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

Abstract

BackgroundThe 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.MethodsWe 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).ResultsOur 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.ConclusionsIn 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.

Molecular Detection of Anaplasma spp. and Ehrlichia spp. in Ruminants from Twelve Provinces of China

“…we appear to have identified based on their 16S rRNA sequences include A. platys , the agent of infectious canine cyclic thrombocytopenia [3], which has been described in dogs in Asia [24, 37] and in sika deer, goats, and cattle in China [4, 19]. The remaining two Anaplasma spp.…”

“…have been described in China, E. chaffeensis , E. canis , E. platys , E. ewingii (granulocytic ehrlichial agent), and also a novel species closely related to E. chaffeensis and A. marginale [14, 15, 33, 40]. The most important Ehrlichia species infecting ruminants, E. ruminantium , is restricted to Africa and some Caribbean islands [19] and has not been reported in China. Of the other Ehrlichia known to infect ruminants (summarized in Zhang et al, 2015 [19]), we only found evidence of infection with E. canis in the domestic ruminants we studied in China.…”

“…The most important Ehrlichia species infecting ruminants, E. ruminantium , is restricted to Africa and some Caribbean islands [19] and has not been reported in China. Of the other Ehrlichia known to infect ruminants (summarized in Zhang et al, 2015 [19]), we only found evidence of infection with E. canis in the domestic ruminants we studied in China. Although E. canis is best known as a very common dog pathogen around the world, infections have also been described in people [41] and in cats [42], and there is thus growing evidence that E. canis has a wider host range than previously thought [2, 19, 43].…”

“…Although E. canis is best known as a very common dog pathogen around the world, infections have also been described in people [41] and in cats [42], and there is thus growing evidence that E. canis has a wider host range than previously thought [2, 19, 43]. Our finding of E. canis and closely related organisms in a goat and cattle in China further supports this evidence and is consistent with the findings of a study showing that E. canis or very closely related organisms are present in domestic ruminants in the Caribbean [19] and also a study showing that E. canis occurs in sika deer in China [4]. Further studies are underway in our laboratory to determine the pathogenicity of E. canis in domestic ruminants.…”

“…[18] and Ehrlichia spp. [19], and the mammalian HMBS gene [20] as an endogenous internal control, were performed on a Roche Light-Cycler 480-II PCR Instrument. Positive PCR products were verified by gel electrophoresis and sequenced using forward and antisense primers (BGI, Shanghai, China).…”