Cloning and Characterization of Trypanosoma congolense and T. vivax Nucleoside Transporters Reveal the Potential of P1-Type Carriers for the Discovery of Broad-Spectrum Nucleoside-Based Therapeutics against Animal African Trypanosomiasis.

African Animal Trypanosomiasis (AAT), caused predominantly by Trypanosoma brucei brucei, T. vivax and T. congolense, is a fatal livestock disease throughout Sub-Saharan Africa. Treatment options are very limited and threatened by resistance. Tubercidin (7-deazaadenosine) analogs have shown activity against individual parasites but viable chemotherapy must be active against all three species. Divergence in sensitivity to nucleoside antimetabolites could be caused by differences in nucleoside transporters. Having previously characterized the T. brucei nucleoside carriers, we here report the functional expression and characterization of the main adenosine transporters of T. vivax (TvxNT3) and T. congolense (TcoAT1/NT10), in a Leishmania mexicana cell line ('SUPKO') lacking adenosine uptake. Both carriers were similar to the T. brucei P1-type transporters and bind adenosine mostly through interactions with N3, N7 and 3'-OH. Expression of TvxNT3 and TcoAT1 sensitized SUPKO cells to various 7-substituted tubercidins and other nucleoside analogs although tubercidin itself is a poor substrate for P1-type transporters. Individual nucleoside EC50s were similar for T. b. brucei, T. congolense, T. evansi and T. equiperdum but correlated less well with T. vivax. However, multiple nucleosides including 7-halogentubercidines displayed pEC50>7 for all species and, based on transporter and anti-parasite SAR analyses, we conclude that nucleoside chemotherapy for AAT is viable.

Brain and pituitary-adrenal lesions of Trypanosoma brucei brucei and Trypanosoma congolense infections in the West African Dwarf rams: Is trypanotolerance overrated?

Trypanosomosis of the West African Dwarf (WAD) sheep is often neglected due to emphasis on trypanotolerance. Nevertheless, significant pathological changes may occur in tissues of infected WAD sheep. The purpose of this study was to evaluate the brain, pituitary, and adrenal lesions of Trypanosoma brucei brucei (Tbb) and Trypanosoma congolense (Tc) infections in WAD rams. Fifteen WAD rams were infected intraperitoneally with Tbb or Tc (106 trypanosomes/animal) or were uninfected controls (5 rams per group). Adrenocorticotrophic hormone (ACTH) and cortisol were assayed in serum by enzyme immunoassay technique. The brain, pituitary, and adrenal glands were processed for histopathology. Serum ACTH levels of infected rams were significantly (P < .05) higher than that of controls on days 14 and 70 post infection (PI). Serum cortisol levels of infected rams were significantly (P < .05) higher than that of controls only on day 14 PI. Mortality was 60% in Tbb- and 40% in Tc-infected rams. The brain of the infected groups showed chromatolysis of cortical neurons and Purkinje cells with severe encephalitis. Degenerative, necrotic, and inflammatory changes were seen in the pituitary and adrenal glands of the infected rams. Adrenal corticomedullary ratio was significantly (P < .05) higher in Tc-infected rams than controls. Based on the high mortality levels, likely due to severe encephalitis, the WAD sheep may not be regarded as trypanotolerant.

IL-27 Negatively Regulates Tip-DC Development during Infection.

Tumor necrosis factor (TNF)/inducible nitric oxide synthase (iNOS)-producing dendritic cells (Tip-DCs) have profound impacts on host immune responses during infections. The mechanisms regulating Tip-DC development remain largely unknown. Here, using a mouse model of infection with African trypanosomes, we show that a deficiency in interleukin-27 receptor (IL-27R) signaling results in escalated intrahepatic accumulation of Ly6C-positive (Ly6C+) monocytes and their differentiation into Tip-DCs. Blocking Tip-DC development significantly ameliorates liver injury and increases the survival of infected IL-27R-/- mice. Mechanistically, Ly6C+ monocyte differentiation into pathogenic Tip-DCs in infected IL-27R-/- mice is driven by a CD4+ T cell-interferon gamma (IFN-γ) axis via cell-intrinsic IFN-γ signaling. In parallel, hyperactive IFN-γ signaling induces cell death of Ly6C-negative (Ly6C-) monocytes in a cell-intrinsic manner, which in turn aggravates the development of pathogenic Tip-DCs due to the loss of the negative regulation of Ly6C- monocytes on Ly6C+ monocyte differentiation into Tip-DCs. Thus, IL-27 inhibits the dual-track exacerbation of Tip-DC development induced by a CD4+ T cell-IFN-γ axis. We conclude that IL-27 negatively regulates Tip-DC development by preventing the cell-intrinsic effects of IFN-γ and that the regulation involves CD4+ T cells and Ly6C- monocytes. Targeting IL-27 signaling may manipulate Tip-DC development for therapeutic intervention.IMPORTANCE TNF/iNOS-producing dendritic cells (Tip-DCs) are at the front line as immune effector cells to fight off a broad range of invading microbes. Excessive development of Tip-DCs contributes to tissue destruction. Thus, identifying master regulators of Tip-DC development is fundamental for developing new therapeutic strategies. Here, we identify Tip-DCs as a terminal target of IL-27, which prevents Tip-DC-mediated early mortality during parasitic infections. We demonstrate that IL-27 inhibits Tip-DC development via a dual-track mechanism involving the complex interactions of effector CD4+ T cells, Ly6C- monocytes, and Ly6C+ monocytes. These findings delineate an in-depth view of mechanisms of Tip-DC differentiation that may have significant implications for the ongoing development of IL-27-based immunotherapy.

In vitro and in vivo antitrypanosomal efficacy of combination therapy of Anogeissus leiocarpus, Khaya senegalensis and potash.

ETHNOPHARMACOLOGICAL RELEVANCE: Pastoralists in Nigeria mix barks of Anogeissus leiocarpus (AL) Khaya senegalensis (KS) and potash (Pt) to treat animal African trypanosomosis. AIM: To evaluate antitrypanosomal potential of A. leiocarpus, K. senegalensis and potash for insights into the traditional claim of antitrypanosomal combination therapy (ATCT). MATERIALS AND METHODS: Fifty microliter each of six different concentrations of AL, KS, Pt, AL + KS, AL + KS + Pt and diminazene aceturate (DA, positive control) was incubated with 50 µL of parasite-laden blood containing 108Trypanosoma congolense cells in a 96-well microtitre plate. Negative control wells were devoid of the extracts and drug but supplemented with phosphate-buffered saline (PBS). Efficacy of treatment was observed at 1 h interval for complete immobilisation or reduced motility of the parasites. Each incubated mixture was inoculated into mouse at the point of complete immobilisation of parasite motility or at the end of 6-h observation period for concentrations that did not immobilise the parasites completely. For in vivo assessment, thirty-five parasitaemic rats were randomly allocated into seven groups of 5 rats each. Each rat in groups I-V was treated with 500 mg/kg of AL, KS, Pt, AL + KS and AL + KS + Pt, respectively, for 7 days. Rats in groups VI and VII were treated with diminazene aceturate 3.5 mg/kg once and PBS 2 mL/kg (7 days), which served as positive and negative controls, respectively. Daily monitoring of parasitaemia through the tail vein, packed cell volume and malondialdehyde were used to assess efficacy of the treatments. RESULTS: The AL + KS + Pt group significantly (p < 0.05) and dose-dependently reduced parasite motility and completely immobilized the parasites at 10, 5 and 2.5 µg/µL with an IC50 of 9.1×10-4 µg/µL. All the mice with conditions that produced complete cessation of parasite motility did not develop parasitaemia within one month of observation. The AL + KS group significantly (p < 0.05) lowered the level of parasitaemia and MDA, and significantly (p < 0.05) maintained higher PCV than PBS group. CONCLUSION: The combination of A. leiocarpus and K. senegalensis showed better antitrypanosomal effects than single drug treatment and offers prospects for ATCT. Our findings support ethnopharmacological use of combined barks of A. leiocarpus and K. senegalensis by pastoralist in the treatment of animal African trypanosomosis in Nigeria.

Validation of ligands targeting metacaspase-2 (MCA2) from Trypanosoma brucei brucei and their application to MCA5 from T. congolense as possible trypanocides.

Metacaspases (MCAs) are ideal drug and diagnostic targets for animal and human African trypanosomiasis, as these cysteine peptidases are absent from the metazoan kingdom and have been implicated in the parasite cell cycle and cell death. Tsetse fly-transmitted trypanosomes that live free in the bloodstream and/or cerebrospinal fluid of the mammalian host cause animal and human African trypanosomiasis (nagana or sleeping sickness respectively). Chemotherapy and chemoprophylaxis are the main forms of control, but in contrast to human trypanocides, the veterinary drugs are old and drug resistance is on the increase. A peptidomimetic library targeting the MCA2 from Trypanosoma brucei brucei has ligands with low IC50 values, some of which were antiparasitic. This study validates the inhibitory activity of these ligands using the protein structure solved by X-ray diffraction after the ligand library was published. Water molecules were shown to be important in substrate binding and strategies to improve the efficacy of these ligands are highlighted. These ligands appear to be pan-specific as they were docked into the active site of the homology modelled MCA5 of animal infective Trypanosoma congolense with similar binding energies and conformations.

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection.

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.

Expression and copper binding properties of the N-terminal domain of copper P-type ATPases of African trypanosomes.

Copper is an essential component of cuproproteins but can be toxic to cells, therefore copper metabolism is very carefully regulated within cells. To gain insight into trypanosome copper metabolism, Trypanosoma spp. genomic databases were screened for the presence of copper-containing and -transporting proteins. Among other genes encoding copper-binding proteins, a copper-transporting P-type ATPase (CuATPase) gene was identified. Sequence and phylogenetic analyses suggest that the gene codes for a Cu+ transporter belonging to the P1B-1 ATPase subfamily that has an N-terminal domain with copper binding motifs. The N-terminal cytosolic domains of the proteins from Trypanosoma congolense and Trypanosoma brucei brucei were recombinantly expressed in Escherichia coli as maltose binding protein (MBP) fusion proteins. These N-terminal domains bound copper in vitro and within E. coli cells, more than the control MBP fusion partner alone. The copper binding properties of the recombinant proteins were further confirmed when they inhibited copper catalysed ascorbate oxidation. Native CuATPases were detected in a western blot of lysates of T. congolense IL3000 and T. b. brucei ILTat1.1 bloodstream form parasites using affinity purified IgY antibodies against N-terminal domain peptides. The CuATPase was also detected by immunofluorescence in T. b. brucei bloodstream form parasites where it was associated with subcellular vesicles. In conclusion, Trypanosoma species express a copper-transporting P1B-1-type ATPase and together with other copper-binding proteins identified in the genomes of kinetoplastid parasites may constitute potential targets for anti-trypanosomal drug discovery.

TLR-2 and MyD88-Dependent Activation of MAPK and STAT Proteins Regulates Proinflammatory Cytokine Response and Immunity to Experimental Trypanosoma congolense Infection.

It is known that Trypanosoma congolense infection in mice is associated with increased production of proinflammatory cytokines by macrophages and monocytes. However, the intracellular signaling pathways leading to the production of these cytokines still remain unknown. In this paper, we have investigated the innate receptors and intracellular signaling pathways that are associated with T. congolense-induced proinflammatory cytokine production in macrophages. We show that the production of IL-6, IL-12, and TNF-α by macrophages in vitro and in vivo following interaction with T. congolense is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) including ERK, p38, JNK, and signal transducer and activation of transcription (STAT) proteins. Specific inhibition of MAPKs and STATs signaling pathways significantly inhibited T. congolense-induced production of proinflammatory cytokines in macrophages. We further show that T. congolense-induced proinflammatory cytokine production in macrophages is mediated via Toll-like receptor 2 (TLR2) and involves the adaptor molecule, MyD88. Deficiency of MyD88 and TLR2 leads to impaired cytokine production by macrophages in vitro and acute death of T. congolense-infected relatively resistant mice. Collectively, our results provide insight into T. congolense-induced activation of the immune system that leads to the production of proinflammatory cytokines and resistance to the infection.

Host Immune Responses and Immune Evasion Strategies in African Trypanosomiasis.

Parasites, including African trypanosomes, utilize several immune evasion strategies to ensure their survival and completion of their life cycles within their hosts. The defense factors activated by the host to resolve inflammation and restore homeostasis during active infection could be exploited and/or manipulated by the parasites in an attempt to ensure their survival and propagation. This often results in the parasites evading the host immune responses as well as the host sustaining some self-inflicted collateral tissue damage. During infection with African trypanosomes, both effector and suppressor cells are activated and the balance between these opposing arms of immunity determines susceptibility or resistance of infected host to the parasites. Immune evasion by the parasites could be directly related to parasite factors, (e.g., antigenic variation), or indirectly through the induction of suppressor cells following infection. Several cell types, including suppressive macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T cells have been shown to contribute to immunosuppression in African trypanosomiasis. In this review, we discuss the key factors that contribute to immunity and immunosuppression during T. congolense infection, and how these factors could aid immune evasion by African trypanosomes. Understanding the regulatory mechanisms that influence resistance and/or susceptibility during African trypanosomiasis could be beneficial in designing effective vaccination and therapeutic strategies against the disease.

CRIg plays an essential role in intravascular clearance of bloodborne parasites by interacting with complement.

Although CRIg was originally identified as a macrophage receptor for binding complement C3b/iC3b in vitro, recent studies reveal that CRIg functions as a pattern recognition receptor in vivo for Kupffer cells (KCs) to directly bind bacterial pathogens in a complement-independent manner. This raises the critical question of whether CRIg captures circulating pathogens through interactions with complement in vivo under flow conditions. Furthermore, the role of CRIg during parasitic infection is unknown. Taking advantage of intravital microscopy and using African trypanosomes as a model, we studied the role of CRIg in intravascular clearance of bloodborne parasites. Complement C3 is required for intravascular clearance of African trypanosomes by KCs, preventing the early mortality of infected mice. Moreover, antibodies are essential for complement-mediated capture of circulating parasites by KCs. Interestingly, reduced antibody production was observed in the absence of complement C3 during infection. We further demonstrate that CRIg but not CR3 is critically involved in KC-mediated capture of circulating parasites, accounting for parasitemia control and host survival. Of note, CRIg cannot directly catch circulating parasites and antibody-induced complement activation is indispensable for CRIg-mediated parasite capture. Thus, we provide evidence that CRIg, by interacting with complement in vivo, plays an essential role in intravascular clearance of bloodborne parasites. Targeting CRIg may be considered as a therapeutic strategy.

Diminazene aceturate (Berenil) downregulates Trypanosoma congolense-induced proinflammatory cytokine production by altering phosphorylation of MAPK and STAT proteins.

Diminazene aceturate (Berenil) is the most commonly used trypanolytic agent in livestock. We previously showed that Berenil downregulates Trypanosoma congolense (T. congolense)-induced cytokine production in macrophages both in vitro and in vivo. Here, we investigated the molecular mechanisms through which the drug alters T. congolense-induced cytokine production in macrophages. We show that pretreatment of macrophages with Berenil significantly downregulated T. congolense-induced phosphorylation of mitogen-activated protein kinase (p38), signal transducer and activator of transcription (STAT) proteins including STAT1 and STAT3, and NFκB activity both in vitro and in vivo. Collectively, our results reveal a mechanistic insight through which Berenil downregulates T. congolense-induced cytokine production in macrophages by inhibiting key signaling molecules and pathways associated with proinflammatory cytokine production.

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing.

Kinetoplastid parasites-trypanosomes and leishmanias-infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition.

Mitonuclear genomics challenges the theory of clonality in Trypanosoma congolense: Reply to Tibayrenc and Ayala.

We recently published the first genomic diversity study of Trypanosoma congolense, a major aetiological agent of Animal African Trypanosomiasis. We demonstrated striking levels of SNP and indel diversity in the Eastern province of Zambia as a consequence of hybridization between divergent trypanosome lineages. We concluded that these and earlier findings in T. congolense challenge the predominant clonal evolution (PCE) model. In a recent comment, Tibayrenc and Ayala claim that there are many features in T. congolense supporting their theory of clonality. While we can follow the reasoning of the authors, we also identify major limitations in their theory and interpretations that resulted in incorrect conclusions. First, we argue that each T. congolense subgroup should be analysed independently as they may represent different (sub)species rather than "near-clades". Second, the authors neglect major findings of two robust population genetic studies on Savannah T. congolense that provide clear evidence of frequent recombination. Third, we reveal additional events of introgressive hybridization in T. congolense by analysing the maxicircle coding region using next-generation sequencing analyses. At last, we pinpoint two important misinterpretations by the authors and show that there are no spatially and temporally widespread clones in T. congolense. We stand by our earlier conclusions that the clonal framework is unlikely to accurately model the population structure of T. congolense. Other theoretical frameworks such as Maynard Smith's epidemic model may better represent the complex ancestry seen in T. congolense, where clones delimited in space and time arise against a background of recombination.

Hybridization in Trypanosoma congolense does not challenge the predominant clonal evolution model. A comment on Tihon et al., 2017, Mol. Ecol.

Tihon et al. have just published in Mol. Ecol. a fine genomic study on Trypanosoma congolense, agent of Animal African Trypanosomiasis. They present very convincing evidence that T. congolense underwent several hybridization events between distinct genetic lines in Zambia. They claim that their data challenge our predominant clonal evolution model (PCE) of micropathogens. We point out the main tenets of our model and show that Tihon et al.'s claim is based on a misinterpretation of the PCE model. Actually, their data strongly support PCE in T. congolense at a microevolutionary level.

Myeloid-Derived Suppressor Cells Contribute to Susceptibility to Trypanosoma congolense Infection by Suppressing CD4+ T Cell Proliferation and IFN-γ Production.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of bone marrow-derived myeloid cells that have immune-suppressive activities. These cells have been reported to suppress T cell immunity against tumors as well as in some parasitic and bacterial infections. However, their role during Trypanosoma congolense infection has not been studied. Given that immunosuppression is a hallmark of African trypanosomiasis, we investigated the role of MDSCs in immunity to T. congolense infection. We found increased numbers of MDSCs in the spleen and liver of infected mice, which correlated with increased parasitemia. Depletion of MDSCs significantly increased the percentage of proliferating and IFN-γ-producing CD4+ T cells from the spleen of T. congolense-infected mice. Furthermore, MDSCs from T. congolense-infected mice directly suppressed CD4+ T cell proliferation in a coculture setting. This suppressive effect was abolished by the arginase-1 inhibitor, Nω-hydroxy-nor-l-arginine (nor-NOHA), indicating that MDSCs suppress CD4+ T cell proliferation and function in an arginase-1-dependent manner. Indeed, depletion of MDSCs during infection led to control of the first wave of parasitemia and prolonged survival of infected mice. This was also associated with increased CD4+ T cell proliferation and IFN-γ production. Taken together, our findings identify an important role of MDSCs in the pathogenesis of experimental T. congolense infection via suppression of T cell proliferative and effector cytokine responses in an arginase-1-dependent manner.

An ATP-Based Luciferase Viability Assay for Animal African Trypanosomes Using a 96-Well Plate.

Cell viability assays using multi-well cell culture plates are frequently used for in vitro drug screening. We herein describe an ATP-based luciferase viability assay for animal African trypanosomes using a 96-well plate. This assay could be further applied to the screening of novel compounds for the treatment of animal African trypanosomiasis.

In vivo effect of Commiphora swynnertonii ethanolic extracts on Trypanosoma congolense and selected immunological components in mice.

BACKGROUND: The search for alternative trypanocidal compounds which can be available at affordable price is of paramount importance for control of trypanosomosis in human and animals. The current study evaluates the in vivo activity of ethanolic stem bark extracts on Trypanosoma congolense and selected immunological components in an inbred Swiss albino mouse model. METHODS: Groups of mice infected with T. congolense were treated with the stem bark extracts at a rate of 1000 mg/kg, 1500 mg/kg, and 2000 mg/kg, twice a day in one set and thrice a day in another setting for three days consecutively. Negative (infected and untreated) and positive (infected treated with diminazene diaceturate at 3.5 mg/kg) control groups were used. Levels of parasitaemia were monitored daily for the first 10 days and thereafter 2-3 times per week to the end of experiment. In the other setting, uninfected mice, randomized in groups were treated with the extract but categorized as: thorough mixed extract (TME) and supernatant extract (SE) each at 500 mg/kg and 1500 mg/kg, in 8 hourly intervals respectively for three days consecutively. Control group was administered with phosphate buffered saline with glucose at 0.1 ml/10 g in a similar manner as for the extract. Whole blood and spleen were taken 24 h after the last treatment for hematological and histopathological analysis. RESULTS: The groups that received the extracts at 8 hourly intervals drastically reduced the parasitaemia. The higher dose of SE significantly reduced the percentage of lymphocytes (P < 0.05). Both high and low dose of TME significantly reduced lymphocytes percent (P < 0.05) while percent of neutrophils and monocytes increased significantly (P < 0.05). Histopathological changes of the spleen in the mice treated with higher concentrations of the extract of C. swynnertonii were suggestive of lymphocytes toxicity. CONCLUSION: The current study has provided evidence that, in vivo trypanocidal activity of ethanolic bark extracts of C. swynnertonii is probably affected by its negative effect on humoral mediated immune response. Further studies are recommended to determine its potential as an alternative source of lead compounds for trypanocidal drug discovery.

Antitrypanosomal activity of Verbascum sinaiticum Benth. (Scrophulariaceae) against Trypanosoma congolense isolates.

BACKGROUND: African Trypanosomiasis is a neglected tropical disease with a large impact on the livelihood of the rural poor in Sub-Saharan Africa. The available drugs for managing this disease are old, expensive and are facing the problem of drug resistance. Thus, the aim of this study was to evaluate in vivo antitrypanosomal efficacy of aqueous and absolute methanol leaf extracts of Verbascum sinaiticum Benth. against Trypanosoma congolense field isolate. METHODS: Verbascum sinaiticum (Local name 'qetetina') is a biennial plant, and 60-150 cm tall. It is traditionally used to treat wound, stomachache, viral infection, cancer, sunstroke, fever, abdominal colic, diarrhea, hemorrhage, anthrax, and hepatitis. The efficacy of aqueous and absolute methanol leaf extracts of V. sinaiticum was evaluated in a randomized experiment with Swiss albino mice infected with T. congolense field isolate. The extracts were administered at doses of 100, 200 and 400 mg/kg by intraperitoneal injection for seven days at 12 Days Post-Infection (DPI) when the peak parasitaemia level was approximately 10(8) trypanosomes/ml. Parasitaemia, Packed Cell Volume (PCV), mean survival time and change in body weight were used as indices for monitoring the efficacy of the extracts. Diminazene (28 mg/kg) was used as a positive control while 2 % Tween was used as the negative control. Phytochemicals screening were conducted following standard methods. RESULTS: The extracts showed no toxicity effect in Swiss albino mice and had LD50 above 2000 mg/kg. The phytochemicals screened in V. sinaiticum were alkaloids, flavonoids, glycoside, saponins, steroids, phenolic compounds, and tannins. The mice treated with absolute methanol leaf extract of V. sinaiticum at 400 mg/kg dose had significantly lower mean parasitaemia (7.20 ± 0.16) (p < 0.001) as compared to the negative control group (8.82 ± 0.12) on day 14 of treatment. Animals treated with the same dose had significant (p < 0.001) higher PCV value and body weight and as well as the highest mean survival time of 40.20 ± 0.31 days as compared to the negative control at the end of the observation period. CONCLUSION: This study established that Verbascum sinaiticum had trypanocidal activity. The crude extracts have partially eliminated trypanosomes in a dose-dependent manner. The study can be a basis for future in-depth analysis of the biologically active chemicals.

MIF-Mediated Hemodilution Promotes Pathogenic Anemia in Experimental African Trypanosomosis.

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis.