Rhipicephalus sanguineus sensu lato (s.l.), commonly known as the domestic dog tick, is a globally distributed tick. This species plays a significant role in human and animal health, as well as in economy due to its ability to infest livestock. The use of shotgun metagenomics has proven valuable in identifying tick-borne pathogens and key members of the tick microbiome, including endosymbionts. However, the application of shotgun metagenomics in R. sanguineus s.l. ticks in Latin America remains largely unexplored. Therefore, our objective aimed to explore and further analyze the metagenome of the tropical lineage of R. sanguineus s.l. ticks in Colombia. Through our analysis, we identified the three most prevalent pathogens harbored by these ticks, namely: Anaplasma phagocytophilum, Francisella tularensis, and Theileria equi. However, the most abundant microorganism detected was not a pathogen but the endosymbiont Coxiella mudrowiae. Interestingly, Coxiella mudrowiae exhibited significant negative correlations with several pathogens and other endosymbionts. Furthermore, we successfully constructed 27 medium-quality metagenome-assembled genomes (MAGs) for this microorganism, enabling us to conduct a pangenome analysis by comparing them with available genomes and identifying proteins of interest, such as those involved in vitamin B synthesis. This study represents the first implementation of shotgun metagenomics as a methodology to expand our understanding of pathogens and endosymbionts in the circulating tropical lineage of R. sanguineus s.l. ticks in Colombia. The findings of this research serve as a foundation for the development of prevention and mitigation strategies against pathogens transmitted by this tick species. Information gained from this study can contribute to the improvement of public health measures and veterinary practices aimed at controlling the impact of tick-borne diseases.

Metagenome, microbiome, R. sanguineus s.l, shotgun-metagenomics, tick

Ticks are blood-feeding arthropods that serve as obligate vectors, capable of transmitting a wide range of pathogens, surpassing other types of vectors in terms of diversity (Liu and Bonnet 2014). Among the hard-bodied ticks belonging to the family Ixodidae, several species have significant implications for public and animal health (Venzal et al. 2003). One such species is Rhipicephalus sanguineus sensu lato (s.l.), commonly known as the brown dog tick, which has a global distribution (Dantas-Torres 2010). This tick species displays endophilic and monotropic behavior, primarily infesting dogs, but it has also been found infesting wildlife, horses, and humans in rare instances (Dantas-Torres et al. 2013). R. sanguineus s.l. holds great importance in both human and animal health, exerting direct and indirect effects on various hosts, including economically significant livestock (Dantas-Torres et al. 2013). Within R. sanguineus s.l., five different lineages have been identified using mitochondrial molecular markers, namely R. sanguineus s.l. (tropical lineage), R. sp. I, R. sp. II (temperate lineage), R. sp. III, and R. sp. IV (Dantas-Torres et al. 2013). Among these lineages, R. sanguineus s.l. and R. sp. II are considered the primary lineages, with R. sanguineus s.l. being predominantly distributed in tropical regions, while R. sp. II is commonly found in temperate zones. However, it should be noted that certain countries exhibit a co-circulation of both lineages. Currently, only the circulation of the tropical lineage has been documented in Colombia (Paez-Triana et al. 2021).

It is widely recognized that ticks have the ability to harbor and transmit a diverse range of microorganisms, making them important vectors for diseases (Walker et al. 2000). While ticks can acquire numerous pathogens during their feeding, not all of them are capable of reproducing and being transmitted to new hosts. However, Rhipicephalus sanguineus s.l. is known to be a competent transmitter of a wide array of pathogens, including bacteria, protozoa, fungi, nematodes, and viruses (Jia et al. 2020). These tick-borne diseases (TBDs) contribute significantly to the overall burden of vector-borne diseases (VBDs), impacting human and animal health as well as national economies, especially in developing regions (Couper and Swei 2018; Ergunay et al. 2022). Therefore, there is a pressing need for comprehensive surveillance to identify pathogens and assess the potential risks to public health (Woolhouse and Gowtage-Sequeria 2005; Ergunay et al. 2022). However, studying the ecological community of ticks involves more than just identifying pathogens. The tick microbiota also includes commensal and endosymbiotic organisms (Pollet et al. 2020), understanding endosymbionts as those organisms that provide a benefit to the tick. These benefits include enhancing reproductive fitness, facilitating nutritional adaptation, influencing development and reproduction, providing defense against environmental stressors, and modulating tick immunity (Pollet et al. 2020). Also, an impact of this community on pathogen transmission has been observed. For example, in Dermacentor andersoni, negative correlations were found between parasites and endosymbionts like Anaplasma marginale and R. belli and positive between Francisella endosymbionts and F. novicida (Gall et al. 2016).The mechanisms involve the secretion of compounds that affect pathogen growth, either positively or negatively or the competition between the two organisms for nutrients, leading to reduced pathogen growth, or even the endosymbiont’s impact on the tick’s immune system results in increased pathogen growth (Gall et al. 2016). Many of these have been identified as endosymbionts based on their genomic characteristics (Gottlieb et al. 2015), the absence of virulence factors indicating their potential non-pathogenic nature, the presence of essential metabolic pathways for tick survival (Buysse and Duron 2021), and even through in-vivo experiments demonstrating the significant benefits of their presence in ticks (Ben-Yosef et al. 2020). Among these endosymbionts are the Coxiella-like endosymbionts (CLE), Wolbachia endosymbionts (WE), Rickettsia endosymbionts (RE), and Francisella endosymbionts (FE), among others (Hussain et al. 2022). Despite research on the potential pathogenicity of some of these organisms, it is currently assumed that they do not cause pathogenicity in humans and is limited to specific animals (Hussain et al. 2022). However, their significance in ticks as endosymbionts has indeed been widely recognized, rendering them a community of great importance for study.

Moreover, comprehensive understanding of the tick microbiota is crucial for unraveling the complex interactions within this community and their implications for tick biology and disease transmission. Traditional methods used to identify microorganisms in ticks have limitations in terms of sensitivity, as they only capture a fraction of the tick’s microbiota (Wade 2002; Batool et al. 2021). These methods primarily focus on the most common pathogens associated with tick-borne diseases. They involve techniques such as culturing, microscopy, and PCR coupled with Sanger sequencing (Batool et al. 2021). In contrast, metagenomic techniques, particularly shotgun metagenomics, offer a more comprehensive understanding of the tick’s microbial composition by identifying all genes and genomes present (Xia et al. 2015). This approach allows for the assessment of microbial diversity, abundance, and the analysis of complete or partial genomes (Xia et al. 2015). By utilizing metagenomics, we can strategically survey tick-borne pathogens, evaluate the associated risks, and gain insights into the mechanisms of disease transmission by these microorganisms. Additionally, this approach enables the identification of other ecologically significant communities, such as endosymbionts. Metagenomics goes beyond taxonomic classification and provides initial insights into the functional roles performed by these microorganisms (Bonnet and Pollet 2021), considering the diverse pathways of interaction between these communities and ticks. Previous studies have effectively employed this methodology. For example, a study conducted in Palestine focused on Rhipicephalus spp. and Haemaphysalis spp., successfully identified various pathogenic species and endosymbionts. They generated genomes for Rickettsia massiliae, Candidatus Rickettsia barbariae, and Coxiella endosymbionts (Ravi et al. 2019). Similarly, investigations involving Amblyomma species have revealed important pathogens such as Coxiella burnetii and Francisella tularensis (Ergunay et al. 2022). In the case of R. sanguineus s.l., several studies have explored its microbiota using amplicon-based sequencing targeting the 16S rRNA gene. Pollet et al. identified Proteobacteria as the dominant phylum, with the genus Rickettsia and the family Coxiellaceae being the most abundant in these ticks (Pollet et al. 2020). Another study by René-Martellet et al. confirmed the prevalence of the genus Rickettsia and observed a high abundance of Coxiella and Bacillus, particularly in the tropical lineage, which exhibited the highest Coxiella abundance (René-Martellet et al. 2017). Additionally, Cassia-Luzzi et al. compared the microbiota composition among different lineages of R. sanguineus s.l. and between embryos and female adults, revealing a significant abundance of Proteobacteria in all lineages and Actinobacteria and Firmicutes in the tropical lineage (Luzzi et al. 2021).

The study of R. sanguineus s.l. ticks in South America, particularly in Colombia, is limited despite its significance in developing and tropical countries. A study by Cotes-Perdomo et al. (2020) conducted PCR analysis and identified Anaplasma marginale, Babesia vogeli, Babesia bigemina, and Coxiella spp. (C. mudrowiae and endosymbionts) in ticks collected from dogs in the Magdalena department. However, the lack of comprehensive research hinders our understanding of the potential impact of this tick species on domestic animals and humans in the country. It is crucial to consider the potential transmission of medically and veterinary important diseases by R. sanguineus s.l., including Rocky Mountain spotted fever, human granulocytic anaplasmosis, Lyme disease, and canine babesiosis. Despite the significant advantages of shotgun metagenomics in identifying a wide range of organisms, this technology has not yet been applied to R. sanguineus s.l. ticks in Latin America. However, this approach could provide valuable insights into the interactions and functions of the entire microbiome of these ticks, considering the diverse interaction pathways between these communities and the tick. It has been observed that pathogens can activate mechanisms and manipulate tick responses to facilitate their infection, while ticks can limit pathogen infection, and high bacterial infections can enhance tick survival. Based on the aforementioned information, our objective is to analyze the metagenome of the tropical lineage of R. sanguineus s.l. ticks in the Santander and Casanare departments in Colombia. By employing shotgun metagenomics, we aim to enhance our understanding of the microbial composition and potential pathogens associated with these ticks, thereby contributing to the development of effective prevention and control strategies for tick-borne diseases in the region.

Ticks are recognized as the second most important vectors of diseases after mosquitoes (Chen et al. 2023). The Center for Disease Control and Prevention (CDC) reports that tick-borne diseases (TBDs) accounted for over 75% of the 650,000 reported vector-borne diseases in the United States between 2004 and 2016 (Rosenberg et al. 2018). The advent of advanced sequencing technologies, such as shotgun metagenomics, has revolutionized disease research by enabling rapid and accurate identification of emerging and re-emerging pathogens (Chen et al. 2023). This approach has also shed light on the diverse ecological communities that exist within ticks, providing a comprehensive understanding of tick microbiomes (Xia et al. 2015). Such comprehensive knowledge is crucial for the prevention and identification of both veterinary and human diseases, offering valuable insights into these disease-carrying vectors. Metagenomics has the potential to drive innovative strategies in vector control and the prevention of TBDs. By leveraging the information obtained through metagenomic studies, we can develop targeted interventions and enhance our ability to identify and combat TBDs effectively. This approach holds great promise for improving the health outcomes of both animals and humans by strengthening our prevention and identification efforts in the field of tick-borne diseases.

Here we employed shotgun metagenomics to characterize the microbiome of 38 R. sanguineus s.l. specimens. The predominant bacterial composition in our samples was found to be from the Coxiellaceae family (Fig. 1A), which is consistent with previous studies on the tropical lineage of R. sanguineus s.l. ticks worldwide (René-Martellet et al. 2017; Portillo et al. 2019; Luzzi et al. 2021). Notably, research by Luzzi et al. (2021) demonstrated a significant similarity in bacterial composition between embryos and adults, suggesting the potential for transovarial transmission of Coxiella spp. within this family. The identification of endosymbionts within the Coxiellaceae family (Fig. 1A) further emphasizes their importance in R. sanguineus s.l. ticks, as they have been consistently observed across different continents and studies. In addition to the Coxiellaceae family, our study identified other genera such as Pseudomonas, Rickettsia, Propionibacterium, and Bacillus, which have been previously associated with this tick species (Lalzar et al. 2012; René-Martellet et al. 2017). Furthermore, the presence of genera like Clostridium, Streptomyces, Burkholderia, Fusobacterium, Lactobacillus, and Campylobacter, although not previously reported in R. sanguineus s.l., has been documented in other tick species (Karim et al. 2017; Papa et al. 2020). It is likely that these bacteria may be acquired from the environment or host, such as the skin or cavities. It is important to note that certain bacteria, such as Bacillus and Rickettsia species, have been reported to exhibit a higher prevalence in one of the sexes (René-Martellet et al. 2017); our study found significant differences in various genera between sexes and departments. Including Rickettsia, where females displayed higher abundance, this observation aligns with the findings of René-Martellet et al. Despite these differences, the fundamental composition of the top 10 genera remains consistent across departments and sexes.

The presence of archaea and viruses in the samples was minimal in terms of abundance, but a diverse range of genera was identified within these groups (Fig. 1B). Archaea in ticks have received limited attention and research due to the scarcity of available data. The genus Methanobacterium has been previously detected in other tick species like Ixodes granulatus (Chen et al. 2023). As far as we know our study provides the first identification of archaea in this tick species using metagenomics. However, it is important to note that their role in ticks remains unclear and further investigation is necessary to understand their potential interactions and contribution to the tick’s bacterial and eukaryotic communities. With this finding it seems that the methane can play a role in the tick. However, a more comprehensive approach is required to explore the involvement of archaea in tick physiology and health and unravel their significance in tick interactions. In addition, this study identified the presence of the Baculoviridae family, which consists of arthropod viruses (Herniou and Jehle 2007). Specifically, the genera Alphabaculovirus and Betabaculovirus, known as lepidopteran-specific nucleopolyhedroviruses (NPVs) (Herniou and Jehle 2007), were found to be dominant among the DNA viruses detected in the samples. The presence of these viruses suggests their potential involvement in tick biology and their interaction with the tick’s natural environment. Further research is needed to elucidate the specific roles and implications.

To identify the pathogens and endosymbionts present in the samples and explore their interactions, a database was created using available genomes. As a result, three major pathogens were detected in the samples: A. phagocytophilum, which causes human granulocytic anaplasmosis (HGA) and anaplasmosis in animals (Karshima et al. 2022); F. tularensis, the causative agent of tularemia (Telford and Goethert 2020); and T. equi, the causative agent of equine piroplasmosis (PE) (Wise et al. 2013). The vector competence of R. sanguineus s.l for these pathogens is currently unknown. However, previous studies have reported the detection of pathogen DNA in R. sanguineus s.l specimens worldwide (Ghafar and Amer 2012; Zhang et al. 2012; Lopes de Carvalho et al. 2016; Cabrera et al. 2022; Rocafort-Ferrer et al. 2022). Moreover, research conducted in Colombia has also identified these pathogens in both hosts and ticks, suggesting their likely circulation within the country (Máttar and Parra 2006; Molina-Guzmán et al. 2019; Santodomingo et al. 2019; Bonilla-Aldana et al. 2022; Cabrera et al. 2022). Considering the significance of these pathogens in terms of animal and human health, including the potential for humans to act as accidental hosts for these diseases (Cabrera et al. 2022), and the potential economic impact on a developing nation (Santodomingo et al. 2019), it is crucial to further investigate the vector competence of R. sanguineus s.l for these pathogens. However, it is important to consider in the case of F. tularensis that, as of now, genomes of FE have not been identified in RefSeq. Moreover, it has been observed that metagenomic studies can lead to confusion in distinguishing endosymbionts from their pathogenic species (Buysse and Duron 2021). Therefore, future research should be directed towards confirming the presence of F. tularensis or FE in these ticks. Even so, long-term surveillance studies focusing on these three tick-borne diseases should be implemented in Colombia. It is important to note that there is a hypothesis suggesting that the reported cases of TBDs in humans within the country may be underestimated due to underreporting (Máttar and Parra 2006). Additionally, considering the tendency of this tick species to feed on domestic animals such as dogs, cattle, and horses, there is a significant likelihood of human-animal contact in Colombia, highlighting the need for further research in this area.

On the other hand, a significant abundance of reads corresponded to various endosymbionts (Fig. 2B). It is well-known that ticks harbor a diverse microbiome primarily composed of non-pathogenic organisms, and studying their ecology and evolutionary dynamics is crucial. In particular, a substantial number of reads aligned with Coxiella endosymbionts. However, some reads were also classified as Wolbachia endosymbionts (WE). Recent studies have reported the presence of Wolbachia in R. sanguineus s.l. ticks (Chao et al. 2021). Nevertheless, it is essential to identify and further investigate these endosymbionts due to the potential implications of Wolbachia in controlling vector-borne diseases in other arthropods (Chao et al. 2021). Therefore, future research on this tick species should expand, encompassing genome sequencing, characterization of their role as endosymbionts, and even exploring their potential experimental use in vector control. Regarding the identified Coxiella spp., a clear dominance of Coxiella mudrowiae (Fig. 2D) was observed, a candidate species previously identified and described as endosymbiont. C. mudrowiae has been detected in various geographical locations and different tick species (Ravi et al. 2019; Rahal et al. 2020). This candidate was recognized as an ongoing endosymbiont based on several genomic features that are characteristic of endosymbionts. These include a notable reduction in genome size when compared to C. burnetii, a pronounced rate of pseudogenization, and the pseudogenization of characteristic virulence genes such as the Dot/Icm Type IVB secretion system (T4BSS), a crucial system for the pathogenicity of C. burnetii (Gottlieb et al. 2015; Tsementzi et al. 2018; Brenner et al. 2021). However, imaging data or clinical evidence to verify whether this organism could potentially become a pathobiont were not found. Consequently, further development of this research area is imperative, given its widespread prevalence worldwide. Nevertheless, since all current evidence suggests its role as an endosymbiont and no pathogenic traits have been identified, it will be classified as an endosymbiont throughout the paper. Additionally, multiple Coxiella-like endosymbionts (CLE) were detected within the same tick individuals, indicating that multiple CLEs can establish themselves within the tick microbiota. This phenomenon has been previously discovered through the analysis of the 16S rRNA gene in tick species, including R. sanguineus s.l. (Machado-Ferreira et al. 2016). It is plausible that each endosymbiont provides a different function to the tick and coexists or that they compete for colonization. However, we observed a negative correlation between C. mudrowiae and the other CLE (Fig. 2E), suggesting that competition for colonization is more likely. Nonetheless, conducting further comprehensive research on both species is crucial to better understand their interactions, whether it be WE-WE, WE-CLE, or CLE-CLE, as well as to comprehend the possible coevolution occurring between different endosymbionts and ticks, and even among the endosymbionts themselves.

It is well-established that endosymbionts engage in ongoing interactions with the transmitted pathogens, which can either facilitate, restrict, or impede their transmission (Bonnet et al. 2017). Examining the correlations, Coxiella mudrowiae showed negative associations with multiple pathogens and endosymbionts. This finding was further supported by observing regions with a higher abundance of this CLE, where pathogen read abundance was reduced. Similarly, negative correlations were identified with other endosymbionts. In the Casanare department, where Coxiella mudrowiae was more prevalent, the remaining endosymbionts showed lower abundance, and vice versa (Fig. 2C, D). Previous studies have highlighted the importance of investigating these interactions. For example, in Dermacentor andersoni ticks, a negative correlation was found between pathogens such as Anaplasma marginale and Rickettsia belli and endosymbionts, while a positive correlation was observed between Francisella endosymbionts and Francisella novicida (Gall et al. 2016). Therefore, further exploration of these negative interactions involving Coxiella mudrowiae, which is known to be abundant in this and other tick species, is crucial. This investigation should encompass not only next-generation sequencing techniques but also biological studies aimed at validating and enhancing our understanding of these interactions.

Similarly, considering the prominence of this endosymbiont in our samples, we assembled multiple MAGs and compared them to the genomes constructed thus far. Currently, two genomes have been characterized, one derived from R. sanguineus s.l and another from R. turanicus (Gottlieb et al. 2015; Tsementzi et al. 2018), both originating from Israel. These genomes highlight the functional role and significance of Coxiella endosymbionts in ticks, as they are involved in vitamin synthesis, cofactor production, and other metabolic processes (Tsementzi et al. 2018). In our comparison of the MAGs to these reference genomes, we observed the absence of several genes (Fig. 3B, C), which could be attributed to their incomplete nature. However, analysis of the clusters of orthologous groups (COGs) revealed that the quantities were highly similar to those in the available genomes, albeit with a reduction in certain functional groups compared to C. burnetii. This reduction has been previously documented and is associated with pseudogenization and gene loss resulting from the obligate endosymbiotic nature of this bacterium (Gottlieb et al. 2015).

Furthermore, the MAGs formed a distinct monophyletic group separate from the assembled genomes. However, when analyzing average nucleotide identity (ANI) percentages and constructing a phylogenetic tree based on 40 informative proteins, the MAGs clustered with known Coxiella species and indicated a low degree of diversity within the country. This aligns with previous research that has attributed the limited intrapopulational diversity of C. mudrowiae to the restricted number of hosts, as represented by the two Coxiella strains derived from different tick species (Gottlieb et al. 2015). These findings are consistent with our observations and the anticipated limited diversity within the country (Paez-Triana et al. 2021). However, the substantial single-nucleotide polymorphism (SNP) differences between our MAGs and the available genomes could be attributed to the considerable geographic distance separating the reported origin countries of these genomes. Therefore, we hypothesize that a distinct strain of C. mudrowiae is circulating within Colombia. Such an undertaking would enable comprehensive genomic and phylogenetic analyses, thereby enhancing our understanding of the co-evolutionary mechanisms between C. mudrowiae and its tick host.

Finally, we conducted an assessment of specific genes that have been previously reported in this endosymbiont (Fig. 4), particularly those involved in vitamin B synthesis (Tsementzi et al. 2018; Brenner et al. 2021). We observed the presence of these genes, consistent with findings from the other two genomes of C. mudrowiae. However, we noted the absence of panD and panE, which have been documented in other Coxiella-like endosymbionts, such as the CLE of Rhipicephalus sanguineus s.l l (Brenner et al. 2021). Nevertheless, it has been postulated that these missing genes may be substituted by other genes present in the genome of C. mudrowiae (Tsementzi et al. 2018). Additionally, previous studies have indicated that these genes are also present in C. burnetii, suggesting their essential role in the pathways required by both organisms and their interaction with their respective hosts (human and tick) (Brenner et al. 2021). However, upon analyzing the protein sequences, we discovered substantial variations that resulted in low similarity between the proteins and even divergence in the protein trees (Fig. 4). Despite previous investigations in proteomics highlighting the significance of these genes and proteins in the endosymbiotic association between C. mudrowiae and its tick host (Cibichakravarthy et al. 2022), it is crucial to assess how these dissimilarities in the proteins might impact their structure and functionality compared to what C. burnetii can produce. Understanding the metabolic aspects of this endosymbiont could potentially contribute to future biotechnological strategies for controlling tick populations and managing tick-borne diseases both within the country and globally. Previous studies have indicated that the absence of Coxiella-like endosymbionts in ticks results in delayed post-feeding development, prolonged feeding periods, and reduced fertility and fecundity. Larvae lacking CLE endosymbionts have been observed to exhibit unsuccessful feeding (Ben-Yosef et al. 2020). Overall, further investigations focusing on the genetic and functional characteristics of these genes and proteins are necessary to fully comprehend their roles in the endosymbiotic relationship between C. mudrowiae and ticks. Such understanding can inform the development of innovative approaches for tick control and the management of tick-borne diseases.

Despite certain limitations, such as competition for genomic resources between ticks and their pathogens, which can limit the number of assigned reads, and challenges associated with taxonomic assignment due to the scarcity of research on vector microbiomes, particularly in ticks, understanding the microbiome of these vectors is of utmost importance. It not only allows us to identify the pathogens that the tropical lineage of R. sanguineus s.l. could transmit or carry to a wide range of animals, including humans due to their close contact with domestic dogs, but it is also essential for the control of diseases transmitted by these ticks. This is particularly crucial considering the increasing emergence of resistance markers in pathogens worldwide. Understanding the dynamics of these microorganisms and their interaction with ticks is essential to effectively address the challenges related to veterinary and animal health. Additionally, it can provide valuable information for implementing more effective prevention and control strategies, thereby minimizing the impact of diseases transmitted by R. sanguineus s.l. on public health and the livestock industry. To gain a comprehensive understanding, it is imperative that future investigations focus on exploring the prevalence and circulation of the different pathogens identified in this study and implement active surveillance measures within the country.

Furthermore, conducting in-depth studies on the endosymbiont C. mudrowiae is crucial. This involves not only comprehending its behavior within the country through complete genome analysis but also assessing potential strategies that could pave the way for future vector control approaches utilizing this endosymbiont. Moreover, to further enhance our understanding of pathogen transmission in R. sanguineus s.l., it is imperative to expand this study to encompass the diverse lineages of this tick species worldwide. This expansion would allow for the identification of distinct pathogens that may be transmitted by different lineages. Additionally, considering the knowledge generated regarding endosymbionts in these ticks, it becomes important to explore their prevalence and potential interactions within each lineage. By doing so, we can develop comprehensive surveillance strategies that encompass the entire R. sanguineus s.l. species complex, thereby enabling a more thorough assessment of pathogen diversity and transmission dynamics. Such an approach would provide valuable insights for the implementation of effective surveillance and control measures targeting this complex species.

Our study has yielded a comprehensive understanding of the pathogens and endosymbionts present in R. sanguineus s.l ticks in Colombia. We have successfully identified three significant pathogens, namely A. phagocytophilum, F. tularensis, and T. equi, which have implications for both animal and human health. Implications ranging from the occurrence of mild symptoms to the death of both humans and animals. This information is invaluable for guiding tick-borne disease surveillance efforts in the country. Furthermore, we have made notable discoveries regarding the presence of highly relevant endosymbionts, including Coxiella mudrowiae, among others, within this tick species. One intriguing finding is the negative correlations observed between the presence of pathogens and endosymbionts. This suggests complex interactions within the tick microbiome that warrant further investigation. In addition, our study represents a milestone as we have successfully assembled multiple MAGs for C. mudrowiae, making them the first MAGs of this endosymbiont reported in Colombia. These MAGs have provided valuable insights into the phylogenetic relationships and functional characteristics of C. mudrowiae in the country. Overall, our findings contribute significantly to the understanding of pathogen and endosymbiont dynamics in R. sanguineus s.l ticks in Colombia. This knowledge serves as a crucial resource for guiding tick-borne disease surveillance efforts and has implications for public health and animal welfare.

We thank the personnel who helped in the collection of the specimens in the field.