Figure. Agarose gel electrophoresis of results of PCR amplification of Ehrlichia chaffeensis nss rRNA gene from whole blood samples of coyotes numbers 9-11.* Lane 1= negative control (no DNA); Lane 2= coyote 9 (+); Lane 3= coyote 10 (+); Lane 4= coyote 11 (-); Lane 5 = positive control (E. chaffeensis-infected DH82 cells). M = 100-bp DNA ladder (Life Technologies, Rockville, MD).* * Ehrlichia forward primer ECC (5'-AGAACGAACGCTGGCGGCAAGC-3') and Ehrlichia reverse primer ECB (5'-CGTATTACCGCGGCTGCTGGCA-3') amplified all Ehrlichia spp (12,18). These reactions (50 l) contained 10 l of template DNA in 10 mM Tris-Cl (pH 8.3), 0.2 mM each deoxynucleoside triphosphate (dNTP), 2 mM MgCl2, 50 mM KCl, 0.5 m each primer, and 1.25 U of Taq DNA polymerase (Promega Corporation, Madison, WI). A hot-start PCR was used in which each enzyme was added to reactions after an initial 3-min denaturation step at 94°C. Reactions consisted of 30 cycles of denaturation at 94°C for 1 min, annealing at 65°C for 2 min, and extension at 72°C for 2 min. Products of this reaction were used as template with species-specific primer sets for three nested reactions. Primers HE1 (5'-CAATTGCTTATAACCTTTTGGTTATAAAT-3') and HE3 (5'-TATAGGTACCGTCATTATCTTCCCTAT-3') (17) were used for E. chaffeensis-specific amplifications. Primers ECAN5 (5'-CAATTATTTATAGCCTCTGGCTATAGGA-3') (12,13) and HE3 were used for E. canis-specific amplifications, and primers EE5 (5'-(CAATTCCTAAATAGTCTCTGACTATTTAG-3') (this study) and HE3 were used for E. ewingii-specific amplifications. Reactions (50 l) contained 10 l of the reaction product with ECC and ECB primers as template, and the remaining reaction components as above. A hot-start PCR was used in which the enzyme was added to reactions after an initial 3-min denaturation step at 94°C. Reactions with species-specific primers were in two stages. The first consisted of three cycles of denaturation at 94°C for 1 min, annealing at 55oC for 2 min, and extension at 72°C for 1.5 min. The second consisted of 37 cycles of denaturation at 92°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 1.5 min. Distilled, deionized water served as a negative control. Positive control DNA samples were purified from E. chaffeensis-infected DH82 cells, blood from a dog experimentally infected with E. canis, and diluted general primer PCR reactions of synovial fluid from a dog experimentally infected with E. ewingii. To prevent contamination of samples, DNA purification, PCR master mix assembly, and amplifications were performed in separate rooms. Positive displacement pipetters and aerosol-free pipette tips were also used as further precautions.