Files
Abstract
Eimeria are a type of parasitic protozoan that can cause coccidiosis, an enteric disease, in various animals. In commercial chickens, several species of Eimeria can cause devastating outbreaks of coccidiosis, resulting in significant financial losses. To detect and differentiate Eimeria species, molecular methods such as polymerase chain reactions (PCR) and next-generation sequencing (NGS) technology can be used. However, a routine and straightforward protocol and bioinformatic platform for using NGS to differentiate Eimeria species has not been developed yet. To address this issue, our laboratory aimed to evaluate the ability of nanopore NGS to sequence PCR amplicons of Eimeria gene regions and create a protocol to sequence, identify, and distinguish Eimeria species in mixed populations. We generated amplicons of Internal Transcribed Spacer-1 (ITS1), Ribosomal 18S DNA (18S), and Cytochrome Oxidase 1 (CO1) genes using pan-species primers and sequenced them with nanopore NGS. Our first study found that nanopore NGS can sequence Eimeria amplicons in coccidiosis vaccines with >97% contig sequence identity across replicates. Additionally, the high amplicon sequencing depth allowed us to create detailed phylogenetic trees with existing reference sequences, identifying all species present. In our second study, we replicated these results with field samples. Nanopore NGS was able to sequence amplicons from oocysts isolated on broiler farms using various coccidiosis control programs. Our protocol was used again to create detailed phylogenetic trees, including field samples, coccidiosis vaccines, and existing references, showing the relatedness between contigs. Finally, we developed a bioinformatic platform to supplement our sequence assembly methods. This platform automates the computational process by subjecting all raw nanopore NGS reads to the Basic Local Alignment Search Tool (BLASTn) and parsing through those results. In conclusion, our work demonstrated the suitability of nanopore NGS to sequence amplicons of identifying gene regions in Eimeria and investigate Eimeria species present in broiler chicken houses under various coccidiosis control programs. The resulting data produced detailed Eimeria gene assemblies and phylogenetic trees, showing the good applicability of nanopore NGS technology for sequencing and discerning mixed species populations from vaccines and field samples.