Babesia microti in Adult Dermacentor reticulatus Ticks from Eastern Poland

Abstract

A total of 468 adult Dermacentor reticulatus ticks (298 females and 170 males) collected by flagging in the forests of Lubelskie province, eastern Poland, were analyzed by PCR and nested-PCR for the presence of Babesia microti DNA. In all, 21 ticks (4.5%) were found to be infected with B. microti. The infection rate in females (5.0%) was slightly greater than in males (3.5%). Detection of B. microti for the first time in adult D. reticulatus ticks suggests that this species should be considered as a potential vector of B. microti in Europe.

Introduction

B abesia microti (Apicomplexa, Piroplasmida) is a tick-borne protozoan parasite that develops in erythrocytes of animals and humans. According to some reviews this species actually consists of a genetically diverse species complex (Gray 2006; Siński et al. 2006; Hunfeld et al. 2008). It occurs in Microtus voles and other small mammals, and is transmitted by bites of Ixodes ricinus ticks in Eurasia, and of Ixodes scapularis and Dermacentor variabilis ticks in North America (Karbowiak 2004). Pieniążek and associates (2006) described the occurrence of B. microti and B. divergens in single individuals of I. ricinus. Human cases of babesiosis caused by B. microti are common in the U.S. (Homer et al. 2000; Kjentrup and Conrad 2000; Skotarczak et al. 2002; Gray 2006; Hunfeld et al. 2008), and recently have also been reported in Europe (Hildebrandt et al. 2007). Thus far, it is not clear which tick species, apart from I. ricinus, could be potential vectors of B. microti in Eurasia. One of the candidates is Dermacentor reticulatus, a species common in eastern Europe (Siuda 1993, 2006), and one that has also been reported in central and western European countries (Nijhof et al. 2007; Dobler et al. 2008). To date, B. microti has been detected in larvae and nymphs of D. reticulatus (Welc-Falęciak et al. 2008), but not in adult specimens of this species (Nijhof et al. 2007; Zygner et al. 2010; Cochez et al. 2011). To study this problem, a sample of questing D. reticulatus ticks collected from lower vegetation in Lublin province, eastern Poland, was examined for the presence of B. microti using polymerase chain reaction (PCR).

Materials and Methods

Collection of ticks

Unfed D. reticulatus ticks (males and females) were collected during the spring and summer of 2008 and 2009 from the forested areas of five districts of the Lublin region. Of these, two districts (Parczew and Włodawa) harbored wet lakeland forests, while the other three districts (Zamość, Puławy, and Lublin) harbored dry upland forests. The ticks were collected by dragging a woolen flag over lower vegetation at the peripheral and inner parts of deciduous and mixed forests, including suburban localities and recreational areas. The ticks were collected alive and stored at −20°C for further investigation.

DNA isolation

A total of 468 D. reticulatus ticks (298 females and 170 males) were examined. Protozoan DNA was isolated from ticks after removal from alcohol by boiling in 100 μL of 0.7 M ammonium hydroxide for 20 min, and evaporating for 15 min, according to the method of Rijpkema and associates (1996), and stored at −20°C for further analysis.

Detection of Babesia microti DNA by PCR and nested-PCR

Tick lysates were examined for the presence of B. microti DNA using amplification by PCR and confirmatory re-amplification by nested-PCR. The following sets of primers were applied: a pair of outer primers: Bab1 (5′-CTT AGT ATA AGC TTT TAT ACA GC-3′) and Bab4 (5′-ATA GGT CAG AAA CTT GAA TGA TAC A-3′); and a pair of inner primers: Bab2 (5′-GTT ATA GTT TAT TTG ATG TTC GTT T-3′) and Bab3 (5′-AAG CCA TGC GAT TCG CTA AT-3′). These primers are specific for a gene encoding the nuclear small subunit ribosomal RNA (SS-rDNA; Stańczak et al. 2004), and were previously described by Persing and colleagues (1992).

Each PCR reaction was done in a 25-μL reaction volume which contained the following mix of reagents: 0.625 U Taq DNA polymerase (QIAGEN, Valencia, CA), 1× PCR buffer containing 15 mM MgCl₂, 0.625 μL 2 mM dNTP (final concentration 0.05 mM; Fermentas, Vilnius, Lithuania), 1 μL 10 μM each of primer Bab1 and Bab4 (Eurogentec, Seraing, Belgium), 2.5 μL of DNA, and nuclease-free water (Applied Biosystems, Foster City, CA). DNA of B. microti merozoites (obtained from the Department of Parasitology, University of Warsaw) was used as a positive control and nuclease-free water as a negative control. The size of the amplified DNA fragment was 238 base pairs (bp).

Nested PCR reactions were carried out under the same conditions with 1 μL of the first amplification product, and with 1 μL 10 μM of each inner primer Bab2 and Bab3. The size of the amplified DNA fragment was 154 bp.

The amplification and re-amplification were carried out in a PTC-150 thermal cycler (MJ Research Inc., Waltham, MA) according to the method of Stańczak and associates (2004). Products of amplification and re-amplification were identified in 2% and 2.5% agarose gel, respectively, after electrophoresis in standard conditions and staining with ethidium bromide solution (2 μg/mL). The amplified fragments were visualized in a transilluminator under UV light (UV-953; JW Electronics, Warsaw, Poland).

DNA sequencing was performed with 38% (8 out of 21) of randomly-selected positive samples with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) using ABI PRISM Big Dye Terminator v. 3.1. Cycle Sequencing Kits, and Big Dye XTerminator Purification Kits (Applied Biosystems). The results were compared with sequences in the GenBank database using the BLAST server at the National Center for Biotechnology Information (Bethesda, MD).

Statistical analysis

The data were analyzed by chi-square testing and Student's t-test with the use of STATISTICA for Windows v. 5.0 (StatSoft Inc., Tulsa, OK).

Results

Out of 468 adult Dermacentor reticulatus ticks, 21 (4.5%) were found to be infected with Babesia microti. The infection was found in 15 females out of 298 (5.0%) and in 6 males out of 170 (3.5%). There was no statistically significant difference between the prevalence of B. microti in females and males of D. reticulatus (p>0.05).

The sequence analysis of positive samples obtained from D. reticulatus confirmed that the amplified products showed 97–99% homology with known B. microti sequences, accession numbers AB366158.1 (99% homology with a B. microti gene for 18S ribosomal RNA, strain: Munich), and EU882727.1 (97–98% homology with a B. microti 18S ribosomal RNA gene isolated from I. ricinus), for 6 and 2 samples, respectively.

Discussion

Dermacentor reticulatus (syn. Dermacentor pictus) occurs in Eurasia in two areas. Its eastern, main area covers northern Asia and northeastern Europe, from the Yenisei River in Siberia to the Vistula River in Poland, while its western, more isolated area covers England, France, the Netherlands, Belgium, Switzerland, and southwestern Germany (Siuda 1993; Nijhof et al. 2007; Cochez et al. 2011). Most likely, the species is migrating from its main area westward, as it was recently detected in western Poland (Nowak 2011), and Austria (Sixl et al. 2003; Dobler et al. 2008). D. reticulatus is a known vector of Babesia species, causing babesiosis (piroplasmosis) in cattle, horses, and dogs (B. bovis, B. bigemina, B. caballi, B. canis, and Theileria [Babesia] equi; Arthur 1963; Wall and Shearer 2001; Sawczuk 2006; Silva et al. 2010), but so far it has not been associated with human babesiosis caused by B. microti or B. divergens. Welc-Falęciak and associates (2008) detected the presence of B. microti in 11.8% of partly-engorged larvae, and 4.0% of partly-engorged nymphs of D. reticulatus feeding on rodents in the Mazury Lakes District of northern Poland. Walter (1982) was not successful in the experimental transmission of B. microti into golden hamsters by infected nymphs of D. reticulatus.

So far, B. microti has not been reported in the adult D. reticulatus ticks that feed on large animals and less commonly on humans. Nijhof and colleagues (2007) did not find B. microti in 72 D. reticulatus ticks in the Netherlands, and Zygner and co-workers (2010) did not detect B. microti in 381 adult D. reticulatus ticks collected from dogs in Warsaw. Recently, Cochez and associates (2011) did not detect Babesia spp. in 282 D. reticulatus ticks collected in Belgium. Thus, Ixodes ricinus is regarded as the most important vector of B. microti and B. divergens in Europe. It has been shown that in Poland 3–15.3% of adult I. ricinus ticks are infected with B. microti (Skotarczak and Cichocka 2001; Skotarczak et al. 2002; Stańczak et al. 2004; Siński et al. 2006; Wójcik-Fatla et al. 2006; Zygner et al. 2010), while in other central and western European countries the incidence is slightly lower, in the range of 0.1–11.0% (Duh et al. 2001; Foppa et al. 2002; Kalman et al. 2003; Hartelt et al. 2004; Nijhof et al. 2007). Among other ticks, Ixodes trianguliceps was suspected as a possible vector of B. microti, but its role seems to be marginal because of the rare occurrence of this species (Karbowiak 2004; Siński et al. 2006).

In the present study, Babesia microti was detected in adult D. reticulatus ticks that feed on humans and large domestic and wild animals (e.g., cattle, deer, and elk). Thus our findings suggest that this tick species should be considered as a potential vector of B. microti that may cause human babesiosis. The research by Plesman and colleagues (1979) indicates that deer are not a reservoir of B. microti, but the role of cattle in this regard is unknown and requires further study.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Arthur

. British Ticks. London: Butterworths, 1963.

Cochez

, Lempereur

, Madder

et al. Foci report on indigenous Dermacentor reticulatus populations in Belgium and a preliminary study of associated babesiosis pathogens. Met Vet Entomol, 2011, DOI:10.1111/j.1365-2915.2011.00988.x.

Dobler

, Essbauer

, Terzioglu

et al. Prevalence of tick-borne encephalitis virus and rickettsiae in ticks of the district Burgenland, Austria. Wien Klin Wochenschr, 2008; 120,Suppl 4:45–48(in German).

Duh

, Petrovec

, Avšič-Zupanc

. Diversity of Babesia infecting European sheep ticks (Ixodes ricinus) J Clin Microbiol, 2001; 39:3395–3397.

Foppa

, Krause

, Spielman

et al. Entomologic and serologic evidence of zoonotic transmission of Babesia microti, eastern Switzerland. Emerg Infect Dis, 2002; 8:722–726.

Gray

. Identity of the causal agents of human babesiosis in Europe. Int J Med Microbiol, 2006; 296,Suppl 40:131–136.

Hartelt

, Oehme

, Frank

et al. Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in Southern Germany. Int J Med Microbiol, 2004; 293,Suppl 37:86–92.

Hildebrandt

, Hunfeld

, Baier

et al. First confirmed autochthonous case of human Babesia microti infection in Europe. Eur J Clin Microbiol Infect Dis, 2007; 26:595–601.

Homer

, Aguilar-Delfin

, Telford III

et al. Babesiosis. Clin Microbiol Rev, 2000; 13:451–469.

10.

Hunfeld

, Hildebrandt

, Gray

. Babesiosis: recent insights into an ancient disease. Int J Parasitol, 2008; 38:1219–1237.

11.

Kalman

, Streter

, Szell

et al. Babesia microti infection of anthropophilic ticks (Ixodes ricinus) in Hungary. Ann Trop Med Parasitol, 2003; 97:317–319.

12.

Karbowiak

. Zoonotic reservoir of Babesia microti in Poland. Pol J Microbiol, 2004; 53,Suppl:61–65.

13.

Kjemtrup

, Conrad

. Human babesiosis: an emerging tick-borne disease. Int J Parasitol, 2000; 30:1323–1337.

14.

Nijhof

, Bodaan

, Postigo

et al. Ticks and associated pathogens collected from domestic animals in the Netherlands. Vector Borne Zoonotic Dis, 2007; 7:585–595.

15.

Nowak

. Discovery of Dermacentor reticulatus (Acari: Amblyommidae) populations in the Lubuskie Province (Western Poland) Exper Appl Acarol, 2011; 54:191–197.

16.

Persing

, Mathiesen

, Marshall

et al. Detection of Babesia microti by polymerase chain reaction. J Clin Microbiol, 1992; 30:2097–2103.

17.

Pieniążek

, Sawczuk

, Skotarczak

. Molecular identification of Babesia parasites isolated from Ixodes ricinus ticks collected in northwestern Poland. J Parasitol, 2006; 92:32–35.

18.

Plesman

, Spielman

, Etkind

et al. Role of deer in the epizootiology of Babesia microti in Massachusetts, USA. J Med Entomol, 1979; 15:537–540.

19.

Rijpkema

, Golubic

, Moelkenboer

et al. Identification of four genomic groups of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in Lyme borreliosis endemic region of northern Croatia. Exp Appl Acarol, 1996; 20:20–30.

20.

Sawczuk

. Babesia. Molecular Biology of Pathogens Transmitted by Ticks. Skotarczak

. Warsaw: Wydawnictwo Lekarskie PZWL, 2006; 221–239(in Polish).

21.

Silva

, Marques

, Oliva

. Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method. Vet Parasitol, 2010; 174:199–205.

22.

Siński

, Bajer

, Welc

et al. Babesia microti: Prevalence in wild rodents and Ixodes ricinus ticks from the Mazury Lakes District of north-eastern Poland. Int J Med Microbiol, 2006; 296:137–143.

23.

Siuda

. Characteristics of ticks (Ixodida) of medical importance in Poland. Molecular Biology of Pathogens Transmitted by Ticks. Skotarczak

. Warsaw: Wydawnictwo Lekarskie PZWL, 2006; 33–42(in Polish).

24.

Siuda

. Ticks of Poland (Acari: Ixodida). Part II. Taxonomy and Geographical Distribution. Warsaw: Polskie Towarzystwo Parazytologiczne, 1993(in Polish).

25.

Sixl

, Petrovec

, Marth

et al. Investigation of Anaplasma phagocytophila infections in Ixodes ricinus and Dermacentor reticulatus ticks in Austria. Ann NY Acad Sci, 2003; 990:94–97.

26.

Skotarczak

, Cichocka

. Isolation and amplification by polymerase chain reaction DNA of Babesia microti and Babesia divergens in ticks in Poland. Ann Agric Environ Med, 2001; 8:187–189.

27.

Skotarczak

, Wodecka

, Cichocka

. Coexistence DNA of Borrelia burgdorferi sensu lato and Babesia microti in Ixodes ricinus ticks from north-western Poland. Ann Agric Environ Med, 2002; 9:25–28.

28.

Stańczak

, Gabre

, Kruminis-Łozowska

et al. Ixodes ricinus as a vector of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti in urban and suburban forests. Ann Agric Environ Med, 2004; 11:109–114.

29.

Wall

, Shearer

. Veterinary Ectoparasites: Biology, Pathology and Control. Oxford: Blackwell Science, 2001.

30.

Walter

. Transmission of Babesia microti by nymphs of Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, Rhipicephalus sanguineus and Ixodes hexagonus. Z Parasitenkd, 1982; 66:353–354(in German).

31.

Welc-Falęciak

, Bajer

, Behnke

et al. Effects of host diversity and the community composition of hard ticks (Ixodidae) on Babesia microti infection. Int J Med Microbiol, 2008; 298:235–242.

32.

Wójcik-Fatla

, Cisak

, Chmielewska-Badora

et al.

Prevalence of Babesia microti in Ixodes ricinus ticks from Lublin region (eastern Poland)

Ann Agric Environ Med, 2006; 13:319–322.

33.

Zygner

, Bąska

, Wiśniewski

et al.

The molecular evidence of Babesia microti in hard ticks removed from dogs in Warsaw (central Poland)

Pol J Microbiol, 2010; 59:95–97.