New Duplex qPCR Test Reveals Hidden Babesia Parasites in Maine Ticks

A team of researchers at the University of Maine Cooperative Extension has developed a new mitochondrial DNA-based quantitative PCR assay that simultaneously detects and differentiates two Babesia species circulating in blacklegged ticks: Babesia microti, the primary cause of human babesiosis in the northeastern United States, and Babesia odocoilei, a cervid-associated parasite that is increasingly recognized in Ixodes scapularis populations. The study, published open access in Parasites & Vectors, reports that the new duplex assay outperformed widely used assays targeting the parasite’s 18S ribosomal RNA gene, detecting substantially more infections and delivering quantification cycle values several cycles lower in matched samples. The findings suggest that some of the surveillance data on tick-borne pathogens collected with 18S-based tools may undercount or misclassify Babesia infections.

Babesia microti is a protozoan parasite transmitted by the same deer tick, Ixodes scapularis, that vectors the agents of Lyme disease, anaplasmosis, and Powassan virus disease in the northeastern United States. In humans, infection can range from asymptomatic to a malaria-like illness marked by fever, fatigue, and hemolytic anemia, and it can be severe or fatal in elderly, immunocompromised, or asplenic patients. Accurate detection of B. microti in ticks and host blood is therefore central to public health surveillance. For years, the workhorse method for this purpose has been quantitative PCR targeting the 18S rRNA gene, a highly conserved region of the parasite nuclear genome that is present in all apicomplexan parasites and is relatively easy to amplify.

That reliance on the 18S gene, however, comes with well-known trade-offs. Because the gene is conserved across many related organisms, primers designed against it can cross-react with non-target species, producing ambiguous or false-positive signals. At the same time, assays that achieve specificity by narrowing the primer-binding sites may sacrifice sensitivity, particularly when parasite loads in individual ticks are low. The Maine team encountered exactly these problems. While screening tick samples for B. microti with multiple published 18S-based assays, they observed inconsistent results between the assays — discrepancies that pointed to possible cross-reactivity with non-target organisms, limited sensitivity, or both. Those observations highlighted the need for a more sensitive, species-specific qPCR assay for B. microti and for better tools to detect and differentiate other Babesia species present in Maine’s ticks and vertebrate hosts.

The first step was to establish which other Babesia species actually mattered in the local tick population. The researchers identified Babesia odocoilei as the Babesia species most commonly found alongside B. microti in Ixodes scapularis ticks in Maine. B. odocoilei primarily infects white-tailed deer and other cervids and is not considered a major human pathogen, but its presence in the same vector ticks creates a genuine diagnostic challenge: an 18S-based assay that cannot cleanly separate the two species risks conflating a deer parasite with a human pathogen, distorting both prevalence estimates and risk assessments. Distinguishing the two reliably requires a genetic target with enough sequence variation between them to support species-specific probing — and enough copies per parasite to support sensitive detection.

Mitochondrial DNA offered exactly that combination. To build the new assay, the authors sequenced approximately 5,000-base-pair regions of the mitochondrial DNA of both B. microti and B. odocoilei, giving them a detailed view of regions where the two species diverge. Mitochondrial genomes are typically present in many copies per cell, far more than the single-copy or low-copy nuclear loci, which means that even small quantities of parasite DNA yield a stronger amplification signal. By designing primers and fluorescent probes against species-discriminating sites within the mitochondrial sequences, the team constructed a duplex reaction: a single qPCR well in which two independent probe systems report simultaneously, one specific for B. microti and one for B. odocoilei. The chemistry follows standard probe-based qPCR principles, with species-specific amplification monitored in real time through distinct fluorescent channels, allowing both detection and differentiation of the two parasites without additional post-PCR steps such as sequencing or melting-curve analysis.

With the assay built, the researchers put it to work on a large surveillance dataset: 4,464 Ixodes spp. ticks collected through both active and passive surveillance in Maine. Active surveillance typically involves field collection of ticks from known habitats, while passive surveillance relies on ticks submitted by members of the public — in this case, Maine residents who sent specimens to the University of Maine Cooperative Extension Tick Lab for identification and testing. Across this sample set, the mitochondrial duplex assay detected B. microti in 449 ticks, corresponding to a prevalence of 10.1 percent, and B. odocoilei in 803 ticks, or 18.0 percent. Notably, 92 ticks — 2.1 percent of the total — carried mixed infections with both species, a finding that a less discriminatory assay could easily have obscured. Prevalence of both parasites varied by collection method, tick life stage, and geographic location, patterns that matter for interpreting where and when human exposure risk is highest.

The most striking validation result came from a direct comparison with the older methodology. In 46 ticks identified as B. microti-positive, the mitochondrial assay produced quantification cycle (Cq) values 5.9 to 7.1 cycles lower than two published 18S rDNA assays, a difference that was highly statistically significant with Dunnett-adjusted p values below 0.0001. In qPCR, each cycle roughly corresponds to a doubling of the target, so a six- to seven-cycle advantage translates to an order-of-magnitude or greater improvement in apparent sensitivity. Lower Cq values mean the assay detects parasite DNA earlier in the amplification run — that is, from smaller starting quantities — which reduces the chance that low-level infections in individual ticks go undetected. For a pathogen like B. microti, which can occur at low densities in field-collected ticks, that sensitivity gain directly affects the accuracy of prevalence estimates.

The duplex design also resolves a diagnostic ambiguity that has practical consequences for surveillance programs. When a single-target 18S assay reacts with multiple Babesia species, the operator may not know which species produced the signal, or whether a strong signal reflects one abundant parasite or two co-infecting ones. The mitochondrial duplex eliminates that uncertainty by assigning each species its own fluorescent channel, and the detection of 92 dual-positive ticks demonstrates that co-infections are not a theoretical possibility but a measurable component of the ecology. Surveillance programs that rely on species-blind assays may, in effect, be blending the epidemiology of a human pathogen with that of a wildlife parasite, and the Maine data suggest how much hidden structure such blending can conceal.

The work was carried out by Ann M. Bryant, Griffin M. Dill, and Thomas F. Rounsville Jr. of University of Maine Cooperative Extension, with validation samples donated by collaborators including Chuck Lubelczyk at the MaineHealth Institute for Research, Pauline Kamath at the University of Maine Wildlife Disease Genetics Lab, and Nicole Chinnici and the Tick Research Lab of Pennsylvania at East Stroudsburg University. Funding came in part from the United States Department of Agriculture Animal and Plant Health Inspection Service, the United States Geological Survey, and the State of Maine. The study’s scope is regional — the samples all come from Maine — but the methodological lesson is general: the choice of genetic target shapes what a surveillance program can see, and mitochondrial targets may offer both the copy number needed for sensitivity and the sequence divergence needed for species-level discrimination in Babesia.

The authors conclude that the mitochondrial duplex qPCR assay provided improved sensitivity and improved detection of both B. microti and B. odocoilei compared with published 18S rDNA assays, and that the results support the use of mitochondrial targets to differentiate co-occurring Babesia species and to strengthen the interpretation of tick-borne pathogen surveillance data. As babesiosis case counts rise in parts of the northeastern United States and ticks continue to expand their range, the tools used to track the parasites behind those numbers matter as much as the surveillance itself. A test that can cleanly tell one Babesia species from another, in a single reaction and at lower detection limits, gives public health and veterinary authorities a sharper picture of which parasites are present, where they are concentrated, and how often ticks carry more than one — information that older, less discriminating assays may have been quietly missing all along.

Subject of Research: Development of a mitochondrial duplex qPCR assay for detecting and differentiating Babesia microti and Babesia odocoilei in Ixodes ticks

Article Title: A novel mitochondrial quantitative PCR duplex for the detection of Babesia microti and Babesia odocoilei

Article References: A novel mitochondrial quantitative PCR duplex for the detection of Babesia microti and Babesia odocoilei. (n.d.). https://doi.org/10.1186/s13071-026-07746-x

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07746-x

Keywords: Babesia microti, Babesia odocoilei, qPCR, mitochondrial DNA, Ixodes scapularis, tick surveillance, human babesiosis, Maine, 18S rRNA gene, diagnostic assay, tick-borne disease, Parasites & Vectors