTiszanána 1 non-gravid female + 1 male NegativeKiszombor 1 male NegativeTószeg 1 male NegativeMindszent 3 non-gravid females + 1 male NegativeHarsány 1 male NegativeMagyarbóly 1 non-gravid female NegativeErdotelek 1 male NegativeSzolnok 1 non-gravid female NegativeSzeged-Szentmihály 1 non-gravid female NegativeSzabadkígyós 1 male NegativeFegyvernek 1 non-gravid female NegativeSáránd 1 non-gravid female + 1 male NegativeBékéscsaba 1 non-gravid female NegativeBerettyóújfalu 2 non-gravid females + 3 males NegativeOrosháza 1 non-gravid female Negative

1

1

Dfa

Golden jackal Hevesaranyos

Székesfehérvár

Please cite this article in press as: Tolnai, Z., et al., Dirofilaria immjackals in Hungary. Vet. Parasitol. (2014), http://dx.doi.org/10.1

Europe has experienced the spreading of D. immitis,irofilaria repens and some other vector-borne helminths

rom the Mediterranean countries towards the northernnd eastern ones in the past decades (Genchi et al., 2005,

male Negativenon-gravid female Negative

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2007; Simón et al., 2012; Otranto et al., 2013a). The firstD. repens endemic region was detected in southern part ofHungary in 1998 (Széll et al., 1999), and D. repens is cur-rently considered a widely distributed emerging parasite

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in Hungary (Szénási et al., 2008). Recently, another filar-ial parasite occurring in the Mediterranean Basin (Otrantoet al., 2013b), Onchocerca lupi has been reported fromHungary (Sréter and Széll, 2008). Although the sample sizewas smaller (n = 100) in our previous study (Sréter et al.,2003), D. immitis was not detected in foxes of Hungary 11years ago. The present study has revealed that Hungarybecame a D. immitis endemic country in the past decade.Several factors including climate change (Genchi et al.,2009, 2011; Simón et al., 2012; Otranto et al., 2013a), globalmovement (Tatem et al., 2006) and nature conservationefforts (Széll et al., 2013) might be in the background ofthese changes. As the veterinary and public health sig-nificance of these parasites are high, further studies areneeded on the distribution of vector-borne helminths ofdogs, cats and humans in other European countries includ-ing Hungary. As wild carnivores are excellent sentinelsfor the spread of D. immitis, and the number of studies islimited in Europe (Simón et al., 2012), further investiga-tions can be encouraged in this field.

Conflict of interest

The authors declare that the research was conducted inthe absence of any commercial and financial relationshipsthat could be construed as potential conflict of interest.

Acknowledgements

We thank János Malinovszki and Zsolt Tóth for their helpin sample collection and Andrea Kollár for her technicalassistance.

References

Casiraghi, M., Bazzocchi, C., Mortarino, M., Ottina, E., Genchi, C., 2001. Asimple molecular method for discriminating common filarial nema-todes of dogs (Canis familiaris). Vet. Parasitol. 141, 368–372.

Corpet, F., 1988. Multiple sequence alignment with hierarchical clustering.Nucleic Acids Res. 16 (1988), 10881–10890.

Genchi, C., Rinaldi, L., Cascone, C., Mortarino, M., Cringoli, G., 2005.Is heartworm really spreading in Europe? Vet. Parasitol. 133,137–148.

Genchi, C., Rinaldi, L., Cringoli, G. (Eds.), 2007. Dirofilaria immitis and D.repens in dog and cat and human infections. Rolando Editore, Zagreb,p. 1211.

Please cite this article in press as: Tolnai, Z., et al., Dirofilaria immjackals in Hungary. Vet. Parasitol. (2014), http://dx.doi.org/10.1

Genchi, C., Rinaldi, L., Mortarino, M., Genchi, M., Cringoli, G., 2009. Climateand Dirofilaria infection in Europe. Vet. Parasitol. 163, 286–292.

Genchi, C., Mortarino, M., Rinaldi, L., Cringoli, G., Traldi, G., Genchi, M.,2011. Changing climate and changing vector-borne disease distribu-tion: the example of Dirofilaria in Europe. Vet. Parasitol. 176, 295–299.

PRESSology xxx (2014) xxx–xxx

Gortázar, C., Castillo, J.A., Lucientes, J., Bianco, J.C., Arriolabengoa, A., Cal-vete, C., 1994. Factors affecting Dirofilaria immitis prevalence in redfoxes in north-eastern Spain. J. Wild. Dis. 30, 545–547.

Gortázar, C., Villafuerte, R., Lucientres, J., Fernández-de-Luco, D., 1998.Habitat related differences in helminth parasites of red foxes in theEbro valley. Vet. Parasitol. 80, 75–81.

Jacsó, O., Mándoki, M., Majoros, G., Pétsch, M., Mortarino, M., Genchi,C., Fok, É., 2009. First autochthonous Dirofilaria immitis (Leidy, 1856)infection in a dog in Hungary. Helminthologia 46, 159–161.

McCall, J.W., Genchi, C., Kramer, L.H., Guerrero, J., Venco, L., 2008. Heart-worm disease in animals and humans. Adv. Parasitol. 66, 193–285.

Magi, M., Calderini, P., Gabrielli, S., Dell’Omodarme, M., Macchioni, M.,Prati, M.C., Cancrini, G., 2008. Vulpes vulpes: a possible wild reservoirfor zoonotic filariae. Vector Borne Zoonotic Dis. 8, 249–252.

Manas, S., Ferrer, D., Castellá, J., López-Martín, J.M., 2005. Cardiopul-monary helminth parasites of red foxes (Vulpes vulpes) in Catalonia,north-eastern Spain. Vet. J. 169, 118–120.

Marconcini, A., Magi, G., Macchioni, G., Sassetti, M., 1996. Filariosis in foxesin Italy. Vet. Res. Commun. 20, 316–319.

Molnár, V., Pazár, P., Rigó, D., Máthé, D., Fok, É., Glávits, R., Vajdovich, P.,Jacsó, O., Balogh, L., Sós, E., 2010. Autochthonous Dirofilaria immitisinfection in a ferret with aberrant larval migration in Europe. J. SmallAnim. Pract. 51, 393–396.

National Center for Biotechnology Information, 2014. BLAST, Accessed at:http://www.ncbi.nlm.nih.gov/blast

Otranto, D., Dantas-Torres, F., Brianti, E., Traversa, D., Petric, D., Genchi,C., Capelli, G., 2013a. Vector-borne helminths of dogs and humans inEurope. Parasites Vectors 6, 16.

Otranto, D., Dantas-Torres, F., Giannelli, A., Latrofa, M.S., Papadopoulos,E., Cardoso, L., Cortes, H., 2013b. Zoonotic Onchocerca lupi infectionin dogs, Greece and Portugal, 2011−2012. Emerg. Infect. Dis. 19,2000–2003.

QGIS Team, 2012. Quantum GIS, Accessed at: http://www.qgis.orgSimón, F., Siles-Lucas, M., Morchón, R., González-Miguel, J., Mellado, I.,

Carretón, E., Montoya-Alonso, J.A., 2012. Human and animal dirofi-lariasis: the emergence of a zoonotic mosaic. Clin. Microbiol. Rev. 25,507–544.

Sergenat, E.S.G., 2014. EpiTools epidemiological calculator. AusVet, Avail-able at: http://epitools.ausvet.com.au

Sréter, T., Széll, Z., 2008. Onchocercosis: a newly recognized disease indogs. Vet. Parasitol. 151, 113.

Sréter, T., Széll, Z., Marucci, G., Pozio, E., Varga, I., 2003. Extraintestinalnematode infections of red foxes (Vulpes vulpes) in Hungary. Vet. Para-sitol. 148, 365–370.

Sréter-Lancz, Z., Széll, Z., Sréter, T., 2007. Molecular genetic comparison ofOnchocerca sp. infecting dogs in Europe with other spirurid nematodesincluding Onchocerca lienalis. Vet. Parasitol. 115, 329–334.

Széll, Z., Sréter, T., Csikós, K., Kátai, Z., Dobos-Kovács, M., Vetési, F., Varga,I., 1999. Autochtonous Dirofilaria repens infection in dogs in Hungary.Magy. Állatorv. Lapja 121, 100–104 (in Hungarian).

Széll, Z., Marucci, G., Bajmóczy, E., Cséplo, A., Pozio, E., Sréter, T., 2008.Spatial distribution of Trichinella britovi, T. pseudospiralis and T. spiralisin red foxes (Vulpes vulpes) in Hungary. Vet. Parasitol. 156, 210–215.

Széll, Z., Marucci, G., Pozio, E., Sréter, T., 2013. Echinococcus multilocularisand Trichinella spiralis in golden jackals (Canis aureus) of Hungary. Vet.Parasitol. 197, 393–396.

itis: An emerging parasite in dogs, red foxes and golden016/j.vetpar.2014.04.004

Szénási, Z., Kovács, H., Pampiglione, S., Fioravanti, M.L., Kucsera, I., Balázs,T., Tiszlavicz, L., 2008. Human dirofilariosis in Hungary: an emergingzoonosis in central Europe. Wien. Klin. Wochensch. 120, 96–102.

Tatem, A.J., Rogers, D.J., Hay, S.I., 2006. Global transport networks andinfectious disease spread. Adv. Parasitol. 62, 293–343.