The research, led by Mariachiara Vardeu and Elena Ghiorzi of the University of Padua together with colleagues at the University of Pavia, was conceived around a central weakness in the existing literature: most tick microbiome studies choose a decontamination method by convention rather than by evidence. Ethanol washes, bleach treatments, and commercial DNA-degrading reagents all appear in published workflows, but until now there has been no direct, controlled comparison of how effectively each removes surface-associated bacteria while preserving the tick’s internal microbial community. Without such a benchmark, differences between laboratories could reflect cleaning chemistry as much as genuine biology, undermining efforts to compare microbiome data across studies, regions, or tick populations.

To build that benchmark, the team designed an elegant experimental trick. Rather than relying on the unknown and variable bacteria naturally present on wild-caught ticks, they deliberately contaminated adult Ixodes ricinus ticks and nymphs with a defined bacterial mock community of known composition. This meant that the researchers knew exactly which bacterial sequences represented surface contamination and could track, with precision, how much of that signal survived each cleaning treatment. Any mock-community reads remaining after decontamination were, by definition, residual surface contamination; any reduction in those reads measured the effectiveness of the protocol. It is a simple idea in principle, but one that transforms decontamination from a matter of faith into a measurable quantity.

The contaminated ticks were then subjected to five decontamination regimens: a wash in 70% ethanol, the traditional mainstay of tick microbiome studies; a 1% bleach treatment; DNA/RNA Zap, a commercial reagent that degrades nucleic acids on external surfaces; and two combination protocols in which a lysozyme pre-treatment, designed to weaken bacterial cell walls, was followed by either bleach or DNA/RNA Zap. Mock-inoculated ticks that received no decontamination served as controls. After cleaning, the researchers characterized the microbial communities using Oxford Nanopore full-length 16S rRNA gene sequencing, a platform that reads the entire bacterial barcoding gene rather than short fragments, providing higher taxonomic resolution for identifying which bacteria survived which treatment.

The results were striking in their clarity. Ethanol, the most widely used decontamination agent in the field, consistently showed the weakest effect, leaving substantial mock-community signal intact and suggesting that many published tick microbiome profiles built on ethanol-washed specimens may carry a meaningful contribution from surface bacteria. By contrast, both bleach and DNA/RNA Zap, whether applied alone or preceded by lysozyme, produced near-complete depletion of the mock-community reads. In other words, the two harsher chemical approaches came closest to erasing the experimental contamination signal, while the gentler ethanol wash barely dented it. Perhaps unexpectedly, the enzymatic lysozyme pre-treatment conferred no measurable advantage over bleach or DNA/RNA Zap used on their own, a finding that simplifies protocol design and removes an unnecessary step from future workflows.

Effectiveness at removing contaminants is only half the story, however. An ideal decontamination protocol must also preserve the tick’s genuine internal microbiota, including endosymbionts that play roles in tick nutrition and reproduction, and any pathogens the tick may harbor. To probe this dimension, the team compared sequencing results from tick legs, which are dominated by surface-associated microbes, with leg-free bodies, which contain the internal organs and their resident communities. This anatomical dissection revealed that Brevibacterium, a bacterial genus frequently reported in tick studies, was predominantly surface-associated in this system, raising the possibility that some taxa previously interpreted as tick symbionts may in fact be exoskeletal passengers. Meanwhile, the endosymbiont Candidatus Midichloria, a bacterium closely associated with Ixodes ticks, remained readily detectable in leg-free bodies even after decontamination, indicating that the effective treatments stripped the surface without erasing the tick’s true internal signature.

The preservation of pathogen signals was equally reassuring. In nymphs that had been experimentally infected with Borrelia, the causative agent of Lyme disease, Borrelia-associated sequences remained readily detectable following surface treatment. This matters enormously for disease surveillance, because ticks are routinely screened for Borrelia and other pathogens using molecular methods, and a decontamination protocol aggressive enough to destroy pathogen DNA would compromise those assays. The study’s findings suggest that bleach- and DNA/RNA Zap-based protocols occupy a valuable middle ground: strong enough to eliminate exoskeletal contamination, yet gentle enough to leave internal microbial and pathogen signals intact and quantifiable.

Beyond the head-to-head protocol comparison, the study also explored how decontamination choice shapes community-level diversity analyses, the statistical frameworks ecologists use to compare microbiomes across samples. These analyses suggested treatment-associated differences in the bacterial profiles recovered from the ticks, hinting that laboratories using different cleaning methods may not be sampling quite the same biological reality. The authors are careful to frame these diversity patterns as exploratory rather than definitive, but the implication is clear: decontamination is not a neutral technical detail but an active variable that can shift the apparent composition of a tick’s microbiome, potentially confounding meta-analyses that pool data from studies using different protocols.

The authors are equally candid about the limits of their benchmark. The mock-community approach, while powerful, was tested under controlled laboratory conditions with a defined and relatively simple surface community. Real ticks collected in the field carry more diverse and unevenly distributed microbial loads, and different tick species present different exoskeleton chemistries and sizes that could influence how reagents penetrate and perform. Validation using field-collected ticks, additional tick species, more complex surface-associated microbial communities, and independent laboratory settings will be required before the community can propose broader methodological standardization. The researchers also emphasize that the optimal decontamination strategy may depend on the biological compartment and microbial fraction of interest, meaning that a one-size-fits-all protocol may never exist.

Nevertheless, the study delivers an immediately actionable message for anyone working on tick microbiomes or tick-borne pathogen surveillance. Under the conditions tested, bleach and DNA/RNA Zap produced the strongest reduction in experimentally introduced surface bacteria while preserving dominant tick-associated taxa and pathogen sequences, whereas ethanol, the field’s default choice, performed worst. Lysozyme pre-treatment added nothing measurable. For a research community racing to understand how tick microbiomes influence vector competence and disease transmission across a changing European landscape, this quantitative framework arrives at an opportune moment. By making surface decontamination a benchmarked, comparable step rather than an unexamined habit, the Padua-led team has taken a meaningful step toward microbiome data that can be trusted, compared, and built upon, and toward a clearer picture of what really lives inside one of Europe’s most medically important arthropods.

Subject of Research: Benchmarking surface decontamination protocols for tick microbiome profiling using 16S rRNA sequencing

Article Title: Benchmarking surface decontamination methods for reliable Ixodes ricinus microbiome profiling

Article References: Benchmarking surface decontamination methods for reliable Ixodes ricinus microbiome profiling. (n.d.). https://doi.org/10.1186/s13071-026-07726-1

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07726-1

Keywords: Ixodes ricinus, tick microbiome, surface decontamination, 16S rRNA sequencing, Oxford Nanopore, mock community, Borrelia, Midichloria, Brevibacterium, Lyme disease, vector competence, Parasites & Vectors

The motivation for the study rests on a growing recognition that Bartonella, Borrelia, and piroplasmid parasites circulate widely among arthropod vectors and animal reservoirs in Brazil, and that people with weakened immune systems may be particularly vulnerable to their consequences. Bartonella species are fastidious, intracellular bacteria best known for causing cat scratch disease in immunocompetent individuals, but in patients with profound immunosuppression they have been linked to severe vascular lesions such as bacillary angiomatosis, a condition historically described in people with advanced AIDS. Borrelia species, the spirochetes responsible for relapsing fever and Lyme-like illness, and piroplasmids, the protozoan parasites that include Babesia, add further layers of diagnostic complexity because they can produce nonspecific febrile symptoms that overlap with many opportunistic infections.

To investigate these possibilities, the researchers enrolled 75 HIV-positive individuals who were symptomatic and living in Brazil, collecting detailed epidemiological histories and medical records for each participant. The team then took a two-pronged diagnostic approach. The first prong was serological: they used indirect immunofluorescence assays, or IFA, to detect IgG antibodies against six distinct Bartonella antigens, namely Bartonella henselae, Bartonella koehlerae, Bartonella quintana, and two genotypes of Bartonella vinsonii subsp. berkhoffii. All assays were performed in a blinded manner, meaning the laboratory staff did not know the clinical status of the samples they were testing, a design feature intended to reduce bias. A reciprocal antibody titer of 1:64 or greater was defined as seroreactivity, the threshold at which a sample was considered to show evidence of prior exposure.

The second prong was molecular. The researchers extracted DNA from blood samples and searched for genetic signatures of Bartonella, Borrelia, and piroplasmid organisms using both quantitative polymerase chain reaction, or qPCR, and digital PCR, or dPCR. Crucially, they did not rely on a single snapshot of the bloodstream. Because Bartonella bacteria are notorious for circulating at extremely low numbers and in a cyclic fashion, the team enriched blood cultures in Bartonella alpha-Proteobacteria Growth Medium, known as BAPGM, for up to 28 days, and then repeated the qPCR testing on the cultured material. This enrichment step is designed to amplify any sparse organisms that might be present below the detection limit of direct blood testing, making it one of the most sensitive protocols available for hunting these bacteria in human blood.

The serological results were striking. Of the 75 participants, 47, or 62.7 percent, were seroreactive to at least one Bartonella antigen. The most frequently detected antibodies were directed against B. henselae, found in 57.3 percent of the cohort, followed by B. vinsonii subsp. berkhoffii genotype II, detected in 44.0 percent. These figures suggest that exposure to Bartonella species is remarkably common among symptomatic HIV-infected individuals in this Brazilian population, a finding consistent with the documented circulation of these bacteria in fleas, ticks, and animal reservoirs across the country. Whether the antibodies reflect true past infection, cross-reactivity with other bacteria, or persistent low-level colonization that evaded molecular detection remains an open question that the authors themselves acknowledge.

Perhaps the most clinically provocative finding was the statistical association between Bartonella seroreactivity and systemic manifestations of disease. Among the 47 seroreactive patients, 11 exhibited systemic signs and symptoms, compared with only 1 of the 28 non-seroreactive patients. This difference translated into an odds ratio of 8.25, with a 95 percent confidence interval spanning 1.00 to 68.2 and a p value of 0.026. In practical terms, seroreactive patients were roughly eight times more likely to present with systemic manifestations than those without detectable antibodies, although the wide confidence interval reflects the modest sample size and signals considerable statistical uncertainty. The association was exploratory in nature, and the authors were careful to frame it as a hypothesis-generating observation rather than proof of causation.

On the molecular front, the picture was far more definitive, and far more negative. Every one of the 75 participants tested negative for Bartonella and Borrelia DNA by both qPCR and dPCR, both before and after the extended BAPGM enrichment culture period. Two participants did show a low positive partition number for piroplasmid DNA by dPCR, a result that initially raised the possibility of Babesia-like infection, but when the samples were retested the signal could not be reproduced. The authors interpreted these non-repeatable findings as most likely representing technical artifacts or contamination rather than true infection, underscoring a central principle of molecular diagnostics: a single low-level positive result, especially near the limit of detection, demands confirmation before it can be considered evidence of active disease.

The disconnect between high seroprevalence and absent molecular detection is the intellectual heart of the study, and it speaks to a broader challenge in the field of vector-borne pathogen research. Antibodies indicate that the immune system has encountered an organism at some point, but they do not distinguish between a resolved infection years ago and a persistent, sequestered infection hiding in tissue reservoirs such as the vascular endothelium, where Bartonella is known to reside. Conversely, negative blood PCR does not exclude infection in tissues, because these bacteria may only intermittently spill into the bloodstream. The BAPGM enrichment culture was designed specifically to address this intermittency, and its failure to yield any PCR-positive samples after nearly a month of incubation provides reasonably strong evidence that active bloodstream infection with these organisms was absent in this cohort at the time of testing.

The clinical implications are nuanced. On one hand, the study provides reassurance that in this population of symptomatic HIV-infected Brazilians, active Bartonella, Borrelia, and piroplasmid infections were not detectable, suggesting that these pathogens are unlikely to be a common hidden driver of unexplained symptoms in similar patients. On the other hand, the strong association between seroreactivity and systemic manifestations hints that prior Bartonella exposure might somehow shape the clinical course of HIV disease, or that both are correlated with shared risk factors such as living conditions, animal contact, or arthropod exposure that were not fully disentangled in this exploratory analysis. The authors conclude that the clinical relevance of Bartonella seroreactivity in this setting remains to be clarified, a candid acknowledgment that serology alone cannot settle the question.

Looking forward, the study lays groundwork for several important next steps. Larger cohorts with longitudinal follow-up would help determine whether seroreactive patients experience different disease trajectories over time. Tissue-based sampling, rather than blood alone, could reveal whether Bartonella organisms persist in vascular niches that blood PCR cannot reach. And the combination of dPCR technology with extended enrichment culture, as deployed here, offers a template for future investigations of other fastidious vector-borne pathogens in immunocompromised populations worldwide. For now, the message for clinicians is measured: exposure to Bartonella appears frequent among symptomatic HIV patients in Brazil, but confirmed active infection with these three pathogen groups was not found, and the meaning of a positive antibody test in this population remains an open scientific question worthy of continued investigation.

Subject of Research: Prevalence of Bartonella, Borrelia, and piroplasmid exposure and infection in symptomatic HIV-infected individuals in Brazil

Article Title: Prevalence of Bartonella spp., Borrelia spp., and piroplasmid in HIV-infected symptomatic individuals in Brazil: a cross-sectional study

Article References: Robveille, C., Sarris, A. B., Breitschwerdt, E. B., André, M. R., do Amaral, R. B., Maggi, R. G., Mussi-Pinhata, M. M., Dias, C. M., & de Carvalho Santana, R. (2026). Prevalence of Bartonella spp., Borrelia spp., and piroplasmid in HIV-infected symptomatic individuals in Brazil: a cross-sectional study. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07584-x

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07584-x

Keywords: Bartonella, Borrelia, piroplasmid, HIV, vector-borne disease, serology, immunofluorescence assay, qPCR, digital PCR, BAPGM culture, Brazil, opportunistic infection

The research team, led by Dieter J. A. Heylen of the Evolutionary Ecology Group at the University of Antwerp, together with colleagues from the Luxembourg Institute of Health and certified bird ringers in Belgium, examined 284 redwings captured during late-autumn migration in 2022 and 2024. Both years were marked by unusually mild autumn conditions, a detail that proved central to interpreting the results. Of the birds sampled, 72.9 percent were infested with ticks, a rate the authors describe as the highest ever reported for this species anywhere in Europe. The overwhelming majority of the parasites collected were Ixodes ricinus, the castor bean tick whose blood-feeding stages are responsible for transmitting the majority of tick-borne infections to humans and domestic animals across the European continent.

The scale of pathogen carriage among those ticks was equally striking. The team screened 698 ticks removed from the migrating birds and found that 12.9 percent carried Borrelia bacteria. The infections were not confined to a single lineage. Genetic screening identified multiple genospecies of the Borrelia burgdorferi sensu lato complex, including strains associated with birds and others associated with mammals, alongside Borrelia miyamotoi, a relapsing-fever spirochete increasingly recognized as a human pathogen. Perhaps most telling, infected ticks were found on nearly one-third of the birds examined, meaning that a large proportion of the migrating population was effectively functioning as a long-distance courier service for viable, pathogen-laden parasites.

Extrapolating from their field measurements to the size of the European redwing population, the researchers estimate that tens of millions of Borrelia-infected ticks may be transported southward each autumn on the backs of these migrating thrushes. Ticks attached to birds can detach at stopover sites hundreds or thousands of kilometers from where they were acquired, seeding new locations with both the arthropods themselves and the pathogens they carry. Because migratory birds travel enormous distances in short periods, they represent one of the most efficient dispersal mechanisms available to ticks, whose own locomotion over a lifetime rarely exceeds a few meters. The study thus adds a seasonal dimension to what ecologists already understood about birds as tick vectors: the phenomenon does not stop when summer ends.

The timing of the infestations is what gives the work its conceptual force. Migratory birds calibrate the onset and pace of their journeys primarily by photoperiod, the changing length of daylight, which is an astronomical cue immune to year-to-year weather. Tick activity, by contrast, is governed largely by ambient temperature, since ticks are ectotherms whose questing behavior halts when conditions become cold. Historically, late-autumn temperatures across much of western and central Europe fell low enough to push ticks into dormancy well before the main passage of redwings, minimizing the overlap between the two biological calendars. The Belgium study, conducted during two exceptionally mild Novembers, documents what happens when that buffer disappears: host and parasite schedules collide, and the birds depart with an unintended cargo.

This divergence between climatically driven parasite activity and photoperiodically driven migration is a textbook example of a phenological mismatch, and the study’s authors argue that continued climate warming is likely to widen it in the parasites’ favor. Migration timing is comparatively stable, anchored to day length, while the tick activity season expands at both ends of the year as winters warm. Each increment of autumn warming therefore extends the period during which questing ticks can board migrating hosts. The consequence, according to the researchers, is a strengthening seasonal window for long-distance parasite dispersal, one that epidemiological models of Lyme disease risk have largely failed to incorporate because they treat late autumn as an epidemiological dead zone.

The age structure of the infestations added a further layer of biological insight. Birds in their first calendar year, that is, individuals hatched the same summer and undertaking their first migration, carried significantly higher tick loads than older birds. The authors suggest several non-exclusive explanations, including the inexperience of young birds in grooming and preening parasites effectively, behavioral differences in habitat use that bring first-year birds into closer contact with questing ticks, and possible physiological differences in skin characteristics that affect how readily ticks attach and feed. Whatever the mechanism, the pattern means that a disproportionate share of the dispersing parasite burden travels on the youngest cohort, the very individuals that make up the bulk of many migratory populations and that colonize wintering areas before returning north in spring.

The pathogens detected on these birds are of more than academic concern. Borrelia burgdorferi sensu lato encompasses the genospecies responsible for the great majority of Lyme borreliosis cases in Europe, an infection that can produce erythema migrans rashes, neurological complications, arthritis and cardiac involvement when untreated. Borrelia miyamotoi, though historically overshadowed by its Lyme-causing relatives, has been implicated in human cases of a relapsing-fever-like illness and, in immunocompromised patients, severe meningoencephalitis. The identification of both avian-associated and mammalian-associated genospecies on migrating redwings underscores the role of birds in maintaining and redistributing genetically diverse pathogen reservoirs, with consequences for the local disease ecology of every stopover and wintering ground along the flyway.

It is important to note that the study does not claim that migrating birds directly infect humans. Ticks feeding on birds do not transmit Borrelia to their hosts in ways that pose immediate public-health danger at the moment of capture. The risk pathway is indirect: infected ticks detach in new environments, and if those environments support the tick’s life cycle and suitable reservoir hosts, the parasites and their pathogens can establish locally. A southbound redwing that drops an infected larva or nymph in a French hedgerow, an Iberian woodland or a Mediterranean scrubland effectively introduces Borrelia genetic material into a new ecological arena, where subsequent feeding cycles on local wildlife can amplify and maintain the pathogen. The study’s contribution is to show that this introduction channel remains open deep into the season previously considered safe.

The work also carries a methodological message for disease surveillance. Monitoring programs for tick-borne pathogens in Europe typically concentrate their efforts on spring and early summer, when human exposure risk peaks and tick activity is maximal. The Belgian findings argue for extending surveillance into the late-autumn migration window, particularly during warm years, to capture a dispersal event of continental significance. With both sampling years of the study falling among the mildest autumns on record, and with climate projections pointing toward more frequent such conditions, the overlap between southbound migration and active, infected ticks may shift from an anomaly to a routine feature of the European disease landscape. What the redwings reveal, in effect, is that the geography and seasonality of Lyme disease risk are being quietly rewritten at altitude, one migrating thrush and one hitchhiking tick at a time.

Subject of Research: Late-autumn dispersal of Borrelia-infected ticks by southward-migrating redwings and its implications for Lyme disease epidemiology under climate warming

Article Title: Lyme beyond the season: late-autumn dissemination of infected ticks by southward migrating redwings (Turdus iliacus)

Article References: Heylen, D. J. A., Ledegen, I., Grimon, M., Reteng, P., Hübschen, J. M., & Krawczyk, A. I. (2026). Lyme beyond the season: late-autumn dissemination of infected ticks by southward migrating redwings (Turdus iliacus). Parasites & Vectors. https://doi.org/10.1186/s13071-026-07673-x

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07673-x

Keywords: Lyme disease, redwing, Turdus iliacus, Ixodes ricinus, Borrelia, bird migration, tick-borne pathogens, phenological mismatch, climate change, parasite dispersal, Borrelia miyamotoi, seasonal surveillance