Frey, C. F., Schuppers, M. E., Müller, N., Ryser-Degiorgis, M. P., and Gottstein, B. (2009). Assessment of the Prevalence of Trichinella Spp

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Frey, C. F., Schuppers, M. E., Müller, N., Ryser-Degiorgis, M. P., and Gottstein, B. (2009). Assessment of the prevalence of Trichinella spp. in red foxes and Eurasian lynxes from Switzerland. Veterinary Parasitology 159: 295-299.

Keywords: 8CH/epidemiology/Eurasian lynx/genotyping/lynx/Lynx lynx/parasites/PCR/ prevalence/red fox/Trichinella/Trichinella britovi/Vulpes vulpes

Abstract: Trichinella spp. larvae have not been detected in Swiss pigs, horses, or wild boar for many decades, whereas the parasite was repeatedly isolated from red foxes and Eurasian lynxes. Whenever the isolated larvae could be subjected to genotyping, T. britovi was found as infective agent. The present study was initiated to re-assess the epidemiological situation of Trichinella infection in Swiss carnivorous wildlife, namely in red foxes and lynxes. Tissue samples from 1,298 foxes were collected between 2006 and 2007, and those of 55 lynxes between 1999 and 2007. All samples were tested by a standard artificial digestionmethod and a multiplex-PCR to determine the species and/or genotypes of recovered larvae. Trichinella larvae were found in 21 foxes (1.6%) and 15 lynxes (27.3%), and T. britovi was identified as infecting species in all cases. The geographic distribution of positive foxes showed two main clusters: one in Central Switzerland and one in the West of the country, where also many lynxes were found to be positive. While the prevalence for Trichinella infection in foxes was not statistically correlated with sex or age class, the prevalence in lynx was significantly higher in males compared to females, and in adults compared to juveniles. Veterinary Parasitology 159 (2009) 295–299

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Veterinary Parasitology

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Assessment of the prevalence of Trichinella spp. in red foxes and Eurasian lynxes from Switzerland

C.F. Frey a,*, M.E. Schuppers b,N.Mu¨ ller a, M.P. Ryser-Degiorgis c, B. Gottstein a a Institute of Parasitology, Vetsuisse Faculty, University of Bern, P.O. Box 8466, 3001 Bern, Switzerland b SAFOSO, Bremgartenstrasse 109A, 3012 Bern, Switzerland c Centre for Fish and Wildlife Health (FIWI), Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, P.O. Box 8466, 3001 Bern, Switzerland

ARTICLE INFO ABSTRACT

Keywords: Trichinella spp. larvae have not been detected in Swiss pigs, horses, or wild boar for many Trichinella britovi decades, whereas the parasite was repeatedly isolated from red foxes and Eurasian lynxes. Red fox Whenever the isolated larvae could be subjected to genotyping, T. britovi was found as Eurasian lynx infective agent. The present study was initiated to re-assess the epidemiological situation Genotyping of Trichinella infection in Swiss carnivorous wildlife, namely in red foxes and lynxes. Tissue Prevalence samples from 1,298 foxes were collected between 2006 and 2007, and those of 55 lynxes Switzerland between 1999 and 2007. All samples were tested by a standard artificial digestion method and a multiplex-PCR to determine the species and/or genotypes of recovered larvae. Trichinella larvae were found in 21 foxes (1.6%) and 15 lynxes (27.3%), and T. britovi was identified as infecting species in all cases. The geographic distribution of positive foxes showed two main clusters: one in Central Switzerland and one in the West of the country, where also many lynxes were found to be positive. While the prevalence for Trichinella infection in foxes was not statistically correlated with sex or age class, the prevalence in lynx was significantly higher in males compared to females, and in adults compared to juveniles. ß 2008 Elsevier B.V. All rights reserved.

1. Introduction studies carried out between 1968 and 1985 in foxes revealed prevalences ranging from 14% to 4% (Ho¨rning, In Switzerland, Trichinella spp. larvae have not been 1976, 1977, 1987; Vanzetti, 1982), suggesting a decrease of found in domestic pigs or horses for more than a century, the prevalence in foxes over time. and in wild boar (Sus scrofa) for several decades (Ho¨rning, While some data exist on Trichinella prevalence in Swiss 1968, 1976; Vanzetti, 1982; Jakob et al., 1994; Gottstein wildlife, little information is available on the species of et al., 1997). However, serological investigations revealed Trichinella involved; a single fox was shown to be infected evidence of positive wild boars (Gottstein et al., 1997), and with T. britovi (Gottstein et al., 1997). In order to assess the larvae have been isolated from red foxes (Vulpes vulpes) risk of infection for domestic pigs and humans in and other carnivorous wildlife, such as lynxes (Lynx lynx). Switzerland, additional knowledge on the epidemiology The most recent studies documented a prevalence of larvae of Trichinella spp. in Swiss wildlife is necessary. of 0.9% in foxes and 30% in lynxes (Gottstein et al., 1997; The aims of the present study were: (1) to determine Schmidt-Posthaus et al., 2002). Interestingly, earlier the current prevalence of Trichinella spp. infection in red fox and lynx in Switzerland in order to detect any change in prevalence in the past years; (2) to determine the genotype * Corresponding author. of the recovered Trichinella larvae; (3) to assess the E-mail address: [email protected] (C.F. Frey). geographical distribution of Trichinella spp.; and (4) to

0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.10.060 296 C.F. Frey et al. / Veterinary Parasitology 159 (2009) 295–299 investigate possible correlations between prevalence of 2.4. Purification of genomic DNA and PCR Trichinella infection and sex and age of red fox and lynx. Multiplex PCR was performed on recovered larvae from all foxes, and from lynxes sampled since 2002. Extraction 2. Materials and methods of genomic DNA of individual larvae (between 2 larvae and 2.1. Fox samples 10 larvae per positive sample) was performed according to Zarlenga et al. (1999). Genotyping of the Trichinella isolates Between January 2006 and June 2007, 1289 tongues was performed by applying a multiplex, 2-step PCR as from hunted foxes were collected either by state game- described (Zarlenga et al., 1999). keepers or local hunters and sent to the Institute of Parasitology in Berne. The geographic origin of the samples 2.5. Statistical analysis collected, the estimated age (juvenile <6 months, sub- adult 6–12 months, adult >12 months), and the sex of each Significance of differences in prevalence was assessed fox were recorded by means of a standardized ques- using a 2-sided Fisher’s exact test (NCSS 2007, Kaysville, tionnaire. The age of the foxes was estimated by the UT, USA). The level of significance was set at P < 0.05. hunters or gamekeepers, based on both body size and season of death. 3. Results The questionnaire was completed for 90% of the samples. The recorded information showed that 56% of 3.1. Fox the samples originated from male and 44% from female Twenty-one of 1289 fox samples were positive for foxes. The age distribution of the sampled foxes was 20% Trichinella larvae, revealing an overall prevalence of 1.6% juvenile, 19% sub-adult, and 61% adult. (95% confidence interval 1.0–2.5%) for Trichinella infection in Swiss red foxes (Table 1). Larvae from all positive 2.2. Lynx samples samples were identified by PCR as T. britovi. The positive samples originated mainly from Central Switzerland and The Eurasian lynx is a protected species in Switzerland. from the Western part of the country (Fig. 1). Information Currently, about 100 adult animals live in Switzerland, on sex and age were available for 17 of the 21 positive mainly in the Jura Mountains and the North-West Swiss animals: 10 were males and 7 were females; 7 foxes were Alps (von Arx et al., 2004; Zimmermann et al., 2005). juveniles, 2 were sub-adults, and 8 were adults. No According to the Swiss Lynx Concept, all carcasses of free- ranging lynxes must be submitted to the Centre for Fish and Wildlife Health (FIWI) in Berne for a complete, standardized post-mortem examination. Between January Table 1 1999 and June 2007, muscle samples (diaphragm and/or Number of positive per investigated animals per canton. Cantons with masseter) were collected from 55 lynxes. Sex and age of positive animals are indicated in boldface. each animal were recorded. Age was estimated according Canton Positive/investigated to body size and tooth wear, and age classes were chosen Foxes Lynxes considering sexual maturity and, therefore, social beha- Aargau 0/36 na* viour (Stubbe and Krapp, 1993): juvenile (<1 year), sub- Appenzell-A. 0/20 na* adult (1 to <2 years for females, and 1 to <3 years for Appenzell-I. 1/20 na* males), and adult (2 or 3 years). Basel-Land 0/106 0/1 Information on sex was available for 54 animals (one Basel-Stadt 0/17 na* carcass could not be specified) and age for all 55 animals; Bern 0/104 6/27 Fribourg 5/148 4/7 23 lynxes were female and 31 were male; 24 were juvenile, Gene`ve na* na* 8 were sub-adult, and 23 were adult. Glarus na* na* Graubu¨ nden 0/23 na* 2.3. Artificial digestion technique Jura 0/47 1/3 Luzern 3/125 na* Neuchaˆtel 0/35 0/2 The outer layer of each fox tongue collected was Nidwalden 3/12 na* removed and the remaining tissue mass divided sagitally Obwalden 1/30 na* into two halves. One half was subjected to a pooled Schaffhausen na* na* * artificial digestion assay consisting of 5 samples (i.e. Schwyz 1/13 na Solothurn 0/2 na* approximately 50 g in total). If larvae were found in pooled St. Gallen 0/38 0/2 samples, the other halves of the tongues of the pool were Thurgau 0/132 na* digested individually. For lynx samples, individual artifi- Ticino 0/99 na* * cial digestion was performed with at least 10 g of Uri 5/35 na Valais 1/75 na* diaphragm and/or masseter muscle. The artificial digestion Vaud 1/49 4/13 (magnetic stirrer) method was carried out according to the Zug 0/35 na* EU-regulation (EG) no. 2075/2005. Recovered larvae were Zu¨ rich 0/86 na* transferred from the digestion fluid to PBS and stored at Total 21/1289 (1.6%) 15/55 (27.3%) À20 8C for further analysis. * No samples available. C.F. Frey et al. / Veterinary Parasitology 159 (2009) 295–299 297

Fig. 1. Map of Switzerland showing the geographical distribution of fox samples. Circles: Trichinella-negative samples, dots: Trichinella-positive samples.

statistically significant correlation was found between age confidence-interval 17.2–40.2%) in Swiss lynxes (Table 1). and sex and the occurrence of Trichinella infection. T. britovi was identified in all eight cases submitted for PCR analysis. The positive lynxes originated both from the Jura 3.2. Lynx Mountains and the North-Western Swiss Alps (Fig. 2). Two of the positive lynxes were females, and 13 were males; 2 Fifteen of the 55 lynxes were positive for Trichinella were juveniles, 2 were sub-adults, and 10 were adults. larvae, revealing an overall prevalence of 27.3% (95% Statistically, there was a significantly higher prevalence of

Fig. 2. Map of Switzerland showing the geographical distribution of lynx samples. Circles: Trichinella-negative samples, dots: Trichinella-positive samples. 298 C.F. Frey et al. / Veterinary Parasitology 159 (2009) 295–299

Trichinella infection in male lynxes compared to females occurrence or absence of the host species in these parts of (P = 0.007), and a statistically lower prevalence in juvenile the country (Zimmermann et al., 2005). Overall, these animals compared to adults (P = 0.008). results indicate that the occurrence of Trichinella sp. in Prevalence in lynxes was significantly higher than in Swiss wildlife is not dependent on the presence of lynx. foxes (P < 0.0001). However, lynx might be a good indicator for the existence of Trichinella infection in wildlife due to its position at the 4. Discussion top of the food-chain and its high life expectancy. The artificial digestion method was used in the present The present study is the first to determine the genotype study. Previous studies on foxes, in which artificial of Trichinella larvae found in a large number of wild digestion was used in parallel with serology, revealed a carnivores in Switzerland. T. britovi was the only species higher prevalence by means of serology than digestion identified in both foxes and lynxes. This species was also (Jakob et al., 1994; Gottstein et al., 1997). Serology also identified in a fox in an earlier study (Gottstein et al., 1997). revealed a seroprevalence of 8.7% in wild boar (Gottstein T. britovi persists in a sylvatic cycle, and its main reservoir et al., 1997), although Trichinella larvae have not been in most regions of the EU is the red fox (Pozio, 1998). found in wild boar for many years (Ho¨rning, 1968, 1976; The observed prevalence of 1.6% in red foxes is similar Vanzetti, 1982; Jakob et al., 1994; Gottstein et al., 1997). to the prevalence of 0.9% and 1.3% (P > 0.05; Gottstein et al. Low infection intensity in some individuals/species may be (1997) and Jakob et al. (1994, respectively), but signifi- sufficient to induce a serum antibody response but not to cantly lower than the prevalences of 9% and 14% reported allow direct demonstration of larvae by using the in the 1970s (P < 0.001; Ho¨rning, 1976, 1977, respec- conventional digestion method. Thus, prevalences tively). The finding of 1.6% prevalence is lower than the obtained by conventional digestion method might be prevalence of 4% determined in 1982 (P = 0.06; N = 118; underestimated. Vanzetti, 1982). Our study revealed that the Trichinella To gain more exact information on the epidemiological prevalence in red foxes has remained stable at a low level situation of Trichinella in Switzerland, the seroprevalence (<2%) during the past decade (Gottstein et al., 1997; Jakob of Trichinella in carnivores and in wild boar needs to be et al., 1994), and thus confirmed the suspected decrease of (re)assessed. Furthermore, investigations to elucidate the prevalence since the 1970s (Ho¨rning, 1976, 1977; Vanzetti, prevalence of Trichinella infections in rodents and other 1982). small omnivorous mammals potentially acting as hosts for Regarding lynx, our prevalence of 27.3% corroborates the parasite are necessary. previous data obtained from a smaller sample size (6 of 20 lynxes collected in 1998–1999; Schmidt-Posthaus et al., Conflict of interest statement 2002). These results indicate that the prevalence of Trichinella sp. in lynxes has also been stable for the past None of the authors (C.F. Frey, M.E. Schuppers, N. 10 years. Two factors may explain the higher Trichinella Mu¨ ller, M.-P. Ryser-Degiorgis, B. Gottstein) has a financial prevalence in lynxes: (1) foxes are a significant prey of lynx or personal relationship with other people or organisations in Switzerland (Jobin et al., 2000), and can therefore be that could inappropriately influence or bias the paper considered as a source of infection for lynxes; (2) lynxes entitled ‘‘Assessment of the prevalence of Trichinella spp. in can live up to 17 years in the wild (U. Breitenmoser, pers. red foxes and Eurasian lynxes from Switzerland.’’. comm.), while most foxes are shot before they reach 3 years of age (Nimmervoll, 2007). Thus, lynxes have a longer Acknowledgements exposure time to potentially Trichinella-infected meat. This hypothesis is further supported by our finding that adult Many thanks go to Verena Eidam, Ramona Graf, and lynxes have a higher prevalence of Trichinella infection Christine Wittwer for excellent technical assistance; and to than juveniles, as was also observed by Oksanen et al. Nadia Robert, Stefan Hoby, Valeria Cafe´-Marçal, Helena (1998) in Finnish lynxes. Nimmervoll, Veronika Sieber, Patrick Rehmann, Martin Interestingly, we found that male lynxes present a Janovsky, and Luca Bacciarini for their contribution to the higher prevalence of infection compared to females. This lynx samples collection. Hunting officers, state game- finding is also in accordance with earlier observations in wardens, hunters, and biologists of the KORA are kindly Finnish lynxes (Oksanen et al., 1998). However, the reason acknowledged for the submission of lynx carcasses and fox for this difference is unknown, since the food habits of samples. We thank Ulrich Weber for his help with the female and male lynxes are comparable (Jobin et al., 2000). graphical figures. This work was funded by the Swiss Geographical differences in the prevalence of infection Federal Veterinary Office and the Swiss Federal Office of in foxes were obvious, with most cases originating from the Environment.. the canton of Fribourg and from Central Switzerland (Fig. 1). Jakob et al. (1994) found a similar geographical References distribution of Trichinella infections among foxes. Inter- Gottstein, B., Pozio, E., Connolly, B., Gamble, H.R., Eckert, J., Jakob, H.P., estingly, many positive lynxes came from the canton of 1997. Epidemiological investigation of trichinellosis in Switzerland. Fribourg and surrounding areas (Fig. 2). However, positive Vet. Parasitol. 72, 201–207. lynxes also originated from regions where all tested foxes Ho¨rning, B., 1968. Zur Naturherd-Problematik der Trichinellose in der Schweiz. Revue Suisse de Zoologie 75, 1063–1066. were negative (Jura Mountains, Bernese Oberland). Data on Ho¨rning, B., 1976. Trichinella spiralis und Trichinellose in der Schweiz. lynxes from other Swiss areas are lacking, due to the sparse Hausdruckerei Institut fu¨ r exakte Wissenschaften, Bern. C.F. Frey et al. / Veterinary Parasitology 159 (2009) 295–299 299

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