Development and Validation of Real-Time PCR for Detecting Anaplasma Bovis–Like Agent in Dermacen

Innovative real-time PCR for detection of Anaplasma bovis–like agent in Dermacentor ticks: Development, validation, and field application

The clinical data associated with human cases were not available in the cited reports, though patient samples were submitted under suspicion of tickborne illness.

bovis–like agent in ticks and potentially in humans.

• A previously described Anaplasma bovis–like agent has been detected in humans in the United States (notably in 2020, with further descriptions in 2023 involving four patients and some Dermacentor tick samples).
• Earlier epidemiologic efforts encountered constraints due to reliance on conventional PCR methods, which, while suitable for genetic characterization, were less efficient for screening large sample sets.
• Against this backdrop, the authors pursued a rapid, targeted molecular assay to enable efficient epidemiologic surveys of the A.

bovis–like agent and related Anaplasma spp.

species, including A.

bovis, A.

capra, A.

centrale, A.

marginale, A.

ovis, A.

phagocytophilum, and A.

platys.

bovis–like agent.

bovis–like DNA samples previously extracted from D.

variabilis ticks.

and other tick- or mammal-associated pathogens (A.

phagocytophilum, A.

marginale, A.

platys, Ehrlichia ewingii, E.

chaffeensis, Rickettsia bellii, and R.

montanensis).

No cross-amplification with these non-target organisms was observed in the tested samples.

bovis–like agent was confirmed via Sanger sequencing and BLAST analysis, aligning with previously reported Dermacentor spp.

tick and human-derived sequences.

• Target and design framework:
• The team mined heat shock chaperon gene groEL sequences from GenBank that encompassed the A.
• They aligned these sequences to generate candidate primer and probe sets, ultimately constructing a single assay designated AboviTM that targets a 125-base-pair fragment of groEL.
• The AboviTM design employed oligonucleotides with intentional mismatches to non-target sequences at the binding sites to favor specificity for the A.
• Synthesis and preliminary testing:
• Custom oligonucleotides for AboviTM were synthesized for TaqMan assays, followed by initial validation using four A.
• Verification included conventional PCR confirmation of amplicons, gel electrophoresis to assess product size, and Sanger sequencing to confirm identity via BLAST analysis.
• Reaction composition and cycling:
• Each 20-μL reaction contained 10 μL of TaqMan Fast Advanced Master Mix, 900 nM of each primer, 250 nM probe, 2 μL of DNA template, and water.
• Thermal cycling consisted of an initial 95°C activation step for 60 seconds, then 45 cycles of 95°C denaturation for 3 seconds, followed by 60°C annealing/extension for 30 seconds.
• Analytical optimization and standards:
• A gBlocks synthetic double-stranded DNA standard matching the target sequence served as the quantitative standard.
• A series of 10-fold dilutions of this standard, spanning 1 to 100,000,000 target copies per reaction, established the assay’s dynamic range and performance.
• The assay was evaluated across varying primer and probe concentrations to identify optimal conditions; optimal performance occurred with 900 nM primers and 250 nM probe.
• Standard curves were generated using two approaches: (a) synthetic target DNA alone, and (b) the same dilution series with approximately 54 ng of noninfected Dermacentor variabilis tick DNA added per reaction to simulate a tick DNA matrix.
• The AboviTM assay was run in triplicate across five independent runs to ensure reproducibility.
• Specificity and analytical limitations:
• Specificity testing included triplicate runs against a panel of genetically related Anaplasma spp.
• The authors report that sequence identity of the amplification products for the A.
• The stated limit of detection (LOD) was 10 copies of target DNA per reaction, with the assay capable of detecting down to 1 copy in some configurations, albeit the practical LOD was designated as 10 copies; there is a parenthetical note suggesting a more favorable signal at lower copy numbers, but the final stated LOD is 10 copies.
• The report emphasizes reliance on a single assay for screening, noting that it could enhance epidemiologic investigations but does not present clinical diagnostic claims.

ticks collected from dogs and cats across the United States during 2021–2023.

bovis–like agent by AboviTM; no positive ticks were reported among other Dermacentor spp.

tested or among the total tested cohort.

Sequence identity analyses demonstrated 100% match to previously reported sequences from Dermacentor ticks and humans in the United States (GenBank accession numbers provided in the report).

bovis–like agent in Dermacentor spp.

ticks.

bovis–like agent detections remains described as likely focal, with prior regional data indicating a higher prevalence in D.

variabilis ticks from Oklahoma (10.1%, 4/38) and no detections in Kansas D.

variabilis ticks (0/93) or in Oklahoma beef cattle (0/140) from that study.

bovis–like agent DNA has, to date, been detected in humans and D.

variabilis ticks from several central US states (Minnesota, Oklahoma, Missouri, Iowa, Nebraska) and in D.

andersoni ticks from Saskatchewan, Canada.

They acknowledge that transmission dynamics and full geographic distribution are not yet fully characterized.

bovis–like agent, potentially enabling more scalable screening of human samples, vectors, and animal hosts.

bovis–like agent in the United States.

• Sample collection and processing:
• The AboviTM assay was applied to 672 Dermacentor spp.
• Veterinary clinics contributed ticks to a centralized repository at Oklahoma State University (OSU) College of Veterinary Medicine for morphological identification and storage in 70% ethanol at −20°C until DNA extraction.
• Ticks were processed by dissection: ticks were immobilized ventral side down on slides and opened along the coronal plane; internal contents were scraped into sterile tubes, with exoskeletons discarded.
• Total genomic DNA was extracted using the Cytiva Blood Genomic Prep Mini Spin Kit.
• Assay application and results:
• One male Dermacentor variabilis tick, collected from a dog in Blaine, Minnesota, tested positive for the A.
• The positive sample underwent confirmation by conventional PCR targeting 16S (rrs), citrate synthase (gltA), and groEL genes, with amplicons validated by gel electrophoresis, subsequently purified and sequenced via Sanger sequencing (unidirectional).
• Prevalence and geographic context:
• The observed prevalence within this tick sampling was 0.1% (1/672) for the A.
• The geographic distribution of A.
• The authors note that A.
• Implications for surveillance:
• The AboviTM assay is presented as a rapid, specific diagnostic tool with high efficiency for detecting the A.
• The authors suggest that applying AboviTM to future large-scale screening efforts could improve the epidemiologic understanding of the A.

bovis–like DNA, conventional PCR confirmation, gel-based amplicon size verification, and Sanger sequencing with BLAST confirmation.

ticks from across the United States) and acknowledges focal distribution tendencies in prior work.

The authors caution that broad generalizations about geographic spread cannot be made from the available data.

bovis–like agent require further investigation.

• Data sources and sequence confirmation:
• The study integrates multiple lines of evidence for assay development and verification, including in silico design with sequence alignments, experimental validation against known A.
• The reported GenBank accession numbers indicate that the positive tick-derived sequences matched previously described sequences from Dermacentor ticks and human cases in the United States.
• Scope of human data:
• Human clinical data were not included in this source beyond references to prior reports and the absence of detailed clinical information for human cases in the cited human investigations.
• Prevalence interpretation:
• The study emphasizes that prevalence findings are limited to the samples tested (672 Dermacentor spp.
• Open questions highlighted by the authors:
• Which other tick species or mammalian hosts contribute to the transmission cycle remains undetermined.
• The broader geographic distribution and the complete epidemiology of the A.
• Additional studies are necessary to define the agent’s movement dynamics between vectors, animals, and humans.

bovis–like agent, designed to minimize cross-reactivity through targeted primer/probe configuration and sequence-based specificity checks.

bovis–like agent in ticks and its detection in human samples in prior reports underscore a zoonotic/vector-borne transmission concern, though clinical data and outcomes are not elaborated in the current report.

bovis–like infections.

In summary, this work delivers a carefully designed real-time PCR assay (AboviTM) with validated specificity and a defined detection limit for identifying an Anaplasma bovis–like agent in Dermacentor spp.

ticks.

The assay was applied to a substantial geographic sample of ticks collected from companion animals in the United States, revealing a single positive instance (0.1%) among 672 ticks and corroborated by conventional PCR and sequencing.

The authors frame AboviTM as a tool to augment epidemiologic surveillance for this agent in ticks and potentially in humans, while acknowledging the current limitations in understanding the agent’s geographic distribution, vector range, and transmission dynamics.

Uncertainties persist regarding the broader epidemiology and clinical correlates, and the work suggests that broader, systematic application of this diagnostic approach could help to refine risk assessments and illuminate transmission networks in future studies.

• Methodological advancement:
• The study reports the development of a probe-based real-time PCR assay (AboviTM) specifically tailored to detect an A.
• The assay demonstrates robust performance characteristics in controlled conditions, with demonstrated specificity against a panel of related pathogens and a defined limit of detection for clinical and surveillance contexts.
• Field applicability:
• The deployment of AboviTM to a broad collection of Dermacentor ticks across the United States demonstrates practical utility for large-scale vector surveillance, enabling a more efficient screening process than conventional PCR approaches.
• The finding of a single positive case among 672 ticks indicates a low observed prevalence in this dataset but also validates the assay's capacity to detect the agent in field-collected specimens.
• Epidemiologic implications:
• The presence of an A.
• Given the apparent focal distribution and limited number of detected cases, the AboviTM assay could become a valuable component of targeted surveillance strategies, potentially contributing to risk assessment and epidemiologic mapping when integrated with broader ecological and sentinel data.
• Limitations and reporting boundaries:
• The source explicitly acknowledges uncertainty regarding the broader geographic distribution and transmission networks due to the modest size of positive detections across a wide range of tick specimens and the incomplete characterization of the human symptomatology associated with A.
• The article refrains from making clinical claims or recommendations regarding management or treatment, and it emphasizes that further research is required to articulate the full public health relevance.