Prevalence of Theileria equi and Babesia caballi Infections in Horses in South Gujarat, India

Equine piroplasmosis (EP) is a tick-borne disease caused by Babesia caballi and Theileria equi that affects horses, mules, donkeys and zebras. Both parasites are transmitted by ticks of genera Dermacentor, Rhipicehalus and Hyalomma. The disease is reported from many parts of India (Sanjeev et al., 2020). In 2004, the OIE approved the competitive Enzyme Linked Immunosorbent Assay (cELISA) for detection of antibodies against T. equi and B. caballi and as a specified test for global horse activity (OIE, 2014).

The prevalence rates of the T. equi were earlier reported by many workers in Gujarat and adjoining states using blood smear examinations and PCR (Sumbria et al., 2016, Vidhyalakshmi et al., 2018; Bharai et al., 2020; Sanjeev et al., 2020). However, a different level of sero-prevalence of antibodies against T. equi was documented from various states of India (Khurana et al., 2014; Dahiya et al., 2018; Bhojani et al., 2021). There is no previous serological study focusing on the occurrence of T. equi infection in horses of south Gujarat in India, therefore, present study was conducted to determine the prevalence of T. equi and B. caballi in horses by blood smears and to identify the presence of T. equi antibodies in the serum of horses with cELISA.

Sample collection
 
The present study was conducted from March, 2016 to March, 2020. Blood samples from horses were collected using vacutainer tubes with or without anticoagulant. The samples were transported under refrigeration to the laboratory and blood smear were prepared. Samples were finally stored at -20^oC for molecular analysis.
 
Serological detection of T. equi and B. caballi by cELISA
 
The blood was collected from jugular vein of horses into a serum clot activators. The blood samples were centrifuged at 3000-3500 rpm for 15-30 minutes. The serum was separated and stored at -20^oC until 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) following the manufacturer’s instructions. The optical density (OD) 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:

Serum samples with ≥40% inhibition were considered positive and samples with <40% inhibition were considered negative as per the manufacturer guidelines.
 
Molecular detection of T. equi by PCR
 
Total 90 blood samples were selected randomly from the horses showed seropositivity. The DNA was extracted from blood samples as per the protocol outlined in the user manual. VetPCR™ detection Kit (VMRD, Inc., Pullman, USA) was used to detect the DNA of T. equi and B. caballi.
 
The products obtained were subjected to electrophoresis (100 volts, 45 minutes) in agarose gels at 2% in TBE with GelRed. Samples that showed amplificationproducts with a size of 499bp (T. equi) were considered as positive (Fig 1). Reaction mixture was prepared for sample, positive control, negative control and internal control by combining the reagents as shown in Table 1 and PCR cycling protocol in Table 2.






 
Statistical analysis
 
The data were subjected to statistical analysis using chi-square test and R-software version 3.6.3 as per the method described by Snedecor and Cochran (1994).

Microscopic examination of blood smears
 
Blood smears were shown to have a low sensitivity (1.35%) to detect Babesia and Theileria parasites, although it is a simple, inexpensive and useful tool to diagnose the disease in the field. Identification of equine piroplasmosis in carrier animals by means of blood smear examination is difficult, inaccurate and not practical, on large-scale; serological methods are preferred (OIE, 2014). Prevalence studies conducted in other areas of India reported different levels of positivity as 4.17 % from Punjab (Deepak et al., 2016), 5.05% from Gujarat (Vidhyalakshmi et al., 2018), 5.71% from Republic of Guinea and India (Diallo et al., 2018), 6.97% from Mathura, U.P. (Sanjeev et al., 2020), 63.41% from Junagadh, Gujarat (Bharai et al., 2020) and only one horse found positive out of 151 blood smears for T. equi using blood smear examination (Bhojani et al., 2021).
 
cELISA for detection of T. equi and B. caballi
 
In the present research, the overall prevalence of equine piroplasmosis was 62.71% (185/295) by cELISA, out of which 61.69% (182/295) for T. equi and 1.02% (03/295) for B. caballi (Table 3). Similarly, very law 0.55% (01/182) prevalence of B. caballi was reported using cELISA in Gujarat by Vidhyalakshmi (2015) whereas Khurana et al., (2014) carried out sero-surveillance of T. equi on 291 samples collected from various states of India and overall prevalence of T. equi was 24.66%. Bhojani et al., (2021) carried out a cross-sectional study on the seroprevalence of T. equi using cELISA in 151 horses from Bikaner, Rajasthan state, out of which 75/151 (49.66%) were found positive for T. equi. The high prevalence of piroplasmosis might be due to the carrier animals responsible for the maintenance of the infection. Warm humid environment favours the tick vectors and high incidence of disease (Patel et al., 2013). The highest positive sample for T. equi by cELISA in the present study could be due to presence of antibodies of particular parasites in animal’s body. These findings are suggestive and supportive that animals might have come in contact with the parasite and developed antibodies approximately ten days after post-infection. Antibodies against B. caballi usually last about four years and in the case of T. equi, developed antibodies remain for life as reported by De Waal (2004).


 
PCR for detection of T. equi and B. caballi
 
Out of 90 seropositive horses screened for T. equi by PCR, 9 horses were found positive indicating an overall prevalence rate of 10.00% by PCR while none of the samples showed the presence of DNA for B. caballi. (Table 3). It was in agreement with that of Vidhyalakshmi et al., (2018) who reported 11.52% horses positive for T. equi from Gujarat State by PCR method. In contrarily to our study, 33.33% prevalence of Theileria species in equines had been reported by (Deepak et al., 2016) from Punjab state. Whereas, 48.14% for B. caballi and 29.63% prevalence rates for T. equi were reported from Saurashtra region of Gujarat by Bharai (2018). Comparable findings were accounted in area of Mathura (Uttar Pradesh) and boarder areas of Rajasthan by Sanjeev et al., (2020) who recorded 10.46% prevalence rate of T. equi by PCR. Similarly, Diallo et al., (2018) carried out molecular analysis of T. equi from various agro-climatic zones of Punjab and reported overall 8.57 % of equid samples positive for T. equi by primary PCR. Moreover, high prevalence of T. equi through PCR in other parts of world had been reported as 27.7% by Ebrahimi et al., (2018) from Iran, 66.0% by (Montes Cotesa et al., 2019) from Spain and 50.00% by Sharon et al., (2020) from Israel.      

cELISA is useful as an efficient diagnostic assay to determined T. equi and B. caballi antibodies from carrier horses. PCR was more useful diagnostic assay for the detection of T. equi than blood smear examination. Further research is needed to compare different diagnostic techniques in different regions, to identify the specific piroplasm and to produce geographic distribution maps of equine piroplasms in Gujarat and elsewhere for better understanding the epidemiology of equine piroplasmosis.