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