Desarrollo de un pipeline bioinformático para el análisis filogenético de Babesia bovis y Babesia bigemina en Rhipicephalus microplus (Ixodidae) mediante secuenciación con nanoporos

Abstract

Bovine babesiosis is a parasitic disease transmitted by Rhipicephalus ticks, posing a significant threat to animal health and agricultural productivity in tropical and subtropical regions. In Latin America, Babesia bovis and Babesia bigemina are the most prevalent species; therefore, precise genetic characterization is essential to understand their diversity, evolution, and epidemiological dynamics. This study aimed to develop a standardized bioinformatic pipeline for the phylogenetic analysis of B. bovis and B. bigemina using sequencing data generated by Oxford Nanopore technology. The workflow was implemented in the Galaxy Australia platform and included quality control with FastQC, adapter trimming with Porechop, genome alignment using Bowtie2 and Samtools, de novo assembly with SPAdes, and specific sequence selection through a custom BLAST database and AWK filtering commands. The selected sequences corresponded to RAP-1a (for B. bovis) and SpeI-AvaI (for B. bigemina). Phylogenetic analyses were performed using PAUP v4.0b169* under a Maximum Parsimony framework, and clade support was assessed using 1,000 bootstrap replicates. Multiple sequence alignments were conducted in MacVector v18.7.6 with ClustalW (GOP: 30.0; GEP: 10.0). The resulting trees revealed contrasting patterns between species: B. bovis displayed a highly supported and genetically homogeneous Ecuadorian clade, suggesting a recent clonal expansion across Napo, Sucumbíos, and Orellana provinces. In contrast, B. bigemina exhibited greater phylogenetic structure, with four well-supported subclades (bootstrap: 88–100%), reflecting higher genetic diversity and possible regional geographic differentiation. These findings highlight the coexistence of local lineages and their divergence from international reference strains. Furthermore, they emphasize the importance of incorporating regional genetic data into diagnostic and control strategies. The developed pipeline proved effective and reproducible, offering a robust foundation for future molecular and epidemiological studies on Babesia spp. in Ecuador. This research contributes significantly to the understanding of the phylogeography and genetic diversity of these hemoparasites, which are key components in the design of surveillance and prevention programs for bovine babesiosis.