Seroprevalence and immunological characterization of Trypanosoma evansi infection in livestock of four agro-climatic zones of Himachal Pradesh, India

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.


Introduction
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 . T. evansi is considered to have developed from T. brucei brucei, due to alterations in its mitochondrial DNA, and has developed the ability to spread mechanically through diverse vector species such as Tabanus and Stomoxys (Desquesnes et al. 2013;Tehseen et al. 2017). Economic losses due to surra in India were estimated to be US $ 671.1 million owing to several direct and indirect losses such as decrease in milk production, reduced growth, reduced drought power, reproductive losses, death, and additional expenses on treatment and maintenance of affected animals ). Human cases of T. evansi infections have also been reported from different parts of the world including India, adding a new dimension to its epidemiology as a rare zoonosis (Joshi et al. 2005;Chau et al. 2016). Surra is endemic in India and has been reported from different parts of the country (Pathak and Chhabra 2011). However, there are limited reports of surra from Himachal Pradesh, India Yadav et al. 2019). Occurrence of 18 species belonging to the 6 genera of family Tabanidae had been reported from Himachal Pradesh (Maity et al. 2015). Tabanids are potential vectors for mechanical transmission of T. evansi infection (Desquesnes et al. 2013). Himachal Pradesh is an agriculture-intensive state with 89.96% of its population being rural and dependent on agriculture for its livelihood (HP Economics andStatistics Department, Economic Survey 2017-2018). However, due to a lack of organized epidemiological study of surra in the state, the prevalence status of the disease is still obscured. Therefore, the present study was designed to determine the epidemiology of surra in Himachal Pradesh in cattle, buffalo, and equines. The data would provide surra disease prevalence status in the state. This will help in drawing the attention of policy makers of Animal Husbandry Department in the state. Accordingly, formulation of surra control strategies may be helpful in the curtailment of disease cases in the target region.

Study area
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 2000). The average annual rainfall of the state is 1111 mm (Asian Development Bank 2010). Due to differences in elevation (450-6500 m), the climate of the state varies greatly from hot and sub-humid tropical in the southern low tracts to cold, alpine, and glacial in the eastern and northern high mountain ranges. The state covers an area of 55,673 km 2 and has a population of 1,826,290 cattle; 646,570 buffalo; and 34,070 equines according to 20th Livestock Census. In: Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture and Farmers Welfare 2019.
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.

Study animals
Animals included in the study were cattle, buffaloes, and equines of different age and sex groups. True-to-breedspecific 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.

Study design, sampling, and sample size
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 (2005) using the formula for finite population: where n = estimated sample size where z = statistic for a level of confidence using a 95% confidence interval which is 1.96 The expected prevalence was taken as 10% on the basis of previous study . The calculated sample was adjusted for a finite population of samples which correlated with 278 buffalo, 412 horses, and 417 cattle samples.

Sample and data collection
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.

Indirect ELISA
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. (2013). The samples were taken in duplicate, and absorbance readings were taken at a wavelength of 450 nm in Multiscan Plus ELISA Reader (Thermo Fisher Scientific, Finland). Relative percent positivity (RPP) value was estimated by analyzing one positive and one negative control serum in each plate in duplicate. Serum samples that showed an RPP value higher than cutoff value were considered positive for the presence of T. evansi antibodies. An RPP value of 15% was used as a cutoff value to evaluate seropositivity of any of the test serum samples of equines and bovines against T. evansi infection .

Immunoblot
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 1970), using Dual Mini Gel Cast Electrophoresis System (Atto Corporation, Japan) to know the polypeptide profile of WCL antigen. Denatured samples of WCL and protein markers were electrophoresed through stacking gel and subsequently through resolving gel at 50 V and 100 V, respectively. Immunoblotting was done as per method defined by Towbin et al. (1979) using serum samples found positive in antibody ELISA. SDS-PAGE (10%)-electrophoresed WCL antigens were trans-blotted on the PVDF membrane using tris-glycine-methanol transfer buffer in semi-dry transfer apparatus (Atto Corporation, Japan) at a constant current of 0.8 mA/cm 2 for a period of 90 min. The membrane was blocked overnight using skimmed milk 7% (w/v) (SM-PBST), and the blocked membrane was then incubated for 1 h with 1:20 dilution of respective serum samples in SM-PBST at a temperature of 37 °C. Furthermore, the membrane was made to react with anti-species secondary IgG (1:250 dilutions) at 37 °C for 1 h. Immunoreactive bands developed on reaction with positive serum were visualized using 3,3′-diaminobenzidine (DAB) substrate. The substrate reaction was then terminated by washing the membranes with distilled water.

Statistical analysis
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 chisquare 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.

Seroprevalence
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.

SDS-PAGE
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.

Immunoblot
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.

Serum biochemistry
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.

Discussion
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. (2013) reported higher seroprevalence against T. evansi infection among equines from Himachal Pradesh; this difference may be due to screening of less number of samples and non-structured sampling from equines. However, there are also reports of significantly higher prevalence of trypanosomosis in mules as compared to horses in north-western regions of India Parashar et al. 2018;Yadav et al. 2019) which may be attributed to work stress and malnourished conditions as mules are mainly used for drought purpose and reared by socioeconomically poor farmers in India. Furthermore, different climatic conditions in different agro-climatic zones affect breeding of vector flies resulting in variation in seroprevalence against T. evansi in different regions of the country. In cattle and buffaloes, the prevalence of surra was observed to be 22.52 and 23.57%, respectively, with no significant difference, although the prevalence was significantly lower in equines. Cattle and buffaloes are usually kept together in a tie-stall housing system, and due to the interrupted feeding habit of tabanid flies, usually, the whole of the herd gets affected which can be related to the higher prevalence of the disease. On the other hand, the equine population is scattered and mostly employed for transportation, resulting in less contact with other affected animals. In addition, equines are kept loose, and their defensive behavior deters tabanids from biting (Muzari et al. 2010). Furthermore, T. evansi infection in equines is thought to be very fatal, with death of most of the infected animals in early stages (Desquesnes et al. 2013), potentially resulting in the reduction in number of sick animals and the creation of an equine population with minimal signs of infection. 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. 2014). In cattle and buffaloes, no significant difference was found in prevalence between zone 2 + 3 and zone 4; however, prevalence was found to be significantly lower in cattle from zone 1 as compared to zone 2 + 3 and zone 4. Lower prevalence in zone 1 can be attributed to its temperate and dry conditions, which are unfavorable for proliferation of different vectors responsible for transmission of surra. A study on effect of climate variables on vector and prevalence of bovine trypanosomosis revealed higher prevalence of flies of the genera Tabanus and Stomoxys in lowland in comparison to midland and highland regions of Ethiopia (Zekarias et al. 2017). The finding of this study was consistent with previous study showing higher seroprevalence of bovine trypanosomosis in zones with higher temperatures (43.02% in the undulating zone) compared to those with lower temperatures (27.36% in the sub-mountain zone) in Punjab (Singla et al. 2013).
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. 2012;Singla et al. 2013;Singh et al. 2016;Parashar et al. 2018). The possibility of both the sexes being equally exposed to the bites of disease-carrying vectors may explain the similar prevalence between them. In contrary, several authors have reported higher prevalence of surra infection in female than male animals and was linked to numerous stress factors such as pregnancy and lactation in female animals, rendering them more susceptible to infection (Agrawal et al. 2013;Sumbria et al. 2017;Sharma et al. 2019;Dodiya et al. 2020).
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. 2017;Batu et al. 2017;Alanazi et al. 2018 andSingh et al. 2019). Previous researchers have revealed varied results regarding the effect of age on the incidence of surra, which contradicts the current data. Prevalence was reported to be higher in older as compared to younger animals (Payne et al. 1991;Singla et al. 2013;Parashar et al. 2018;Gangwar et al. 2019;Sharma et al. 2019;Dodiya et al. 2020). In contrast, young equines were found to be at significantly higher risk of infection as compared to the adults (Sumbria et al. 2017). In bovines, increasing seropositivity with age could be attributable to antibody persistence due to the chronic nature of disease, rather than an age-specific component.
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. 2011, Chavda et al. 2015. BUN levels rise as a result of parasite damage during pathogenesis, which causes mononuclear infiltration of the renal glomeruli, interstitial nephritis, tubular degeneration, and tissue deterioration in the visceral organs (Hilali et al. 2006;Bal et al. 2014). The mean levels of both AST and ALT/GGT enzymes were found to be significantly higher in T. evansi-infected group of animals. These findings in the present study are in consonance with those reported in previous studies (Sivajothi et al. 2015;Amin et al. 2020). The increase in AST levels might be attributed to tissue damage induced by trypanosomes, as well as the destruction of the parasite by the host immune system, resulting in the release of trypanosomal AST (Takeet and Fagbemi 2009;Pandya et al. 2018a). This rise in ALT levels could be due to hepatic damage caused by the parasite (Akinseye et al. 2020). The glucose levels in infected animals were considerably lower than those in the non-infected group. Significant reduction in glucose levels in infected as compared to the control group is also reported by several workers (Cadioli et al. 2006), Singh et al. (2011), Sivajothi et al. (2015, and Pandya et al. (2018b). This phenomenon of hypoglycemia can be attributed to direct utilization of glucose by the trypanosomes. In addition, fever and hepatocellular damage associated with trypanosome infection which causes an increase in metabolic rate and therefore greater utilization of glucose by the host (Von Brand 1973;Opperdoes et al. 1987). Contrary, Aquino and coworkers (2002) observed no significant reduction in glucose levels of infected animals. The polypeptide pattern of WCL antigen prepared was identical to earlier reports . Major polypeptides found were in molecular weight range of 66.2-38 kDa in addition to some high and low molecular weight polypeptides. These observations are similar to those reported in previous study in which a comparable polypeptide pattern of T. evansi was observed with WCL antigen of buffalo, horse, and cow isolates (Laha and Sasmal 2008). Similarly, major polypeptides of T. evansi of equine isolate were observed in the range of 62-66 kDa, 52-55 kDa, and 41-43 kDa ). Immunoblot analysis of T. evansi-positive field serum of equines revealed major immunogenic polypeptides present in molecular weight range of 62-66 kDa and some minor polypeptide band in the range of 72-25 kDa. The polypeptide clusters identified in our study were similar to those observed by previous researchers (Aquino et al. 2010;Yadav et al. 2013). In bovines, immunodominant polypeptide bands ranging from 85 to 32 kDa were detected. However, the main polypeptide bands seen in all of the bovine serum samples were between 62-66-, 52-55-, and 46-48-kDa range. Immunodominant proteins of molecular weight range of 178-24 kDa were observed with antigen prepared from the T. evansi isolate of horse, cattle, and buffalo (Laha and Sasmal 2008). The 46-48-and 38-kDa bands were recognized in both cattle and buffalo which are similar to those reported by Aquino et al. (2010) mainly in later stages of infection suggesting the chronic nature of the disease in bovines. The presence of these polypeptides in the chronic stages of infection might be due to the release of internal antigens following the destruction of parasites by VSG-specific antibodies (Aquino et al. 2010). The 62-66-kDa polypeptide cluster was found to be recognized by the immune serum of all the infected animals including cattle, buffalo, and equines. Immunodominant bands in a similar molecular weight range have been reported previously in different studies (Giardina et al. 2003;Laha and Sasmal 2008;Aquino et al. 2010;Yadav et al. 2013). Stage of infection, strain variation, and variation in immunological response of different host might be the factors responsible for variation in the detection of immunogenic polypeptides. Detailed studies are required at molecular level to explain these findings including host-parasite interaction, immunogenic characterization of polypeptides at amino acid, and epitope level in different parasitic strains.

Conclusion
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.