Targeting schistosome cholinesterases for vaccine and drug development

The nervous system of schistosomes has been successfully targeted by anthelmintic drugs but the use of many of these has discontinued because of toxic side effects and so there is a need to better understand key neuronal processes at a molecular level to develop safer and more effective intervention strategies that target this vital system. Cholinesterases - acetylcholinesterases (AChE)s and butyrylcholinesterases (BChE)s - are key enzymes that play a pivotal role in the nervous system of schistosomes by regulating neurotransmission through acetylcholine hydrolysis and, accordingly, are an example of such an intervention target. The first results chapter (chapter two) of this thesis investigated the anti-schistosome efficacy of polypyridylruthenium (II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb₁₂-tri and Rubb₇-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb₁₂-tri) and 10 mg/kg (Rubb₇-tnl). Mice treated with Rubb₁₂-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb₁₂-tri - 68%, Rubb₇-tnl - 56%) and were significantly morphologically altered (Rubb₁₂-tri - 62% abnormal, Rubb₇-tnl - 35% abnormal). I hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway. Chapter three of this thesis provided the first comprehensive molecular characterization of three S. mansoni cholinesterases (SmChEs), designated as SmAChE1, SmBChE1 and SmAChE3, which were identified from the interrogation of the now wholly annotated S. mansoni genome. Anti-SmChE antibodies localized the proteins to the tegument and neuromusculature of adults and schistosomula and developmental expression profiling differed among the molecules, suggestive of functions extending beyond traditional cholinergic signaling for each of them. I also reported the presence of ChE activity in parasite ES products for the first time and proteomically identified the molecules responsible (SmAChE1 and SmBChE1). Functional recombinant versions of the three SmChEs were produced in Pichia pastoris and enzyme nomenclature (AChE or BChE) was verified based on substrate preference. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosomal AChE inhibitor dichlorvos whereas SmBChE1-silenced parasites displayed increased sensitivity to dichlorvos. SmChEs were further characterized by RNAi-based experiments in chapter four of this thesis. RNAi-mediated silencing of individual SmChEs, or simultaneous silencing of all three SmChEs, significantly suppressed transcript and protein expression levels and AChE activity in parasites. In a dissection of the hypothesis that tegumental AChE mediates exogenous glucose scavenging by the parasite, I showed that RNAi-mediated knockdown of SmAChE1 and SmAChE3, but not SmBChE1, significantly reduced glucose uptake by schistosomes. Parasite survivability in vitro and in vivo was significantly impaired with the silencing of SmChEs, either individually or in combination, attesting to the essentiality of these molecules. Chapter five of this thesis explored the vaccine potential of SmChEs. When treated in vitro with anti-SmChE IgG, parasites displayed significantly decreased ChE activity, which eventually resulted in death. Vaccination with individual SmChEs, or a combination of all three SmChEs, significantly reduced worm burdens (28% - 38%, averaged across two independent trials) compared to controls. Liver egg burdens were significantly decreased for all mice across both trials (13% - 46%) except those vaccinated with SmAChE1 in trial 1. Egg viability, as determined by egg hatching from liver homogenates, was significantly reduced in the groups vaccinated with the SmChE cocktail (40%) and SmAChE3 (46%). Surviving worms from each vaccinated group were significantly stunted and depleted of glycogen stores, compared to controls. In conclusion, this thesis has identified the burgeoning potential of a new class of antischistosome drugs that, at least in part, target the nervous system of the parasite and provided a comprehensive characterization of a family of ChEs from S. mansoni, giving compelling evidence for the essentiality of the proteins and their utility as intervention targets against schistosomiasis

Sequential cropping of Vertisols in the Ethiopian highlands using a broadbed-and-furrow system

Investigates the effects of improved surface drainage on the productivity of Vertisols in a wheat-and-chickpea cropping trial conducted in 1987 at ILCA;s Debre Zeit research site in the Ethiopian highlands. Chickpea plots were subjected to four irrigation treatments, viz, no irrigation, irrigation at planting, 35 days and 70 days after planting. The trial showed that with a starter irrigation to aid the germination of a second crop, sequential cropping of two crops in the same growing season is feasible in the Debre Zeit

Development of coordinated research efforts

The experience of ICRISAT with the use of Vertisols in semi-arid India indicates that the key to drastically improved productivity of Vertisols is the effective control of surface soil water which then enables rational use of the land for food and feed production. ILCA, therefore, developed and tested on-station and on-farm research in various highland Vertisol areas an animal-drawn implement for broadbed and furrow construction which was to combine technical efficiency of surface drainage construction with economic viability. Substantial increments in grain and biomass outputs due to enhanced surface drainage were recorded along with convincing economic returns to the farm in the application of this technology. In this chapter, history, objectives, rational, strategies, target areas and structure and organisation of the Vertisol project are presented; and the tasks of the advisory and technical committees, division the tasks of the advisory and technical committees, division of responsibilities between participating institutions, terms of reference for the technical committee, project phases and funding, and assistance by ICRISAt and IBSRAM are discussed

Grain, fodder and residue management

Although this document is intended to report research on Vertisols, livestock do not respect this boundary as they graze communal land. Hence a general situation of the available feed resources in the highlands is also highlighted which is followed by specific efforts of collaborative partners of the Joint Vertisol Project. In this paper the improvement of native pasture; crop residues as main animal feed and possibilities for increased production; fodder improvement in the Ethiopian highland Vertisols, traditional management and cropping patterns and calendar of highland Vertisols are discussed. This paper examines the possibility of early planting of improved wheat varieties on drained Vertisols as Opposed to the traditional late planting, towards the end of the rainy season, and evaluates wheat varieties in terms of grain and straw yields and quality across highland drained Vertisols sites

Multi-stakeholder Platforms Strengthening Selection and use of Fodder Options in Ethiopia: Lessons and Challenges

N° ISBN - 978-2-7380-1284-5International audienceAlthough existing literature eloquently elaborates the role of an “innovation systems perspective” in rural development and provides theoretical insights into the concepts of the approach, there are few practical lessons emerging from application of the approach in research for development projects in various contexts. This paper analyzes a project designed to strengthen the ability of smallholders to innovate in ways that improved the returns to fodder use in Ethiopia. The paper applies an innovation systems perspective to the innovation process as a means of describing the contribution of a project-driven multi-stakeholder platform. A number of key lessons were gleaned from our research. Participatory selection of technologies that addressed farmers' priority problems and demonstrating tangible economic benefits were found to be effective in winning the trust of farmers and drawing the attention of a wider group of stakeholders. The benefits from the technologies needed to carry limited risk and accrue early economic gains to be attractive to farmers. Linking forage technologies with a range of value chain issues in livestock enterprises was essential for successful adoption of forage technologies by farmers. Engaging diverse actors in stakeholder platforms, including local decision makers, along the dairy/fattening value chain was found to be instrumental in turning the wealth of knowledge surrounding fodder technologies and practices into action and creating immediate benefits to poor livestock keepers. On the other hand we found that nurturing collective capacity of a network of organizations and individuals was a major challenge for small projects with limited mandate and resources. For sustainability, organizations with a long term commitment and strong decision-making power need to play a leading role in facilitating innovation processes. An effective agricultural innovation system requires a cadre of professionals with a new skill set and mind set (markets, agribusiness, rural institutions, rural microfinance, facilitation, system analysis, conflict management, etc.). This implies the need for research and development organizations to re-skill, and the need for the reform of university curricula to include skills in agribusiness, communication and partnership facilitation. Large scale institutional and policy change requires piloting of the concept and practices of facilitating innovation through stakeholder platforms in different contexts, documenting and sharing experiences, building on successes and engaging policy makers in the research process

Dry and wet season performance of selected herbaceous legumes in Uganda

The dry matter production of 9 herbaceous legume species was evaluated over 3 years for their suitability as forage species in the subhumid regions of Uganda. Dry matter production of Centrosema pascuorum (6774), Chamaecrista rotundifolia (Wynn), Clitoria ternatea (9291), Macroptilium atropurpureum (Siratro), Stylosanthes guianesis (Cook), S. guianensis (163), S. hamata (Verano), S. scabra (441) and S. scabra (Seca) was assessed after 3-,6-,9- and 12- weeks regrowth in the wet season and after 6- and 12- weeks regrowth in the dry season. The Stylosanthes species were most productive in both wet and dry seasons. Appropriate seed production technologies need to be developed to make seed available for further studies and for distribution to farmers

Effect of seedbed methods and time of harvest on the yield and nutritive value of some forage crops grown on Vertisol at Debre Zeit, Ethiopia

The influence of the improved drainage broadbed and furrow (BBF), as opposed to the traditional flat seedbed over the growing season on the dry matter yield and nutritive value of Avena sativa, Vigna unguiculata, Lablab purpureus, Vida dasycarpa, Trifolium steudneri and Sesbania sesban were studied on Vertisol. Up to 7 t/ha dry matter yield was recorded for Avena sativa and Lablab purpureus when planted on Vertisols with improved drainage. Dry matter yield of forage crops also increased with advance in stage of maturity or subsequent harvests. On both improved and traditional flat seedbed methods, the chemical analysis of forage crops showed similar declines in crude protein content and in vitro dry matter digestibility (IVDMD) levels as the maturity of forage crops progressed

Hypertension in Chronic Kidney Disease: Navigating the Evidence

Hypertension is both an important cause and consequence of chronic kidney disease. Evidence from numerous clinical trials has demonstrated the benefit of blood pressure control. However, it remains unclear whether available results could be extrapolated to patients with chronic kidney diseases because most studies on hypertension have excluded patients with kidney failure. In addition, chronic kidney disease encompasses a large group of clinical disorders with heterogeneous natural history and pathogenesis. In this paper, we review current evidence supporting treatment of hypertension in various forms of chronic kidney disease and highlight some of the gaps in the extant literature