Analysis of Ehrlichia species by polymerase chain reaction

Six new or putatively new Ehrlichia species have been described in the last decade. These veterinary and human pathogens are obligate intraleukocytic parasites. Two novel human pathogens, Ehrlichia chaffeensis and the unnamed etiologic agent of human granulocytotropic ehrlichiosis (HGE), appear to be tick transmitted. These agents cause a non-specific flu-like syndrome which confounds diagnosis and delays treatment which can lead to more severe illness. Because seroconversion does not occur until 7-21 days after the onset of illness, non-serologic tests are needed that can detect Ehrlichia sp. soon after infection.;Molecular techniques based on PCR amplification of pathogen DNA provide the best opportunity for developing efficient new diagnostic tests. Repetitive element PCR (rep-PCR) is one technique used to distinguish among various bacterial species and strains. Rep-PCR was performed on seven species of Ehrlichia and the agent of HGE. Banding patterns were species-specific and bands ranged in size from approximately 50 to 1000 base pairs.;Another PCR-based method, intergenic spacer (ITS) analysis, uses length or sequence polymorphisms between the 16S and 23S rRNA genes to distinguish among species or strains. The ITS sequences for E. chaffeensis (Arkansas strain), and two strains of the HGE agent (NCH-1 strain from Massachusetts and EPCR105 strain from New York) were determined. For each agent a single spacer was amplified and sequenced. An unexpected level of similarity in ITS length (585 nucleotides) and nucleotide sequence (97.9% homology) between E. chaffeensis and HGE strain EPCR105 was found. However, the ITS from HGE strain NCH-1 was 900 nucleotides in length and included a tRNA;In searching for wildlife reservoirs of these emerging ehrlichial diseases a new Ehrlichia-like agent was identified in white-tailed deer. Discovery of this novel agent and emergence of other ehrlichial pathogens emphasizes the need for high-resolution techniques for characterizing Ehrlichia spp. The rep-PCR and ITS analysis techniques developed here hold substantial promise for enhancing ehrlichial identification, and with refinement, could aid epidemiologic studies and routine diagnostics.