Control of Trypanosoma brucei brucei infections in tsetse, Glossina morsitans

Abstract Numbers of immature Trypanosoma brucei brucei within a tsetse midgut remain remarkably constant after establishment throughout the course of an infection, irrespective of whether the infection eventually matures. These results suggest a system of self regulation of the parasite population in the insect gut based on a form of programmed cell death which would carry advantages for both the parasite and the vector.     

Cleavage of trypanosome surface glycoproteins by alkaline trypsin-like enzyme(s) in the midgut of Glossina morsitans

EP and GPEET procyclin, the major surface glycoproteins of procyclic forms of Trypanosoma brucei, are truncated by proteases in the midgut of the tsetse fly Glossina morsitans morsitans. We show that soluble extracts from the midguts of teneral flies contain trypsin-like enzymes that cleave the N-terminal domains from living culture-derived parasites. The same extract shows little activity against a variant surface glycoprotein on living bloodstream form T. brucei (MITat 1.2) and none against glutamic acid/alanine-rich protein, a major surface glycoprotein of Trypanosoma congolense insect forms although both these proteins contain potential trypsin cleavage sites. Gel filtration of tsetse midgut extract revealed three peaks of tryptic activity against procyclins. Trypsin alone would be sufficient to account for the cleavage of GPEET at a single arginine residue in the fly. In contrast, the processing of EP at multiple sites would require additional enzymes that might only be induced or activated during feeding or infection. Unexpectedly, the pH optima for both the procyclin cleavage reaction and digestion of the trypsin-specific synthetic substrate Chromozym-TRY were extremely alkaline (pH 10). Direct measurements were made of the pH within different compartments of the tsetse digestive tract. We conclude that the gut pH of teneral flies, from the proventriculus to the hindgut, is alkaline, in contradiction to previous measurements indicating that it was mildly acidic. When tsetse flies were analysed 48 h after their first bloodmeal, a pH gradient from the proventriculus (pH 10.6+/-0.6) to the posterior midgut (pH 7.9+/-0.4) was observed.     

Spatio-temporal distribution of tsetse and other biting flies in the Mouhoun River basin, Burkina Faso

In the Mouhoun River basin, Burkina Faso, the main vectors of African animal trypanosomoses are Glossina palpalis gambiensis Vanderplank and Glossina tachinoides Westwood (Diptera: Glossinidae), both of which are riverine tsetse species. The aim of our study was to understand the impact of landscape anthropogenic changes on the seasonal dynamics of vectors and associated trypanosomosis risk. Three sites were selected on the basis of the level of disturbance of tsetse habitats and predominant tsetse species: disturbed (Boromo, for G. tachinoides) and half-disturbed (Douroula for G. tachinoides and Kadomba for G. p. gambiensis). At each of these sites, seasonal variations in the apparent densities of tsetse and mechanical vectors and tsetse infection rates were monitored over 17 months. Tsetse densities differed significantly between sites and seasons. Of 5613 captured tsetse, 1897 were dissected; 34 of these were found to be infected with trypanosomes. The most frequent infection was Trypanosoma vivax (1.4%), followed by Trypanosoma congolense (0.3%) and Trypanosoma brucei (0.05%). The mean physiological age of 703 tsetse females was investigated to better characterize the transmission risk. Despite the environmental changes, it appeared that tsetse lived long enough to transmit trypanosomes, especially in half-disturbed landscapes. A total of 3021 other biting flies from 15 species (mainly Tabanidae and Stomoxyinae) were also caught: their densities also differed significantly among sites and seasons. Their relative importance regarding trypanosome transmission is discussed; the trypanosomosis risk in cattle was similar at all sites despite very low tsetse densities (but high mechanical vector densities) in one of them.     

A global sensitivity analysis for African sleeping sickness

African sleeping sickness is a parasitic disease transmitted through the bites of tsetse flies of the genus Glossina. We constructed mechanistic models for the basic reproduction number, R0, of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, respectively the causative agents of West and East African human sleeping sickness. We present global sensitivity analyses of these models that rank the importance of the biological parameters that may explain variation in R0, using parameter ranges based on literature, field data and expertize out of Uganda. For West African sleeping sickness, our results indicate that the proportion of bloodmeals taken from humans by Glossina fuscipes fuscipes is the most important factor, suggesting that differences in the exposure of humans to tsetse are fundamental to the distribution of T. b. gambiense. The second ranked parameter for T. b. gambiense and the highest ranked for T. b. rhodesiense was the proportion of Glossina refractory to infection. This finding underlines the possible implications of recent work showing that nutritionally stressed tsetse are more susceptible to trypanosome infection, and provides broad support for control strategies in development that are aimed at increasing refractoriness in tsetse flies. We note though that for T. b. rhodesiense the population parameters for tsetse - species composition, survival and abundance - were ranked almost as highly as the proportion refractory, and that the model assumed regular treatment of livestock with trypanocides as an established practice in the areas of Uganda experiencing East African sleeping sickness.     

A new transmission risk index for human African trypanosomiasis and its application in the identification of sites of high transmission of sleeping sickness in the Fontem focus of southwest Cameroon

A new index for the risk for transmission of human African trypanosomiasis was developed from an earlier index by adding terms for the proportion of tsetse infected with Trypanosoma brucei gambiense group 1 and the contribution of animals to tsetse diet. The validity of the new index was then assessed in the Fontem focus of southwest Cameroon. Averages of 0.66 and 4.85 Glossina palpalis palpalis (Diptera: Glossinidae) were caught per trap/day at the end of one rainy season (November) and the start of the next (April), respectively. Of 1596 tsetse flies examined, 4.7% were positive for Trypanosoma brucei s.l. midgut infections and 0.6% for T. b. gambiense group 1. Among 184 bloodmeals identified, 55.1% were from pigs, 25.2% from humans, 17.6% from wild animals and 1.2% from goats. Of the meals taken from humans, 81.5% were taken at sites distant from pigsties. At the end of the rainy season, catches were low and similar between biotopes distant from and close to pigsties, but the risk for transmission was greatest at sites distant from the sties, suggesting that the presence of pigs reduced the risk to humans. At the beginning of the rainy season, catches of tsetse and risk for transmission were greatest close to the sties. In all seasons, there was a strong correlation between the old and new indices, suggesting that both can be used to estimate the level of transmission, but as the new index is the more comprehensive, it may be more accurate.     

Discovery of 2-(1H-imidazo-2-yl)piperazines as a new class of potent and non-cytotoxic inhibitors of Trypanosoma brucei growth in vitro

The identification of a new series of growth inhibitors of Trypanosoma brucei rhodesiense, causative agent of Human African Trypanosomiasis (HAT), is described. A selection of compounds from our in-house compound collection was screened in vitro against the parasite leading to the identification of compounds with nanomolar inhibition of T. brucei growth. Preliminary SAR on the hit compound led to the identification of compound 34 that shows low nanomolar parasite growth inhibition (T. brucei EC₅₀ 5?nM), is not cytotoxic (HeLa CC₅₀?>?25,000?nM) and is selective over other parasites, such as Trypanosoma cruzi and Plasmodium falciparum (T. cruzi EC₅₀ 8120?nM, P. falciparum EC₅₀ 3624?nM).     

Does isometamidium chloride treatment protect tsetse flies from trypanosome infections during SIT campaigns?

African animal trypanosomosis is a major pathological constraint to cattle breeding across 10 million km2 of sub-Saharan West African countries infested by tsetse flies, their cyclic vectors. The release of sterile males (sterile insect technique [SIT]) is a potentially important control technique aimed at eliminating the vectors. Prior to release, tsetse are generally treated with isometamidium chloride, a trypanocide, to prevent them from transmitting parasites. The present study investigated the preventive action of isometamidium chloride (0.5 mg/L) on the subsequent susceptibility of tsetse released into the wild. A total of 1755 Glossina palpalis gambiensis Vanderplank and 744 Glossina tachinoides Westwood were released, of which 50 and 48, respectively, were recaptured 22-43 days after release. Their probosces were analysed by polymerase chain reaction to identify mature infections with three trypanosome species (Trypanosoma vivax, Trypanosoma brucei sensu lato and Trypanosoma congolense savannah type). Two mature infections with T. vivax and four with T. congolense were detected, indicating that the use of this treatment regimen in an SIT campaign would not totally prevent sterile males from transmitting trypanosomes.     

Trypanosoma brucei 29-13 strain is inducible in but not permissive for the tsetse fly vector

Using green fluorescent protein as a reporter, we have shown that the strain 29-13 of Trypanosoma brucei, widely used for inducible down-regulation of mRNA, is inducible in, but not permissive for the tsetse flies Glossina palpalis gambiensis and Glossina morsitans morsitans. Within two weeks post-infection, 42% males and females of teneral and non-teneral tsetse flies harboured intestinal infections, yet not a single infection progressed into the salivary glands.     

Entomological survey in the historical focus of human African trypanosomiasis of Bendje (Gabon)

The situation of human African trypanosomiasis (sleeping sickness) is poorly known in Gabon. Most of the historical foci have not been investigated for more than 15 years. Few cases are passively recorded from the historical focus of Bendjé; they involved mainly fishermen but determining their contamination site is difficult because of their mobility due to their activity. The presence of these cases in that focus could favour its reactivation if the vector is still there. In order to assess a potential transmission risk in that area, an entomological survey has been carried out in it. Traps were set up during four days in different habitats used by humans during their daily activities. Three species of tsetse flies (Glossina palpalis palpalis, G. pallicera newsteadi and G. caliginea) were caught and two species of trypanosomes (Trypanosoma vivax and T. brucei s.l.) were identified by PCR. These results suggest the presence of an animal transmission cycle. Human-flies contact was confirmed in all type of habitats but no transmission was quantified in the mangrove.     

Identification of selective tubulin inhibitors as potential anti-trypanosomal agents

The potency of a series of sulfonamide tubulin inhibitors against the growth of Trypanosoma brucei (T. brucei), as well as human cancer and primary fibroblast cells were evaluated with the aim of determining whether compounds that selectively inhibit parasite proliferation could be identified. Several compounds showed excellent selectivity against T. brucei growth, and have the potential to be used for the treatment of Human African trypanosomiasis. A T. brucei tubulin protein homology model was built based on the crystal structure of the bovine tubulin. The colchicine-binding domain, which is also the binding site of the tested sulfonamide tubulin inhibitors, showed clear differences between the tubulin structures and presumably explained the selectivity of the compounds.     

Cyclical transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by tsetse flies infected with culture-form procyclic trypanosomes

Culture procyclic forms of Trypanosoma brucei rhodesiense and Trypanosoma congolense were fed to Glossina morsitans morsitans through artificial membranes. A very high percentage of the flies so fed produced established midgut infections, a proportion of which went on to develop into mature metacyclic trypanosomes capable of infecting mammalian hosts. The method offers a safe, clean way of infecting tsetse flies with African trypanosomes which reduces the need for trypanosome-infected animals in the laboratory.     

Inhibition of the DNA amplification of trypanosomes present in tsetse flies midguts: implications for the identification of trypanosome species in wild tsetse flies

The present study was carried out in order to investigate if there was really a failure of PCR in identifying parasitologically positive tsetse flies in the field. Tsetse flies (Glossina palpalis gambiensis and Glossina morsitans morsitans) were therefore experimentally infected with two different species of Trypanosoma (Trypanosoma brucei gambiense or Trypanosoma congolense). A total of 152 tsetse flies were dissected, and organs of each fly (midgut, proboscis or salivary glands) were examined. The positive organs were then analysed using PCR. Results showed that, regardless of the trypanosome species, PCR failed to amplify 40% of the parasitologically positive midguts. This failure, which does not occur with diluted samples, is likely to be caused by an inhibition of the amplification reaction. This finding has important implications for the detection and the identification of trypanosome species in wild tsetse flies.     

The population genetics of Trypanosoma brucei and the origin of human infectivity

The African trypanosome, Trypanosoma brucei, is a zoonotic parasite transmitted by tsetse flies. Two of the three subspecies, T. brucei gambiense and T.b. rhodesiense, cause sleeping sickness in humans whereas the third subspecies, T.b. brucei, is not infective to humans. We propose that the key to understanding genetic relationships within this species is the analysis of gene flow to determine the importance of genetic exchange within populations and the relatedness of populations. T.brucei parasites undergo genetic exchange when present in infections of mixed genotypes in tsetse flies in the laboratory, although this is not an obligatory process. Infections of mixed genotype are surprisingly common in field isolates from tsetse flies such that there is opportunity for genetic exchange to occur. Population genetic analyses, taking into account geographical and host species of origin, show that genetic exchange occurs sufficiently frequently in the field to be an important determinant of genetic diversity, except where particular clones have acquired the ability to infect humans. Thus, T. brucei populations have an 'epidemic' genetic structure, but the better-characterized human-infective populations have a 'clonal' structure. Remarkably, the ability to infect humans appears to have arisen on multiple occasions in different geographical locations in sub-Saharan Africa. Our data indicate that the classical subspecies terminology for T. brucei is genetically inappropriate. It is an implicit assumption in most infectious disease biology that when a zoonotic pathogen acquires the capability to infect humans, it does so once and then spreads through the human population from that single-source event. For at least one major pathogen in tropical medicine, T. brucei, this assumption is invalid.     

VSG 117 gene is conservatively present and early expressed in Trypanosma evansi YNB stock

African trypanosomes, including Trypanosoma brucei and the closely related species Trypanosoma evansi, are flagellated unicellular parasites that proliferate extracellularly in the mammalian bloodstream and tissue spaces. They evade host immune system by periodically switching their variant surface glycoprotein (VSG) coat. Each trypanosome possesses a vast archive of VSGs with distinct sequence identity and different strains contain different archive of VSGs. VSG 117 was reported as a widespread VSG detected in the genomes of all the T. brucei strains. In this study, the presence and expression of VSG 117 gene was observed in T. evansi YNB stock by RT-PCR with VSG-specific primers. We further confirmed that this VSG tends to be expressed in the early stage of T. evansi infections (on day 12-15) by immuno-screening the previously isolated infected blood samples. It is possible that the VSG 117 gene evolved and spread through the African trypanosome population via genetic exchange, before T. evansi lost its ability to infect tsetse fly. Our finding provided an evidence of the close evolutionary relationship between T. evansi and T. brucei, in the terms of VSG genes.     

Effects of anti-protozoal drugs and histopathological studies on trypanosome species

The trypanosomostatic and trypanosomicidal effects of four anti-protozoal drugs, namely halofantrine hydrochloride, chloroquine phosphate, benzoylmetronidazole and pyrimethamine, on species of trypanosomes, viz. Trypanosoma brucei brucei (MBOS/NG/94/NITR) Bassa strain, T. congolense (MBOS/NG/93/NVRI) Zaria strain and T. brucei gambiense (MHOM/NG/92/NITR) Abraka strain, were investigated. In vitro and in vivo studies on these drugs vis-a-vis the parasites were carried out. The histopathological changes in organs and tissues of experimentally infected rats were also studied. Results from the in vitro studies indicated that halofantrine hydrochloride, chloroquine phosphate, benzoylmetronidazole and pyrimethamine appeared to be effective trypanosomicidal agents against T. brucei brucei (Bassa strain), T. congolense (Zaria strain) and T. brucei gambiense (Abraka strain). The in vivo studies showed that these drugs were sub-curative by prolonging the survival period of the trypanosome-infected rats, but not necessarily curing the infection. Histopathological findings indicated inflammatory reactions characterised by infiltration to variable degrees in the majority of tissues, mostly in the lungs and liver. The most consistent lesions were interstitial pneumonia, multifocal necrosis and oedema. Pathological findings showed the T. brucei brucei and T. brucei gambiense strains studied to be both intravascular and extravascular parasites. These results suggest that halofantrine hydrochloride, chloroquine phosphate, benzoylmetronidazole and pyrimethamine could be used as supportive, suppressive and/or synergistic/additive drugs in the treatment of African trypanosomiasis. Their effects on species of trypanosomes have been studied and are reported for the first time.     

Cyclical Transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by Tsetse Flies Infected with Culture-Form Procyclic Trypanosomes*

SYNOPSIS. Culture procyclic forms of Trypanosoma brucei rhodesiense and Trypanosoma congolense were fed to Glossina morsitans morsitans through artificial membranes. A very high percentage of the flies so fed produced established midgut infections, a proportion of which went on to develop into mature metacyclic trypanosomes capable of infecting mammalian hosts. The method offers a safe, clean way of infecting tsetse flies with African trypanosomes which reduces the need for trypanosome-infected animals in the laboratory.     

Trypanosoma brucei gambiense: study of population genetic structure of Central African stocks using amplified fragment length polymorphism (AFLP)

To understand the maintenance and resurgence of historical Human African Trypanosomiasis (HAT) foci, AFLP was used to genotype 100 Central African Trypanosoma brucei s.l. stocks. This technique confirmed the high genetic stability of T. b. gambiense group 1 stocks and the micro genetic variability within Central African T. b. gambiense stocks. It revealed several T. b. gambiense genotypes and allowed the identification of minor and major genotypes in HAT foci. The coexistence of these genotypes in the same focus suggests that clustering of stocks according to HAT focus does not provide the true genetic picture of trypanosome circulating within the disease focus because the minor genotypes are generally underestimated. The presence of minor and major genotypes in HAT foci may explain the persistence and the resurgence of Central African sleeping sickness foci.     

Trypanosoma brucei brucei: Effects of ferrous iron and heme on ecto-nucleoside triphosphate diphosphohydrolase activity

Trypanosoma brucei brucei is the causative agent of animal African trypanosomiasis, also called nagana. Procyclic vector form resides in the midgut of the tsetse fly, which feeds exclusively on blood. Hemoglobin digestion occurs in the midgut resulting in an intense release of free heme. In the present study we show that the magnesium-dependent ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of procyclic T. brucei brucei is inhibited by ferrous iron and heme. The inhibition of E-NTPDase activity by ferrous iron, but not by heme, was prevented by pre-incubation of cells with catalase. However, antioxidants that permeate cells, such as PEG-catalase and N-acetyl-cysteine prevented the inhibition of E-NTPDase by heme. Ferrous iron was able to induce an increase in lipid peroxidation, while heme did not. Therefore, both ferrous iron and heme can inhibit E-NTPDase activity of T. brucei brucei by means of formation of reactive oxygen species, but apparently acting through distinct mechanisms.     

Factors affecting trypanosome maturation in tsetse flies

Trypanosoma brucei brucei infections which establish successfully in the tsetse fly midgut may subsequently mature into mammalian infective trypanosomes in the salivary glands. This maturation is not automatic and the control of these events is complex. Utilising direct in vivo feeding experiments, we report maturation of T. b. brucei infections in tsetse is regulated by antioxidants as well as environmental stimuli. Dissection of the maturation process provides opportunities to develop transmission blocking vaccines for trypanosomiasis. The present work suggests L-cysteine and/or nitric oxide are necessary for the differentiation of trypanosome midgut infections in tsetse.