title: Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands.
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 (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.
authors.affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, 225009, P. R. China. zhangjilei0103@163.com.
authors.forename: Jilei
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Ross University School of Veterinary Medicine, Basseterre, St. Kitts and Nevis. pkelly@rossvet.edu.kn.
authors.forename: Patrick
authors.initials: P
authors.lastname: Kelly
authors.affiliation: Anhui Science and Technology University College of Animal Science, Anhui, China. weina0925@126.com.
authors.forename: Weina
authors.initials: W
authors.lastname: Guo
authors.affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, 225009, P. R. China. clxu@yzu.edu.cn.
authors.forename: Chuanling
authors.initials: C
authors.lastname: Xu
authors.affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, 225009, P. R. China. weilanjing@yahoo.com.
authors.forename: Lanjing
authors.initials: L
authors.lastname: Wei
authors.affiliation: Utrecht Centre for Tick-borne Diseases (UCTD), FAO Reference Centre for Ticks and Tick-borne Diseases, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands. F.Jongejan@uu.nl.|Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa. F.Jongejan@uu.nl.
authors.forename: Frans
authors.initials: F
authors.lastname: Jongejan
authors.affiliation: Ross University School of Veterinary Medicine, Basseterre, St. Kitts and Nevis. adloftis@gmail.com.
authors.forename: Amanda
authors.initials: A
authors.lastname: Loftis
authors.affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, 225009, P. R. China. wangcm@yzu.edu.cn.
authors.forename: Chengming
authors.initials: C
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002418:Cattle Diseases / Q000453:epidemiology / Q000469:parasitology*; D004536:Ehrlichia / Q000145:classification / Q000302:isolation & purification; D016873:Ehrlichiosis / Q000453:epidemiology / Q000662:veterinary*; D031541:Fluorescence Resonance Energy Transfer; D015511:Goat Diseases / Q000453:epidemiology / Q000382:microbiology*; D006041:Goats; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000453:epidemiology / Q000382:microbiology*; D014900:West Indies / Q000453:epidemiology