Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease, Ehrlichia ruminantium, from the Panola Mountain Ehrlichia

Summary Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick‐borne disease of ca...

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Veröffentlicht in:	Transboundary and emerging diseases Jg. 63; H. 6; S. e260 - e269
Hauptverfasser:	Sayler, K. A., Loftis, A. D., Mahan, S. M., Barbet, A. F.
Format:	Journal Article
Sprache:	Englisch
Veröffentlicht:	Germany Blackwell Publishing Ltd 01.12.2016 John Wiley & Sons, Inc
Schlagworte:	Africa Animal diseases Animals Caribbean Cattle Cattle Diseases - epidemiology cross reaction diagnostic qPCR disease surveillance DNA DNA Primers Economic importance Ehrlichia - genetics Ehrlichia - isolation & purification Ehrlichia ruminantium Ehrlichia ruminantium - genetics Ehrlichia ruminantium - isolation & purification emerging diseases Enzyme-Linked Immunosorbent Assay epidemiological studies Epidemiology foreign animal disease genes Goats heartwater Heartwater Disease - diagnosis Heartwater Disease - epidemiology islands Livestock livestock losses Outbreaks Panola Mountain Ehrlichia pathogens Peptides Polymerase chain reaction quantitative polymerase chain reaction Real-Time Polymerase Chain Reaction sequence homology Sheep Sub-Saharan Africa United States United States - epidemiology Caribbean United States Sub-Saharan Africa heartwater diagnostic qPCR livestock losses Panola Mountain Ehrlichia foreign animal disease Ehrlichia ruminantium
ISSN:	1865-1674, 1865-1682
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Abstract	Summary Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick‐borne disease of cattle, sheep and goats responsible for stock losses in sub‐Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross‐reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP‐1B peptides for ER and PME, we tested the cross‐reactivity of this assay using sera from infected livestock. The MAP‐1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual‐plex Taqman™ qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease‐causing organism.
AbstractList	Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick‐borne disease of cattle, sheep and goats responsible for stock losses in sub‐Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross‐reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP‐1B peptides for ER and PME, we tested the cross‐reactivity of this assay using sera from infected livestock. The MAP‐1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual‐plex Taqman™ qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease‐causing organism. Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick-borne disease of cattle, sheep and goats responsible for stock losses in sub-Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross-reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP-1B peptides for ER and PME, we tested the cross-reactivity of this assay using sera from infected livestock. The MAP-1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual-plex Taqman qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease-causing organism. Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick-borne disease of cattle, sheep and goats responsible for stock losses in sub-Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naive livestock. The serologic assay of choice to diagnose heartwater is cross-reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP-1B peptides for ER and PME, we tested the cross-reactivity of this assay using sera from infected livestock. The MAP-1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual-plex Taqman super((TM)) qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease-causing organism. Summary Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick‐borne disease of cattle, sheep and goats responsible for stock losses in sub‐Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross‐reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP‐1B peptides for ER and PME, we tested the cross‐reactivity of this assay using sera from infected livestock. The MAP‐1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual‐plex Taqman™ qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease‐causing organism. Summary Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick-borne disease of cattle, sheep and goats responsible for stock losses in sub-Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross-reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP-1B peptides for ER and PME, we tested the cross-reactivity of this assay using sera from infected livestock. The MAP-1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual-plex Taqman(TM) qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease-causing organism.
Author	Sayler, K. A. Mahan, S. M. Barbet, A. F. Loftis, A. D.
Author_xml	– sequence: 1 givenname: K. A. surname: Sayler fullname: Sayler, K. A. email: saylerk@ufl.edu, K. A. Sayler. University of Florida College of Veterinary Medicine, 2015 SW 16th Avenue, Veterinary Academic Building 1017, Room V2-240, Gainesville, FL, USA.Tel.: +352 871 3259; Fax: +352 392 9704;, saylerk@ufl.edu organization: Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA – sequence: 2 givenname: A. D. surname: Loftis fullname: Loftis, A. D. organization: Department of Microbiology and Immunology, Midwestern University, AZ, Glendale, USA – sequence: 3 givenname: S. M. surname: Mahan fullname: Mahan, S. M. organization: Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA – sequence: 4 givenname: A. F. surname: Barbet fullname: Barbet, A. F. organization: Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, FL, Gainesville, USA
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Snippet	Summary Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is... Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely... Summary Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is...
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SubjectTerms	Africa Animal diseases Animals Caribbean Cattle Cattle Diseases - epidemiology cross reaction diagnostic qPCR disease surveillance DNA DNA Primers Economic importance Ehrlichia - genetics Ehrlichia - isolation & purification Ehrlichia ruminantium Ehrlichia ruminantium - genetics Ehrlichia ruminantium - isolation & purification emerging diseases Enzyme-Linked Immunosorbent Assay epidemiological studies Epidemiology foreign animal disease genes Goats heartwater Heartwater Disease - diagnosis Heartwater Disease - epidemiology islands Livestock livestock losses Outbreaks Panola Mountain Ehrlichia pathogens Peptides Polymerase chain reaction quantitative polymerase chain reaction Real-Time Polymerase Chain Reaction sequence homology Sheep Sub-Saharan Africa United States United States - epidemiology
Title	Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease, Ehrlichia ruminantium, from the Panola Mountain Ehrlichia
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