Cepharanthine, a bioactive alkaloid dervived from the plant Stephania cephalantha has recently gained significant attention for its promising therapeutic potential. In view of the increasing resistance and toxicity reports associated with current treatments for Trypanosoma evansi, the present study evaluated the anti-trypanosomal activity of different concentrations of Cepharanthine against T. evansi under both in vitro and in vivo conditions. Cepharanthine exhibited significant anti-trypanosomal activity, with an IC50 value of 1131 nM at 24 h. Additionally, high specific selectivity indices (SSI) of 78.84 on Vero cell line and 107.78 on equine PBMCs, indicates high safety profile. In the mice model, Cepharanthine cleared parasitemia within 10 days and extended survival period of infected mice by more than 30 days when administered at a dose of 12.5 mg/kg body weight, followed by additional dosages at 48- and 72-hour intervals. In transcript analysis, the upregulated mRNA expression of trypanothione reductase (2.15- fold, p < 0.05) and aurora kinase (1.27-folds, p < 0.05) suggested the disturbance in redox homeostasis and cell cycle regulation by Cepharanthine. On the other hand, trans-sialidase, oligopeptidase, arginine kinase, topoisomerase II, calcium ATPase 1 and ornithine decarboxylase remained unaffected after 24 h of exposure. Collectively, these findings indicate that Cepharanthine exert potent anti-T. evansi activity and may serve as safer alternative to existing conventional anti-trypanosomal drugs against T. evansi.

Trypanosoma evansi, the causative agent of Surra, is a significant emerging zoonotic kinetoplastid protozoan parasite with wide host range and limited chemotherapeutic options. Arginine kinase is mainly responsible for providing metabolic plasticity and maintaining cellular homeostasis of trypanosome under adverse conditions. This study investigated the in vitro anti-trypanosomal activity of Vanilloid derivative, Capsaicin against T. evansi and its effect on mRNA expression of arginine kinase enzyme in T. evansi. Herein, we determined the anti-trypanosomal activity of Capsaicin against T. evansi and its safety towards mammalian cells by estimating the selectivity index. From the dataset generated during the experiments, the IC50 values of capsaicin was determined as 115 µM, respectively. Cytotoxicity assays showed that Capsaicin exhibited moderate cytotoxicity against equine PBMC and Vero cells line with CC50 value of 309.8 µM and 308.3 µM, respectively giving a selectivity index of around 2.68 at the therapeutic dose against T. evansi. Real-time PCR analysis revealed that Capsaicin showed significant alteration of mRNA expression of arginine kinase 1 gene at 24 and 48 h of exposure with IC50 of drug. The study suggests that Capsaicin may be a potential approach for treating T. evansi infections, warranting further in vivo investigations.

We report on the in vitro and in vivo success of Imidocarb dipropionate (IDP), an aromatic diamidine compound, as an effective drug against Surra-causing organism, Trypanosoma evansi. IDP is a clinically acclaimed drug for the treatment of babesiosis and anaplasmosis. We have also investigated cytotoxic effect of IDP on equine Peripheral mononuclear cells (PBMCs) and Vero cell line. The data generated indicate that the IC50 of IDP (2.73 µM) was significantly lower than its cytotoxic concentration (32.66 and 80.70 µM against PBMCs and Vero cell line, respectively). We have recorded significant downregulation in mRNA expression of genes responsible for cellular regulatory activities such as cell division, glycolysis, nucleic acid synthesis, immune evasion strategy and redox homeostasis. During mice experimentation, dose of 20 mg/kg body weight was found to prolong the survival period of T. evansi infected mice by 20 days following two subsequent intra-peritoneal injections administered 72 h apart. Hematological, biochemical and histopathological investigations showed the effect of IDP on mice kidney and liver at four times multiples of the therapeutic dosage. Further, clinical trials on higher-order organisms are required before recommendation of IDP against T. evansi in the clinical host.

The monomorphic, non-cyclic, extracellular haemoprotozoan parasite, Trypanosoma evansi leads to Surra disease in domesticated animals. Currently, diminazene aceturate (DA) and isometamidium chloride (ISM) are the most used chemotherapeutic agents for the treatment of Surra in animals. There is still little knowledge on the anti- trypanosomal mechanism of action of DA and ISM. The work addresses a significant gap in the understanding of the anti-typanosomal mechanism of DA and ISM by investigating their effects on mRNA expression profiles of 13 genes of T. evansi. The half maximal inhibitory concentration (IC50) of DA and ISM for a pony isolate of T. evansi was estimated as 335.3 nM and 308.6 nM, respectively. Transcript analysis of DA and ISM exposed T. evansi population showed its effects on the metabolic machinery of T. evansi by down-regulating the mRNA expression of all the 13 targeted genes. However, ISM exposure did not affect mRNA expression of Expression site-associated genes 8 (ESAG8), oligopeptidase B and ornithine decarboxylase genes. The finding provides valuable insights into the molecular action of these drugs, which is crucial for developing more effective treatment of Surra disease. Further, comprehensive transcriptome and proteomic analysis could provide a deeper insight into precise molecular pathway of these medications against T. evansi.

Trypanosoma evansi, a hemoflagellate poses huge economic threat to the livestock industry of several countries of Asia, Africa, South America and Europe continents of the world. Limited number of available chemical drugs, incidents of growing drug resistance, and related side effects encouraged the use of herbal substitutes. In the present investigation, the impact of six alkaloids of quinoline and isoquinoline group was evaluated on the growth and multiplication of Trypanosoma evansi and their cytotoxic effect was examined on horse peripheral blood mononuclear cells in an in vitro system. Quinine, quinindine, cinchonine, cinchonidine, berbamine and emetine showed potent trypanocidal activities with IC50/24 h values 6.631 & PLUSMN; 0.244, 8.718 & PLUSMN; 0.081, 16.96 & PLUSMN; 0.816, 33.38 & PLUSMN; 0.653, 2.85 & PLUSMN; 0.065, and 3.12 & PLUSMN; 0.367 & mu;M, respectively, which was comparable to the standard anti-trypanosomal drug, quinapyramine sulfate (20 & mu;M). However, in the cytotoxicity assay, all the drugs showed dose dependent cytotoxic effect and quinine, berbamine and emetine showed selectivity index more than 5, based of ration of CC50 to IC50. Among the selected alkaloids, quinidine, berbamine and emetine exhibited higher apoptotic effects in T. evansi. Likewise, drug treated parasites showed a dose-dependent and time-dependent increase in reactive oxygen species (ROS) production. Therefore, increased apoptosis in combination with ROS generation could be responsible for the observed trypanocidal effect which could be further evaluated in T. evansi-infected mice model.

Current chemotherapy against the Surra organism, Trypanosoma evansi has several limitations in terms of efficacy, toxicity, availability and emerging resistance. These reasons make the search of new chemo-preventive and chemo-therapeutic agent with high potency and low toxicity. Alkaloid phyto-molecules, berberine has shown promising anti-kinetoplastid activity against T. cruzi, T. congolense, T. brucei, Leishmania donovani and L. tropica. However, till date, there is no investigation of therapeutic efficacy of berberine chloride (BC) against T. evansi. The IC50 value of BC for growth inhibition of T. evansi at 24 h of culture was calculated as 12.15 µM. The specific selectivity index (SSI) of BC was calculated as 19.01 and 10.43 against Vero cell line and Equine PBMC's, respectively. Thirteen drug target genes affecting various metabolic pathways were studied to investigate the mode of trypanocidal action of BC. In transcript analysis, the mRNA expression of arginine kinase 1 remained refractory to exposure with BC, which provides metabolic plasticity in adverse environmental conditions. In contrary, rest all the drug target gene were down-regulated, which indicates that drug severely affect DNA replication, cell proliferation, energy homeostasis, redox homeostasis and calcium homeostasis of T. evansi, leading to the death of parasite in low concentrations. It is the first attempt to investigate in vitro anti-trypanosomal activity of BC against T. evansi. These data imply that phytochemicals as alternative strategies can be explored in the future as an alternative treatment for Surra in animal.

The kinetoplastid protozoan parasite, Trypanosoma evansi causes a fatal disease condition known as Surra in equines throughout the globe. Disease condition being acute in nature, entrust a huge economic and health impact on the equine industry. Till date, quinapyramine methyl sulphate (QPS) is the first line of treatment and a panacea for the T. evansi infection in equines. Still after the >70 years of its discovery, there is no clue about the mode of action of QPS in T. evansi. The establishment of in vitro cultivation of T. evansi in HMI-9 media has provided opportunity to study the alteration in mRNA expression of parasite on exposure to the drug. With this research gap, the present study aimed to investigate the relative mRNA expression of 13 important drug target genes to elucidate the anti-trypanosomal activity of QPS against T. evansi. The IC50 of QPS against a pony isolate of T. evansi was determined as 276.4 nM(147.21 ng/ mL) in the growth inhibitory assay. The in vitro cultured T. evansi population were further exposed to IC50 of QPS and their relative mRNA expression was studied at 12 h, 24 h and 48 h interval.The mRNA expression of several genes such as hexokinase, trypanothione reductase, aurora kinase, oligopeptidase B and ribonucleotide reductase II were found refractory (non-significant, p > 0.1234) to the exposure of QPS. Significant up-regulation of trans-sialidase (p < 0.0001), ESAG8 (p < 0.0021), ribonucleotide reductase I (p < 0.0001), ornithine decarboxylase (p < 0.0001), topoisomerase II (p < 0.0021) and casein kinase I (p < 0.0021) were recorded after exposure with QPS. The arginine kinase 1 and calcium ATPase I showed highly significant (p < 0.0001) down-regulation in the drug kinetics. Therefore, the arginine kinase 1 and calcium ATPase I can be explored further to elucidate the trypanocidal activity of QPS. The preliminary data generated provide the potential of arginine kinase 1 and calcium ATPase I mRNA mediated pathway of trypanocidal action of QPS. Further, transcriptomics approach is required to investigate the possible mechanism of action of drugs at molecular level against the targeted organism.

Naphthoquinone is a heterocyclic moiety whose natural derivatives are present as bioactive compounds in many plants and have stimulated a resurgence of interest in the past decades due to their wide range of pharmacological activities. Naphthoquinone agents have dynamic pharmacophores and privileged sub-structures in the chemistry of medicine. They have received much interest in drug discovery as trypanocidal because naphthoquinone and their derivatives revealed massive significance potential against the trypanosomes. Among natural naphthoquinones, lapachol, β-lapachone and its α-isomer exhibited useful trypanocidal activities. Some naphthoquinones have already been used commercially as an antiparasitic agent. Several naphthoquinones with diverse structural motifs have been synthesized and evaluated mainly against Trypanosoma cruzi and some studies have also been reported against Trypanosoma brucei and Trypanosoma evansi. This review summarized various mechanisms of action of naphthoquinone like reductive activation of quinone by the production of the semiquinone, generation of reactive oxygen species and free radicals such as superoxide anion radical and H2O2, and oxidative stress in the parasite. The information assembled in this review will help to understand the mechanism behind the activity and may also be useful to find the bio-efficacy of naphthoquinone compounds upon substitution against trypanosomatids.

Over the past decades, a number of 1,4-naphthoquinones have been isolated from natural resources and several of naphthoquinone derivatives with diverse structural motif have been synthesized; they possess a multitude of biochemical properties and modulate numerous pharmacological roles that offer new targets for addressing the challenges pertaining to novel drug developments. Among natural naphthoquinones, lapachol, α-lapachone, β-lapachone, lawsone, juglone, and plumbagin have been evaluated for its potential as antitrypanosomal activities. The chemotherapeutic drugs available for combating human trypanosomiasis, that is, American trypanosomiasis and African trypanosomiasis caused by Trypanosoma cruzi and Trypanosoma brucei, respectively, and animal tripanosomosis caused by Trypanosoma evansi have a problem of drug resistance and several toxic effect. Therefore, search of alternative effective drug molecules, without toxic effects, have enthused the researchers for searching new drug entity with potential clinical efficacy. In the search for new antitrypanosomal compound, this review focuses on different natural quinones and their synthetic derivatives associated with antitrypanosomal studies. In this context, this review will be useful for the development of new antitrypanosomal drugs mainly based on different structural modification of natural and synthetic naphthoquinones.

Surra caused by an extracellular hemoflagellate, Trypanosoma evansi, leads to severe economic loss to livestock productivity in India. Among the various mammalian pathogenic trypanosomes, T. evansi has the widest host range.Usually, species specific conjugates are used in conventional indirect immunosorbent assay (ELISA) for diagnosis of T. evansi infection in different animal species. The aim of the study was to explore the use of non-species specific conjugates viz., protein A, G and chimeric protein A/G instead of species specific conjugates for development of indirect ELISAs. These assays were used for detection of antibodies against T. evansi infection in multiple animal species viz., equine, cattle, buffalo, dog, pig and camel. The functional affinities of serum immunoglobulins of six different animal species with different conjugates were determined by estimation of relative avidity index (RAI). The species specific conjugate based whole cell lysate- T. evansi antigen ELISA was considered as reference assay for comparison of sensitivity and specificity of non-species specific conjugates based ELISAs optimized in the present study. Data showed that serodiagnosis of T. evansi can be carried out by using chimeric protein A/G conjugate in multiple hosts viz., equine, buffalo, camel, pig and dog; protein G conjugate in equine and buffalo and protein A conjugate in camel, pig and dog. The relative diagnostic sensitivity and specificity for chimeric protein A/G conjugate varied from 60 to 100% and 79-100%, respectively for different livestock species. This approach might be helpful in monitoring and surveillance of T. evansi infection in multiple hosts in particular when host specific secondary antibody conjugates are not available. Investigations should be made in wild animals and other warm-blooded vertebrates to validate this hypothesis.

Trypanosoma evansi is a causative agent of chronic wasting and fatal disease of livestock and wild animals known as surra. In this study, repurposing approach based on drug target was used to investigate the efficacy of kinase inhibitors (Barasertib-HQPA, BAR and Palbociclib isethionate, PAL) and protease inhibitors (Z-pro-prolinal, Z-PRO and Leupeptin hemisulphate, LEU) against T. evansi in HMI-9 medium. BAR, PAL and Z-PRO exhibited IC50 values of 13.52 µM, 0.6375 µM and 63.20 µM against T. evansi in terms of growth inhibition, in the contrary, LEU failed to exhibit a significant growth inhibition at any time interval. Furthermore, oligopeptidase B and aurora kinase genes of T. evansi were targeted to determine the effect of these drugs on quantitative mRNA expression, which showed significant (p < 0.01) up-regulation of both genes in the BAR and PAL-exposed population at 12 h of exposure, whereas, Z-PRO showed only significant (p < 0.05) up-regulation of aurora kinase gene at 12 h interval. In cytotoxicity assay, BAR exhibited 52

Trypanosoma evansi, a hemoflagellate protozoan, leads to wasting disease, surra in livestock animals causing huge economic losses. Currently, the preferred assay for surra diagnosis is whole cell lysate (WCL) based ELISA, which requires the use of rodents for WCL preparation. To avoid use of laboratory animals, we used recombinant DNA technology to express T. evansi invariable surface glycoprotein (ISG) in E. coli. The potential of recombinant ISG65 (rISG65) as a diagnostic antigen was investigated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The results indicated that rISG65 reacted with horse T. evansi positive serum giving two bands of approximately 48 kDa and 96 kDa. T. evansi-specific antibodies were detected as early as 10 and 14 days post infection using immunoblot and indirect ELISA, respectively using rISG65 antigen. No cross-reactivity was observed in ELISA and immunoblot with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions were observed between 30 and 100 kDa in T. evansi isolate of horse origin indicating the existence of multiple copies of ISG protein in a single trypanosome. The recombinant ISG has proven to be good candidate antigen to be used in ELISA for serodiagnosis of T. evansi infection in different animals.

High cytotoxicity and increasing resistance reports of existing chemotherapeutic agents against T. evansi have raised the demand for novel, potent, and high therapeutic index molecules for the treatment of surra in animals. In this regard, repurposing approach of drug discovery has provided an opportunity to explore the therapeutic potential of existing drugs against new organism. With this objective, the macrocyclic lactone representative, ivermectin, has been investigated for the efficacy against T. evansi in the axenic culture medium. To elucidate the potential target of ivermectin in T. evansi, mRNA expression profile of 13 important drug target genes has been studied at 12, 24, and 48 h interval. In the in vitro growth inhibition assay, ivermectin inhibited T. evansi growth and multiplication significantly (p < 0.001) with IC50 values of 13.82 μM, indicating potent trypanocidal activity. Cytotoxicity assays on equine peripheral blood mononuclear cells (PBMCs) and Vero cell line showed that ivermectin affected the viability of cells with a half-maximal cytotoxic concentration (CC50) at 17.48 and 22.05 μM, respectively. Data generated showed there was significant down-regulation of hexokinase (p < 0.001), ESAG8 (p < 0.001), aurora kinase (p < 0.001), casein kinase 1 (p < 0.001), topoisomerase II (p < 0.001), calcium ATPase 1 (p < 0.001), ribonucleotide reductase I (p < 0.05), and ornithine decarboxylase (p < 0.01). The mRNA expression of oligopeptidase B remains refractory to the exposure of the ivermectin. The arginine kinase 1 and ribonucleotide reductase II showed up-regulation on treatment with ivermectin. The ivermectin was found to affect glycolytic pathways, ATP-dependent calcium ATPase, cellular kinases, and other pathway involved in proliferation and maintenance of internal homeostasis of T. evansi. These data imply that intervention with alternate strategies like nano-formulation, nano-carriers, and nano-delivery or identification of ivermectin homologs with low cytotoxicity and high bioavailability can be explored in the future as an alternate treatment for surra in animals.

Polymerase spiral reaction (PSR) opens new avenues for specific diagnosis of pathogens known for cryptic infection at field level and its application is still unexplored in the field of parasitology. The present study aimed to explore and optimize colorimetric based PSR technique for the detection of Trypanosoma evansi in the blood of the host by targeting the 196bp Invariable Surface Glycoprotein (ISG) gene of Trypanosoma evansi. The specificity of the test was determined against Theileria equi, Theileria annulata, Babesia caballi, Burkholderia mallei and Equine herpes virus. The T. evansi DNA was extracted from purified parasites and serially diluted from 2.8ng to 2.8 × 10-8 pg. The detection limit of PSR was found to be as low as 2.8 × 10-6 pg of T. evansi DNA, which will aid in detection of Surra infection. The duration of reaction for determination of result of field sample is 1h and result can be read by naked eyes. In addition, PSR assay was also performed on DNA extracted from 28 field equine samples; out of which 1 was found positive by microscopy and ISG-196 targeted PCR assay and 2 were recorded positive by PSR assay. Data generated shows colorimetric PSR is convenient, rapid, sensitive and specific tool for diagnosis and monitoring of Surra infection in livestock at field level. Further, visual PSR assay has wide scope for application in government policies aimed at detection of early infection, sub-clinical cases, drug-efficacy studies, control and elimination of Surra organism from livestock animals.

Serodiagnosis of surra, caused by Trypanosoma evansi, is still based on native antigens purified from bloodstream form of T. evansi grown in rodents. In order to investigate prospective diagnostic possibilities as an alternative for native antigens, we cloned, expressed 26 kDa calflagin protein containing 218 amino acids from T. evansi (Indian Strain) in Escherichia coli. The potential of recombinant calflagin (rCLF) protein as diagnostic antigen was evaluated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The antibodies against T. evansi were detected with rCLF antigen in serum samples of experimentally infected equines as early as 10 days and 14 days post infection, using immunoblot and ELISA respectively. No cross-reactivity was observed with rCLF antigen in ELISA with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions ranging from 10 to 28 kDa were detected using distinct T. evansi isolates (pony, cattle, donkey and camel origin) indicating presence of multiple calflagin family members in a single trypanosome. Indirect immunofluorescence antibody test with anti-CLF rabbit hyperimmune serum showed localisation of native immunogenic protein near attachment of flagellum. The rCLF protein was found to be a potential diagnostic candidate for distinguishing T. evansi positive and negative equine serum sample, suggesting that it could be used for serological surveys in animals for surra. In addition, it could be used with other potential diagnostic candidates to improve the diagnostic efficiency.

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.

Equine trypanosomosis has negative impacts on health and working efficiency of equines in different areas of the world includingIndia.A cross sectional survey was conducted in thirteen districts of eastern plane zone of Uttar Pradesh, India during August to November, 2020 to evaluate the molecular and serological prevalence, relative risk factors and hemato-biochemical alterations of equine trypanosomosis.A total of 285 equine blood samples of both sex and different age groups were collected for the study.The overall prevalence of T. evansi infection in equines was 15.08 percent by Invariable surface glycoprotein (ISG) PCR and 19.29 percent by indirect-ELISA.The relative risk factors among different animals associated with PCR and ELISA techniques indicated that donkeys/mules were 27.08 and 34.65 times more prone to infection than horses.The young animals as well as parasite infected animals were also more susceptible to this infection than adult and parasite naive animals.Significant alterations in hemato-biochemical parameters (viz.Hb, PCV, TEC, MCV, MCHC, absolute lymphocyte count, TSP, albumin, globulin, serum glucose, serum enzymes, blood urea, creatinine and uric acid) were observed in infected animals as compared to infection free control.

Trypanosoma evansi, an extracellular haemoflagellate, has a wide range of hosts receptive and susceptible to infection, in which it revealed highly inconsistent clinical effects. Drugs used for the treatment of trypanosomosis have been utilized for more than five decades and have several problems like local and systemic toxicity. In the present investigation, imatinib and sorafenib were selected as drugs as they are reported to have the potential to cause reactive oxygen species (ROS)–mediated effect in cancer cells. Both have also been reported to have potential against T. brucei, T. cruzi and Leishmania donovani. To date, imatinib and sorafenib have not evaluated for their growth inhibitory effect against T. evansi. Imatinib and sorafenib showed significant (p < 0.001) inhibition on parasite growth and multiplication with IC50 (50% inhibitory concentration) values 6.12 μM and 0.33 μM respectively against T. evansi. Both the drug molecules demonstrated for the generation of ROS in T. evansi and were found up to 65% increased level of ROS as compared with negative control in the axenic culture system. Furthermore, different concentrations of imatinib and sorafenib were found non-toxic on horse peripheral blood mononuclear cells and Vero cell lines. Also, in conclusion, our results demonstrated that imatinib- and sorafenib-induced generation of ROS contributed inhibitory effect on the growth of Trypanosoma evansi in an axenic culture system.