Análisis del metagenoma y viroma del linaje tropical de la garrapata Rhipicephalus sanguineus sensu lato (s.l.) en los departamentos de Santander y Casanare en Colombia
Thesis
Ticks, globally recognized as vectors of paramount significance, are characterized by their capacity to transmit an extensive array of pathogens to diverse vertebrate species. Among these ticks, Rhipicephalus sanguineus sensu lato (s.l.), known as the brown dog tick, holds a position of great importance in both animal and human health. R. sanguineus s.l. is a monophyletic, endophilic, three-host tick species, notable for its widespread global distribution. This tick species can act as a vector for various pathogens, serving as mechanical or biological transmitters of bacteria, protozoa, fungi, nematodes, and viruses. However, ticks do not operate in isolation; they harbor a complex microbiota comprising pathogens transmitted by them, commensals, and endosymbionts. These endosymbionts can offer a range of advantages to ticks, influencing their biological fitness and, consequently, their population dynamics. To identify these microorganisms, particularly those transmitted by ticks, various methodologies have been employed. These encompass conventional techniques such as microscopic analysis, culture, and gene amplification by PCR, combined with Sanger sequencing. However, these methods have limitations, including their inability to provide sensitivity and specificity for individual pathogens. In recent times, metagenomics, specifically the shotgun metagenomics approach, has expanded the scope of these techniques. It enables the simultaneous identification of multiple pathogens and the detection of other ecologically significant communities, such as endosymbionts. Despite the potential advantages of shotgun metagenomics, it has not yet been applied to Rhipicephalus sanguineus s.l. in Colombia, despite its critical role as a disease vector for both human and animal health on a global scale. This tick species has direct and indirect impacts on various hosts, including economically significant ones such as livestock, where it transmits a diverse range of pathogens. In Colombia, the situation is further exacerbated, as the majority of identified pathogens have been detected using the polymerase chain reaction (PCR) technique. PCR is limited in its capacity to amplify only a small number of pathogens, thereby failing to provide insight into the full spectrum of pathogens that R. sanguineus s.l. may carry. Consequently, this knowledge gap impedes our understanding of the potential effects this tick species may be having on domestic animals and humans in the country, particularly in relation to medical and veterinary relevant diseases. These diseases include Rocky Mountain spotted fever, caused by R. rickettsia, human granulocytic anaplasmosis, caused by A. phagocytophilum, Lyme disease caused by B. burgdorferi, and canine babesiosis, primarily caused by B. vogeli. Given the overall lack of knowledge about this tick species in Colombia, as well as its impact on disease transmission and its wide range of potential hosts, the central question arises: What communities of microorganisms can be identified in R. sanguineus s.l.? Conducting this research enabled us to detect circulating pathogenic organisms within the country via this tick and contribute to a better understanding of endosymbionts. Most importantly, it advanced our comprehension of the microbiome of R. sanguineus s.l., laying the foundation for future strategies to control tick-borne diseases based on microbiome modifications. In light of the above, the overarching objective of this study was to analyze the metagenome of the tropical lineage of the tick Rhipicephalus sanguineus sensu lato (s.l.) in the regions of Boyacá and Casanare in Colombia. This objective is further detailed through two specific aims: 1. Describe the composition of DNA microbial communities in R. sanguineus s.l. using a metagenomic approach. 2. Analyze the composition of RNA viruses in R. sanguineus s.l. through virome assessment using Oxford Nanopore sequencing technology. Each of these specific objectives corresponds to a distinct chapter within the thesis. In the first chapter of the thesis, DNA was extracted from ticks, which was subsequently subjected to Illumina's Novoseq 6000 sequencing platform, capable of handling 6 gigabytes per sample. For the bioinformatic analysis, two approaches were used: direct sequence assignment and metagenomic assembly (MAGs). Through these techniques, microorganisms were identified, including endosymbionts and pathogens. Among the microorganisms identified, the three most abundant in relative terms were Anaplasma phagocytophilum, Francisella tularensis, and Theileria equi. Additionally, endosymbionts belonging to the Coxiella, Rickettsia, and Wolbachia genera were identified. Notably, MAGs were successfully assembled for the species Coxiella mudrowiae, enabling comparative and functional genomics analysis. Furthermore, correlations between the various microorganisms identified were explored, with particular emphasis on the negative correlations observed between C. mudrowiae and other endosymbionts and pathogens. In the second chapter of the thesis, RNA was extracted from the samples, followed by the elimination of host RNA using RiboZero treatment and viral enrichment via SMART9N. Sequencing was carried out using Oxford Nanopore's MinION device. A similar approach to the first chapter was followed, including direct sequence assignment and the generation of MAGs for the complete set of samples, all while maintaining geographical and gender-specific restrictions. As a result of this research, six different viruses constituting the virome of this tick species were identified. These viruses include a species closely related to the Rhipicephalus-associated Flavi virus, the tick Mogiana virus, a virus from the Iflaviridae family, the tick Jingmen virus, Bole tick virus 4, Mivirus sp. Successful assembly of two of these viruses facilitated phylogenetic and comparative analyses based on available genomes from various regions around the world.
