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

The fox was first treated with ivermectin for the mange and with amoxicillin combined with clavulanic acid for the suspected urinary infection. Instead of recovering, the animal worsened, growing weaker and paler. Only a month after admission, in September 2024, did the clinical team suspect a blood parasite and run a complete blood count, serum biochemistry, urinalysis and faecal examinations. The haematology revealed a regenerative anaemia marked by anisocytosis, polychromasia, Howell-Jolly bodies, rouleaux formation and increased reticulocytes, alongside leukocytosis with neutrophilia, a left shift and lymphocytosis. Biochemistry showed elevated alanine aminotransferase and alkaline phosphatase, increased total bilirubin, reduced total proteins with hypoalbuminemia, raised cholesterol, amylase and lipase, and a reduced sodium-to-potassium ratio. Urinalysis detected haemoglobinuria at 50 micromoles per litre together with haematuria. A blood smear then delivered the decisive clue: intraerythrocytic inclusions morphologically consistent with small piroplasmids.

Molecular confirmation followed quickly. DNA extracted from whole blood was analysed by conventional PCR targeting the 18S ribosomal DNA gene, and sequencing of the roughly 510-base-pair amplicon, compared against GenBank using BLAST, showed 99.6 per cent nucleotide identity with Babesia banethi. Crucially, the fox screened negative for antibodies against Leishmania infantum, Ehrlichia canis and Anaplasma phagocytophilum/platys, negative for Dirofilaria immitis antigen and negative for Rickettsia species DNA, ruling out common co-infections. B. banethi is a recently described small Babesia species, genetically distinct from the dominant fox piroplasm Babesia vulpes. While B. vulpes belongs to clade I of the piroplasmids, within the B. microti-like group, phylogenetic analyses place B. banethi in clade III, the so-called Western group, well apart from the large Babesia sensu stricto species such as B. canis, B. vogeli and B. gibsoni that infect domestic and wild carnivores. The species had previously been detected in foxes from Israel and Iraq, and its competent tick vector remains unknown, although parasite DNA has been found in Ixodes kaiseri ticks collected from infected animals.

With no guidelines for managing babesiosis in wildlife, the veterinary team turned to standard canine practice. At the end of September 2024 the fox received imidocarb dipropionate at 6.6 milligrams per kilogram subcutaneously, in two doses spaced 28 days apart, the most routinely used regimen for canine babesiosis. Fifteen days after the first dose the fox showed genuine clinical and haematological improvement, with only a persistent lymphocytosis marring the blood picture. Yet the parasitological follow-up told a different story: blood smears and PCR both remained positive, indicating persistent parasitaemia. Two weeks after the second imidocarb dose the animal continued to improve clinically and the parasitaemia and lymphocytosis had disappeared on smear, but PCR was still positive for B. banethi DNA. The drug had suppressed the disease without eradicating the parasite.

The relapse came roughly three months after the second imidocarb dose, in February 2025. The fox began showing mild lethargy and pale mucous membranes again, and laboratory work confirmed the return of anaemia, lymphocytosis, thrombocytosis and microscopically visible parasitaemia. Subsequent examinations at follow-up points roughly 255 and 281 days after the initial treatment recorded a consistent decline in the animal’s condition, with B. banethi detectable by both blood smear and PCR. Almost a full year after the original diagnosis, the team switched strategy. They initiated a combination of atovaquone and proguanil hydrochloride, the antimalarial sold as Malarone, at 20 milligrams per kilogram twice daily by mouth, together with azithromycin at 10 milligrams per kilogram once daily subcutaneously, for ten days, following the general regimen used against small-form Babesia infections in dogs.

The response was striking. At follow-up examinations roughly 331 and 352 days after the initial diagnosis, the fox showed marked clinical and haematological recovery, with a negative blood smear, although PCR positivity persisted. About two months after the end of the Malarone course the animal remained parasitaemic by PCR but was clinically stable, albeit with lingering laboratory abnormalities including lymphocytosis and hypergammaglobulinemia; a repeat SNAP Leish 4Dx test again came back negative. In January 2026 the fox received a second course of the atovaquone-proguanil and azithromycin combination, and one month after that therapy ended the animal was clinically stable, with only persistent lymphocytosis and elevated liver enzymes remaining, and for the first time tested both cytologically and molecularly negative for B. banethi.

The longitudinal record offers several lessons about how small Babesia parasites behave in their hosts and how treatment should be judged. PCR positivity soon after drug administration, the authors note, should not be read as treatment failure, because it may reflect the temporary persistence of parasite DNA rather than active infection; molecular testing is generally recommended no earlier than two months after completing therapy for small Babesia species. When PCR positivity lingers beyond three months, however, clinicians must consider sequestration of the parasite in target organs such as the spleen and in circulating erythrocytes, with the potential for reactivation after immunosuppressive events. For rescued wildlife destined for release, the authors argue that the primary indicator of therapeutic success should be clinical stabilisation and cytological negativity rather than complete clearance of parasite DNA, since foxes should spend as little time as possible in rehabilitation facilities.

The haematological details also distinguish this infection from better-known canine babesiosis. The mild thrombocytosis observed during the acute and relapse phases likely reflects rebound stimulation of the bone marrow in response to haemolytic anaemia, a pattern that contrasts with the thrombocytopenia commonly described in dogs infected with B. vulpes. Most other abnormalities, including anaemia, elevated liver enzymes and increased total bilirubin, mirrored those reported in dogs with small Babesia infections. Recurrent lymphocytosis, hypergammaglobulinemia and hypoalbuminemia pointed to chronic immune stimulation, while the raised ALT and ALP activities with hyperbilirubinaemia and brown urine probably reflected hepatocellular lesions secondary to haemolysis, anoxia and systemic inflammation. No hepatoprotective or additional supportive therapy was given, a limitation the authors acknowledge when interpreting the persistent liver enzyme changes. The team also stresses the complementary value of blood smear examination and molecular testing: smears identify acute, chronic and relapsing phases, while PCR confirms the species and documents persistent infection.

Why did this fox become visibly ill at all, when foxes are generally regarded as asymptomatic reservoirs of piroplasm infections? The severe sarcoptic mange and poor nutritional state may have impaired the animal’s immune response and prolonged the clinical course, but the absence of co-infections with other vector-borne pathogens or gastrointestinal parasites, together with the repeated recurrence of signs and parasitaemia, supports a primary pathogenic role for B. banethi itself. That possibility carries a broader warning. If a parasite can establish a persistent, treatment-refractory infection in a species considered relatively resistant to vector-borne pathogens, the clinical impact on more susceptible hosts, particularly domestic dogs sharing the same environment and exposed to the same potentially competent ticks, could be considerable. The case also highlights practical realities of wildlife medicine: follow-up intervals were stretched to limit repeated handling of the animal, which may have delayed detection of parasitaemia recurrences, and an earlier switch to atovaquone-proguanil with azithromycin as a first-line protocol might have shortened the disease course. Given the off-label use of atovaquone, the authors add that efficacy must be balanced against the risk of fostering antimicrobial resistance, a concern already documented for B. gibsoni in dogs. All clinical assumptions, they caution, now require validation in a larger series of B. banethi cases.

Subject of Research: Clinical course and treatment of Babesia banethi infection in a red fox

Article Title: Clinical Case and Follow‐Up of Babesia banethi Infection in a Red Fox

Article References: Clinical Case and Follow‐Up of Babesia banethi Infection in a Red Fox. (n.d.). https://doi.org/10.1002/vms3.71184

Image Credits: AI Generated

DOI: 10.1002/vms3.71184

Keywords: Babesia banethi, babesiosis, red fox, Vulpes vulpes, tick-borne pathogens, piroplasmids, wildlife medicine, imidocarb dipropionate, atovaquone-proguanil, azithromycin, regenerative anaemia, Italy