New Duplex Real-Time PCR Test Tracks Two Fox Babesia Parasites and Their Tick Vectors

A new diagnostic tool promises to sharpen the way scientists track two closely related blood parasites of wild canids. Researchers at the University of Bari in Italy, working with collaborators, have developed and validated a duplex quantitative real-time PCR (dqPCR) assay that can simultaneously detect and differentiate Babesia banethi and Babesia vulpes in DNA extracted from animal tissues and ticks. The study, published in the open-access journal Parasites & Vectors, addresses a growing need in veterinary parasitology, where the boundaries between wildlife, domestic animals, and humans are increasingly blurred and where precise molecular tools are essential for understanding how vector-borne pathogens circulate in nature.

Babesia species are single-celled piroplasmid parasites that infect red blood cells of a wide range of mammals and are transmitted by ticks. In domestic dogs, some Babesia species cause babesiosis, a disease that can range from mild anemia to a rapidly fatal hemolytic condition. Among the piroplasmids that infect wild canids, Babesia vulpes has emerged as a parasite widely distributed in red foxes, the most abundant wild canid across much of Europe and other temperate regions. Foxes live in close proximity to urban and peri-urban environments, which makes them potentially important sentinels and reservoirs for parasites that could spill over into domestic dogs or, in some cases, into humans.

Babesia banethi, by contrast, is a much more recent addition to scientific knowledge. Since its description, many fundamental aspects of its biology have remained poorly understood, including its epidemiology, the range of hosts it can infect, the tissues in which it resides, and which tick species might transmit it. This gap in knowledge is not merely academic. Without knowing which animals harbor the parasite and which ticks can carry it, public and animal health authorities cannot assess the risk it poses or design surveillance programs to monitor its spread. The new assay was developed specifically to close this methodological gap and to support future investigations on both Babesia species.

The technical design of the assay reflects a careful balance between breadth and specificity. The researchers employed generic primers, which bind to conserved regions of the 18S ribosomal DNA gene shared by these parasites, paired with species-specific TaqMan-MGB probes that distinguish the two Babesia species by producing separate fluorescent signals. Because the two reactions run in the same tube, a single sample can be screened for both parasites at once, halving the labor, reagent cost, and sample consumption compared with running separate single-species assays. The 18S rDNA gene is a standard target in piroplasmid diagnostics because it is present in multiple copies in the parasite genome, which enhances sensitivity, while still containing enough species-discriminating variation for probe-based differentiation.

Validation was carried out on a diverse panel of real-world samples. The researchers tested the assay using blood and organ samples from naturally infected red foxes, samples from other vertebrate hosts, and DNA extracted from various tick species. This breadth matters because a diagnostic assay that performs well only on purified plasmid controls may fail when confronted with the complex matrix of a tissue homogenate or a tick crushed and extracted whole. The duplex assay proved capable of detecting both Babesia species whether they occurred individually or in simulated mixed infections, a scenario that is biologically plausible in wild foxes that may be exposed to multiple parasite species through overlapping tick bites.

The analytical performance figures reported in the study are striking. The dqPCR achieved high analytical specificity, showing no cross-reactivity with other hemoparasites that infect carnivores, a critical property since fox blood and organs frequently contain DNA from other tick-borne organisms such as Hepatozoon, Theileria, or other Babesia species that could otherwise generate false positives. Sensitivity reached as low as ten to the power of zero plasmid copies per reaction, meaning the assay could detect a single copy of the target sequence in the reaction mixture. At that level of sensitivity, the assay approaches the theoretical detection limit of any PCR-based method and allows researchers to quantify very low parasite burdens, which is essential when screening ticks, where parasite DNA may be present only in trace amounts.

Perhaps the most compelling demonstration of the assay’s practical value came from its application to field-collected ticks. The researchers detected Babesia banethi DNA in an engorged Ixodes kaiseri tick and Babesia vulpes DNA in Haemaphysalis erinacei. These findings do not by themselves prove that these tick species are biological vectors capable of transmitting the parasites, because detecting parasite DNA in a tick could reflect a recent blood meal from an infected host rather than a true infection of the tick itself. However, they provide exactly the kind of lead that vector investigations require. By identifying candidate tick species that carry the parasite DNA, the assay allows researchers to prioritize which ticks to study in greater depth, for example through experimental transmission studies or by examining tick tissues to determine whether the parasite invades the salivary glands, a hallmark of true vector competence.

The ability to quantify parasite burden in tissues opens additional research avenues. For a recently described parasite like Babesia banethi, whose tissue tropism remains poorly characterized, the assay can be used to screen a range of organs from infected foxes to map where the parasite concentrates in the body. Such information is valuable for understanding pathogenesis, for selecting the most appropriate diagnostic sample types, and for interpreting the results of future surveys. Similarly, quantitative data from blood samples can reveal whether infected foxes carry high or low parasite loads, which has implications for how likely they are to infect feeding ticks and thus contribute to transmission cycles.

The broader context of this work is the reshaping epidemiology of vector-borne diseases. Increasing interactions among wildlife, domestic animals, vectors, and humans, driven by urbanization, climate change, and changing land use, are altering the dynamics of pathogens such as Babesia. Red foxes, in particular, have adapted remarkably well to human-modified landscapes and now live at high densities in and around cities across Europe. As fox populations expand and tick distributions shift, parasites once confined to remote wild habitats can move closer to pets and people. Tools that allow rapid, sensitive, and species-specific screening of large numbers of samples are therefore foundational for surveillance networks that monitor these changes in real time.

The research team, led by Mariaelisa Carbonara and including Giada Annoscia, Maria Stefania Latrofa, Antonio Camarda, and senior author Domenico Otranto, a well-known figure in veterinary parasitology with affiliations spanning the University of Bari, City University of Hong Kong, and Chulalongkorn University in Bangkok, credits the Apulian Regional Wildlife Rescue Center for its collaboration in animal sampling. The work was funded through the Italian PN Ricerca, Innovazione e Competitività program for the green and digital transition, with the funders having no role in study design, data collection, or publication decisions. The assay’s developers describe it as a rapid, sensitive, and species-specific diagnostic tool suited for large-scale epidemiological studies, screening of candidate vertebrate hosts and tick vectors, assessment of parasite burden in tissues, and detection of mixed infections. As foxes continue to share our cities and ticks continue to expand their ranges, this duplex real-time PCR assay provides the parasitology community with a precise new lens through which to watch two enigmatic Babesia species move through the wild.

Subject of Research: Development and validation of a duplex real-time PCR assay for detecting and differentiating Babesia banethi and Babesia vulpes in foxes and ticks

Article Title: Detection and differentiation of Babesia banethi and Babesia vulpes in foxes and potential tick vectors by a duplex real-time PCR assay

Article References: Carbonara, M., Annoscia, G., Latrofa, M. S., Camarda, A., & Otranto, D. (2026). Detection and differentiation of Babesia banethi and Babesia vulpes in foxes and potential tick vectors by a duplex real-time PCR assay. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07668-8

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07668-8

Keywords: Babesia banethi, Babesia vulpes, duplex real-time PCR, red foxes, ticks, Ixodes kaiseri, Haemaphysalis erinacei, piroplasmids, vector-borne disease, 18S rDNA, wildlife parasitology, diagnostics