Total 295 blood samples from the horses were collected for diagnosis of equine piroplasmosis through cELISA test. The blood was collected from jugular vein of horses into a serum clot activators. The blood was centrifugated at 30003500 rpm for 15-30 minutes. The serum was separated and stored at−20°C until further use for performing cELISA.

The stored serum samples were assessed for the presence of antibodies to T. equi and B. caballi using a commercial cELISA test kit (VMRD, Inc., Pullman, USA) methods following the manufacturer’s instructions. The optical density (OD) values of the controls and samples were measured at 630 nm wave length using an automatic microplate reader (Cyberlab, R01, USA) and the percentage of inhibition (%) was calculated as follows: I (%) =100-(sample OD ×100)/ (mean OD of three negative controls). Serum samples with ≥40% inhibition were considered positive and samples with <40% inhibition were considered negative as recommended by the manufacturer guidelines.

Overall prevalence of piroplasmosis by cELISA is presented in table 1. In our study, the overall infection rate of EP was 62.71% (185/295) amongst them 61.69% (182) of T. equi infection whereas 1.02% (03) of horses found positive for B. caballi through cELISA. The low prevalence of piroplasmosis from Gujarat state has been reported as 16.67% by Khurana et al. (2014), and 11.52% by Vidhyalakshmi et al. (2018). Similar studies conducted in other parts of country where they observed that seroprevalence of T. equi in horses was 32.65 % (Kumar et al., 2013), 24.66% (Khurana et al., 2014), 49.66% (Bhojani, 2016). The reason for higher prevalence of T. equi in this study might be the antibodies against T. equi are usually remains life-long whereas antibodies against B. caballi generally remain for 4-5 years. (Rüegg et al., 2008).

Breed wise distribution of piroplasmosis by cELISA is presented in table 1. Sero-prevalence of piroplasmosis infection in this study was found significantly (P<001) associated with different breeds of horses. Non-significant difference of equine piroplasmosis according to breed of horses has been reported by Dodiya et al. (2016) and Bharai et al. (2020) from Gujarat. These differences could be due to different management conditions and practices adopted by horse owner (Sevinc et al., 2008). Elisa et al. (2016) stated that native breed of horses and mixed breed ones showed 3 times and 3.16 times respectively more risk to be seropositive to T. equi by cELISA than foreign breed horses. Higher prevalence of equine piroplasmosis in non-descript breed of horse as observed in the present study may be due to the larger population in studied area than any other breed. Breed wise prevalence of infection might also be depended on immune status of animals and management practice followed by the owner.

Age wise distribution of piroplasmosis by cELISA is presented in table 1. The age-wise prevalence was found to be non significant. Our findings were contradictory to Bharai et al. (2020) who reported that in Gujarat state the age wise prevalence of equine piroplasmosis in horses were higher in adult horses (68.29%) followed by yearlings (17.07%) whereas Dodiya et al. (2016) carried out a retrospective survey and reported that the age wise incidence of equine piroplasmosis was higher in adult group (2-18 years) of horses compared to other aged groups from Saurashtra region of Gujarat. Bhojani (2016) carried out epidemiological survey for equine piroplasmosis in Rajasthan (India) and confirmed that seroprevalence of equine piroplasmosis was higher in 1-5 year age group followed by 6-10 years age group. However, other epidemiological studies reported that the age wise prevalence of equine piroplasmosis in horses were older than 5 years were more likely to be seropositive for T. equi by Turaki et al. (2014), 2 to 4 years horses by Bahrami et al. (2014), between 6 and 10 years of aged horse by Hussain et al. (2014). Moreover, Ebrahimi et al. (2018) also reported prevalence of T. equi was higher in older animals than young equines.

Sex wise distribution of piroplasmosis by cELISA is presented in table 1. Gender was a definite risk factor, with females showing more positivity (63.90%) to equine piroplasmosis than males (61.11%). However, the sex/ gender wise prevalence was statistically non-significant. Similarly, Salib et al. (2013) and Turaki et al. (2014) reported a higher sero-positivity in stallions/male as compared mares which could be due to the different level of care, grooming and attention these animals were getting according to their importance for owners. However, mare is more prone to EP than stallion has been reported by Javed et al. (2014), Dodiya et al. (2016), Ebrahimi et al. (2018), Zeynel and Bekir (2019) and Bharai et al. (2020). Moreover, Bahrami et al. (2014) reported that both male and female horses have the same susceptibility of infection. The numerically higher sero-positivity found in females as compared to males in our study could be due to the large proportion of female equines enrolled for sampling as compared to males.

The risk factor associated data with equine piroplasmosis are presented in table 2.

Highly significant (P<0.01) difference was observed in pacca house and tick control adopted by owner compared to kaccha house and tick control not adopted by owner Moreover, there was no any significance differences observed in organized farm and unorganized farm, kept with other animal species and kept without other animals. Similar studies have been conducted by Bhojani (2016) who reported comparable data of risk factors associated with T. equi infection. Other study suggested that chances of contracting the piroplasmosis more higher in tick infestation major and equines have greater interaction with each other or other domestic animals which favors the multiply of tick infestation (Elisa et al., 2016, Ebrahimi et al., 2018 and Ali et al, 2019).