Author ORCID Identifier

https://orcid.org/0009-0007-8513-6124

Date Available

12-20-2024

Year of Publication

2024

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Agriculture, Food and Environment

Department/School/Program

Veterinary Science

Faculty

Dr. Martin K. Nielsen

Faculty

Dr. Martin K. Nielsen

Abstract

Strongylus vulgaris is the most pathogenic nematode parasite of grazing horses worldwide. This gastrointestinal parasitic nematode is in the family Strongylidae under the subfamily Strongylinae, or Strongylins. While the epidemiology, complete life cycle, and high pathogenicity of the fourth and fifth larval stages of S. vulgaris are well established, we understand very little about its biology. This parasite is renowned for its unique life cycle and capacity to cause arterial damage, leading to significant health and economic impacts for the equine industry. It also is the most intensively documented Strongylin; however, S. vulgaris has yet to gain drug resistance as nearly all other helminth parasites of horses have, including species in the second subfamily of Strongylidae, the Cyathostomins. Modern parasite management plans treat horses less frequently to preserve any remaining efficacy. As a result, numerous documented increases in the prevalence of S. vulgaris exist in the literature for a parasite with limited species-specific diagnostic tests available. Genomics has proved an important tool in humans, providing for biotechnological innovations in diagnostics and treatments of pathogen-derived diseases. Currently, no high-quality reference genome exists for a Strongylin. This study represents a comprehensive genomic and transcriptomic investigation into S. vulgaris, providing the first high-quality de novo genome assembly and comparative life stage-specific transcriptomic analysis. Utilizing high-quality de novo genome assembly with PacBio HiFi long-read sequencing, we generated a 705 Mb assembly with 99.2% completeness and annotated 21,825 protein-coding genes. Approximately 37% of the genome consists of DNA repeat regions, in line with other high-quality genomes of parasitic strongylids. Transcriptomic profiling and gene expression analyses were performed on the pathogenic life stages (L4 and ML5) using PacBio Iso-Seq and Illumina RNA-Seq, resulting in high-resolution insights into life stage-specific gene expression. L4 larvae exhibited greater genome utilization (52%) than ML5 females (45%), with differential expression analyses identifying stage-specific genes linked to collagen synthesis, energy metabolism, and immune responses. The L4 stage uniquely transcribed genes involved in developmental regulation, displayed higher transcriptional diversity by transcribing 7,000 more genes, and exhibited activation of innate immune response genes. These results underscore this life stage’s role in host tissue invasion and arterial damage. In contrast, ML5 stages exhibited metabolic and sensory adaptations critical for transitioning to adulthood and showed increased expression of genes related to sensory signaling and oxidative stress mitigation. Differential gene expression highlighted stage-specific activation of collagen synthesis, oxidative stress responses, adaptive immune response, and energy metabolism, providing insights into the molecular mechanisms driving pathogenicity and how these differentiate between sex and life stage. The integration of genome and transcriptome data enabled the identification of key genes associated with the parasite's survival, host interactions, and developmental transitions in the pathogenic and adult life stages. Variant analyses revealed a higher density of single nucleotide polymorphisms (SNPs) and insertions in the L4 stage (1 SNP/9 kb and 1 insertion/174 kb) compared to the ML5 stage (1 SNP/14 kb and 1 insertion/295 kb), suggesting elevated transcriptional plasticity. This work also sheds light on the evolutionary dynamics of S. vulgaris within the Strongylidae family. Comparative mitochondrial genome analyses revealed unique nematode features such as AT-rich tRNA adaptations and alternate codon usage, suggesting lineage-specific evolutionary pressures. Phylogenetic analyses showed a close relationship between S. vulgaris and other strongyles; however, S. vulgaris clustered independently when compared to 15 Cyathostomins and 2 Strongylins. These findings indicate the potential need for taxonomic revision, with the Strongylin subfamily being reclassified into separate subfamilies to better reflect their evolutionary relationships. Results show strong genetic links to other hematophagous and migratory parasitic nematodes, supporting the utility of S. vulgaris as a model for parasitic nematode research, including human hookworm studies. This study establishes a critical genomic resource for S. vulgaris, advancing our understanding of its biology and pathogenicity. These findings aid in developing diagnostic, therapeutic, and preventative alternatives to current anthelmintic drugs, and this assembly provides the genomic foundation needed for future studies of the most pathogenic parasitic nematode of horses.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.524

Funding Information

This study was supported by the Department of Biochemistry and Molecular Genetics at the University of Louisville and the Clay Fellowship, part of the Gluck Equine Research Foundation at the University of Kentucky, both in 2023.

Share

COinS