Circulation of Toxocara spp. in suburban and rural ecosystems in the Slovak Republic

Veterinary Parasitology 126 (2004) 317 324 www.elsevier.com/locate/vetpar Circulation of Toxocara spp. in suburban and rural ecosystems in the Slovak Republic D. Antolová a, *, K. Reiterová a, M. Miterpáková a, M. Stanko b, P. Dubinský a a Parasitological Institute of the Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice, Slovak Republic b Institute of Zoology, Slovak Academy of Sciences, Löfflerova 10, 040 01 Košice, Slovak Republic Received 19 February 2004; received in revised form 29 July 2004; accepted 10 August 2004 Abstract Toxocara spp., the common roundworms of domestic and wild animals, are the causative agents of larval toxocarosis in humans. The migration of Toxocara larvae in men causes clinical syndrome, called larva migrans visceralis or larva migrans ocularis. The objective of the present work was to investigate the prevalence of toxocarosis in dogs, red foxes (Vulpes vulpes), and small mammals in the Slovak Republic. T. canis infection was diagnosed in 16.6% out of 145 dogs examined. Young animals showed significantly higher positivity (50.0%) than adults (12.4%). Coprological investigation of 310 red foxes showed 8.1% prevalence of T. canis, with higher occurrence in animals from rural conditions (8.6%) than from suburban environment (5.6%). Out of 710 small mammals examined, anti-toxocara antibodies were detected in 7.7% of the animals. The most frequently seropositive species was Apodemus agrarius (15.9%). Seropositivity of small mammals from suburban localities was higher (10%) compared with rural areas (5.8%), with adult animals exhibiting higher seropositivity (8.0%) than subadults (6.8%). Our results have confirmed the importance of dogs, red foxes and small mammals in circulation of this serious helminthozoonosis in suburban and rural ecosystems. # 2004 Elsevier B.V. All rights reserved. Keywords: Toxocara canis; Dog; Red fox; Small mammals; Epidemiology; Larva migrans * Corresponding author. Tel.: +421 55 633 14 11. E-mail address: halas@saske.sk (D. Antolová). 0304-4017/$ see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.08.005

318 D. Antolová et al. / Veterinary Parasitology 126 (2004) 317 324 1. Introduction Larval toxocarosis is a serious helmithozoonosis caused by larval stages of Toxocara canis and Toxocara cati, frequent parasites of domestic and wild carnivores. The life cycle of these parasites is direct but in their circulation paratenic hosts also play a significant role. Adult worms, in the intestinal tract of definitive host, shed eggs via the faeces into the environment where, after the development, they are ingested by definitive or paratenic hosts. In definitive hosts (carnivores), the larvae of Toxocara spp. overcome tracheal or somatic migration. They reach the small intestine in young animals, while in adults they usually stay in the tissues as somatic or dormant larvae. In the intestine of paratenic hosts larvae hatch from the eggs, migrate via blood vessels and then remain as somatic larvae in the tissues (Overgaauv and van Knapen, 2000). This migration in man causes the complex of symptoms called larva migrans visceralis (VLM) or, if the eyes are damaged, larva migrans ocularis (OLM). Milder form of toxocarosis, called covert toxocarosis, is also very common. The most common wild carnivore in the Slovak Republic is red fox (Vulpes vulpes), definitive host of Toxocara spp. In the wildlife, about 50% of its diet consists of small mammals, frequent paratenic hosts of Toxocara spp. A territory of a red fox usually spans over 5 50 km 2, with optimal population density being 2 individuals per 1000 ha (Reichholf, 1996). According to the Associations of Hunters, the current population density of this animal in the Slovak Republic is excessive for seven to eight times that markedly contributes to its spread into suburban and urban areas. Thus the infected red foxes and small mammals, except domestic carnivores seem to be another risk factor in circulation of toxocarosis. From the aspect of urbanisation of foxes is important to point out the fact that T. canis eggs isolated from red foxes showed significant higher infectivity compared with those isolated from dogs (Epe et al., 1999). The aim of this study was to determine the prevalence of toxocarosis in hosts, which present the major risk factors of larval toxocarosis in humans, i.e. in dogs, red foxes and small mammals. 2. Material and methods 2.1. Red foxes and dogs A total of 310 red foxes collected in the period of October 2002 February 2003 within the monitoring of occurrence of Echinococcus spp. and Trichinella spp. in the Slovak Republic were included in this study. The capture site of every fox was recorded. Samples of faeces from the animals negative for the rabies were stored at 80 8C at least for 7 days to minimise the risk of Echinococcus multilocularis infection. The localities of the origin of these animals were characterised as follows: suburban locality peripheral part of cities, elevation of 220 450 m above sea level; and rural locality countryside settlements, elevation of 150 200 m above sea level. Faecal samples were obtained from 145 stray and pet dogs. The data as sex, age, and place of keeping were collected. Animals aged up to 8 months were classified as young.

2.2. Small mammals Free-living small mammals were trapped during 2000 2002 in localities with different degree of human involvement. They were caught in the standard live and snap traps set in lines. The traps were spaced 5 m apart, and each line was exposed usually in two nights. Trap lines were checked regularly in the morning. The wick soaked in oil and nut mixture were used as baits. Examined small mammals came from six areas of the East Slovakia, namely Košická kotlina basin (alluvial habitats, windbreaks, acacia belts, fields, forest ecotones, gardens, deer parks, 190 280 m above sea level a.s.l.), Východoslovenská rovina plain (fields, windbreaks, field forests, shrubs, gardens, lowland forests in protected areas, 100 120 m a.s.l.), Beskydské predhorie Mountains (alluvial habitats in agricultural landscape, forest ecotones, 270 280 m a.s.l.), Ondavská vrchovina Mountains (alluvial habitats in agricultural landscape, 190 220 m a.s.l.) and small part of mammals (1.4%) was caught in other areas of the East Slovakia (Valaškovce and Úhorná). Examined material consisted of the following species: Sorex araneus common shrew (Linnaeus, 1758), Sorex minutus pygmy shrew (Linnaeus, 1766), Neomys fodiens water shrew (Pennant, 1771), Clethrionomys glareolus bank vole (Schreber, 1780), Microtus arvalis common vole (Pallas, 1778), Microtus subterraneus common pine vole (de Sélys-Longchaps, 1836), Apodemus flavicollis yellow-necked mouse (Melchior, 1834), Apodemus sylvaticus wood mouse (Linnaeus, 1758), Apodemus agrarius striped field mouse (Pallas, 1711) and Apodemus microps (Kratochvíl and Rosický, 1952). The captured animals were zoologically identified and subjected to necropsy. The hearts with its blood clot were cut open, eluted in 1 ml of saline solution for 24 h and then centrifuged at 500 g for 2 min (Treml and Nesňálová, 1993). Eluates were subsequently used for serological examination. 2.3. Coprological examination D. Antolová et al. / Veterinary Parasitology 126 (2004) 317 324 319 In total, 310 red foxes and 145 dogs faecal samples were investigated for the presence of intestinal parasites by the standard flotation method using the Sheather solution (saturated sucrose solution, specific gravity 1.3). 2.4. Determination of anti-toxocara antibodies by ELISA T. canis larval excretory secretory antigens were prepared by the method of De Savigny (1975). Antibodies were determined in the eluates from the hearts of small mammals using an ELISA modified by Havasiová-Reiterová et al. (1995). Microtitre plates were coated overnight at 4 8C with antigen containing 1 mg/ml protein diluted in carbonate buffer, ph 9.6. The plates were washed three times with phosphate buffer saline ph 7.2 containing Tween 20 (PBS-T20). The eluates (diluted 1:2 in 5% skimmed milk in PBS-T20) were placed to plates in a volume 100 ml per well. After the 1 h incubation at 37 8C the plates were repeatedly washed three times. Horse-radish peroxidase-labelled anti-mouse immunoglobulin (Anti-mouse Polyvalent Immunoglobulins IgG, IgA, IgM; SIGMA) diluted 1:8000 in a volume 100 ml was used as conjugate. After incubation for 1 h plates

320 D. Antolová et al. / Veterinary Parasitology 126 (2004) 317 324 were washed three times and 100 ml of substrate (o-phenylenediamin with 0.05% H 2 O 2 ) was added. The reaction was stopped after 20 min of incubation in the dark at the room temperature by 50 ml of2mh 2 SO 4 and the optical density was measured spectrophotometrically at 490 nm (Thermo Labsystems Opsys MR, USA). 2.5. Data analysis The significance of the differences in prevalence of T. canis according to the place of the origin, age and species of animals was evaluated by the chi-square test with Yates correction. 3. Results 3.1. Red foxes Out of 310 examined red foxes, T. canis was detected in 25 animals (8.1%). In addition, Toxascaris leonina was found in 47.1% of foxes investigated (146/310). When comparing the occurrence of T. canis and T. leonina in rural and suburban ecosystems, the higher prevalence was recorded in rural foxes (8.6 and 48.4%, respectively) than in foxes captured on the town periphery (5.6 and 40.1%, respectively). No significant association between infection rate and capture site was observed. (Table 1). 3.2. Dogs Results of coprological investigation of dogs, related to the age and the origin of dogs, are shown in Table 2. T. canis eggs were detected in 24 out of 145 investigated dogs, representing the incidence of 16.6%. The higher positivity was recorded in young dogs (50%) than in dogs older than 8 months (12.4%). This difference in the rate of infection is statistically significant (P < 0.01). Stray dogs were infected significantly more frequently (32.1%) (P < 0.01) than pet ones (7.6%). T. leonina eggs were detected only in one adult of stray dog. 3.3. Small mammals Examination of 710 small mammals revealed 55 positive individuals, representing the prevalence of 7.7%. Trapped animals were classified into 10 species. Among them, the Table 1 Occurrence of Toxocara canis and Toxascaris leonina in red foxes according to the place of origin Locality Toxocara canis Toxascaris leonina N/n Positivity (%) N/n Positivity (%) Suburban 3/54 5.6 22/54 40.7 Rural 22/256 8.6 124/256 48.4 Total 25/310 8.1 146/310 47.1 N number of positive samples, n number of examined samples.

D. Antolová et al. / Veterinary Parasitology 126 (2004) 317 324 321 Table 2 Occurrence of Toxocara canis a Toxascaris leonina in dogs according to their role and age Helminths Age of dogs Role of dogs Total positivity of dogs Young Adult Stray Pet N/n % N/n % N/n % N/n % N/n % T. canis 8/16 50.0 16/129 12.4 17/53 32.1 7/92 7.6 145/24 16.6 T. leonina 0/16 0.0 1/129 0.8 1/53 1.9 0/92 0.0 145/1 0.7 N number of positive samples, n number of examined samples, % positivity. Table 3 Species representation of small mammals and occurrence of anti-toxocara antibodies according to the place of origin Small mammals Suburban Rural Total positivity N/n % N/n % N/n % Apodemus agrarius 24/115 20.9 22/175 12.6 46/290 15.9 Apodemus flavicollis 4/121 3.3 3/79 3.8 7/200 3.5 Clethrionomys glareolus 1/41 2.4 1/32 3.1 2/73 2.7 Microtus arvalis 0/1 0 0/70 0 0/71 0 Apodemus microps 0/3 0 0/52 0 0/55 0 Sorex araneus 0/2 0 0/9 0 0/11 0 Apodemus sylvaticus 0/5 0 0/1 0 0/6 0 Microtus subterraneus 0/2 0 0/2 0 Sorex minutus 0/1 0 0/1 0 Neomys fodiens 0/1 0 0/1 0 Total 29/289 10.0 26/421 6.2 55/710 7.7 N number of positive samples, n number of examined samples, % positivity. most numerous species was Apodemus agrarius with the highest number of infected animals (46 out of 290 individuals) accounting for the 15.9% seroprevalence. Less numerous was species A. flavicollis with 3.5% positivity. Out of 73 representatives of the species Clethrionomys glareolus, only 2 animals were positive (2.7%). The remaining less frequent species were free of toxocarosis (Table 3). A statistically significant higher occurrence of anti-toxocara antibodies was recorded only in the A. agrarius (P < 0.05) when compared to another positive species. Table 4 Occurrence of anti-toxocara antibodies in small mammals according to the age Age category N n Positivity (%) Adult 30 375 8.0 Subadult 18 263 6.8 Unknown 7 72 9.7 Total 55 710 7.7 N number of positive samples, n number of examined samples.

322 D. Antolová et al. / Veterinary Parasitology 126 (2004) 317 324 In accordance to the place of the origin, the higher incidence of toxocarosis was observed in suburban (10%) than in rural conditions (6.2%). However, there was no significant difference observed between these amounts (Table 3). The number of anti- Toxocara positive animals was higher in adult hosts (8%) than in subadult ones (6.8%), with no significant difference recorded (Table 4). 4. Discussion Relatively expansive spread of red foxes and their expansion into town areas implicate the risk of dissemination of T. canis and also of other parasite eggs. Deposition of faeces on public places can lead to infection, especially in children. Since Toxascaris leonina is able to circulate between the red foxes and small mammals (its paratenic hosts), we have also surveyed its prevalence in the Slovak region. Our results indicate that T. leonina is a more prevalent nematode (47.1%) in red foxes in the Slovak Republic than T. canis (8.1%). Similar data were recently acquired in the Spanish territory (52.2 and 4.4%, respectively) (Criado-Fornelio et al., 2000). However, our observations regarding prevalence do not correspond with the previous report from the Slovak Republic presented by Letková et al. (2001) (14.9 and 27.7%, respectively) and reports from other European countries. For example, Balicka-Ramisz et al. (2003) observed T. leonina in 0.9% and T. canis in 39.8% of red foxes in Poland and Smith et al. (2003) found 0.3 and 62%, respectively, positive foxes in Great Britain. A possible explanation of striking variation in prevalence might be attributed to the fact that T. canis is more common in young animals than in older animals in which larvae remain as somatic larvae in their tissues and T. leonina infects mainly adult carnivores (Jurášek and Dubinský, 1993). The adult foxes prevailed in our epidemiological study. In addition, the research of Letková et al. (2001) was carried out not in the whole territory of Slovakia, but only in the east Slovak region. The occurrence of both, T. canis and T. leonina, was higher in rural localities in comparison to suburban areas. This is in agreement with observations of Richards et al. (1995) who found higher positivity in red foxes originating from rural areas (76%) than from urban areas (61.1%). Dogs represent another important host implying environmental contamination with T. canis eggs. Our study revealed the 16.6% prevalence of T. canis and 0.7% of T. leonina, that is similar to results of Barutzki and Schaper (2003) who observed prevalences of 22.4 and 1.8%, respectively, in Germany. Significant differences were recorded between the occurrence of T. canis in young dogs (50%) and adult dogs (12.4%), that corresponds to the findings of Habluetzel et al. (2003) who found prevalences of 42.7 and 15.7%, respectively, in Italy. Stray dogs were more than four times positive for T. canis than domestic ones. This fact is due to the lack or complete absence of deworming of free-living animals. We detected 7.7% prevalence of anti-toxocara antibodies in small mammals. The majority of captured micromammals belonged to the Order Rodentia (A. agrarius, A. flavicollis, A. microps, A. sylvaticus, C. glareolus, P. subterraneus, M. arvalis), remaining originated from the Order Insectivora. Anti-Toxocara antibodies were recorded only in the rodent species. The most abundant species with the highest number of seropositive animals (15.9%) was that of striped field mouse (Apodemus agrarius). This species is very abundant in mammal communities of East Slovakia with strong non-cyclic

D. Antolová et al. / Veterinary Parasitology 126 (2004) 317 324 323 changes of densities (Stanko, 1994) and frequent occurrence in the suburban and urban areas of mentioned territory (Mošanský, 1984; Stanko et al., 1990). A. agrarius was also found as one of the most numerous and seropositive (30.4%) species under Slovak conditions in a study of Dubinský et al. (1995). They found a higher seropositivity (15.1%) compared with our data. Small mammals as paratenic hosts serve as the significant reservoir of Toxocara larvae, which can survive in their tissues for several months or years. Daily intake of red fox is approximately 0.5 1 kg of meaty food that corresponds to about 20 50 micromammals (Sládek and Mošanský, 1985). Localities with a higher density of small mammals are more frequently visited by foxes what results in accumulation of their faeces containing parasite eggs in such places. Lactogenic transfer of T. canis larvae from mothers to the progeny of paratenic hosts is an important epidemiological phenomenon that contributes to the maintenance of their high prevalence in the rural and urban regions (Tomašovičová et al., 1993). Presence of larval toxocarosis in paratenic hosts is influenced by the age composition of population, with higher occurrence in adults than in young animals, as it was confirmed by our data. The food structure has also apparently played an important role in the pattern of infection since omnivores showed a higher prevalence than insectivores. Changes in the number of small mammals throughout different years depend on climatic conditions (Stanko, 1994) and might substantially influence the prevalence of toxocarosis in definitive hosts. Consequently, we suppose that prevalence of larval toxocarosis in paratenic hosts may indicate the degree of environmental contamination. The present study has pointed out the crucial role of red foxes, dogs and small mammals in the circulation and maintenance of toxocarosis in suburban and rural localities. The circulation constantly present in examined ecosystems throughout the studied period implicate major public health threat. Findings of many authors suggest the importance and zoonotic potential of toxocarosis. The study by Havasiová et al. (1993) revealed 13.65% seropositivity in healthy blood donors in the Slovak Republic, Demirci et al. (2002) found seropositivity of 19.4% in patients without eosinophilia in Turkey, and Deutz et al. (2003) recorded the 17% seroprevalence in a group of 149 hunters in Austria. Acknowledgements This study was financed by the Agency for Support Science and Technology of the Slovak Republic through the support of the project APVT-51-004702, and partially financed by the Slovak Grant Committee VEGA, Grant No. 2/4179/04. References Balicka-Ramisz, A., Ramisz, A., Pilarczyk, B., Bienko, R., 2003. Fauna of gastro-intestinal parasites in red foxes in Western Poland. Med. Weter. 59, 922 925. Barutzki, D., Schaper, R., 2003. Endoparasites in dogs and cats in Germany 1999 2002. Parasitol. Res. 90, 148 150. Criado-Fornelio, A., Gutierrez-Garcia, L., Rodriguez-Caabeiro, F., Reus-Garcia, E., Roldan-Soriano, M.A., Diaz- Sanchez, M.A., 2000. A parasitological survey of wild red foxes (Vulpes vulpes) from the province of Guadalajara, Spain. Vet. Parasitol. 92, 245 251.

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