Abstract:
BACKGROUND : The Ehrlichia are obligate intracellular Gram-negative tick-borne bacteria that are important human
and animal pathogens. There is a need for assays to rapidly and reliably detect and differentiate the five generally
recognized species into groups in a single reaction: E. canis, E. chaffeensis, E. ewingii, E. muris and E. ruminantium.
METHODS : We developed primers and probes against the 16S rRNA gene to enable us to reliably detect the five
major Ehrlichia spp. in a single FRET-qPCR. We tested the Ehrlichia FRET-qPCR on reference strains and on DNA from
the blood of domestic ruminants from five Caribbean islands. The Ehrlichia present were determined using melting
point analysis and by sequencing the Ehrlichia FRET-qPCR products as well as those of a nested PCR against the
citrate synthase gene (gltA).
RESULTS : Our Ehrlichia FRET-qPCR was negative for the closely related Anaplasma marginale and A. phagocytophilum
but gave positive reactions with reference strains of the most generally recognized species and with other less
characterized Ehrlichia of domestic ruminants, mainly E. ovina, the Panola Mountain Ehrlichia, and Ehrlichia sp.
BOV2010. Melting point analysis revealed 4 distinct groups: E. ruminantium (Tm ~55.8 °C); E. chaffeensis and E.
ewingii (Tm ~57.7 °C); E. canis, E. muris, E. ovina and Ehrlichia sp. BOV 2010 (Tm ~62.0 °C); and the Panola Mountain
Ehrlichia (Tm ~65.5 °C). The detection limit of the FRET-qPCR was ~ 5 gene copies in a reaction and the sequences
of the FRET-qPCR products were as expected. With DNA from domestic ruminants from the Caribbean we found
12.2 % (134/1,101) positive: cattle (76/385; 19.7 %), sheep (45/340; 13.2 %) and goats (13/376; 3.5 %). Melting point
analysis and sequencing of the FRET-qPCR and nested PCR gltA products showed the Ehrlichia we detected were E.
canis or very closely related organisms.
CONCLUSIONS : In a single reaction, our Ehrlichia FRET-qPCR can detect the Ehrlichia spp. we studied and differentiate
them into four groups. Domestic ruminants in the Caribbean are not uncommonly exposed to Ehrlichia, possibly E.
canis or very closely related organisms.
