title: PCR detection of acute Ehrlichia canis infection in dogs.
abstract: A polymerase chain reaction (PCR)-based detection assay that specifically detected Ehrlichia canis in dogs with acute infections was developed. A region of the 16S ribosomal RNA gene of E. canis was targeted for PCR amplification and chemiluminescent hybridization (CH) with a complementary internal 287-base pair (bp) oligonucleotide probe. The CH improved the PCR assay sensitivity 1,000-fold as compared with visualization on ethidium bromide-stained agarose gels. The PCR assay with CH (PCR/CH) detected as little as 30 fg of E. canis genomic DNA, the equivalent of approximately 150 E. canis organisms. The 495-bp product defined by the specific primers was not detected when genomic DNA from E. platys, E. chaffeensis, E. risticii, and E. equi were used in the PCR/CH assay. The PCR/CH assay was tested with unfractionated blood samples collected from 9 dogs experimentally infected with E. canis. The PCR/CH assay had greater detection sensitivity than did cell culture isolation (CCI) from infected blood. PCR/CH detected E. canis 7 days prior to CCI in 4 of 6 experimentally infected dogs. The results obtained with the PCR/CH assay otherwise consistently matched the results obtained by CCI. This PCR/CH assay is a rapid, sensitive, and specific method for E. canis detection with sensitivity comparable to or exceeding that of CCI. A diagnosis of E. canis using this PCR/CH assay can be made in 2 days as compared with 1-4 weeks for CCI. The PCR/CH assay appears to be an acceptable alternative or complement to current diagnostic techniques.
authors.affiliation: Department of Veterinary Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis 95616, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: McBride
authors.forename: R E
authors.initials: RE
authors.lastname: Corstvet
authors.forename: S D
authors.initials: SD
authors.lastname: Gaunt
authors.forename: J
authors.initials: J
authors.lastname: Chinsangaram
authors.forename: G Y
authors.initials: GY
authors.lastname: Akita
authors.forename: B I
authors.initials: BI
authors.lastname: Osburn
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004283:Dog Diseases; D004285:Dogs; D004536:Ehrlichia / Q000235:genetics / Q000302:isolation & purification*; D016873:Ehrlichiosis / Q000175:diagnosis / Q000662:veterinary*; D005798:Genes, Bacterial; D008163:Luminescent Measurements; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S