Annals of Tropical Medicine & Parasitology, Vol. 97, No. 3, 317–319 (2003)
Short Communication Babesia microti infection of anthropophilic ticks ( Ixodes ricinus) in Hungary The incidence of human babesiosis has appeared to increase exponentially over the last few decades (Goren ot et al., 1998; Homer et al., 2000; Kjemptrup and Conrad, 2000). Although this upward trend has been attributed to the ecological changes resulting from reforestation, increasing awareness of the disease, and/or an increasing number of susceptible individuals (mainly as the result of HIV infection), the exact cause remains unclear (Homer et al., 2000; Kjemptrup and Conrad, 2000). The parasites causing most cases of human babesiosis in the U.S.A. and Japan appear to be Babesia microti (e.g. of the US, Kobe and Hobetsu types) or B. microti-like (of the WA1 and related types) and normally infect rodents and probably other wild mammals (Homer et al., 2000; Kjemptrup and Conrad, 2000; Tsuji et al., 2001; Wei et al., 2001). In contrast, most of the human babesial infections detected in Europe have been attributed to B. divergens (Goren ot et al., 1998; Kjemptrup and Conrad, 2000), although the identi cation of the species, generally based solely on morphology, may not always have been accurate. Babesia microti infects small mammals in Europe (Sebek et al., 1977), and B. microti-like parasites have recently been detected in European dogs (Zahler et al., 2000; Camacho et al., 2001, 2002, 2003). The canine parasites, which can be distinguished from true B. microti on the basis of the sequences of the 18S ribosomal rRNA (18S rDNA) gene (Zahler et al., 2000), have never been detected in man. Babesia microti of rodents has not been implicated as a cause of human illness in Europe, because its main enzootic vector on the continent is Ixodes trianguliceps, a © 2003 The Liverpool School of Tropical Medicine DOI: 10.1179/000349803235002272
rodent-feeding, nest-dwelling tick that does not bite humans (Randolph, 1995). In Slovenia (Duh et al., 2001) and Switzerland (Floppa et al., 2002), however, B. microti has recently been detected in the anthropophilic tick, Ixodes ricinus, and there is some evidence of human exposure to B. microti in Switzerland and Germany (Krampitz et al., 1986; Floppa et al., 2002; Hunfeld et al., 2002). As the relevant studies only covered small geographical areas (Duh et al., 2001; Floppa et al., 2002), it remains unclear how commonly European I. ricinus are infected with B. microti. The main aim of the present study was to determine the prevalence of B. microti in I. ricinus collected from 16 Hungarian counties, together covering an area of about 70,000 km2. Ticks were removed from the carcasses of 100 red foxes ( Vulpes vulpes) that had been sent to the Central Veterinary Institute (CVI) in Budapest, in January– September 2002. The origins, transportation and storage of these foxes, which were sent to the CVI as part of a programme of rabies immunization and control, have already been described (Sre´ter et al., 2003). The ticks identi ed, from the key of Babos (1965), as I. ricinus were stored in 70% ethanol in microcentrifuge tubes, in separate pools (of fewer than ve ticks each) from each fox, until they could be examined further. DNA was extracted from each pool (Sre´ter et al., 2000) after the ticks had been crushed using disposable needles and homogenized using a pellet pestle (Sigma). The DNA samples were then tested in PCR using two primers (Piro-A and Piro-B) designed to amplify fragments of the 18S rDNA of Babesia spp. (Armstrong et al., 1998). Each sample found
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positive with this primer set was then tested in another PCR, using a primer pair (Bab-1 and Bab-4) that speci cally targets the 18S rDNA of B. microti (Persing et al., 1992). All of the PCR were performed in a GeneAmp 2400 PCR system (Perkin Elmer, Foster City, CA). The products were separated by electrophoresis in 1.5%-agarose gels and then detected by staining with ethidium bromide. To provide an objective and precise means of identi cation, the amplicons produced with the Bab-1 and Bab-4 primers were further characterized by sequence analysis (Sre´ter et al., 2000). The nucleotide sequences observed were compared with those in the GenBank sequence database, using the blast programme of the National Center for Biotechnology Information (Bethesda, MD), via the Center’s website (http://www.ncbi.nlm.nih.gov/blast). Of the 112 pools of I. ricinus investigated (together containing 452 nymphs and adults), B. microti-like DNA was detected in four pools, of ticks collected from four diVerent foxes. The sequences of the ampli ed DNA from each of these four pools, which were completely (100%) homologous with the published B. microti sequences (GenBank accession M93660; Duh et al., 2001) and diVerent from those of the B. microtilike parasites of dogs (GenBank accession AF188001; Zahler et al., 2000), provided the rst evidence for the occurrence of B. microti in Hungary (Sebek et al., 1977). The four foxes infested with I. ricinus carrying B. microti came from four diVerent areas of Hungary: from Budapest and the counties of Pest, Heves and Borsod-Abau´j-Zemple´n. Together, these observations and those made earlier in Slovenia (Duh et al., 2001) and Switzerland (Floppa et al., 2002) show that B. microti infection is far more common and wide-spread in the I. ricinus of Central Europe than previously realised. Ixodes ricinus can transmit B. microti, at least experimentally (Gray et al., 2002), and there is serological evidence of human exposure to B. microti in Central Europe (Krampitz et al., 1986; Floppa et al., 2002; Hunfeld et al., 2002). It
seems likely, therefore, that human infections with B. microti do occur in Europe but are not recognized, partly, perhaps, because the clinical signs of such infections are nonspeci c (Homer et al., 2000; Kjemptrup and Conrad, 2000) or because the European strains of B. microti are simply less pathogenic to humans than American or Japanese strains. acknowledgements. The authors thank A. Uzsoki, for her invaluable technical assistance in the sample preparation and PCR assays, and Z. To´th and N. Strinovich, for their help in the collection of samples. D. Ka’lma’n Veterinary Research Institute of the Hungarian Academy of Sciences, H-1143 Budapest, Hunga´ria krt. 21, Hungary T. Sre’ter Z. Sze’ll Department of Wildlife Diseases and Parasitology, Central Veterinary Institute, H-1149 Budapest, Ta´bornok u. 2, Hungary L. Egyed Veterinary Research Institute of the Hungarian Academy of Sciences, H-1143 Budapest, Hunga´ria krt. 21, Hungary Received 10 January 2003, Revised 21 February 2003, Accepted 24 February 2003 Reprint requests to: T. Sre´ter. E-mail:
[email protected]; fax: +36 1 252 5177.
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