The investigation was conducted in January 2025 in the Altay Mountain area of Wenquan County, Xinjiang Uygur Autonomous Region, at an elevation of 2,200 metres and at coordinates 44°47′30″ N, 80°53′30″ E. Investigators collected 89 fleas from the body surfaces of 23 free-ranging sheep, taking two to six fleas from each animal. Six representative specimens—three males and three females—were selected for morphological identification. The remaining 83 fleas, all female, were processed individually for DNA extraction and pathogen screening. Three female samples were also used to confirm the flea species by sequencing the mitochondrial cytochrome c oxidase subunit II, or COII, gene.

Morphological examination supported the identification of V. alakurt. The specimens had dark-brown heads and thoraxes, developed eyes and round antennal clubs with seven segments. Their labial palpi contained 10 to 15 segments and extended beyond the forecoxae, while their hind tibiae each had six notches. Male body length ranged from 3.7 to 4.9 millimetres, and female body length ranged from 5.6 to 7.5 millimetres. Fatty tissue occupied the posterior three-quarters of the female abdomen. The COII sequences shared 99.41 percent identity across 674 of 678 base pairs with the V. alakurt reference sequence OP433454.1, providing molecular support for the morphological assignment.

Researchers used separate genetic markers to screen for the four pathogen genera: the 16S ribosomal RNA gene for Anaplasma, the Com1 gene for Coxiella, and the 18S ribosomal RNA gene for Babesia and Theileria. Each PCR run included distilled water as a negative control and positive controls from the investigators’ laboratories. Amplified products were purified, sequenced and compared with reference sequences in GenBank. The authors reported that the molecular identifications were confirmed after three repetitions. Anaplasma ovis was detected in 6 of 83 fleas, a proportion of 7.23 percent; Coxiella burnetii in 37, or 44.58 percent; Babesia in 8, or 9.64 percent; and Theileria annulata in 4, or 4.82 percent. The Babesia category comprised seven detections designated as Babesia sp. and one assigned to Babesia ovis.

Phylogenetic analyses placed the detected sequences in clades associated with their respective pathogen groups. The local Anaplasma ovis sequences clustered with a sheep-derived Xinjiang reference strain, PX682654, and showed genetic divergence from sequences attributed to Iran, Spain and Kenya. The authors interpreted this pattern as evidence for locally differentiated A. ovis lineages in alpine pastoral areas along the China–Kazakhstan border. Their analysis did not directly measure how widely any particular genotype circulated among livestock or fleas, however, and the phylogenetic result alone does not describe population prevalence. The study sequences were deposited in GenBank under accession numbers PZ263706 and PZ263708 for the Anaplasma 16S rRNA target.

Coxiella burnetii sequences clustered with strains obtained from ticks, fleas, humans and animals in Xinjiang and Kazakhstan. The closest reported homology was to a Hyalomma sp. strain, PV976747, from Shihezi in Xinjiang, while the flea-derived sequences differed from reference strains from Europe, the United States, Japan, South Korea and India. The authors proposed that the close relationship between the flea sequences and local tick isolates was consistent with long-term co-circulation involving ticks, fleas and livestock in the border mountain pastures. The two Com1 sequences generated in the study were deposited as PZ444052 and PZ390894. Even so, genetic similarity between organisms found in different arthropod groups does not by itself establish a transmission route or show that the flea maintained the organism during development.

The Babesia and Theileria comparisons also matched the study sequences with reference organisms from domestic and foreign sources. Theileria annulata sequences were associated with records from Xinjiang and Kazakhstan, including references MG645811 and PQ056497, while Babesia ovis clustered with sheep-derived reference material. The broader Babesia sp. sequences corresponded with an organism reported from Ornithodoros lahorensis and from ticks, including reference sequences OM438140.1 and KY464046.1. In the analysis, clear genetic boundaries separated the target organisms from Theileria equi and Babesia caballi. The relevant 18S rRNA sequences were deposited in GenBank as PZ429426, PZ263724, PZ263721, PZ429427 and PZ263716. These comparisons support the molecular assignments, but sequence matching cannot establish that fleas acquired the organisms by feeding, replicated them in their tissues or passed them to vertebrate hosts.

Several fleas carried more than one detected pathogen. Coxiella burnetii occurred together with Anaplasma ovis in fleas numbered 25, 27 and 37. A second group—fleas numbered 3, 45 and 75—contained C. burnetii together with Babesia and Theileria species. The authors noted that concurrent infections have been documented in other blood-feeding arthropods, including simultaneous detections of Borrelia burgdorferi and Anaplasma phagocytophilum in human fleas and co-detections of C. burnetii, Borrelia species and Babesia microti in ticks collected from camels. The co-detections in the present study show that several targeted organisms were present together in individual V. alakurt, adding to the documented diversity of pathogen profiles encountered in fleas and ticks.

V. alakurt belongs to the endemic flea family Vermipsyllidae, whose recognized species are distributed mainly across alpine regions of China and neighboring countries. The species was first recorded in Xinjiang in 1965, and adults are mainly active from December through March in alpine pastures. It primarily infests sheep, with goats, cattle, yaks, horses and red deer listed as secondary hosts. Heavy infestations can produce pruritus, emaciation, progressive anaemia and abortion in pregnant females and can cause mortality in severe cases, resulting in economic losses for local animal husbandry. The complete life cycle includes egg, three larval instars, pupa and adult; larvae and pupae live freely in soil, marmot burrows and environmental debris, whereas adults parasitize animals and take blood meals mainly in winter.

The results do not establish that V. alakurt is an effective vector for any of the detected pathogens. The researchers examined only adult fleas, leaving eggs, larvae and pupae untested, and they did not screen the source sheep for infection. Consequently, pathogen DNA in a flea could have arrived in ingested host blood. The study could not distinguish replication within flea tissues from transient persistence in the digestive tract, determine the developmental stage at which acquisition occurred, or show that the organisms passed onward to sheep or humans. The interactions among ticks, fleas and livestock also remained unresolved. The observed genetic closeness between the flea C. burnetii sequences and local tick-derived isolates, together with the co-detection of several pathogens, is an epidemiological signal that warrants further study, but it is not direct evidence of pathogen transmission through the flea.

Taken together, the study expands the known pathogen spectrum associated with V. alakurt and documents Anaplasma, Coxiella, Babesia and Theileria in fleas collected from sheep in the China–Kazakhstan border area. Its strongest finding concerns carriage and co-detection: the sampled female fleas contained DNA from four pathogen genera, with C. burnetii detected most frequently and several fleas harbouring multiple targets. Determining whether the findings represent a distinct epidemiological role for V. alakurt will require screening its immature stages, surveying the livestock from which fleas are collected, and experimentally establishing whether the organisms survive, replicate or move through the flea–host interface. Until those steps are completed, the results should be understood as the first molecular evidence of these four pathogen genera in this flea species rather than proof that it transmits them.

Subject of Research: Molecular detection of tick-associated pathogen DNA in the alpine livestock flea Vermipsylla alakurt

Article Title: Anaplasma spp., Coxiella, Babesia and Theileria Infections in Vermipsylla Alakurt Fleas

Article References: Jiushikun, J., Yizigeleke, T., Fan, Y., Li, W., Zhang, Y., Zhao, G., Tang, L., & Wang, Y. (2026). Anaplasma spp., Coxiella, Babesia and Theileria Infections in Vermipsylla Alakurt Fleas. Acta Parasitologica, 71(6), Article 240. https://doi.org/10.1007/s11686-026-01428-5

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01428-5

Keywords: Vermipsylla alakurt, alpine fleas, vector-borne pathogens, Coxiella burnetii, Anaplasma ovis, Babesia, Theileria annulata, livestock health, Xinjiang, molecular surveillance, Anaplasma, Coxiella

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