Grace Murilla

Wolbachia, Sodalis and trypanosome co-infections in natural populations of Glossina austeni and Glossina pallidipes

BackgroundTsetse flies harbor at least three bacterial symbionts: Wigglesworthia glossinidia, Wolbachia pipientis and Sodalis glossinidius. Wigglesworthia and Sodalis reside in the gut in close association with trypanosomes and may influence establishment and development of midgut parasite infections. Wolbachia has been shown to induce reproductive effects in infected tsetse. This study was conducted to determine the prevalence of these endosymbionts in natural populations of G. austeni and G. pallidipes and to assess the degree of concurrent infections with trypanosomes.MethodsFly samples analyzed originated from Kenyan coastal forests (trapped in 2009–2011) and South African G. austeni collected in 2008. The age structure was estimated by standard methods. G. austeni (n=298) and G. pallidipes (n= 302) were analyzed for infection with Wolbachia and Sodalis using PCR. Trypanosome infection was determined either by microscopic examination of dissected organs or by PCR amplification.ResultsOverall we observed that G. pallidipes females had a longer lifespan (70 d) than G. austeni (54 d) in natural populations. Wolbachia infections were present in all G. austeni flies analysed, while in contrast, this symbiont was absent from G. pallidipes. The density of Wolbachia infections in the Kenyan G. austeni population was higher than that observed in South African flies. The infection prevalence of Sodalis ranged from 3.7% in G. austeni to about 16% in G. pallidipes. Microscopic examination of midguts revealed an overall trypanosome infection prevalence of 6% (n = 235) and 5% (n = 552), while evaluation with ITS1 primers indicated a prevalence of about 13% (n = 296) and 10% (n = 302) in G. austeni and G. pallidipes, respectively. The majority of infections (46%) were with T. congolense. Co-infection with all three organisms was observed at 1% and 3.3% in G. austeni and G. pallidipes, respectively. Eleven out of the thirteen (85%) co-infected flies harboured T. congolense and T. simiae parasites. While the association between trypanosomes and Sodalis infection was statistically significant in G. pallidipes (P = 0.0127), the number of co-infected flies was too few for a definite conclusion.ConclusionsThe tsetse populations analyzed differed in the prevalence of symbionts, despite being sympatric and therefore exposed to identical environmental factors. The density of infections with Wolbachia also differed between G. austeni populations. There were too few natural co-infections detected with the Sodalis and trypanosomes to suggest extensive inter-relations between these infections in natural populations. We discuss these findings in the context of potential symbiont-mediated control interventions to reduce parasite infections and/or fly populations.

Genetic diversity and population structure of Trypanosoma brucei 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.

Genome-Wide Comparative Analysis of Chemosensory Gene Families in Five Tsetse Fly Species

For decades, odour-baited traps have been used for control of tsetse flies (Diptera; Glossinidae), vectors of African trypanosomes. However, differential responses to known attractants have been reported in different Glossina species, hindering establishment of a universal vector control tool. Availability of full genome sequences of five Glossina species offers an opportunity to compare their chemosensory repertoire and enhance our understanding of their biology in relation to chemosensation. Here, we identified and annotated the major chemosensory gene families in Glossina. We identified a total of 118, 115, 124, and 123 chemosensory genes in Glossina austeni, G. brevipalpis, G. f. fuscipes, G. pallidipes, respectively, relative to 127 reported in G. m. morsitans. Our results show that tsetse fly genomes have fewer chemosensory genes when compared to other dipterans such as Musca domestica (n>393), Drosophila melanogaster (n = 246) and Anopheles gambiae (n>247). We also found that Glossina chemosensory genes are dispersed across distantly located scaffolds in their respective genomes, in contrast to other insects like D. melanogaster whose genes occur in clusters. Further, Glossina appears to be devoid of sugar receptors and to have expanded CO2 associated receptors, potentially reflecting Glossinas obligate hematophagy and the need to detect hosts that may be out of sight. We also identified, in all species, homologs of Ir84a; a Drosophila-specific ionotropic receptor that promotes male courtship suggesting that this is a conserved trait in tsetse flies. Notably, our selection analysis revealed that a total of four gene loci (Gr21a, GluRIIA, Gr28b, and Obp83a) were under positive selection, which confers fitness advantage to species. These findings provide a platform for studies to further define the language of communication of tsetse with their environment, and influence development of novel approaches for control.

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.

Kenya Trypanosomiasis Research Institute Cryobank for Human and Animal Trypanosome Isolates to Support Research: Opportunities and Challenges

Human African trypanosomiasis (HAT) is classified in the category of the most neglected tropical diseases. In man, the disease is caused by two tsetse (Glossina spp.)-transmitted trypanosome subspecies: Trypanosoma brucei gambiense, which is responsible for the chronic form of HAT in West and Central Africa, and T. b. rhodesiense, which causes acute disease in eastern and southern Africa. African animal trypanosomiasis (AAT) is caused by various trypanosome species, the major ones being T. vivax, T. congolense, and T. evansi [1]. Current diagnostic tools are inadequate and diagnosis is complicated, whereas the drugs for treatment are highly toxic and not very effective; patients die if untreated [2]. In 2005, an annual prevalence of 50,000–70,000 cases per year and incidence rates of 15,000–17,000 cases per year were reported [3]. Although recent data from the World Health Organization (WHO) shows that the number of reported cases of HAT declined to less than 10,000 in 2009, leading to speculation that the disease could be eliminated [4], [5], there is great need to maintain vigilance. The East African Trypanosomiasis Research Organization (EATRO) was established to carry out research and develop technologies for effective control of trypanosomiasis. In view of this, a trypanosome cryobank was established in Tororo, Uganda, in the mid-1950s to provide materials for research. At that time, dry ice was used as a refrigerant, but in 1977 it was replaced with liquid nitrogen. Following the collapse of the East African Community in 1977, the Kenya Trypanosomiasis Research Institute (KETRI) was established to take over the functions of tsetse and trypanosomiasis research in Kenya. The cryobank was therefore transferred to KETRI during this period. In 2003, following a reorganization of research institutions by the Government of Kenya, KETRI was merged with the Kenya Agricultural Research Institute (KARI) and renamed the Trypanosomiasis Research Centre (KARI-TRC). KARI-TRC continued with all the research programmes and activities that were being carried out under KETRI, including collection and preservation of trypanosome stabilates. The institution developed a policy on stabilate collection by scientists and clinicians for cryopreservation. We describe the establishment of the cryobank and procedures used in cryopreservation of stabilates and summarize the data (numbers and types) on trypanosome species stored in the cryobank, which are available for use in research by the scientific community. The cryobank contains 2,347 stabilates, including 1,747 primary isolates, out of which there are 42 mixed infections and one miscellaneous Herpetomonas muscorum, and 600 derivatives, including six mixed infections. Primary isolates were collected mainly from countries in the eastern Africa region, including Kenya, Uganda, Tanzania, Sudan, and Ethiopia. However, collections or donations from countries outside the region, including Nigeria, Mozambique, Botswana, Germany, and South America, have been added as part of collaborations between KARI-TRC and other institutions around the world. The stabilates were isolated between 1934 and 2010. The majority of the stabilates were recovered between 1960 and 1970 (Figure 1), the same period when some of the worst epidemics occurred, after which the numbers added have been on the decline. The period from 1940 to 1949 coincided with World War II, when the work on trypanosomiasis research and control stalled: the laboratories in eastern Africa that were the source of isolates were closed, only to resume after 1945 when the war came to an end. Figure 1 Number of primary trypanosome stabilates collected, preserved, and stored at the Kenya Trypanosomiasis Research Institute cryobank. Trypanosomes Trypanosomes are extracellular protozoan parasites which cause disease in humans and animals. Isolation and cryopreservation of new trypanosome strains from patients in different HAT foci ensures availability of these stabilates for use in parasitological, biochemical, molecular, serological, and pharmacological studies many years after their isolation from the host. Brun et al. [1] observed that one of the major obstacles in the elucidation of the factors responsible for relapses after melarsopol treatment was lack of recent T. b. gambiense isolates from patients from various endemic areas where the problem had been reported. The WHO steering committee on human African trypanosomiasis treatment and the East African Network for Tsetse and Trypanosomiasis (EANETT) have therefore recommended that collection of stabilates be a continuous activity in order to monitor the occurrence and spatial distribution of treatment failure [6]. Since its inception, KETRI has established an institutional policy of encouraging collection of stabilates by scientists and clinicians for cryopreservation. In this paper, we describe the establishment of the cryobank and summarize the data (numbers and types) on trypanosome species stored in the cryobank, which are available for research by the wider scientific community.

Infectra®-kit: A device for restraining mice and confining tsetse flies during trypanosome infection transmission experiments

Chemical (anaesthesia) and manual techniques are commonly used to restrain mice during vector-mediated parasite transmission experiments in the laboratory. Chemical restraint may interfere with natural fly vector-mouse interactions and therefore potentially affect the outcome of transmission experiments. Conversely, manual restraint is labour-intensive and exposes laboratory animals to excessive restraining-related discomfort. We report development of a mouse restraining device (Infectra(®)-kit) that allows essential transmission studies to be carried out with minimal human manipulation and without the need for anaesthesia. Infectra(®)-kit can be used as a single unit for restraining one mouse or as eight-assembled units, thus significantly improving efficiency of a single operator in comparison to manual restraint. The kit was validated by comparing feeding success in tsetse flies fed on mice restrained using Infectra(®)-kit (Group I) to those manually restrained (Group II). The mean±SE % feeding success was 75.0±8.2% and 82.1±8.2% for tsetse flies in Groups I and II respectively. Statistical analysis using two sample t-test showed no significant difference between the two groups at p≤0.05, indicating that Infectra(®)-kit as a restraining device was as good as the conventional manual restraint method. The main benefits of using Infectra(®)-kit for transmission studies therefore include reduction of man-hours and animal restraining-related discomfort. In addition, the risk of accidental injury to laboratory personnel by either mice or tsetse flies is minimized, which is an important consideration when working with zoonotic parasites.

Expression profiling of Trypanosoma congolense genes during development in the tsetse fly vector Glossina morsitans morsitans

BackgroundThe tsetse transmitted parasitic flagellate Trypanosoma congolense causes animal African trypanosomosis (AAT) across sub-Saharan Africa. AAT negatively impacts agricultural, economic, nutritional and subsequently, health status of the affected populace. The molecular mechanisms that underlie T. congolense’s developmental program within tsetse are largely unknown due to considerable challenges with obtaining sufficient parasite cells to perform molecular studies.MethodsIn this study, we used RNA-seq to profile T. congolense gene expression during development in two distinct tsetse tissues, the cardia and proboscis. Indirect immunofluorescent antibody test (IFA) and confocal laser scanning microscope was used to localize the expression of a putative protein encoded by the hypothetical protein (TcIL3000_0_02370).ResultsConsistent with current knowledge, genes coding several variant surface glycoproteins (including metacyclic specific VSGs), and the surface coat protein, congolense epimastigote specific protein, were upregulated in parasites in the proboscis (PB-parasites). Additionally, our results indicate that parasites in tsetse’s cardia (C-parasites) and PB employ oxidative phosphorylation and amino acid metabolism for energy. Several genes upregulated in C-parasites encoded receptor-type adenylate cyclases, surface carboxylate transporter family proteins (or PADs), transport proteins, RNA-binding proteins and procyclin isoforms. Gene ontology analysis of products of genes upregulated in C-parasites showed enrichment of terms broadly associated with nucleotides, microtubules, cell membrane and its components, cell signaling, quorum sensing and several transport activities, suggesting that the parasites colonizing the cardia may monitor their environment and regulate their density and movement in this tissue. Additionally, cell surface protein (CSP) encoding genes associated with the Fam50 ‘GARP’, ‘iii’ and ‘i’ subfamilies were also significantly upregulated in C-parasites, suggesting that they are important for the long non-dividing trypomastigotes to colonize tsetse’s cardia. The putative products of genes that were upregulated in PB-parasites were linked to nucleosomes, cytoplasm and membrane-bound organelles, which suggest that parasites in this niche undergo cell division in line with prior findings. Most of the CSPs upregulated in PB-parasites were hypothetical, thus requiring further functional characterization. Expression of one such hypothetical protein (TcIL3000_0_02370) was analyzed using immunofluorescence and confocal laser scanning microscopy, which together revealed preferential expression of this protein on the entire surface coat of T. congolense parasite stages that colonize G. m. morsitans’ proboscis.ConclusionCollectively, our results provide insight into T. congolense gene expression profiles in distinct niches within the tsetse vector. Our results show that the hypothetical protein TcIL3000_0_02370, is expressed on the entire surface of the trypanosomes inhabiting tsetse’s proboscis. We discuss our results in terms of their relevance to disease transmission processes.

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

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

Differential virulence and tsetse fly transmissibility of Trypanosoma congolense and Trypanosoma brucei 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.

Correction: Genome-Wide Comparative Analysis of Chemosensory Gene Families in Five Tsetse Fly Species

[This corrects the article DOI: 10.1371/journal.pntd.0004421.].