Seroprevalence and Risk Factor Associated with Endemicity of Theileria equi Infection in Horses in Rajasthan State, India

Equine piroplasmosis is a tick-borne disease caused by intraerythrocytic protozoa, Theileria equi (Mehlhorn and Schein, 1998) and Babesia caballi (Nuttal and Strickland, 1910). The disease is endemic in the tropical and sub-tropical area of the world (Shkap et al., 1998; Uilenberg, 2006; Vial and Gorenflot, 2006). Multiple tick’s species of genera are transmitting both the protozoa. Hyalomma, Dermacentor, Amblyomma and Rhipicephalus are distributed worldwide and considered as a vector for these protozoa (de Waal, 1992; Ali et al., 1996). These tick species are most prevalent in different Indian climatic zones (Geevarghese et al., 1997). In Indian regions, Hyalomma anatolicum anatolicum transmits T. equi and Theileria annulata in equines and bovine, respectively (Chaudhuri et al., 1969; Bhattacharyulu et al., 1975). Theileria equi infection is endemic in India in many agro-climatic regions (Kumar et al., 2013; Dahiya et al., 2018; Sumbria et al., 2018) and sporadic clinical cases have been reported (Gautam, 1976; Sharma et al., 1982).

Equines infected with piroplasmosis show different symptoms like high fever, haemoglobinuria, pale mucus membrane, icterus, petechial haemorrhage on the nictitating membrane, peripheral oedema and occasionally death (De Waal, 1992; Ambawat et al., 1999; de Waal and van Heerden, 2004; Radostits et al., 2006; Onyiche et al., 2019). The disease condition observes as per-acute, acute, sub-acute or chronic (Uilenberg, 2006; Zobba et al., 2008). In endemic areas, the subclinical condition is more common, as infected equids recover from the disease and become a latent carrier (de Waal and van Heerden, 2004). Transplacental transmission of T. equi infection from latently infected pregnant mare to naïve unborn neonate has been reported (Phipps and Otter, 2004; Allsopp et al., 2007; Georges et al., 2011; Chhabra et al., 2012).

Direct and indirect methods are in vogue for diagnosis of equine piroplasmosis. These methods include microscopic blood smears examination, serological assays, cell culture (microaerophilous stationary phase, MASP technique) and molecular techniques viz. PCR/qPCR assays (Kumar et al., 2009; Tirosh-Levy et al., 2020). Direct diagnosis includes the demonstration of an intraerythrocytic form of protozoa in Giemsa stained blood or organ smear in the acute stage of infection (Nagore et al., 2004). Nevertheless, it is challenging to demonstrate T. equi parasite in blood smears prepared from latently infected equines. Therefore, various serological assays developed to increase diagnostic specificity and sensitivity. These indirect serological methods prescribed for large scale epidemiological and seroprevalence studies on T. equi infection. Office International des Epizooties (OIE) prescribed competitive inhibition enzyme-linked immunosorbent assay (cELISA) for international trade and transportation of equines (OIE, 2004). EMA-2 recombinant antigen of T. equi has widely used in ELISA for seroepidemiological studies (Huang et al., 2003; Kumar et al., 2013).

Theileria equi infection reported to be endemic in Rajasthan (Kumar et al., 2013), but its epidemiological information is limited. This study aimed to investigate the seroepidemiology and risk factors associated with the endemicity of T. equi infection in Rajasthan state in India. 

Animals and sampled area
 
One hundred fifty one horses were screened for equine piroplasmosis. Samples from these animals were collected from different areas of Rajasthan including Ajmer, Barmer, Bikaner, Nagaur and Pali districts (Fig 1A). The animals were sampled as per guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) after due approval from the Institute Animals Ethics Committee (IAEC).


 
Sample collection and serological examination
 
Blood was withdrawn aseptically from the jugular vein and collected in sterile clot activator vacutainers. Serum was separated after centrifugation at 4000 g for 25 min and kept immediately at -20^oC till further processing. Blood smears from each horse were prepared on a glass slide at the time of blood collection and fixed with methanol. Blood smears were stained using Giemsa staining (Himedia Laboratories, India) and processed further for microscopic examination for any evidence of haemoparasites including T. equi (Henry, 1992).

Serum samples were screened by ELISA, developed at the equine piroplasmosis laboratory of National Research Centre on Equines (NRCE) for detection of antibodies against T. equi (Kumar et al., 2013; Bhagwan et al., 2015). Briefly, ELISA plate coated with recombinant protein (rEMA) and incubated overnight at 4^oC. Next day the plate was blocked with 3% BSA-PBS for 1 h followed by washing with PBS containing 0.05% Tween-20 (PBS-T) four times. The test serum samples including control positive or negative were diluted in 1% BSA-PBS (1:200 dilution) and 50 µl added was to ELISA plate in duplicate wells. The plate was incubated at room temperature (RT) for 1 h. Thereafter it was washed with PBS-T. Rabbit anti-horse IgG HRP conjugated antibody was used as secondary antibody. It was diluted in 1% BSA-PBS and added to the wells of the ELISA plate. The plate was incubated at RT for 1 h. After that, the plate was  washed again with PBS-T. The substrate solution of O-phenylenediamine dihydrochloride, OPD (Sigma Aldrich) was prepared as per manufacturer instructions and added to each well of the ELISA plate. The ELISA plate was incubated in the dark for 5 min at RT. The development of orange-brown colour was stopped by adding 50 µl of 3M H2SO4. The ELISA plate was read at an absorbance of 492 nm (OD492) in ELISA plate reader (BioTek, USA). The ELISA OD492 cutoff point was determined by calculating the relative per cent positivity (RPP) as per the following formula. Test serum sample showing RPP >20 was considered as positive.
 
 

 

Haemato-Biochemical analysis
 
Haematological parameters on the collected whole blood were analysed manually. Haemoglobin (Hb); packed cell volume (PCV); total erythrocytes count (TEC) and total leukocytes count parameters (TLC) were estimated as per prescribed methodology. Biochemical parameters such as aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin and gamma-glutamyl transferase (GGT) were analysed using commercial kits (Transasia Biomedicals Ltd., India).
 
Risk factors analysis
 
A customised questionnaire was prepared and information related to sampled area, age, sex and managemental practices were collected from the equine owners at the time of sample collection. The questionnaire also included a description of ‘unorganised farms’ and ‘organised farms’. At ‘unorganised farms’ equine owners practise inappropriate managemental practices viz. stables with kaccha (imperfect and crude) floor, neglected sanitation and unstable feeding programmes; while ‘organised farms’ owners pursue appropriate technical managemental schedule.
 
Statistical analysis
 
The data were statistically analysed and compared by GraphPad Prism version 5.0 software (San Diego California, USA). The association of seroprevalence of T. equi with respects to different sampled geographic areas of Rajasthan and epidemiological risk factors were statistically analysed by Pearson’s chi-square test. 

Microscopic examination of blood smears
       
Stained blood smears were microscopically examined for the presence of T. equi parasites, if any. A total 151 blood smears were examined, only one blood smears was positive for T. equi protozoa, which was clinically infected (Fig 1B).
 
Clinical findings and seroprevalence
 
The symptoms such as fever, haemoglobinuria, mild colic, anaemia and icteric conjunctival mucous membrane were observed in horses with clinical infection by T. equi parasite. Out of 151 serum samples collected from the study area, 75 (49.66%) were seropositive to T. equi infection. In Ajmer area maximum percentage of equids were seropositive to T. equi infection, followed by Nagaur, Bikaner, Pali and Barmer area (Fig 1C).
       
Haemato-biochemical analysis
 
Most of the parameters of. Haemato-biochemical parameters (Hb, PCV, TEC, GGT, ALP and total bilirubin, Table 1) did not differ significantly among apparently healthy and T. equi sero-positive horse. Whereas, values of TLC and AST differ significantly among these two groups (Table 1). However, these haemato-biochemical parameters in T. equi clinically infected horse differ significantly from apparently healthy and T. equi seropositive equines (Table 1). These observations in T. equi clinically infected horse were indicative of anaemic and liver damage condition. 


 
Risk factors analysis
 
Relative risk factors involved in T. equi infected/seropositive equines were analysed (Table 2). In Ajmer area, a more significant number of T. equi seropositivity was observed as compared to the other sampling areas. The equines at Barmer area were at least relative risk (0.083 times) than at Ajmer area for T. equi infection.



Analysis of age-related data indicated higher seroprevalence of T. equi in horses of age 1-5 year. Whereas, ≥  ten years old horses were at least risk. Seroprevalence of T. equi was higher in foals of 4 to 12 months age (n=10). Whereas, neonate foals (0 – 2 and 3 - 4 months) were at least risk.

A high T. equi seroprevalence was observed in female horses (n=63; 54.13%) as compared to male horses (n=12; 34.28%) indicating that females are significantly more at risk than male equine population.

Equine farm management practices influence the incidence of T. equi infection remarkably. The equines reared at the organised farm were significantly at lower risk of contracting infection with T. equi as compared to equines reared at the unorganised farm. Likewise, equines were significantly at less risk (0.503 times) when kept with other animal species than reared without any other animal species. The equines infested with tick-vectors were significantly two times (2.188) more inclination of getting T. equi infection as compared to those who were not infested with ticks. Flooring in the stable is a significant risk factor towards maintaining proper drainage and sanitation. Equines reared at kaccha house were more at risk than equines kept on pacca floor. The risk of getting infected with T. equi decreased significantly in equines, where owners adopted control measures such as anti-tick spray and bathing of equines etc. 

Equine piroplasmosis is the economically significant disease of horses, donkeys, mules and zebras. Tick vectors are ubiquitous and responsible for the spread of parasite in the equine population. A T. equi infected horse showing clinical signs of the disease condition was observed in the present study. Similar clinical observations were recorded by other researchers also (Hailat et al., 1997; Radostits et al., 2006; Balkaya et al., 2010; Garba et al., 2011; Behera et al., 2012; Hussain et al., 2014).

Very high seroprevalence of T. equi infection has been reported from Rajasthan state (Kumar et al., 1997; Kumar et al., 2013). In the present study also very high T. equi seropositivity was recorded in the samples collected from differrnt areas of Rajasthan state (Fig 1). Theileria equi seroprevalence was higher in the horses of Ajmer district followed by Nagaur, Bikaner, Pali and Barmerareas. Ajmer area has the highest livestock density among the sampled region (Livestock Census All India Report, 2019). High livestock density help in the propagation of the infected tick vectors, which may be responsible for the highest seroprevalence of T. equi infection. Livestock density also influences tick biology and tick’s control measures. Similar observation reported by different researchers (Salim et al., 2008; Kumar et al., 2013; Hussain et al., 2014). Decreased haemoglobin concentration, packed cell volume and total erythrocytic counts indicated anaemic condition in T. equi clinically infected horse (Table 1). Theileria equi infection inflicts lipid peroxidation of infected erythrocytes membrane, making it more fragile. This process eventually leads to its lysis (Ambawat et al., 1999). That may be the reason for the anaemic condition of the T. equi infected horse. An increased concentration of liver enzymes (AST, GGT and ALP) in T. equi infected horse (Table 1) is associated with hepatocytes necrosis and centrilobular degeneration. These findings are in agreement with earlier reports (Camacho, et al., 2005; Zobba et al., 2008; Kumar et al., 2008).

Seropositivity of T. equi infection among different regions was analysed based on age, gender and managemental practices (Table 2). The female equine population was more seropositive to T. equi infection as compared to the male’s population. Males (stallion) are more scattered as compared to female (mare) population, as stallion usually used for the breeding purpose only. Hence, the comparatively male equine population is at lesser risk than females.  

We also collected serum samples from a dam and their foals. Theileria equi antibodies observed in neonates (0 to 4 months of age) in the present study, indicating trans-colostrum transfer of specific antibodies. Equine neonates are naïve at birth and they acquire T. equi immunity from colostrum of their preimmune dam (de Waal and van Heerden, 1994; Kumar et al., 2008 ). This passive immunity is transitory and disappears after some time. Kumar et al., (2008) observed antibodies titre in naïve foals up to 63 to 77 days post-foaling. 
  
A significantly higher incidence of T. equi infection at unorganised farms may be attributed to unhygienic management practices e. g. open grazing system, absence of grooming practices etc. This managemental practice increases the probability of getting infested with T. equi infected ticks. (Kouam et al., 2010; Moretti et al., 2010; Abutarbush et al., 2012; Steinman et al., 2012; Peckle et al., 2013). Nevertheless, infected ticks’ infestation is responsible for making the equids T. equi seropositive (Bhagwan et al., 2015).  Kaccha housing conditions are responsible for the propagation of ticks breeding.

Theileria equi is endemic among horses of the Rajasthan region and clinical infection is common. Theileria equi seroprevalence was higher in the horses of Ajmer district followed by Nagaur, Bikaner, Pali and Barmer areas. Significantly, higher incidence of T. equi infection was observed at unorganised stud farms. This managemental practice increases the probability of getting infested with T. equi infected ticks. Risk factors play a vital role in maintaining the endemicity of T. equi infection.

This manuscript is a part of master’s research work of the first author. Authors wish to acknowledge their gratitude to the Director, ICAR-National Research Centre on Equines (Indian Council of Agricultural Research), Hisar, Haryana, India for providing all the necessary facilities for conducting this study and to the Head, Department of Epidemiology and Preventive Veterinary Medicine, College of Veterinary and Animal Science, the Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India for managing the administrative matters of the first author as a student of the department. This study was conducted under Consortium of Research Platform on Vaccine and Diagnostics, a ICAR funded research project.

The authors declare that they have no conflict of interest.

RB and VD performed the experiments. AC collected samples. and KM and SC perform risk analysis, RK performed ELISA. YP performed statistical calculations and prepared the graphs SK conceptualised, designed and supervised the whole study. Also drafted the final version of manuscript. All authors read and approved the final manuscript.