Examensarbete i ämnet naturvårdsbiologi 20 poäng

Presence of Trichinella spp. in Swedish Lynx Anna Strömqvist 2004

Handledare: Göran Hartman, Dan Christensson Institutionen för naturvårdsbiologi SLU Box 7002 750 07 Uppsala

Nr 134 Uppsala 2004

1

Examensarbeten utförda vid institutionen för naturvårdsbiologi, SLU (förteckning över tidigare arbeten kan fås från institutionen) 114. Andersson, Sofia. 2003. The utilization of grass-covered clearcuts by hares - effects of spatial configuration and habitat composition. Handledare: Gunnar Jansson & Åke Pehrson, Examinator: Henrik Andrén. 115. Sehlberg, Ulrika. 2004. Naturvärdesbedömning av produktionsbestånd och bestånd avsatta enligt FSCstandard på Lycksele region, Holmen Skog. Handledare och examinator: Lena Gustafsson. 116. Westby, Anders. 2004. Wolf predation and habitat use in relation to moose density during winter. Handledare: Håkan Sand, Examinator: Olof Liberg. 117. Halvarsson, Mattias. 2004. Kärlväxter i Natura 2000-habitatet åstallskog - Samband mellan populationsoch biotopparametrar. Handledare och examinator: Tommy Lennartsson. 118. Sandkvist, Martin. 2004. The effect of winter cereals and landscape composition on local abundance of breeding farmland birds. Handledare och examinator: Tomas Pärt. 119. Nilsson, Karin. 2004. Flavocetraria cucullata och F. nivalis, två i Uppland minskande lavarter. Handledare och examinator: Göran Thor. 120. HansErs, Mona. 2004. Population changes of lynx (Lynx lynx) and roe deer (Capreolus capreolus) in southcentral Sweden. Handledare: Henrik Andrén & Olof Liberg, Examinator: Henrik Andrén. 121. Benediktson, Gry. 2004. Development of digital photography as a means to estimate species composition and species cover in tundra vegetation. Handledare: Anders Glimskär, Examinator: Roger Svensson. 122. Wärnbäck, Jan. 2004. The importance of human settlements on farmland birds breeding in semi-natural pastures. Handledare och examinator: Tomas Pärt. 123. Christensen, Carin. 2004. Seed set, seed predation and parasitism at different spatial scales in Lotus corniculatus. Handledare: Tommy Lennartsson & Aina Pihlgren, Examinator: Tommy Lennartsson. 124. Pettersson, Maria. 2004. Hur går det för gentianorna? Gentianella amarella och G. campestris i Uppland 1992 och 2003. Handledare och examinator: Tommy Lennartsson. 125. Eriksson, Alexander. 2004. Habitat selection of a nursery colony of Barbastellus barbastellus in south Sweden. Handledare: Johnny de Jong, Ingemar Ahlén, Examinator: Åke Berg. 126. Pettersson, Monica. 2004. Territory size and habitat preference of the Eurasian crane Grus grus L. during late breeding season in South Central Sweden. Handledare: Mikael Hake, Examinator: Henrik Andrén. 127. Lifvendahl, Zahrah. 2004. Fodervärde på fuktiga naturbetesmarker – analyser av fem vegetationsbildande arter. Handledare och examinator: Åke Berg. 128. Karlsson, Henrik. 2004. Causes for differences in arrival time and reproductive performance of Red-backed Shrikes in farmland grasslands and on forest clearcuts. Handledare och examinator: Bo Söderström. 129. Lindgren, Joakim. 2004. Individual character traits in male European Woodcoock, Scolopax rusticula, roding song. Handledare Göran Hartman, Jonas Lemel, Examinator: Göran Hartman. 130. Gabrielsson, Charlotte. 2004. Effekter på älg och rådjur av kalk- och askspridning. Handledare och examinator: Göran Hartman. 131. Westling, Ulrika. 2004. Läkeväxter i Sverige: En studie i biologisk mångfald. Handledare: Håkan Tunón, Roger Svensson, Examinator: Roger Svensson. 132. Wittern, Askia. 2004. Habitat use of North Island Robin (Petroica longipes) during natal dispersal. Handledare: Åsa Berggren, Bo Söderström, Examinator: Tomas Pärt. 133. Jensen, Magnus. 2004. Movements and habitat use of brown hares (Lepus europaeus) in forest dominated landscapes. Handledare: Gunnar Jansson, Åke Pehrson, Examinator: Henrik Andrén. 134. Strömqvist, Anna. 2004. Presence of Trichinella spp. in Swedish Lynx. Handledare: Göran Hartman, Dan Christensson, Examinator: Göran Hartman.

I denna serie publiceras examensarbeten utförda vid institutionen för naturvårdsbiologi, Sveriges Lantbruksuniversitet (SLU). Tidigare nummer i serien kan i mån av tillgång beställas från institutionen. Institutionen för naturvårdsbiologi SLU Box 7002 750 07 UPPSALA

2

Presence of Trichinella spp. in Lynx lynx in Sweden

3

Abstract Thirty six lynxes (Lynx lynx) were investigated for Trichinella spp. using artificial digestion, the magnetic stirrer method. The animals were of both sexes, various ages and collected during the first half of 2004 at different locations in Sweden. Three animals (8.3 %), all adult males from the county of Jämtland, were found to be infected with Trichinella spp. The level of infection was in the different cases 0.054, 0.063 and 0.12 larvae per gram muscle tissue, (lpg). Identification of Trichinella species was successfully confirmed by polymerase chain reaction, PCR, in one of the lynxes. The species identified was T. spiralis. Low expected frequencies precluded statistical analysis. The differences in frequency of trichinellosis in Swedish lynx, red fox (3.3 %) and brown bear (0%) are discussed. The differences in Trichinella infection in Swedish wildlife is compared to considerably higher frequencies in Finland. Introduction Trichinella spp. are parasitic nematodes that cause trichinellosis, a world-wide zoonosis. (Urquhart et al., 1996; Gottstein et al., 1997). The famous German pathologist Rudolph Virchow, was one of the first to describe the transmission of trichinellosis in the midst 1800s (www.trichinella.org). The parasites are transmitted from animal to animal by ingestion of infected meat (Vercammen et al., 2002). A wide range of vertebrates may be infected by Trichinella spp. (Dupouy-Camet, 2000). Principally, the most common ones are mammal carnivores that have cannibalistic and scavenger behaviour (Fink-Gremmels et al., 2001). Generally, the red fox (Vulpes vulpes) appears to be the main reservoir in Europe (Ronéus and Christensson, 1979; Fink-Gremmels et al., 2001; Vercammen et al., 2002). Characteristic of Trichinella spp. is the lifecycle. This includes two larval generations and an adult stage, all occurring in one host. After ingestion, digestive enzymes in the stomach of the host liberate the Trichinella larvae from the muscle tissue in which the larvae were encapsulated. When liberated, the larvae invade the intestinal mucosa and during a four week period they develop into adult stage and mate. The male dies after copulation. A week after copulation the female gives birth to new larvae that enter the lymphatic vessels and are distributed everywhere in the body via the circulation of the blood. Just the larvae that are transported to muscle fibres of the striated muscles will develop further. After getting occupied by a larva, the muscle fibre will be transformed by microvascularization into a nurse cell (Fig. 1), which is encapsulated by a host-derived membrane. (Urquhart et al., 1996; 4

Kassai, 1999; Dupouy-Camet, 2000; Fink-Gremmels et al., 2001; Kapel, 2002; Bowman, 2003). The encystment is completed in 14 – 16 days (www.trichinella.org). The nurse cell will keep the larva nourished and viable for many years (Kapel, 2002). The period of time the larvae survive and are infective in the host, range from a few months up to 30 years in human (Fink-Gremmels et al., 2001). When the larvae die, the wall of the cysts becomes calcified (Kassai, 1999; Kapel, 2002; Bowman, 2003). However, there are some exceptions of Trichinella species that do not form cysts (Dupouy-Camet, 2000; Dick and Pozio, 2001).

Fig.1. Schematic drawing of a Trichinella larva in its nurse cell with microvascularization. Adapted from Kasai (1999) with modification.

So far, ten species of Trichinella have been described world-wide (Dupouy-Camet, 2000; Fink-Gremmels et al., 2001). Until recently, the discovered species of Trichinella in Sweden were T. spiralis, T. nativa and T. britovi. In a recent study, Christensson and Pozio (2004) found T. pseudospiralis in both wild boar (Sus scrofa) and in European lynx (Lynx lynx), all shot in Sweden. T. pseudospiralis is the only species found in Sweden that do not form cysts. This makes the larvae of T. pseudospiralis more difficult to detect using trichinoscopy, than other Trichinella species that form cysts. Trichinoscopy is a classic method for detecting Trichinella larvae in meat. Briefly, muscle samples are divided into small pieces and pressed between two microscope slides. The Trichinella larvae are supposed to be detected in the muscle sample by means of microscopy. A larva of T. pseudospiralis that remain stretched out at full length is hard to distinguish from a muscle fibre. This can be one explanation to why T. pseudospiralis had not been discovered in Sweden until recently. (Christensson and Pozio, 2004). Another method in detecting Trichinella larvae is the digestive method. By

5

using an artificial gastric juice including HCl and pepsin during specified time and temperature, the larvae are released from the muscle tissue. This method gives better accuracy while detecting non-encapsulating Trichinella larvae such as T. pseudospiralis. The sensitivity in detecting Trichinella larvae is linked to the size of the muscle sample and the predilection site. The probability to detect larvae in an animal, infected with a low amount of larvae, increases with sample size. (Fink-Gremmels et al., 2001). The predilection site depends on the host and seems to be related to high motor activity and blood flow in a specific muscle in a specific host. For example, the predilection sites in mobile mammal carnivores are mostly limited to the extremities. (Kapel et al., 1994). There are no significant morphological differences between larvae of diverse Trichinella species regardless of ability to initiate capsule development or not. Hence, to differentiate diverse Trichinella species, DNA-analysis has to be used. (Zarlenga, 1999). In December 2003, the Swedish Environmental Protection Agency made a resolution (based on local estimates of the lynx density), permitting regulated hunting of lynxes in restricted areas in Sweden during the winter 2004 (www.naturvardsverket.se). Lynxes, killed by hunters or dead by other cause, were to be sent to the Swedish National Veterinary Institute (SVA). This provided an opportunity to investigate lynxes on a larger scale than before. The investigation included Trichinella analysis since little is known about the lynx’s importance as a carrier of Trichinella infection in Sweden. The aim of this study is to present the current status of Trichinella in free-living lynxes in Sweden with consideration to frequency of various Trichinella species, infection level, presence of Trichinella in relationship to geographical location and gender. Materials and Methods Animal samples Most of the investigated lynxes were killed during regulated hunting, others were killed in traffic and some were found dead in nature. The geographical origination of the lynxes was known. Sex was established. Age was first determined at SVA using parameters such as bodyweight, bodylength and the circumference of the skull. Age of infected lynxes was further stated by ocular examination of the cranium at the Swedish Museum of Natural 6

History. Parameters applied were the size of the cranium, growth and calcification of the sutures and the wear of teeth Parasitological methods Artificial digestion (the magnetic stirrer method), in line with the directive of the Swedish National Food Administration (SLV SF 1996:32), was used to detect Trichinella in the animals. Approximately, a total 50 g of muscle tissue per animal was taken from the tongue, the diaphragm and the forelimbs, all known to be predilection sites in carnivores (Kapel, C.M. et al., 1994). As such, all muscles sampled from one animal were digested together. Discovered larvae per gram of muscle tissue were counted. Molecular methods QIAamp DNA Micro kit was used by the instructions for genomic DNA isolation and purification of Trichinella DNA. Distilled water (20 µl) was used as elution. The expansion segment V (ESV) region of the ribosomal DNA and the mitochondrial cytochrome c-oxidase subunit I gene (COX1) was amplified by polymerase chain reaction (PCR) according to formerly published articles by Zarlenga et al. (1999) respective Nagano et al. (1999) with some modifications due to routine at SVA. The amplicons were controlled by electrophoresis in a 1 % agarose gel with ethidium bromide (∼0.8 µl/ml). GenomiPhi™ DNA Amplification Kit was used, according to instructions to amplify the remaining Trichinella DNA in two of the DNA isolates. For these two, PCR and DNA determination on gel was repeated. In the next step, the remaining amplicons of the three DNA isolates were purified using QIAquick® PCR Purification Kit.

The purified amplicons were subjected to BigDye®

Terminator v3.1 sequencing reagents. The products were determined using an automatic cycle sequencer model ABI 3100 (Applied Biosystems). The sequences were edited using the Vector NTI Suite 9 software. The sequences were thereafter compared with previously characterized genes at non-redundant data bases using BLAST, Basic Local Alignment Search Tool, at the home page of National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov/). Results A total sample of 36 lynxes was examined for the presence of Trichinella spp. The animals were killed between January and June in 2004 and the distribution limited to eight out of the

7

21 counties of Sweden. Among the 36 lynxes were 24 males and 12 females. Age determination showed that eight animals were juveniles and 28 were adults, i.e. older than one year. (table 1, fig.2).

Table 1. Distribution of investigated lynxes due to geographical location, sex and age. County

Registration

Number of adults

Number of juveniles

Total number of

Males / females

Males / females

investigations

Jämtland

Z

9/4

1/1

15

Västerbotten

AC

5/2

1/1

9

Värmland

S

1/1

0/1

3

Västernorrland

Y

2/1

0/0

3

Västra Götaland

O

1/1

0/0

2

Dalarna

W

0/0

2/0

2

Västmanland

U

1/0

0/0

1

Örebro

T

0/0

1/0

1

Three animals, or 8.3 %, were found to be infected with Trichinella. The level of infection was in the different cases 0.054, 0.063, and 0.12 larvae per gram of muscle tissue, lpg. No PCR products were found in two of the samples after the first PCR-analysis. It was assumed to be too small amount of Trichinella DNA in these cases. Therefore, it was necessary to amplify the DNA using GenomiPhi™ DNA Amplification Kit. Identification of Trichinella species was confirmed in one of the three infected lynxes. The species identified was T. spiralis. The distribution of lynxes with trichinellosis was limited to the county of Jämtland (county registration Z (fig. 2)), and to adult males.

8

Fig. 2. Map of Sweden. The letters indicate county registration.

Low expected frequencies precludes statistical analysis. Discussion According to the present study, the frequency of trichinellosis in lynx in Sweden appears to be 8.3 %. In addition, investigation of Trichinella infection in other Swedish, carnivorous mammals, such as red fox (Vulpes vulpes) and brown bear (Ursus arctos), have been made at SVA. The frequency of trichinellosis in 484 red foxes, collected in the years of 2000 –2003, was measured to 3.3 % (unpublished data, SVA). This prevalence is considerably lower than data from an earlier study by Ronéus and Christensson (1979) that showed a frequency of 19.6 % (n=1114). The change could be explained by the fact that great parts of the red fox population were eliminated during an outbreak of sarcoptic mange in the 1970-1980’s (www.jagareforbundet.se). Therefore, the Trichinella population might not, so far, have had time to recover. None of the investigated brown bears (n= 14), examined during the second half of 2003 and the first half of 2004, were Trichinella infected (unpublished data, SVA). This is in accordance with the study by Oivanen et al. (2002), where a similar pattern of Trichinella infection in the Finnish populations of lynx, red fox and brown bear was shown. These differences could be a result of feeding habits. The food can be divided in to low– and high-risk food. Low-risk food includes hares, ungulates and birds whereas carnivores and rodents are counted as high-risk food (Oksanen et al., 1998). Plants and insects are not included in any group because they do not play any part in the spread of Trichinella. Brown bears are omnivores that mainly feed on ants, berries, and ungulates (Sandegren and Swenson, 9

1997). The diet rarely includes mammal carnivores. This might minimise the risk for the brown bears of getting infected by Trichinella. Red foxes are omnivores feeding on rodents, hares, insects as well as berries and carcasses. Hence, the red fox has a higher potential risk, than brown bear, to receive trichinellosis by eating infected meat. Lynx are strictly carnivorous. The lynx is dependent on ungulates, hares and galliform birds. It is also comparably more known that lynxes regularly kill and eat domestic cats, rodents and red foxes. (Oksanen et al., 1998). This can make them very exposed to Trichinella infection. This in turn, could be a possible explanation to the fact that lynx have the highest frequency of trichinellosis found in the three compared species. However, the frequencies were considerably higher in Finland compared to Sweden, giving; lynx 53 % (n=96), red fox 37 % (n=158) and brown bear 9 % (n=150). The difference between Trichinella infection in Sweden and in Finland may be due to the growing population of raccoon dog (Nyctereutes procyonoides) in Finland. The raccoon dog, originating from the Far East, was introduced to the former Soviet Union and from there, it spread to Finland. The colonisation of raccoon dogs in Finland coincides with an increase of trichinellosis in Finnish wildlife. The raccoon dog is an easy prey both for lynx and red fox. It has been found to be well receptive to Trichinella infection (lpg range; 0.1-500) and is regarded as one of the most important reservoirs of Trichinella spp. in Finland. It is an omnivore with similar feeding preferences as the red fox. It is not as well adapted for hunting as the red fox. The frequency of trichinellosis in raccoon dogs in Finland was measured to 38 %. This number is in the same range as for the Finnish red foxes, which further could support the link between trichinellosis and feeding habits. (Oivanen et al., 2002). Currently, no established raccoon dog population is known in Sweden. Moreover, the spread of trichinellosis, from Finland to Sweden, may be halted due to limited connections between Finnish and Swedish populations of possible carriers (Söderberg, pers. comm. 2004). Even though examinations in this study have been made on common predilection sites in mammal carnivores, limited investigations have been made on species-specific predilection sites for lynx. This can therefore be more examined. According to the result of this study, the infection level in the three different cases with Trichinella, ranged between 0.052-0.12 lpg. In Finland, Oivanen et al. (2002) found an infection level in lynx ranging between 0.02-43 (median 0.7) lpg. As above, the frequency of 10

trichinellosis in wildlife is much higher in Finland compared to Sweden. A high frequency of infected animals means a high risk of transmission of the infection. The present study indicates that trichinellosis in Swedish lynx is limited to adult males in the county of Jämtland. Yet, the number of subjects is too small for such a conclusion. Despite this fact, different explanations can be discussed. While most of the investigated lynxes were killed during regulated hunting in restricted areas, the collection of lynxes is not a random sample of the entire Swedish lynx population. Areas with high lynx density are overrepresented. Actual studies of the geographical dispersal of trichinellosis in other Swedish, carnivorous mammals are lacking. It could be interesting to find out whether trichinellosis in one species is geographically connected to infection in other species, as is the case in Finland (Oivanen et al 2002). The result of this study shows a possibility that males runs a higher risk to become infected by Trichinella. Oksanen et al. (1998) found a similar pattern. Male lynxes are in general larger and heavier than females. Consequently, they can more easily kill other carnivorous mammals that could be carriers of trichinellosis. (Oksanen et al., 1998). The fact that, in this study, only lynxes over the age of one year were Trichinella infected is in accordance with the Finnish study by Oksanen et al., (1998) and the Swedish study by Ronéus and Christensson (1979). The occasions for an animal to be exposed to infection increases with age. The identification of Trichinella species was successful in one lynx out of three. This individual was the one with the highest lpg. This can explain why this was the case where identification was accomplished. The amount of DNA in the two other samples was probably too low and was therefore amplified. GenomiPhi™ DNA Amplification Kit is an amplification product that is unspecific due to genomes. This means that it amplifies all genomes in a sample regardless origin. While trying to amplify DNA from a subject isolated from a host, it is a relatively high risk that the sample will be contaminated with host DNA. Nevertheless, the species identified was T. spiralis. Christensson and Pozio (2004) found a lynx shot in Sweden in 1998, infected with T. pseudospiralis and Oivanen et al. (2002) found three lynxes infected with T. nativa. This indicates that lynxes are susceptible towards diverse Trichinella species. However, the infectivity of Trichinella may differ depending on Trichinella species and host. Kapel (2001) made a study on wild boar and Trichinella infectivity, which showed that T. 11

spiralis was highly infective in wild boar whereas, for example, T. nativa has low adaptation in this host species. It would be interesting to find out whether the infectivity of diverse Trichinella species differs in lynx. To achieve this, studies on larger number of subjects have to be made. The status of trichinellosis in the Swedish wild fauna is of growing importance, especially since the population of wild boar is increasing. Wild boar is a potential source of trichinellosis in humans. Spread of trichinellosis in the wild fauna will affect the population of wild boars and thereby even humans if the handling of meat is made improperly. Acknowledgement I would like to acknowledge my principle supervisor Göran Hartman, SLU, for good advice and for bringing a more ecological perspective to my work. Dan Christensson at the Department of Parasitology, SVA, for making this study possible and for sharing his experience and knowledge with me. Torsten Mörner at the Department of Wildlife, SVA, for introducing me to the subject. Arne Söderberg at the Department of Wildlife, for all your great answers. I am most greatful to Bodil Christensson and Annie Engström at the Department of Parasitology for all your time and assistance during the practical laboratory work. I would also like to thank everyone at the Department of Parasitology for creating the best working atmosphere! References Bowman D.D., 2003. Georgies’ Parasitology for Veterinarians 8th ed. Saunders, St Louis. Christensson, D., Pozio, E., 2004. Ny trikinart i Sverige.Svensk veterinär tidning. 56:1, 21-23. Dick, T.A., Pozio, E., 2001. Trichinella spp. and Trichinellosis. In Samuel W.M., Pybus, M.J., Kocan, A.A. (editors). Parasitic Diseases of Wild Mammals 2nd ed. Pages 380-396. Iowa state University Press, Iowa. Dupouy-Camet, J., 2000. Trichinellosis: a worldwide zoonosis. Veterinary Parasitology. 93, 191-200. Fink-Gremmels, J., van Knapen, F., Boireau, P., Dupouy-Camet, J., Geerts, S., Kapel, C., Noeckler, K., Pozio, E., 2001. Opinion of the Scientific Committee on Veterinary Measures relating to Public Health on Trichinellosis, epidemiology, methods of detecting Trichinella – free pig production. European Commission. http://europa.eu.int/comm/food/fs/sc/scv/out47_en.pdf 2003-12-09. 12

Gottstein, B., Pozio, E., Connolly, B., Gamble, H.R., Eckert, J., Jacob, H.-P., 1997. Epidemiological investigation of trichinellosis in Switzerland. Veterinary Parasitology. 72, 201-207. Kapel, C.M., Henriksen, S.A., Dietz, H.H.,Henriksen, P., Nansen, P., 1994. A Study on the Predilection Sites of Trichinella spiralis Muscle Larvae in Experimentally Infected Foxes (Alopex lagopus, Vulpes vulpes). Acta Veterinaria Scandinavica. 35, 125-132. Kapel, C.M.O., 2001. Sylvatic and domestic Trichinella spp. In wild boars; infectivity, muscle larvae distribution, and response. The Journal of Parasitology. 87:2, 309314. Kapel, C., 2002. Trikinen – et tilltagende problem i Europa. Dansk Veterinærtidsskrift. 2, 1115. Kassai, T., 1999. Veterinary Helminthology. Butterworth-Heinemann, Oxford. Nagano, I., Wu, Z., Matsuo, A., Pozio, E., Takahashi, Y., 1999. Identification of Trichinella isolates by polymerase chain reaction-restriction fragment length polymorphism of the mitochondrial cytocrome c-oxidase subunit I gene. International Journal for Parasitology. 29, 1113-1120. Oivanen, L., Kapel, C.M.O., Pozio, E., La Rosa, G., Mikkonen, T., Sukura, A., 2002. Associations between Trichinella species and host species in Finland. The Journal of Parasitology. 88, 84-88. Oksanen, A., Lindgren, E., Tunkkari, P., 1998. Epidemiology of trichinellosis in lynx in Finland. Journal of Helminthology. 72, 47-53. Ronéus, O., Christensson, D., 1979. Presence of Trichinella Spiralis in free-living red foxes (Vulpes vulpes) in Sweden related to Trichinella infection in swine and man. Acta Veterinaria Scandinavica. 20, 583-594. Sandegren, F., Swenson, J., 1999. Björnen – Viltet, Ekologin och Människan 2nd ed. Svenska Jägareförbundet, Uppsala. SLV FS 1996:32. http://www.slv.se/upload/dokument/Lagstiftning/1996-1999/1996_32.pdf 2004-10-15. Söderberg , A., 2004. Pers. Comm. The Swedish National Veterinary Institute. Urquhart, G.M., Armour, J., Duncan, J.L., Dunn, A.M., Jennings, F.W., 1996. Veterinary Parasitology 2nd ed. Blackwell science Ltd, Oxford.

13

Vercammen, F., Vervaeke, M., Dorny, P., Brandt, J., Brochier, B., Geerts, S., Verhagen, R., 2002. Survey for Trichinella spp. in red foxes (Vulpes vulpes) in Belgium. Veterinary Parasitology. 103, 83-88. Zarlenga, D.S., Chute, M.B., Martin, A., Kapel, C.M.O., 1999. A multiplex PCR for unequivocal differentiation of all encapsulated and non-encapsulated genotypes of Trichinella. International Journal for Parasitology. 29, 1859-1867. http://naturvardsverket.se/dokument/press/2003/december/p031218.htm 2004-06-08. http://www.ncbi.nlm.nih.gov/ 2004-06-18. http://www.jagareforbundet.se/viltvetande/detaljeradeartfakta/rodravforsta.asp 2004-06-09. http://www.trichinella.org 2004-08-09.

14