Je. Barlough et al., NESTED POLYMERASE CHAIN-REACTION FOR DETECTION OF EHRLICHIA-EQUI GENOMIC DNA IN HORSES AND TICKS (IXODES PACIFICUS), Veterinary parasitology, 63(3-4), 1996, pp. 319-329
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.