Tsetse fly (Glossina pallidipes) midgut responses to Trypanosoma brucei challenge

Abstract Background Tsetse flies (Glossina spp.) are the prominent vector of African trypanosome parasites (Trypanosoma spp.) in sub-Saharan Africa, and Glossina pallidipes is the most widely distributed species in Kenya. This species displays strong resistance to infection by parasites, which are typically eliminated in the midgut shortly after acquisition from the mammalian host. Although extensive molecular information on immunity for the related species Glossina morsitans morsitans exists, similar information is scarce for G. pallidipes. Methods To determine temporal transcriptional responses of G. pallidipes to Trypanosoma brucei brucei challenge, we conducted Illumina based RNA-seq on midgut organ and carcass from teneral females G. pallidipes at 24 and 48 h post-challenge (hpc) with T. b. brucei relative to their respective controls that received normal blood meals (without the parasite). We used a suite of bioinformatics tools to determine differentially expressed and enriched transcripts between and among tissues, and to identify expanded transcripts in G. pallidipes relative to their orthologs G. m. morsitans. Results Midgut transcripts induced at 24 hpc encoded proteins were associated with lipid remodelling, proteolysis, collagen metabolism, apoptosis, and cell growth. Midgut transcripts induced at 48 hpc encoded proteins linked to embryonic growth and development, serine endopeptidases and proteosomal degradation of the target protein, mRNA translation and neuronal development. Temporal expression of immune responsive transcripts at 48 relative to 24 hpc was pronounced, indicative of a gradual induction of host immune responses the following challenge. We also searched for G. m. morsitans orthologous groups that may have experienced expansions in the G. pallidipes genome. We identified ten expanded groups in G. pallidipes with putative immunity-related functions, which may play a role in the higher refractoriness exhibited by this species. Conclusions There appears to be a lack of strong immune responses elicited by gut epithelia of teneral adults. This in combination with a compromised peritrophic matrix at this stage during the initial phase of T. b. brucei challenge may facilitate the increased parasite infection establishment noted in teneral flies relative to older adults. Although teneral flies are more susceptible than older adults, the majority of tenerals are still able to eliminate parasite infections. Hence, robust responses elicited at a later time point, such as 72 hpc, may clear parasite infections from the majority of flies. The expanded G. m. morsitans orthologous groups in G. pallidipes may also be functionally associated with the enhanced refractoriness to trypanosome infections reported in G. pallidipes relative to G. m. morsitans

Pharmacology of DB844, an Orally Active aza Analogue of Pafuramidine, in a Monkey Model of Second Stage Human African Trypanosomiasis

Novel drugs to treat human African trypanosomiasis (HAT) are still urgently needed despite the recent addition of nifurtimox-eflornithine combination therapy (NECT) to WHO Model Lists of Essential Medicines against second stage HAT, where parasites have invaded the central nervous system (CNS). The pharmacology of a potential orally available lead compound, N-methoxy-6-{5-[4-(N-methoxyamidino) phenyl]-furan-2-yl}-nicotinamidine (DB844), was evaluated in a vervet monkey model of second stage HAT, following promising results in mice. DB844 was administered orally to vervet monkeys, beginning 28 days post infection (DPI) with Trypanosoma brucei rhodesiense KETRI 2537. DB844 was absorbed and converted to the active metabolite 6-[5-(4-phenylamidinophenyl)-furanyl-2-y​l]-nicotinamide(DB820), exhibiting plasma Cmax values of 430 and 190 nM for DB844 and DB820, respectively, after the 14th dose at 6 mg/kg qd. A 100-fold reduction in blood trypanosome counts was observed within 24 h of the third dose and, at the end of treatment evaluation performed four days post the last drug dose, trypanosomes were not detected in the blood or cerebrospinal fluid of any monkey. However, some animals relapsed during the 300 days of post treatment monitoring, resulting in a cure rate of 3/8 (37.5%) and 3/7 (42.9%) for the 5 mg/kg×10 days and the 6 mg/kg×14 days dose regimens respectively. These DB844 efficacy data were an improvement compared with pentamidine and pafuramidine both of which were previously shown to be non-curative in this model of CNS stage HAT. These data show that synthesis of novel diamidines with improved activity against CNS-stage HAT was possible.This investigation received financial support from the Bill and Melinda Gates Foundation through the Consortium for Parasitic Drug Development

Multiple evolutionary origins of Trypanosoma evansi in Kenya

Trypanosoma evansi is the parasite causing surra, a form of trypanosomiasis in camels and other livestock, and a serious economic burden in Kenya and many other parts of the world. Trypanosoma evansi transmission can be sustained mechanically by tabanid and Stomoxys biting flies, whereas the closely related African trypanosomes T. brucei brucei and T. b. rhodesiense require cyclical development in tsetse flies (genus Glossina) for transmission. In this study, we investigated the evolutionary origins of T. evansi. We used 15 polymorphic microsatellites to quantify levels and patterns of genetic diversity among 41 T. evansi isolates and 66 isolates of T. b. brucei (n = 51) and T. b. rhodesiense (n = 15), including many from Kenya, a region where T. evansi may have evolved from T. brucei. We found that T. evansi strains belong to at least two distinct T. brucei genetic units and contain genetic diversity that is similar to that in T. brucei strains. Results indicated that the 41 T. evansi isolates originated from multiple T. brucei strains from different genetic backgrounds, implying independent origins of T. evansi from T. brucei strains. This surprising finding further suggested that the acquisition of the ability of T. evansi to be transmitted mechanically, and thus the ability to escape the obligate link with the African tsetse fly vector, has occurred repeatedly. These findings, if confirmed, have epidemiological implications, as T. brucei strains from different genetic backgrounds can become either causative agents of a dangerous, cosmopolitan livestock disease or of a lethal human disease, like for T. b. rhodesiense

Genome sequence of the tsetse fly (Glossina morsitans):Vector of African trypanosomiasis

Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein-encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology.IS

NRMU final technical report : human/wildlife conflicts in Transmara, Kenya

KARI-TRC technical report submitted to ECAPAPA-ASARECAIn the Transmara District, residents claimed that elephants, baboons, leopards, hippos, lions, hyenas, buffaloes, wild pigs and zebras, in that order, were the most destructive and dreaded wild animals in the study area. Most of these species of wildlife inhabit the same areas as agro-pastoralists who live in mixed forestlands bordering nature reserves. 77% of survey respondents reported that some livestock (cattle, sheep, goats) had been killed by wild animals within a period of one year. A wide range of questions were posed to selected respondents regarding Wildlife Compensation schemes. Most had no idea what the schemes were for, or felt that politicians were the beneficiaries

Genetic diversity and population structure of <i>Trypanosoma brucei</i> in Uganda: implications for the epidemiology of sleeping sickness and Nagana.

BACKGROUND:While Human African Trypanosomiasis (HAT) is in decline on the continent of Africa, the disease still remains a major health problem in Uganda. There are recurrent sporadic outbreaks in the traditionally endemic areas in south-east Uganda, and continued spread to new unaffected areas in central Uganda. We evaluated the evolutionary dynamics underpinning the origin of new foci and the impact of host species on parasite genetic diversity in Uganda. We genotyped 269 Trypanosoma brucei isolates collected from different regions in Uganda and southwestern Kenya at 17 microsatellite loci, and checked for the presence of the SRA gene that confers human infectivity to T. b. rhodesiense. RESULTS:Both Bayesian clustering methods and Discriminant Analysis of Principal Components partition Trypanosoma brucei isolates obtained from Uganda and southwestern Kenya into three distinct genetic clusters. Clusters 1 and 3 include isolates from central and southern Uganda, while cluster 2 contains mostly isolates from southwestern Kenya. These three clusters are not sorted by subspecies designation (T. b. brucei vs T. b. rhodesiense), host or date of collection. The analyses also show evidence of genetic admixture among the three genetic clusters and long-range dispersal, suggesting recent and possibly on-going gene flow between them. CONCLUSIONS:Our results show that the expansion of the disease to the new foci in central Uganda occurred from the northward spread of T. b. rhodesiense (Tbr). They also confirm the emergence of the human infective strains (Tbr) from non-infective T. b. brucei (Tbb) strains of different genetic backgrounds, and the importance of cattle as Tbr reservoir, as confounders that shape the epidemiology of sleeping sickness in the region

Phylogeography and population structure of the tsetse fly Glossina pallidipes in Kenya and the Serengeti ecosystem.

Glossina pallidipes is the main vector of animal African trypanosomiasis and a potential vector of human African trypanosomiasis in eastern Africa where it poses a large economic burden and public health threat. Vector control efforts have succeeded in reducing infection rates, but recent resurgence in tsetse fly population density raises concerns that vector control programs require improved strategic planning over larger geographic and temporal scales. Detailed knowledge of population structure and dispersal patterns can provide the required information to improve planning. To this end, we investigated the phylogeography and population structure of G. pallidipes over a large spatial scale in Kenya and northern Tanzania using 11 microsatellite loci genotyped in 600 individuals. Our results indicate distinct genetic clusters east and west of the Great Rift Valley, and less distinct clustering of the northwest separate from the southwest (Serengeti ecosystem). Estimates of genetic differentiation and first-generation migration indicated high genetic connectivity within genetic clusters even across large geographic distances of more than 300 km in the east, but only occasional migration among clusters. Patterns of connectivity suggest isolation by distance across genetic breaks but not within genetic clusters, and imply a major role for river basins in facilitating gene flow in G. pallidipes. Effective population size (Ne) estimates and results from Approximate Bayesian Computation further support that there has been recent G. pallidipes population size fluctuations in the Serengeti ecosystem and the northwest during the last century, but also suggest that the full extent of differences in genetic diversity and population dynamics between the east and the west was established over evolutionary time periods (tentatively on the order of millions of years). Findings provide further support that the Serengeti ecosystem and northwestern Kenya represent independent tsetse populations. Additionally, we present evidence that three previously recognized populations (the Mbeere-Meru, Central Kenya and Coastal "fly belts") act as a single population and should be considered as a single unit in vector control

Characterization of a composite with enhanced attraction to savannah tsetse flies from constituents or analogues of tsetse refractory waterbuck (Kobus defassa) body odor.

Savannah tsetse flies avoid flying toward tsetse fly-refractory waterbuck (Kobus defassa) mediated by a repellent blend of volatile compounds in their body odor comprised of δ-octalactone, geranyl acetone, phenols (guaiacol and carvacrol), and homologues of carboxylic acids (C5-C10) and 2-alkanones (C8-C13). However, although the blends of carboxylic acids and that of 2-alkanones contributed incrementally to the repellency of the waterbuck odor to savannah tsetse flies, some waterbuck constituents (particularly, nonanoic acid and 2-nonanone) showed significant attractive properties. In another study, increasing the ring size of δ-octalactone from six to seven membered ring changed the activity of the resulting molecule (ε-nonalactone) on the savannah tsetse flies from repellency to attraction. In the present study, we first compared the effect of blending ε-nonalactone, nonanoic acid and 2-nonanone in 1:1 binary and 1:1:1 ternary combination on responses of Glossina pallidipes and Glossina morsitans morsitans tsetse flies in a two-choice wind tunnel. The compounds showed clear synergistic effects in the blends, with the ternary blend demonstrating higher attraction than the binary blends and individual compounds. Our follow up laboratory comparisons of tsetse fly responses to ternary combinations with different relative proportions of the three components showed that the blend in 1:3:2 proportion was most attractive relative to fermented cow urine (FCU) to both tsetse species. In our field experiments at Shimba Hills game reserve in Kenya, where G. pallidipes are dominant, the pattern of tsetse catches we obtained with different proportions of the three compounds were similar to those we observed in the laboratory. Interestingly, the three-component blend in 1:3:2 proportion when released at optimized rate of 13.71mg/h was 235% more attractive to G. pallidipes than a combination of POCA (3-n-Propylphenol, 1-Octen-3-ol, 4-Cresol, and Acetone) and fermented cattle urine (FCU). This constitutes a novel finding with potential for downstream deployment in bait technologies for more effective control of G. pallidipes, G. m. morsitans, and perhaps other savannah tsetse fly species, in 'pull' and 'pull-push' tactics

Differential virulence and tsetse fly transmissibility of <i>Trypanosoma congolense</i> and <i>Trypanosoma brucei</i> strains

African animal trypanosomiasis causes significant economic losses in sub-Saharan African countries because of livestock mortalities and reduced productivity. Trypanosomes, the causative agents, are transmitted by tsetse flies (Glossina spp.). In the current study, we compared and contrasted the virulence characteristics of five Trypanosoma congolense and Trypanosoma brucei isolates using groups of Swiss white mice (n = 6). We further determined the vectorial capacity of Glossina pallidipes, for each of the trypanosome isolates. Results showed that the overall pre-patent (PP) periods were 8.4 ± 0.9 (range, 4–11) and 4.5 ± 0.2 (range, 4–6) for T. congolense and T. brucei isolates, respectively (p < 0.01). Despite the longer mean PP, T. congolense–infected mice exhibited a significantly (p < 0.05) shorter survival time than T. brucei–infected mice, indicating greater virulence. Differences were also noted among the individual isolates with T. congolense KETRI 2909 causing the most acute infection of the entire group with a mean ± standard error survival time of 9 ± 2.1 days. Survival time of infected tsetse flies and the proportion with mature infections at 30 days post-exposure to the infective blood meals varied among isolates, with subacute infection–causing T. congolense EATRO 1829 and chronic infection–causing T. brucei EATRO 2267 isolates showing the highest mature infection rates of 38.5% and 23.1%, respectively. Therefore, our study provides further evidence of occurrence of differences in virulence and transmissibility of eastern African trypanosome strains and has identified two, T. congolense EATRO 1829 and T. brucei EATRO 2267, as suitable for tsetse infectivity and transmissibility experiments

Data from: Temporal genetic differentiation in Glossina pallidipes tsetse fly populations in Kenya

Background: Glossina pallidipes is a major vector of both Human and Animal African Trypanosomiasis (HAT and AAT) in Kenya. The disease imposes economic burden on endemic regions in Kenya, including south-western Kenya, which has undergone intense but unsuccessful tsetse fly control measures. We genotyped 387 G. pallidipes flies at 13 microsatellite markers to evaluate levels of temporal genetic variation in two regions that have been subjected to intensive eradication campaigns from the 1960s to the 1980s. One of the regions, Nguruman Escarpment, has been subject to habitat alteration due to human activities, while the other, Ruma National Park, has not. In addition, Nguruman Escarpment is impacted by the movement of grazing animals into the area from neighboring regions during the drought season. We collected our samples from three geographically close sampling sites for each of the two regions. Samples were collected between the years 2003 and 2015, spanning ~96 tsetse fly generations. Results: We established that allelic richness averaged 3.49 and 3.63, and temporal Ne estimates averaged 594 in Nguruman Escarpment and 1120 in Ruma National Park. This suggests that genetic diversity is similar to what was found in previous studies of G. pallidipes in Uganda and Kenya, implying that we could not detect a reduction in genetic diversity following the extensive control efforts during the 1960s to the 1980s. However, we did find differences in temporal patterns of genetic variation between the two regions, indicated by clustering analysis, pairwise FST, and Fisher’s exact tests for changes in allele and genotype frequencies. In Nguruman Escarpment, findings indicated differentiation among samples collected in different years, and evidence of local genetic bottlenecks in two locations previous to 2003, and between 2009 and 2015. In contrast, there was no consistent evidence of differentiation among samples collected in different years, and no evidence of local genetic bottlenecks in Ruma National Park. Conclusion: Our findings suggest that, despite extensive control measures especially between the 1960s and the 1980s, tsetse flies in these regions persist with levels of genetic diversity similar to that found in populations that did not experience extensive control measures. Our findings also indicate temporal genetic differentiation in Nguruman Escarpment detected at a scale of > 80 generations, and no similar temporal differentiation in Ruma National Park. The different level of temporal differentiation between the two regions indicates that genetic drift is stronger in Nugruman Escarpment, for as-yet unknown reasons, which may include differences in land management. This suggests land management may have an impact on G. pallidipes population genetics, and reinforces the importance of long term monitoring of vector populations in estimates of parameters needed to model and plan effective species-specific control measures