RESEARCH Figure 2. Seasonal variation in samples that were positive on poly- merase chain reaction (PCR) for Anaplasma phagocytophilum. negative on the more specific secondary PCR. Five of these samples were sequenced (no. AY776167); none was positive for A. phagocytophilum, but all had high homolo- gy (99%) with a sequence amplified from I. ricinus ticks collected from humans in Italy and previously deposited in GenBank as Rickettsiales bacterium it86 (no. AF525482.1). R. helvetica and TBE-complex Virus All 237 samples were tested by PCR for R. helvetica, but none were found positive (95% CI 0%–1.6%). Seroprevalence of TBE-complex virus was evaluated on 229 samples. Twenty samples (8.7%) were positive on the HI test. The verifying neutralization test was positive for 14 (6.1%) samples, but 4 could not be evaluated because of cell monolayer destruction, and 2 samples were not available for neutralization testing because of lack of mate- rial. Positive samples came from Bornholm (n = 6, 30%), Zealand and Falster (n = 3, 15%), and Jutland (n = 11, 55%) (Figure 3). Significant differences in seroprevalence could not be shown for sex or season, but TBE-complex virus–positive animals were significantly more common in the young adult–adult group than in the fawn–yearling group (p = 0.014). Discussion In the present study, roe deer were used as sentinels. They are widely distributed throughout Denmark and out- number other large wild animals. They play a central role in the life cycle of I. ricinus by feeding large numbers of the tick at all 3 life stages (12). Density of I. ricinus is strongly related to the abundance of roe deer (13,14). Roe deer are considered incompetent as reservoirs of Borrelia spp. (15); however, their central role in the life cycle of the tick and the fact that they can respond immunologically to Borrelia infection renders them useful as sentinels for bor- reliosis (16). The finding of a Borrelia seroprevalence of 36.6% is lower than that found in the study by Webster and Frandsen (7) from 1994 (52%), but in the previous study all roe deer samples came from Zealand, and when this regional difference is taken into account the difference is not significant (50% vs. 52%, p = 0.773). The regional dif- ferences found correlate well with the known tick density in Denmark (Figure 1). The lack of seropositive roe deer from 3 districts in northern Jutland may, however, be due to the low sample size from these districts. The role of ticks and roe deer in the transmission of Bartonella infections is still uncertain. Schouls et al. found that 60% of ticks collected from infested roe deer carried Bartonella spp. (17). B. henselae has also recently been found in ticks infesting humans in Italy (18), and B. quin- tana has been recovered from ticks in California (19). In 2001, McGill et al. found that 31% of elite orienteers (par- ticipants in the competitive sport of finding the fastest route between defined checkpoints with only a compass and map), a high-risk group for tick bites, were seroposi- tive to Bartonella elizabethae (20). The recent finding of patients in the United States with Borrelia burgdorferi. and B. henselae co-infection also supports the idea that ticks play a role as a vector for Bartonella spp. (21). Our find- ing of no B. henselae– or B. quintana–seropositive roe deer does not support this idea. Whether roe deer are seropositive to other Bartonella spp., like B. elizabethae, remains to be evaluated, as well as the human pathogenic potential of B. elizabethae. Roe deer are thought to be competent reservoirs of A. phagocytophilum (22), and a high seroprevalence has been found in previous studies in Europe. We found a sero- prevalence of 95.6%, which is similar to findings from Norway (96%) and Slovenia (94%) (23,24). Larger varia- tion has been found in the number of PCR-positive roe deer, from 12.5% in Czech Republic to 85.6% in Slovenia (24,25). Although differences in PCR protocols may to some extent explain this difference, the seasonal variation in the number of PCR-positive roe deer, as shown in this study, may also play a role (Figure 2). As the probability of roe deer being rickettsemic changes through the season, serology represents a better surveillance tool than PCR. The high proportion of roe deer that are bacteremic throughout the tick season and the importance of roe deer in the life cycle of I. ricinus may indicate that roe deer are the main reservoir of A. phagocytophilum in Europe. Although regional variation was found in the number of PCR-positive animals, A. phagocytophilum is widespread in Denmark and seems correlated to tick and roe deer den- sity. Limited variation was found among the 16S RNA sequences analyzed. Reliable differentiation of possible A. phagocytophilum strains in Denmark would be better accomplished by sequencing genes with higher variation than the conserved sequence of 16S RNA; among better candidates are the Ank-gene and the groESL operon. 1058 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 7, July 2005