Transcriptomes of newly-isolated Trypanosoma brucei rhodesiense reveal hundreds of mRNAs that are co-regulated with stumpy-form markers

Background: During natural Trypanosoma brucei infections, the parasites differentiate spontaneously into a non-dividing “stumpy” form when a certain level of parasitaemia is attained. This form is metabolically adapted for rapid further differentiation into procyclic forms upon uptake by Tsetse flies. Results: We describe here four central Ugandan isolates of Trypanosoma brucei rhodesiense that have undergone only three rodent passages since isolation from human patients. As expected, SNP analysis shows that these isolates are more closely related to each other than to the commonly used strains Lister 427, Antat1.1, and TREU927. TREU927 generally has smaller copy numbers of repeated genes than the other strains, while Lister 427 trypanosomes with a 30-year history of in vitro culture and cloning have more histone genes than the other isolates. The recently isolated trypanosomes were grown in rats, and their transcriptomes characterised. In comparison with cultured procyclic and bloodstream forms, there were increases in mRNAs encoding the stumpy-form markers ESAG9 and PIP39, with coordinated alterations in the levels of over 600 additional mRNAs. Numerous mRNAs encoding proteins of no known function were either increased or decreased. The products of the mRNAs that were increased in parallel with PIP39 included not only enzymes of procyclic-form metabolism, but also components of the translational and RNA control machineries. Many of the mRNAs that were decreased in cells with elevated PIP39 reflected reduced cell division. Conclusions: These transcriptomes suggest new avenues for research into the regulation of trypanosome differentiation

Lay-up optimization for the hull of a racing sailing yacht

Deformability and buckling load of yacht hulls with fiber reinforced plastic sandwich structure depend on the stack sequence of the skins. In this work an optimization of fiber directions of the laminae for a racing yacht is proposed. This procedure has been divided into three parts (i.e. material characterization, surface model definition, lay-up optimization). First of all a set of unidirectional specimens has been realized, by using the same fibers and matrix (carbon/epoxy) used for the hull as well as the same procedure and workers, in order to characterize the material according to American Society for Testing and Materials (ASTM) Standard D3039, employing strain gage technique. In the second part, by means of an original software in Turbo-Pascal (which uses the half-width value matrix as an input) linked to Pro/ENGINEER, it has been possible to obtain the body plan and surface and finite element (FE) models of the sailing yacht for the subsequent analyses. In the third step, an optimization procedure that uses the results of FE structural analyses in three different sailing configurations is performed, with the aim of obtaining the fiber directions that are able to minimize the yacht deformability, also taking into account the buckling loads. An approximate analytical model has been used in conjunction with a sweep technique in order to evaluate the best of the solutions

Characterization of evolutionarily conserved Trypanosoma cruzi NatC and NatA- N-terminal acetyltransferase complexes

Protein N-terminal acetylation is a co- and post-translational modification, conserved among eukaryotes. It determines the functional fate of many proteins including their stability, complex formation and subcellular localization. N-terminal acetyltransferases (NATs) transfer an acetyl group to the N-termini of proteins, and the major NATs in yeast and humans are NatA, NatB and NatC. In this study, we characterized the Trypanosoma cruzi (T. cruzi) NatC and NatA protein complexes, each consisting of one catalytic subunit and predicted auxiliary subunits. The proteins were found to be expressed in the three main life cycle stages of the parasite, formed stable complexes in vivo, and partially co-sedimented with the ribosome in agreement with a co-translational function. An in vitro acetylation assay clearly demonstrated that the acetylated substrates of the NatC catalytic subunit from T. cruzi were similar to those of yeast and human NatC, suggesting evolutionary conservation of function. An RNAi knockdown of the Trypanosome brucei (T. brucei) NatC catalytic subunit indicated that reduced NatC-mediated N-terminal acetylation of target proteins reduce parasite growth

AcSDKP is down-regulated in anaemia induced by Trypanosoma brucei infection in mice

Background Anaemia commonly results from destruction of erythrocytes in the peripheral blood and failure of the bone marrow haematopoietic cells to replenish the erythrocytes. The mechanisms involved in trypanosoma-induced anaemia, including the role of the bone marrow haematopoietic cells are incompletely understood. We studied the responses of a tetrapeptide, AcSDKP, and IL-10, and their association with bone marrow nucleated cells in a Trypanosoma brucei brucei GVR35 experimental infection model.Methods Mouse infection was done intraperitoneally with 1 × 103 trypanosomes/mL. Mice were either infected or left uninfected (N = 100). At days 0, 9, 16, 23, 30, 37, and 44 post-infection, mice were euthanised and blood was collected by cardiac puncture to examine for parasitaemia and packed cell volume (PCV) and then centrifuged for plasma, which was used for cytokine ELISA. The mice’s femurs were also dissected and bone marrow was collected for femur cellularity.Results PCV dropped from 39.6% to 27% in infected animals by day 9 and remained low (relative to uninfected mice) for the duration of the experiment. AcSDKP levels decreased from day 0 (11.5 × 104 pg/mL) to day 16 (10 × 104), and increased by day 30 (12.6 × 104). There was a significant difference at day 16 (P = 0.023) between the infected and uninfected groups. By contrast, expression of IL-10 markedly increased between day 0 (18.6 pg/mL) and day 16 (145 pg/mL) and decreased by day 30 (42.8 pg/mL). There was also a significant difference in IL-10 expression between infected and uninfected mice at day 16 (P < 0.001). Bone marrow nucleated cells were significantly reduced during periods of low plasma AcSDKP and high plasma IL-10 concentrations (5.4 × 106 infected vs 6.2 × 106 on day 0 and 4.9 × 106 infected vs 10 × 106 uninfected on day 16).Conclusions These data unravel a possible negative feedback interaction between AcSDKP and IL-10 in trypanosome infection. More importantly, this study implicates an IL-10/AcSDKP cytokine network in the regulation of bone marrow nucleated cells and provides a new potential mechanism in the pathogenesis of trypanosoma-induced anaemia. Further mechanistic blocking experiments on AcSDKP and IL-10 are recommended to further clarify understanding of the interaction

Candidate genes-based investigation of susceptibility to Human African Trypanosomiasis in Cote d'Ivoire

Human African Trypanosomiasis (HAT) or sleeping sickness is a Neglected Tropical Disease. Long regarded as an invariably fatal disease, there is increasing evidence that infection by T. b. gambiense can result in a wide range of clinical outcomes, including latent infections, which are long lasting infections with no parasites detectable by microscopy. The determinants of this clinical diversity are not well understood but could be due in part to parasite or host genetic diversity in multiple genes, or their interactions. A candidate gene association study was conducted in Côte d’Ivoire using a case-control design which included a total of 233 subjects (100 active HAT cases, 100 controls and 33 latent infections). All three possible pairwise comparisons between the three phenotypes were tested using 96 SNPs in16 candidate genes (IL1, IL4, IL4R, IL6, IL8, IL10, IL12, IL12R, TNFA, INFG, MIF, APOL1, HPR, CFH, HLA-A and HLA-G). Data from 77 SNPs passed quality control. There were suggestive associations at three loci in IL6 and TNFA in the comparison between active cases and controls, one SNP in each of APOL1, MIF and IL6 in the comparison between latent infections and active cases and seven SNP in IL4, HLA-G and TNFA between latent infections and controls. No associations remained significant after Bonferroni correction, but the Benjamini Hochberg false discovery rate test indicated that there were strong probabilities that at least some of the associations were genuine. The excess of associations with latent infections despite the small number of samples available suggests that these subjects form a distinct genetic cluster different from active HAT cases and controls, although no clustering by phenotype was observed by principle component analysis. This underlines the complexity of the interactions existing between host genetic polymorphisms and parasite diversity

Candidate gene polymorphisms study between human African trypanosomiasis clinical phenotypes in Guinea

Human African trypanosomiasis (HAT), a lethal disease induced by Trypanosoma brucei gambiense, has a range of clinical outcomes in its human host in West Africa: an acute form progressing rapidly to second stage, spontaneous self-cure and individuals able to regulate parasitaemia at very low levels, have all been reported from endemic foci. In order to test if this clinical diversity is influenced by host genetic determinants, the association between candidate gene polymorphisms and HAT outcome was investigated in populations from HAT active foci in Guinea.Samples were collected from 425 individuals; comprising of 232 HAT cases, 79 subjects with long lasting positive and specific serology but negative parasitology and 114 endemic controls. Genotypes of 28 SNPs in eight genes passed quality control and were used for an association analysis. IL6 rs1818879 allele A (p = 0.0001, OR = 0.39, CI95 = [0.24-0.63], BONF = 0.0034) was associated with a lower risk of progressing from latent infection to active disease. MIF rs36086171 allele G seemed to be associated with an increased risk (p = 0.0239, OR = 1.65, CI95 = [1.07-2.53], BONF = 0.6697) but did not remain significant after Bonferroni correction. Similarly MIF rs12483859 C allele seems be associated with latent infections (p = 0.0077, OR = 1.86, CI95 = [1.18-2.95], BONF = 0.2157). We confirmed earlier observations that APOL1 G2 allele (DEL) (p = 0.0011, OR = 2.70, CI95 = [1.49-4.91], BONF = 0.0301) is associated with a higher risk and APOL1 G1 polymorphism (p = 0.0005, OR = 0.45, CI95 = [0.29-0.70], BONF = 0.0129) with a lower risk of developing HAT. No associations were found with other candidate genes.Our data show that host genes are involved in modulating Trypanosoma brucei gambiense infection outcome in infected individuals from Guinea with IL6 rs1818879 being associated with a lower risk of progressing to active HAT. These results enhance our understanding of host-parasite interactions and, ultimately, may lead to the development of new control tools

Identity of Fusarium species associated with collar rot and wilt in passion fruit (Passiflora edulis)

Background: Despite the immense contribution of passion fruits to people’s livelihood on a global scale, the crop’s productivity remains low owing to fungal diseases causing up to 100% loss. Fungi are highly variable and the identity of species or variates responsible for recently devastating passion fruit wilt and collar rot diseases had not been characterized. This study was aimed at identifying pathogens causing wilt and collar rot symptoms in passion fruits. Methodology: Fungi were isolated from diseased samples collected from three locations in Central Uganda to identify Fusarium spp associated with collar rot and wilting of passion fruit. This was established by differentiating mycelium pigmentation on Potato Dextrose Agar (PDA), examining slides at X40 magnification under a light microscope for specific macro and microconidia, and amplification with specific Transcription Elongation Factor-1α, TEF 1α primers for identification of Fusarium spp. Results: It was revealed that wilting was associated with a single species, out of 6 selected isolates from the suspected wilted plant, 3 were Fusarium spp associated with the disease in the field but only one of these isolates was proved to be a pathogenic type Fusarium oxysporium. Collar rot was associated with one pathogenic Fusarium spp out of the 6 selected isolates. Conclusion: The results indicate that collar rot and Fusarium wilt are each caused by specific strains of Fusarium pathogens. Recommendation: The identification of pathogenic Fusarium in farmers’ orchards is a starting point for designing effective disease management measures against the predominant fungal pathogenic variants in passion fruits. 

Polymerase chain reaction identification of Trypanosoma brucei rhodesiense in wild tsetse flies from Nkhotakota Wildlife Reserve, Malawi

Background: Trypanosoma brucei rhodesiense is the causative agent of acute human African trypanosomiasis. Identification of T. b. rhodesiense in tsetse populations is essential for understanding transmission dynamics, assessng human disease risk, and monitoring spatiotemporal trends and impact of control interventions. Accurate detection and characterisation of trypanosomes in vectors relies on molecular techniques. For the first time in Malawi, a molecular technique has been used to detect trypanosomes in tsetse flies in Nkhotakota Wildlife Reserve.Methods: A polymerase chain reaction (PCR) technique was used to identify the serum resistance associated (SRA) gene of T. b. rhodesiense in tsetse flies. Of 257 tsetse flies that were randomly caught, 42 flies were dissected for microscopic examination. The midguts of 206 flies were positive and were individually put in eppendorf tubes containing phosphate-buffered saline (PBS buffer) for DNA extraction. Internal transcribed spacer (ITS)-PCR was first used to isolate all trypanosome species from the flies. TBR PCR was then used to isolate the Trypanozoon group. T. brucei-positive samples were further evaluated by SRA PCR for the presence of the SRA gene.Results: Of 257 flies caught, 185 (72%) were Glossina morsitans morsitans and 72 (28%) were Glossina pallidipes. Three were tenerals and 242 were mature live flies. Of the 242 flies dissected, 206 were positive, representing an 85.1% infection rate. From 206 infected flies, 106 (51.5%) were positive using ITS-PCR, 68 (33.0%) being mixed infections, 18 (8.7%) T. brucei, 9 (4.4%) Trypanosoma vivax, 4 (1.9%) Trypanosoma godfrey, 3 (1.5%) Trypanosoma congolense savanna, 3 (1.5%) Trypanosoma simae, and 1 (0.4%) Trypanosoma simaetsavo. When subjected to TBR PCR, 107(51.9%) were positive for T. brucei. Of the 107 T. bruceipositive samples, 5 (4.7%) were found to have the SRA gene.Conclusions: These results suggest that wild tsetse flies in Malawi are infected with human-infective trypanosomes that put communities around wildlife reserves at risk of human African trypanosomiasis outbreaks. Further studies need to be done to identify sources of blood meals for the flies and for surveillance of communities around wildlife reserves

<i>Trypanosoma brucei rhodesiense</i> transmitted by a single tsetse fly bite in vervet monkeys as a model of human African trypanosomiasis

Sleeping sickness is caused by a species of trypanosome blood parasite that is transmitted by tsetse flies. To understand better how infection with this parasite leads to disease, we provide here the most detailed description yet of the course of infection and disease onset in vervet monkeys. One infected tsetse fly was allowed to feed on each host individual, and in all cases infections were successful. The characteristics of infection and disease were similar in all hosts, but the rate of progression varied considerably. Parasites were first detected in the blood 4-10 days after infection, showing that migration of parasites from the site of fly bite was very rapid. Anaemia was a key feature of disease, with a reduction in the numbers and average size of red blood cells and associated decline in numbers of platelets and white blood cells. One to six weeks after infection, parasites were observed in the cerebrospinal fluid (CSF), indicating that they had moved from the blood into the brain; this was associated with a white cell infiltration. This study shows that fly-transmitted infection in vervets accurately mimics human disease and provides a robust model to understand better how sleeping sickness develops

Molecular identification of Anopheles gambiae sensu stricto Giles (formerly Anopheles gambiae Savannah Form) in Kamuli District, Uganda

Anopheles gambiae sensu stricto Giles (formerly A. gambiae S molecular form), the largely anthropophilic species, is reportedly the most important malaria vector in Uganda among the A. gambiae complex species. Indoor and outdoor human-biting mosquitoes were caught for four consecutive nights in each of 48 households in Kamuli district using human-baited bed net traps for subsequent identification of the principal Anopheles sibling species responsible for transmitting malaria. Sibling species under the A. gambiae complex were characterized by polymerase chain reaction using species specific single nucleotide polymorphism (SNPs) in the intergenic spacer region (IGS) with primers specific for A. gambiae s.s., Anopheles arabiensis, Anopheles melas, Anopheles merus and Anopheles quadriannulatus. Molecular forms of the A. gambiae s.s. were further discriminated using primers specific for Mopti and Savannah forms. Out of 300 A. gambiae s.l. amplified, 98% (n= 294) were A. gambiae s.s. Out of 142 A. gambiae s.s. samples analyzed for molecular forms, 78.9% (n=112) were identified as A. gambiae s.s. Giles (A. gambiae Savannah (S) form, while the other 21.1% were not identifiable. the presence of A. gambiae s.s. Giles in Kamuli was also reported. Considering the anthropophilic, endophagic and endophilic behavior of A. gambiae s.s. (and of the molecularly similar A. gambiae s.s. Giles), the combined use of insecticide-treated nets (ITNs), indoor residual spraying, larval source management and improved house design in the context of integrated vector management, may be the appropriate vector control strategies in the area. There is also need for regular monitoring of the vector species composition, distribution and behavior for proper planning of appropriate vector control interventions in the future.Key words: Sibling species, molecular forms, Anopheles gambiae complex, anthropophily, IPM