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Theileria equi and Babesia caballi infection of equids in Punjab, India: a serological and molecular survey Deepak Sumbria, Lachhman Das Singla & Amrita Sharma

Tropical Animal Health and Production ISSN 0049-4747 Volume 48 Number 1 Trop Anim Health Prod (2016) 48:45-52 DOI 10.1007/s11250-015-0917-1

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Author's personal copy Trop Anim Health Prod (2016) 48:45–52 DOI 10.1007/s11250-015-0917-1

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Theileria equi and Babesia caballi infection of equids in Punjab, India: a serological and molecular survey Deepak Sumbria 1 & Lachhman Das Singla 1 & Amrita Sharma 1

Received: 21 July 2015 / Accepted: 8 September 2015 / Published online: 19 September 2015 # Springer Science+Business Media Dordrecht 2015

Abstract A cross-sectional study was conducted in Submountain undulating, Undulating plain, Western and Western plain agro-climatic zones of Punjab province, India, to determine the prevalence, agreement between diagnostic tests and associated related risk factors of Theileria equi and Babesia caballi infection in equids (horses, donkey, mules). An overall prevalence of 14.14 and 0.0 % of T. equi and B. caballi was recorded by multiplex polymerase chain reaction targeting 18S ribosomal RNA (rRNA) for both the parasites and 75 and 1.11 % by competitive enzyme-linked immunosorbent assay in a representative sample of 180 animals. Only two animals with positive antibody titre from B. caballi and none with PCR indicated T. equi as the predominant haemoprotozoan responsible for equine piroplasmosis in the study area. Among the PCR-positive animals, presence of tick vectors in farm vicinity was the most influential associated with T. equi infection (P=0.002; odds ratio (OR) 9.30; 95 % confidence interval (CI)=3.32–27.10). For animals with higher anti-T. equi antibody titres, strong association of seroprevalence for T. equi was recorded with age, sex, usage, tick infestation and deworming/vaccination status of host animals and farm management strategies. The study has demonstrated the possible absence of B. caballi in both conducive and nonconducive areas of Punjab and demonstrated T. equi as the potential agent of equine piroplasmosis in Punjab.

* Lachhman Das Singla [email protected] 1

Department of Veterinary Parasitology, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141004, India

Keywords Theileria equi . Babesia caballi . Multiplex PCR . cELISA . Punjab . Risk factors

Introduction Equine piroplasmosis (EP) in equids (horses, donkeys and mules) is caused by Babesia (Theileria) equi and/or Babesia caballi, the two intracellular haemoprotozoan parasites of phylum Apicomplexa, order Piroplasmida. The distribution of piroplasmosis depends on the presence of ixodid tick vectors, and the disease is endemic in tropical and subtropical regions (Brüning 1996). The disease has impact on the equid industry worldwide (De Waal, 1992). In India, isolated clinical cases of T. equi have been reported (Chhabra et al. 2011); along with some outbreak (Gautam and Dwivedi 1976) on the other hand, no confirmed case of B. caballi has been reported by molecular and serological methods. The clinical signs are variable and often non-specific (Bashiruddin et al. 1999). It can be peracute, acute, subacute, or chronic (Rampersad et al., 2003). The peracute form is observed in neonatal foals following infection in utero, mainly in T. equi but rarely in B. caballi (Chhabra et al. 2011; Sumbria et al. 2014b); moreover, there is an abrupt onset of signs, which lead to collapse and sudden death. In acute case, animal suffers from fever, inappetence, gastrointestinal disturbance, swaying, depression, pale mucous membranes, or icterus, dyspnoea, anaemia and haemoglobinuria (primarily in T. equi but rare in B. caballi). The subacute form is characterized by anorexia, weight loss, intermittent pyrexia, anaemia, weight loss, limb oedema, lethargy, pink mucus membranes and poor performance. Anaemia may be absent or minimal in the chronic form. For B. caballi, imidocarb dipropionate given subcutaneously at 0.5–1 mg/kg is effective (Soulsby 1982); on the other hand, it can eliminate T. equi from horses at 4 mg/kg

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intra-muscularly on four occasions at 72-h intervals (Berlin et al. 2010), but this dose is lethal to donkeys (Soulsby 1982). The animals may recover from the disease (T. equi) and become long-term carriers (de Waal and Van Heerden 1994). The causative agents cannot be differentiated on the basis of non-specific clinical signs alone. Generally, the disease is routinely diagnosed by conventional parasitological techniques (stained thin blood smears), serological techniques and/or molecular techniques. Conventional techniques are the gold standard method to detect piroplasms in infected equids with acute signs (Böse et al. 1995). Serological methods detect both past and current infections simultaneously and thus used for screening the equids in international trades (OIE 2008). Among all the serological tests, competitive inhibition enzyme-linked immunoassay (cELISA) is the test of choice and is recommended by the World Organization for Animal Health (OIE 2008). This test distinguishes between T. equi and B. caballi (GarciaBocanegra et al. 2013). For the diagnosis of active infection, molecular techniques (PCR) also give the promising result. Diagnosis by PCR has been found sensitive enough to detect parasite DNA from 2.5-μl blood sample with a parasitaemia of 0.000001 %. Thus, for cross-sectional study on epidemiological and carrier status of EP, a combination of serological and molecular diagnostic method tests (Machado et al. 2012; Munkhjargal et al. 2013) was adopted in the present study. The scarcity of enough literature on the epidemiology of these two causative agents of EP in Punjab state province (India) has prompted authors to undertake this pioneer investigation. Moreover, the assessment of associated risk factors may be helpful for epidemiologists as well as clinicians.

Materials and methods Ethical aspects (consent statements) The ethics committee for animal experiments of Guru Angad Dev Veterinary and Animal Sciences University granted an approval (IAEC/2014/46-73) for the conduction of work. Prior consent was taken from the owners of the equids. Complete care and measures were taken to avoid any accidental injury to the equids while collecting the blood samples. Study areas and sampling frame Punjab province of India covers a total area of 50,362 km2 between 29″ 30′ N and 32″ 32′ N latitude, 73″ 55′ E and 76″ 50′ E longitudes and 213 and 959 m altitude (http://punjabjudiciary. gov.in/index.php?trs=gurdist). Punjab state is further stratified into five major agro-climatic zones (Submountain undulating zone, Undulating plain zone, Central plain zone, Western zone, Western plain zone) based on climatic, edaphic and agricultural

Trop Anim Health Prod (2016) 48:45–52

pattern. A representative equid blood sample collection was done from the six districts of four major agro-climatic zones of Punjab. Blood (∼5 ml) was collected from jugular vein of each animal into anticoagulant-coated and non-coated vacutainers for nucleic acid extraction and serum collection. To study the status of molecular prevalence of the concurrent infection, the expected prevalence of 20 % with confidence limits of 95 % and a desired absolute precision of 5 % to collect maximum number of samples was considered (Thrusfield 2005). The number of samples thus calculated was adjusted for finite population and was correlated with 180 samples collected. A predesigned epidemiological questionnaire addressing the horse breed, age, sex and deworming/vaccination; management and presence of ticks; and other domestic animals on farm was maintained to calculate the risk associated with parasitic infection. The farms with inappropriate management system having raw/without concrete (kacha) floor, poor sanitation and open grazing system were classified as ‘unorganized farms’ while those with proper scientific management conditions were considered ‘organized farms’. The animals were thoroughly screened for the ticks especially in ear, mane, groin and dock. Competitive inhibition enzyme-linked immunoassay Serum obtained by centrifuging the collected blood samples (3500 rpm, 10 min) was stored at −20 °C. In order to detect antibodies against T. equi and B. caballi, commercial cELISA test kits based on equine merozoite antigen 1 (EMA1) and rhoptry-associated protein (RAP1) were carried out respectively according to the manufacturer’s instructions (VMRD Inc., Pullman, WA, USA). The optical density values were obtained using an automatic plate reader at 630-nm wavelength. Samples associated with percent inhibition values ≥40 % were considered positives. Calculation of % inhibition (% I) was done by formula: % I=100 [1−(Sample OD÷ Negative Control OD)]. DNA extraction and multiplex PCR assay Genomic DNA was extracted from the blood samples as per the protocol of DNeasy Blood & Tissue Kit (QIAGEN). Reverse primers for T. equi (EquiR: 5′-TGCCTTAAACTT CCTTGCGAT-3′) and B. caballi (CabR: 5′-CTCGTT CATGATTTAGAATTGC-3′) and common forward primer (UFP: 5′-TCGAAGACGATCAGATACC GTCG-3′) specifically targeting 18S ribosomal RNA (rRNA) gene (Abedi et al. 2014) were procured from Bangalore Genei (India) Pvt. Ltd. In the current study, the primers were used for targeting 18S rRNA gene of T. equi as this gene is evolutionarily stable, with limited intra-species sequence variation (Kim et al. 2008). PCR reaction mixture (25 μl) was constituted by 12.5 μl of KAPA2G ® Fast HotStart ReadyMix (2× containing KAPA2G® Fast HotStart DNA polymerase, KAPA2G® Fast

Author's personal copy 26 (14.14) [10.01–20.32]

11.03 10.83*

7 (26.92) [13.70–46.08]

25.28* 20.97*

133 (73.89) [67.02–79.76]

3.94

10.97

2 (1.11) [−0.21 to 2.43] 180

*Significant value

Total

χ2

15 (24.59) [15.51–36.68] 8 (22.86) [12.07–39.02] 0 [0]

0 [0]

0 [0] 54 (88.52) [81.63–95.42] 31 (88.57) [79.48–97.66]

23 (88.46) [77.87–99.05] 26

35 61

Fazilka

Firozpur Western plain zone

4 (14.29) [5.70–31.49]

5 (15.15) [6.65–30.92] 0 [0] 22 (66.67) [52.80–80.53] 33 Muktsar

1 (5) [−4.55 to 14.55] 1 (5) [−4.55 to 14.55] 0 [0]

2 (3.28) [−0.57 to 7.13] 2 (7.14) [−1.08 to 15.37] 9 (14.74) [5.84–23.63]

0 [0]

28

8 (40) [21.49–58.51] 8 (40) [21.49–58.51] 20

Moga

61 Western zone

47 (77.04) [65.09–85.81] 25 (89.29) [72.80–96.26]

1 (2.63) [−2.46 to 7.71] 24 (63.16) [49.93–76.38] 24 (63.16) [49.93–76.38] 38

Submountain Hoshiarpur 38 undulating zone Undulating plain zone SBS Nagar 20

0 [0]

0 [0]

1 (2.63) [−2.46 to 7.71]

Positive (%) [95 % CI] Positive (%) [95 % CI]

Positive (%) [95 % CI]

Mixed sero-prevalence of T. equi and B. caballi Prevalence of T. equi by multiplex PCR Sero-prevalence of T. equi

Each set of the primers was specific for the respective parasite DNA, and non-target DNA amplification was not seen in negative controls. The custom sequence products obtained (accession number LC042536.1) revealed the amplicon length of 430 bp of T. equi amplification product with 99 % identity to T. equi 18S rRNA strain from South Africa (EU642508.1) and 584 bp of B. caballi with 99 % homology with B. caballi genotype B2_CABRBEQ115 18S rRNA gene obtained from blood of equine South Africa (EU642514.1) on the BLASTn analysis. Molecular diagnosis revealed 14.14 % (26, 95 % confidence interval (CI)=10.01–20.32) positivity for T. equi by multiplex PCR, which varied significantly among all the zones. The highest prevalence of T. equi was reported from district Fazilka (26.92 %) of Western plain zone and the lowest from district Hoshiarpur (2.63 %) of Submountain undulating zone (Table 1). The molecular prevalence of T. equi was in a

Total samples examined

Results

Districts

The products obtained by the primers specific for haemoparasite were custom sequenced from Xcelris Genomics, Ahmedabad, India. The nucleotide sequences were subjected to BLASTn analysis (Altschul et al. 1990) for determining the similarity with the sequences present in the nucleotide database. The association of prevalence of haemoparasites with various geographic areas understudy was statistically analyzed by chi-square test at P≤0.05 as the values representing a significant difference. Analysis of risk factor and agreement between the diagnostic tests was done on WinEpiscope software V 0.1.

Zones

Nucleotide sequence and statistical analysis

Table 1

HotStart PCR buffer, 0.2 mM dNTP each, 1.5 mM MgCl2), 1.5 μl of 10 pmol EquiR/CabR primers and 2.5 μl of UFP primer along with 5-μl DNA template suspended in 2 μl of nuclease-free water. The reaction was set in automated thermocycler (Eppendorf AG 2233/Hamburg, Germany) with the following programme: initial denaturation at 96 °C (10 min), 36 cycles of denaturation at 96 °C (1 min), annealing at 60.5 °C (1 min), and extension at 72 °C (1 min) and final extension at 72 °C (5 min). The amplified PCR products were separated by electrophoresis on 1 % agarose gel and visualized under UV Transilluminator (Syngene, InGenius LHR [STIGLER/1394, UK]) for detection of the bands for T. equi (430 bp) and B. caballi (540 bp) (Alhasan et al. 2005). Positive DNA of T. equi and B. caballi was obtained from the National Research Centre on Equines, India, and Department of Pathology and Parasitology, University of Veterinary and Pharmaceutical Sciences, Czech Republic (no confirm report of B. caballi in India), respectively.

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Prevalence of Theileria equi and mixed infection of Theileria equi and Babesia caballi in various agro-climatic zones and districts of Punjab by cELISA and multiplex PCR

Trop Anim Health Prod (2016) 48:45–52

Author's personal copy 48 Table 2

Trop Anim Health Prod (2016) 48:45–52 Comparison results of multiplex PCR and cELISA for the detection of T. equi and B. caballi T. equi PCR (percentage)

T. equi ELISA (percentage) + − Total

B. caballi ELISA (percentage)

+ 22 (12.22) 4 (2.22)

− 113 (62.78) 41 (22.78)

Total 135 (75) 45 (25)

+ 2 (1.11) 0 (0)

− 133 (73.89) 45 (25)

Total 135 (75) 45 (25)

26 (14.44)

154 (85.56)

180

2 (1.11)

178 (98.89)

180

significantly increasing trend from northeast (Submountain undulating zone: 2.63 %, 95 % CI=−2.46–7.71) to southwest (Western plain zone: 24.59 %, 95 % CI = 15.51–36.68) Punjab. No positive reaction was observed by multiplex PCR for B. caballi infection. Out of 180 samples screened, 133 (73.89 %, 95 % CI=67.02–79.76) showed sero-positivity for T. equi by cELISA; the prevalence varies in a significant manner among all the districts and zones. The highest seroprevalence of T. equi was recorded in district Moga (89.29 %) of Western zone and the lowest in district SBS Nagar (40 %)

of Submountain undulating zone (Table 1). The seroprevalence of T. equi was in a significantly increasing trend from north-eastern (Undulating plain zone: 40 %, 95 % CI= 21.49–58.51) to south-western (Western plain zone: 88.52 %, 95 % CI=81.63–95.42) of Punjab. Only two (1.11 %, 95 % CI=−0.21–2.43) samples showed positive titres for mixed infection of both the haemoparasites (Table 1). Out of 180 samples examined, 62.78 % (113/180) samples were ELISA-positive for T. equi, but negative by PCR, 22.78 % (22/180) samples were positive by both techniques,

Table 3 Distribution of variables identified to determinate the risk factors associated with T. equi prevalence (molecular) in equids (horses, donkeys/ mules) in Punjab, India Factor

Climatic factors

Animal (horses, donkeys/mules)

Risk factor

Samples

T. equi (%)

β

SE

P value

Odds ratio (95 % CI)

122 58

−2.112

0.918

0.021

6.87 (1.49–43.66)

Species

χ2 Horses Donkeys/mules

24 (19.67) 2 (3.45) 8.37*

160 20

1.512

0.788

0.055

3.40 (1.25–12.26)

Age

χ2 2 years

19 (11.88) 7 (35) 7.69*

38 142

3 (7.89) 23 (16.20) 1.67

0.873

0.947

0.303

2.25 (0.59–10.05)

Management

Deworming/vaccinations

χ2 Male Female χ2 Organized Unorganized

110 70

9 (8.18) 17 (24.29) 8.98*

1.376

0.570

0.016

3.60 (1.40–9.45)

95 85

1.518

0.493

0.002

4.56 (1.61–13.53)

χ2 Yes No

6 (6.32) 20 (23.53) 16.96*

51 129

6 (11.76) 20 (15.50) 0.41

0.079

0.678

0.907

1.38 (0.48–4.12)

68 112

13 (19.12) 13 (11.61) 1.93

−0.678

0.615

0.270

1.80 (0.72–4.49)

155 25

14 (9.03) 12 (48) 26.45*

−2.104

0.663

0.002

9.30 (3.32–27.10)

105 75

9 (8.57) 17 (22.67) 7.03*

–.096

0.707

0.892

3.13 (1.22–8.18)

χ2 Domestic animals (cattle/buffalo)

Ticks (H. a anatolicum)

Use of equids

*Significant value

Multiplex PCR

High/low Low/high

Temperature/humidity

Sex

Farm practices

Parameter

Yes No χ2 Absent Present χ2 Recreational Commercial χ2

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whereas 2.22 % (4/180) samples were positive by PCR, but were negative by ELISA, and 22.78 % (41/180) samples were negative by both the techniques (Table 4). On the other hand, only 1.11 % (2/180) samples were sero-positive by both ELISAs, while 73.89 % (133/180) samples were positive for T. equi only (Table 2). The results of molecular diagnosis of T. equi when correlated with various physical and biological risk factors revealed a significant association of disease prevalence with various risk factors, namely, species (P = 0.055; odds ratio (OR) 3.40; 95 % CI=1.25–12.26), sex (P=0.016; OR 3.60; 95 % CI=1.40–9.45), management (P=1.082; OR 4.56; 95 % CI= 1.61–13.53), ticks (P = 0.002; OR 9.30; 95 % CI= 3.32– 27.10), and use of equids (P=0.892; OR 3.13; 95 % CI= 1.22–8.18) (Table 3). In case of B. caballi, the sero-positivity was seen only in two animals with the positivity titre in the range of 42.40– 47.74 %. It was observed that, overall, there was no significant Table 4

difference in the risk factor (except the sex, presence of domestic animals on farm and use of equids) on the basis of seropositivity for T. equi, but a notable significant difference was seen in various risk factors, namely, age, sex, management, deworming/vaccinations, ticks and use of equine (Recreational/Commercial) (Table 4, Fig. 1) among the animals classified under high sero-positivity titre group.

Discussion Current investigation is the first epidemiological report on T. equi and B. caballi by molecular and serological methods in Punjab. Multiplex PCR-based diagnosis showed the overall 14.14 % prevalence of T. equi, with no case of B. caballi, whereas by cELISA, 73.89 and 1.11 % prevalence for T. equi and mixed infection of both haemoprotozoans were recorded. These results justify the greater specificity of

Risk factors associated with sero-prevalence range of T. equi

Factor

Risk factor

Climatic factor

Temperature/humidity

Animal (horses, donkeys/mules)

Species

Age

Sex

Farm practices

Management

Deworming/vaccination

Domestic animals (cattle/buffalo)

Parameter

Samples

Use of equids

Total ELISA positive (%)

High/low

122

58 (64)

43 (79)

101 (82.79)

58

χ2 Horses

23 (35) 0.96

9 (49) 7.45*

32 (55.17) 15.54*

160

61 (38.13)

56 (35)

117 (73.13)

Donkeys/mules

20

χ2 2 years

142

χ2 Male

48 (33.80) 1.55

59 (41.55) 4.07*

107 (75.35) 0.75

110

45 (40.91)

25 (22.73)

70 (63.64)

Female

70

23 (32.86) 1.18

40 (57.14) 21.96*

63 (90) 15.41*

χ2 Organized

95

35 (36.84)

33 (34.74)

68 (71.58)

Unorganized

85

χ2 Yes

22 (25.88) 2.49

43 (50.59) 4.62*

65 (76.47) 0.56

51

28 (54.90)

9 (17.65)

37 (72.55)

No

129

χ2 Yes

38 (29.46) 10.19*

58 (44.96) 11.6*

96 (74.42) 0.07

68

31 (45.59)

27 (39.71)

58 (85.29)

No

112

χ Absent Present

37 (30.04) 2.84

38 (33.93) 0.61

75 (66.96) 7.37*

155 25

χ2 Recreational

57 (36.77) 5 (20) 2.68

56 (36.13) 15 (60) 5.13*

113 (72.90) 20 (80) 0.56

105

43 (40.95)

25 (23.81)

68 (64.76)

75

25 (33.33) 1.08

40 (53.33) 16.5*

65 (86.67) 10.88*

Commercial χ2 Total *Significant value

Positivity between 70 and 100 %

Low/high

2

Ticks (H. a anatolicum)

Positivity between 40 and 70 %

180

133

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Trop Anim Health Prod (2016) 48:45–52

Fig. 1 Relative sero-prevalence of T. equi by cELISA among the various districts of Punjab

multiplex PCR over serological method. Highest T. equi infection in arid/semi-arid south-western part of Punjab (Table 1, Fig. 1) contrary to humid conditions of northeastern part may be corroborated with the tick vector proliferation (Haque et al. 2011). As 113 sero-positive animals did not show positive amplification in PCR for T. equi, this may be attributed to the presence of PCR inhibitors or parasite clearance from the circulating blood of the hosts (Allsopp et al. 2007; Bhoora 2009; Bhoora et al. 2010; Baptista et al. 2013). ELISA is an antibody-based test, and it is a well known fact that antibodies persist for many weeks after active infection, but PCR targets the DNA of contemporary parasite. About four samples (2.22 %) of animals were in the early infection period wherein the antibody titres had not reached the detectable levels (Jaffer et al. 2009; Bhoora et al. 2010; Baptista et al. 2013). Sampling time plays a critical role in the detection of circulating parasites, as the prepatent period for T. equi infections is 12– 14 days (Mehlhorn and Schein 1998; Baptista et al. 2013). Only two cases showed low but positive titre for B. caballi, indicating that these equids may have exposure of parasite in their lifetime (Table 2). Furthermore in India, this was the first time that serological testing of B. caballi has been done. The current data should not be generalized unless molecular positivity of B. caballi is confirmed by molecular methods in future studies, as in a recent study in Punjab (Pakistan), sero-prevalence of B. caballi was about 21.6 % (Hussain et al. 2014) so there may be a chance of occurrence of B. caballi in India also.

Analysis of host species as a risk factor revealed that as temperature increases and relative humidity decreases from north-east to south-west of Punjab province, so infection rate increases toward the south-west both by molecular and serological techniques (19.67 and 82.79 %, respectively) because these conditions are favourable for the tick (Hyalomma anatolicum anatolicum) to propagate (Haque et al. 2011). The donkeys/mules possessed significantly higher prevalence of T. equi (35 %) than horses (11.88 %), as, generally, donkeys/mules are kept outdoors under poor living condition for daily transport and farm activities, thereby being more exposed to ticks (Kouam et al. 2010; Sumbria et al. 2015). A similar trend was noted in sero-positive animals for T. equi, though non-significant (Table 4). Moreover, thoroughbred horses receive balanced meals and proper veterinary care and thus are at lesser risk of tick infestation. A tenuous relation association of the disease prevalence was observed with the age of host animals by both molecular and serological methods (Shkap et al. 1998; Karatepe et al. 2009; Mujica et al. 2011; Steinmana et al. 2012); utmost care is taken for equids in Punjab state, as they (only horses) are primarily utilized for breeding and recreational purposes. A contrary scenario has been reported by Rüegg et al. (2007) and Kouam et al. (2010). Significantly higher prevalence of T. equi was observed in mares as compared stallions by both the techniques (24.29, 57.14 and 90 %, respectively) (Tables 3 and 4); the possible reason is the strict living standard provided to the stallion utilized for breeding purposes as also reported by Shkap et al. (1998), Rebiro et al. (1999) and Rüegg et al.

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(2007) elsewhere. Moreover, there is also a partial selection of females over male counterpart for their draught and breeding potential (Sumbria et al. 2014a; Moretti et al. 2010). Regarding various farm practices under study, due to the breech in management practices in the unorganized farm (Table 3), the incidence of direct contact with tick vectors increases resulting in significant amplification of infection (Kouam et al. 2010; Moretti et al. 2010; Abutarbush et al. 2012; Steinmana et al., 2012; Peckle et al. 2013; Sumbria et al. 2015). Similar significant result was noted by serological testing also (Table 4). PCR and serology depicted that herds with no proper deworming/vaccination programs showed high level of infection as reported by Garcia-Bocanegra et al. (2013) (Tables 3 and 4). Overall, in both molecularand serological-positive animals, an elevated trend of T. equi infection was seen when kept with domestic ruminants (cattle/ buffalo); similar results were documented by GarciaBocanegra et al. (2013). By both techniques (Tables 3 and 4), in the present study, tick presence was significantly associated with T. equi infection; these results are concordant with those of Garcia-Bocanegra et al. (2013). Advanced management and disease control programs reduce the chance of infection in equids kept for recreational purposes by both molecular and serological methods (22.67 and 10.88 %, respectively), while open grazing practices in equids used for commercial purposes enhance the chance of T. equi infection (Moretti et al. 2010). In international trading, cELISA is considered as official test because it shows both current and past exposures of the parasite; on other hand, molecular test targets only the DNA of parasite, so for epidemiological studies, combination of both should be used (Munkhjargal et al. 2013) because PCR shows the presence of parasite and cELISA explores the history of animal regarding the disease. Moreover, the current study has depicted the possible absence of B. caballi in both conducive and non-conducive areas of Punjab. Thus, the potent cause of EP in Punjab is T. equi, and this data will be invaluable for the awareness among farmers, equine owners and veterinarian practitioners, for strategizing control measures against this vector-borne disease and for planning preventive therapy to the exposed animals.

Acknowledgments The funds for conducting of work were obtained from UGC project entitled BDevelopment of control strategies based on molecular epidemiology and drug efficacy for equine piroplasmosis in Punjab^ (C-VPS-UGC-24). We are very grateful to Dr. Sanjay Kumar, Principal Scientist, Veterinary Medicine, National Research Centre on Equines, India, and Dr. Moneeb Qablan, Department of Pathology and Parasitology, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 61241, Brno, Czech Republic, for providing positive DNA samples. Conflict of interest The authors declare that they have no conflict of interest.

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