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African trypanosomiasis and malaria are among the most severe health challenges to humans and livestock in Africa and new drugs are needed. Leaves of Hyptis suaveolens Kuntze (Lamiaceae) and Momordica charantia L. (Cucurbitaceae) were extracted with hexane, ethyl acetate, and then methanol, and subjected to silica gel column chromatography. Structures of six isolated compounds were elucidated through NMR and HR-EIMS spectrometry. Callistrisic acid, dehydroabietinol, suaveolic acid, suaveolol, and a mixture of suaveolol and suaveolic acid (SSA) were obtained from H. suaveolens, while karavilagenin D and momordicin I acetate were obtained from M. charantia. The isolated biomolecules were tested against trypomastigotes of Trypanosoma brucei brucei and T. congolense, and against Plasmodium falciparum. The most promising EC 50 values were obtained for the purified suaveolol fraction, at 2.71 ± 0.36 μg/mL, and SSA, exhibiting an EC 50 of 1.56 ± 0.17 μg/mL against T. b. brucei trypomastigotes. Suaveolic acid had low activity against T. b. brucei but displayed moderate activity against T. congolense trypomastigotes at 11.1 ± 0.5 μg/mL. Suaveolol and SSA were also tested against T. evansi, T. equiperdum, Leishmania major and L. mexicana but the antileishmanial activity was low. Neither of the active compounds, nor the mixture of the two, displayed any cytotoxic effect on human foreskin fibroblast (HFF) cells at even the highest concentration tested, being 200 μg/mL. We conclude that suaveolol and its mixture possessed significant and selective trypanocidal activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Animal African trypanosomosis (AAT) is an important global disease of livestock that causes economic losses of up to 4.5 billion US dollars per year. Thus, eliminating AAT in endemic countries will improve agricultural productivity and economic growth. To prevent AAT, vector control and the development of prophylactic drugs are crucial. Ascofuranone (AF) is a bioactive fungal compound with proven in vitro trypanocidal potency and in vivo treatment efficacy. However, the complex stereoselective synthesis of AF has prevented its cost-effective industrial production. Recently, a genetically modified strain of Acremonium egyptiacum fungus that produces a high yield of AF was developed. Therefore, we hypothesized that the oral administration of the AF-producing fungus itself may be effective against AAT. Hence, this study aimed to evaluate the prophylactic activity of orally administered dry-heat-sterilized A. egyptiacum against Trypanosoma congolense IL3000 infection using a mouse model. The survival rate was significantly prolonged (p = 0.009), and parasitemia was suppressed in all AF-fungus-treated groups (Group 1-9) compared with that in the untreated control group (Group 10). Hence, the trypanocidal activity of AF was retained after dry-heat-sterilization of the AF-producing fungus and that its oral administration effectively prevented AAT. Since AAT is endemic to rural areas with underdeveloped veterinary infrastructure, dry-heat-sterilized A. egyptiacum would be the most cost-effective potential treatment for AAT., Competing Interests: Declaration of competing interest The authors declare no competing interests in association with this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: African Animal Trypanosomosis (AAT) caused by Trypanosoma congolense is a parasitic disease affecting the livestock industry in sub-Saharan Africa and usually results in severe anemia, organ damage, and ultimately the death of the infected host. The present study was designed to investigate the possible chemotherapeutic effect of eugenol on T. congolense infections and its inhibitory effect on the trans-sialidase (TconTS) gene expression., Methods: Animals were infected with T. congolense and treated with 15 and 30 mg/kg body weight (BW) of eugenol for ten (10) days., Results: The eugenol (15 mg/kg BW) significantly (P < 0.05) reduced the T. congolense proliferation, increased animal survival, and reduced serum urea level. However, both dosages of eugenol significantly (P < 0.05) ameliorated T. congolense-induced anemia, renal hypertrophy, splenomegaly, and reduced total damage score in the liver and kidney of infected animals. In addition, the compound significantly (P < 0.05) downregulated the expression levels of TconTS1, TconTS2, TconTS3, and TconTS4 but the effect was more pronounced (sevenfold reduction) on TconTS1., Conclusions: The oral administration of eugenol suppressed T. congolense proliferation and prevented some major pathologies associated with trypanosomiasis infection. The reversal of renal hypertrophy and splenomegaly by the compound in addition to the reduction in the expression level of the TconTS gene variants could explain the observed anemia ameliorative potential of the compound., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Sub-Saharan Africa is profoundly challenged with African Animal Trypanosomiasis and the available trypanocides are faced with drawbacks, necessitating the search for novel agents. Herein, the chemotherapeutic potential of phloroglucinol on T. congolense infection and its inhibitory effects on the partially purified T. congolense sialidase and phospholipase A 2 (PLA 2 ) were investigated. Treatment with phloroglucinol for 14 days significantly ( p < 0.05) suppressed T. congolense proliferation, increased animal survival and ameliorated anemia induced by the parasite. Using biochemical and histopathological analyses, phloroglucinol was found to prevent renal damages and splenomegaly, besides its protection against T. congolense -associated increase in free serum sialic acids in infected animals. Moreover, the compound inhibited bloodstream T. congolense sialidase via mixed inhibition pattern with inhibition binding constant (Ki) of 0.181 µM, but a very low uncompetitive inhibitory effects against PLA 2 (Ki > 9000 µM) was recorded. Molecular docking studies revealed binding energies of -4.9 and -5.3 kcal/mol between phloroglucinol with modeled sialidase and PLA 2 respectively, while a 50 ns molecular dynamics simulation using GROMACS revealed the sialidase-phloroglucinol complex to be more compact and stable with higher free binding energy (-67.84 ± 0.50 kJ/mol) than PLA 2 -phloroglucinol complex (-77.17 ± 0.52 kJ/mol), based on MM-PBSA analysis. The sialidase-phloroglucinol complex had a single hydrogen bond interaction with Ser453 while none was observed for the PLA 2 -phloroglucinol complex. In conclusion, phloroglucinol showed moderate trypanostatic activity with great potential in ameliorating some of the parasite-induced pathologies and its anti-anemic effects might be linked to inhibition of sialidase rather than PLA 2 .

Background: Trypanosomiasis is one of the neglected tropical diseases of both humans and animals which decreases their productivity and causes death in the worst scenario. Unavailability of vaccines, the low therapeutic index of trypanocidal drugs, and the development of resistance lead to the need for research focused on developing alternative treatment options especially from medicinal plants. The present study was aimed to investigate antitrypanosomal activities of leaves of Cymbopogon citratus and seeds of Lepidium sativum in in-vivo mice model., Methods: The plant extracts were prepared by maceration using 80% methanol and reconstituted with 10% dimethyl sulfoxide (DMSO) to have the desired concentration. The test doses were adjusted to 100, 200 and 400 mg/kg based on the toxicity profile. The plants extracts were administered to the respective groups of mice after the 12 th day of field isolate T. congolense inoculation for seven consecutive days. The level of parasitemia, bodyweight, packed cell volume (PCV), and differential white blood cell counts were measured., Results: The in -vivo test results revealed that both plant extracts had dose-dependent antitrypanosomal activity. Both crude extracts showed a significant reduction in parasite load (P < 0.05), increased or prevent the fall of PCV value (P < 0.05), decreased lymphocytosis and increased neutrophil counts (p < 0.05) and improved bodyweight but significant bodyweight increment (P < 0.05) was observed only in C. citratus treated mice compared to the negative and positive controls., Conclusion: The present study concluded that the crude extracts of leaves of C. citratus and seeds of L. sativum had antitrypanosomal effects. Both plants extracts reduced parasitemia level, prevented anemia and improved bodyweight of treated mice. Comparative results from all tested parameters showed that the best activities were observed with C. citratus treated groups of mice., (© 2021. The Author(s).)

Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross-resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogs DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [ 3 H]-diminazene was slow with low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene-resistant Trypanosoma brucei brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [ 3 H]-diminazene transport studies, whole-genome sequencing, and RNA-seq found no major changes in diminazene uptake, folate transporter sequence, or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential Ψm. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions., Competing Interests: The authors have declared that no competing interests exist.

Trypanothione reductase is a key enzyme that upholds the redox balance in hemoflagellate protozoan parasites such as T. congolense. This study aims at unraveling the potency of Kolaviron against trypanothione reductase in T. congolense infection using Chrysin as standard. The experiment was performed using three different approaches; in silico, in vitro and in vivo. Kolaviron and Chrysin were docked against trypanothione reductase, revealing binding energies (-9.3 and -9.0 kcal/mol) and K i of 0.211μM and 0.151μM at the active site of trypanothione reductase as evident from the observed strong hydrophobic/hydrogen bond interactions. Parasitized blood was used for parasite isolation and trypanothione reductase activity assay using standard protocol. Real-time PCR (qPCR) assay was implored to monitor expression of trypanothione reductase using primers targeting the 177-bp repeat satellite DNA in T. congolense with SYBR Green to monitor product accumulation. Kolaviron showed IC 50 values of 2.64μg/ml with % inhibition of 66.78 compared with Chrysin with IC 50 values of 1.86μg/ml and % inhibition of 53.80. In vivo studies following the administration of these compounds orally after 7 days post inoculation resulted in % inhibition of Chrysin (57.67) and Kolaviron (46.90). Equally, Kolaviron relative to Chrysin down regulated the expression trypanothione reductase gene by 1.352 as compared to 3.530 of the infected group, in clear agreement with the earlier inhibition observed at the fine type level. Overall, the findings may have unraveled the Kolaviron potency against Trypanosoma congolense infection in rats.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism., Competing Interests: The authors have declared that no competing interests exist.

The search for novel therapeutic candidates against animal trypanosomiasis is an ongoing scientific endevour because of the negative impacts of the disease to the African livestock industry. In this study, the in vivo therapeutic potentials of phytol toward Trypanosoma congolense infection and the inhibitory effects on trypanosomal sialidase were investigated. Rats were infected with T. congolense and administered daily oral treatment of 50 and 100 mg/kg BW of phytol. Within the first 10 days of the treatment, no antitrypanosomal activity was recorded but a moderate trypanostatic activity was observed from day 17-day 21 pi. However, at 100 mg/kg BW, phytol demonstrated a significant (p < 0.05) ameliorative potentials toward T. congolense-induced host-associated pathological damages such as anaemia, hepatic and renal damages; and the data was comparable to diminazine aceturate. Moreover, the T. congolense caused a significant (p < 0.05) increase in free serum sialic acid level which was significantly (p < 0.05) prevented in the presence of phytol (100 mg/kg BW). In an in vitro analysis, phytol inhibited partially purified T. congolense sialidase using an uncompetitive inhibition pattern with inhibition binding constant of 261.24 μmol/mL. Subsequently, molecular docking revealed that the compound binds to homology modelled trypanosomal sialidase with a binding free energy of -6.7 kcal/mol which was mediated via a single hydrogen bond while Trp324 and Pro274 were the critical binding residues. We concluded that phytol has moderate trypanostatic activity but with a great potential in mitigating the host-associated cellular damages while the anaemia amelioration was mediated, in part, through the inhibition of sialidase., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A chemical examination of an extract from the aerial part of Oxytropis lanata led to the isolation and identification of 36 compounds, including saponins, isoflavonoids, oxazoles, and glycosides. The three among them were previously unreported oleanane-type saponins. In trypanocidal screening, 5,7,4'-trihydroxyisoflavone showed inhibitory activity against Trypanosoma congolense (IC 50  = 10.5 μM), the causative agent of African trypanosomosis in animals; this activity was similar to that of active compounds from the roots of this plant. O. lanata is known to be a traditional medicinal plant in Mongolia for the treatment of inflammatory diseases. The anti-hyaluronidase effect of saponins 3, 5, 8, and 9, (IC 50  = 0.15-0.22 mM) was stronger than that of sodium cromoglicate, which was used as a reference drug (IC 50  = 0.37 mM). The chemical structures of the new saponins were determined based on HRFABMS, 1 H and 13 C NMR, 1 H- 1 H COSY, HMQC, HMBC, and ROESY spectroscopic data along with chemical procedures., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

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 10 8 Trypanosoma 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 IC 50 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., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Tsetse flies (Diptera: Glossinidae) and tabanids (Diptera: Tabanidae) are haematophagous insects of medical and veterinary importance due to their respective role in the biological and mechanical transmission of trypanosomes. Few studies on the distribution and relative abundance of both families have been conducted in Mozambique since the country's independence. Despite Nicoadala, Mozambique, being a multiple trypanocidal drug resistance hotspot no information regarding the distribution, seasonality or infection rates of fly-vectors are available. This is, however, crucial to understanding the epidemiology of trypanosomosis and to refine vector management., Methods: For 365 days, 55 traps (20 NGU traps, 20 horizontal traps and 15 Epsilon traps) were deployed in three grazing areas of Nicoadala District: Namitangurine (25 traps); Zalala (15 traps); and Botao (15 traps). Flies were collected weekly and preserved in 70% ethanol. Identification using morphological keys was followed by molecular confirmation using cytochrome c oxidase subunit 1 gene. Trap efficiency, species distribution and seasonal abundance were also assessed. To determine trypanosome infection rates, DNA was extracted from the captured flies, and submitted to 18S PCR-RFLP screening for the detection of Trypanosoma., Results: In total, 4379 tabanids (of 10 species) and 24 tsetse flies (of 3 species), were caught. NGU traps were more effective in capturing both the Tabanidae and Glossinidae. Higher abundance and species diversity were observed in Namitangurine followed by Zalala and Botao. Tabanid abundance was approximately double during the rainy season compared to the dry season. Trypanosoma congolense and T. theileri were detected in the flies with overall infection rates of 75% for tsetse flies and 13% for tabanids. Atylotus agrestis had the highest infection rate of the tabanid species. The only pathogenic trypanosome detected was T. congolense., Conclusions: Despite the low numbers of tsetse flies captured, it can be assumed that they are still the cyclical vectors of trypanosomosis in the area. However, the high numbers of tabanids captured, associated to their demonstrated capacity of transmitting trypanosomes mechanically, suggest an important role in the epidemiology of trypanosomosis in the Nicoadala district. These results on the composition of tsetse and tabanid populations as well as the observed infection rates, should be considered when defining strategies to control the disease.

African animal trypanosomiases (AAT) remain the major constraint for livestock production, agriculture and food security in Africa. Although several control measures have been developed to fight AAT, the use of trypanocides remains the main strategy in most affected poor and rural communities. However, several studies have highlighted drug-resistant-trypanosome infections in many African countries, though this phenomenon is still not well described. This study aims to detect trypanosome species and the molecular profiles of drug-resistant-trypanosomes in naturally infected domestic animals of Yoko in the centre region of southern Cameroon. Therefore, in October 2017, 348 animals were blood sampled. The level of packed cell volume (PCV) was evaluated in each animal and trypanosome infections were investigated with the capillary tube centrifugation technique (CTC). Thereafter, DNA was extracted from blood samples and different trypanosome species were identified by PCR. The resistant/sensitive molecular profiles of trypanosomes for diminazene aceturate (DA) and isometamidium chloride (ISM) were investigated by PCR-RFLP. About 18.4% (64/348) of animals analyzed by PCR were found with trypanosome infections including Trypanosoma vivax, Trypanosoma brucei s.l. and Trypanosoma congolense forest and savannah. Trypanosoma congolense savannah was the predominant species with an infection rate of 15.2%. Between villages, significant (p˂0.0001) differences were found in the overall trypanosome infection rates. No molecular profile for ISM resistant-trypanosomes was identified. Conversely, about 88.9% (40/45) of T. congolense positive samples have shown molecular profiles of DA-resistant strains while the remaining 11.1% (5/45) showed mixed molecular profiles of resistant/sensitive strains. Results showed that the molecular profiles of DA-resistant strains of T. congolense in domestic animals of Yoko were widespread. This data needs to be confirmed by testing in vivo the drug susceptibilities of the trypanosome strains herein detected. In conclusion, appropriate future control measures are required. In addition to the intensification of vector control, ISM is advised for the treatment of animals infected by trypanosomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Vegetable oils are frequently used as solvents for lipophilic materials; accordingly, the effects of their components should be considered in animal experiments. In this study, the effects of various vegetable oils on the course of Trypanosoma congolense infection were examined in mice. C57BL/6J mice were orally administered four kinds of oils (i.e., coconut oil, olive oil, high oleic safflower oil, and high linoleic safflower oil) with different fatty acid compositions and infected with T. congolense IL-3000. Oil-treated mice infected with T. congolense showed significantly higher survival rates and lower parasitemia than those of control mice. Notably, coconut oil, which mainly consists of saturated fatty acids, delayed the development of parasitemia at the early stage of infection. These results indicated that vegetable oil intake could affect T. congolense infection in mice. These findings have important practical implications; for example, they suggest the potential effectiveness of vegetable oils as a part of the regular animal diet for controlling tropical diseases and indicate that vegetable oils are not suitable solvents for studies of the efficacy of lipophilic agents against T. congolense., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Extracts of 35 samples of European propolis were tested against wild type and resistant strains of the protozoal pathogens Trypanosoma brucei, Trypanosoma congolense and Leishmania mexicana. The extracts were also tested against Crithidia fasciculata a close relative of Crithidia mellificae, a parasite of bees. Crithidia, Trypanosoma and Leishmania are all members of the order Kinetoplastida. High levels of activity were obtained for all the samples with the levels of activity varying across the sample set. The highest levels of activity were found against L. mexicana. The propolis samples were profiled by using liquid chromatography with high resolution mass spectrometry (LC-MS) and principal components analysis (PCA) of the data obtained indicated there was a wide variation in the composition of the propolis samples. Orthogonal partial least squares (OPLS) associated a butyrate ester of pinobanksin with high activity against T. brucei whereas in the case of T. congolense high activity was associated with methyl ethers of chrysin and pinobanksin. In the case of C. fasciculata highest activity was associated with methyl ethers of galangin and pinobanksin. OPLS modelling of the activities against L. mexicana using the mass spectrometry produced a less successful model suggesting a wider range of active components.

Trypanosoma congolense is an important pathogen that wreaks havoc in the livestock industry of the African continent. This study evaluated the in vivo antitrypanosomal activity of geranylacetone and its ameliorative effect on the disease-induced anaemia and organ damages as well as its inhibitory effects against trypanosomal sialidase using in vitro and in silico techniques. Geranylacetone was used to treat T. congolense infected rats, at a dose of 50 and 100 mg/kg BW, for 14 days where it was found to reduce the parasite burden in the infected animals. Moreover, 100 mg/kg BW of geranylacetone significantly (p < 0.05) ameliorated the anaemia, hepatic and renal damages caused by the parasite. This is in addition to the alleviation of the parasite-induced hepatosplenomegaly and upsurge in free serum sialic acid levels in the infected animals which were associated with the observed anaemia amelioration by the compound. Consequently, bloodstream T. congolense sialidase was partially purified on DEAE cellulose column and inhibition kinetic studies revealed that the enzyme was inhibited by geranylacetone via an uncompetitive inhibition pattern. In silico analysis using molecular docking with Autodock Vina indicated that geranylacetone binds to trypanosomal sialidase with a minimum free binding energy of -5.8 kcal/mol which was mediated by 26 different kinds of non-covalent interactions excluding hydrogen bond whilst Asp163 and Phe421 had the highest number of the interactions. The data suggests that geranylacetone has trypanostatic activity and could protect animals against the T. congolense-induced anaemia through the inhibition of sialidase and/or the protection of the parasite-induced hepatosplenomegaly., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Eight isovaleryllignans (1-4 and 8-11), three isovalerylphenylpropanoids (5-7), three known lignans (12-14), and four known compounds were isolated from an extract of the aerial part of Brachanthemum gobicum. The structures of the isolated compounds were elucidated based on NMR and MS data analyses. The enantiomers of compounds 1-3, 5, 8, and 9 were isolated using chiral-phase HPLC, and the absolute configurations of 1a/1b-3a/3b, 5a/5b, 8a/8b, and 9a/9b were elucidated from their optical rotations and ECD spectra; the other lignans were assumed to be racemic or scalemic by chiral-phase HPLC analyses and optical rotation data. Some of the acylated lignans (racemic mixtures) (1-4, 8, 9, and 12-14) exhibited moderate inhibitory activities against Trypanosoma congolense, the causative agent of nagana disease in animals.

The four components present in the trypanocidal treatment Samorin, the commercially available formulation of isometamidium, were separated and purified by column chromatography. These compounds as well as the Samorin mixture and the other phenanthridine trypanocide, homidium, were tested on Trypanosoma congolense and wild type, diamidine- and isometamidium-resistant Trypanosoma brucei brucei strains using an Alamar blue drug sensitivity assay. EC 50 values obtained suggest that M&B4180A (2) was the most active of the components, followed by M&B38897 (1) in all the strains tested, whereas M&B4596 (4) was inactive. Samorin was found to be significantly more active than any of the individual components alone, against T. congolense and all three T. b, brucei strains. Samorin and all its active constituents displayed reduced activity against the previously characterised isometamidium-resistant strain ISMR1., (Copyright © 2019. Published by Elsevier Ltd.)

Animal African trypanosomiasis (AAT) is a significant socioeconomic burden for sub-Saharan Africa because of its huge impact on livestock health. Existing therapies including those based on minor groove binders (MGBs), such as the diamidines, which have been used for decades, have now lost efficacy in some places because of the emergence of resistant parasites. Consequently, the need for new chemotherapies is urgent. Here, we describe a structurally distinct class of MGBs, Strathclyde MGBs (S-MGBs), which display excellent in vitro activities against the principal causative organisms of AAT: Trypanosoma congolense, and Trypanosoma vivax. We also show the cure of T. congolense-infected mice by a number of these compounds. In particular, we identify S-MGB-234, compound 7, as curative by using two applications of 50 mg/kg intraperitoneally. Crucially, we demonstrate that S-MGBs do not show cross-resistance with the current diamidine drugs and are not internalized via the transporters used by diamidines. This study demonstrates that S-MGBs have significant potential as novel therapeutic agents for AAT.

Human and animal African trypanosomiasis (HAT & AAT, respectively) remain a significant health and economic issue across much of sub-Saharan Africa. Effective control of AAT and potential eradication of HAT requires affordable, sensitive and specific diagnostic tests that can be used in the field. Small RNAs in the blood or serum are attractive disease biomarkers due to their stability, accessibility and available technologies for detection. Using RNAseq, we have identified a trypanosome specific small RNA to be present at high levels in the serum of infected cattle. The small RNA is derived from the non-coding 7SL RNA of the peptide signal recognition particle and is detected in the serum of infected cattle at significantly higher levels than in the parasite, suggesting active processing and secretion. We show effective detection of the small RNA in the serum of infected cattle using a custom RT-qPCR assay. Strikingly, the RNA can be detected before microscopy detection of parasitaemia in the blood, and it can also be detected during remission periods of infection when no parasitaemia is detectable by microscopy. However, RNA levels drop following treatment with trypanocides, demonstrating accurate prediction of active infection. While the small RNA sequence is conserved between different species of trypanosome, nucleotide differences within the sequence allow generation of highly specific assays that can distinguish between infections with Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax. Finally, we demonstrate effective detection of the small RNA directly from serum, without the need for pre-processing, with a single step RT-qPCR assay. Our findings identify a species-specific trypanosome small RNA that can be detected at high levels in the serum of cattle with active parasite infections. This provides the basis for the development of a cheap, non-invasive and highly effective diagnostic test for trypanosomiasis., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Yvonne Freund was employed by Anacor Pharmaceuticals. Benzoxaboroles were supplied by Anacor Pharmaceuticals (YF) which was later taken over by Pfizer. This does not alter our adherence to all the PLOS Pathogens policies on sharing data and materials.

Trypanosoma congolense is a protozoan parasite that is transmitted by tsetse flies, causing African Animal Trypanosomiasis, also known as Nagana, in sub-Saharan Africa. Nagana is a fatal disease of livestock that causes severe economic losses. Two drugs are available, diminazene and isometamidium, yet successful treatment is jeopardized by drug resistant T. congolense. Isothermal microcalorimetry is a highly sensitive tool that can be used to study growth of the extracellular T. congolense parasites or to study parasite growth inhibition after the addition of antitrypanosomal drugs. Time of drug action and time to kill can be quantified in a simple way by real time heat flow measurements. We established a robust protocol for the microcalorimetric studies of T. congolense and developed mathematical computations in R to calculate different parameters related to growth and the kinetics of drug action. We demonstrate the feasibility and benefit of the method exemplary with the two standard drugs, diminazene aceturate and isometamidium chloride. The method and the mathematical approach can be translated to study other pathogenic or non-pathogenic cells if they are metabolically active and grow under axenic conditions., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

African trypanosomiasis is a neglected parasitic disease that is still of great public health relevance, and a severe impediment to agriculture in endemic areas. The pathogens possess certain unique metabolic features that can be exploited for the development of new drugs. Notably, they rely on an essential, mitochondrially-localized enzyme, Trypanosome Alternative Oxidase (TAO) for their energy metabolism, which is absent in the mammalian hosts and therefore an attractive target for the design of safe drugs. In this study, we cloned, expressed and purified the physiologically relevant form of TAO, which lacks the N-terminal 25 amino acid mitochondrial targeting sequence (ΔMTS-TAO). A new class of 32 cationic and non-cationic 4-hydroxybenzoate and 4-alkoxybenzaldehyde inhibitors was designed and synthesized, enabling the first structure-activity relationship studies on ΔMTS-TAO. Remarkably, we obtained compounds with enzyme inhibition values (IC 50 ) as low as 2 nM, which were efficacious against wild type and multidrug-resistant strains of T. brucei and T. congolense. The inhibitors 13, 15, 16, 19, and 30, designed with a mitochondrion-targeting lipophilic cation tail, displayed trypanocidal potencies comparable to the reference drugs pentamidine and diminazene, and showed no cross-resistance with the critical diamidine and melaminophenyl arsenical classes of trypanocides. The cationic inhibitors 15, 16, 19, 20, and 30 were also much more selective (900 - 344,000) over human cells than the non-targeted neutral derivatives (selectivity >8-fold). A preliminary in vivo study showed that modest doses of 15 and 16 reduced parasitaemia of mice infected with T. b. rhodesiense (STIB900). These compounds represent a promising new class of potent and selective hits against African trypanosomes., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

African animal trypanosomosis is a debilitating tsetse-transmitted parasitic disease of sub-Saharan Africa. Therapeutic and prophylactic drugs were introduced more than 50 years ago, and drug resistance is increasingly reported. In a cross-sectional study, 467 cattle were microscopically screened for trypanosomes. Samples were collected in May-July 2014 from five villages (Botao, Mungama, Zalala-Electrosul, Zalala-Madal, and Namitangurine) in Nicoadala district, Zambezia province. To evaluate treatment efficacy, trypanosome-positive animals in each village were randomly assigned to two groups, one treated with 0.5 mg/kg b.w. isometamidium (Inomidium®), the second with 3.5 mg/kg b.w. diminazene (Inomazene®). Cattle were microscopically monitored at days 0, 14, and 28 post-treatment. At day 28, trypanocides were swapped to investigate single or multiple resistance. Microscopically negative samples from the monitoring days were tested using 18S-PCR-RFLP. 22.9% (107/467) was found positive on day 0. On day 14, nine animals in Botao and seven in Mungama were positive. On day 28, in Botao, four animals from the diminazene group and four from the isometamidium group were positive. In Mungama, four animals from the diminazene group were positive on day 28. On day 42, six animals (9%) in Botao and two (9.5%) in Mungama remained positive after drug swap. No relapses occurred in Namitangurine. The 18S-PCR-RFLP consistently detected more positive than microscopy: indeed, positives reached 12, 13, and 8 in Botao and 9, 7, and 4 in Mungama, at days 14, 28, and 42, respectively. Single- and multi-drug resistance in Nicoadala district, Zambezia province, is thus here confirmed. This should be considered when choosing control options.

Novel l-valinate amide benzoxaboroles and analogues were designed and synthesized for a structure-activity-relationship (SAR) investigation to optimize the growth inhibitory activity against Trypanosoma congolense (T. congolense) and Trypanosoma vivax (T. vivax) parasites. The study identified 4-fluorobenzyl (1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-l-valinate (5, AN11736), which showed IC 50 values of 0.15 nM against T. congolense and 1.3 nM against T. vivax, and demonstrated 100% efficacy with a single dose of 10 mg/kg against both T. congolense and T. vivax in mouse models of infection (IP dosing) and in the target animal, cattle, dosed intramuscularly. AN11736 has been advanced to early development studies., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Context: Control of African trypanosomiasis relies on chemotherapy, but the development of resistance and the problem of drug residues require research for alternatives. Triterpenes and phenolics, the major constituents of Pleurotus sajor-caju (Fr.) Singer (Pleurotaceae), are reported to be effective against trypanosomiasis., Objective: Trypanocidal effect of whole Pleurotus sajor-caju aqueous extract was investigated in vivo against Trypanosoma congolense., Materials and Methods: Mice (25-32 g) were divided into seven groups of six animals. Mice in groups A-F received 2.5 × 10 4 trypanosomes, while group G was uninfected. Extracts (100-250 mg/kg) were administered intraperitoneally for 5 days to groups A-D while diminazine aceturate (group E) and normal saline (group F) served as positive and negative controls, respectively. Parasitemia, survival time, body weight and haematological parameters were monitored for 60 days post-treatment., Results: Parasitemia decreased significantly (p < 0.01) post-treatment with 200 and 250 mg/kg of the extract and became undetectable by day 16 and 12 post-infection, respectively; the ED 50 was 221.5 mg/kg. The packed cell volume (PCV) and the weight of mice treated with 250 mg/kg extract were 46.20 ± 2.6% and 32.05 ± 3.63 g, respectively, which is higher than the group treated with diminazine aceturate. The mean survival time of animals in groups D and E was >60 days, while that of group F was <4 days. Differential leucocyte count on day 68 post-infection in groups C, D and E were not significantly different., Conclusion: Pleurotus sajor-caju therefore could be a potential source of new trypanocidal drugs.

Isometamidium Chloride (ISM) is one of the principal drugs used to counteract Trypanosoma congolense infection in livestock, both as a prophylactic as well as a curative treatment. However, numerous cases of ISM resistance have been reported in different African regions, representing a significant constraint in the battle against Animal African Trypanosomiasis. In order to identify genetic signatures associated with ISM resistance in T. congolense, the sensitive strain MSOROM7 was selected for induction of ISM resistance in a murine host. Administered ISM concentrations in immune-suppressed mice were gradually increased from 0.001 mg/kg to 1 mg/kg, the maximal dose used in livestock. As a result, three independent MSOROM7 lines acquired full resistance to this concentration after five months of induction, and retained this full resistant phenotype following a six months period without drug pressure. In contrast, parasites did not acquire ISM resistance in immune-competent animals, even after more than two years under ISM pressure, suggesting that the development of full ISM resistance is strongly enhanced when the host immune response is compromised. Genomic analyses comparing the ISM resistant lines with the parental sensitive line identified shifts in read depth at heterozygous loci in genes coding for different transporters and transmembrane products, and several of these shifts were also found within natural ISM resistant isolates. These findings suggested that the transport and accumulation of ISM inside the resistant parasites may be modified, which was confirmed by flow cytometry and ex vivo ISM uptake assays that showed a decrease in the accumulation of ISM in the resistant parasites., (Copyright © 2017. Published by Elsevier Ltd.)

The primate-specific serum protein apolipoprotein L1 (APOL1) is the only secreted member of a family of cell death promoting proteins 1-4 . APOL1 kills the bloodstream parasite Trypanosoma brucei brucei, but not the human sleeping sickness agents T.b. rhodesiense and T.b. gambiense 3 . We considered the possibility that intracellular members of the APOL1 family, against which extracellular trypanosomes could not have evolved resistance, could kill pathogenic T. brucei subspecies. Here we show that recombinant APOL3 (rAPOL3) kills all African trypanosomes, including T.b. rhodesiense, T.b. gambiense and the animal pathogens Trypanosoma evansi, Trypanosoma congolense and Trypanosoma vivax. However, rAPOL3 did not kill more distant trypanosomes such as Trypanosoma theileri or Trypanosoma cruzi. This trypanolytic potential was partially shared by rAPOL1 from Papio papio (rPpAPOL1). The differential killing ability of rAPOL3 and rAPOL1 was associated with a distinct dependence on acidic pH for activity. Due both to its instability and toxicity when injected into mice, rAPOL3 cannot be used for the treatment of infection, but an experimental rPpAPOL1 mutant inspired by APOL3 exhibited enhanced trypanolytic activity in vitro and the ability to completely inhibit T.b. gambiense infection in mice. We conclude that pH dependence influences the trypanolytic potential of rAPOLs.

Trypanosoma congolense is one of the most prevalent pathogens which causes trypanosomosis in African animals, resulting in a significant economic loss. In its life cycle, T. congolense is incapable of synthesizing purine nucleotides via a de novo pathway, and thus relies on a salvage pathway to survive. In this study, we identified a gene from T. congolense, TcIL3000&#95;5&#95;1940, as a guanosine 5'-monophosphate reductase (GMPR), an enzyme that modulates the concentration of intracellular guanosine in the pathogen. The recombinant protein was expressed in Escherichia coli, and the gene product was enzymatically confirmed as a unique GMPR, designated as rTcGMPR. This enzyme was constitutively expressed in glycosomes at all of the parasite's developmental stages similar to other purine nucleotide metabolic enzymes. Mycophenolic acid (MPA) was found to inhibit rTcGMPR activity. Hence, it is a potential lead compound for the design of trypanocidal agents, specifically GMPR inhibitor., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Animal trypanosomosis is a devastating parasitic disease that is of economic importance to livestock production. The infection includes animal African trypanosomosis, surra, and dourine. The treatment is based solely on few compounds that were discovered decades ago and which are associated with severe toxicity. Furthermore, it is likely that the parasite has developed resistance towards them. Thus, there is an urgent need for new, accessible, and less toxic drugs. Azithromycin is an antibiotic with documented efficacy against Toxoplasma, Babesia, and Plasmodium. The current study investigated its effects against animal trypanosomes. An in vitro system was used to determine the trypanocidal effects of azithromycin against Trypanosoma congolense, Trypanosoma brucei brucei, and Trypanosoma evansi, and cytotoxicity in Madin-Darby bovine kidney (MDBK) and NIH 3T3 cells. Furthermore, the trypanocidal effects of azithromycin were investigated in T. congolense-infected mice. In vitro, azithromycin had an IC 50 of 0.19 ± 0.17; 3.69 ± 2.26; 1.81 ± 1.82 μg/mL against T. congolense, T. b. brucei, and T. evansi, respectively. No cytotoxic effects were observed in MDBK and NIH 3T3 cells. The efficacy of orally administered azithromycin was investigated in short-term and long-term treatment protocols. Although the short-term treatment protocol showed no curative effects, the survival rate of the mice was significantly prolonged (p < 0.001) in comparison to the control group. The long-term treatment yielded satisfying curative effects with doses of 300 and 400 mg/kg achieving 80 and 100% survival, respectively. In conclusion, long-term oral azithromycin treatment has trypanocidal effects against T. congolense.

Background: The search for novel antitrypanosomal agents had previously led to the isolation of ellagic acid as a bioactive antitrypanosomal compound using in vitro studies. However, it is not known whether this compound will elicit antitrypanosomal activity in in vivo condition which is usually the next step in the drug discovery process., Purpose: Herein, we investigated the in vivo activity of ellagic acid against bloodstream form of Trypanosoma congolense and its ameliorative effects on trypanosome-induced anemia and organ damage as well as inhibitory effects on trypanosomal sialidase., Methods: Rats were infected with T. congolense and were treated with 100 and 200mg/kg body weight (BW) of ellagic acid for fourteen days. The levels of parasitemia, packed cell volume and biochemical parameters were measured. Subsequently, T. congolense sialidase was partially purified on DEAE cellulose column and the mode of inhibition of ellagic acid on the T. congolense sialidase determined. Molecular docking study was also conducted to determine the mode of interaction of the ellagic acid to the catalytic domain of T. rangeli sialidase., Results: At a dose of 100 and 200mg/kg (BW), ellagic acid demonstrated significant (P < 0.05) trypanosuppressive effect for most of the 24 days experimental period. Further, the ellagic acid significantly (P < 0.05) ameliorated the trypanosome-induced anemia, hepatic and renal damages as well as hepatomegaly, splenomegaly and renal hypertrophy. The trypanosome-associated free serum sialic acid upsurge alongside the accompanied membrane bound sialic acid reduction were also significantly (P < 0.05) prevented by the ellagic acid treatment. The T. congolense sialidase was purified to a fold of 6.6 with a yield of 83.8%. The enzyme had a K M and Vmax of 70.12mg/ml and 0.04µmol/min respectively, and was inhibited in a non-competitive pattern by ellagic acid with an inhibition binding constant of 1986.75μM. However, in molecular docking study, ellagic acid formed hydrogen bonding interaction with major residues R 39 , R 318 , and W 124 at the active site of T. rangeli sialidase with a predicted binding free energy of -25.584kcal/mol., Conclusion: We concluded that ellagic acid possesses trypanosuppressive effects and could ameliorate the trypanosome-induced pathological alterations., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

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.

African animal trypanosomosis (AAT) is caused by the tsetse fly-transmitted protozoans Trypanosoma congolense and T. vivax and leads to huge agricultural losses throughout sub-Saharan Africa. Three drugs are available to treat nagana in cattle (diminazene diaceturate, homidium chloride, and isometamidium chloride). With increasing reports of drug-resistant populations, new molecules should be investigated as potential candidates to combat nagana. Dicationic compounds have been demonstrated to have excellent efficacy against different kinetoplastid parasites. This study therefore evaluated the activities of 37 diamidines, using in vitro and ex vivo drug sensitivity assays. The 50% inhibitory concentrations obtained ranged from 0.007 to 0.562 μg/ml for T. congolense and from 0.019 to 0.607 μg/ml for T. vivax On the basis of these promising results, 33 of these diamidines were further examined using in vivo mouse models of infection. Minimal curative doses of 1.25 mg/kg of body weight for both T. congolense - and T. vivax -infected mice were seen when the diamidines were administered intraperitoneally (i.p.) over 4 consecutive days. From these observations, 15 of these 33 diamidines were then further tested in vivo , using a single bolus dose for administration. The total cure of mice infected with T. congolense and T. vivax was seen with single i.p. doses of 5 and 2.5 mg/kg, respectively. This study identified a selection of diamidines which could be considered lead compounds for the treatment of nagana., (Copyright © 2017 American Society for Microbiology.)

Ethnopharmacological Relevance: Leaves from the plant species studied herein are traditionally used in northern Nigeria against various protozoan infections. However, none of these herbal preparations have been standardized, nor have their toxicity to mammalian cells been investigated. In search of improved and non-toxic active antiprotozoal principles that are not cross-resistant with current anti-parasitics, we here report the results of the in vitro screening of extracts from seven selected medicinal plant species (Centrosema pubescens, Moringa oleifera, Tridax procumbens, Polyalthia longifolia, Newbouldia laevis, Eucalyptus maculate, Jathropha tanjorensis), used traditionally to treat kinetoplastid infections in Nigeria, and the isolation of their bioactive principles., Aim of the Study: To investigate the efficacies of medicinal plant extracts, and of compounds isolated therefrom, against kinetoplastid parasites, assess cross-resistance to existing chemotherapy, and assay their toxicity against mammalian cells in vitro., Material and Methods: Plants were extracted with hexane, ethyl acetate and methanol. Active principles were isolated by bioassay-led fractionation, testing for trypanocidal activity, and identified using NMR and mass spectrometry. EC 50 values for their activity against wild-type and multi-drug resistant Trypanosoma brucei were obtained using the viability indicator dye resazurin., Results: Seven medicinal plants were evaluated for activity against selected kinetoplastid parasites. The result shows that crude extracts and isolated active compounds from Polyalthia longifolia and Eucalyptus maculata, in particular, display promising activity against drug-sensitive and multi-drug resistant Trypanosoma brucei. The EC 50 value of a clerodane (16α-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide) isolated from Polyalthia longifolia was as low as 0.38µg/mL, while a triterpenoid (3β,13β-dihydroxy-urs-11-en-28-oic acid) isolated from Eucalyptus maculata displayed an EC 50 of 1.58µg/mL. None of the isolated compounds displayed toxicity towards Human Embryonic Kidney cells at concentrations up to 400µg/mL. In addition, the isolated compounds were active against Leishmania mexicana, as well as against T. congolense., Conclusion: We have isolated a clerodane compound from Polyalthia longifolia that shows low toxicity, no cross-resistance with current treatments, and promising activity against both human-infective and veterinary Trypanosoma species., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

African Animal Trypanosomosis (AAT) is a major disease of cattle in Togo and its control is essentially based on chemotherapy. However, because of excessive use of trypanocides during the past decades, chemo-resistance in the parasites has developed. In order to assess the current situation of AAT and resistance to trypanocidal drugs in Northern Togo, a study was conducted on cattle from December 2012 to August 2013 in the regions of Kara and Savanes. An initial cross-sectional survey was carried out in 40 villages using the Haematocrit Centrifugation Technique (HCT). Out of these, 5 villages with a trypanosome prevalence of >10% were selected for a block treatment study (BT) with diminazene diaceturate (DA: 3.5mg/kg for a 14-day follow-up) and isometamidium chloride (ISM: 0.5mg/kg for a 28-day follow-up). Positive blood samples collected during the parasitological surveys and an equivalent number of negatives were further analyzed by PCR-RFLP for trypanosome species confirmation and molecular diagnosis of resistance to DA in Trypanosoma congolense. The results from 1883 bovine blood samples confirmed a high overall trypanosome prevalence of 10.8% in Northern Togo. PCR-RFLP revealed that T. congolense is the dominant pathogenic trypanosome species (50.5%) followed by T. vivax (27.3%), and T. brucei (16.2%). The BT showed varying levels of treatment failures ranging from 0 to 30% and from 0 to 50% for DA and for ISM respectively, suggesting the existence of resistant trypanosome populations in the study area. Our results show that AAT still represents a major obstacle to the development of cattle husbandry in Northern Togo. In areas of high AAT risk, a community-based integrated strategy combining vector control, rational use of trypanocidal drugs and improving the general condition of the animals is recommended to decision makers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We investigated a chemical strategy to boost the trypanocidal activity of 2,4-dihydroxybenzoic acid (2,4-DHBA)- and salicylhydroxamic acid (SHAM)-based trypanocides with triphenylphosphonium and quinolinium lipophilic cations (LC). Three series of LC conjugates were synthesized that were active in the submicromolar (5a-d and 10d-f) to low nanomolar (6a-f) range against wild-type and multidrug resistant strains of African trypanosomes (Trypanosoma brucei brucei and T. congolense). This represented an improvement in trypanocidal potency of at least 200-fold, and up to >10 000-fold, compared with that of non-LC-coupled parent compounds 2,4-DHBA and SHAM. Selectivity over human cells was >500 and reached >23 000 for 6e. Mechanistic studies showed that 6e did not inhibit the cell cycle but affected parasite respiration in a dose-dependent manner. Inhibition of trypanosome alternative oxidase and the mitochondrial membrane potential was also studied for selected compounds. We conclude that effective mitochondrial targeting greatly potentiated the activity of these series of compounds.

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.

Eleven 2,5-diphenyloxazole derivatives (1-11), together with six known isoflavonoid derivatives, were isolated from the roots of Oxytropis lanata. The 2,5-diphenyloxazole (1) obtained proved to be identical to a standard sample used as a scintillator and liquid laser dye. The other oxazole derivatives isolated were found to have one to four hydroxy and/or O-methyl groups in their phenyl rings. Seven of the oxazole derivatives obtained are new (3-9). The inhibitory activity of the isolated compounds was evaluated against Trypanosoma congolense, the causative agent of African trypanosomosis in animals. Oxazoles with di- and trihydroxy groups showed trypanocidal activity, and 2-(2',3'-dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (4) exhibited the most potent inhibitory activity (IC 50 1.0 μM).

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.

Background: African trypanosomosis is the disease caused by extracellular protozoan parasites of the genus Trypanosoma transmitted by tsetse flies. The current study has evaluated the trypanocidal activity of Commiphora swynnertonii extracts on Trypanosoma congolense., Methods: The effect of ethanolic stem bark and resinous extracts on motility of T. congolense was evaluated by in vitro study at concentrations of 2 mg/ml and 4 mg/ml. Then, trypanocidal activity was evaluated by drug incubation infectivity test using mice at concentrations of 0.4 mg/ml and 2 mg/ml. In both studies negative (without drug) and positive (diminazene diaceturate) controls were used., Results: The in vitro study showed that, ethanolic stem bark extract of C. swynnertonii at concentration of 4 mg/ml caused complete cessation of motility for T. congolense in 30 min. However, resinous ethanolic extract had delayed effect on cessation of motility of T. congolense observed at 90 and 100 min post-incubation at concentrations of 4 mg/ml and 2 mg/ml respectively. The drug incubation infectivity test study depicted that ethanolic stem bark extract at concentration of 2 mg/ml significantly (p = 0.000) reduced the infectivity of T. congolense in mice. However, it did not vary significantly (P =0.897) with group treated with diminazene diaceturate incubated mixture., Conclusion: The current study has provided evidence that, ethanolic stem bark extract of C. swynnertonii possess trypanocidal activity against T. congolense. Based on these findings, further studies are recommended to determine its potential as a lead to trypanocidal drug discovery.

This study aimed to evaluate the trypanocidal activity of mycophenolic acid (MPA) and its derivatives for Trypanosoma congolense The proliferation of T. congolense was completely inhibited by adding <1 μM MPA and its derivatives. In addition, the IMP dehydrogenase in T. congolense was molecularly characterized as the target of these compounds. The results suggest that MPA and its derivatives have the potential to be new candidates as novel trypanocidal drugs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: African trypanosomiasis affects both humans and livestock in sub-Saharan countries including Ethiopia. Due to limitations to current chemotherapy, there is an urgent need for the development of new, safe, cheap and effective drugs. In the present study, the leaf of Dovyalis abyssinica was tested for its in vivo antitrypanosomal activity against Trypanosoma congolense field isolate on mice., Methods: The leaf of D. abyssinica was macerated using dichloromethane and methanol. The extracts at doses of 250, 200, 150 and 100 mg/kg body weight were administered intraperitonealy daily for 7 days to mice infected with T. congolense. Following administration, parasitemia, packed cell volume, rectal temperature, body weight and survival time were monitored., Results: Administration of dichloromethane and methanol extracts at 250 and 200 mg/kg reduced (p<0.05) parasitemia and rectal temperature, and improved (p<0.05) PCV, mean body weight, and mean survival time compared to dimethylsulfoxide treatment., Conclusion: Crude dichloromethane and methanol leaf extracts of D. abyssinica displayed anti-trypanosomal activity that may serve as lead for the development of effective alternative antitrypanosomal drugs.

African Animal Trypanosomosis is threatening the agricultural production and cattle breeding more severely than any other livestock disease in the continent, even more since the advent of drug resistance. A longitudinal study was conducted from November 2012 to May 2013 in the Ghibe valley to evaluate diminazene aceturate (DA) resistance and assess livestock owner's perception of trypanocidal drug use. Four Peasant Associations (PAs) were purposively selected and the cattle randomly sampled in each PAs. At the beginning of the study (t0), 106 bovines positive for trypanosomes by the haematocrit centrifugation technique (HCT) and 119 negative control animals were recruited for six months follow-up using HCT, 18S-PCR-RFLP, DpnII-PCR-RFLP and microsatellite analysis. Prevalence of trypanosomosis was 18.1% based on the HCT technique and the mean PCV value was 23.6±5.1% for the 587 sampled cattle. Out of the 106 HCT positive, 64 (60.4%) were positive for the presence of trypanosomes using the 18S-PCR-RFLP. Species detection showed 38 (59.4%) Trypanosoma congolense savannah, 18 (28.1%) Trypanosoma vivax, 5 (7.8%) Trypanosoma theileri and 3 (4.7%) T. congolense Kilifi. Among the T. congolense savannah samples, 31 (81.6%) showed a DA resistant RFLP profile, 2 (5.3%) a mixed profile and 5 did not amplify using the DpnII-PCR-RFLP. A positive HCT had a significant effect on PCV (p<0.001) with the mean PCV value equal to 24.4±0.2% in the absence of trypanosomes and to 20.9±0.3% in the presence of trypanosomes. PCV increased significantly (p<0.001) with 4.4±0.5% one month after treatment. All T. congolense savannah type were analyzed using microsatellite markers TCM1, TCM3 and TCM4. The main events were new infections (40.0%) and relapses (37.5%) with cures lagging at 22.5%. In 10 purposively selected PAs a semi-structured questionnaire was used. The average herd size was the highest in Abelti PA (6.7±1.8 TLU) and the mean herd size was statistically different (p=0.01) in the 10 PAs. Trypanosomosis was designated as the main disease affecting cattle by 97% of the respondents. DA was used by 95.5% of the farmers though more than half of them (51.9%) were not familiar with isometamidium (ISM). There was a trend to overdose young small animals and to underdose large ones. Oxen were treated very frequently (nearly 20 times/year) and calves almost never. To improve the situation in the Ghibe valley, extension messages should be delivered to promote a rational drug use, improved livestock management and the application of strategic vector control methods., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Animal African trypanosomosis (AAT), caused by Trypanosoma congolense, is widespread throughout sub-Saharan Africa. There are significant concerns related to the current drugs available for the treatment of AAT due to their limited effectiveness across species and their adverse effects. Moreover, drug resistant trypanosomes have recently been reported in the field. High throughput screening (HTS) of large chemical compound library collections is a promising approach for identifying novel drug candidates. While HTS for Trypanozoon trypanosomes, T. brucei sspp. and T. evansi is well established, no assays have been developed for T. congolense. In the present study, the authors developed an ATP-based luciferase viability assay for T. congolense in a 96-well plate format. The calculated 50% inhibitory concentration (IC50) values for pentamidine and diminazene were 10-100 times higher in T. congolense than in T. brucei. This result suggests that the transporters for the 2 tested compounds differ between T. congolense and T. brucei. This assay could further be applied to screen novel chemical compounds for the treatment of AAT caused by T. congolense.

Since the 1950s, the chemotherapy of animal African trypanosomosis in cattle has essentially relied on only two compounds: isometamidium chloride (ISM), a phenanthridine, and diminazene aceturate, an aromatic diamidine. The commercial formulations of ISM, including Veridium(®) and Samorin(®), are a mixture of different compounds: ISM is the major component, mixed with the red isomer, blue isomer and disubstituted compound. To investigate the pharmacological effects of these individual compounds ISM, the blue and red isomers and the disubstituted compound were synthesised and purified by HPLC. The activity of each compound was analysed both in vitro, and in mice in vivo. For the in vitro analysis, a drug sensitivity assay was developed in 96-well tissue culture plates to determine the effective concentration which killed 50% of trypanosome population within 48 h of drug exposure (IC50). All compounds tested in vitro possessed trypanocidal activity, and purified ISM was the most active. Veridium(®) and Samorin(®) had similar IC50 values to purified ISM for both Trypanosoma congolense and Trypanosoma brucei brucei. The disubstituted compound had the highest IC50 values whereas intermediate IC50 values were obtained for the blue and red isomers. In vivo, single-dose tests were used to evaluate the trypanocidal and prophylactic activity against T. congolense. Interestingly, the prophylactic effect two months post treatment was as efficient with ISM, Veridium(®), Samorin(®) and the disubstituted compound at the highest dose of 1mg/kg whereas the red and blue isomers both showed much lower prophylactic activity. This study on T. congolense implies that it is necessary to limit the quantity of the blue and red isomers in the commercial mixture. Finally, the in vitro sensitivity assay may be useful for screening new trypanocides but also for the testing and detection of resistant trypanosome isolates., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Treatment of trypanosomosis is currently facing a number of problems including toxicity of trypanocidal drugs and development of resistance by the parasites. These limitations have prompted the search for alternative active substances (such as of natural origin). The purpose of the study was to investigate the effect of extracts of Moringa stenopetala and Artemisia absinthium on Trypanosoma congolense in mice., Methods: Swiss white male mice aged 8-12 weeks were divided into six experimental groups of six animals. Water and methanol extracts of the two plants were prepared. T. congolense was isolated from cattle at Ghibe valley (Ethiopia). All experimental mice received approximately 1 x 10(5) trypanosomes in 0.2 ml of blood. Plant extracts were given orally to four groups (2 plant species and two extraction methods) at 400 mg/kg body weight for seven consecutive days. One group remained as distilled water treated control and the other as diminzene aceturate treated control. The effect of the extracts on levels of parasitaemia, body weight, packed cell volume (PCV) and mice survival was monitored for 25 days., Results: All treatments have significantly reduced parasitaemia and helped improve body weight, PCV and survival of mice compared to the water-treated control (P < 0.01 in all cases). These effects were comparable to that with diminazene aceturate. No significant difference was observed in the reduction of parasitaemia between plant extract treatment groups. However, mice with extracts of A. absinthium had significantly higher body weight than those with extracts of M. stenopetala (P < 0.05)., Conclusions: The two plants have antitrypanosomal potential against T. congolense by reducing the levels of parasitaemia, maintaining good PCV and body weight, and prolonging the lives of infected animals.

Background: There is an urgent need for the development of new, cheap, safe and highly effective drugs against African trypanosomiasis that affects both man and livestock in sub-Saharan Africa including Ethiopia. In the present study the exudate of Aloe gilbertii, an endemic Aloe species of Ethiopia, aloin, aloe-emodin and rhein were tested for their in vitro and in vivo antitrypanosomal activities against Trypanosoma congolense field isolate. Aloin was prepared from the leaf exudate of A. gilbertii by acid catalyzed hydrolysis. Aloe-emodin was obtained by oxidative hydrolysis of aloin, while rhein was subsequently derived from aloe-emodin by oxidation. In vitro trypanocidal activity tests were conducted on parasites obtained from infected mice, while mice infected with T. congolense were used to evaluate in vivo antitrypanosomal activity of the test substances., Results: Results of the study showed that all the test substances arrested parasites motility at effective concentration of 4.0 mg/ml within an incubation period ranging from 15 to 40 min. Moreover, the same concentration of the test substances caused loss of infectivity of the parasites to mice during 30 days observation period. Among the tested substances, rhein showed superior activity with minimum inhibitory concentration (MIC) of 0.4 mg/ml. No adverse reactions were observed when the test substances were administered at a dose of 2000 mg/kg. Rhein at doses of 200 and 400 mg/kg, and the exudate, aloin and aloe-emodin at a dose of 400 mg/kg reduced the level of parasitaemia significantly (P < 0.05) and improved anaemia., Conclusion: The results obtained in this investigation indicate that aloin and its derivatives particularly rhein have the potential to be used as a scaffold for the development of safe and cost effective antitrypanosomal drugs that can be useful in the continuing fight against African trypanosomiasis.

African trypanosomosis is a major threat to livestock production in sub-Saharan Africa. Although the disease mainly concerns cattle, dogs can also be infected by Trypanosoma spp. transmitted by tsetse flies. Between 1997 and 2003, the parasite Trypanosoma congolense was identified in French military dogs sent to Africa. On infected dogs, the diagnosis was made during the mission or just after the return to France, depending on when the symptoms appeared. The high incidence and mortality rate among these dogs led veterinarians of the French Health Service to implement a systematic chemoprophylaxis beginning in 2004. Between 2004 and 2011, the chemoprophylaxis was carried out on more than 400 military dogs. The protocol of chemoprophylaxis relies on the use of isometamidium chloride (Trypamidium(®), Merial). The drug has been used successfully at the dosage of 1mg/kg body weight by deep intramuscular injection, every two or three months. In addition, dogs are given collars impregnated with deltamethrin (Scalibor(®), MSD Animal Health). Isometamidium chloride was also used successfully in the treatment of military dogs infected with T. congolense, with a full recovery and without any relapses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A cross-sectional study was carried out in the Ghibe valley from August to October 2010. 411 head of cattle were sampled in eight villages for buffy coat examination (BCE) and blood spots were collected from each animal for trypanosomose diagnosis by 18S-PCR-RFLP and diminazene aceturate (DA) resistance by Ade2-PCR-RFLP. Three villages were selected in a zone where trypanosomosis control operations are currently on-going whereas the other 5 villages were located outside these control operations. Twenty-four samples (5.84%) were diagnosed positive for Trypanosoma congolense by BCE and injected in mice for further characterization. Twelve of those isolates successfully multiplied in mice and were tested by an in vivo mouse test for diminazene (DA) (10 and 20mg/kg B.W.) and isometamidium (ISM) (1mg/kg B.W.) resistance. All were shown to be resistant to both drugs at all doses. The use of the Ade2-PCR-RFLP on these isolates confirmed their DA-resistance profile. Seventy-three of the collected blood spots (17.8%) were diagnosed positive for T. congolense by 18S-PCR-RFLP of which 37 (50.7%) gave amplification products with the Ade2-PCR-RFLP. Here, 35 (94.6%) showed a resistant profile, 1 (2.7%) a sensitive profile and 1 (2.7%) a mixed profile. The data were analysed by logistic regression model and the relapsing time in mice tests was assessed using the Cox regression model. There was no significant intervention effect (P=0.83) with odds ratio equal to 1.21 when using the BCE data. 18S-PCR-RFLP test also showed no significant intervention effect (P=0.60) with odds ratio equal to 1.43. The hazard ratio of getting parasitaemic after treatment with DA at 20mg/kg B.W. compared to the control group was 0.38 which differs significantly from one (P<0.001). Relapsing time after treatment with DA 10mg/kg B.W. or ISM 1mg/kg B.W. was also significantly longer than the prepatent period of the control group. The situation of drug resistance in the Ghibe valley is further discussed., (Copyright © 2012 Elsevier B.V. All rights reserved.)