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: Bryant, A. M., Dill, G. M., & Rounsville, T. F., Jr. (2026). A novel mitochondrial quantitative PCR duplex for the detection of Babesia microti and Babesia odocoilei. Parasites & Vectors. 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

The logic behind the approach rests on the peculiar ecology of Lyme disease in the northeastern United States. The blacklegged tick, Ixodes scapularis, passes the bacterium Borrelia burgdorferi sensu stricto through its life stages, but the pathogen’s persistence in a given landscape depends heavily on one particular host: the white-footed mouse, Peromyscus leucopus. These abundant rodents serve as the principal natural reservoir of the spirochete, and larval ticks that feed on infected mice are far more likely to acquire the bacterium than larvae feeding on deer, birds, or other mammals. When those larvae molt into nymphs the following year, the infected nymphs become the primary threat to humans, delivering the bite that transmits Lyme disease in the vast majority of cases.

Interrupting the cycle at the mouse-tick interface has therefore long been an attractive target for researchers. Earlier laboratory and simulated field studies had already demonstrated that a bait containing just 0.005 percent fipronil could systemically control Ixodes scapularis ticks parasitizing treated white-footed mice. Fipronil, the same active ingredient found in some companion-animal flea and tick products, works systemically: after a mouse ingests the bait, the compound and its metabolites circulate in the animal’s blood, and ticks that attach and begin feeding are killed before they can complete their blood meal. The question the Connecticut study set out to answer was whether that laboratory promise would survive contact with real backyards, real weather, and real wildlife.

Between 2022 and 2024, the research team deployed reusable rodent bait stations on residential properties during the period when blacklegged ticks are active in their nymphal and larval stages, roughly May through August. The stations dispensed the fipronil-treated bait to free-ranging mice, and the team monitored consumption, replenishing bait as needed to keep the system running through each season. On average, each property received 4.9 kilograms of bait per year, which translates to approximately 247 milligrams of fipronil—a remarkably small total quantity of active ingredient distributed across an entire property over an entire season.

To confirm that the mice were actually being exposed to the compound, the researchers captured white-footed mice during the larval season each year on both treated and untreated control properties and analyzed their blood plasma. The results were striking: fipronil sulfone, a metabolite that serves as a marker of exposure, was detectable in 100 percent of plasma samples analyzed in both 2022 and 2023, and in 83.3 percent of samples in 2024. In other words, the bait stations were doing their job, delivering the active ingredient to the target animals with high consistency even under fully natural field conditions.

The effect on the ticks themselves was even more impressive. On treated properties, the number of larvae parasitizing captured mice fell by up to 84.2 percent compared with control properties, and the number of larvae that successfully engorged—those that managed to complete a blood meal, which is the critical step for both tick survival and pathogen acquisition—dropped by up to 92.5 percent. A larval tick that fails to engorge on a mouse not only fails to acquire Borrelia from that mouse; it also faces greatly reduced odds of surviving to molt into the next life stage. The bait, in effect, turned the most important host in the Lyme disease system into a lethal trap for its parasites.

The benefits extended to the pathogen as well. Molecular assays used to detect Borrelia burgdorferi infection in the captured mice showed that infection prevalence in white-footed mice on treated properties was significantly reduced by up to 34.1 percent relative to controls. That reduction matters because fewer infected mice means fewer larval ticks acquiring the spirochete, which in principle should mean fewer infected nymphs questing for human blood the following spring. The study thus demonstrated a measurable dent in the reservoir competence of the local mouse population, achieved not by reducing mouse numbers but by breaking the tick-mouse transmission loop.

The picture was more nuanced, however, when the researchers turned to the stage of the tick life cycle that matters most for human health. Drag sampling and flagging during the nymphal seasons were used to estimate the abundance of host-seeking nymphs on the properties, and molecular testing assessed how many of those nymphs carried Borrelia. While both nymph density and nymphal infection showed reductions on treated properties, neither difference reached statistical significance compared with the control sites. The authors suggest that the bait application rate during the larval season may need to be increased to push these downstream effects over the threshold of detectability—a reminder that intervening at one link of a complex transmission chain does not automatically translate into reduced risk at every other link.

That caveat aside, the study represents one of the most thorough field evaluations to date of a systemic acaricide bait aimed at the wildlife reservoir of Lyme disease. Its design—multi-year, property-level, with matched controls, bait-consumption monitoring, pharmacokinetic confirmation, and molecular pathogen testing on both rodents and questing ticks—addresses many of the weaknesses that have plagued earlier tick-control trials. The extremely low dose of active ingredient involved also raises the prospect of an environmentally targeted intervention: rather than broadcasting insecticides across vegetation, the treatment is delivered only to the small mammals that seek out the bait stations, and only in quantities measured in hundreds of milligrams per property per year.

The researchers, based at Genesis Laboratories in Colorado and the Connecticut Agricultural Experiment Station, were supported by a contract with the United States Centers for Disease Control and Prevention, and the work benefited from molecular detection assistance from CDC scientists. They emphasize that additional field studies are needed to confirm the findings and to explore what large-scale treatment across neighborhoods or landscapes might achieve, since tick populations on untreated properties can continually seed treated areas. Still, the core result stands: a bait box, a few hundred milligrams of fipronil, and the natural foraging behavior of white-footed mice were enough to strip the vast majority of larval ticks from the animals that sustain Lyme disease in the northeastern United States. As integrated tick management strategies evolve, the humble mouse bait station may prove to be one of the most quietly powerful tools in the fight against America’s most common vector-borne disease.

Subject of Research: Field efficacy of low-dose fipronil bait for controlling blacklegged ticks on white-footed mice and reducing Lyme disease transmission

Article Title: Efficacy of a low-dose fipronil bait in controlling blacklegged tick (Ixodes scapularis) larvae parasitizing white-footed mice (Peromyscus leucopus) under field conditions in the northeastern United States

Article References: Poché, D. M., Williams, S. C., Linske, M. A., & Poché, R. M. (2026). Efficacy of a low-dose fipronil bait in controlling blacklegged tick (Ixodes scapularis) larvae parasitizing white-footed mice (Peromyscus leucopus) under field conditions in the northeastern United States. Parasites & Vectors, 19(1), Article 423. https://doi.org/10.1186/s13071-026-07676-8

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07676-8

Keywords: Lyme disease, Ixodes scapularis, Peromyscus leucopus, fipronil, tick control, Borrelia burgdorferi, vector-borne disease, systemic acaricide, bait stations, integrated tick management, Connecticut, public health