Theileria equi sensu lato genetic diversity and phylogeny: new insights from mitochondrion, apicoplast and nucleus multilocus gene analysis.

Overview

• This study investigates the genetic diversity and evolutionary relationships within the Theileria equi sensu lato group, a tick-borne parasite affecting horses, by analyzing multiple genes from different cellular compartments.
• The findings highlight distinct lineages and sub-lineages that may represent separate species, underscoring the need for refined taxonomy and better diagnostic tools.

Background

• Equine piroplasmosis is a widespread disease impacting horses globally, caused by two main parasite species, including Theileria equi.
• Theileria equi has undergone various taxonomic changes, previously known as Babesia equi, and currently considered part of the Equus group, distinct from Babesia and Theileria genera.
• Genetic studies revealed substantial diversity within T. equi, leading to the recognition of Theileria haneyi as a separate lineage and suggesting multiple cryptic species exist within the broader T. equi sensu lato group.
• This diversity complicates disease diagnosis and international regulations concerning horse movement.

Objectives

• To better understand genetic diversity within T. equi sensu lato by studying multiple genes from different cellular genomes (nuclear, mitochondrial, and apicoplast).
• To clarify phylogenetic relationships among recognized lineages and their sub-divisions.
• To assess implications of this diversity for current molecular and serological diagnostic methods.

Methodology

• Designed primers targeting five genes: hsp70, clamp, and ama-1 (nuclear), cox1 (mitochondrial), and tufA (apicoplast).
• Analyzed 36 samples that were mono-infected with T. equi sensu lato lineages A, C (Theileria haneyi), and E.
• Used multilocus gene sequencing and phylogenetic analyses to confirm lineage separations based on previous 18S rRNA data.
• Conducted phylogenetic and haplotype network analyses on genes used in diagnostics: ema-1 and ema-11.

Key Findings

• Confirmed the division of T. equi sensu lato into lineages A, C (T. haneyi), and E using multilocus data.
• Within lineage A, identified two distinct sub-lineages A1 and A2 across all five genes, with a third sub-lineage A3 supported by nuclear and mitochondrial markers.
• Genetic divergences, especially in mitochondrial gene cox1, suggest these sub-lineages may warrant species status.
• Phylogenetic/haplotype analyses of diagnostic genes (ema-1 and ema-11) showed:
  • Lineage E DNA is not detected by current molecular assays targeting these genes.
  • Sub-lineages A1 and A3 correspond with known wide-ranging ema-1 clades A and B.
  • Sub-lineage A2, mainly found in France, relates to a new ema-1 clade D, also present in Eurasian regions like Croatia, Mongolia, and China.
  • A fifth ema-1 clade E was confirmed as well.
  • Lineage C shows at least two sub-lineages, supported by ema-11 sequence diversity, phylogenetics, and haplotype networks.

Implications

• The study emphasizes that multiple cryptic species exist within the T. equi sensu lato complex, but formal taxonomical changes require more comprehensive global genetic data.
• Recognition of distinct species or sub-lineages is crucial because different lineages may not be equally detectable by current diagnostic tests, affecting disease surveillance and control.
• Diagnostic tools targeting ema-1 and ema-11 genes may fail to detect certain lineages, especially lineage E, which poses challenges for regulatory compliance and international horse movement.
• Understanding the phylogenetic diversity will guide the development of improved molecular and serological tests for equine piroplasmosis.

Conclusion

• Theileria equi sensu lato comprises multiple genetically distinct lineages and sub-lineages, some of which may qualify as separate species.
• Multilocus gene analysis from nuclear, mitochondrial, and apicoplast genomes confirms and refines earlier phylogenetic divisions.
• The study calls for expanded worldwide genetic surveys before revising the taxonomy and adjusting diagnostic tools to ensure accurate detection and control of equine piroplasmosis.