The rationale for targeting mucins rests on their central role in tick biology. Mucins are heavily glycosylated proteins that, in ticks, contribute to the formation of the cement cone that anchors the mouthparts into the host skin during feeding, help modulate the host immune response at the bite site, and participate in assembling the peritrophic membrane that lines the tick gut and protects it from ingested blood and microbes. Because these functions are essential to feeding and survival, proteins involved in them are attractive vulnerabilities: antibodies circulating in the host’s blood would be ingested by the tick and could interfere with these processes from within the parasite.

Before committing to laboratory production, the team characterized Mucin-6 using bioinformatic tools. The predictions indicated a hydrophilic but intrinsically unstable protein with a molecular mass of 24.9 kilodaltons after removal of its signal peptide, the short N-terminal sequence that normally directs secretion. Importantly for vaccine design, the sequence was predicted to be rich in linear B-cell epitopes, the short stretches of amino acids that antibodies can recognize on a denatured or soluble protein. Abundant epitopes increase the likelihood that vaccination will elicit a robust and specific humoral response, which is the arm of immunity most relevant for an antigen that will be encountered by the tick through a blood meal.

To confirm that the gene is biologically relevant throughout the tick life cycle, the researchers used quantitative PCR to measure Mucin-6 expression across developmental stages and feeding states. The gene was transcribed at every stage examined, from larva to adult, with significantly higher expression in eggs. This broad and consistent expression profile matters for vaccine utility: an antigen expressed only in a narrow stage or feeding condition would leave windows in which the parasite is unprotected, whereas a constitutively expressed protein offers a target throughout the tick’s interaction with the vaccinated host.

With the sequence validated, the team moved to recombinant production. The Mucin-6 coding sequence was cloned into the pET28a expression vector and transformed into BL21(DE3) Escherichia coli, a standard laboratory strain engineered for high-level protein expression under T7 promoter control. On induction, the recombinant protein accumulated in inclusion bodies, the dense aggregates of misfolded protein that bacteria often form when expressing foreign or disulfide-rich proteins. The researchers recovered the protein from these bodies, renatured it to restore a soluble conformation, and purified it using nickel-nitrilotriacetic acid affinity chromatography, which exploits the histidine tag encoded by the pET28a vector. On SDS-PAGE, the purified recombinant Mucin-6 migrated at approximately 41 kilodaltons, larger than the predicted 24.9 kilodaltons of the mature protein, a discrepancy consistent with the tag and the anomalous electrophoretic behavior typical of mucin-like, compositionally biased proteins.

The immunization trial used six New Zealand White rabbits randomly assigned to a vaccinated group of three and a phosphate-buffered saline control group of three. Each vaccinated rabbit received 500 micrograms of recombinant Mucin-6 formulated with Freund’s adjuvant in three subcutaneous injections spaced 14 days apart. Enzyme-linked immunosorbent assays tracked the antibody response, revealing titers that reached 1:512,000 by day 35, an exceptionally high level indicating strong recognition of the recombinant antigen. Fourteen days after the final booster, the researchers challenged each rabbit with 30 adult ticks, 15 males and 15 females, and measured the parameters that define tick vaccine efficacy: engorgement weight, egg mass, blood-feeding duration and egg-hatching rate.

The results showed a consistent, statistically significant burden on the parasites. Ticks that fed on immunized rabbits had engorged body weights reduced by 13.6 percent and egg masses reduced by 14.7 percent compared with ticks fed on control rabbits, both differences significant at P less than 0.05. Blood-feeding duration was prolonged by 8.3 percent, suggesting that antibodies disrupted the efficiency of feeding, forcing ticks to remain attached longer to obtain a full blood meal. Combining the effects on tick weight and fecundity, the authors calculated an overall vaccine efficacy of 26.3 percent. While modest compared with the best commercial anti-tick formulations, this figure represents a meaningful proof of concept for a single antigen tested in its first host species.

A critical question for any recombinant antigen is whether antibodies raised against the laboratory-produced protein also recognize the native version in the parasite. The team addressed this by western blot, incubating tick lysates with serum from the immunized rabbits. The anti-rMucin-6 serum bound native Mucin-6 in tick extracts, confirming that the recombinant protein preserved epitopes present in the authentic tick protein and that the antibodies generated in the vaccinated animals could, in principle, engage their target during a natural blood meal. This cross-recognition underpins the observed phenotypic effects and strengthens the case that Mucin-6, rather than an artifact of immunization, is the operative antigen.

The study sits within a broader effort to develop anti-tick vaccines that reduce both parasite loads and pathogen transmission without the drawbacks of chemical acaricides. The only commercially available tick vaccine, based on the gut antigen Bm86 against Rhipicephalus microplus, demonstrated decades ago that vaccinating the host can starve and sterilize ticks from within, but its efficacy varies across tick species and regions, driving the search for new antigens. Secreted proteins such as mucins, which are exposed to host antibodies in the ingested blood and are functionally indispensable, form a promising class of candidates. The Hainan team’s work adds Mucin-6 to this pipeline and demonstrates a complete workflow, from epitope prediction and expression profiling through prokaryotic production and a controlled animal challenge.

Limitations remain before Mucin-6 could approach practical deployment. The trial involved a small number of animals, a single antigen and a single host species, and Freund’s adjuvant is unsuitable for veterinary or human use, so future work would need to test the antigen with licensed adjuvants, in larger cohorts, and ideally in combination with other antigens to push efficacy higher. The authors, led by corresponding author Qingfeng Guan with co-first authors Jie Chen and Yongchuan Zhu, conclude that Mucin-6 merits further evaluation as a candidate vaccine antigen against R. linnaei. Given that tick-borne diseases continue to expand their geographic range with changing climates and livestock movements, even incremental gains from antigen discovery of this kind carry substantial implications for animal health and the economics of tick control.

Subject of Research: Evaluation of the tick protein Mucin-6 from Rhipicephalus linnaei as a recombinant anti-tick vaccine antigen

Article Title: Prokaryotic expression and vaccine potential of Mucin-6 from Rhipicephalus linnaei

Article References: Chen, J., Zhu, Y., Zhao, J., Wang, J., Han, Q., & Guan, Q. (2026). Prokaryotic expression and vaccine potential of Mucin-6 from Rhipicephalus linnaei. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07675-9

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07675-9

Keywords: Rhipicephalus linnaei, Mucin-6, anti-tick vaccine, recombinant protein, prokaryotic expression, vaccine efficacy, tick control, humoral immunity, B-cell epitopes, Parasites & Vectors, livestock health, vector biology

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