Novel Minor Groove Binders Cure Animal African Trypanosomiasis in an in Vivo Mouse Model.

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.

Trypanosoma congolense: In Vitro Culture and Transfection.

Trypanosoma congolense, together with T. vivax and T. brucei, causes African animal trypanosomiasis (AAT), or nagana, a livestock disease carried by bloodsucking tsetse flies in sub-Saharan Africa. These parasitic protists cycle between two hosts: mammal and tsetse fly. The environment offered by each host to the trypanosome is markedly different, and hence the metabolism of stages found in the mammal differs from that of insect stages. For research on new diagnostics and therapeutics, it is appropriate to use the mammalian life cycle stage, bloodstream forms. Insect stages such as procyclics are useful for studying differentiation and also serve as a convenient source of easily cultured, non-infective organisms. Here, we present protocols in current use in our laboratory for the in vitro culture of different life cycle stages of T. congolense-procyclics, epimastigotes, and bloodstream forms-together with methods for transfection enabling the organism to be genetically modified. © 2019 by John Wiley & Sons, Inc.

A gene expression comparison of Trypanosoma brucei and Trypanosoma congolense in the bloodstream of the mammalian host reveals species-specific adaptations to density-dependent development.

In the bloodstream of mammalian hosts Trypanosoma brucei undergoes well-characterised density-dependent growth control and developmental adaptation for transmission. This involves the differentiation from proliferative, morphologically 'slender' forms to quiescent 'stumpy' forms that preferentially infect the tsetse fly vector. Another important livestock trypanosome, Trypanosoma congolense, also undergoes density-dependent cell-cycle arrest although this is not linked to obvious morphological transformation. Here we have compared the gene expression profile of T. brucei and T. congolense during the ascending phase of the parasitaemia and at peak parasitaemia in mice, analysing species and developmental differences between proliferating and cell-cycle arrested forms. Despite underlying conservation of their quorum sensing signalling pathway, each species exhibits distinct profiles of gene regulation when analysed by orthogroup and cell surface phylome profiling. This analysis of peak parasitaemia T. congolense provides the first molecular signatures of potential developmental competence, assisting life cycle developmental studies in these important livestock parasites. Furthermore, comparison with T. brucei identifies candidate molecules from each species that may be important for their survival in the mammalian host, transmission or distinct tropism in the tsetse vector.

Shape-shifting trypanosomes: Flagellar shortening followed by asymmetric division in Trypanosoma congolense from the tsetse proventriculus.

Trypanosomatids such as Leishmania and Trypanosoma are digenetic, single-celled, parasitic flagellates that undergo complex life cycles involving morphological and metabolic changes to fit them for survival in different environments within their mammalian and insect hosts. According to current consensus, asymmetric division enables trypanosomatids to achieve the major morphological rearrangements associated with transition between developmental stages. Contrary to this view, here we show that the African trypanosome Trypanosoma congolense, an important livestock pathogen, undergoes extensive cell remodelling, involving shortening of the cell body and flagellum, during its transition from free-swimming proventricular forms to attached epimastigotes in vitro. Shortening of the flagellum was associated with accumulation of PFR1, a major constituent of the paraflagellar rod, in the mid-region of the flagellum where it was attached to the substrate. However, the PFR1 depot was not essential for attachment, as it accumulated several hours after initial attachment of proventricular trypanosomes. Detergent and CaCl2 treatment failed to dislodge attached parasites, demonstrating the robust nature of flagellar attachment to the substrate; the PFR1 depot was also unaffected by these treatments. Division of the remodelled proventricular trypanosome was asymmetric, producing a small daughter cell. Each mother cell went on to produce at least one more daughter cell, while the daughter trypanosomes also proliferated, eventually resulting in a dense culture of epimastigotes. Here, by observing the synchronous development of the homogeneous population of trypanosomes in the tsetse proventriculus, we have been able to examine the transition from proventricular forms to attached epimastigotes in detail in T. congolense. This transition is difficult to observe in vivo as it happens inside the mouthparts of the tsetse fly. In T. brucei, this transition is achieved by asymmetric division of long trypomastigotes in the proventriculus, yielding short epimastigotes, which go on to colonise the salivary glands. Thus, despite their close evolutionary relationship and shared developmental route within the vector, T. brucei and T. congolense have evolved different ways of accomplishing the same developmental transition from proventricular form to attached epimastigote.

Isothermal microcalorimetry - A quantitative method to monitor Trypanosoma congolense growth and growth inhibition by trypanocidal drugs in real time.

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.

Molecular characterization of tsetse's proboscis and its response to Trypanosoma congolense infection.

Tsetse flies (Glossina spp.) transmit parasitic African trypanosomes (Trypanosoma spp.), including Trypanosoma congolense, which causes animal African trypanosomiasis (AAT). AAT detrimentally affects agricultural activities in sub-Saharan Africa and has negative impacts on the livelihood and nutrient availability for the affected communities. After tsetse ingests an infectious blood meal, T. congolense sequentially colonizes the fly's gut and proboscis (PB) organs before being transmitted to new mammalian hosts during subsequent feedings. Despite the importance of PB in blood feeding and disease transmission, little is known about its molecular composition, function and response to trypanosome infection. To bridge this gap, we used RNA-seq analysis to determine its molecular characteristics and responses to trypanosome infection. By comparing the PB transcriptome to whole head and midgut transcriptomes, we identified 668 PB-enriched transcripts that encoded proteins associated with muscle tissue, organ development, chemosensation and chitin-cuticle structure development. Moreover, transcripts encoding putative mechanoreceptors that monitor blood flow during tsetse feeding and interact with trypanosomes were also expressed in the PB. Microscopic analysis of the PB revealed cellular structures associated with muscles and cells. Infection with T. congolense resulted in increased and decreased expression of 38 and 88 transcripts, respectively. Twelve of these differentially expressed transcripts were PB-enriched. Among the transcripts induced upon infection were those encoding putative proteins associated with cell division function(s), suggesting enhanced tissue renewal, while those suppressed were associated with metabolic processes, extracellular matrix and ATP-binding as well as immunity. These results suggest that PB is a muscular organ with chemosensory and mechanosensory capabilities. The mechanoreceptors may be point of PB-trypanosomes interactions. T. congolense infection resulted in reduced metabolic and immune capacity of the PB. The molecular knowledge on the composition and putative functions of PB forms the foundation to identify new targets to disrupt tsetse's ability to feed and parasite transmission.

Interspecies quorum sensing in co-infections can manipulate trypanosome transmission potential.

Quorum sensing (QS) is commonly used in microbial communities and some unicellular parasites to coordinate group behaviours 1,2 . An example is Trypanosoma brucei, which causes human African trypanosomiasis, as well as the livestock disease, nagana. Trypanosomes are spread by tsetse flies, their transmission being enabled by cell-cycle arrested 'stumpy forms' that are generated in a density-dependent manner in mammalian blood. QS is mediated through a small (<500 Da), non-proteinaceous, stable but unidentified 'stumpy induction factor' 3 , whose signal response pathway has been identified. Although QS is characterized in T. brucei, co-infections with other trypanosome species (Trypanosoma congolense and Trypanosoma vivax) are common in animals, generating the potential for interspecies interactions. Here, we show that T. congolense exhibits density-dependent growth control in vivo and conserves QS regulatory genes, of which one can complement a T. brucei QS signal-blind mutant to restore stumpy formation. Thereafter, we demonstrate that T. congolense-conditioned culture medium promotes T. brucei stumpy formation in vitro, which is dependent on the integrity of the QS signalling pathway. Finally, we show that, in vivo, co-infection with T. congolense accelerates differentiation to stumpy forms in T. brucei, which is also QS dependent. These cross-species interactions have important implications for trypanosome virulence, transmission, competition and evolution in the field.

Adenosine-uridine-rich element is one of the required cis-elements for epimastigote form stage-specific gene expression of the congolense epimastigote specific protein.

It is known that gene expression in kinetoplastida is regulated post-transcriptionally. Several previous studies have shown that stage-specific gene expression in trypanosomes is regulated by cis-elements located in the 3' untranslated region (UTR) of each mRNA and also by RNA binding proteins. Our previous study revealed that gene expression of congolense epimastigote specific protein (cesp) was regulated by cis-elements located in the 3'UTR. In the present study, we identified the adenosine and uridine rich region in the cesp 3'UTR. Using transgenic trypanosome cell lines with different egfp expression cassettes, we showed that this adenosine and uridine rich region is one of the regulatory elements for epimastigote form (EMF) stage-specific gene expression via the regulatory cis-element of the eukaryotic AU rich element (ARE). Therefore this required element within the cesp 3'UTR was designated as T. congolense ARE. This required cis-element might selectively stabilize mRNA in the EMF stage and destabilize mRNA in other stages. By RNA electro mobility shift assay, unknown stage-specific RNA binding proteins (RBPs) whose sequences specifically interacted with the required cis-element were found. These results indicate that EMF stage specific cis-element and RBP complexes might specifically stabilize cesp mRNA in EMF.

Flying tryps: survival and maturation of trypanosomes in tsetse flies.

Survival in and colonization of the tsetse fly midgut are essential steps in the transmission of many species of African trypanosomes. In the fly, bloodstream trypanosomes transform into the procyclic stage within the gut lumen and later migrate to the ectoperitrophic space, where they multiply, establishing an infection. Progression of the parasite infection in the fly depends on factors inherent to the biology of trypanosomes, tsetse, and the bloodmeal. Flies usually eradicate infection early on with both pre-existing and inducible factors. Parasites, in contrast, respond to these stimuli by undergoing developmental changes, allowing a few to both survive and migrate within the tsetse. Here we discuss parasite and fly factors determining trypanosome colonization of the tsetse, focusing mainly on the midgut.

The life cycle of Trypanosoma (Nannomonas) congolense in the tsetse fly.

BACKGROUND: The tsetse-transmitted African trypanosomes cause diseases of importance to the health of both humans and livestock. The life cycles of these trypanosomes in the fly were described in the last century, but comparatively few details are available for Trypanosoma (Nannomonas) congolense, despite the fact that it is probably the most prevalent and widespread pathogenic species for livestock in tropical Africa. When the fly takes up bloodstream form trypanosomes, the initial establishment of midgut infection and invasion of the proventriculus is much the same in T. congolense and T. brucei. However, the developmental pathways subsequently diverge, with production of infective metacyclics in the proboscis for T. congolense and in the salivary glands for T. brucei. Whereas events during migration from the proventriculus are understood for T. brucei, knowledge of the corresponding developmental pathway in T. congolense is rudimentary. The recent publication of the genome sequence makes it timely to re-investigate the life cycle of T. congolense. METHODS: Experimental tsetse flies were fed an initial bloodmeal containing T. congolense strain 1/148 and dissected 2 to 78 days later. Trypanosomes recovered from the midgut, proventriculus, proboscis and cibarium were fixed and stained for digital image analysis. Trypanosomes contained in spit samples from individually caged flies were analysed similarly. Mensural data from individual trypanosomes were subjected to principal components analysis. RESULTS: Flies were more susceptible to infection with T. congolense than T. brucei; a high proportion of flies infected with T. congolense established a midgut and subsequent proboscis infection, whereas many T. brucei infections were lost in the migration from foregut to salivary glands. In T. congolense, trypomastigotes ceased division in the proventriculus and became uniform in size. The trypanosomes retained trypomastigote morphology during migration via the foregut to the mouthparts and we confirmed that the trypomastigote-epimastigote transition occurred in the proboscis. We found no equivalent to the asymmetric division stage in T. brucei that mediates transition of proventricular trypomastigotes to epimastigotes. In T. congolense extremely long epimastigotes with remarkably elongated posterior ends were observed in both the proboscis and cibarium; no difference was found in the developmental stages in these two organs. Dividing trypomastigotes and epimastigotes were recovered from the proboscis, some of which were in transition from trypomastigote to epimastigote and vice versa. It remains uncertain whether these morphological transitions are mediated by cell division, since we also found non-dividing cells with a variously positioned, juxta-nuclear kinetoplast. CONCLUSIONS: We have presented a detailed description of the life cycle of T. congolense in its tsetse fly vector. During development in the fly T. congolense shares a common migratory pathway with its close relative T. brucei, culminating in the production of small metacyclic trypanosomes that can be inoculated with the saliva. Despite this outward similarity in life cycle, the transitional developmental stages in the foregut and mouthparts are remarkably different in the two trypanosome species.

Differential protein expression throughout the life cycle of Trypanosoma congolense, a major parasite of cattle in Africa.

Trypanosoma congolense is an important pathogen of livestock in Africa. To study protein expression throughout the T. congolense life cycle, we used culture-derived parasites of each of the three main insect stages and bloodstream stage parasites isolated from infected mice, to perform differential protein expression analysis. Three complete biological replicates of all four life cycle stages were produced from T. congolense IL3000, a cloned parasite that is amenable to culture of major life cycle stages in vitro. Cellular proteins from each life cycle stage were trypsin digested and the resulting peptides were labeled with isobaric tags for relative and absolute quantification (iTRAQ). The peptides were then analyzed by tandem mass spectrometry (MS/MS). This method was used to identify and relatively quantify proteins from the different life cycle stages in the same experiment. A search of the Wellcome Trust's Sanger Institute's semi-annotated T. congolense database was performed using the MS/MS fragmentation data to identify the corresponding source proteins. A total of 2088 unique protein sequences were identified, representing 23% of the ∼9000 proteins predicted for the T. congolense proteome. The 1291 most confidently identified proteins were prioritized for further study. Of these, 784 yielded annotated hits while 501 were described as "hypothetical proteins". Six proteins showed no significant sequence similarity to any known proteins (from any species) and thus represent new, previously uncharacterized T. congolense proteins. Of particular interest among the remainder are several membrane molecules that showed drastic differential expression, including, not surprisingly, the well-studied variant surface glycoproteins (VSGs), invariant surface glycoproteins (ISGs) 65 and 75, congolense epimastigote specific protein (CESP), the surface protease GP63, an amino acid transporter, a pteridine transporter and a haptoglobin-hemoglobin receptor. Several of these surface disposed proteins are of functional interest as they are necessary for survival of the parasites.

Complete in vitro life cycle of Trypanosoma congolense: development of genetic tools.

BACKGROUND: Animal African trypanosomosis, a disease mainly caused by the protozoan parasite Trypanosoma congolense, is a major constraint to livestock productivity and has a significant impact in the developing countries of Africa. RNA interference (RNAi) has been used to study gene function and identify drug and vaccine targets in a variety of organisms including trypanosomes. However, trypanosome RNAi studies have mainly been conducted in T. brucei, as a model for human infection, largely ignoring livestock parasites of economical importance such as T. congolense, which displays different pathogenesis profiles. The whole T. congolense life cycle can be completed in vitro, but this attractive model displayed important limitations: (i) genetic tools were currently limited to insect forms and production of modified infectious BSF through differentiation was never achieved, (ii) in vitro differentiation techniques lasted several months, (iii) absence of long-term bloodstream forms (BSF) in vitro culture prevented genomic analyses. METHODOLOGY/PRINCIPAL FINDINGS: We optimized culture conditions for each developmental stage and secured the differentiation steps. Specifically, we devised a medium adapted for the strenuous development of stable long-term BSF culture. Using Amaxa nucleofection technology, we greatly improved the transfection rate of the insect form and designed an inducible transgene expression system using the IL3000 reference strain. We tested it by expression of reporter genes and through RNAi. Subsequently, we achieved the complete in vitro life cycle with dramatically shortened time requirements for various wild type and transgenic strains. Finally, we established the use of modified strains for experimental infections and underlined a host adaptation phase requirement. CONCLUSIONS/SIGNIFICANCE: We devised an improved T. congolense model, which offers the opportunity to perform functional genomics analyses throughout the whole life cycle. It represents a very useful tool to understand pathogenesis mechanisms and to study potential therapeutic targets either in vitro or in vivo using a mouse model.

Tsetse EP protein protects the fly midgut from trypanosome establishment.

African trypanosomes undergo a complex developmental process in their tsetse fly vector before transmission back to a vertebrate host. Typically, 90% of fly infections fail, most during initial establishment of the parasite in the fly midgut. The specific mechanism(s) underpinning this failure are unknown. We have previously shown that a Glossina-specific, immunoresponsive molecule, tsetse EP protein, is up regulated by the fly in response to gram-negative microbial challenge. Here we show by knockdown using RNA interference that this tsetse EP protein acts as a powerful antagonist of establishment in the fly midgut for both Trypanosoma brucei brucei and T. congolense. We demonstrate that this phenomenon exists in two species of tsetse, Glossina morsitans morsitans and G. palpalis palpalis, suggesting tsetse EP protein may be a major determinant of vector competence in all Glossina species. Tsetse EP protein levels also decline in response to starvation of the fly, providing a possible explanation for increased susceptibility of starved flies to trypanosome infection. As starvation is a common field event, this fact may be of considerable importance in the epidemiology of African trypanosomiasis.

From clonal to sexual: a step in T. congolense evolution?

Although clearly demonstrated in Trypanosoma brucei, genetic exchange remains controversial in other trypanosome species. Recently, Morrison and co-workers applied a population-genetics analysis, and established the existence of mating in Trypanosoma congolense. Starting from this original discovery, we focus here on the important question of how mating is induced during the trypanosome life cycle and discuss the use of statistics to evidence this type of non-obligatory biological process.

Analysis of expressed sequence tags from the four main developmental stages of Trypanosoma congolense.

Trypanosoma congolense is one of the most economically important pathogens of livestock in Africa. Culture-derived parasites of each of the three main insect stages of the T. congolense life cycle, i.e., the procyclic, epimastigote and metacyclic stages, and bloodstream stage parasites isolated from infected mice, were used to construct stage-specific cDNA libraries and expressed sequence tags (ESTs or cDNA clones) in each library were sequenced. Thirteen EST clusters encoding different variant surface glycoproteins (VSGs) were detected in the metacyclic library and 26 VSG EST clusters were found in the bloodstream library, 6 of which are shared by the metacyclic library. Rare VSG ESTs are present in the epimastigote library, and none were detected in the procyclic library. ESTs encoding enzymes that catalyze oxidative phosphorylation and amino acid metabolism are about twice as abundant in the procyclic and epimastigote stages as in the metacyclic and bloodstream stages. In contrast, ESTs encoding enzymes involved in glycolysis, the citric acid cycle and nucleotide metabolism are about the same in all four developmental stages. Cysteine proteases, kinases and phosphatases are the most abundant enzyme groups represented by the ESTs. All four libraries contain T. congolense-specific expressed sequences not present in the Trypanosoma brucei and Trypanosoma cruzi genomes. Normalized cDNA libraries were constructed from the metacyclic and bloodstream stages, and found to be further enriched for T. congolense-specific ESTs. Given that cultured T. congolense offers an experimental advantage over other African trypanosome species, these ESTs provide a basis for further investigation of the molecular properties of these four developmental stages, especially the epimastigote and metacyclic stages for which it is difficult to obtain large quantities of organisms. The T. congolense EST databases are available at: http://www.sanger.ac.uk/Projects/T_congolense/EST_index.shtml. The sequence data have been submitted to EMBL under the following accession numbers: FN263376-FN292969.

Nutritional stress of adult female tsetse flies (Diptera: Glossinidae) affects the susceptibility of their offspring to trypanosomal infections.

The epidemiology of tsetse-transmitted trypanosomiasis depends, among other factors, on the proportion of infected flies in a tsetse population. A wide range of intrinsic and extrinsic factors seem to determine the ability of a tsetse fly to become infected and to transmit the parasite. In this paper, we investigated the effect of nutritional stress of reproducing female Glossina morsitans morsitans on the susceptibility of their offspring to trypanosomal infections. Adult female flies that were nutritionally stressed by feeding only once a week, produced pupae with a significant lower weight and offspring with a significant lower fat content as well as a lower baseline immune peptide gene expression. Moreover, infection experiments showed that the emerging teneral flies were significantly more susceptible to a Trypanosoma congolense or Trypanosoma brucei brucei infection than flies emerging from non-starved adult females. These findings suggest that in the field, substantial nutritional stress of adult tsetse flies, as is often experienced during the hot dry season, can increase significantly the vectorial capacity of the emerging teneral flies and thus result in an increased infection rate of the tsetse population.

Establishment of an in vitro transgene expression system in epimastigotes of Trypanosoma congolense.

Trypanosoma congolense epimastigote forms (EMFs) adhere to the tsetse fly proboscis, proliferate, and differentiate into animal-infective metacyclic forms (MCFs). This differentiation step, called metacyclogenesis, is indispensable for the cyclical transmission of the parasite. Although an in vitro metacyclogenesis culture system was established several decades ago, few genetic tools have been utilized to investigate the molecular mechanisms underlying T. congolense metacyclogenesis. This study established a transgene expression system using an in vitro derived EMF of T. congolense IL3000, and the transgenic EMF successfully underwent metacyclogenesis in vitro. The newly constructed expression vector pSAK was designed for integration into the alpha-beta tubulin locus, which is tandemly arranged in the T. congolense genome. The expression cassette of pSAK/enhanced green fluorescent protein (eGFP) was transfected into the EMF by electroporation. An EMF expressing eGFP was successfully generated and differentiated into an MCF that constitutively expressed eGFP. The in vitro metacyclogenesis system in combination with the transgenic EMF technique will be important tools to investigate the molecular mechanisms of metacyclogenesis.

Identification and molecular characterization of a novel stage-specific surface protein of Trypanosoma congolense epimastigotes.

The cattle pathogen Trypanosoma congolense expresses life cycle stage-specific surface molecules involved in adaptation to different host and vector environments. Here we report the discovery and molecular characterization of a novel stage-specific GPI-anchored surface glycoprotein that is selectively expressed in the epimastigote (EMF) life cycle stage of T. congolense. Culture supernatants of EMF but not of procyclic culture forms (PCFs) promoted adhesion of PCF parasites in an in vitro assay. Biosynthetic labeling experiments showed that these EMF culture supernatants contained a 100kDa trypanosome-derived protein that was not present in supernatants from PCF. We named this molecule "congolense epimastigote-specific protein" (CESP). The gene encoding CESP was isolated from an EMF cDNA library after immunoscreening. The multicopy gene had a 2070-bp open reading frame that encodes a polypeptide of 689 amino acids with a predicted mass of 72.9kDa. The discrepancy between the predicted (72.9kDa) and observed (100kDa) masses may be explained partially by glycosylation of the molecule which has six potential N-glycosylation sites and a predicted GPI anchor. Indeed, metabolic labeling of CESP with [(3)H] ethanolamine revealed that CESP was a GPI-anchored protein. Confocal laser scanning microscopy showed that CESP was expressed only on the surface of the EMF stage of the parasite. The identification of CESP as a unique component of culture supernatants from EMF and that such supernatants can confer plastic-adhesive ability on PCF suggest that CESP is worth further investigation as an adhesion molecule that perhaps allows T. congolense EMF to adhere to the tsetse proboscis.

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.

Investigations on the transmissibility of Trypanosoma congolense by the tsetse fly Glossina morsitans morsitans during its development in a mammalian host.

Experiments were conducted to investigate the effect of the developmental stage of a monomorphic T. congolense IL1180 strain, in a vertebrate host, on its transmissibility by the tsetse fly Glossina morsitans morsitans Westwood (Diptera: Glossinidae). Batches of 160 male teneral tsetse flies were given a single bloodmeal on mice infected with this T. congolense strain 4, 5, 6, 7 or 10 days post-infection. The proportion of infected flies in each of those batches showed that the stage of development of the trypanosome does affect the proportion of flies that develop a mature or immature infection with immature and mature infection rates of flies infected on days 5 or 10 significantly higher. The proportion of infected flies was not affected by the parasitaemia at the moment of infection. Results show that tsetse flies can become infected at any phase of the development of the T. congolense IL 1180 strain but the ease with which trypanosomes develop in the fly depends on the phase in the parasite's development in the host. Those observations suggest that in analogy with the pleomorphic T. brucei s.l. adaptation of the monomorphic T. congolense to development in the fly may also determine the parasite's transmissibility. Moreover, the findings stress the importance of standardising experiments in which the vectorial capacity of tsetse flies is determined and compared.