First Molecular Detection of Anaplasma phagocytophilum in European Brown Bear ( Ursus arctos )

Z oonotic tick-borne diseases are an increasing health burden in Europe and there is a speculation that this is partly due to global changes (climate and socioeconomic) that affect vector biology, ecology, and disease transmission (Daniel et al. 2003). Data on the vector tick Ixodes ricinus suggest that an extension of its range is accompanied by an emergence or re-emergence of several pathogenic vector-transmissible microorganisms (McMichael et al. 1996).

Anaplasma phagocytophilum (order Rickettsiales, family Anaplasmataceae) is an obligate intracellular bacterium that attacks granulocytes of mammalian hosts (Woldehiwet and Scott 1993). This bacterium is responsible for nonspecific febrile illness in humans (human granulocytic anaplasmosis) and animals (equine and canine anaplasmosis and pasture fever in ruminants) (Rikihisa 1991).

In Europe, enzootic circulation and maintenance of A. phagocytophilum in natural foci is ensured by biological transmission among infected vector ticks (I. ricinus) and hosts—competent as well as susceptible (Liz et al. 2002, Swanson et al. 2006).

Lately, many studies regarding the reservoir competence have indicated that besides wild rodents, free-ranging ungulates (roe deer and red deer) could also, in a long term, play a role as the source of infection in European natural foci (Stuen 2007). Since the first detection of the agent causing tick-borne fever in animals in 1940, and the first molecular identification of human granulocytic anaplasmosis agent in 1994 in the United States (Chen et al. 1994) and in 1996 in Europe (Petrovec et al. 1997), circulation of biologically, clinically, and geographically distinct A. phagocytophilum variants was registered in a great variety of mammalian species (Stuen 2007). The distinct strains with a variation in virulence were isolated even from the same host species (Levin et al. 2004). To date, the presence of antibodies against Anaplasma and bacterial DNA was recorded in several domestic and wild animals including carnivorous, herbivorous, as well as omnivorous mammals, birds, and reptiles (especially lizards) (Majláthová, unpublished data) worldwide (Stuen 2007). In the natural foci of Slovakia, the presence of A. phagocytophilum was for the first time detected in questing I. ricinus ticks in the Western and later in the Eastern Slovakia at the localities considered highly endemic for Lyme borreliosis and subsequently was recorded in ticks and numerous hosts all across Slovakia (Špitálska and Kociánová 2002, Derdáková et al. 2003) (Víchová, unpublished data). The main aim of our study was to investigate the presence of A. phagocytophilum infection in free-ranging European brown bears (Ursus arctos) (order Carnivora, suborder Caniformia, family Ursidae). The bears were shot legally in Slovakia to determine whether the brown bear plays any role in the circulation of A. phagocytophilum, because these host–parasite interactions have not been studied yet.

In total, tissue samples (muscle, liver, or spleen) from 92 European brown bears (U. arctos) shot at the different localities in geographically dispersed montane and submontane areas of Central Slovakia were collected by the professional hunters from May 2004 to December 2007. Samples were stored in plastic vials with 70% ethanol until the isolation of the DNA using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, with a previous tissue digestion with proteinase K (Promega, Madison, WI) at 56°C, overnight. To verify whether genomic DNA had been isolated successfully from each tissue sample, polymerase chain reaction (PCR) amplification of 145-bp orthologous fragment of the vertebrate mitochondrial 12S rRNA gene was performed in a total of 25 μL reaction mixture volume of MasterTaq DNA polymerase kit (Eppendorf AG, Hamburg, Germany) (Humair et al. 2007). Vertebrate DNA was used as template. Nuclease-free water was added instead of DNA as negative control. Only samples with amplified fragment of 12S rRNA gene were considered as those with successfully isolated genomic DNA and were stored at −20°C for further analysis. Seventy-four (25 females, 39 males, and 10 of unknown sex) samples were tested for the presence of bacterial DNA. Molecular searching for A. phagocytophilum was based on PCR amplification of 16S rRNA gene, as published previously (Kolbert 1996, Kawahara et al. 2006). PCR products were electrophoresed on 1% agarose gels stained with GoldView Nucleic Acid Stain (Bejing SBS Genetech, Beijing, China). The size of amplified fragments was compared with 100-bp DNA ladder. Differences in the prevalence of A. phagocytophilum between sexes were evaluated statistically with the two-tailed χ ² test (degrees of freedom). A value of p < 0.05 was considered statistically significant. Of the 74 samples, a total of 18 (3 females, 14 males, and 1 of unknown sex) tested positive for A. phagocytophilum DNA by PCR amplification of the 16S rRNA gene. An overall infection prevalence of 24.3% (confidence interval: 0.151–0.357) was recorded. The infection was significantly higher in males than females (p < 0.05, degrees of freedom = 1).

The DNA was purified from a few randomly selected 16S rRNA PCR amplicons using a QIAquick PCR purification kit (Qiagen). To confirm the presence of A. phagocytophilum, sequencing was performed at the Department of Molecular Biology (Faculty of Natural Sciences Comenius University, Bratislava, Slovakia). The complementary strands of sequenced products were manually assembled. The sequences were compared with GenBank entries by Blast N2.2.13 (Altschul et al. 1997).

Two partial sequences of 16S rRNA gene with length of 248 bp (EU165367) and 586 bp (GQ122210) were submitted to GenBank database. Phylogenetic analysis revealed that our isolate GQ122210 shares 100% identity with A. phagocytophilum under accession number AY527214 as well as isolate EU165367 that shows 99% similarity to A. phagocytophilum FJ968661.

A. phagocytophilum has been detected in vertebrate hosts and vector ticks in nearly all European countries (Strle 2004). The intensity of infection varies because of geographical or environmental differences and is influenced by the presence of intermediate host species or reservoirs (Blanco and Oteo 2002). The role of mammals in the maintenance and propagation of A. phagocytophilum in nature is crucial (Liz et al. 2002). The potential role of the black bears (Ursus americanus) in the ecology of A. phagocytophilum in the United States has already been investigated. Serological as well as molecular studies suggested that bears are exposed to infection and naturally infected with A. phagocytophilum (Schultz et al. 2002, Barbet et al. 2006). Parola et al. (2003) have also recorded the presence of Anaplasma sp. strain closely related to A. bovis in Haemaphysalis ticks collected from Malayan sun bear (Helarctos malayanus). Up to now, there are no data available regarding the presence of the pathogen in ursine carnivores in Europe.

Our study suggests that enzootic circulation of A. phagocytophilum between I. ricinus ticks and the largest of all the land carnivores from the family Ursidae—European brown bear (U. arctos)—is established in the natural conditions of Central Europe. Our results demonstrate that bears are susceptible and naturally infected with A. phagocytophilum and indicate the potential possibility for this animal species to serve as the next wildlife reservoir for the pathogen. Nevertheless, for the clarification of the exact role of these animals in enzootic circulation of A. phagocytophilum in natural foci, further investigations are needed.