Red foxes, an important source of zoonotic parasites in Romania

Red foxes, an important source of zoonotic parasites in Romania Diana Onac 1, Miruna Oltean 1, Viorica Mircean 1, Adriana Jarca 2, Vasile Cozma 1 1 University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Veterinary Medicine, Department of Parasitology and Parasitic Diseases, Cluj-Napoca 400372, Romania. 2 2nd Medical Department, University of Medicine and Pharmacy Iuliu Haţieganu, Victor Babeş Street no. 8, Cluj- Napoca, Romania. Correspondence: Tel. +40264-596384, Fax+40264-593792, E-mail dianaonac@yahoo.com Abstract. The role of red foxes in maintaining and spreading important diseases in some geographical areas is very important and has direct consequences in the developing of zoonoses control programs. Our study was aimed to determine the current prevalence of certain parasites in red foxes from a few counties of Romania and identifies the presence of Giardia spp. In total 217 red foxes were examined in our laboratory, foxes provided by Veterinary Sanitary Offices or from road accidents. The results of this study revealed that 87.09% of 217 red foxes in the nine counties of Romania had coccidian oocysts and helminth eggs in their faeces. Three protozoan species and thirteen helminth species including one trematode, two cestode and ten nematode species were detected by coprological examination. Echinococcus spp. was not observed by microscopic examination of the intestinal scrapings. Other fourteen species of transited parasites were observed: Oxiuris eggs, Siphacia muris, Anoplocephala spp., Metastrongylus elongatus, Trichisonoides crassicauda, Demodex sp., Eimeria spp., Heterakis gallinae, Hymenolepis diminuta, Hymenolepis nana, Listroforus gibus, Dicrocelium lanceolatum, Aspiculuris tetraptera Monocystis agilis. Eight of these parasite taxa have zoonotic potential: Sarcocystis spp., Giardia spp., Capillaria spp., Ancylostoma caninum, Toxocara canis, Taenia spp., Mesocestoides spp., Hymenolepis diminuta. Ancylostoma caninum was the most common helminth species found in this survey. It is concluded that the red foxes from Romania is a host range of intestinal helminth species. Keywords: Red fox; Parasites; Zoonotic; Romania. Received 15/08/2015. Accepted 12/09/2015. Introduction Foxes obviously play a key role in the interface between wildlife, pets and humans. Reasons for this include the increasing population density of foxes, their susceptibility to relevant pathogens, their hunting preference for small mammals which leads to frequent ingestion of intermediate hosts, and their wide distribution and vicinity to human settlements as a 112

consequence of their synanthropic lifestyle (Wandeler et al., 2003; Deplazes et al., 2004; Duscher et al., 2005, 2006; Torina et al., 2013). The red fox was the main reservoir for sylvatic rabies in Central Europe, which was very common and a threat to human and animal health before the oral fox vaccination campaign (Müller et al., 2009). The vaccination against rabies is held responsible for the increasing fox population in Central Europe (Romig et al., 1999; Chautan et al., 2000; Deplazes et al., 2004; Duscher et al., 2006). Therefore the foxes are held responsible for harboring and transmitting a wide range of vector-borne and zoonotic diseases in Europe, including in Romania. More than 75% of human diseases are of zoonotic origin and are related to wildlife and domestic animals (Taylor et al., 2001). Several studies on the helminths of red foxes have been undertaken in Europe but only a few originate from Eastern Europe. Reports from Belarus (Shimalov and Shimalov, 2003), Hungary (Sréter et al., 2003), Estonia (Moks et al., 2005), and Latvia (Bagrade et al., 2008) and also Romania (Siko Barabasi et al., 2010) have demonstrated high helminth prevalence and a varied helminth fauna in red foxes. The epidemiological data regarding the intestinal parasitical fauna of red foxes are of great importance by maintaining outbreaks of zoonotic diseases in different geographic areas and limiting the implementation of control measures. The present study estimates the prevalence of the important zoonotic parasites among red foxes. Material and methods Animals and samples Between December 2011 and January 2015, 217 samples were collected from red foxes originated in 7 counties from Romania. Samples (gastrointestinal tract) were collected one time from foxes provided of Sanitary Veterinary Institutions following organized hunters and also corpses resulted in car accidents. All the foxes investigated at the laboratory of Parasitology and Parasitical diseases were free of rabies. The corpses were maintained at -20 C until examination. We registered for each fox the following data: age, gender and area. Coproparasitological examination For the examination of the gastrointestinal tracts it was made the necropsy. The intestine was open along its entire length and examined for the presence of the parasites and also it were collected faecal samples for flotation and sedimentation methods. Statistical analyses Descriptive epidemiology was performed using EpiInfo 2000 software. Frequency, prevalence, 95% confidence interval and the p value were established. Age (youth and adults), sex (males and females), origin (county) of foxes provided the basis to analyze the relations between these factors and the presence of zoonotic parasites. Results Among 217 faecal samples examined, in 186 samples (85.7%) helminth eggs were found. Three protozoan species and thirteen helminth species including one trematode, two cestode and ten nematode species were detected by coprological examination. Seven of these parasite taxa have zoonotic potential: Sarcocystis spp., Giardia spp., Capillaria spp., Ancylostoma caninum, Toxocara canis, Taenia spp. and Mesocestoides lineatus. Monospecific infection was found in 54 (24.4%) of the samples as follows: Ancylostoma caninum (10/4.6), Alaria alata (8/3.68), Taenia spp. (7/3.2), Capillaria spp. (7/3.2), Isospora spp. (7/3.2), Sarcocystis spp. (4/1.8), Uncinaria stenocephala (3/1.4), Toxocara canis (2/0.9), Trichocephalus vulpis (2/0.9), Toxascaris leonina (1/0.4), Mesocestoides lineatus (1/0.4) and Crenosoma vulpis (1/0.4) (table 1). Poliparasitism was observed in 75.6% of the samples (n=164), Ancylostoma caninum was the most common helminth specie found in this 113

survey (30.4%) followed by Alaria alata (28.6%) and Isospora spp. (26.3%) (table 2). During the examination of the intestine there were not observed adult parasites from the Genus Echinococcus. Regarding the distribution of the parasites according to the variables taken into the study, females were more affected than males, youth foxes than the adults, and the prevalence of the infection according to the area of provenience vary between 73.5% in CV to 100% in AB County (table 3). Through coproparasitological examination, other fourteen species of transited parasites were observed: Oxiuris spp., Siphacia muris, Anoplocephala spp., Metastrongylus elongatus, Trichisonoides crassicauda, Demodex spp., Eimeria spp., Heterakis gallinae, Hymenolepis diminuta, Hymenolepis nana, Listroforus gibus, Dicrocelium lanceolatum, Aspiculuris tetraptera and Monocystis agilis. From this, Hymenolepis diminuta represent also a zoonotic parasite (table 4). Species of the parasite Table 1. The prevalence and 95%CI for monospecific infections Number of foxes with monospecific infection Prevalence reported to total number of foxes 95%CI reported to the number of foxes infected with species Isospora spp. 7 3.2 7.0-31.4 Sarcocystis spp. 5 2.3 4.2-26.8 Alaria alata 9 4.4 9.8-35.3 Taenia spp. 7 3.2 7.0-31.4 Mesocestoides lineatus 1 0.4 0.1-14.5 Ancylostoma caninum 10 4.6 11.2-37.1 Uncinaria stenocephala 3 1.4 1.7-21.4 Toxocara canis 2 0.9 0.7-18.2 Toxascaris leonina 1 0.4 0.1-14.5 Capillaria spp. 7 3.2 7.0-31.4 Trichocephalus vulpis 2 0.9 0.7-18.2 Crenosoma vulpis 1 0.4 0.1-14.5 TOTAL 54 24.4 - Table 2. The prevalence and 95%CI of the parasites found through coproparasitological examination Phylum Parasite Prevalence (n/%) 95%CI Isospora spp. 57/26.3 20.5-32.7 Protozoa Sarcocystis spp. 40/18.4 13.5-24.2 Giardia spp. 6/2.8 1.0-5.9 Trematoda Alaria alata 62/28.6 22.7-35.1 Cestoda Taenia spp. 41/18.9 13.9-24.7 Mesocestoides lineatus 10/4.6 2.2-8.3 Toxocara canis 20/9.2 5.7-13.9 Ancylostoma caninum 66/30.4 24.4-37.0 Uncinaria stenocephala 26/12.0 8.0.-17.1 Toxascaris leonina 15/16.9 3.9-11.1 Nematoda Trichocephalus vulpis 9/4.1 1.9-7.7 Crenosoma vulpis 6/2.8 1.0-5.9 Rictularia affinis 4/1.8 0.5-4.7 Capillaria spp. 49/22.6 17.2-28.7 Spirocerca lupi 1/0.5 0.0-2.5 Syphacia obvelata 2/0.9 0.1-3.3 114

Table 3. The prevalence of parasitism according to sex, age and provenience of the foxes Sex Age County Variable Prevalence (n/%) 95%CI Females 77/87.5 78.7-93.6 Males 109/84.5 77.1-90.3 Youth 57/100 100 Adults 129/80.6 73.6-86.4 AB 8/100 100 BH 49/89.1 77.8-95.9 CJ 16/76.2 52.8-91.8 CV 36/73.5 58.9-85.1 HD 37/94.9 82.7-99.4 HG 6/75.0 34.9-96.8 SM 34/91.9 78.1-98.3 Table 4. The prevalence and 95%CI of transited parasite species Transited parasite species Prevalence (n/%) 95%CI Oxiuris spp. 3/1.4 0.3-4.0 Siphacia muris 2/0.9 0.1-3.3 Anoplocephala spp. 1/0.5 0.0-2.5 Metastrongylus elongatus 2/0.9 0.1-3.3 Trichisonoides crassicauda 1/0.5 0.0-2.5 Demodex spp. 1/0.5 0.0-2.5 Eimeria spp. 9/4.1 1.9-7.7 Heterakis gallinae 1/0.5 0.0-2.5 Hymenolepis diminuta 7/3.2 1.3-6.5 Hymenolepis nana 1/2.5 0.0-2.5 Listroforus gibus 1/0.5 0.0-2.5 Dicrocelium lanceolatum 3/1.4 0.3-4.0 Aspiculuris tetraptera 1/0.5 0.0-2.5 Monocystis agilis 5/2.3 0.8-5.3 Discussions High rates of parasitic infections are endemic especially in the Southeastern Europe where the economy was ravaged by war and the recent economy downturn in selected former of Sovietic bloc not yet fully recovered after the fall of Communism. The overall prevalence of intestinal helminth infections in Romanian foxes (85.7%) is in good accordance with values reported for foxes from various parts of the Europe. Di Cerbo et al. (2008) observed that from 645 examined foxes, 84.5% were infected with different parasite species. Of the 219 foxes examined in Belgium the infection level was 62.6% (Reperant, 2005), in Switzerland (n=267) of 95.1% (Vervaeke et al., 2005), in Denmark 98.3% (Al-Sabi et al., 2014), in Poland among 604 examined foxes, 94.5% were found infected. In Romania, in a study performed by Siko Barabasi et al. (2010), the prevalence of the parasitism among 561 foxes examinated was 93%. In that study, the most prevalent parasite was Toxocara canis (29.4%), followed by Mesocestoides lineatus (28.7%) and Trichocephalus vulpis (27.2%), more than in our study. The prevalence of Ancylostoma caninum, which was the most prevalent in our study (30.4%) was only of 18.2% in the study of Siko Barabasi et al. (2010). High prevalences of cestodes are reported in all the studies performed around the world with emphasis over zoonotic taxa, especially E. granulosus and E. multilocularis. Regarding alveolar and cystic echinococcosis, reports from several countries provide documented evidence for the emergence or reemergence in recent years. High prevalence of Echinococcus in Wild and Domestic Animals is associated with disease incidence in humans. Pastoralism is the occupation with the highest risk of being infected with the both kinds of echinococcosis 115

due to the proximity of livestock, dogs, and wildlife host species. In our study further analyses are needed to evaluate the presence of Echinococcus spp. between eggs of Taeniidae (Eckert et al., 2000, 2001). The presence of Alaria alata in red foxes was also identified in several countries in Europe: Germany (28.3-29.7%), Austria (18.4%), Poland (76.5-88.0%), former Yugoslavia (64.8%) and Bulgaria (2.1%). The high prevalence of this parasite represent a zoonotic risk because of the fact that the larval stageare known to produce viable muscular cysts in several mammalian hosts (pig, wild boar) (Möhl et al., 2009). In our study the prevalence of 28.6% of Alaria alata is higher that in the study performed by Siko Barabasi et al. (2010) (15%). Protozoan species were also described in different papers but not in Romania. Vergles Rataj et al. (2013), in a study performed in Poland described 2 protozoan species Isospora spp. and Sarcocystis spp., but with a lower prevalence of 0.4%, respectively 2.8%. Also in a study performed in Iran by Shamsollahi et al. (2014), it was observed also Isospora spp. (9.67%) and Giardia spp. (11.29%). In our study the prevalence of the protozoan species was 26.3% for Isospora spp., 18.4% for Sarcocystis spp. and 2.8% for Giardia spp. Giardia intestinalis is a cosmopolitan pathogen with a very wide host range, including humans, domestic animals, and wild animal species (Cacciò et al., 2008; Thompson et al., 1993).The high prevalence of Giardia spp. was attributed to the fact that this parasite can colonize niche previously occupied by parasites such as T. canis and D. caninum, and most of the anthelmintics do not interfere in the development of Giardia spp. (Bugg et al., 1999). Most of this parasites observed in this study are known to be present in dog, and for this reason, the possibility of foxes serving as a reservoir for all the referred parasites might represent a significant risk for dogs and owners in the same time. Conclusions The presence of these zoonotic parasites in a region with a very high human population density urges a close surveillance of these parasites as they may lead to expansions of zoonoses in Romania. Finally, this study emphasizes the need to study the prevalence of intestinal parasites in regions that are recently colonised by red foxes since such studies may add to our understanding of the emergence, the temporal spread and the persistence of zoonoses in Europe. The potential threat of red foxes to public health is of increasing concern. It is concluded that the red foxes from Romania is a host range of intestinal helminth species. Acknowledgements This paper was financed under the frame of European Social Fund, Human Resources Development Operational Programme 2007-2013, project no. POSDRU/159/1.5/S/136893. References Al-Sabi M., Halasa T., Kapel C. 2014. Infections with cardiopulmonary and intestinal helminths and sarcoptic mange in red foxes from two different localities in Denmark. Acta Parasitol. 59(1):98-107. Bagrade G., Šnabel V., Romig T., Ozolinš J., Hüttner M., Miterpakov M., Ševcova D. Dubinsky P. 2008. Echinococcus multilocularis is a frequent parasite of red foxes (Vulpes vulpes) in Latvia. Helminthology 45:157-161. Bugg R.J., Robertson I.D., Eliot A.D, Thompson R.C.A. 1999. Gastrointestinal parasites of urban dogs in Perth, Western Australia. Vet. J. 157(3):295-301. Cacciò S.M., Beck R., Lalle M., Marinculic A., Pozio E. 2008. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. Int. J. Parasitol. 38:1523-1531. Chautan M., Pontier D., Artois M. 2000. Role of rabies in recent demographic changes in red fox (Vulpes vulpes) populations in Europe. Mammalia 64:391-410. 116

Deplazes P., Hegglin D., Gloor S., Romig T. 2004. Wilderness in the city: the urbanization of Echinococcus multilocularis. Trends Parasitol. 20:77-84. Di Cerbo A.R., Manfredi M.T., Trevisiol K., Bregoli M., Ferrari N., Pirinesi F., Bazzoli S. 2008. Intestinal helminth communities in the red fox (Vulpes vulpes L.) in the Italian Alps. Acta Parasitol. 53:302-311. Duscher G., Winkelmayer R., Prosl H. 2005. Schwarzwildverbreitung in Gebieten mit Trichinellen- funden bei Füchsen in Österreich. Wien. Tierarztl. Monatsschr. 92:315-321. Duscher G., Pleydell D., Prosl H., Joachim A. 2006. Echinococcus multilocularis in Austrian foxes from 1991 until 2004. J. Vet. Med. B Infect. Dis. Vet. Public Health. 53:138-144. Eckert J., Conraths F.J., Tackmann K. 2000. Echinococcosis: an emerging or re-emerging zoonosis? Int. J. Parasitol. 30(12-13):1283-1294. Eckert J., Deplazes P., Gottstein B., Craig P.S., Heath D., Jenkins D.J., Kamiya M., Lightowlers M.W.L. 2001. Echinococcosis in animals: clinical aspects, diagnosis and treatment. In: Eckert J., Gemmell M.A., Meslin F.-X., Pawlowski Z.S., 2001. WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern, World Health Organisation for Animal Health, Paris, France, pp. 72-99. Möhl K., Grosse K., Hamedy A., Wüste T., Kabelitz P., Lücker E. 2009. Biology of Alaria spp. and human exposition risk to Alaria mesocercariaea review. Parasitol. Res. 105:1. Moks E., Saarma U., Valdmann H. 2005. Echinococcus multilocularis in Estonia. Emerg. Inf. Dis. 11:1973-1974. Müller T., Bätza H.-J., Beckert A., Bunzenthal C., Cox J.H., Freuling C.M. 2009. Analysis of vaccinevirus-associated rabies cases in red foxes (Vulpes vulpes) after oral rabies vaccination campaigns in Germany and Austria. Arch. Virol. 154:1081-1091. Reperant L.A. 2005. Ecologie parasitaire d Echinococcus multilocularis et autres helminthes du système renard roux rongeurs en milieu urban et peri-urbain. Implications zoonotiques dans le canton de Geneve, Suisse. École Nationale Vétérinaire de Lyon. Thèse n. 52 pour obtenir le grade de Docteur Vétérinaire. École Nationale Vétérinaire de Lyon, 105 pp. Romig T., Bilger B., Dinkel A., Merli M., Mackenstedt U. 1999. Echinococcus multilocularis in animal hosts: new data from western Europe. Helminthologia 36:185-191. Shamsollahi M., Razmjoo M., Bahrami A.M. 2014. Seroepidemiological survey of important parasitic infections of wild carnivores. Int. J. Adv. Biol. Biom. Res. 2(3):783-792. Shimalov V.V., Shimalov V.T. 2003. Helminth fauna of the red fox (Vulpes vulpes Lenneaus, 1758) in southern Belarus. Parasitol. Res. 89:77-78. Sikó Barabasi S., Fok E., Gubányi A., Mészáros F., Cozma V. 2010. Helminth fauna of the small intestine in the European red fox, Vulpes vulpes with notes on the morphological identification of Echinococcus multilocularis. Sci. Parasitol. 11(3):141-151. Sréter T., Széll Z., Marucci G., Pozio E., Varga I. 2003. Extraintestinal nematode infections of red foxes (Vulpes vulpes) in Hungary. Vet. Parasitol. 115:329-334. Taylor L.H., Latham S.M., Woolhouse M.E. 2001. Risk factors for human disease emergence. Philos. T. Roy. Soc. B 356(1411):983-989. Thompson R.C., Reynoldson J.A., Mendis A.H. 1993. Giardia and giardiasis. Adv. Parasit. 32:71-160. Torina A., Blanda V., Antoci F., Scimeca S., D'Agostino R., Scariano E. 2013. A molecular survey of Anaplasmaspp., Rickettsia spp., Ehrlichia canis and Babesia microti in foxes and fleas from Sicily. Transbound. Emerg. Dis. 60(Suppl. 2):125-130. Vergles Rataj A., Posedi J., Žele D., Vengušt G. 2013. Intestinal parasites of the red fox (Vulpes vulpes) in Slovenia. Acta Veterinaria Hungarica 61(4):454-462. Vervaeke M., Dorny P., De Bruyn L., Vercammen F., Jordaens K., Van Den Berge K., Verhagen R. 2005. A survey of intestinal helminths of red foxes (Vulpes vulpes) in Northern Belgium. Acta Parasitol. 50:221-227. Wandeler P., Funk S.M., Largiadèr C.R., Gloor S., Breitenmoser U. 2003. The city-fox phenomenon: genetic consequences of a recent colonization of urban habitat. Mol. Ecol. 12:647-656. 117