The brown dog tick, commonly called R. sanguineus s.l., has a global distribution and is primarily associated with domestic dogs, although it has also been found on wildlife and horses and, rarely, on people. The species complex can carry bacteria, protozoa, fungi, nematodes and viruses associated with disease in animals and humans. Mitochondrial markers have distinguished five lineages within the complex, including a tropical lineage distributed mainly in tropical regions and a temperate lineage found more often in cooler zones, although both can circulate in some countries. Before this investigation, only the tropical lineage had been documented in Colombia. Its close association with dogs creates opportunities for encounters with people and other domestic animals, making characterization of the tick microbiome relevant to veterinary and public-health surveillance.

For the analysis, researchers collected 20 ticks in Yopal, Casanare, including nine females and 11 males, and 18 in Puente Nacional, Santander, including eight females and 10 males. Ticks were removed manually from clearly tick-caused lesions and preserved in a DNA/RNA shielding solution. Researchers used taxonomic keys to identify them morphologically as members of the tropical R. sanguineus s.l. lineage. One longitudinal half of every tick was washed in phosphate-buffered saline, and DNA was extracted with a commercial tissue kit, with the disruption step extended to 12 hours at 56 °C. DNA concentration was measured with a fluorometer and quality was evaluated by agarose-gel electrophoresis. Each sample was sequenced on an Illumina NovaSeq 6000 with paired-end 150-base reads, producing approximately 6 gigabytes of raw data per tick.

After quality control and adapter removal, the investigators filtered reads with minimum length of 150 bases, average quality of 20 and trailing-quality threshold of 20. Host-associated reads were removed by alignment to the available R. sanguineus s.l. genome, and remaining reads were assigned taxonomically with Centrifuge and Kraken reporting tools. Pathogen and endosymbiont screening used a custom database containing 2,153 relevant genomes, from which 524 nonrelevant entries were removed. The retained database included genomes assigned to Anaplasma, Rickettsia, Francisella, Ehrlichia, Borrelia, Coxiella, Babesia, Theileria and selected endosymbionts. Wilcoxon tests compared read abundance by sex and department, while Spearman rank correlations with Benjamini-Hochberg correction assessed relationships among the ten most abundant pathogens and endosymbionts. Separate analyses searched for virulence-associated sequences and antibiotic-resistance markers.

Host sequences represented a mean of 88.5% of reads, with a standard deviation of 6%. Among the remaining reads, an average of 19% was classified, with a standard deviation of 5%, leaving about 81% unclassified. Bacteria accounted for most classified sequences, ranging from 3% to 24%, while archaea represented 0.07% to 0.09% and viruses 0.02% to 0.04%. Coxiellaceae was the predominant bacterial family in every sample, and a Coxiella endosymbiont contributed between 48% and 95% of classified bacterial reads. Other detected genera included Bacillus, Clostridium, Pseudomonas, Rickettsia, Streptomyces, Propionibacterium, Burkholderia, Fusobacterium, Lactobacillus and Campylobacter. The broad microbial composition was similar across departments and sexes, although individual genera differed significantly. Clostridium, Fusobacterium and Rickettsia were more abundant in Santander, while Corynebacterium and Propionibacterium were more abundant in Casanare.

The three leading pathogen signals were F. tularensis, A. phagocytophilum and T. equi, with mean relative abundances of 23%, 7% and 7%, respectively. The standard deviations were 8, 1 and 3 percentage points. The three were generally more abundant in Santander than in Casanare. Within Casanare, male ticks carried significantly higher relative abundances of F. tularensis and T. equi than females, while the same pattern in Santander was not statistically significant. A. phagocytophilum showed the reverse pattern in Santander, where females had significantly higher abundance. Other pathogens in the genera Babesia, Ehrlichia, Borrelia and Coxiella occurred at lower levels, ranging from 2% to 0.02%, and additional species had relative abundances below 0.02% and very few reads. F. tularensis causes tularemia, A. phagocytophilum causes human granulocytic anaplasmosis and anaplasmosis in animals, and T. equi is associated with equine piroplasmosis. Detection of their DNA does not, however, demonstrate vector competence, and metagenomic screening may have difficulty distinguishing F. tularensis from Francisella endosymbionts.

C. mudrowiae accounted for a mean of 47% of reads assigned against the endosymbiont database, with a standard deviation of 18 percentage points. Other endosymbionts included Wolbachia, Coxiella-like and Rickettsia endosymbionts, but their relative abundances ranged from approximately 0.02% to 3%. C. mudrowiae was more abundant in Casanare than in Santander, whereas most other endosymbionts were more abundant in Santander. It was also negatively associated with 11 organisms: three other endosymbionts and eight pathogens. Most other pairs showed strong positive correlations within or between the two ecological groups. These correlations describe patterns in read abundance and do not establish the mechanisms responsible. The inverse relationship is consistent with several possible interactions, including competition for nutrients or genomic resources, but the study did not test whether C. mudrowiae restricts pathogens or whether both respond to shared environmental and host conditions.

The viral component was small but included several families. Reads were assigned to Baculoviridae, Myoviridae and Siphoviridae, with Baculoviridae dominating the viral signal and most of its reads assigned to the genera Alphabaculovirus and Betabaculovirus. These genera contain nucleopolyhedroviruses associated with lepidopteran insects. Other viral families and genera were represented by very few reads, making precise taxonomic assignment difficult. The results therefore show that viral sequences were recoverable from the tick samples, but they do not demonstrate active viral replication, persistence or transmission in R. sanguineus s.l. Archaea likewise accounted for only 0.07% to 0.09% of classified sequences. The detected families included Methanobacteriaceae, Methanocaldococcaceae, Methanosarcinaceae, Thermoplasmataceae and Sulfolobaceae. The authors described this as the first metagenomic identification of archaea in this tick species, while emphasizing that their biological role in ticks remains unclear.

Sample-by-sample assembly and genome binning produced 27 medium-quality C. mudrowiae MAGs, with completeness ranging from 87.21% to 89.44% and contamination from 0.58% to 1.2%. Taxonomic assignments were checked with pubMLST. Four assemblies were unusually long and contained many genes absent from the other MAGs and the two available C. mudrowiae genomes; one contained 2,907 unique genes, largely annotated as hypothetical proteins, and no plasmids were identified. Because these outliers distorted the shared and variable genome estimates, the researchers excluded them from a second pangenome analysis. That analysis identified a core genome of 1,089 genes, or 41% of the pangenome, and an accessory genome of 1,574 genes, or 59%. The recovered MAGs clustered separately from the two previously available genomes in core-genome and single-nucleotide-polymorphism analyses. However, average nucleotide identity exceeded 95%, and a phylogeny based on 40 conserved proteins grouped the new assemblies with C. mudrowiae rather than with other Coxiella endosymbionts or C. burnetii.

Functional annotation showed that the MAGs contained complete pathways for synthesizing biotin, or vitamin B7, and riboflavin, or vitamin B2, as well as the cofactors coenzyme A and flavin adenine dinucleotide. These capabilities are consistent with a proposed role in supplying nutrients that may be scarce in a tick’s blood meals. The genes panE and panD, associated with folic-acid and vitamin B6 biosynthesis, and phoA were not identified, although related pathways have been reported in other Coxiella endosymbionts and C. burnetii. Comparisons showed low protein similarity between the Colombian MAGs and C. burnetii, with phylogenetic separation of the species. The study could not determine how these differences affect protein activity or the endosymbiont’s contribution to tick nutrition, development, reproduction or pathogen acquisition. It also identified resistance markers at much higher levels than virulence-associated reads, with five markers accounting for approximately 80% to 90% of the resistance-marker signal, but the analysis did not establish that the markers were expressed or conferred resistance in the ticks.

Together, the results provide a metagenomic baseline for the tropical R. sanguineus s.l. complex in two Colombian departments while exposing important interpretive limits. Host DNA dominated each library, approximately 81% of non-host reads remained unclassified, and reference genomes were unavailable or inadequate for some organisms. The sample was limited to 38 adult ticks removed from dogs in two municipalities, and the detected pathogens and endosymbionts were not experimentally tested for replication, transmission or pathogenic effects. The negative correlations involving C. mudrowiae therefore require biological validation rather than being treated as evidence of pathogen suppression. The study’s clearest contribution is the simultaneous documentation of pathogen signals, a diverse microbiome and multiple C. mudrowiae genomes in Colombian tropical-lineage ticks. It also identifies priorities for further work: targeted confirmation of F. tularensis and related Francisella endosymbionts, long-term surveillance of F. tularensis, A. phagocytophilum and T. equi, functional testing of the endosymbiont’s metabolic and immune interactions, and broader sampling of the tick complex within Colombia.

Subject of Research: Shotgun metagenomic profiling of pathogens, viruses and bacterial endosymbionts in Colombian tropical-lineage Rhipicephalus sanguineus s.l. ticks.

Article Title: Metagenomic exploration of endosymbionts and pathogens in the tropical lineage of Rhipicephalus sanguineus sensu lato (s.l.) ticks in Colombia

Article References: Paez-Triana, L., Herrera, G., Vega, L., García-Corredor, D., Pulido Medellín, M. O., Paniz-Mondolfi, A., Muñoz, M., & Ramírez, J. D. (2023). Metagenomic exploration of endosymbionts and pathogens in the tropical lineage of Rhipicephalus sanguineus sensu lato (s.l.) ticks in Colombia. Metabarcoding and Metagenomics, 7, Article e109085. https://doi.org/10.3897/mbmg.7.109085

Image Credits: AI Generated

DOI: 10.3897/mbmg.7.109085

Keywords: shotgun metagenomics, brown dog tick, Rhipicephalus sanguineus, tick microbiome, Coxiella mudrowiae, tick-borne pathogens, Francisella tularensis, Anaplasma phagocytophilum, Theileria equi, bacterial viruses, Colombia, Metagenomic

The study, published in the open-access journal Parasites & Vectors, was led by Artharee Rungrojn of the Mahidol Oxford Tropical Medicine Research Unit at Mahidol University in Bangkok, working with colleagues from Chulalongkorn University, Thailand’s Veterinary Research and Development Center, the French National Centre for Scientific Research, and the University of Oxford. The team collected 131 brown dog ticks from dogs across five Thai provinces, deliberately choosing sampling sites that spanned different geographical regions and contrasting ecological settings, from dense urban environments to rural landscapes. This design allowed them to ask a deceptively simple question with powerful implications: which forces, environmental or host-related, actually shape the community of bacteria that lives inside a tick?

To answer it, the researchers turned to a technology that has been transforming microbiome science. Rather than using conventional short-read sequencing, which reads only fragments of a gene and often leaves bacterial species ambiguously identified, they employed Oxford Nanopore Technology to sequence the full-length bacterial 16S rRNA gene. This gene, present in all bacteria, serves as a molecular barcode, and reading it in its entirety allows far more precise identification of which bacterial genera and species are present. For a vector like R. linnaei, where distinguishing a harmless symbiont from a dangerous pathogen matters enormously, that extra resolution is not a luxury but a necessity.

The headline finding concerns a single dominant organism. Candidatus Coxiella mudrowiae, a bacterium related to the genus that includes the agent of Q fever, was detected in every single tick examined. This kind of universal presence is the signature of a vertically transmitted symbiont, a microbe passed from mother to offspring through the generations rather than picked up from the environment. Such symbionts are increasingly recognized as essential partners in tick biology, often supplying vitamins and other nutrients that the tick’s blood-only diet lacks. The complete dominance of the R. linnaei microbiome by this one bacterium suggests a deep, ancient co-evolutionary relationship that persists regardless of where the ticks live.

Yet beneath that shared foundation, the researchers found substantial variation, and the drivers of that variation were unambiguous. Using a battery of statistical tools, including alpha diversity metrics that measure how many bacterial types are present, Bray-Curtis dissimilarities that quantify how different communities are from one another, principal coordinates analysis to visualize those differences, and PERMANOVA to test whether the patterns are statistically significant, the team showed that geography, habitat type, and blood-feeding status all left clear fingerprints on the tick microbiome. Ticks from different regions of Thailand harbored measurably different bacterial communities, and so did ticks from urban versus rural settings.

One of the most intriguing results concerns the urban-rural divide. Ticks collected in cities showed lower bacterial diversity than their rural counterparts. This pattern echoes findings from human microbiome research, where urban lifestyles and environments are repeatedly associated with reduced microbial diversity, but seeing the same signature in a tick population raises fascinating ecological questions. Urban ticks may encounter fewer environmental microbes, feed on dogs with different health and treatment profiles, or experience pressures such as acaricide exposure and habitat fragmentation that prune their microbial communities. Whatever the mechanism, the implication is that urbanization does not just change the landscape a tick inhabits; it changes the microscopic ecosystem the tick carries within it.

Blood-feeding status told a complementary story. Ticks that had engorged themselves with a blood meal showed altered microbial diversity compared with ticks that were still actively feeding or unfed. A blood meal is a transformative event in a tick’s life, triggering physiological changes, immune responses, and digestive activity, and the new data suggest it also reshuffles the bacterial residents of the tick’s body. This has direct relevance to disease transmission, because the ability of a tick to acquire, maintain, and pass on a pathogen, its so-called vector competence, is increasingly understood to depend on the microbial community that competes with or facilitates that pathogen. A feeding-induced shift in the microbiome could therefore influence whether a tick that picks up an infection while biting one host successfully transmits it to the next.

By contrast, the effects of tick sex and developmental stage were less pronounced, a somewhat surprising result given that these factors have been shown to matter in other tick species. The researchers also used ANCOM-BC2, a modern differential abundance method designed to handle the compositional quirks of microbiome data, to pinpoint specific bacterial taxa whose numbers rose or fell in association with ecological and host-related factors. Among the low-frequency taxa they detected were putative tick-associated pathogens, including species of Anaplasma and Ehrlichia, two genera of intracellular bacteria responsible for significant disease in dogs and, in some cases, in humans. Their detection at low frequency underscores both the value of sensitive full-length sequencing and the importance of continued surveillance in a region where tick-borne disease burden remains incompletely characterized.

The significance of this work extends beyond Thailand’s borders. R. linnaei belongs to the broader brown dog tick complex, a group of morphologically similar species whose taxonomy has only recently been untangled with molecular tools, and whose members transmit pathogens such as Rickettsia, Ehrlichia, and Babesia worldwide. Understanding what shapes the microbiome of this vector in Southeast Asia, a region of intense biodiversity, rapid urbanization, and close human-animal contact, provides a baseline for predicting how tick-borne disease risk might shift as landscapes change. If urban environments consistently simplify tick microbiomes, and if that simplification affects pathogen dynamics, then cities may be quietly engineering new patterns of disease risk that current surveillance systems are not designed to detect.

Methodologically, the study also makes a case that will resonate with microbiome researchers everywhere: full-length 16S rRNA sequencing on Nanopore platforms is a practical and powerful approach for vector microbiome investigations. The work emerged from Rungrojn’s doctoral research at Mahidol University and received support from the Wellcome Trust, MOTIP, and the Royal Society of Tropical Medicine and Hygiene, reflecting the growing international investment in understanding the hidden microbial dimensions of vector-borne disease. As sequencing technology becomes faster and cheaper, studies of this kind could move from snapshot surveys to longitudinal monitoring, tracking how tick microbiomes respond seasonally and as cities expand. For now, the message from Thailand’s dogs and their tiny passengers is clear: the microbes inside a tick are not a fixed inheritance but a living community, responsive to geography, habitat, and the simple act of taking a blood meal, and that responsiveness may hold keys to controlling the diseases these ticks spread.

Subject of Research: Environmental and host-associated factors shaping the bacterial microbiome of the brown dog tick Rhipicephalus linnaei in Thailand

Article Title: Environmental and host-associated determinants of microbiome variation in the brown dog tick Rhipicephalus linnaei in Thailand

Article References: Rungrojn, A., Chaisiri, K., Thaipadungpanit, J., Batty, E. M., Taweethavonsawat, P., Thempachana, O., Kongkaew, W., Morand, S., & Blacksell, S. D. (2026). Environmental and host-associated determinants of microbiome variation in the brown dog tick Rhipicephalus linnaei in Thailand. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07656-y

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07656-y

Keywords: Rhipicephalus linnaei, brown dog tick, microbiome, 16S rRNA, Oxford Nanopore sequencing, Candidatus Coxiella mudrowiae, Thailand, tick-borne pathogens, Anaplasma, Ehrlichia, urban ecology, vector competence

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: Carbonara, M., Prioletti, M., Camarda, A., Castellana, S., Menga, G., Girardi, G., Baneth, G., & Otranto, D. (2026). Clinical Case and Follow‐Up of Babesia banethi Infection in a Red Fox. Veterinary Medicine and Science, 12(5), Article e71184. 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

Haemaphysalis ticks belonging to the subgenus Kaiseriana are recognized as important vectors of tick-borne pathogens throughout tropical Asia. Yet, despite their medical and veterinary significance, data on which tick species infest captive wildlife in East Malaysia have remained scarce. Captive animals such as Sambar deer, a large cervid native to South and Southeast Asia, can act as amplifying hosts for ticks and the agents they transmit, and facilities like bioparks and teaching farms create conditions in which ticks, wildlife, livestock, and people come into close and repeated contact. Understanding exactly which tick species are present, and which pathogens they harbor, is therefore a prerequisite for any meaningful risk assessment. The new study set out to close that gap by combining classical morphological identification with DNA-based species confirmation and pathogen screening.

Between 2021 and 2022, the researchers collected a total of 60 ticks from five Sambar deer at the two Malaysian sites. Each specimen was first identified morphologically using standard taxonomic keys, an approach that relies on subtle differences in body structures such as the palps, scutum, and spiracular plates. The team then attempted to extract DNA from 49 of the specimens. Here the study encountered a practical constraint familiar to anyone working with field-collected arthropods: specimen degradation. Because the ticks had been preserved and stored under conditions that compromised their genetic material, only seven samples retained DNA of sufficient quality and quantity for downstream molecular analysis. Rather than discarding the morphological work, the researchers treated the seven genetically tractable ticks as a focused molecular window onto the broader collection.

For species identification, the team sequenced the mitochondrial cytochrome c oxidase subunit I gene, known as cox1, a workhorse marker in animal DNA barcoding. The resulting sequences were compared against reference databases and subjected to phylogenetic analysis using maximum likelihood methods, which infer evolutionary trees by finding the branching pattern that best explains the observed genetic differences. Two Haemaphysalis species emerged with strong statistical support. Sequences assigned to Haemaphysalis shimoga showed 97.88 to 99.27 percent identity to reference material, while those assigned to Haemaphysalis bispinosa were even more convincing at 99.5 to 100 percent identity. Crucially, each species resolved in its own well-supported monophyletic clade on the phylogenetic trees, meaning that all sequences of each species descended from a common ancestor to the exclusion of the other, a pattern that reinforces the species-level assignments.

The identification of H. shimoga in Malaysia is particularly noteworthy. The taxon was originally described from southern India as a subspecies of Haemaphysalis cornigera, and its presence on Sambar deer in Malaysian Borneo extends the known geographic footprint of this tick considerably. H. bispinosa, by contrast, has a long and somewhat complicated history in the region. Work dating back to the late 1960s argued, on biogeographic grounds, that H. bispinosa should be considered an introduced species in the Malay Peninsula and Borneo, likely arriving with domesticated animals. Its confirmation here on captive Sambar deer, at sites on both sides of the country, is consistent with that picture of a tick that thrives wherever its preferred hosts and suitable climatic conditions coincide, including in managed wildlife settings.

With the tick species confirmed, the researchers turned to the pathogens. They screened the seven molecularly characterized specimens for bacteria of the genus Anaplasma using sequencing of the 16S ribosomal DNA gene, and for protozoan parasites of the genus Theileria using the 18S ribosomal DNA gene. Both gene targets are standard markers for detecting and differentiating these organisms, and the resulting sequences were again placed into phylogenetic frameworks by maximum likelihood analysis. The results were remarkable for a sample set of just seven ticks. Five of the seven, comprising four H. shimoga and one H. bispinosa, were infected with Theileria species. Sequences matched Theileria capreoli at 98.83 to 100 percent identity and Theileria cervi at 95.07 to 99.74 percent identity, two protozoans associated with cervid hosts in various parts of the world.

The single H. bispinosa specimen proved to be the study’s most consequential finding. It tested positive for Anaplasma phagocytophilum, with sequence identities of 99.53 to 100 percent, and it simultaneously harbored both Theileria cervi and Theileria capreoli, making it a documented triple co-infection in a single tick. Anaplasma phagocytophilum is the agent of human granulocytic anaplasmosis as well as tick-borne fever in ruminants, and it is regarded as a widespread, multi-host pathogen with highly adaptive strategies that allow it to persist across ticks, wild and domestic mammals, and occasionally humans. Its detection in a Haemaphysalis tick infesting captive deer in Malaysia raises the possibility that the ecological circuitry needed to maintain this bacterium, competent tick vectors and susceptible mammalian hosts in close proximity, is already in place in the country.

Co-infections of this kind are increasingly understood to be the rule rather than the exception in tick microbiology. Individual ticks frequently carry multiple pathogens at once, and mixed infections can influence disease severity in hosts, complicate diagnosis, and alter transmission dynamics in ways that single-pathogen models fail to capture. The Malaysian finding, in which one tick carried two protozoan parasites and one bacterial pathogen simultaneously, is a vivid local illustration of that global pattern. It also underscores why molecular screening of even small numbers of well-characterized ticks can be informative: morphological identification alone would have revealed which tick species were present, but only DNA sequencing exposed the pathogen community hiding inside them.

The study’s authors are careful about the limits of their data. With only seven specimens yielding usable DNA, the prevalence estimates that could be calculated are statistically fragile, and the degraded condition of most of the collection means the true infection rates among the 60 ticks remain unknown. Detection of pathogen DNA in a tick also does not by itself demonstrate that the tick can biologically transmit the agent, nor does it confirm that the deer were infected. Those questions would require blood testing of the hosts, experimental vector competence studies, and larger, systematically designed tick surveys. What the study does establish, firmly, is a set of firsts: the first molecular confirmation of H. shimoga and H. bispinosa on captive Sambar deer in Malaysia, and the first molecular detection of A. phagocytophilum alongside Theileria co-infection in ticks from the country.

The practical implications extend in several directions. For wildlife managers and veterinarians responsible for captive deer, the results argue for routine tick surveillance and acaricide-based control at facilities where animals are held in semi-natural enclosures. For public health authorities, the presence of A. phagocytophilum DNA in a local tick vector warrants attention, particularly given that people working at bioparks, farms, and slaughterhouses occupy exactly the interfaces where tick bites are most likely. The researchers have deposited all their sequence data in GenBank, making the cox1, 16S, and 18S sequences available for future comparative studies across Southeast Asia. As climate change, land-use change, and the wildlife trade continue to reshuffle the distributions of ticks and their hosts in the tropics, baseline datasets of this kind, anchored in both morphology and molecular phylogenetics, will become only more valuable for tracking how tick-borne pathogen landscapes evolve in Malaysia and beyond.

Subject of Research: Molecular identification of Haemaphysalis tick species and detection of Anaplasma and Theileria co-infections in captive Sambar deer in Malaysia

Article Title: First Molecular Report of Haemaphysalis shimoga and Haemaphysalis bispinosa Infesting Captive Sambar Deer (Rusa unicolor) in Malaysia, with Detection of Anaplasma phagocytophilum and Theileria Co-infection

Article References: Altwaim, S. A., Kamaludeen, J., Mohammed, M. A., Suif, Z., Mustafa, S., Paul, B. T., Syed-Hussain, S. S., Aziz, N.-A. A., Numan, M., & Ali, A. (2026). First Molecular Report of Haemaphysalis shimoga and Haemaphysalis bispinosa Infesting Captive Sambar Deer (Rusa unicolor) in Malaysia, with Detection of Anaplasma phagocytophilum and Theileria Co-infection. Acta Parasitologica, 71(5), Article 212. https://doi.org/10.1007/s11686-026-01392-0

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01392-0

Keywords: Haemaphysalis shimoga, Haemaphysalis bispinosa, Sambar deer, Anaplasma phagocytophilum, Theileria capreoli, Theileria cervi, tick-borne pathogens, co-infection, Malaysia, cox1 barcoding, tick surveillance, captive wildlife

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