Abstract
Background:
Equine piroplasmosis (EP), caused by intraerythrocytic parasites (Babesia caballi and Theileria equi), is an emerging equine tick-borne disease of great veterinary significance to equine health globally.

Aim:
The present study aimed to investigate the molecular prevalence and associated risk factors of EP in Punjab, Pakistan.

Methods:
A total of 600 equines (n = 600), consisting of horses (n1 = 200), mules (n2 = 200), and donkeys (n3 = 200), were examined year-round in the districts of Sargodha and Mandi Bahauddin, Punjab, Pakistan, using microscopy, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR).

Results:
Overall PCR-confirmed positivity % was 45.0% (270/600), while ELISA and microscopy detected 56% (336/600) and 33.0% (198/600) positivity, respectively. There was no noticeable difference in the positivity % of PCR between equine species (p > 0.05). Analysis of district-level data showed significantly higher PCR positivity in Sargodha than in Mandi Bahauddin (p = 0.049). Seasonal variation was significant (p < 0.001), with PCR positivity highest in spring (62.8%) and lowest in winter (20.0%), whereas ELISA seropositivity peaked during summer (65.8%) and was lowest during winter (17.3%). Univariate analysis revealed significant differences in PCR positivity across gender, age group, gelding status, and stress (p < 0.05). Multivariable logistic regression identified stress as a consistent independent predictor of PCR-confirmed infection across all species, while seasonal effects remained significant in several species-specific models (p < 0.05).

Conclusion:
The current study demonstrated that stress and seasonal factors play an important role in the epidemiology of equine piroplasmosis. PCR, ELISA, and microscopy were useful diagnostic tools; however, PCR remained the most definitive method for EP detection.

Key words: Equine piroplasmosis; PCR; Prevalence; Risk factors.