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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  ( T  m  ~55.8 °C);  E. chaffeensis  and  E. ewingii  ( T  m  ~57.7 °C);  E. canis ,  E. muris ,  E. ovina  and  Ehrlichia  sp. BOV 2010 ( T  m  ~62.0 °C); and the Panola Mountain  Ehrlichia  ( T  m  ~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.

Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands