Reemergence of Human African Trypanosomiasis Caused by Trypanosoma brucei rhodesiense, Ethiopia.

We report 4 cases of human African trypanosomiasis that occurred in Ethiopia in 2022, thirty years after the last previously reported case in the country. Two of 4 patients died before medicine became available. We identified the infecting parasite as Trypanosoma brucei rhodesiense. Those cases imply human African trypanosomiasis has reemerged.

Symbiotic bacteria Sodalis glossinidius, Spiroplasma sp and Wolbachia do not favour Trypanosoma grayi coexistence in wild population of tsetse flies collected in Bobo-Dioulasso, Burkina Faso.

BACKGROUND: Tsetse flies, the biological vectors of African trypanosomes, have established symbiotic associations with different bacteria. Their vector competence is suggested to be affected by bacterial endosymbionts. The current study provided the prevalence of three tsetse symbiotic bacteria and trypanosomes in Glossina species from Burkina Faso. RESULTS: A total of 430 tsetse flies were captured using biconical traps in four different collection sites around Bobo-Dioulasso (Bama, Bana, Nasso, and Peni), and their guts were removed. Two hundred tsetse were randomly selected and their guts were screened by PCR for the presence of Sodalis glossinidius, Spiroplasma sp., Wolbachia and trypanosomes. Of the 200 tsetse, 196 (98.0%) were Glossina palpalis gambiensis and 4 (2.0%) Glossina tachinoides. The overall symbiont prevalence was 49.0%, 96.5%, and 45.0%, respectively for S. glossinidius, Spiroplasma and Wolbachia. Prevalence varied between sampling locations: S. glossinidius (54.7%, 38.5%, 31.6%, 70.8%); Spiroplasma (100%, 100%, 87.7%, 100%); and Wolbachia (43.4%, 38.5%, 38.6%, 70.8%), respectively in Bama, Bana, Nasso and Peni. Noteworthy, no G. tachnoides was infected by S. glossinidius and Wolbachia, but they were all infected by Spiroplasma sp. A total of 196 (98.0%) harbored at least one endosymbionts. Fifty-five (27.5%) carried single endosymbiont. Trypanosomes were found only in G. p. gambiensis, but not G. tachinoides. Trypanosomes were present in flies from all study locations with an overall prevalence of 29.5%. In Bama, Bana, Nasso, and Peni, the trypanosome infection rate was respectively 39.6%, 23.1%, 8.8%, and 37.5%. Remarkably, only Trypanosoma grayi was present. Of all trypanosome-infected flies, 55.9%, 98.3%, and 33.9% hosted S. glossinidius, Spiroplasma sp and Wolbachia, respectively. There was no association between Sodalis, Spiroplasma and trypanosome presence, but there was a negative association with Wolbachia presence. We reported 1.9 times likelihood of trypanosome absence when Wolbachia was present. CONCLUSION: This is the first survey reporting the presence of Trypanosoma grayi in tsetse from Burkina Faso. Tsetse from these localities were highly positive for symbiotic bacteria, more predominantly with Spiroplasma sp. Modifications of symbiotic interactions may pave way for disease control.

Identification of Semiochemical Candidates Involved in Glossina Palpalis Gambiensis Larviposition Site Selection and Behavioural Responses of Adult Gravid Females.

Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of human and animal trypanosomes. This viviparous insect develops and produces a single larva at 10-day intervals deposited in specific sites. In some species aggregation of larvae has been shown and seems to be mediated by both physical factors and volatile semiochemicals of larval origin. In this context, this study aims to identify chemicals emitted during the pupariation process in Glossina palpalis gambiensis. Volatile Organic Compounds (VOCs) emitted by larvae were identified using static headspace solid-phase microextraction and gas-chromatography mass-spectrometry (GC-MS) analysis. Electrophysiology and behavioural assays were performed on gravid females to confirm VOCs behavioural activity and attractiveness. GC-MS results revealed ten chemicals emitted during the pupariation process of G. p. gambiensis larvae. Among these chemicals, gravid females were shown to detect nine of them during coupled gas chromatography - electroantennographic detection tests. Behavioural assays highlighted two compounds were as attractive as pupae and one compound and a blend of four compounds were more attractive than pupae. Although the larval origin of some of them needs to be confirmed as they may also likely produced by micro-organisms, these compounds induced significant behavioural responses in the laboratory. Further experiments have to explore the biological activity and competitiveness of these compounds in the field. This work opens interesting opportunities for behavioural manipulation and control of tsetse flies.

Tsetse fly density and trypanosoma infection rate in Bedele and Dabo Hana districts of Buno Bedele Zone, Southwest Ethiopia.

BACKGROUND: Trypanosomiasis is an infectious disease caused by parasitic protozoa of the genus Trypanosome and primarily transmitted by tsetse flies. This study aimed to determine the density of tsetse flies and the rate of trypanosome infection in the Bedele and Dabo Hana districts of the Buno Bedele Zone in Ethiopia. RESULTS: A cross-sectional study was conducted from January to February 2023 to catch tsetse flies, determine tsetse density, and estimate the trypanosome infection rate. We used 100 traps (40 NGU, 30 pyramidal, and 30 biconical) to catch the flies. The following standard procedures were followed to identify the specific trypanosome species in the collected tsetse flies: The flies were dissected, and the salivary glands were removed. We placed the salivary glands in a drop of saline solution on a microscope slide. A coverslip was placed over the salivary glands, the slide was examined under a microscope, and the trypanosomes were identified based on their morphology. A total of 3,740 tsetse flies were captured from 100 traps, resulting in an overall apparent density of 18.7 flies per trap per day. Within the study area, only one species of tsetse fly, Glossina tachinoides, was identified. Of the 1,320 dissected Glossina tachinoides, 1.82% were found to be infected with trypanosome parasites. Among these infections, 58.33% were attributed to Trypanosoma congolense, while the remaining 41.67% were caused by Trypanosoma brucei. The infection rate of trypanosomes was significantly higher in female tsetse flies (87.5%) as compared to male flies (12.5%). Furthermore, a significantly higher infection rate was observed in flies older than 20 days (83.33%) and in hunger stage 1 flies (58.33%) compared to hunger stages 2, 3, and 4. CONCLUSIONS: This study highlights the necessity of implementing control and suppression measures targeting the vector (tsetse flies) and the parasite (trypanosomes) to effectively manage and prevent pathogenic animal trypanosomiasis.

The internal transcribed spacer 1 sequence polymorphism brings updates to tsetse species distribution in the northern Cameroon: Importance in planning efficient vector control.

Vector control remains one of the best strategies to prevent the transmission of trypanosome infections in humans and livestock and, thus, a good way to achieve the elimination of human African trypanosomiasis and animal African trypanosomiasis. A key prerequisite for the success of any vector control strategy is the accurate identification and correct mapping of tsetse species. In this work, we updated the tsetse fly species identification and distribution in many geographical areas in Cameroon. Tsetse flies were captured from six localities in Cameroon, and their species were morphologically identified. Thereafter, DNA was extracted from legs of each tsetse fly and the length polymorphism of internal transcribed spacer-1 (ITS1) region of each fly was investigated using PCR. ITS1 DNA fragments of each tsetse species were sequenced. The sequences obtained were analysed and compared to those available in GenBank. This enabled to confirm/infirm results of the morphologic identification and then, to establish the phylogenetic relationships between tsetse species. Morphologic features allowed to clearly distinguish all the tsetse species captured in the South Region of Cameroon, that is, Glossina palpalis palpalis, G. pallicera, G. caliginea and G. nigrofusca. In the northern area, G. morsitans submorsitans could also be distinguished from G. palpalis palpalis, G. tachinoides and G. fuscipes, but these three later could not be distinguished with routine morphological characters. The ITS1 length polymorphism was high among most of the studied species and allowed to identify the following similar species with a single PCR, that is, G. palpalis palpalis with 241 or 242 bp and G. tachinoides with 221 or 222 bp, G. fuscipes with 236 or 237 bp. We also updated the old distribution of tsetse species in the areas assessed, highlighting the presence of G. palpalis palpalis instead of G. fuscipes in Mbakaou, or in sympatry with G. morsitans submorsitans in Dodeo (northern Cameroon). This study confirms the presence of G. palpalis palpalis in the Adamawa Region of Cameroon. It highlights the limits of using morphological criteria to differentiate some tsetse species. Molecular tools based on the polymorphism of ITS1 of tsetse flies can differentiate tsetse species through a simple PCR before downstream analyses or vector control planning.

Trypanosomes and gut microbiota interactions in triatomine bugs and tsetse flies: A vectorial perspective.

Triatomines (kissing bugs) and tsetse flies (genus: Glossina) are natural vectors of Trypanosoma cruzi and Trypanosoma brucei, respectively. T. cruzi is the causative agent of Chagas disease, endemic in Latin America, while T. brucei causes African sleeping sickness disease in sub-Saharan Africa. Both triatomines and tsetse flies are host to a diverse community of gut microbiota that co-exist with the parasites in the gut. Evidence has shown that the gut microbiota of both vectors plays a key role in parasite development and transmission. However, knowledge on the mechanism involved in parasite-microbiota interaction remains limited and scanty. Here, we attempt to analyse Trypanosoma spp. and gut microbiota interactions in tsetse flies and triatomines, with a focus on understanding the possible mechanisms involved by reviewing published articles on the subject. We report that interactions between Trypanosoma spp. and gut microbiota can be both direct and indirect. In direct interactions, the gut microbiota directly affects the parasite via the formation of biofilms and the production of anti-parasitic molecules, while on the other hand, Trypanosoma spp. produces antimicrobial proteins to regulate gut microbiota of the vector. In indirect interactions, the parasite and gut bacteria affect each other through host vector-activated processes such as immunity and metabolism. Although we are beginning to understand how gut microbiota interacts with the Trypanosoma parasites, there is still a need for further studies on functional role of gut microbiota in parasite development to maximize the use of symbiotic bacteria in vector and parasite control.

Application of biomolecular techniques on tsetse fly puparia for species identification at larvipostion sites.

Puparia are commonly found in tsetse fly larviposition sites during studies on larval ecology. This chitinous shell is representative of past or ongoing exploitation of these sites by tsetse flies. The morphological characteristics of the puparium are not sufficiently distinctive to allow identification of the species. This study explores the applicability of biomolecular techniques on empty puparia for tsetse fly species identification. Five techniques were compared for DNA extraction from tsetse fly puparia, 1/Chelex® 100 Resin, 2/CTAB, 3/Livak's protocol, 4/DEB + proteinase K and 5/QIAamp® DNA Mini kit, using two homogenisation methods (manual and automated). Using a combination of two primer pairs, Chelex, CTAB, and DEB + K proved the most efficient on fresh puparia with 90, 85, and 70% samples identified, respectively. Shifting from fresh to one- to nine-month-old puparia, the Chelex method gave the best result allowing species identification on puparia up to seven months old. The subsequent testing of the Chelex extraction protocol identified 152 (60%) of 252 field-collected puparia samples at species level. The results show that reliable genetic identification of tsetse flies species can be performed from empty puparia, what can prove of great interest for future ecological studies on larviposition sites. The Chelex technique was the most efficient for DNA extraction, though the age-limit of the samples stood at seven months, beyond which DNA degradation probably compromises the genetic analysis.

Tsetse flies (Glossina morsitans morsitans) choose birthing sites guided by substrate cues with no evidence for a role of pheromones.

Tsetse flies significantly impact public health and economic development in sub-Saharan African countries by transmitting the fatal disease African trypanosomiasis. Unusually, instead of laying eggs, tsetse birth a single larva that immediately burrows into the soil to pupate. Where the female chooses to larviposit is, therefore, crucial for offspring survival. Previous laboratory studies suggested that a putative larval pheromone, n-pentadecane, attracts gravid female Glossina morsitans morsitans to appropriate larviposition sites. However, this attraction could not be reproduced in field experiments. Here, we resolve this disparity by designing naturalistic laboratory experiments that closely mimic the physical characteristics found in the wild. We show that gravid G. m. morsitans were neither attracted to the putative pheromone nor, interestingly, to pupae placed in the soil. By contrast, females appear to choose larviposition sites based on environmental substrate cues. We conclude that, among the many cues that likely contribute to larviposition choice in nature, substrate features are a main determinant, while we failed to find evidence for a role of pheromones.

Why are biting flies attracted to blue objects?

Diurnal biting flies are strongly attracted to blue objects. This behaviour is widely exploited for fly control, but its functional significance is debated. It is hypothesized that blue objects resemble animal hosts; blue surfaces resemble shaded resting places; and blue attraction is a by-product of attraction to polarized light. We computed the fly photoreceptor signals elicited by a large sample of leaf and animal integument reflectance spectra, viewed under open/cloudy illumination and under woodland shade. We then trained artificial neural networks (ANNs) to distinguish animals from leaf backgrounds, and shaded from unshaded surfaces, in order to find the optimal means of doing so based upon the sensory information available to a fly. After training, we challenged ANNs to classify blue objects used in fly control. Trained ANNs could make both discriminations with high accuracy. They discriminated animals from leaves based upon blue-green photoreceptor opponency and commonly misclassified blue objects as animals. Meanwhile, they discriminated shaded from unshaded stimuli using achromatic cues and never misclassified blue objects as shaded. We conclude that blue-green opponency is the most effective means of discriminating animals from leaf backgrounds using a fly's sensory information, and that blue objects resemble animal hosts through such mechanisms.

Molecular detection of Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts in wild population of tsetse flies collected in Cameroon, Chad and Nigeria.

BACKGROUND: Tsetse flies are cyclical vectors of African trypanosomiasis (AT). The flies have established symbiotic associations with different bacteria that influence certain aspects of their physiology. Vector competence of tsetse flies for different trypanosome species is highly variable and is suggested to be affected by bacterial endosymbionts amongst other factors. Symbiotic interactions may provide an avenue for AT control. The current study provided prevalence of three tsetse symbionts in Glossina species from Cameroon, Chad and Nigeria. RESULTS: Tsetse flies were collected and dissected from five different locations. DNA was extracted and polymerase chain reaction used to detect presence of Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts, using species specific primers. A total of 848 tsetse samples were analysed: Glossina morsitans submorsitans (47.52%), Glossina palpalis palpalis (37.26%), Glossina fuscipes fuscipes (9.08%) and Glossina tachinoides (6.13%). Only 95 (11.20%) were infected with at least one of the three symbionts. Among infected flies, six (6.31%) had Wolbachia and Spiroplasma mixed infection. The overall symbiont prevalence was 0.88, 3.66 and 11.00% respectively, for Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts. Prevalence varied between countries and tsetse fly species. Neither Spiroplasma species nor S. glossinidius were detected in samples from Cameroon and Nigeria respectively. CONCLUSION: The present study revealed, for the first time, presence of Spiroplasma species infections in tsetse fly populations in Chad and Nigeria. These findings provide useful information on repertoire of bacterial flora of tsetse flies and incite more investigations to understand their implication in the vector competence of tsetse flies.

Protein abundance in the midgut of wild tsetse flies (Glossina palpalis palpalis) naturally infected by Trypanosoma congolense s.l.

Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches have been developed to control the disease, among which is the anti-vector Sterile Insect Technique. Another approach to anti-vector strategies could consist of controlling the fly's vector competence through hitherto unidentified regulatory factors (genes, proteins, biological pathways, etc.). The present work aims to evaluate the protein abundance in the midgut of wild tsetse flies (Glossina palpalis palpalis) naturally infected by Trypanosoma congolense s.l. Infected and non-infected flies were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteomes from the guts of parasite-infected flies were compared to that of uninfected flies to identify quantitative and/or qualitative changes associated with infection. Among the proteins with increased abundance were fructose-1,6-biphosphatase, membrane trafficking proteins, death proteins (or apoptosis proteins) and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed the highest increased abundance. The present study, together with previous proteomic and transcriptomic studies on the secretome of trypanosomes from tsetse fly gut extracts, provides data to be explored in further investigations on, for example, mammal host immunisation or on fly vector competence modification via para-transgenic approaches.

Improved estimates of abortion rates in tsetse (Glossina spp.).

Abortion rates were assessed among 170, 846 tsetse (154,228 Glossina pallidipes and 19,618 Glossina morsitans morsitans) sampled in Zimbabwe in 1988-1999. The study produced improved estimates of abortion rates and how these varied with fly age and size and temperatures experienced during pregnancy. An abortion was diagnosed if the uterus was empty and the largest oocyte <0.82 of the expected mature length. Abortion rates for G. pallidipes and G. m. morsitans were 0.64% (95% ci: 0.59-0.69) and 0.83% (0.62-1.10) for trapped flies and 2.03% (1.77-2.31) and 1.55% (1.20-1.98) for flies from artificial refuges. Abortion rates increased with increasing temperature and decreased with increasing wing length and wing fray. Contrary to laboratory findings, abortion rates did not increase in the oldest flies. Percentages of tsetse with empty uteri, regardless of abortion status, were significantly higher than estimated abortion percentages. For tsetse from traps, 4.01% (95% ci: 3.90-4.13) of G. pallidipes and 2.52% (2.14-2.95) of G. m. morsitans had empty uteri; for flies from artificial refuges, the percentages were 12.69% (12.07-13.34) and 14.90% (13.82-16.02), respectively. Abortion losses are small relative to losses at all other stages of life.

A Bayesian maximum entropy model for predicting tsetse ecological distributions.

BACKGROUND: African trypanosomiasis is a tsetse-borne parasitic infection that affects humans, wildlife, and domesticated animals. Tsetse flies are endemic to much of Sub-Saharan Africa and a spatial and temporal understanding of tsetse habitat can aid surveillance and support disease risk management. Problematically, current fine spatial resolution remote sensing data are delivered with a temporal lag and are relatively coarse temporal resolution (e.g., 16 days), which results in disease control models often targeting incorrect places. The goal of this study was to devise a heuristic for identifying tsetse habitat (at a fine spatial resolution) into the future and in the temporal gaps where remote sensing and proximal data fail to supply information. METHODS: This paper introduces a generalizable and scalable open-access version of the tsetse ecological distribution (TED) model used to predict tsetse distributions across space and time, and contributes a geospatial Bayesian Maximum Entropy (BME) prediction model trained by TED output data to forecast where, herein the Morsitans group of tsetse, persist in Kenya, a method that mitigates the temporal lag problem. This model facilitates identification of tsetse habitat and provides critical information to control tsetse, mitigate the impact of trypanosomiasis on vulnerable human and animal populations, and guide disease minimization in places with ephemeral tsetse. Moreover, this BME analysis is one of the first to utilize cluster and parallel computing along with a Monte Carlo analysis to optimize BME computations. This allows for the analysis of an exceptionally large dataset (over 2 billion data points) at a finer resolution and larger spatiotemporal scale than what had previously been possible. RESULTS: Under the most conservative assessment for Kenya, the BME kriging analysis showed an overall prediction accuracy of 74.8% (limited to the maximum suitability extent). In predicting tsetse distribution outcomes for the entire country the BME kriging analysis was 97% accurate in its forecasts. CONCLUSIONS: This work offers a solution to the persistent temporal data gap in accurate and spatially precise rainfall predictions and the delayed processing of remotely sensed data collectively in the - 45 days past to + 180 days future temporal window. As is shown here, the BME model is a reliable alternative for forecasting future tsetse distributions to allow preplanning for tsetse control. Furthermore, this model provides guidance on disease control that would otherwise not be available. These 'big data' BME methods are particularly useful for large domain studies. Considering that past BME studies required reduction of the spatiotemporal grid to facilitate analysis. Both the GEE-TED and the BME libraries have been made open source to enable reproducibility and offer continual updates into the future as new remotely sensed data become available.

Improved models for the relationship between age and the probability of trypanosome infection in female tsetse, Glossina pallidipes Austen.

Between 1990 and 1999, at Rekomitjie Research Station, Zambezi Valley, Zimbabwe, 29,360 female G. pallidipes were dissected to determine their ovarian category and trypanosome infection status. Overall prevalences were 3.45 and 2.66% for T. vivax and T. congolense, respectively, declining during each year as temperatures increased from July - December. Fits to age-prevalence data using Susceptible-Exposed-Infective (SEI) and SI compartmental models were statistically better than those obtained using a published catalytic model, which made the unrealistic assumption that no female tsetse survived more than seven ovulations. The improved models require knowledge of fly mortality, estimated separately from ovarian category distributions. Infection rates were not significantly higher for T. vivax than for T. congolense. For T. congolense in field-sampled female G. pallidipes, we found no statistical support for a model where the force of infection was higher at the first feed than subsequently. The long survival of adult female tsetse, combined with feeding at intervals ≤3 days, ensures that post-teneral feeds, rather than the first feed, play the dominant role in the epidemiology of T. congolense infections in G. pallidipes. This is supported by estimates that only about 3% of wild hosts at Rekomitjie were harbouring sufficient T. congolense to ensure that tsetse feeding off them take an infected meal, so that the probability of ingesting an infected meal is low at every meal.

Single-cell RNA sequencing of Trypanosoma brucei from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens.

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly's salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.

Seasonal variation in tsetse fly apparent density and Trypanosoma spp. infection rate and occurrence of drug-resistant trypanosomes in Lambwe, Kenya.

Tsetse flies are major arthropod vectors of trypanosomes that cause debilitating African animal trypanosomiasis. The emergence of drug-resistant trypanosomes is a common problem in sub-Saharan Africa. This study aimed to identify tsetse flies' seasonal variation in apparent densities and their infection rates and the occurrence of drug-resistant trypanosomes. Tsetse flies were collected from Lambwe, Kenya, during May and September 2021. Genomic DNA was extracted from them, and the ITS1 gene was amplified to detect Trypanosoma infection with subsequent species determination. Transporter genes DMT, E6M6, TbAT/P2, and TcoAde2 were targeted to detect polymorphisms associated with drug-resistance, using sequencing and comparison to drug-sensitive trypanosome species referenced in Genbank. A total of 498 tsetse flies and 29 non-tsetse flies were collected. The apparent density of flies was higher in wet season 6.2 fly per trap per density (FTD) than in the dry season 2.3 FTD (P = 0.001), with n = 386 and n = 141 flies caught in each season, respectively. Male tsetse flies (n = 311) were more numerous than females (n = 187) (P = 0.001). Non-tsetse flies included Tabanids and Stomoxys spp. Overall, Trypanosoma infection rate in tsetse was 5% (25/498) whereby Trypanosoma vivax was 4% (11/25), Trypanosoma congolense 36% (9/25), and Trypanosoma brucei 20% (5/25) (P = 0.186 for the distribution of the species), with infections being higher in females (P = 0.019) and during the wet season (P < 0.001). Numerous polymorphisms and insertions associated with drug resistance were detected in DMT and E6M6 genes in two T. congolense isolates while some isolates lacked these genes. T. brucei lacked TbAT/P2 genes. TcoAde2 sequences in three T. congolense isolates were related to those observed in trypanosomes from cattle blood in our previous study, supporting tsetse fly involvement in transmission in the region. We report Trypanosoma associated with trypanocidal drug-resistance in tsetse flies from Lambwe, Kenya. Female tsetse flies harbored more Trypanosoma infections than males. Tsetse transmission of trypanosomes is common in Lambwe. Risk of trypanosome infection would seem higher in the wet season, when tsetse flies and Trypanosoma infections are more prevalent than during the dry season. More efforts to control animal trypanosome vectors in the region are needed, with particular focus on wet seasons.

Intraflagellar transport during assembly of flagella of different length in Trypanosoma brucei isolated from tsetse flies.

Multicellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle. Trypanosoma brucei assembles flagella of 3 to 30 µm during its development in the tsetse fly. This provides an opportunity to examine how cells naturally modulate organelle length. Flagella are constructed by addition of new blocks at their distal end via intraflagellar transport (IFT). Immunofluorescence assays, 3D electron microscopy and live-cell imaging revealed that IFT was present in all T. brucei life cycle stages. IFT proteins are concentrated at the base, and IFT trains are located along doublets 3-4 and 7-8 and travel bidirectionally in the flagellum. Quantitative analysis demonstrated that the total amount of flagellar IFT proteins correlates with the length of the flagellum. Surprisingly, the shortest flagellum exhibited a supplementary large amount of dynamic IFT material at its distal end. The contribution of IFT and other factors to the regulation of flagellum length is discussed.

Microbe Profile: Wigglesworthia glossinidia: the tsetse fly's significant other.

Wigglesworthia glossinidia is an obligate, maternally transmitted endosymbiont of tsetse flies. The ancient association between these two organisms accounts for many of their unique physiological adaptations. Similar to other obligate mutualists, Wigglesworthia's genome is dramatically reduced in size, yet it has retained the capacity to produce many B-vitamins that are found at inadequate quantities in the fly's vertebrate blood-specific diet. These Wigglesworthia-derived B-vitamins play essential nutritional roles to maintain tsetse's physiological homeostasis as well as that of other members of the fly's microbiota. In addition to its nutritional role, Wigglesworthia contributes towards the development of tsetse's immune system during the larval period. Tsetse produce amidases that degrade symbiotic peptidoglycans and prevent activation of antimicrobial responses that can damage Wigglesworthia. These amidases in turn exhibit antiparasitic activity and decrease tsetse's ability to be colonized with parasitic trypanosomes, which reduce host fitness. Thus, the Wigglesworthia symbiosis represents a fine-tuned association in which both partners actively contribute towards achieving optimal fitness outcomes.

Prevalence of trypanosomes and selected symbionts in tsetse species of eastern Zambia.

Insect symbionts have attracted attention for their potential use as anti-parasitic gene products in arthropod disease vectors. While tsetse species of the Luangwa valley have been extensively studied, less is known about the prevalence of symbionts and their interactions with the trypanosome parasite. Polymerase chain reaction was used to investigate the presence of Wolbachia and Sodalis bacteria, in tsetse flies infected with trypanosomes (Trypanosoma vivax, Trypanosoma congolense and Trypanosoma brucei). Out of 278 captured tsetse flies in eastern Zambia, 95.3% (n = 265, 95% CI = 92.8­97.8) carried endosymbionts: Wolbachia (79.1%, 95% CI 73.9­83.8) and Sodalis (86.3%, 95% CI 81.7­90.1). Overall, trypanosome prevalence was 25.5% (n = 71, 95% CI = 20.4­30.7), 10.8% (n = 30, 95% CI 7.1­14.4) for T. brucei, 1.4% (n = 4, 95% CI = 0.4­3.6) for both T. congolense and T. vivax, and 0.7% (n = 2, 95% CI 0.1­2.6) for T. b. rhodesiense. Out of 240 tsetse flies that were infected with Sodalis, trypanosome infection was reported in 40 tsetse flies (16.7%, 95% CI = 12.0­21.4) while 37 (16.8%, 95% CI 11.9­21.8) of the 220 Wolbachia infected tsetse flies were infected with trypanosomes. There was 1.3 times likelihood of T. brucei infection to be present when Wolbachia was present and 1.7 likelihood of T. brucei infection when Sodalis was present. Overall findings suggest absence of correlation between the presence of tsetse endosymbionts and tsetse with trypanosome infection. Lastly, the presence of pathogenic trypanosomes in tsetse species examined provided insights into the risk communities face, and the importance of African trypanosomiasis in the area.

Genetic connectivity of trypanosomes between tsetse-infested and tsetse-free areas of Kenya.

The prevalence rates of trypanosomes, including those that require cyclical transmission by tsetse flies, are widely distributed in Africa. Trypanosoma brucei and Trypanosoma congolense are actively maintained in regions where there are no tsetse flies although at low frequencies. Whether this could be due to an independent evolutionary origin or multiple introduction of trypanosomes due to continuous movement of livestock between tsetse-free and -infested areas is not known. Thus, the aim of the study was to carry out microsatellite genotyping to explore intra-specific genetic diversity between T. (Trypanozoon), T. congolense and Trypanosoma vivax from the two regions: tsetse infested and tsetse free. Microsatellite genotyping showed geographical origin-based structuring among T. (Trypanozoon) isolates. There was a clear separation between isolates from the two regions signalling the potential of microsatellite markers as diagnostic markers for T. brucei and Trypanosoma evansi isolates. Trypanosoma vivax isolates also clustered largely based on the sampling location with a significant differentiation between the two locations. However, our results revealed that T. congolense isolates from Northern Kenya are not genetically separated from those from Coastal Kenya. Therefore, these isolates are likely introduced in the region through animal movement. Our results demonstrate the occurrence of both genetic connectivity as well as independent evolutionary origin, depending on the trypanosome species between the two ecologies.