18 software (Teramecs, Kyoto, Japan), and positive real-time reactions were determined by taking into account the time taken for the turbidity value to increase above a predetermined threshold value of 0.1 [29]. To confirm that each LAMP amplified the correct target, the product was electrophoresed in a 2.0% agarose gel stained with Gel-Red TM (Biotium, Hayward, CA) or visualized under UV light, as described below. LAMP specificity assays were conducted using 18 different isolates of E. ruminantium, isolates of 5 closely related rickettsial bacteria, and tick DNA samples positive for 3 different species of USA ehrlichiae (described below). Detection of LAMP products In addition

to monitoring turbidity and gel electrophoresis, we used a common dsDNA-binding dye for the detection of LAMP products. One microliter of the dsDNA-dye mixture, consisting of 25% AZD2281 price (v/v) glycerol and Gel-Red TM (1:50 dilution of a 10,000× stock solution), was put inside the

lid of LAMP reaction tubes. To prevent dye mixture from dripping with vapor, the reaction mixture was overlaid with one drop of mineral oil. After the reaction terminated, the tubes were inverted several times, and LAMP products were visualized under UV light. pCS20 PCR and pCS20 real-time PCR assays To compare the specificity and sensitivity of the LAMP, conventional PCR and real-time PCR to amplify the pCS20 gene was R788 conducted using primers HH1F and HH2R [16], and CowF, CowR and Cow™ probe [20], respectively (Figure 2). PCR was performed Cell press with either the KAPA Blood

PCR kit (Kapabiosystems, Boston, MA) or the AmpliTaq Gold PCR kit (Applied Biosystem). In order to reduce the effect of PCR inhibitors in the templates, the KAPA Blood PCR kit was used for the analysis of field samples. PCR products were electrophoresed in a 1.2% agarose gel stained with Gel-Red TM. The real-time PCR was performed with THUNDERBIRD qPCR Mix (Toyobo, Osaka, Japan) and analyzed on Stratagene Mx3000 QPCR System (Stratagene, La Jolla, CA). A. americanum samples harbouring DNA from Ehrlichia species This study employed 17 DNA samples from A. americanum ticks recovered from people in the USA between 2004 and 2006, in which zoonotic Ehrlichia (E. ewingii, E. chaffeensis, or PM Ehrlichia) were detected by conventional PCR for the P28 antigen gene (E. ewingii) or nested PCR based on the 16S rRNA gene (E. chaffeensis) or citrate synthase gene (PM Ehrlichia), as described elsewhere [42, 45]. Collection details are shown in Table 4. Acknowledgements The cattle and goat owners are greatly acknowledged for their cooperation. We are thankful to all personnel who assisted in collection of field samples in Uganda, Tanzania, and Zambia. We also thank Dr. Amanda Loftis for her facilitating work with the USA ehrlichiae and for her assistance editing this manuscript. The first author was supported by a research grant fellowship from the Japanese Society for the Promotion of Science (JSPS) for young scientists.