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,...
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BMC
2015-10-01
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| Series: | Parasites & Vectors |
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| Online Access: | https://doi.org/10.1186/s13071-015-1118-5 |
| _version_ | 1797811742269505536 |
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| author | Jilei Zhang Patrick Kelly Weina Guo Chuanling Xu Lanjing Wei Frans Jongejan Amanda Loftis Chengming Wang |
| author_facet | Jilei Zhang Patrick Kelly Weina Guo Chuanling Xu Lanjing Wei Frans Jongejan Amanda Loftis Chengming Wang |
| author_sort | Jilei Zhang |
| collection | DOAJ |
| description | 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. |
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| language | English |
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| spelling | doaj.art-3c21b7f1f0b940bca08c478efd47c3f62023-06-04T11:15:02ZengBMCParasites & Vectors1756-33052015-10-01811810.1186/s13071-015-1118-5Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islandsJilei Zhang0Patrick Kelly1Weina Guo2Chuanling Xu3Lanjing Wei4Frans Jongejan5Amanda Loftis6Chengming Wang7Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary MedicineRoss University School of Veterinary MedicineAnhui Science and Technology University College of Animal ScienceJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary MedicineJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary MedicineUtrecht Centre for Tick-borne Diseases (UCTD), FAO Reference Centre for Ticks and Tick-borne Diseases, Faculty of Veterinary Medicine, Utrecht UniversityRoss University School of Veterinary MedicineJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary MedicineAbstract 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.https://doi.org/10.1186/s13071-015-1118-5EhrlichiaFRET-qPCRDomestic ruminantsCaribbean |
| spellingShingle | Jilei Zhang Patrick Kelly Weina Guo Chuanling Xu Lanjing Wei Frans Jongejan Amanda Loftis Chengming Wang Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands Parasites & Vectors Ehrlichia FRET-qPCR Domestic ruminants Caribbean |
| 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 | Ehrlichia FRET-qPCR Domestic ruminants Caribbean |
| url | https://doi.org/10.1186/s13071-015-1118-5 |
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