The Trypanosoma brucei MISP family of invariant proteins is co-expressed with BARP as triple helical bundle structures on the surface of salivary gland forms, but is dispensable for parasite development within the tsetse vector.

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected tsetse flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of glycosylphosphatidylinositol (GPI)-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate's skin.

A multi-adenylate cyclase regulator at the flagellar tip controls African trypanosome transmission.

Signaling from ciliary microdomains controls developmental processes in metazoans. Trypanosome transmission requires development and migration in the tsetse vector alimentary tract. Flagellar cAMP signaling has been linked to parasite social motility (SoMo) in vitro, yet uncovering control of directed migration in fly organs is challenging. Here we show that the composition of an adenylate cyclase (AC) complex in the flagellar tip microdomain is essential for tsetse salivary gland (SG) colonization and SoMo. Cyclic AMP response protein 3 (CARP3) binds and regulates multiple AC isoforms. CARP3 tip localization depends on the cytoskeletal protein FLAM8. Re-localization of CARP3 away from the tip microdomain is sufficient to abolish SoMo and fly SG colonization. Since intrinsic development is normal in carp3 and flam8 knock-out parasites, AC complex-mediated tip signaling specifically controls parasite migration and thereby transmission. Participation of several developmentally regulated receptor-type AC isoforms may indicate the complexity of the in vivo signals perceived.

Targeting the tsetse-trypanosome interplay using genetically engineered Sodalis glossinidius.

Sodalis glossinidius, a secondary bacterial symbiont of the tsetse fly, is currently considered as a potential delivery system for anti-trypanosomal components interfering with African trypanosome transmission (i.e. paratransgenesis). Nanobodies (Nbs) have been proposed as potential candidates to target the parasite during development in the tsetse fly. In this study, we have generated an immune Nb-library and developed a panning strategy to select Nbs against the Trypanosoma brucei brucei procyclic developmental stage present in the tsetse fly midgut. Selected Nbs were expressed, purified, assessed for binding and tested for their impact on the survival and growth of in vitro cultured procyclic T. b. brucei parasites. Next, we engineered S. glossinidius to express the selected Nbs and validated their ability to block T. brucei development in the tsetse fly midgut. Genetically engineered S. glossinidius expressing Nb_88 significantly compromised parasite development in the tsetse fly midgut both at the level of infection rate and parasite load. Interestingly, expression of Nb_19 by S. glossinidius resulted in a significantly enhanced midgut establishment. These data are the first to show in situ delivery by S. glossinidius of effector molecules that can target the trypanosome-tsetse fly crosstalk, interfering with parasite development in the fly. These proof-of-principle data represent a major step forward in the development of a control strategy based on paratransgenic tsetse flies. Finally, S. glossinidius-based Nb delivery can also be applied as a powerful laboratory tool to unravel the molecular determinants of the parasite-vector association.

Prevalence of Trypanosoma and Sodalis in wild populations of tsetse flies and their impact on sterile insect technique programmes for tsetse eradication.

The sterile insect technique (SIT) is an environment friendly and sustainable method to manage insect pests of economic importance through successive releases of sterile irradiated males of the targeted species to a defined area. A mating of a sterile male with a virgin wild female will result in no offspring, and ultimately lead to the suppression or eradication of the targeted population. Tsetse flies, vectors of African Trypanosoma, have a highly regulated and defined microbial fauna composed of three bacterial symbionts that may have a role to play in the establishment of Trypanosoma infections in the flies and hence, may influence the vectorial competence of the released sterile males. Sodalis bacteria seem to interact with Trypanosoma infection in tsetse flies. Field-caught tsetse flies of ten different taxa and from 15 countries were screened using PCR to detect the presence of Sodalis and Trypanosoma species and analyse their interaction. The results indicate that the prevalence of Sodalis and Trypanosoma varied with country and tsetse species. Trypanosome prevalence was higher in east, central and southern African countries than in west African countries. Tsetse fly infection rates with Trypanosoma vivax and T. brucei sspp were higher in west African countries, whereas tsetse infection with T. congolense and T. simiae, T. simiae (tsavo) and T. godfreyi were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in G. tachinoides. Double and triple infections with Trypanosoma taxa and coinfection of Sodalis and Trypanosoma were rarely observed but it occurs in some taxa and locations. A significant Chi square value (< 0.05) seems to suggest that Sodalis and Trypanosoma infection correlate in G. palpalis gambiensis, G. pallidipes and G. medicorum. Trypanosoma infection seemed significantly associated with an increased density of Sodalis in wild G. m. morsitans and G. pallidipes flies, however, there was no significant impact of Sodalis infection on trypanosome density.

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

Evaluation of the relative roles of the Tabanidae and Glossinidae in the transmission of trypanosomosis in drug resistance hotspots in Mozambique.

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.

Catalase compromises the development of the insect and mammalian stages of Trypanosoma brucei.

Catalase is a widespread heme-containing enzyme, which converts hydrogen peroxide (H2 O2 ) to water and molecular oxygen, thereby protecting cells from the toxic effects of H2 O2 . Trypanosoma brucei is an aerobic protist, which conspicuously lacks this potent enzyme, present in virtually all organisms exposed to oxidative stress. To uncover the reasons for its absence in T. brucei, we overexpressed different catalases in procyclic and bloodstream stages of the parasite. The heterologous enzymes originated from the related insect-confined trypanosomatid Crithidia fasciculata and the human. While the trypanosomatid enzyme (cCAT) operates at low temperatures, its human homolog (hCAT) is adapted to the warm-blooded environment. Despite the presence of peroxisomal targeting signal in hCAT, both human and C. fasciculata catalases localized to the cytosol of T. brucei. Even though cCAT was efficiently expressed in both life cycle stages, the enzyme was active in the procyclic stage, increasing cell's resistance to the H2 O2 stress, yet its activity was suppressed in the cultured bloodstream stage. Surprisingly, following the expression of hCAT, the ability to establish the T. brucei infection in the tsetse fly midgut was compromised. In the mouse model, hCAT attenuated parasitemia and, consequently, increased the host's survival. Hence, we suggest that the activity of catalase in T. brucei is beneficial in vitro, yet it becomes detrimental for parasite's proliferation in both invertebrate and vertebrate hosts, leading to an inability to carry this, otherwise omnipresent, enzyme.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

The Tsetse Fly Displays an Attenuated Immune Response to Its Secondary Symbiont, Sodalis glossinidius.

Sodalis glossinidius, a vertically transmitted facultative symbiont of the tsetse fly, is a bacterium in the early/intermediate state of its transition toward symbiosis, representing an important model for investigating how the insect host immune defense response is regulated to allow endosymbionts to establish a chronic infection within their hosts without being eliminated. In this study, we report on the establishment of a tsetse fly line devoid of S. glossinidius only, allowing us to experimentally investigate (i) the complex immunological interactions between a single bacterial species and its host, (ii) how the symbiont population is kept under control, and (iii) the impact of the symbiont on the vector competence of the tsetse fly to transmit the sleeping sickness parasite. Comparative transcriptome analysis showed no difference in the expression of genes involved in innate immune processes between symbiont-harboring (Gmm Sod+ ) and S. glossinidius-free (Gmm Sod-) flies. Re-exposure of (Gmm Sod-) flies to the endosymbiotic bacterium resulted in a moderate immune response, whereas exposure to pathogenic E. coli or to a close non-insect associated relative of S. glossinidius, i.e., S. praecaptivus, resulted in full immune activation. We also showed that S. glossinidius densities are not affected by experimental activation or suppression of the host immune system, indicating that S. glossinidius is resistant to mounted immune attacks and that the host immune system does not play a major role in controlling S. glossinidius proliferation. Finally, we demonstrate that the absence or presence of S. glossinidius in the tsetse fly does not alter its capacity to mount an immune response to pathogens nor does it affect the fly's susceptibility toward trypanosome infection.

Innate immunity in the tsetse fly (Glossina), vector of African trypanosomes.

Tsetse flies (Glossina sp.) are medically and veterinary important vectors of African trypanosomes, protozoan parasites that cause devastating diseases in humans and livestock in sub-Saharan Africa. These flies feed exclusively on vertebrate blood and harbor a limited diversity of obligate and facultative bacterial commensals. They have a well-developed innate immune system that plays a key role in protecting the fly against invading pathogens and in modulating the fly's ability to transmit African trypanosomes. In this review, we briefly summarize our current knowledge on the tsetse fly innate immune system and its interaction with the bacterial commensals and the trypanosome parasite.

Combining paratransgenesis with SIT: impact of ionizing radiation on the DNA copy number of Sodalis glossinidius in tsetse flies.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of the causative agents of African Trypanosomosis, which has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. The sterile insect technique (SIT) has shown to be a powerful method to manage tsetse fly populations when used in the frame of an area-wide integrated pest management (AW-IPM) program. To date, the release of sterile males to manage tsetse fly populations has only been implemented in areas to reduce transmission of animal African Trypanosomosis (AAT). The implementation of the SIT in areas with Human African Trypanosomosis (HAT) would require additional measures to eliminate the potential risk associated with the release of sterile males that require blood meals to survive and hence, might contribute to disease transmission. Paratransgenesis offers the potential to develop tsetse flies that are refractory to trypanosome infection by modifying their associated bacteria (Sodalis glossinidius) here after referred to as Sodalis. Here we assessed the feasibility of combining the paratransgenesis approach with SIT by analyzing the impact of ionizing radiation on the copy number of Sodalis and the vectorial capacity of sterilized tsetse males. RESULTS: Adult Glossina morsitans morsitans that emerged from puparia irradiated on day 22 post larviposition did not show a significant decline in Sodalis copy number as compared with non-irradiated flies. Conversely, the Sodalis copy number was significantly reduced in adults that emerged from puparia irradiated on day 29 post larviposition and in adults irradiated on day 7 post emergence. Moreover, irradiating 22-day old puparia reduced the copy number of Wolbachia and Wigglesworthia in emerged adults as compared with non-irradiated controls, but the radiation treatment had no significant impact on the vectorial competence of the flies. CONCLUSION: Although the radiation treatment significantly reduced the copy number of some tsetse fly symbionts, the copy number of Sodalis recovered with time in flies irradiated as 22-day old puparia. This recovery offers the opportunity to combine a paratransgenesis approach - using modified Sodalis to produce males refractory to trypanosome infection - with the release of sterile males to minimize the risk of disease transmission, especially in HAT endemic areas. Moreover, irradiation did not increase the vector competence of the flies for trypanosomes.

Towards improving tsetse fly paratransgenesis: stable colonization of Glossina morsitans morsitans with genetically modified Sodalis.

BACKGROUND: Tsetse flies (Glossina sp.) refractory to trypanosome infection are currently being explored as potential tools to contribute in the control of human and animal African trypanosomiasis. One approach to disrupt trypanosome transmission by the tsetse fly vector involves the use of paratransgenesis, a technique that aims to reduce vector competence of disease vectors via genetic modification of their microbiota. An important prerequisite for developing paratransgenic tsetse flies is the stable repopulation of tsetse flies and their progeny with its genetically modified Sodalis symbiont without interfering with host fitness. RESULTS: In this study, we assessed by qPCR analysis the ability of a chromosomally GFP-tagged Sodalis (recSodalis) strain to efficiently colonize various tsetse tissues and its transmission to the next generation of offspring using different introduction approaches. When introduced in the adult stage of the fly via thoracic microinjection, recSodalis is maintained at high densities for at least 21 days. However, no vertical transmission to the offspring was observed. Oral administration of recSodalis did not lead to the colonization of either adult flies or their offspring. Finally, introduction of recSodalis via microinjection of third-instar larvae resulted in stably colonized adult tsetse flies. Moreover, the subsequent generations of offspring were also efficiently colonized with recSodalis. We show that proper colonization of the female reproductive tissues by recSodalis is an important determinant for vertical transmission. CONCLUSIONS: Intralarval microinjection of recSodalis proves to be essential to achieve optimal colonization of flies with genetically modified Sodalis and its subsequent dissemination into the following generations of progeny. This study provides the proof-of-concept that Sodalis can be used to drive expression of exogenous transgenes in Glossina morsitans morsitans colonies representing a valuable contribution to the development of a paratransgenic tsetse fly based control strategy.

Drug quality analysis of isometamidium chloride hydrochloride and diminazene diaceturate used for the treatment of African animal trypanosomosis in West Africa.

BACKGROUND: Diminazene diaceturate (DA) and isometamidium chloride hydrochloride (ISM) are with homidium bromide, the main molecules used to treat African Animal Trypanosomosis (AAT). These drugs can be purchased from official suppliers but also from unofficial sources like local food markets or street vendors. The sub-standard quality of some of these trypanocides is jeopardizing the efficacy of treatment of sick livestock, leading thus to economic losses for the low-resource farmers and is contributing to the emergence and spread of drug resistance. The objective of this study was to assess the quality of trypanocidal drugs sold in French speaking countries of West Africa. In total, 308 drug samples including 282 of DA and 26 of ISM were purchased from official and unofficial sources in Benin, Burkina Faso, Côte d'Ivoire, Mali, Niger and Togo. All samples were analysed at LACOMEV (Dakar, Senegal), a reference laboratory of the World Organisation for Animal Health, by galenic inspection and high performance liquid chromatography. RESULTS: The results showed that 51.90% of the samples were non-compliant compared to the standards and were containing lower quantity of the active ingredient compared to the indications on the packaging. The non-compliances ranged from 63.27% in Togo to 32.65% in Burkina Faso (61.82% in Benin, 53.84% in Mali, 50% in Côte d'Ivoire, 47.36% in Niger). The rates of non-compliance were not statistically different (P = 0.572) from official or unofficial suppliers and ranged from 30 to 75% and from 0 to 65% respectively. However, the non-compliance was significantly higher for ISM compared to DA (P = 0.028). CONCLUSIONS: The high non-compliance revealed in this study compromises the efficacy of therapeutic strategies against AAT, and is likely to exacerbate chemoresistance in West Africa. Corrective actions against sub-standard trypanocides urgently need to be taken by policy makers and control authorities.

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

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

Neutrophils enhance early Trypanosoma brucei infection onset.

In this study, Trypanosoma brucei was naturally transmitted to mice through the bites of infected Glossina morsitans tsetse flies. Neutrophils were recruited rapidly to the bite site, whereas monocytes were attracted more gradually. Expression of inflammatory cytokines (il1b, il6), il10 and neutrophil chemokines (cxcl1, cxcl5) was transiently up-regulated at the site of parasite inoculation. Then, a second influx of neutrophils occurred that coincided with the previously described parasite retention and expansion in the ear dermis. Congenital and experimental neutropenia models, combined with bioluminescent imaging, indicate that neutrophils do not significantly contribute to dermal parasite control and elicit higher systemic parasitemia levels during the infection onset. Engulfment of parasites by neutrophils in the skin was rarely observed and was restricted to parasites with reduced motility/viability, whereas live parasites escaped phagocytosis. To our knowledge, this study represents the first description of a trypanosome infection promoting role of early innate immunological reactions following an infective tsetse fly bite. Our data indicate that the trypanosome is not hindered in its early development and benefits from the host innate responses with the neutrophils being important regulators of the early infection, as already demonstrated for the sand fly transmitted Leishmania parasite.

Resistance to trypanocidal drugs in cattle populations of Zambezia Province, Mozambique.

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.

Evidence for viable and stable triploid Trypanosoma congolense parasites.

BACKGROUND: Recent whole genome sequencing (WGS) analysis identified a viable triploid strain of Trypanosoma congolense. This triploid strain BANANCL2 was a clone of the field isolate BANAN/83/CRTRA/64 that was collected from cattle in Burkina Faso in 1983. RESULTS: We demonstrated the viability and stability of triploidy throughout the complete life-cycle of the parasite by infecting tsetse flies with the triploid clone BANANCL2. Proboscis-positive tsetse flies efficiently transmitted the parasites to mice resulting in systemic infections. WGS of the parasites was performed at all life-cycle stages, and a method based on a block alternative allele frequency spectrum was developed to efficiently detect the ploidy profiles of samples with low read depth. This approach confirmed the triploid profile of parasites throughout their life-cycle in the tsetse fly and the mammalian host, demonstrating that triploidy is present at all stages and is stable over time. CONCLUSION: The presence of viable field-isolated triploid parasites indicates another possible layer of genetic diversity in natural T. congolense populations. The comparison between triploid and diploid parasites provides a unique model system to study the impact of chromosome copy number variations in African trypanosomes. In addition, the consequences of triploidy can be further investigated using this stable triploid model.

Genomic analysis of Isometamidium Chloride resistance in Trypanosoma congolense.

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.

Nanobodies As Tools to Understand, Diagnose, and Treat African Trypanosomiasis.

African trypanosomes are strictly extracellular protozoan parasites that cause diseases in humans and livestock and significantly affect the economic development of sub-Saharan Africa. Due to an elaborate and efficient (vector)-parasite-host interplay, required to complete their life cycle/transmission, trypanosomes have evolved efficient immune escape mechanisms that manipulate the entire host immune response. So far, not a single field applicable vaccine exists, and chemotherapy is the only strategy available to treat the disease. Current therapies, however, exhibit high drug toxicity and an increased drug resistance is being reported. In addition, diagnosis is often hampered due to the inadequacy of current diagnostic procedures. In the context of tackling the shortcomings of current treatment and diagnostic approaches, nanobodies (Nbs, derived from the heavy chain-only antibodies of camels and llamas) might represent unmet advantages compared to conventional tools. Indeed, the combination of their small size, high stability, high affinity, and specificity for their target and tailorability represents a unique advantage, which is reflected by their broad use in basic and clinical research to date. In this article, we will review and discuss (i) diagnostic and therapeutic applications of Nbs that are being evaluated in the context of African trypanosomiasis, (ii) summarize new strategies that are being developed to optimize their potency for advancing their use, and (iii) document on unexpected properties of Nbs, such as inherent trypanolytic activities, that besides opening new therapeutic avenues, might offer new insight in hidden biological activities of conventional antibodies.

Discovery and genomic analyses of hybridization between divergent lineages of Trypanosoma congolense, causative agent of Animal African Trypanosomiasis.

Hybrid populations and introgressive hybridization remain poorly documented in pathogenic micro-organisms, as such that genetic exchange has been argued to play a minor role in their evolution. Recent work demonstrated the existence of hybrid microsatellite profiles in Trypanosoma congolense, a parasitic protozoan with detrimental effects on livestock productivity in sub-Saharan Africa. Here, we present the first population genomic study of T. congolense, revealing a remarkable number of single nucleotide polymorphisms (SNPs), small insertions/deletions (indels) and gene deletions among 56 parasite genomes from ten African countries. One group of parasites from Zambia was particularly diverse, displaying a substantial number of heterozygous SNP and indel sites compared to T. congolense parasites from the nine other sub-Saharan countries. Genomewide 5-kb phylogenetic analyses based on phased SNP data revealed that these parasites were the product of hybridization between phylogenetically distinct T. congolense lineages. Other parasites within the same region in Zambia presented a mosaic of haplotypic ancestry and genetic variability, indicating that hybrid parasites persisted and recombined beyond the initial hybridization event. Our observations challenge traditional views of trypanosome population biology and encourage future research on the role of hybridization in spreading genes for drug resistance, pathogenicity and virulence.