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 horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from < = 7.6 but > 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of the PCR was examined using a panel of rickettsiae, of which only E. equi 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 three through 14 post-inoculation. In a separate study, three of six adult I. pacificus that as nymphs had been fed on an experimentally infected horse were found to be PCR-positive for E. equi.
Barlough
J E
JE
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis 95616, USA.
Madigan
J E
JE
DeRock
E
E
Bigornia
L
L
D000818:Animals; D001483:Base Sequence; D001773:Blood Cells / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D004536:Ehrlichia / Q000235:genetics* / Q000302:isolation & purification*; D016873:Ehrlichiosis / Q000175:diagnosis / Q000662:veterinary; D005069:Evaluation Studies as Topic; D005260:Female; D006734:Horse Diseases / Q000175:diagnosis; D006736:Horses / Q000382:microbiology* / Q000469:parasitology*; D006801:Humans; D018884:Ixodes / Q000382:microbiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial