Trypanosoma evansi, a hemoflagellate protozoan parasite, causes wasting disease called surra in wide range of animals. Although the organism has been reported from various parts of India, data generated from organized epidemiological study is still in infancy in majority states of India. In the present study, livestock of Himachal Pradesh, India, was targeted for epidemiological investigation of T. evansi infections. A total of 440 equines and 444 cattle serum samples were collected from four agro-climatic zones. Furthermore, serum samples of 280 buffaloes from three different agro-climatic zones of Himachal Pradesh were also collected and evaluated for the presence of T. evansi infection by indirect ELISA. Data generated showed higher prevalence in buffalo (23.57%) followed by cattle (22.52%) and equines (1.82%). Disease was found to be more prevalent (P < 0.01) in cattle of lower altitude as compared to those of higher altitudes. No significant variation was seen in prevalence of disease on the basis of age and sex of the animals. Serum biochemical analysis revealed increased levels of BUN in T. evansi–infected equines. Levels of liver function enzymes such as ALT/GGT and AST were found to be significantly elevated (P < 0.01) in seropositive animals whereas glucose levels were significantly lower in surra-seropositive animals as compared to seronegative animals. Immunoblot analysis of whole cell lysate (WCL) antigen of T. evansi using surra-seropositive samples of equines showed immunodominant bands in the range of 100–25 kDa. Bovine-seropositive samples recognized polypeptide bands in the range of 85–32 kDa, including protein clusters of 52–55 and 48–46 kDa. Polypeptide cluster of 62–66 kDa was found common in seropositive samples of bovines and equines from all agro-climatic zones. T. evansi was found to be highly prevalent in livestock of Himachal Pradesh, and thus, there is dire need for designing of proper control strategies against surra.
Trypanosoma evansi, a unicellular, hemoflagellate parasite, is responsible for causing a highly debilitating disease termed as surra in various host species and has a significant negative impact on livestock industry (Kumar et al. [
The study was carried out in all the districts of Himachal Pradesh, India (Fig. 1). The state is situated between 30° 22′ 40″ to 33° 12′ 40″ N latitude and 75° 47′ 55″ to 79° 04′ 22″ E longitude in northern India, at altitudes ranging from 350 to 6975 m above sea level (Jithendran and Bhat [
Graph: Fig. 1Different agro-climatic zones of Himachal Pradesh, India
Agro-climatically, the state is divided into four zones, viz. high hills temperate dry zone (zone 1), high hills temperate wet zone (zone 2), mid hill sub-humid zone (zone 3), and sub-mountain and low hill subtropical zone (zone 4). Out of these four zones, distribution of zones 2 and 3 could not be separated into different districts, and thus, samples were collected considering both the zones as a single zone, i.e., zone 2 + 3. Equine and cattle serum samples were collected from all four agro-climatic zones, whereas due to negligible population of buffaloes in zone 1, their samples were collected from the remaining three zones only.
Animals included in the study were cattle, buffaloes, and equines of different age and sex groups. True-to-breed-specific data were not available on cattle, buffaloes, and equines used for sampling in the present study. Equines comprised horses, mules, and donkeys in the study area. In the study area, equines were kept in the traditional extensive system in all the four agro-climatic zones, whereas bovines were mostly reared in the intensive system.
A cross-sectional study was carried out from August 2020 to March 2021 to assess the prevalence of equine and bovine trypanosomosis in Himachal Pradesh. The study animals were chosen using a simple random sampling method. Animals were randomly selected from different regions of different districts without targeting suspected or animal showing clinical signs. Furthermore, it was taken care that samples were not collected from multiple animals from the same owner. The sample size for epidemiological studies was calculated according to Thrusfield ([
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where n = estimated sample size
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where z = statistic for a level of confidence using a 95% confidence interval which is 1.96
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The expected prevalence was taken as 10% on the basis of previous study (Kumar et al. [
Approximately 5 ml of blood was collected in plain vials from jugular vein of each animal under septic conditions for extraction of serum which was stored at – 20 °C for subsequent analysis. The species, age, and sex of selected animals were recorded to assess the risks associated with the prevalence of surra in bovines and equines in the study area. The grouping of animals was done into three age groups: < 2 years, 2–5 years, and > 5 years, keeping in view variation of productive and reproductive performances and economic significance in these age groups.
The collected serum samples were examined for presence of anti T. evansi antibodies by indirect ELISA using whole cell lysate (WCL) antigen prepared from purified trypanosomes. ELISA was performed as per the method standardized by Kumar et al. ([
For characterization of immunodominant antigens, immunoblot analysis was carried out using WCL antigen of T. evansi and serum samples of equines, cattle, and buffaloes. SDS-PAGE was performed under reducing conditions according to the method of (Laemmli [
The biochemical parameters were studied in representative T. evansi–seropositive and T. evansi–seronegative animals from different agro-climatic zones. Serum biochemical parameters such as total protein (TP), albumin (ALB), globulin (GLO), total bilirubin (TBIL), direct and indirect bilirubin (DBIL and IBIL), glucose (GLU), blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) were analyzed using ERBA biochemical diagnostics kits (standard protocol as per supplier). Biochemical analysis was done at standard wavelength using the Automated Clinical Chemistry Analyzer-EM 200 (Transasia Bio-Medicals Ltd., India).
Statistical analysis of all quantitative data was done using Graph Pad Prism software version 8.0.2 (San Diego, CA, USA). Different agro-climatic zones associated with prevalence of T. evansi were statistically compared using the chi-square test. Furthermore, species-wise comparison of prevalence of trypanosomosis in livestock of Himachal Pradesh was also carried out using F test and chi-square test. Differences in the serum biochemical levels of seropositive and non-seropositive groups were compared using descriptive statistics and post hoc t-test. The p values below 0.05 were considered statistically significant.
Out of 1164 (440 equines, 444 cattle, and 280 buffalo) serum samples examined, 174 (14.95%) were found to be positive for antibodies against T. evansi (Fig. 2). Overall, seroprevalence of surra was found to be 1.82% (08/440) in equines (Table 1, Fig. 2). No significant difference was reported in prevalence between horses (3.14%) and mules (1.07%) (Table 1). Prevalence was found to be higher in cattle and buffaloes as compared to equines. Out of 444 cattle tested, 100 (22.52%) were found to be positive for T. evansi antibodies by Indirect ELISA. In buffaloes, 66 were found to be positive among 280 examined with seroprevalence rate of 23.57% (Table 1). In cattle population, significantly higher prevalence was observed in zone 4 (31.13%) and zone 2 + 3 (26.39%) as compared to zone 1 (10.07%). In buffaloes, prevalence was found to be 20.14% in zone 2 + 3 and 26.95% in zone 4. However, in equines, no significant difference was reported in prevalence among different zones (Table 2, Fig. 2). Also, age and sex of the animals were not found to affect prevalence of T. evansi in equines, bovines and cattle.
Graph: Fig. 2Seroprevalence of surra in different agro-climatic zone of Himachal Pradesh, India
Table 1 Species wise comparison of prevalence of surra in livestock of Himachal Pradesh, India
Species Number examined Number found positive % prevalence (range at 95% CI) Chi-square value, df E H M H M H M 2.67, 1NS 0.1021 149 281 05 03 (0.44–5.86) (0.0–2.27) 440 08 (0.57–3.07) 74.93, 1** < 0.0001 Bo (C/B) 724 166 (19.87–25.99) C 444 100 (18.64–26.41) 0.06690, 1NS 0.7959 B 280 66 (18.60–28.54) Total (Bo, E) 1164 174 (12.90–17.00)
E equine, C cattle, B buffalo, Bo bovine, H horse, M mule NS, nonsignificant (P > 0.05), *significant (P < 0.05), **highly significant (P < 0.01)
Table 2 Comparative statement of the prevalence of surra in equine, cattle, and buffaloes of different agro-climatic zones of Himachal Pradesh, India
Zone Number examined Number found positive % prevalence (range at 95% CI) Chi-square value, df E Zone 1 139 02 (0–3.42) 1.207, 2 NS 0.5470 Zone 2 + 3 140 4 (0.10–5.62) Zone 4 161 02 (0–2.95) C Zone 1 149 15 (5.24–14.90) 13.87, 2** 0.0010 Zone 2 + 3 144 38 (19.19–33.59) Zone 4 151 47 (23.74–38.51) B Zone 2 + 3 139 28 (13.48–26.81) 1.115, 1 NS 0.2911 Zone 4 141 38 (19.63–34.27)
E equine, C cattle, B buffalo NS non significant (P > 0.05), *significant (P < 0.05), **highly significant (P < 0.01)
SDS-PAGE analysis of WCL antigen of T. evansi revealed multiple visible polypeptides bands in range of 60–18.5 kDa. Among all these, major polypeptide bands were observed in the range of 66.2–38 kDa. Minor polypeptide bands were seen in the range of 18.5–30 kDa. Closely migrated clusters of polypeptides that were not separated by SDS-PAGE were also found in the range of 40–45 kDa.
On immunoblot analysis, immunodominant bands identified in serum samples from T. evansi–infected equines were in the molecular weight range of 100–25 kDa. Polypeptide cluster of 62–66 kDa was recognized by serum samples of equines from all the zones. Other polypeptide bands recognized were of 57, 62, and 68 kDa from zone 2 + 3 and 7 polypeptide bands of 72, 57, 50, 47, 40, 45, and 25 kDa from zone 4.
Serum samples of cattle and buffaloes from all regions also recognized immunodominant polypeptide cluster of 62–66 kDa. Polypeptide cluster of 55–52 kDa was identified by serum samples of cattle of zone 1 and zone 2 + 3. Serum samples of cattle from zone 2 + 3 and zone 4 detected another polypeptide cluster in the range of 48–46 kDa and 68–70 kDa, respectively. Individual polypeptides recognized by cattle were of approximately 70, 66, 55, 36, and 32 kDa from zone 1; 70 and 40 kDa from zone 2 + 3; and 55, 40, 38, and 35 kDa from zone 4.
Immunodominant polypeptides of 85, 80, 55, 48, 35, 38, and 15 kDa were detected in buffaloes of zone 2 + 3, whereas only 39-, 38-, and 35-kDa bands were observed in serum sample of zone 4 buffaloes. In addition, polypeptide clusters in the range of 52–55 kDa and 68–70 kDa were also recorded in serum samples of buffaloes from zone 2 + 3 and zone 4, respectively.
A total of 110 representative serum samples were analyzed for estimation of biochemical parameters. Thirty samples from equines (5 positive, 25 negative), 50 samples from cattle (7 positive, 43 negative), and 30 samples from buffaloes (9 positive, 21 negative) were evaluated. Animals were clubbed into seropositive and non-seropositive groups for comparative analysis. Serum biochemical values of T. evansi–seropositive and T. evansi–non-seropositive animals (horses, cattle, and buffaloes) are shown in the Table 3. The mean values of ALT and AST in T. evansi–seropositive group were found to be significantly higher as compared to non-seropositive group of cattle, buffaloes, and equines. Mean serum glucose levels in T. evansi–seropositive animals was found to be reduced significantly in comparison to non-seropositive animals. In equines, significant drop in mean albumin level was observed in T. evansi–seropositive animals. In comparison to non-seropositive group, mean serum globulin levels of infected cattle group was found to be significantly higher. Mean BUN value showed a significant rise in T. evansi–seropositive equines as compared to the non-seropositive group.
Table 3 Comparison of biochemical parameters between Trypanosoma evansi serological-positive and serological-negative samples in equines, cattle, and buffaloes
Parameters Equine Cattle Buffalo Serological-negative Serological-positive Serological-negative Serological-positive Serological-negative Serological-positive Total protein 7.09 ± 0.09 7.61 ± 0.42 1.21NS 0.2918 7.21 ± 0.13 8.18 ± 0.29 2.71** 0.0092 6.88 ± 0.13 7.09 ± 0.37 0.52NS 0.6101 Albumin 3.02 ± 0.05 2.56 ± 0.16 3.03** 0.0050 2.74 ± 0.06 2.73 ± 0.13 0.03NS 0.9756 2.58 ± 0.06 2.51 ± 0.11 0.62NS 0.5379 Globulin 4.07 ± 0.08 5.05 ± 0.41 2.34NS 0.0789 4.48 ± 0.106 5.45 ± 0.37 3.20** 0.0023 4.29 ± 0.11 4.58 ± 0.30 0.87NS 0.4008 Total bilirubin 0.27 ± 0.03 0.24 ± 0.07 1.60NS 0.1606 0.124 ± 0.00 0.20 ± 0.08 1.01NS 0.3491 0.07 ± 0.00 0.10 ± 0.04 0.80NS 0.4397 Direct bilirubin 0.09 ± 0.01 0.14 ± 0.07 0.62NS 0.5567 0.04 ± 0.00 0.05 ± 0.01 0.12NS 0.9019 0.03 ± 0.00 0.02 ± 0.00 0.27NS 0.7884 Indirect bilirubin 0.17 ± 0.03 0.19 ± 0.06 1.75NS 0.1300 0.07 ± 0.00 0.15 ± 0.06 1.15NS 0.2924 0.04 ± 0.00 0.08 ± 0.04 0.86NS 0.4074 Blood urea nitrogen 21.0 ± 2.00 50.9 ± 10.5 2.76* 0.050 18.78 ± 1.70 61.3 ± 19.02 2.22NS 0.0674 18.89 ± 2.51 23.75 ± 4.44 1.01NS 0.3206 GGT 20.42 ± 0.60 32.96 ± 1.75 8.04** 0.0000 21.87 ± 1.76 63.57 ± 8.01 5.07** 0.0014 25.40 ± 2.30 60.9 ± 4.90 7.30** 0.0000 AST 138.62 ± 4.75 209.98 ± 17.61 5.48** 0.0000 81.9 ± 4.70 143.2 ± 8.00 6.38** 0.0000 106.81 ± 5.77 150.40 ± 8.36 4.19** 0.0002 Glucose 92.47 ± 1.69 57.34 ± 4.63 8.18** 0.0000 77.85 ± 2.84 48.7 ± 4.76 5.03** 0.0000 82.93 ± 4.05 56.15 ± 6.20 3.61** 0.0011
NS non significant (P > 0.05), *significant (P < 0.05), **highly significant (P < 0.01)
Surra is one of the most important hemoprotozoan diseases of livestock with varying prevalence between countries and regions. The study involved cattle, buffaloes, and equines of different agro-climatic zones of Himachal Pradesh. Overall, seroprevalence of T. evansi shown by indirect ELISA in equines was 1.82%. Among equines, disease seroprevalence between mules and horses was not found to differ significantly in the present study. Contrary to this, Kumar et al. ([
Prevalence of surra in equines from zones 1 (1.43%), 2 + 3 (2.86%), and 4 (1.24%) showed no significant differences. Similarly, no significant variation was observed in prevalence of surra in equines between the Western and Central Plain Zone of Punjab (Sumbria et al. [
In the present study, prevalence of T. evansi infection was not found to be affected by sex of animals which is in agreement with previous studies (Tafese et al. [
Similar to sex, age of the animals was not found to have any significant effect on prevalence of surra in equines, cattle, and buffaloes suggesting that all age groups of animals are equally exposed to and affected by T. evansi. This corroborates previous observations, which demonstrated similar prevalence of surra in all age group animals (Tehseen et al. [
In T. evansi–infected equines, the BUN value was observed to be significantly higher when compared with the non-infected group. This finding is in agreement with earlier reports (Singh et al. [
Overall seroprevalence of surra of 14.95% in livestock of Himachal Pradesh suggested that the disease is endemic in the region, and, consequently, proper control strategies aimed at regular monitoring and treatment of surra infection in identified pockets of Himachal Pradesh should be designed and implemented in order to prevent further spread and economic losses caused by this disease. Also, the additional studies for characterization of immuno-dominant antigens of T. evansi using antibodies of different host species may help in better understanding of the host-parasite relationship and provide some clarity regarding immunogenicity and pathogenicity of this parasite. The specific immunogenic polypeptides discovered from the parasite could be tested further for application in the diagnosis of animal trypanosomosis.
The authors gratefully thank the Director, ICAR-National Research Centre on Equines, Hisar, India, for providing all the necessary facilities for conducting this study. The authors are highly grateful to the Director, Animal Husbandry, Himachal Pradesh, and all the members of Disease Investigation Laboratory, Shimla and Mandi (Himachal Pradesh), for their support and cooperation.
RK and SK designed the research proposal, DS and KS conceived the research. SG helped in preparation of the draft of the manuscript. All authors have read and approved the manuscript.
The financial support from ICAR, New Delhi, is duly acknowledged.
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
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Prior approval was taken for animal experimentation in the present study from the Institutional Animal Ethics Committee of ICAR-NRCE, Hisar (NRCE/CPCSEA/2019–20 dated 15.02.2020 and NRCE/CPCSEA/2020–21 dated 20.01.2021).
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The authors declare no competing interests.
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