DOI: 10.2478/s11686-011-0022-1 © W. Stefan´ski Institute of Parasitology, PAS Acta Parasitologica, 2011, 56(2), 232–235; ISSN 1230-2821

RESEARCH NOTE

First report of britovi in

Jelena Cvetkovic1, Vlado Teodorovic2, Gianluca Marucci 3, Dragan Vasilev2, Sasa Vasilev1, Dusko Cirovic4 and Ljiljana Sofronic-Milosavljevic1* 1Department of Immunology and Immunoparasitology, Institute for the Application of Nuclear Energy – INEP, University of , Banatska 31b, 11080 Belgrade, Serbia; 2Department for Food Hygiene and Technology, Faculty of Veterinary Medicine, University of Belgrade, Bulevar Oslobodjenja 18, 11000 Belgrade, Serbia; 3Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy; 4Department of Ecology and Geography, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia

Abstract In Europe, Serbia ranks among countries with a high prevalence of Trichinella infection in pigs, which continues to be a seri- ous human health problem. While in some Balkan countries, more than one Trichinella species/genotype has been described in both the sylvatic and domestic cycles, these data are lacking for Serbia. To date, only a few Serbian isolates of Trichinella have been genetically specified, and all were classified as T. spiralis. Although transmission of Trichinella from domestic pigs to wildlife could be assumed, neither the infection status nor the species of Trichinella circulating among wildlife in Serbia has been investigated. This study shows the presence of two Trichinella species, T. spiralis and T. britovi, in wild originating from five districts in Serbia, where Trichinella infections in domestic pigs and humans have been recorded. Trichinella spiralis was detected in jackals (n = 3), red foxes (n = 2) and a wild cat (n = 1). We also established that wolves (n = 4) and red foxes (n = 2) serve as sylvatic reservoirs for T. britovi. This is the first report on the presence of T. britovi in Serbia.

Keywords Trichinella britovi, Trichinella spiralis, wildlife, epidemiology, Serbia

In Serbia, parasites of the Trichinella genus were detected ini- still ranks among those countries with a high prevalence of tially in pig carcasses in 1918, while the first cases of human Trichinella infections in both domestic pigs and humans. infection were recorded in 1923 (Djordjevic et al. 2003). Im- Molecular identification of Trichinella species by PCR plementation of mandatory meat inspection began in 1958 and was introduced in the early 1990’s, the method has since then resulted in a decrease of Trichinella prevalence in swine to been considerably improved in terms of specificity and sim- 0.009% nadir during the 1970s. Nevertheless, 100–200 human plicity (Dick et al. 1992, Bandi et al. 1993, Soule et al. 1993, cases/year continued to be reported during that time Wu et al. 1998, Appleyard et al. 1999, Zarlenga et al. 1999, (Cuperlovic et al. 2005). Beginning in the early 1990s, an Pozio and La Rosa 2003). Species identification using molec- increasing trend was observed in the number of Trichinella in- ular techniques relies on the nomenclature suggested by Pozio fected swine and cases of human trichinellosis. In Serbia, the et al. (2009b). Application of this technique enabled insight period between 1990 and 1999 was characterized by civil wars into Trichinella species distribution in many countries across and political and economic upheavals that indirectly con- the world, and has contributed to a better understanding of tributed to the re-emergence of Trichinella infections (0.06– parasite flow between the domestic and sylvatic cycles (Pozio 0.17% in swine, 500 human cases on average/year) (Cuper- 2000b, 2007a; Pozio et al. 2009a). In Europe, four Trichinella lovic et al. 2005). In the 21st century, the infection rate had species have been identified: T. spiralis, T. nativa, T. britovi markedly declined: for swine from a prevalence of 0.16% to and T. pseudospiralis (Oivanen et al. 2002, Oivanen and Ok- 0.052% between 2000 and 2009; and for humans from 500 to sanen 2009, Pozio et al. 2009a, b) but in Serbia only T. spiralis less than 200 cases per year over the same interval (Sofronic- has been recognized, probably because only four Trichinella Milosavljevic, unpublished data). Despite this decline, Serbia isolates from Serbia had been identified to the species level at

*Corresponding author: [email protected] Trichinella britovi in Serbia 233

the International Trichinella Reference Center of Rome, Italy T. britovi. T. spiralis was found in six isolates: three from jack- (ITRC, www.iss.it/site/Trichinella/index.asp). Of these, three als, two from red foxes and one from a wild cat, and T. britovi isolates were from domestic pigs and one from a red fox in the other six isolates: four from wolves and two from red (Pozio et al. 2009a). foxes (Table I). The highest LPG was detected in golden jack- The role of wildlife as a reservoir of Trichinella spp. and als (range: 1.9–21.4 LPG, mean: 9.8) and wolves (9–13.4 the existence of a sylvatic cycle was recognized in the former LPG, mean: 9.6) while lower LPG were found in red foxes Yugoslavia (the break up of the country took place in 1991), (3.5–12.9 LPG, mean: 6.9) and the wild cat (5.4 LPG). The as a result of sporadic investigations (Brglez 1988). However, geographical origin of the twelve Trichinella spp. isolates is the prevalence of Trichinella infection in game animals in Ser- shown in Table I. bia has not been investigated since and therefore no informa- The present study has revealed that at least two Trichinella tion is available on Trichinella species currently circulating species, T. britovi and T. spiralis are present in Serbia. While among wildlife. The aim of our work was to isolate and iden- the presence of T. spiralis has been previously reported (Cu- tify Trichinella species infecting wild animals in Serbia. This perlovic et al. 2005, Pozio et al. 2009a), this is the first find- study confirms the existence of T. spiralis and reveals, for the ing of T. britovi in Serbia. This finding was not unexpected, first time, the presence of T. britovi in Serbia. since T. britovi and T. spiralis are the two most common Trichinelloscopic examination of tongue muscle (0.5 g) Trichinella species occurring in Europe (Pozio et al. 2009a). from wild animals, hunted in Serbia, was carried out by local Among the countries that border Serbia, T. britovi presence veterinary services. Positive findings of Trichinella larvae has been detected in Hungary, Romania, Bulgaria, Macedo- were confirmed by the Department for Food Hygiene and nia and Croatia (Pozio 2007a). Technology, Faculty of Veterinary Medicine, Belgrade and Although the species were not identified, Trichinella in- Trichinella-positive muscle samples were stored at –20°C. fection in wildlife was recognized previously in Yugoslavia Eleven samples collected during the regular hunting sea- (Brglez 1988). Thus between 1960 and 1980 the prevalence son of 2008–2009 and one sample obtained in 2005 were de- of Trichinella infections was 47% in lynxes (Lynx lynx), 44% livered to the Department for Immunology and Immuno- in wolves (Canis lupus), 25% in badgers (Meles meles), 16.8% parasitology, Institute for the Application of Nuclear Energy – in brown bears (Ursus arctos), 5.5% in red foxes (Vulpes INEP, Belgrade, for isolation and molecular typing of larvae. vulpes), 4.51% in wild cats (Felis sylvestris) and 4.3% in wild Samples originated from: one wild cat (Felis sylvestris), boars (Sus scrofa). golden jackals (Canis aureus) (n = 3), red foxes (Vulpes In this study, T. spiralis was isolated from a wild cat, vulpes) (n = 4) and wolves (Canis lupus) (n = 4), hunted in golden jackals and red foxes hunted in three districts (Bel- five Serbian districts (Belgrade, Podunavlje, Branicevo, Zla- grade, Podunavlje and Branicevo), where Trichinella infec- tibor and Pirot). tions were present in domestic pigs. The prevalence of Trichinella spp. larvae were collected from infected mus- Trichinella infections in domestic pigs in these districts, dur- cles by artificial digestion of tongue muscle tissue (25 g) ac- ing 2008 was: 0.009%, 0.03% and 0.37%, respectively cording to a standard protocol (Gamble et al. 2000). After (Sofronic-Milosavljevic, unpublished data). In 1999 and 2003, digestion, Trichinella spp. larvae were recovered and the the Ministry of Agriculture of Serbia declared the district of worm burden was expressed as number of larvae per gram of Branicevo as an endemic region for Trichinella infections muscle tissue (LPG). At least 20 larvae from each isolate were (Zivojinovic et al. 2009, 2010). The presence of the so-called stored in absolute ethyl alcohol at –4°C for molecular typing. “domestic” Trichinella species in wildlife is not surprising, Trichinella larvae were identified at the species level by a considering the occurrence of T. spiralis in the domestic cycle multiplex PCR analysis both at the ITRC and at the INEP, ac- of Serbia and human behavior which supports the transmis- cording to the test accredited by the European Union Re- sion of this pathogen species from the domestic to the sylvatic ference Laboratory for Parasites, Rome, Italy (http://www.iss.it/ cycle and vice versa. Our understanding of the transmission of crlp). Briefly, DNA was extracted from a single muscle larvae Trichinella spp. in the domestic and sylvatic cycles (Pozio and 10 µl of larval DNA was used for the PCR. The PCR con- 2007b), points to many risk factors in Serbia including: 1) in- ditions consisted of an initial denaturation step at 95°C for tentional feeding of pigs with food waste containing 4 min followed by 35 cycles at 95°C for 10 s, 55°C for 30 s and scraps; 2) scavenging on garbage dumps by pigs and dogs; 72°C for 30 s. After a final extension step at 72°C for 3 min, and 3) disposal of carcasses in the open, which allows sylvatic PCR-amplified products were separated by electrophoresis in animals to scavenge on carcasses of infected domestic swine a 2% agarose gel (Sigma-Aldrich Chemie GmbH, Steinheim, (Pozio 2007b, Zivojinovic et al. 2010). Our observations on ) with TAE buffer and stained with ethidium bro- the presence of T. spiralis among jackals and wild cats in Ser- mide. Single larvae from two encapsulated and one non-en- bia support the suggestion that other wild animals associated capsulated reference strains were used as controls (T. spiralis, with farms and settlements may also be involved in the infec- ISS3; T. britovi, ISS2; and T. pseudospiralis, ISS13). tion cycle, transmitting the infection to pigs and also from pigs Trichinella larvae collected from muscle samples of twelve to other hosts (Murrell et al. 1987). Our data regarding wild carnivores from Serbia were identified as T. spiralis or Trichinella infections in domestic pigs in particular districts 234 Jelena Cvetkovic et al.

Table I. Trichinella species detected in wildlife in Serbia Trichinella spp. LPG1 Locality of origin Latitude, (district) Longitude Wild cat T. spiralis 5.36 Surcin (Belgrade) 20,3393°E 44,7768°N Red fox T. britovi 3.52 Dobanovci (Belgrade) 20,2522°E 44,8147°N Red fox T. britovi 4.4 Progar (Belgrade) 20,2004°E 44,7515°N Red fox T. spiralis 1 Progar (Belgrade) 20,1703°E 44,6902°N Red fox T. spiralis 12.88 Pozezeno (Branicevo) 21,5746°E 44,7268°N Wolf T. britovi 5.88 Bela Palanka (Pirot) 22,2731°E 43,2085°N Wolf T. britovi 13.36 () 19,9695°E 43,2474°N Wolf T. britovi 8.96 Sjenica (Zlatibor) 19,9315°E 43,2858°N Wolf T. britovi 10.96 Sjenica (Zlatibor) 19,9695°E 43,2474°N T. spiralis 1.88 Osipaonica (Podunavlje) 21,1275°E 44,5482°N Golden jackal T. spiralis 5.88 Zatonje (Branicevo) 21,3564°E 44,7685°N Golden jackal T. spiralis 21.36 Smederevo (Podunavlje) 21,0608°E 44,6372°N

1Number of larvae per gram averaged over three counts. of Serbia, and those presented in this study on wildlife agree T. britovi has already been recorded (Kurdova et al. 2004). with the opinion of Gottstein et al. (2009) that the sylvatic and Since no T. spiralis infection was found in wolves, even domestic cycles are not exclusive but can exist in parallel and though Trichinella infection occurred in pigs in both of these even overlap. districts (0.07% infected pigs for Zlatibor and 0.127% for Pirot This study, represents the first investigation on the pres- districts in 2008, Sofronic-Milosavljevic, unpublished data), it ence of Trichinella spp. in wildlife from Serbia, and reveals appears that this sylvatic cycle occurs in wildlife only, inde- that among the five districts from which infected carnivores pendent of the domestic cycle. originated, both species of Trichinella coexist in the district The mean larval burden in T. britovi infected animals was of Belgrade. Co-existence of these two species was observed similar to that in T. spiralis infected animals (7.84 larvae/g in vulpine populations in other European countries (Pozio et and 8.06 larvae/g, respectively), but due to the small number al. 2000a). This study confirmed previous observations (Pozio of animals analyzed, this observation cannot be statistically 2000b) suggesting that, wild canides play an important role in supported. the transmission of Trichinella infection between the domes- tic and the sylvatic cycles due to their synanthropic behavior Acknowledgements. This work was supported in part by project No. and because they are reservoirs of Trichinella in wildlife. 143047, Ministry of Science and Technological Development, R. Ser- Our results show the existence of a wildlife reservoir for bia and in part by FP6 project INCO-CT-2006-043702-SERBPAR T. britovi in Serbia, involving wolves and red foxes. This is in ZOON. We would like to thank Dr Edoardo Pozio, Head of the Eu- agreement with the recent finding (Pozio et al. 2009b) which ropean Union Reference Laboratory for Parasites for his technical shows that wild canids are the main reservoir of T. britovi. The support to the Serbian National Reference Laboratory for Trichinel- losis and for assistance in the manuscript preparation. isolation of T. britovi in foxes shot in the Belgrade district which is close to pig farms, has to be considered as a risk and suggests a need to rear pigs in confinement systems which avoid contact with wildlife. References Trichinella britovi-infected wolves, were found in two dis- Appleyard G.D., Zarlenga D., Pozio E., Gajadhar A.A. 1999. Differ- tricts of Serbia (Zlatibor and Pirot). Both are located in a entiation of Trichinella genotypes by polymerase chain reac- mountainous region, especially in the Pirot District, which is tion using sequence-specific primers. Journal of Parasitology, close to the border with Bulgaria where the presence of 85, 556–559. DOI: 10.2307/3285795. Trichinella britovi in Serbia 235

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(Accepted January 27, 2011)