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

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. chaff...

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Autores principales: Zhang, Jilei, Kelly, Patrick, Guo, Weina, Xu, Chuanling, Wei, Lanjing, Jongejan, Frans, Loftis, Amanda, Wang, Chengming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4595018/
https://www.ncbi.nlm.nih.gov/pubmed/26438311
http://dx.doi.org/10.1186/s13071-015-1118-5
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author Zhang, Jilei
Kelly, Patrick
Guo, Weina
Xu, Chuanling
Wei, Lanjing
Jongejan, Frans
Loftis, Amanda
Wang, Chengming
author_facet Zhang, Jilei
Kelly, Patrick
Guo, Weina
Xu, Chuanling
Wei, Lanjing
Jongejan, Frans
Loftis, Amanda
Wang, Chengming
author_sort Zhang, Jilei
collection PubMed
description 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.
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spelling pubmed-45950182015-10-07 Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands Zhang, Jilei Kelly, Patrick Guo, Weina Xu, Chuanling Wei, Lanjing Jongejan, Frans Loftis, Amanda Wang, Chengming Parasit Vectors Research 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. BioMed Central 2015-10-06 /pmc/articles/PMC4595018/ /pubmed/26438311 http://dx.doi.org/10.1186/s13071-015-1118-5 Text en © Zhang et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Zhang, Jilei
Kelly, Patrick
Guo, Weina
Xu, Chuanling
Wei, Lanjing
Jongejan, Frans
Loftis, Amanda
Wang, Chengming
Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands
title Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands
title_full Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands
title_fullStr Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands
title_full_unstemmed Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands
title_short Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands
title_sort development of a generic ehrlichia fret-qpcr and investigation of ehrlichioses in domestic ruminants on five caribbean islands
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4595018/
https://www.ncbi.nlm.nih.gov/pubmed/26438311
http://dx.doi.org/10.1186/s13071-015-1118-5
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