NESTED POLYMERASE CHAIN-REACTION FOR DETECTION OF EHRLICHIA-EQUI GENOMIC DNA IN HORSES AND TICKS (IXODES PACIFICUS)

A nested polymerase chain reaction for detecting Ehrlichia equi in hor ses and ticks (Ixodes pacificus) was developed, A major second-round P CR product of 928 bp could be readily visualized in ethidium bromide-s tained agarose minigels, An internal probe was used to verify the iden tity of the amplified product by non-radioactive (digoxigenin-based) S outhern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicat ed that DNA derived from less than or equal to 7.6 but > 3 infected ne utrophils was sufficient to generate a PCR signal, The specificity of the PCR was examined using a panel of rickettsiae, of which only E. eq ui and the closely-related human granulocytotropic ehrlichia produced PCR bands. In an in vivo infection study, E. equi DNA was detected in blood buffy-coat cells of an experimentally-infected horse on days thr ee through 14 post-inoculation. In a separate study, three of six adul t I. pacificus that as nymphs had been fed on an experimentally infect ed horse were found to be PCR-positive for E. equi.