Comparative Genomic Analysis of six Glossina Genomes, Vectors of African Trypanosomes

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

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

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

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

Development and optimization of enzyme linked immunoassays as readout system for activation of theileria parva-specific cytotoxic T lymphocytes.

Immunity against Theileria parva in cattle is mediated by cytotoxic T lymphocytes (eTL) targeted at schizont-infected cells. Antigens expressed by the schizont stage of the parasite that induce protective immune response are possible candidates for sub-unit vaccine against ECF. Upon recognition and antigenic activation, CTLs releases cytokines and cytoplasmic granules containing granzyme and perf orin. Therefore the aim of this study was to develop enzyme linked immunosorbent assays (ELISA) to monitor release of cytokines and other cellular factors as a readout system for the activation of T. parva specific cytotoxic T lymphocytes. T. parva specific cytotoxic T lympocytes (CTL) were generated in the laboratory by repeated stimulation of PBMCs from immunized cattle with autologous irradiated T. parva infected cells. These cells were of CD8+ phenotype and lysed autologous T. parva infected cells as targets in 4hr 51chromium release assays. Presence of intracellular IFN-y, TNF-a and perf orin in these cells was demonstrated using intracellular staining. ELISA for detection of secreted forms of these cytokines were then developed and optimized using cytokine specific monoclonal antibodies and recombinant cytokines. IFN-y ELISA that was developed was capable of detecting recombinant bovine IFN-y as low as 120pglml. We compared IFN-y ELISA with IFN-y bioassay and it was found out that the sensitivity IFN-y ELISA was comparable to that of bioassay (100pglml). The TNF-a ELISA that was developed was not sensitive, since the detection limit of recombinant bovine TNF-a was 3.9nglml. Perf orin ELISA was developed using reagents against human perf orin and there was only an indication that perf orin was present in tissue culture supernatants from co-cultures of CTLs and 100% TpM. Attempts to develop a bovine Granzyme B (GrB) ELISA were not successful as the reagents against human GrB did not cross react with bovine as indicated in lack of intracellular staining of CTLs. Results showed that IFN-y ELISA can be used to detect IFN-y in supernatants after co-culture of CTLs and TpMs, in serum and lymph samples. TNF-a ELISA needs further development by testing more anti-bovine TNF-a reagents to improve the sensitivity. Development of sensitive perf orin and GrB ELISA will depend on availability of specific anti-bovine reagents

Loop-mediated isothermal amplification test for Trypanosoma vivax based on satellite repeat DNA

Trypanosoma vivax is major cause of animal trypanosomiasis and responsible for enormous economic burden in Africa and South America animal industry. T. vivax infections mostly run low parasitaemia with no apparent clinical symptoms, making diagnosis a challenge. This work reports the design and evaluation of a loop-mediated isothermal amplification (LAMP) test for detecting T. vivax DNA based on the nuclear satellite repeat sequence. The assay is rapid with results obtained within 35min. The analytical sensitivity is ∼1trypanosome/ml while that of the classical PCR tests ranged from 10 to 10 trypanosomes/ml. The T. vivax LAMP test reported here is simple, robust and has future potential in diagnosis of animal trypanosomiasis in the field

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

Enhancing vector refractoriness to trypanosome infection : achievements, challenges and perspectives

With the absence of effective prophylactic vaccines and drugs against African trypanosomosis, control of this group of zoonotic neglected tropical diseases depends the control of the tsetse fly vector. When applied in an area-wide insect pest management approach, the sterile insect technique (SIT) is effective in eliminating single tsetse species from isolated populations. The need to enhance the effectiveness of SIT led to the concept of investigating tsetse-trypanosome interactions by a consortium of researchers in a five-year (2013-2018) Coordinated Research Project (CRP) organized by the Joint Division of FAO/IAEA. The goal of this CRP was to elucidate tsetse-symbiome-pathogen molecular interactions to improve SIT and SIT-compatible interventions for trypanosomoses control by enhancing vector refractoriness. This would allow extension of SIT into areas with potential disease transmission. This paper highlights the CRP's major achievements and discusses the science-based perspectives for successful mitigation or eradication of African trypanosomosis.</p

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