Authors

R Thomas⁵; MC Silva-de la Fuente⁶; A Bacigalupo³; N Quiroga⁴; A Santodomingo⁵; M Muñoz⁸; L Vega²; N Luna²; C Hernández²; JD Ramírez²; MS Llewellyn¹; C Botto-Mahan⁴; S Muñoz-Leal⁵; L Moreno⁷;  ¹ School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, UK;  ² Centro de Investigaciones en Microbiología y Biotecnología-UR (CIMBIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Colombia;  ³ School of Biodiversity, One Health and Comparative Medicine, University of Glasgow, UK;  ⁴ Facultad de Ciencias, Universidad de Chile, Chile;  ⁵ Facultad de Ciencias Veterinarias, Universidad de Concepción, Chile;  ⁶ Facultad de Ciencias Agrarias y Forestales, Escuela de Medicina Veterinaria, Universidad Católica del Maule, Chile;  ⁷ Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Chile;  ⁸ Institute of Biotechnology, Universidad Nacional de Colombia, Colombia

Discussion

As vectors of many neglected diseases, blood-sucking arthropods pose an unseen threat until symptomatic cases in humans or animals emerge, or when local epidemics occur. With the current climate change scenario creating an environment conducive to the emergence, re-emergence, and spread of arthropod-borne infectious agents, continuous surveillance and timely identification of microorganisms with pathogenic potential become permanent tasks. Nanopore sequencing has emerged as a powerful tool for epidemiological surveillance in diverse settings, offering rapid and real-time processing of a relatively large number of samples. Our study utilises long-read sequencing to enhance our understanding of the potential vectorial role that blood-sucking arthropods - fleas, lice, mites, ticks and triatomines - play across Chilean ecosystems regarding Bacteria.

To achieve this goal, we extracted DNA from the guts or the whole body of 186 blood-sucking arthropods. Bacterial DNA was enriched by conventional PCR using universal 16S rRNA primers. The amplicons were sequenced in R10.4.1 flow cells on a Oxford Nanopore MinION MK1C. Bioinformatic analyses were conducted using a custom workflow to analyse metabarcoding data on reads above 900 bp, focusing on finding bacterial agents of medical and veterinary relevance.

As result, 97.85% of the samples yielded 16S rRNA long reads for bacterial DNA, which allowed us to identify potentially pathogenic arthropod-borne bacteria at the genus level. Orientia DNA was detected in fleas (1,242 reads) and triatomines (two reads), while Rickettsia DNA from the spotted fever group I (SFGI) was found in ticks (51 reads), fleas (29 reads), lice (11 reads), and mites (three reads). Additionally, Borrelia DNA from the relapsing fever group (RFG) was detected in mites (2 reads).

Although the detection of Orientia DNA in fleas and triatomines was unexpected, the identification of Borrelia DNA in mites and Rickettsia DNA in fleas, lice, and mites has been previously reported. In this sense, our next steps involve the use of taxa-specific primers to confirm our findings. 16S rRNA gene amplicon sequencing is a promising method for profiling arthropod-borne infectious agents, capturing not only a snapshot of potential pathogens circulating among these arthropods but also establishing the foundation for future genetic analysis.

Funding: Fomento a la Vinculación Internacional para Instituciones de Investigación, Agencia Nacional de Investigación y Desarrollo (ANID): “Fortalecimiento en el análisis metagenómico de patógenos transmitidos por vectores de importancia en salud pública” (FOVI220125); ANID Programa Becas: Doctorado Becas Chile 2019-72200391, Doctorado Nacional 2019-21190078, and 2020-21200182; Fondecyt 1221045, 11220177; Anillo ATE230025.