Infection with Anaplasma phagocytophila in cervids from Slovenia: evidence of two genotypic lineages.

OBJECTIVE: Human granulocytic ehrlichiosis (HGE) was recently recognized as an emerging tick-borne infection in Europe. The disease is caused by Anaplasma (previously Ehrlichia) phagocytophila. The first confirmed acute human disease caused by A. phagocytophila was reported from Slovenia in 1998. The tick Ixodes ricinus was identified as the likely vector for this pathogen of humans and animals in Europe. In order to assess the possibility that roe and red deer in Slovenia serve as potential reservoir hosts for A. phagocytophila, materials from both species were examined.
METHODS: Samples were obtained from 32 red deer (Cervus elaphus) and 56 roe deer (Capreolus capreolus). Polyvalent antibodies to the USG3 isolate of Anaplasma phagocytophila were detected by indirect immunofluorescence assay (IFA). DNA was extracted from spleen tissue. The 16S rRNA gene and a portion of the groESL heat shock operon were used for PCR detection and subsequent direct sequencing of amplified products.
RESULTS: Serological and PCR results indicated that high proportions of roe and red deer were infected with A. phagocytophila. Infection was confirmed in 74% of the animals by IFA and in 86% of animals by PCR. While similar prevalences by PCR were seen in the two species (approximately 86%), the prevalence of antibodies was much higher in roe deer (94% vs. 35% in red deer). Sequence analysis of a 1256-bp fragment of the groESL operon revealed genetic diversity among collected samples. Identity of sequences ranging from 98% to 100%. None of the A. phagocytophila groESL and 16S rRNA gene sequences from red or roe deer were identical to the sequences previously obtained from human patients with ehrlichiosis from Slovenia or elsewhere in the world. All red deer sequences clustered with those obtained from humans, whereas all but two sequences from roe deer clustered separately.
CONCLUSIONS: The results of our study indicate that a high percentage of red deer and roe deer in Slovenia are infected with A. phagocytophila. Analysis of groESL and 16S rRNA gene sequences revealed two distinct genetic lineages. Among deer, one variant was primarily associated with roe deer. Although none of the sequences from red deer was identical to those found in humans, they were more closely related.