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A real-time PCR assay for differentiating pathogenic Anaplasma phagocytophilum from an apathogenic, woodrat-adapted genospecies from North America.
Abstract: Anaplasma phagocytophilum is a tick-transmitted bacterial pathogen of humans and animals comprising strains that cause clinical disease in people, dogs and horses (the pathogenic A. phagocytophilum "genospecies") and more distantly related strains. A rodent-adapted genospecies named DU1, found primarily in woodrats, is unable to infect horses. We developed a real-time PCR (RT-PCR) assay, which targets an 85 base pair region of the ank gene and is specific for the pathogenic genospecies of A. phagocytophilum from North America. Thirty DNA samples from A. phagocytophilum RT-PCR-positive rodents and ticks for which the ank gene had previously been sequenced and had been identified as either the pathogenic genospecies or DU1 were used for validation. All nine samples with the pathogenic genospecies tested positive using the new RT-PCR and all 21 samples with the DU1 genospecies tested negative. Two strains from which the whole genome has been sequenced and are known to be pathogenic (A. phagocytophilum, strain HZ isolated from a human from New York and strain MRK isolated from a horse from California) both tested positive using the new RT-PCR assay. We also tested blood from 20 horses and six dogs that were RT-PCR-positive using a previously validated RT-PCR protocol: all were PCR-positive using the new assay as well. The assay has high efficiency and reproducibility and there was no cross-reaction with related and tick-borne bacteria tested, although the assay cross-reacts with the ungulate-adapted genospecies Ap-Variant 1. This new RT-PCR assay will aid in future research on the ecology and epidemiology of A. phagocytophilum by allowing researchers to easily identify the pathogenic genospecies in reservoir hosts and vectors.
Copyright © 2015 Elsevier GmbH. All rights reserved.
Publication Date: 2015-07-06 PubMed ID: 26188998DOI: 10.1016/j.ttbdis.2015.07.003Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research report discusses the development and validation of a real-time PCR (polymerase chain reaction) assay designed to differentiate between harmful and non-harmful species of the bacterium Anaplasma phagocytophilum, a common pathogen found in humans and animals which is transmitted primarily through ticks.
Objective of the Research
- The primary goal of this research was to develop a test that can distinguish between the pathogenic (harmful) genospecies of Anaplasma phagocytophilum and an apathogenic (harmless), woodrat-adapted strain named DU1.
Methodology and Approach
- The developed real-time PCR (RT-PCR) assay targets an 85 base pair region of the ank gene, which is specific for the pathogenic genospecies of A. phagocytophilum in North America.
- For validation, the researchers used thirty DNA samples from A. phagocytophilum RT-PCR-positive rodents and ticks. These samples had already been identified as either the pathogenic genospecies or DU1 based on prior sequencing of the ank gene.
Results of the Validation and Testing
- All nine samples containing the pathogenic genospecies tested positive using the new RT-PCR assay, while all 21 samples with the DU1 genospecies tested negative.
- A couple of fully sequenced A. phagocytophilum strains, known to be pathogenic, also tested positive in the assay.
- Fimilar results were obtained from blood from 20 horses and six dogs that had been diagnosed as RT-PCR-positive using a previously validated protocol.
- The assay demonstrated high efficiency and reproducibility. It showed no cross-reaction with related or tick-borne bacteria, although it did cross-react with the ungulate-adapted genospecies Ap-Variant 1.
Implications of the Research
- This newly developed RT-PCR assay can assist further research into the ecology and epidemiology of A. phagocytophilum.
- It will serve as an efficient tool for researchers to identify the pathogenic genospecies in hosts and vectors, aiding the understanding of the bacterium’s transmission and its potential effects on human and animal health.
Cite This Article
APA
Stephenson N, Hodzic E, Mapes S, Rejmanek D, Foley J.
(2015).
A real-time PCR assay for differentiating pathogenic Anaplasma phagocytophilum from an apathogenic, woodrat-adapted genospecies from North America.
Ticks Tick Borne Dis, 6(6), 774-778.
https://doi.org/10.1016/j.ttbdis.2015.07.003 Publication
Researcher Affiliations
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, USA. Electronic address: nstephenson@ucdavis.edu.
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, USA.
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, USA.
- Wildlife Health Center, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, USA.
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, USA.
MeSH Terms
- Anaplasma phagocytophilum / classification
- Anaplasma phagocytophilum / genetics
- Animals
- California / epidemiology
- Computer Simulation
- DNA, Bacterial / genetics
- DNA, Bacterial / isolation & purification
- Ehrlichiosis / epidemiology
- Ehrlichiosis / microbiology
- Ehrlichiosis / veterinary
- North America
- Real-Time Polymerase Chain Reaction / methods
- Real-Time Polymerase Chain Reaction / veterinary
- Reproducibility of Results
- Rodent Diseases / epidemiology
- Rodent Diseases / microbiology
- Sensitivity and Specificity
- Sigmodontinae
Citations
This article has been cited 2 times.- André MR. Diversity of Anaplasma and Ehrlichia/Neoehrlichia Agents in Terrestrial Wild Carnivores Worldwide: Implications for Human and Domestic Animal Health and Wildlife Conservation. Front Vet Sci 2018;5:293.
- Stephenson N, Foley J. Parallelisms and Contrasts in the Diverse Ecologies of the Anaplasma phagocytophilum and Borrelia burgdorferi Complexes of Bacteria in the Far Western United States. Vet Sci 2016 Sep 22;3(4).
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