[Show abstract][Hide abstract] ABSTRACT: CpG oligodeoxynucleotides (CpG-ODN) stimulate immune cells from a wide spectrum of mammalian species. Class C CpG-ODN is relatively stable and has the combined immune effects of both A and B classes of CpG-ODN. Trypanosoma evansi produces the state of immuno-suppression in the infected hosts. The current chemotherapeutic agents against this parasite are limited in number and usually associated with severe side effects. The present work aimed to determine the immunostimulatory effects of CpG-ODN class C in T. evansi infected rabbits. Rabbits inoculated with CpG C and challenged with T. evansi resulted in delayed onset of clinical signs with reduced severity in comparison to that of T. evansi infected rabbits. The treatment also enhanced humoral immune responses. Histopathological findings in liver and spleen revealed enhancement of mononuclear cell infiltration and secondary B cell follicles. These results demonstrate that CpG-ODN class C, has immunostimulatory properties in rabbit model of trypanosomosis. The use of booster doses or sustained delivery of CpG-ODN will further elucidate the prolonged CpG-ODN generated immune responses.
PLoS ONE 06/2015; 10(6):e0127437. DOI:10.1371/journal.pone.0127437 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study is an observation of transplacental transmission of T. evansi in a donkey neonatal foal. One experimentally infected pregnant donkey mare gave a normal birth to a foal after three months of experimental infection. No trypanosome was seen in wet blood film on microscopic examination in the experimentally infected donkey mare at time of birth of foal, however in serum a significant level of antitrypanosmal IgG antibodies in ELISA and many immunodominant polypeptide bands on immunoblotting were observed. In neonatal foal, live moving trypanosomes were observed in wet blood film just after birth before colostrum feeding. The foal serum sample (collected before colostrum feeding) was found negative in ELISA and immunoblotting indicating that IgG antibodies have not crossed placental barrier in mother donkey mare. After 24 h of birth, the clinical symptoms appeared in foal showing recumbancy, unable to stand and suckle, and poor reflexes. The foal was administered fluid therapy, but could survive only up to 36 h after birth. The study indicated transplacental transmission of T. evansi in donkey but the mechanism responsible for crossing the placental barrier need to be further elucidated.
[Show abstract][Hide abstract] ABSTRACT: The present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most closely related to T. brucei brucei and T. brucei gambiense, whereas T. congolense HSP70 laid in separate clade. The two partial HSP70 sequences (HSP-1 from N-terminal region and HSP-2 from C-terminal region) were expressed and evaluated as diagnostic antigens using experimentally infected equine serum samples. Both recombinant proteins detected antibody in immunoblot using serum samples from experimental infected donkeys with T. evansi. Recombinant HSP-2 showed comparable antibody response to Whole cell lysate (WCL) antigen in immunoblot and ELISA. The initial results indicated that HSP70 has potential to detect the T. evansi infection and needs further validation on large set of equine serum samples.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT: Trypanosoma evansi is the causative agent of surra, which is the most common and widespread trypansomal disease. The infection is mainly restricted to animals, but it has also been documented in human. Trypanosomes possess the thick immunogenic surface coat known as variant surface glycoprotein (VSG). The parasite modifies the VSG constantly resulting in continuous antigenic variations and thus evades the host immune response. Due to antigenic variations, vaccination against trypanosomosis is not useful. Therefore, alternate strategies to augment the immune response are required. CpG-ODN class-C has combined immune effects of both A and B classes of CpG-ODN. In this study, we observed that CpG-ODN class-C stimulated horse peripheral blood mononuclear cells (PBMC) induce the expression of interferon-α (IFN-α), tumor necrosis factor-α (TNF-α), IL-12 and nitric oxide (NO) indicating enhanced innate immune response. We have for the first time demonstrated that co-culture of CpG-ODN with T. evansi antigen induces lymphocyte proliferative responses and result in a synergistic effect in eliciting the immune response.
International Immunopharmacology 07/2014; 22:366-370. DOI:10.1016/j.intimp.2014.07.016 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In present communication, we report an outbreak of Trypanosoma evansi in equine herd n = 30 (horse and mules) which, were reared in fly proof stables as well as in open paddock maintained under semi-intensive system of management, and its effective control using trypanocidal drug. The infection was monitored by antibody ELISA up to 180 days post-treatment (PT). A total of 8 out of 14 equines (57.14 %) which were maintained only in open paddocks were found positive with T. evansi infection parasitologically. The infected animals were treated with quinapyramine methyl sulphate and chloride combination administered at the prescribed dose rate on 3rd day of screening. The parasite could not be detected from any treated animals from day-3 PT up to 6 month. Further, we also could not observe relapse of infection, neither in treated group nor in equine herd maintained at the farm. Sero-conversion was observed in all eight animals by 10th day of screening, indicating that immune response was due to recent infection as the animals became chronologically positive. The antibody titre reached at the peak by 10–14th day in all infected animals, and started declining by 17th day of screening, further reached to near cut off level by 180 days. Since, antibodies persisted up to 6 month PT and antibody detection assays are not able to differentiate between current and past infections in treated cases. The detection of circulating antigen assay and parasitological techniques in combination may be performed for effective diagnosis and management of T. evansi infection.
Journal of parasitic diseases 03/2014; 38(1). DOI:10.1007/s12639-012-0204-2
[Show abstract][Hide abstract] ABSTRACT: Aim:
To reduce the dose, toxic effects and to ensure sustained release of quinapyramine sulfate (QS), a highly effective drug against Trypanosoma evansi.
Materials & methods:
QS-loaded sodium alginate nanoparticles (QS-NPs) were formed by emulsion-crosslinking technology using dioctyl-sodium-sulfosuccinate and sodium alginate. The formulation was characterized for size, stability, morphology and functional groups by a zetasizer, scanning electron microscopy, atomic force microscopy, transmission electron microscopy and Fourier transform infrared spectroscopy. In vitro safety and toxicity studies were performed by metabolic assay in Vero cell lines, and in vivo efficacy was evaluated in mice.
QS-NPs were <60 nm with 96.48% entrapment efficiency and 3.70% drug loading. The formulation showed an initial burst effect followed by slow drug release in accordance with quasi-Fickian Higuchi diffusion mechanism. QS-NPs were much less toxic and able to clear the parasite at a much lower concentration than QS.
The QS-NPs synthesized are safe, less toxic and highly effective compared with QS.
[Show abstract][Hide abstract] ABSTRACT: Trypanosoma evansi infection typically produces wasting disease, but it can also develop into a neurological or meningoencephalitis form in equids. Trypanosomiasis in horses was treated with quinapyramine sulfate, and all the 14 infected animals were recovered clinically. After clinical recovery, four animals developed a neurological form of the disease at various intervals. Two of these animals treated with diminazene aceturate recovered temporarily. Repeated attempts failed to find the parasite in the blood or the cerebrospinal fluid (CSF), but all of the animals were positive in enzyme-linked immunosorbent assay. The calculation of the antibody index (AI) in the serum and the CSF and polymerase chain reaction (PCR) analysis of the CSF and brain tissue were carried out to confirm the neuro-infection. We found PCR and AI analyses of the CSF to be useful tools in the diagnosis of the neurological form of trypanosomiasis when the organism cannot be found in the blood or CSF. The increased albumin quotient is indicative of barrier leakage due to neuroinflammation. The biochemical changes in the CSF due to nervous system trypanosomiasis include increases in the albumin quotient, total protein, and urea nitrogen. It seems to be the first report on relapse of the nervous form of trypanosomiasis in equids even after quinapyramine treatment in endemic areas.
Tropical Animal Health and Production 11/2013; 46(2). DOI:10.1007/s11250-013-0498-9 · 0.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Equine piroplasmosis is a tick-transmitted protozoan disease caused by Theileria equi and/or Babesia caballi. In the present study, we expressed a 53kDa protein from the truncated EMA-2 gene of T. equi (Indian strain) and developed EMA-2ELISA using this expressed protein. This ELISA is able to detect T. equi-specific antibodies in experimentally infected animals as early as 9 days post-infection. The assay developed was validated with the OIE recommended competitive ELISA (cELISA) on 120 serum samples and significant agreement (kappa=0.93) was observed between results of both the ELISAs which indicates suitability of EMA-2ELISA for use in sero-diagnosis. Diagnostic sensitivity and specificity of EMA-2ELISA - as compared with cELISA - were 0.97 and 0.96, respectively. Analysis of 5651 equine serum samples - collected during 2007-2012 from 12 states of India representing eight agro-climatic zones - by EMA-2ELISA revealed 32.65% seroprevalence of T. equi in India. In conclusion, the EMA-2ELISA developed using the T. equi EMA-2 recombinant protein as antigen for detecting T. equi-specific antibodies has good diagnostic potential for sero-epidemiological surveys.
[Show abstract][Hide abstract] ABSTRACT: The present study was undertaken to establish an optimal medium for primary culture initiation and maintenance of T. evansi isolated from different mammalian hosts of diverse geographical regions of India viz. donkey/1 (Hardoi, Uttar Pradesh), donkey/2 (Junagarh, Gujarat), pony/1 (Hisar, Haryana), camel/1 (Bikaner, Rajasthan) which represented isolates 1, 2, 3 and 4, respectively. Primary cultures were initiated with all four isolates in five different in vitro cultivation media with seeding density of 1 × 106 trypanosomes/ml. The parasites of all four isolates could remain viable only for 48 h in medium E (Alsever’s solution) and for 72 h in medium A, C and D. Parasites reached to a maximum density (2.5–3.75 × 106/ml) within 24 h and thereafter, a sharp decline (0.5–0.75 × 106/ml) in the next 72 h was observed in 1, 2 and 3 isolates cultured in medium B. In isolate 4, parasite counts got more than doubled in 24 h and then decreased gradually up to sixth day post initiation of cultivation which thereafter increased gradually up to 34 days and a constant parasite number of 105/ml could be achieved for 90 days in medium B. During this prolonged culture the trypanosomes retained their long slender morphology and infectivity to mice.
Journal of parasitic diseases 06/2013; 39(2). DOI:10.1007/s12639-013-0314-5
[Show abstract][Hide abstract] ABSTRACT: Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immuno blot. Three immuno dominant clusters of proteins i.e. 62-66kDa, 52-55kDa and 41-43kDa were identified based on their consistent reactivity with donkey sequential serum experimentally infected T. evansi up to 280days post infection (dpi). The protein cluster of 62-66kDa was purified in bulk in native form and comparatively evaluated with whole cell lysate antigen (WCL). ELISA and immuno blot showed that polypeptide of this cluster is 100% sensitive in detection of early and chronic infection. Further, this protein cluster was also found immuno reactive against hyper immune serum raised against predominantly 66kDa exo antigen, revealed that this is a common immunodominant moieties in proteome and secretome of T. evansi.
Research in Veterinary Science 05/2013; 95(2). DOI:10.1016/j.rvsc.2013.04.030 · 1.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The importance of Trypanosoma evansi as the etiological agent for surra is often overlooked due to difficulty in accurate diagnosis of the disease. In the present study, an antibody-ELISA was developed using whole cell lysate antigen prepared from purified trypanosomes and used for seroprevalence study of T. evansi in equids. A total of 3695 equids were surveyed and blood samples were collected from each animal during September 2009 to August 2011. Out of these, 420 serum samples were found positive for presence of antibodies against T. evansi collected from equids of six agro-climatic zones of North and North-western regions of India comprising eight states viz., Gujarat (36/479), Haryana (11/275), Himachal Pradesh (14/83), Jammu and Kashmir (32/221), Punjab (1/38), Rajasthan (90/1148), Uttarakhand (141/753), and Uttar Pradesh (65/330). The maximum seroprevalence (19.69%) for T. evansi infection was observed in equids of Uttar Pradesh state with an overall seroprevalence of 11.36% in North and North-western regions of India. The results indicated that surra is endemic in equids of North and North-western parts of India.
[Show abstract][Hide abstract] ABSTRACT: Surra is an economically important disease of livestock caused by an extracellular haemoprotozoan
parasite, Trypanosoma evansi. In this study, genomic DNA was extracted from six Trypanosoma isolates
collected from different geographical areas, and twelve microsatellite loci were successfully amplified in
polymerase chain reaction (PCR) with selected microsatellite primers. For most of the microsatellite loci,
the PCR amplicon size in Indian isolates was found similar to that of Trypanosoma brucei TREU927, except
for three microsatellite loci (TB11/1, TB10/1 and TB7/12). For the microsatellite loci TB11/1, TB10/1 and TB7/
12, the PCR amplicon size in T. evansi isolates was found to be 160, 210 and 200 (bp), respectively. Similar
amplicon size patterns using twelve microsatellite primers were observed in all six Indian T. evansi isolates
used in this study.
[Show abstract][Hide abstract] ABSTRACT: Trypanosoma evansi is a causative agent of 'surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28-41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.
Parasitology Research 02/2011; 109(3):559-65. DOI:10.1007/s00436-011-2284-9 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.
[Show abstract][Hide abstract] ABSTRACT: Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of
its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a
preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment
of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s;
its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90
ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90
multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found
a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating
parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another
protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies
supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.
Journal of Biological Chemistry 12/2010; 285(49):37964-37975. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Equine influenza is a contagious viral disease that affects all members of the family Equidae, i.e., horses, donkeys and mules. The authors describe the pattern of equine influenza outbreaks in a number of states of India from July 2008 to June 2009. The disease was first reported in June 2008 in Katra (Jammu and Kashmir) and spread to ten other states within a year. All outbreaks of equine influenza in the various states were confirmed by laboratory investigations (virus isolation and/or serological confirmation based on haemagglutination inhibition [HI] assays of paired samples) before declaring them as equine influenza virus-affected state(s). The virus (H3N8) was reported from various locations in the country including Katra, Mysore (Karnataka), Ahmedabad (Gujarat), Gopeshwar and Uttarkashi (Uttarakhand) and was isolated in 9- to 11-day-old embryonated chicken eggs. The virus was confirmed as H3N8 by HI assays with standard serum and amplification of full-length haemagglutinin and neuraminidase genes by reverse transcriptase-polymerase chain reaction. Serum samples (n = 4 740) of equines from 13 states in India screened by HI revealed 1074 (22.65%) samples as being positive for antibodies to equine influenza virus (H3N8).
Veterinaria italiana 10/2010; 46(4):449-58. · 0.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trypanosoma evansi infections, commonly called 'surra', cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO) prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS).
Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF) mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more.
Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a glimpse into the biology of this neglected disease, our study is the first step towards identification of diagnostic biomarkers, novel drug targets as well as potential vaccine candidates to fight against T. evansi infections.
PLoS ONE 03/2010; 5(3):e9796. DOI:10.1371/journal.pone.0009796 · 3.23 Impact Factor