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

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.