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Journal of Clinical Microbiology, September 2001, p. 3395-3397, Vol. 39, No. 9
Institute of Microbiology and Immunology,
Medical Faculty, Ljubljana, Slovenia
Received 1 May 2001/Returned for modification 31 May 2001/Accepted 23 June 2001
Questing Ixodes ricinus (Acari: Ixodidae) adult and
nymphal ticks collected in various parts of Slovenia were tested for
the presence of babesial parasites with a PCR assay based on the
nuclear small subunit rRNA gene (nss-ribosomal DNA [rDNA]). Thirteen
of 135 ticks were found to contain babesial DNA. Sequence determination and analysis of amplified portions of nss-rDNA revealed their identity
with Babesia microti and a high degree of homology with Babesia odocoilei and Babesia divergens.
The results of this study represent the first genetic evidence of
B. microti and B.
divergens-like parasites in I. ricinus ticks in Europe.
Babesiosis, caused by infection with
intraerythrocytic parasites of the genus Babesia, is a
well-recognized disease of veterinary importance in cattle, horses, and
dogs. However, babesiosis is gaining increasing attention as an
emerging tick-borne disease in humans (8). The first
confirmed case of human babesiosis was documented in the former
Yugoslavia in 1957 Ixodes ricinus is the most prevalent and widely distributed
tick in Slovenia. As in other European countries, I. ricinus
is the main vector of the causative agents of Lyme borreliosis,
tick-borne encephalitis, and the recently described agent of human
granulocytic ehrlichiosis in Slovenia (13, 18; D. Tovornik, data presented at Symposium of Tick-Borne Encephalitis,
Slovenian Medical Society, Celje, Slovenia, 1973). The aim of this
study was to determine whether I. ricinus ticks collected in
Slovenia were infected with babesial parasites.
In the summer of 1997, 69 nymphs and 70 adult I. ricinus
ticks were collected by flagging vegetation in the Prealpine and Dinaric regions of Slovenia. The species, stage, and gender were determined by a professional entomologist. DNA was extracted from a
single tick by using the QIAamp DNA Mini kit (Qiagen, GmbH, Hilden,
Germany). The efficiency of DNA extraction was confirmed by PCR assay,
which amplifies the mitochondrial 16S rRNA gene (ribosomal DNA
[rDNA]) of tick origin (3). Each sample was tested with
the primers PIRO-A and PIRO-B, which were designed to amplify 407-, 408-, and 435-bp fragments of the nuclear small subunit rRNA gene
(nss-rDNA) of B. odocoilei, B. divergens, and B. microti, respectively (2, 10). All
samples that demonstrated a positive reaction with the PIRO-A and -B
primer set were additionally tested with BAB-1 and -4 primers, specific
for nss-rDNA of B. microti, as described by Persing et
al. (11).
The mitochondrial 16S rDNA of tick origin was amplified in 135 (97.1%)
of the 139 samples tested. Four samples in which we were unable to
amplify tick DNA were excluded from further analysis. Among 135 I. ricinus ticks, 13 (9.6%; 4 adults and 9 nymphs) were positive when tested with the PIRO-A and -B primer set. Ten of them
(7.4%; 3 adults and 7 nymphs), with DNA sizes of 435 bp, were
additionally positive when tested with BAB-1 and -4 primers.
To provide an objective and precise means of identification, amplicons
were further characterized by sequence analysis (1) (Fig.
1). Both strand sequences were
determined for seven amplicons (three 407 bp, four 435 bp) obtained
with PIRO-A and -B primers and for four amplicons of 238 bp obtained
with the BAB-1 and -4 primers.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3395-3397.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Diversity of Babesia Infecting European Sheep
Ticks (Ixodes ricinus)
ABSTRACT
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approximately 100 km from the study sites described
in this report (17). Since then, three distinctly
different babesial parasites have been recognized as the primary agents
of human disease. In Europe, 31 cases of human babesial infection were
mainly caused by Babesia divergens (4). Since
1982, over 200 cases due to Babesia microti have been
reported in the eastern United States (23). In the western
United States, seven cases in humans were attributed to the WA1 type
(12). Babesial parasites require both a competent vertebrate host and nonvertebrate host to maintain transmission cycles.
All babesial parasites described to date are transmitted by ixodid
ticks to their vertebrate hosts (6).
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FIG. 1.
Phylogenetic relationships among
Babesia species and tested samples
inferred from multiple sequence alignment of 190 and 364 bp (primer
sequences were removed) of nss-rDNA. (a) Samples 4a, 5a, 6a, and 7a
were obtained by amplification with the BAB-1 and -4 primers. (b)
Samples 1, 2, 3, 4, 5, 6, and 7 were obtained by amplification with the
PIRO-A and -B primers. The sequences obtained were aligned with those
of B. divergens (GenBank accession no. U07885),
B. odocoilei (U16369), B.
caballi (Z15104), B. bigemina (X59607),
B. canis (L19079), B.
microti (M93660, U09833), B. gibsoni
(AF158702), B. rodhaini (M87565), and
Plasmodium falciparum (M19172). Units at the bottom of
the phylogenetic tree indicate the percentage of nucleotide
substitutions.
Three sequences have shown a high degree of homology with B. odocoilei (U16369) (94.5%) and B. divergens (U07885) (93.7%), respectively. Four gene sequences obtained from 364-bp fragments (PIRO-A and -B primers) showed 99.7% identity with B. microti (U09833) and 100% identity with B. microti (M93660) when amplicons obtained with BAB-1 and -4 primers were compared.
In Europe, I. ricinus ticks serve as a vector for many tick-transmitted pathogens (i.e., borreliae, ehrlichiae, tick-borne encephalitis virus, and babesiae), although this has not been proven by molecular techniques for any Babesia sp. (4, 8). Most human infections with babesiae in Europe are believed to be caused by B. divergens. Even though we did not find an exactly identical sequence of B. divergens, experimental data showed that the human strain of B. divergens can be transmitted to cattle and gerbils by I. ricinus (9). However, we should note that in all European human infections described to date, researchers used light microscopy for identification of the implicated Babesia species. There is no deposited sequence of B. divergens causing disease in humans or animals in mainland Europe. Human cases of babesiosis caused by B. microti in Europe are rarely described and were most likely acquired abroad (4). B. microti has also been documented as a parasite of rodents in different parts of Europe (5, 7, 22). The tick that is probably involved in maintenance of B. microti in rodent populations in Europe is Ixodes trianguliceps. It is a nest-dwelling tick that does not readily bite humans (14-16). Walter has described isolation of B. microti from free-living nymphs of I. ricinus by xenodiagnosis with golden hamsters (21). The results of our study indicate for the first time that I. ricinus can indeed carry different babesiae. The obtained prevalence rate (7.4%) for B. microti determined in our study is high and is comparable to the prevalence rate for B. microti in Ixodes scapularis in the United States (19, 20). From these findings, important questions arise. Why are there no indigenous human cases of babesiosis caused by B. microti reported from Europe? Our findings do not support the explanation that this is caused by the selective feeding behavior of the proposed vector I. trianguliceps (8).
In conclusion, our results represent the first study in Europe in which B. microti and B. divergens-like parasites were directly identified in nymphs and adult I. ricinus ticks by PCR and subsequent sequence analysis.
Nucleotide sequence accession numbers. The unique sequences determined in this study were deposited in GenBank and may be accessed under accession no. AF373331, AF373332, and AF373333.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Microbiology and Immunology, Medical Faculty, Zaloska 4, 1000 Ljubljana, Slovenia. Phone: 386 1 543 7450. Fax: 386 1 543 7401. E-mail: tatjana.avsic{at}mf.uni-lj.si.
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