Molecular Survey of Babesia microti in Wild Rodents in Central Croatia

H uman babesiosis, caused by several Babesia species, is an emerging zoonosis. Babesia microti and B. microti-like parasites are the main causative agents of human babesiosis in the United States, whereas in Europe the predominant agents are Babesia divergens and B. divergens-like organisms (Hunfeld et al. 2008). The first human infection with B. microti was confirmed in Germany in a female patient (Hildebrandt et al. 2007). Seroepidemiological studies on humans suggest that exposure to B. microti occurs more often in Europe than previously recognized. The prevalence varied from 1% in eastern Croatia (Topolovec et al. 2003), 1.5% in Switzerland (Foppa et al. 2002), up to 13% in midwestern Germany (Hunfeld et al. 1998).

Although in Europe the presence of B. microti isolates from ticks have been constantly reported (Duh et al. 2001, Foppa et al. 2002, Wielinga et al. 2009), molecular characterization of B. microti isolates from reservoir hosts in Europe is limited. In only two molecular epizootiological studies, nonzoonotic isolates of B. microti strain were detected in Microtus arvalis (12.8%), Microtus oeconomicus (42%), and Apodemus flavicollis (1%) in Poland (Siński et al. 2006), and potentially zoonotic isolates were detected in Myodes glareolus (15.9%) and A. flavicolis (11.8%) in Slovenia (Duh et al. 2003).

Identification of reservoir hosts is a prerequisite for effective prevention of tick-borne zoonoses. Therefore, we conducted this study to determine the presence of B. microti infection in small wild rodents using polymerase chain reaction (PCR) and sequencing methods.

A total of 120 wild rodent adults were captured in central part of Croatia. The animals were captured in Sherman live traps. We followed animal experimentation guidelines approved by the American Society of Mammalogists—Animal Care and Use Committee (1998). Captured live animals were anesthetized in bags containing ether-soaked cotton. Deeply anesthetized animals were quickly and painlessly euthanized by cervical dislocation as described in the guidelines. Dead animals were aseptically dissected, and the tissue samples for DNA extraction were frozen at −80°C for several days until use.

DNA was extracted from 20 mg of spleen tissue using the DNA blood and tissue kit (Qiagen, Hilden, Germany). Amplification of a fragment (∼560 bp) of the small subunit ribosomal RNA gene was obtained by using the forward primer 5′-GTCTTGTAATTGGAATGATGG-3′ and the reverse primer 5′-CCAAAGACTTTGATTTCTCTC-3′ (Adaszek and Winiarczyk 2008). PCR conditions started with an initial denaturation step at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 60 s, and a final extension was done at 72°C for 7 min, followed by a hold step at 4°C. PCR products were purified using the Qiaquick purification kit (Qiagen) and sequenced in both directions (Macrogen, Seoul, Korea). Fisher exact test was used for statistical analysis.

B. microti was detected in 8 of 120 animals analyzed (6.6%). Sequencing of the amplified portion of the 18S rDNA from each parasite revealed that two (1.6%) of eight isolates had sequences identical to Munich strain from Mus musculus (AB071177) and isolate from Clethrionomys rutilus from Russia (AY943958). Sequences obtained from six samples (5%) were identical to human isolate “Jena” of B. microti (EF413181) and isolates detected in M. glareolus from Germany (AB085191), Ixodes ricinus from Germany (AF231349), Lagurus luteus from China (AB083375), I. ricinus from Switzerland (AY144692), and M. glareolus from Slovenia (AY149572). Infection rate from each rodent species and B. microti isolates are shown in Table 1. No statistically significant difference was observed in infection rate between males (two animals) and females (six animals).

Wild rodents play an important role in nature as reservoir hosts for many pathogens including zoonotic piroplasm B. microti. This survey demonstrates that two species of wild rodents, A. flavicollis and M. glareolus, have been infected with B. microti, whereas Apodemus sylvaticus and Apodemus agrarius were free from the parasite (Table 1). Infection of A. flavicollis and M. glareolus, but not another examined rodent species, is in agreement with results from Duh et al. (2003). Of note, A. flavicollis was more frequently infected (16.2%) in this study than M. glareolus (6%), although the difference was not statistically significant. In contrast, Siński et al. (2006) have found infection in a single A. flavicollis out of 85 examined in Poland, and also the prevalence in M. arvalis (12.8%) and M. oeconomus (42%) was higher. It seems that rodents from North Poland do not represent potential risk to public health because all animals were harboring nonzoonotic Munich strain of B. microti (AB071177). The same strain was confirmed in our study in one A. flavicollis and one M. glareolus. Interestingly, nonzoonotic strain was less frequently detected than the zoonotic Jena isolate (Table 1). Although females were more frequently infected, the prevalence was not statistically significant. In contrast, in other studies, males were more often infected than females (Duh et al. 2003), which maybe because testosterone reduces both innate and acquired resistance to tick feeding (Hughes and Randolph 2001). Phylogenetic analyses confirmed that B. microti is a complex species, consisting of genetically diverse isolates that constitute three clades (Goethert and Telford 2003). Within clades, rodent isolates are subdivided into zoonotic and nonzoonotic clades. Higher prevalence of zoonotic Jena isolate confirmed in this study clearly demonstrates a potential public health risk. Contribution to the presence of transmission risk is the detection of antibodies to B. microti in the serum from a patient with a tick-borne encephalitis in Croatia (Topolovec et al. 2003). Although Škrabalo and Deanović (1957) described the first case of fatal human babesiosis in Croatia, little is known about reservoirs and prevalence in humans. Presence of zoonotic isolate of B. microti in A. flavicollis and M. glareolus emphasizes the need for more serious consideration about Babesia infection in humans.