A real-time PCR assay for differentiating pathogenic Anaplasma phagocytophilum from an apathogenic, woodrat-adapted genospecies from North America

Nicole Stephenson, Emir Hodzic, Samantha Mapes, Daniel Rejmanek, Janet E Foley

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)774-778
Number of pages5
JournalTicks and Tick-borne Diseases
Volume6
Issue number6
DOIs
StatePublished - Sep 1 2015

Fingerprint

Anaplasma phagocytophilum
North America
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
assays
Horses
Ticks
ticks
horses
Rodentia
rodents
Dogs
disease reservoirs
dogs
Cross Reactions
ungulates
Ecology
cross reaction
sampling
Base Pairing

Keywords

  • Human granulocytic anaplasmosis
  • Hydrolysis probe
  • Ixodes pacificus
  • Neotoma fuscipes
  • RT-PCR
  • Tick-borne disease

ASJC Scopus subject areas

  • Infectious Diseases
  • Insect Science
  • Parasitology
  • Microbiology

Cite this

A real-time PCR assay for differentiating pathogenic Anaplasma phagocytophilum from an apathogenic, woodrat-adapted genospecies from North America. / Stephenson, Nicole; Hodzic, Emir; Mapes, Samantha; Rejmanek, Daniel; Foley, Janet E.

In: Ticks and Tick-borne Diseases, Vol. 6, No. 6, 01.09.2015, p. 774-778.

Research output: Contribution to journalArticle

@article{614c0794bdf94ca18071bf53eb9a7871,
title = "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.",
keywords = "Human granulocytic anaplasmosis, Hydrolysis probe, Ixodes pacificus, Neotoma fuscipes, RT-PCR, Tick-borne disease",
author = "Nicole Stephenson and Emir Hodzic and Samantha Mapes and Daniel Rejmanek and Foley, {Janet E}",
year = "2015",
month = "9",
day = "1",
doi = "10.1016/j.ttbdis.2015.07.003",
language = "English (US)",
volume = "6",
pages = "774--778",
journal = "Ticks and Tick-borne Diseases",
issn = "1877-959X",
publisher = "Elsevier GmbH",
number = "6",

}

TY - JOUR

T1 - A real-time PCR assay for differentiating pathogenic Anaplasma phagocytophilum from an apathogenic, woodrat-adapted genospecies from North America

AU - Stephenson, Nicole

AU - Hodzic, Emir

AU - Mapes, Samantha

AU - Rejmanek, Daniel

AU - Foley, Janet E

PY - 2015/9/1

Y1 - 2015/9/1

N2 - 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.

AB - 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.

KW - Human granulocytic anaplasmosis

KW - Hydrolysis probe

KW - Ixodes pacificus

KW - Neotoma fuscipes

KW - RT-PCR

KW - Tick-borne disease

UR - http://www.scopus.com/inward/record.url?scp=84942326567&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84942326567&partnerID=8YFLogxK

U2 - 10.1016/j.ttbdis.2015.07.003

DO - 10.1016/j.ttbdis.2015.07.003

M3 - Article

C2 - 26188998

AN - SCOPUS:84942326567

VL - 6

SP - 774

EP - 778

JO - Ticks and Tick-borne Diseases

JF - Ticks and Tick-borne Diseases

SN - 1877-959X

IS - 6

ER -