First report of Borrelia burgdorferi sensu lato in two threatened carnivores: the Marbled polecat, Vormela peregusna and the European mink, Mustela lutreola (Mammalia: Mustelidae)
© Gherman et al.; licensee BioMed Central Ltd. 2012
Received: 17 April 2012
Accepted: 13 August 2012
Published: 18 August 2012
Lyme disease is a widespread cosmopolitan zoonosis caused by species belonging to the genus Borrelia. It is transmitted from animal reservoir hosts to humans through hard - ticks of genus Ixodes which are vectors of the disease.
Borrelia burgdorferi sensu lato infection was identified in a marbled polecat, Vormela peregusna, and two European minks, Mustela lutreola, from Romania, by PCR. RFLP revealed the presence of a single genospecies, Borrelia burgdorferi sensu stricto.
This is the first report of the Lyme disease spirochetes in the two mentioned hosts.
KeywordsBorrelia burgdorferi s.s First report Mustela lutreola Vormela peregusna Romania
Borrelia burgdorferi sensu lato is the causative agent of Lyme borreliosis, the most widespread vector-borne disease in the cool-temperate regions of the Northern hemisphere. The medical importance of this pathogen is generally restricted to humans and few domestic species. However, as for many vector-borne diseases, the key to the understanding of the epidemiology of Lyme borreliosis consists in revealing the ecological relationships that exist between pathogens, vectors and wildlife hosts . All 18 currently recognized genotypes  circulate in nature between vectors (several ticks of genus Ixodes) and reservoir hosts (various vertebrates) which are able to maintain and transmit the spirochetes .
The collection localities of threatened Mustelidae specimens used in this study
European mink (Mustela lutreola)
Canal Litcov (TL)
European mink (Mustela lutreola)
Canal Litcov (TL)
European mink (Mustela lutreola)
Canal Litcov (TL)
Marbled polecat (Vormela peregusna)
Marbled polecat (Vormela peregusna)
During the necropsy of these animals, tissue samples (myocardium) were collected and processed for DNA extraction (Qiagen, DNeasy Blood & Tissue Kit). An extraction blank was included in each extraction procedures to control the cross-contamination between extracts. The crude DNA was analyzed by a PCR protocol according to Priem et al. (1997)  using primers (Generi Biotech) for Borrelia burgdoferi sensu lato (5’-GGGAATAGGTCTAATTTAGCC-3’, 5’-CACTAATTGTTAAAGTGGAAGT-3’) targeting the OspA gene . Each time the PCR was performed including negative control samples. The positive PCR products were further analyzed by RFLP using two restriction enzymes Alwl (BsPI) and MseI (Tru1 I) (Fermentas), according to the manufacturers protocol. PCR analysis revealed positivity to Borrelia burgdorferi sensu lato in three samples (ML2, ML3 and VP1) belonging to the two host species: M. lutreola and V. peregusna.
Check-list of Ixodid tick species associated with mustelids
Martes sp. 
Several authors have previously reported the presence of Borrelia burgdorferi s.l. in Ixodes ticks removed from mustelids: European badger (Meles meles) , American mink (Mustela vison) , weasel (Mustela nivalis) , beach marten (Martes foina), European polecat (Mustela putorius), and stoat (Mustela ermina) . In Italy, none of the I. hexagonus and I. ricinus collected from beech marten was positive by PCR for B. burgdorferi s.l. .
An interesting study from Switzerland, where questing ticks were analyzed for the host of the previous feeding stage by Reverse Line Blotting revealed the presence of Borrelia burgdorferi s.l. DNA in specimens positive for Meles meles and Mustela putorius probes. The authors suggest the role of mustelids as reservoir hosts for Borrelia burgdorferi.
Other authors examined mustelids for the presence of anti-Borrelia burgdorferi s.l. antibodies. One least weasel caught in forests of the western part of France was seropositive for B. burgdorferi at 1/50 antibody titer , however stoats from Canada infested with I. Scapularis (the most important vector of Lyme disease in North America) were seronegative .
To our knowledge, there is a single study showing the presence of B. burgdorferi s.l. DNA in tissues of mustelids. McDonald and Lariviere (2001)  found the infection in 10 of 45 stoats from Great Britain. However, no information was provided on the tissues examined or the genospecies identified. Here we report the first identification of B. burgdorferi s.l. from the myocardium of a marbled polecat and two European minks. The Marbled polecat, Vormela peregusna is widespread from Southeastern Europe to Russia, China and northern Africa . It is listed by IUCN as vulnerable. The European mink, Mustela lutreola is a semi-aquatic species of mustelid native to Europe and it is listed by IUCN as Critically Endangered. Its current distribution includes small isolated populations in northern Spain, western France and Eastern Europe (Latvia, Estonia, Belarus, Ukraine, central regions of European Russia, the Danube Delta in Romania and northwestern Bulgaria) . In Romania, both species are present in the Southeastern part of the country, Dobrogea [20, 21].
Detection of Lyme spirochete is relatively rare in mustelids, suggesting the limited role of these hosts in natural foci. The detection of pathogens in tissues or in ticks feeding on various vertebrates does not confer to these hosts the status of reservoir host, but rather carrier hosts. Hence, although it has been suggested that mustelids are reservoir hosts for B. burgdorferi s.l., experimental transmission is required to clarify this aspect.
This study was funded from grant IDEI-PCCE CNCSIS 84, 7/2010, while ADS was supported by a research grant POSDRU/88/1.5/S/60185.
Special thanks to the three anonymous referees, who commented on an earlier version of the manuscript.
- Gern L, Humair P: Ecology of Borrelia burgdorferi sensu lato in Europe. Lyme Borreliosis: Biology, Epidemiology and Control. Edited by: Gray JS KO, Lane RS, Stanek G. New York: CABI Publishing; 2002:149–174.View ArticleGoogle Scholar
- Stanek G, Reiter M: The expanding Lyme Borrelia complex-clinical significance of genomic species?. Clin Microbiol Infect. 2011, 17 (4): 487-493. 10.1111/j.1469-0691.2011.03492.x.View ArticlePubMedGoogle Scholar
- Kahl O, Gern L, Eisen L, Lane RS: Ecological research on Borrelia burgdorferi sensu lato: terminology and some methodological pitfalls. Lyme Borreliosis: Biology, Epidemiology and Control. Edited by Gray JS KO, Lane RS, Stanek G. New York: CABI Publishing; 2002:29–46.View ArticleGoogle Scholar
- Kranz A, Toman A, Marinov M, Kiss JB: European mink research: results of the spring 2004 expedition. Sci Ann Danube Delta Inst Res Develop. 2005, 11: 42-44.Google Scholar
- Priem S, Rittig MG, Kamradt T, Burmester GR, Krause A: An optimized PCR leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme Borreliosis. J Clin Microbiol. 1997, 35 (3): 685-690.PubMed CentralPubMedGoogle Scholar
- Floris R, Menardi G, Bressan R, Trevisan G, Ortenzio S, Rorai E, Cinco M: Evaluation of a genotyping method based on the ospA gene to detect Borrelia burgdorferi sensu lato in multiple samples of Lyme borreliosis patients. New Microbiol. 2007, 30: 399-410.PubMedGoogle Scholar
- Gern L, Toutoungi LN, Hu CM, Aeschlimann A: Ixodes (Pholeoixodes) hexagonus , an efficient vector of Borrelia burgdorferi in the laboratory. Med Vet Entomol. 1991, 5: 431-435. 10.1111/j.1365-2915.1991.tb00571.x.View ArticlePubMedGoogle Scholar
- Gern L, Rouvinez E, Toutoungi LN, Godfroid E: Transmission cycles of Borrelia burgdorferi sensu lato involving Ixodes ricinus and/or I. hexagonus ticks and the European hedgehog, Erinaceus europaeus , in suburban and urban areas in Switzerland. Folia Parasitol. 1997, 44: 309-314.PubMedGoogle Scholar
- Gern L, Sell K: Isolation of Borrelia burgdorferi sensu lato from the skin of the European badger (Meles meles) in Switzerland. Vector Borne Zoonotic Dis. 2009, 9 (2): 207-208. 10.1089/vbz.2008.0050.View ArticlePubMedGoogle Scholar
- Magnarelli LA, Swihart RK: Spotted fever group Rickettsiae or Borrelia burgdorferi in Ixodes cookei (Ixodidae) in Connecticut. J Clin Microbiol. 1991, 29 (7): 1520-1522.PubMed CentralPubMedGoogle Scholar
- Gassner F, Verbaarschot P, Smallegange RC, Spitzen J, Van Wieren SE, Takken W: Variations in Ixodes ricinus density and Borrelia infections associated with cattle introduced into a woodland in The Netherlands. Appl Environ Microbiol. 2008, 74 (23): 7138-10.1128/AEM.00310-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Piesman J, Gern L: Lyme borreliosis in Europe and North America. Parasitol. 2004, 129 (Suppl1): S191-S220.View ArticleGoogle Scholar
- Lorusso V, Lia RP, Dantas-Torres F, Mallia E, Ravagnan S, Capelli G, Otranto D: Ixodid ticks of road-killed wildlife species in southern Italy: new tick-host associations and locality records. Exp Appl Acarol. 2011, 55: 293-300. 10.1007/s10493-011-9470-4.View ArticlePubMedGoogle Scholar
- Morán Cadenas F, RAIS O, Humair PF, Douet V, Moret J, Gern L: Identification of host bloodmeal source and Borrelia burgdorferi Sensu Lato in field-collected Ixodes ricinus ticks in Chaumon (Switzerland). J Med Entomol. 2007, 44 (6): 1109-1117. 10.1603/0022-2585(2007)44[1109:IOHBSA]2.0.CO;2.View ArticlePubMedGoogle Scholar
- Doby JM, Betremieux C, Lambert MC, Lorvelec O, Rolland C, Costil C: Les micromammiferes forestiers reservoirs de germes pour Borrelia burgdorferi agent de la borreliose de Lyme ? Etude serologique de 296 animaux dans l’ouest de la France. Rev Med Vet. 1991, 142 (10): 737-742.Google Scholar
- Bouchard C, Beauchamp G, Nguon S, Trudel L, Milord F, Lindsay LR, Bélanger D, Ogden NH: Associations between Ixodes scapularis ticks and small mammal hosts in a newly endemic zone in southeastern Canada: Implications for Borrelia burgdorferi transmission. Ticks Tick Borne Dis. 2011, 2: 183-190. 10.1016/j.ttbdis.2011.03.005.View ArticlePubMedGoogle Scholar
- McDonald RA, Lariviere S: Diseases and pathogens of Mustela spp., with special reference to the biological control of introduced stoat Mustela erminea populations in New Zealand. J Royal Soc New Zealand. 2001, 31 (4): 721-744. 10.1080/03014223.2001.9517671.View ArticleGoogle Scholar
- Schreiber A, Wirth R, Riffel M, Van Rompaey H: Weasels, Civets, Mongooses and their relatives: an action plan for the conservation of mustelids and viverrids. Broadview: Kelvyn Press Inc; 1989.Google Scholar
- Maran T, Skumatov D, Palazón S, Gomez A, Põdra M, Saveljev A, Kranz A, Libois R, Aulagnier S: Mustela lutreola. In IUCN 2011. IUCN Red List of Threatened Species. Version 2011.2. Downloaded on 04 April 2012. 2012, www.iucnredlist.org.Google Scholar
- Călinescu R: [Mammals of Romania. Distribution and biogeographical-economic problems]. Buletinul Ministerului Agriculturii Domeniilor. 1931, 251: 19-81. [in Romanian].Google Scholar
- Cuzic M, Murariu D: [Data on the presence of the Marbled Polecat ( Vormela peregusna Güld. 1770) in south-east Romania]. Studii Cercetări Știinţele Naturii Muzeologie. 2006, 3: 193-198.Google Scholar
- Santos-Silva MM, Beati L, Santos AS, De Sousa R, Núncio MS, Melo P, Santos-Reis M, Fonseca C, Formosinho P, Vilela C, Bacellar F: The hard-tick fauna of mainland Portugal (Acari: Ixodidae): an update on geographical distribution and known associations with hosts and pathogens. Exp Appl Acarol. 2011, 55: 85-121. 10.1007/s10493-011-9440-x.View ArticlePubMedGoogle Scholar
- Page RJC, Langton SD: The occurrence of lxodid ticks on wild mink Mustela vison in England and Wales. Med Vet Entomol. 1996, 10: 359-364. 10.1111/j.1365-2915.1996.tb00757.x.View ArticlePubMedGoogle Scholar
- Murrell BP, Durden LA, Cook JA: Host associations of the tick, Ixodes angustus (Acari: Ixodidae), on Alaskan Mammals. J Med Entomol. 2003, 40 (5): 682-685. 10.1603/0022-2585-40.5.682.View ArticlePubMedGoogle Scholar
- Carey AB, Krinsky WL, Main J: Ixodes dammini (Acari: Ixodidae) and associated did ticks in south-central Connecticut, USA. J Med Entomol. 1980, 17: 89-99.View ArticlePubMedGoogle Scholar
- Whitaker JO, Goff R: Ectoparasites of wild carnivora of Indiana. J Med Entomol. 1979, 15 (5–6): 425-430.View ArticlePubMedGoogle Scholar
- Kollars TM, Oliver JH: Host associations and seasonal occurrence of Haemaphysalis leporispalustris, Ixodes brunneus, I. cookei, I. dentatus, and I. texanus (Acari: Ixodidae) in southeastern Missouri. J Med Entomol. 2003, 40 (1): 103-107. 10.1603/0022-2585-40.1.103.View ArticlePubMedGoogle Scholar
- Caeiro V, Santos-Silva MM, Simōes AL, Núncio MS: Ixodes (Pholeoixodes) canisuga Johnston, 1949 uma espécie nova na ixodofauna em Portugal. Rev Port Cinc Vet. 1997, 92: 120-123.Google Scholar
- Sleeman DP: Ectoparasites of the Irish stoat. Med Vet Entomol. 1989, 3: 213-218. 10.1111/j.1365-2915.1989.tb00217.x.View ArticlePubMedGoogle Scholar
- Corbet GB, Harris S: The Handbook of British Mammals. Oxford: Blackell Scientific Publications; 1991.Google Scholar
- Von Liebisch A, Walter G: Untersuchungen von Zecken bei Haus- und Wildtieren in Deutschland: Zum Vorkommen und zur Biologie der Igelzecke (Ixodes hexagonus) und der Fuchszecke (Ixodes canisuga). Dtsch Tierarztl Wschr. 1986, 93: 377-464.Google Scholar
- Lindquist EE, Wu KW, Redner JH: A new species of the tick genus Ixodes (Acari: Ixodidae) parasitic on mustelids (Mammalia: Carnivora) in Canada. Can Entomol. 1999, 131: 151-170. 10.4039/Ent131151-2.View ArticleGoogle Scholar
- Lubelczyka CB, Hanson T, Lacombe EH, Holman MS, Keirans JE: First U.S. Record of the Hard Tick Ixodes (Pholeoixodes) gregsoni Lindquist, Wu, and Redner. J Parasitol. 2007, 93 (3): 718-719. 10.1645/GE-943R.1.View ArticleGoogle Scholar
- Dias JATS, Núncio MS, Gongalves ACB: Contribuição para a elaboração de um inventário da fauna ixodoideológica (Acarina-Ixodoidea) de Portugal. Garcia Orta Sér Zool. 1994, 20: 49-68.Google Scholar
- Dominguez G: North Spain (Burgos) wild mammals ectoparasites. Parasite. 2004, 11: 267-272.View ArticlePubMedGoogle Scholar
- Toutoungi LN, Gern L, Aeschlimann A, Debrot S: A propos du genre Pholeoixodes , parasite des carnivores en Suisse. Acarologia. 1991, 32 (4): 311-328.Google Scholar
- Anderson JF, Magnarelli LA: Epizootiology of Lyme Disease-Causing Borreliae. Clin Dermatol. 1993, 11: 339-351. 10.1016/0738-081X(93)90088-T.View ArticlePubMedGoogle Scholar
- Dias JATS, Santos-Reis M: A carraqa Ixodes ventalloi Gil Collado, 1936 como principal ectoparasita de uma populao de doninhas (Mustela nivalis Linnaeus, 1766) em Portugal. Garcia Orta Sér Zool. 1989, 14: 35-50.Google Scholar
- Beichel E, Petney TN, Hassler D, Brückner M, Maiwald M: Tick infestation patterns and prevalence of Borrelia burgdorferi in ticks collected at a veterinary clinic in Germany. Vet Parasitol. 1996, 65 (1–2): 147-155.View ArticlePubMedGoogle Scholar
- Walker ED, Stobierski MG, Poplar ML, Smith TW, Murphy AJ, Smith PC, Schmitt SM, Cooley TM, Kramer CM: Geographic distribution of ticks (Acari: Ixodidae) in Michigan, with special emphasis on Ixodes scapularis and Borrelia burgdorferi. J Med Entomol. 1998, 35: 872-882.View ArticlePubMedGoogle Scholar
- Kim HC, Han SH, Chong ST, Klein TA, Choi CY, Nam HY, Chae HY, Lee H, Ko S, Kang JG, Chae JS: Ticks collected from selected mammalian hosts surveyed in the Republic of Korea during 2008–2009. Korean J Parasitol. 2011, 49 (3): 331-335. 10.3347/kjp.2011.49.3.331.PubMed CentralView ArticlePubMedGoogle Scholar
- Furman DE, Loomis EC: The ticks of California (Acari: Ixodida). Bull Calif Insect Surv. 1984, 25: 1-239.Google Scholar
- Kolonin GV: World distribution of ixodid ticks (genus Ixodes). Nauka: Moscow; 1981 [In Russian].Google Scholar
- Kitaoka S, Suzuki H: Studies on the parasite fauna of Thailand. 5. Parasitic ticks on mammals and description of Ixodes siamensis sp. n. and Rhipicephalus tetracornus sp. n. (Acarina: Ixodidae). Trop Med. 1983, 25: 205-219.Google Scholar