Comprehensive molecular characterization of Theileria equi: Detection, genetic diversity, and risk factors assessment.

Overview

• This study conducted a thorough molecular investigation of Theileria equi, the parasite responsible for equine piroplasmosis, focusing on detecting infection rates, assessing genetic diversity, and identifying key risk factors associated with infection in horses, particularly in India and in a global context.

Study Background and Objectives

• Equine piroplasmosis is a tick-borne disease affecting horses worldwide, caused mainly by Theileria equi.
• While global studies are abundant, genetic diversity and molecular characterization of T. equi in India have been underexplored.
• The study aimed to detect the presence of T. equi in horses, characterize its genetic diversity using molecular tools, and identify risk factors contributing to infection.

Methods and Molecular Detection

• Blood samples were collected from 306 horses for analysis.
• Two diagnostic methods were used:
  • Microscopy to detect parasites, resulting in a 10.78% infection rate.
  • Polymerase Chain Reaction (PCR), a more sensitive technique, resulting in a 21.24% infection rate.
• Universal primers targeting the 18S rRNA gene, which is conserved across T. equi and Babesia caballi (another piroplasm), were applied for initial amplification.
• Species-specific primers then amplified a 435 base-pair fragment specific to T. equi for confirmation.
• Positive PCR samples were sequenced to analyze genetic similarity and diversity.

Genetic Diversity and Phylogenetic Analysis

• Sequence analysis showed high genetic similarity among Indian T. equi isolates (99.52-100%) and globally (95.89-100%).
• All Indian isolates belonged to genotype A, indicating a possibly limited genetic pool in that region.
• Phylogenetic trees based on:
  • 18S rRNA gene
  • EMA-1 gene (a known T. equi antigen gene)

  confirmed consistent genotype classification and genetic relationships.

Global Genetic Diversity Insights

• A median-joining network analysis of the 18S rRNA gene sequences from global samples identified 46 distinct haplotypes.
• Hap_1 was the most common haplotype and clustered by geographic region, suggesting localized evolutionary patterns.
• Distribution of haplotypes by country:
  • Brazil: 13 haplotypes (highest diversity)
  • Sri Lanka: 11 haplotypes
  • Israel: 9 haplotypes
  • South Africa and China: 6 haplotypes each
  • Switzerland: 4 haplotypes
  • India: 3 haplotypes (limited diversity)
• The star-like shape of the network suggests a rapid population expansion and recent emergence of new haplotypes worldwide.
• Genetic diversity metrics:
  • Low overall nucleotide diversity (π = 0.172 ± 0.007): indicates small differences in genetic sequences at the nucleotide level.
  • High haplotype diversity (Hd = 1.0 ± 0.005): indicates many different haplotypes exist despite small differences between them.

  This pattern reinforces the idea of recent rapid expansion with minor diversification.

Population Structure Analysis

• Analysis of Molecular Variance (AMOVA) quantified genetic variation:
  • 78.61% of genetic variation occurred between genotypes.
  • 21.39% of variation occurred within genotype groups.
• Fst values higher than 0.33 indicated significant genetic differentiation and population structure among T. equi groups globally.

Risk Factor Assessment for T. equi Infection

• A logistic regression model was used to identify factors significantly associated with T. equi infection in horses.
• Significant predictors included:
  • Summer season (Odds Ratio (OR) = 3.129; p = 0.012): higher infection risk during summer.
  • Tick infestation (OR = 2.988; p = 0.031): presence of ticks increased risk nearly threefold.
  • Presence of ruminants on the farm (OR = 5.865; p = 0.004): farms with cattle or goats had significantly higher risk, possibly due to shared tick hosts.
  • Anaemia in horses (OR = 4.231; p < 0.001): infected horses were more likely to exhibit anaemia, a clinical sign of the disease.
  • Red urine (OR = 4.892; p = 0.017): a symptom reflecting possible hemolysis caused by infection, linked to increased infection likelihood.
• Non-significant factors (no statistical association with infection):
  • Rainy season
  • Age and sex of horse
  • Management practices
  • Body condition score

Conclusions and Implications

• The study highlights a moderate prevalence of T. equi infection in Indian equids, with molecular tools revealing greater detection sensitivity than microscopy.
• Genetic diversity analyses demonstrate that Indian isolates are genetically uniform compared to higher diversity in other regions, suggesting localized control or recent introduction.
• Global haplotype distribution implies rapid recent expansions and geographical structuring of T. equi populations.
• Environmental and management factors identified as risk contributors can inform targeted control strategies, such as tick control during summer and management of mixed-species farms.
• The findings contribute essential baseline data for disease monitoring, vaccine design, and management of equine piroplasmosis.