Epizootiology and Molecular Identification of Trypanosome Species in Livestock Ruminants in the Gambia.

INTRODUCTION: African Animal Trypanosomiasis (AAT) or nagana in animals, is caused by the blood-borne parasitic protozoa called trypanosomes, and is potentially fatal. It is estimated that Africa loses $4‒5 billion annually due to the death of livestock to nagana in the tsetse belt. PURPOSE: Although The Gambia lies within this belt, there is scanty data regarding the epizootiology of nagana in The Gambia. Here, records of reported cases of nagana for the period 2010-2019 at the International Trypanotolerance Centre (ITC) in The Gambia were analyzed retrospectively. METHODS: For insights into the current prevalence of AAT, blood samples of 384 cattle, 42 goats, and 59 sheep from the Central River Region (CRR) and Lower River Region (LRR) were analyzed microscopically for parasite identification. Furthermore, trypanosomes were characterized by polymerase chain reaction (PCR) using a panel of primers that identify trypanosomes to the level of the species and subspecies by targeting a portion of the internally transcribed spacer-one (ITS-1) of the ribosomal RNA. RESULTS: The retrospective study indicates that Trypanosoma vivax (66%) and T. congolense (33.4%) were the predominant species. Based on the archive records of ITC, the villages Touba, Misera, and Sambel Kunda all in the CRR of the Gambia are the most burdened with AAT. Microscopic examination of blood samples from cattle showed a prevalence of 1.56%, whereas the PCR-based analysis gave a higher prevalence of 12.5%. The molecular analysis revealed the presence of T. vivax (3.65%), T. congolense kilifi (2.6%), T. b. brucei (1.3%), T. congolense savannah/forest (0.52%), T. b. gambiense (0.52%). Interestingly, 4.43% of mixed infections i.e. multiple trypanosome species in individual animals were recorded. In 18% of the mixed infection cases, T. godfreyi, T. simiae were coinfecting cattle alongside T. congolense. The molecular identification including the phylogenetic analysis implicated T. congolense as the most predominant trypanosome species infecting animals in The Gambia. CONCLUSION: The incidence of nagana in The Gambia is documented and the prevalent trypanosomes identified to be T. vivax, different types of T. congolense, and T. brucei including the gambiense subspecie. Finally, nagana is less profound in sheep and goats compared to cattle, with seasonal and regional variations playing a significant role in the disease dynamics.

Bioassay-guided isolation of active principles from Nigerian medicinal plants identifies new trypanocides with low toxicity and no cross-resistance to diamidines and arsenicals.

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. EC50 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 EC50 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 (3ß,13ß-dihydroxy-urs-11-en-28-oic acid) isolated from Eucalyptus maculata displayed an EC50 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.

Antitrypanosomal Activity of a Novel Taccalonolide from the Tubers of Tacca leontopetaloides.

INTRODUCTION: Several taccalonolides with various bioactivities have been isolated from Tacca species but no studies to isolate taccalonolides with anti-trypanosomal activity from Tacca leontopetaloides have been reported. OBJECTIVES: To analyse extracts of the roots of Tacca leontopetaloides, purify the extracts by column chromatography and identify isolated compounds by spectroscopic methods. The compounds and fractions will be tested for antitrypanosomal activity in vitro against Trypanosoma brucei brucei. MATERIAL AND METHODS: Dried roots or tubers of Tacca leontopetaloides, chromatographic separation and spectroscopic identification. RESULTS: A novel taccalonolide A propanoate and some known taccalonolides were isolated and their structures were determined by NMR and mass spectrometry CONCLUSION: Several taccalonolides were isolated from Tacca leontopetaloides and were found to have in vitro antitrypanosomal activity against Trypanosoma brucei brucei and EC50 values for the isolated compounds were from 0.79 µg/mL. Copyright © 2016 John Wiley & Sons, Ltd.

Chemical and Antimicrobial Profiling of Propolis from Different Regions within Libya.

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 µg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 µg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts.

Chemical characterisation of Nigerian red propolis and its biological activity against Trypanosoma Brucei.

INTRODUCTION: A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria (RSN), with regards to chemical composition and activity against Trypanosoma brucei in comparison with other African propolis. OBJECTIVE: To carry out fractionation and biological testing of Nigerian propolis in order to isolate compounds with anti-trypanosomal activity. To compare the composition of the RSN propolis with the composition of Brazilian red propolis. METHODOLOGY: Profiling was carried out using HPLC-UV-ELSD and HPLC-Orbitrap-FTMS on extracts of two samples collected from RSN with data extraction using MZmine software. Isolation was carried out by normal phase and reversed phase MPLC. Elucidation of the compounds with a purity > 95% was performed by 1D/2D NMR HRMS and HRLC-MS(n) . RESULTS: Ten phenolic compounds were isolated or in the case of liquiritigenin partially purified. Data for nine of these correlated with literature reports of known compounds i.e. one isoflavanone, calycosin (1); two flavanones, liquiritigenin (2) and pinocembrin (5); an isoflavan, vestitol (3); a pterocarpan, medicarpin (4); two prenylflavanones, 8-prenylnaringenin (7) and 6-prenylnaringenin (8); and two geranyl flavonoids, propolin D (9) and macarangin (10). The tenth was elucidated as a previously undescribed dihydrobenzofuran (6). The isolated compounds were tested against Trypanosoma brucei and displayed moderate to high activity. Some of the compounds tested had similar activity against wild type T. brucei and two strains displaying pentamidine resistance. CONCLUSION: Nigerian propolis from RSN has some similarities with Brazilian red propolis. The propolis displayed anti-trypanosomal activity at a potentially useful level.