RESEARCH blood samples were sent by mail to the laboratory. Because of limited amounts of material received from some dis- tricts, not all samples were available for all serologic tests. B. burgdorferi serologic tests were performed by using indirect immunofluorescence assay (IFA). B. burgdorferi strain DK 6 was used as an antigen, and the conjugate was fluorescein isothiocyanate (FITC)-labeled rabbit anti-deer immunoglobulin (Ig) G (Kierkegaard & Perry Laboratories Inc, Guildford, UK). The cutoff point was 1:64. B. henselae and B. quintana serologic tests were per- formed by using IFA. Slides coated with Vero cells infect- ed with B. henselae and B. quintana (Focus Technologies, Cypress, CA, USA) were used as antigen, and the conju- gate was FITC-labeled rabbit anti-deer IgG (Kierkegaard & Perry Laboratories Inc). The cutoff point was 1:64. A. phagocytophilum serologic testing was performed by using IFA. Slides with HL-60 cells infected with a human isolate of A. phagocytophilum (Focus Technologies) were used as antigen, and FITC-labeled rabbit anti-deer IgG was used as conjugate. The cutoff point was 1:128. Slides were considered borderline positive when a definite-but-dim fluorescence equal to that observed for the positive control at its reference endpoint titer was found. Moderate-to- intense fluorescence of the morula was graded positive. TBE-complex virus serologic testing was performed at the Institute of Virology, University of Vienna, Austria. Samples were first tested by using hemagglutination inhi- bition (HI) test. Positive samples were verified by using neutralization test. DNA Extraction, PCR, and Sequencing Total DNA was extracted from blood samples by using a QIAamp Blood kit (Qiagen, Albertslund, Denmark), according to manufacturer’s instructions. Amplifications were performed in a Perkin Elmer GeneAmp PCR system 9600 (Perkin-Elmer Corp, Norwalk, CT, USA), and real- time PCR was performed in Bio-Rad iCycler iQ Quantitative thermal cycler (Bio-Rad, Herlev, Denmark). DNA amplification was done in a 25-µ L reaction volume by using ReddyMix PCR Master Mix (ABgene, Epsom, United Kingdom), with 5 µ L of sample DNA in each reac- tion. Cycling conditions included initial 3 min of denatura- tion at 96°C, followed by 39 cycles, each consisting of 15 s denaturation at 96°C, 15 s annealing at 58°C, and 15 s extension at 72°C. These 39 cycles were followed by an extension period of 3 min at 72°C. Real-time PCR was performed by using HotStartTaq Master Mix kit (Qiagen). Reaction volume was 25 µ L with 5 µ L sample DNA. Cycling conditions for Anaplasma real-time PCR included an initial activation of Taq polymerase at 95°C for 10 min, followed by 40 cycles, each consisting of 15 s denaturation at 95°C followed by 1 min annealing-extension at 60°C. R. helvetica real-time PCR included an initial activation of Taq polymerase at 95°C for 10 min followed by 45 cycles of 30 s denaturation at 95°C followed by 45 s annealing- extension at 52°C. Specimen processing, PCR setup, and amplification and detection procedures were all conducted in separate areas to minimize the potential for cross-con- tamination. Anaplasma infection was detected with primers that specifically target the 16S rRNA gene of the A. phagocy- tophilia genogroup (9): forward primer 5′-GGTACCY- ACAGAAGAAGTCC and reverse primer 5′-TAGCAC- TCATCGTTTACAGC. PCR products were detected on 3% agarose gels stained with ethidium bromide. If samples were found positive, a second real-time PCR was per- formed. Primers specific for the A. phagocytophilum msp2 gene were used (10): ApMSP2f (5′-ATGGAAGGTAGT- GTTGGTTATGGTATT), ApMSP2r (5′-TTGGTCTTGA- AGCGCTCGTA), and a TaqMan probe ApMSP2p-HEX (5′-TGGTGCCAGGGTTGAGCTTGAGATTG). Primers were labeled at the 5′ and 3′ ends with hexachloro-6- carboxy-fluorescein (HEX) and Black Hole Quencher (BHQ), respectively. R. helvetica infection was detected with primers that specifically target the 23S rRNA gene of R. helvetica: Rhf (5′-ATAGGGAGGAATTTGAAGGA) and Rhr (5′- GGTAATTTGTACGTCGATCC) and a TaqMan probe Rhpr-TR (5′-CGGAACACAGAACCGTAGCG). Primers were labeled at the 5′ and 3′ ends with Texas Red and BHQ, respectively. For quality control, negative and positive con- trols were included each time a PCR was performed. PCR products used for DNA sequencing were purified by using GFX PCR DNA and Gel Band purification kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). For DNA sequencing reaction, ABI Prism Big Dye Terminator v3.0 kit was used (Perkin Elmer, Applied Biosystems Division). Removal of unincorporated dye ter- minators was performed by using DyeEx kit (Qiagen) Samples were run on ABI 373A sequencer (Perkin Elmer, Applied Biosystems Division). Sequences were compared with public domain database by using the Blast software. Sequences obtained are available in GenBank under acces- sion nos. AY776165, AY776166, and AY776167. Statistical Analysis Data were analyzed by using STATA 8.2 (StataCorp LP, College Station, TX, USA). For analysis of seroepidemio- logic results, Fisher exact test and the Mantel-Haenszel method were used. Values of p<0.05 were considered sig- nificant. Results A total of 237 blood samples from roe deer were col- lected from 22 of 25 state forest districts (Figure 1). Blood samples from 112 animals were collected in the summer 1056 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 7, July 2005