Diversity of Rickettsia Species Found Through Metagenomics in Ticks Collected on a University Campus

Ticks of the genus Amblyomma are ectoparasites of public health relevance, because some species are vectors of Brazilian Spotted Fever, and may also be involved in the epidemiology of other zoonoses. Climate changes and anthropogenic activities can contribute to their geographic expansion and population increase, amplifying risks of pathogen transmission. Metagenomics is a useful tool in microorganism surveillance in vectors. This study aimed to investigate Rickettsia species in ticks collected on a university campus. In 2022, 541 free-living ticks were captured with CO 2 traps at two points on a university campus: an anthropized area with a lake and high human circulation (A) and a preserved cerrado area with ecological trails (B). All specimens were identified as Amblyomma sculptum and grouped into pools by site and stage: 356 adults (A1, B2–B6) and 185 nymphs (A2, B1). Pools were pre-treated with high concentrations of DNase to remove host and bacterial DNA and concentrated with LPA Genelute Carrier. DNA extraction was performed with the Roche Large Volume Kit and samples were submitted to reverse transcription with Superscript III. Amplification was performed with the QuantiTect Whole Transcriptome Kit (QIAGEN) and libraries were prepared with the DNA Prep Library Kit and sequenced on the Illumina NextSeq 2000. Bioinformatic analysis included a standard pipeline for taxonomy. Reads were evaluated for quality using fastq and only sequences >30 were used. Human read removal was done by BWA mapping and taxonomic analysis was performed with Kraken2 and phylogenetic alignment with MAFFT. Metagenomics revealed multiple Rickettsia species, with diversity and variable abundance among pools, with a higher number of reads and species in area B. R. amblyommatis and R. rhipicephali were the most prevalent. R. parkeri and R. typhi also stood out, both associated with disease in the country. R. bellii, R. massiliae, among others, and symbionts were detected. Notably, R. rickettsii was identified in pool B6. A. sculptum is an aggressive, anthropophilic tick. The presence of species such as R. rickettsii, R. parkeri, and R. typhi in areas of human-wildlife interaction indicates zoonotic risk. This metagenomics identified pathogens, in addition to symbionts, which may be useful for control and prevention measures.