RESEARCH Schistosomiasis in Zambia

SEES Fellows: The effects of anthropogenic nutrients onparasitic diseases

JOHN MISCHLERDept. of Ecology and Evolutionary Bio., University of Colorado, Boulder, CO, USA

Contact email: [email protected]

1 Project Summary

Recently, parasites that utilize multiple species (schistosomiasis, fasciolosis) have been foundto respond positively to nutrient enrichment. This project aims to explore the ecological interac-tions between the first intermediate hosts of these neglected tropical diseases (aquatic snails) andanthropogenic nutrient enrichment. Work will be conducted at the Nakambala Sugar Estates incentral Zambia where these diseases are endemic, the aquatic snail hosts are present, and hugeamounts of anthropogenic nutrient loading of aquatic ecosystems occur. Snail densities and in-fection prevalences will be evaluated in along with nutrients in and around the sugar estates andhuman schistosomiasis and cattle fasciolosis data will be collected. The partner mentor, Dr. JamesMwansa (UTH Zambia), will manage the human disease testing, drug treatment, and communityeducation as part of his ongoing treatment program while Vincent Lilanda, (chief public health of-ficer at the sugar estates), will provide access. Lab and mesocosm components will take place atthe University of Colorado in Boulder in collaboration with Dr. Pieter Johnson (CU Boulder).Mechanisms indicated in the field will be explored in greater detail under controlled conditions todetermine which nutrients (carbon, nitrogen, phosphorus) are likely to elevate disease risk, how,and at what concentrations. Hotspots of nutrient release will be targeted and pilot wetlands willbe constructed to provide cost effective nutrient management. Reduced nutrient flows will reducesnail host densities and thus disease risk. This approach, along with alterations to nutrient man-agement and continued drug treatments, will provide a local solution to a global problem and reducethe negative effects of agricultural expansion while not slowing production. Additionally, John Mis-chler will receive training through both this research project and interactions as well as developingand teaching an international disease ecology course in Zambia. John Mischler’s PhD training hasbridged the areas of biogeochemical cycling and disease ecology. This proposed work will put him incontact with both industrial, veterinarian, and health care partners. John Mischler will be exposedto the economic concerns and constraints inherent within industry while also getting experiencewith disease monitoring and the social and community aspects of disease prevention. These newperspectives will expand his understanding of coupled human-environmental systems and help himto target his science to where it will do its most good within real-world frameworks.

No study has set out to rigorously determine the conditions and concentrations under whichdisease risk is elevated in real-world systems relevant to human health and well-being. This project isa crucial first step in applying the basic science within systems (sugar plantations) that are notoriousfor high disease prevalences and high host snail densities. This project pulls together expertisefrom biogeochemistry, parasitology, invertebrate zoology, human health, veterinary sciences, andepidemiology and involves individuals from academia, industry, and health care to ensure that thebyproducts of economic growth (here excess nutrients) do not damage aquatic ecosystems’ abilityto mitigate disease risk. Economic factors, environmental factors, and human well-being are allconsidered towards addressing aspects of this coupled agricultural and economic system that inhibit

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John Mischler Nutrients and Disease November 26, 2013

sustainable development, thus preserving ecosystem services that maintain health and well-being forthose living in and around this and other agriculturally-impacted systems. In addition to immediatemetrics, the real test of success for this work is the long-term ability of these nutrient remediationmechanisms to maintain nutrients at levels below those that would enhance disease risk.

This study will catalyze cooperation between academia, industry, and the health professionstowards a goal of improving the well-being of individuals in society. Two American undergraduates,one Zambian MS student, and one American postdoctoral scholar will benefit from these synergiesand the curriculum that will be developed for the Zambian field course will also be helpful for othereducators to use as case studies for the classroom.

2 Project Description

2.1 Objectives

Anthropogenic nutrient enrichment has been shown to lead to increased disease risk in certainsystems (McKenzie and Townsend, 2007). Disease caused by trematode parasites with complexmulti-organism life cycles have proven to be particularly sensitive to nutrient enrichment, especiallyin the first intermediate snail host (Johnson et al., 2007). Trematode parasites cause disease inboth humans (schistosomiasis) and livestock (fasciolosis); thus a mechanistic understanding of thelinks between nutrients and the life cycle of these parasites is vital for mitigating disease riskwithin nutrient-enriched environments. We will combine fieldwork at Nakambala Sugar Estateswith focused lab and mesocosm studies at CU Boulder to determine the effects of added carbon,nitrogen, and phosphorus to disease risk. Our aim is to use this knowledge to provide appropriateand cost-effective nutrient remediation solutions to encourage sustainable agricultural developmentwhile avoiding increases in disease risk.

2.2 Background

Digenean trematode parasites with complex multi-host life cycles are responsible for sig-nificant reductions in economic prosperity and quality of life in many regions of the world. Thetrematode blood flukes that cause the disease schistosomiasis (Schistosoma mansoni and Schist-soma haematobium) trigger chronic inflammatory health effects which contribute to anaemia andundernutrition, which can lead to growth stunting, poor school performance, poor work productiv-ity, and continued poverty (King, 2010; King et al., 2007). Cattle infected with the trematode liverflukes that cause fasciolosis (Fasciola gigantica and Fasciola hepatica) suffer subclinical impairmentof feed efficiency, growth, and fertility, which can have an important impact on productivity (Ka-plan, 2001). Schistosomiasis infects almost 240 million people worldwide with an additional 460million at risk of infection (WHO) while fasciolosis currently infects 2.4 million people with another180 million at risk (WHO) and is responsible for livestock losses approaching $3.2 billion per year(Spithill et al., 1999). Few countries in the most affected regions have undertaken successful andsustainable control programs; in fact the construction of water schemes to meet the power andagricultural requirements for development have lead to increasing rates of transmission of schisto-somiasis (Chitsulo et al., 2000). Meanwhile, the number of reports of humans infected with Fasciolahepatica has increased significantly since 1980 (Mas-Coma et al., 1999). In Zambia, both S. haema-tobium and S. mansoni are endemic with prevalences in elementary school children reaching as highas 90% and 77% respectively (Mwansa, 2005). Fasciolosis is prevalent in cattle communities and isa source of economic loss (Phiri et al., 2007, 2005). Climatic and ecological changes are expectedto change transmission dynamics for fasciolosis and schistosomiasis (Mas-Coma et al., 2008), but

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there are large uncertainties concerning when and how disease risk may be affected. Currently,national and international programs focus solely on drug treatments to reduce disease prevalence.However, drug treatments alone have proven insufficient, with high rates of reinfection occurring(Erko et al., 2003).

Figure 1: Pictures taken at the Nakambala Sugar Estates in 2009. Upper Left: Cattle using astream carrying effluent on the margins of the sugar estates. Upper Right: Effluent routed via acanal to the off of the sugar estates and onto the Kafue Flats. Lower Left: Snails (mainly Bulinusglobosus) thriving in sugar refinery effluent. Lower Right: A eutrophic drainage canal on the sugarestates.

The persistence of these diseases (and others like them) is due in large part to the parasites’complex multi-host life cycles. In almost all species of digenean trematodes the first intermediatehost in the life cycle is an aquatic snail. Infected aquatic snails release hundreds to thousands oflarval parasites (cercariae) that swim through the water and either directly infect the definitivehost by burrowing through the skin (schistosomiasis) or encyst and are trophically transmittedto the definitive host (fasciolosis). Once inside the definitive host the parasite develops to sexualmaturity and produces eggs that are passed back into the aquatic environment through feces/urinewhere they hatch and infect aquatic snails to complete the life cycle. Even when the definitive host(humans, livestock, etc.) has been cleared of infection, the original source of infection (the waterbody with associated infected snails) remains as an independent source of disease transmission.Therefore, successful disease control depends on a dual approach involving drug treatment of thedefinitive host as well as control programs targeting intermediate host aquatic snail populations.

Aquatic systems are extremely sensitive to anthropogenic nutrient additions from domes-tic sewage treatment, industrial sources, and agricultural sources (Howarth, 2008; Howarth et al.,2005; Galloway et al., 2004; Howarth et al., 1995). Nutrient enrichment causes fundamental changesin ecosystem structure and function (Smith and Schindler, 2009; Smith et al., 2006; Smith, 1998;

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Howarth et al., 1988; Hecky and Kilham, 1988; Vollenweider, 1968) which may in turn alter snail-parasite interactions. Indeed digenean trematodes become more abundant with low to moderatelevels of eutrophication (?Lafferty, 1997). Nutrients can alter patterns in disease transmission di-rectly through changes in snail intermediate host food and habitat availability which can reduceinfected snail host mortality and raise parasite output per snail host (Johnson et al., 2007). Inaddition to food quantity, food quality (nutrient stoichiometry) affects cercarial production rates(Bernot, 2013). Nutrients may also reduce competition between susceptible and non-susceptiblesnail hosts, and in some cases, favor survival of the susceptible species over the non-susceptiblespecies (Giovanelli et al., 2005; Okere and Odaibo, 2005), thereby increasing snail infection rates(Johnson et al., 2009; Bakry and El-Monem, 2005). Recent work has even suggested that trema-todes can enhance local or ecosystem-wide nutrient cycling through modulations in host excretion,providing an accelerating feedback that further facilitates parasite transmission (Mischler in prep).Because many of the types of aquatic snails that host trematodes feed mainly on fine detritus, anynutritive particles that settle out of the water column can become snail forage (Madsen, 1992).Therefore, while added nitrogen (N) and phosphorus (P) can alter periphyton nutrients throughgrowth effects, particulate matter containing large amounts of proteins (N) or carbohydrates (C)can similarly affect snail forage quantity and quality.

Infection-induced metabolic and physiological changes to the snail intermediate host havebeen studied extensively in the Biomphalaria/S. mansoni system. The larval development of S.mansoni causes an intense drainage of nutrients from its aquatic snail host that mimics a starvationresponse. Nutrients are continuously withdrawn from the haemolymph of the host snail for parasitereproduction and growth, causing severe decreases in haemolymph glucose (Cheng and Lee, 1971;Stanislawshi and Becker, 1979) as well as decreases in haemolymph amino acids and proteins(Anteson and Williams, 1975; Becket and Hirtbach, 1975; Gilbertson et al., 1967; Gress and Cheng,1973; Lee and Cheng, 1972; Stanislawshi and Becker, 1979; Stanislawsky et al., 1979). AdditionallyMoore and Halton (1973) found similarities between Lymnaea truncatulata infected with Fasciolahepatica and starved snails, using both morphologic and histologic methods.

A holistic and sustainable approach to control trematode-caused diseases within a nutrient-enriched world depends on an integrated approach that considers pathways through which nutrientsaffect the first intermediate host snails. The use of synthetic and natural molluscicides has beenattempted to control snail hosts (Chimbari and Ndlela, 2001) but problems persist regarding cost,access, and unintended effects on aquatic ecosystems. In addition, only one synthetic mollusci-cide is currently approved for use (niclosamide) which makes resistance a concern. Alternatively,in environments with high anthropogenic nutrient loads (i.e. agricultural systems) adequate snailcontrol may be achieved through reductions in nutrient runoff. However, in order to focus efforts toavoid costly and possibly unnecessary nutrient control measures, specific nutrient-disease linkagesmust be evaluated to determine which nutrients (C, N, P) produce functional changes in diseasetransmission and how this change in disease risk arises. These results can then be used to imple-ment focused site-specific nutrient control protocols and community education programs to providelasting and sustainable reductions in disease risk while engineering the system to protect importantecosystem services. While management plans must be site specific, the process of plan design canbe replicated at other sites relying on these original fundamental mechanisms.

2.2.1 Sugar Plantations and Trematodes

Large sugarcane growing operations (sugar plantations) are ideal sites to examine nutrientcontrols on schistosomiasis and fasciolosis transmission because (1) they are located throughouttropical areas where these diseases are endemic, (2) they are areas of high risk for trematode infec-

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tions, specifically schistosomiasis, (3) their extensive irrigation canal networks provide water bodieswithout variations in geometry and hydrology, and (4) large amounts of nutrients are dischargedinto this canal system at discrete points thus providing gradients in nutrient concentrations. Addi-tionally, the impacts of anthropogenic nutrient loading on disease risk in the surrounding ecosystemscan be determined by comparing nutrient discharge points to adjacent non-effluent affected areas.Sugar plantations have been determined to be foci of schistosomiasis infection in Brazil (Barbosaand DaCosta, 1981), Malawi (Crossland, 1963), Sudan (Gasmelseed and Babiker, 2008), Zimbabwe(Ndamba et al., 1991), Ethiopia (Simonsen et al., 1990), Tanzania (Fenwick and Figenschou, 1972),and Senegal (Talla et al., 1990). Host snails thrive in these nutrient-rich canals (Mischler unpub-lished, (Crossland, 1963; Gasmelseed and Babiker, 2008; Pieri et al., 1995; Sturrock et al., 2001) byvirtue of abundant food resources and perennially stable water levels (Moyo and Taonameso, 2005;Sturrock et al., 2001); as a consequence human morbidity due to schistosomiasis is very commonon sugar plantations (Ndamba et al., 1991; Fenwick and Figenschou, 1972; Simonsen et al., 1990).However, the use of drug treatments alone to control schistosomiasis in sugar plantations achievesminimal reductions in disease prevalence and, following treatment, infection prevalence returns topre-treatment levels when snail control is ignored (Erko et al., 2003). Even when a synthetic mol-luscicide is used in conjunction with drug treatment, only partial and temporary disease controlis achieved (Chimbari and Ndlela, 2001) due to lapses in the snail control program (Moyo andTaonameso, 2005). In order for molluscicide to be successful sustained monthly applications areneeded to reduce snail numbers (Pieri et al., 1995) emphasizing the vigilance needed to suppresssnail densities with a molluscicide to avoid population rebound (Crossland, 1963).

Our goal in this study is to explore the mechanistic links between C, N, and P loads and thetransmission of trematode-caused diseases, specifically schistosomiasis and fasciolosis. Within thenutrient-enriched context of a sugar plantation, our aim is to determine the mechanisms linkingnutrient loading to disease risk through the intermediate snail host. This knowledge will then enableus to make recommendations for nutrient management within the sugar plantation which will worksynergistically with existing drug treatment and community education programs to reduce diseaserisk in and around these human-impacted systems. This approach eliminates the need to use costlyand hard to acquire molluscicides therefore increasing the likelihood that snail suppression will besustained in the long-term. The intention of this work is to provide resources to achieve sustainableagricultural and industrial development while avoiding the negative effects of nutrient enrichmenton disease transmission.

2.2.2 Relationship to SEES

This project concentrates on the interactions between anthropogenically-induced ecologicalchanges and disease dynamics. As such it is extremely cross-disciplinary, pulling on expertisefrom biogeochemistry, parasitology, invertebrate zoology, human health, veterinary sciences, andepidemiology. This work involves individuals from academia, industry, and health care workingto ensure that the byproducts of economic growth (here excess nutrients) do not damage aquaticecosystems’ ability to mitigate disease risk. We consider economic factors, environmental factors,and human well-being and aim to address aspects of this coupled agricultural and economic systemthat inhibit sustainable development, thus preserving ecosystem services that maintain health andwell-being for those living in and around this and other agriculturally-impacted systems.

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Figure 2: Upper Left: Satellite image of the Nakambala Sugar Estates with yellow pins placed atsurface water sampling points from the Environmental Consul of Zambia’s 1997/1998 samplingcampaign. Upper Right: Satellite image of the Nakambala Sugar Estates with yellow pins placedat groundwater sampling points from the Environmental Consul of Zambia’s 1997/1998 samplingcampaign. Bottom: Average results from the ECZ’s 1997/1998 sampling campaign.

2.3 Plan of Work

2.3.1 Field Area

All of our field work will take place on and around the Nakambala Sugar Estates alongthe Kafue River in central Zambia (Figure 1 and Figure 2). The property currently spans 13,413hectares and produces over 200,000 tons of sugar annually. Potential snail habitat within the sugarestates consists of: (i) feeding canals which carry Kafue River water to the cane fields, (ii) drainagecanals which collect nutrient-rich water from fields and the refinery and routes it off the property(Figure 1 LL, LR, UR), (iii) natural through-flowing streams coming from the town of Mazabuka,and (iv) retention ponds containing either river, drainage, or waste water that is discharged to thecanal system. This collection of canals and streams which mix within the Nakambala Sugar Estatesprovides a nutrient concentration gradient within which to compare snail size, density, and infectionprevalence without confounding variables related to water course geometry and hydrology.

This is a critical time to do this work in the Nakambala Sugar Estates. South African sugarproducer Illovo has just heavily invested in the sugar estates and they are currently in the processof doubling sugar production to 400,000 tons per year. This expansion includes the constructionof 29.7 km of canals and 265 km of roads; and the laying of 36.8 km of pipelines and 650 km of

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pivot tracks. With such drastic development plans in progress it is especially crucial to determinewhat effects the new production areas might have on disease transmission in and around the sugarestates.

Table 1: Snail species found in canals at Nakambala Sugar Estates organized by water source

Site Water Source Bul. g.a Biom. p.a Lym. n.b Mel. t. Bul. t. Cleo. n. Bel. c.

1 Mazabuka 0 0 0 0 0 0 12 Mazabuka 0 0 0 0 0 0 03 river 0 0 0 11 152 21 244 river, refinery 2 2 4 11 0 0 05 refinery 13 2 18 9 0 0 06 fields, refinery 55 24 2 1 0 0 07 fields 74 0 16 1 0 0 0

a denotes snails species that serve as intermediate hosts for human Schistosomiasisb denotes snails species that serve as intermediate hosts for Fasciolosis

2.3.2 Preliminary Data

We conducted preliminary investigations at the Nakambala Sugar Estates in 2009 to establishits suitability for a large-scale study of the effects of nutrient enrichment on schistsomiasis andfasciolosis. Seven different drainage canals were surveyed for snail populations and water nutrients.Snail species that serve as intermediate hosts for schistsomiasis and fasciolosis are found throughoutthe property but they dominate in both fertilizer-enriched field runoff (Figure 1 LR) as well aseffluent from the sugar refinery (Table 1). Ammonium (NH4) concentrations tend to be higher (upto 3 times higher) in fertilizer and effluent-impacted waters than in natural waters (Figure 3A).Water NH4, NO3, and P concentrations measured by the Environmental Consul of Zambia (ECZ- Figure 2) corroborate these data by showing enriched nutrients in the ditch waters compared tothe river water. Total dissolved organic carbon (TDOC) is reliably higher in waters containingsugar refinery effluent with concentrations over 4 times higher than those in natural waters (Figure3B). High TDOC in refinery effluent is likely a result of residual sugarcane-derived carbohydratesremaining in the waste water following processing as pieces of sugarcane pulp were visible in thewastewater.In two adjacent water bodies, (one receiving a large amount of effluent from the sugar refineryand the other receiving irrigation water directly from the Kafue River), snail size was significantlylarger for those snails living in sugar refinery effluent versus those living in water directly from theKafue River (Figures 4 and 5). Larger snails are known to increase disease risk through increasedcercarial shedding rates and shedding duration (Johnson et al., 2007). B. globosus (vector for S.haematobium) specifically seemed to be more numerous in effluent water (Figure 1 LL). Thesepreliminary results are consistent with the importance of amino acids/proteins and carbohydratesin maintaining infected snails (described above) and indicate the Nakambala Sugar Estates as asuitable system to test mechanistic linkages between nutrient enrichment and disease risk. Thisfield system presents a unique chance to not only test the effects of N and P enrichment on diseaserisk (which have been described in other systems), but also carbohydrate enrichment. Addition-ally, based on the physiology of infection, there may be significant interactions between N and Penrichment and carbohydrate enrichment that greatly increase disease risk in these systems.

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Figure 3: Differences in water nutrient concentrations between water sourced mainly from the KafueRiver and surrounding streams (blue) versus water containing fertilzer runoff and effluent from thesugar refinery (red) for NH4 (left) and total dissolved organic carbon (right).

2.3.3 Study Design

This study will combine field, laboratory, and mesocosm components to determine mechanisticlinks between agricultural nutrient leakage and disease transmission over 3 years. All field workwill be conducted at the Nakambala Sugar Estates in collaboration with Dr. James Mwansa fromthe University Teaching Hospital (UTH) in Zambia, Mr. Vincent Lilanda - chief public healthofficer of the Nakambala Sugar Estates, Mr. Andrew Phiri of the University of Zambia (UNZA),Dr. Charles Michelo (UNZA), and myself with the University of Colorado Boulder (CU Boulder)department of Ecology and Evolutionary Biology (EBIO). The lab and mesocosm components willbe conducted at CU Boulder in collaboration with Dr. Pieter Johnson (EBIO).

2.3.4 Field Study

Question: How do fertilizer and sugar refinery effluent alter the size, distribution, and infectionprevalence of snail intermediate hosts of fasciolosis and schistosomiasis and how does this impactinfection in humans and cattle?Rationale: Our preliminary data suggest that snails that spread schistosomiasis and fasciolosis aremore abundant and larger when found in fertilizer and effluent-impacted water, suggesting theseareas may be hotspots for disease transmission.Hypothesis: We hypothesize that added N and P from fertilizers and added carbohydrates fromrefinery effluent leads to higher quality periphyton and detritus that, when grazed by snails, in-creases snail growth rates and survival and enhances cercarial shedding.

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Figure 4: Differences in snail size between intermediate host snails found in river water (belowruler) versus sugar refinery effluent (above ruler). Snail species are as follows: left - Biomphalariapfeifferi (host for S. mansoni), center - Bulinus globosus (host for S. haematobium), right - Lymnaeanatalensis (host for F. gigantica).

Figure 5: Differences in snail size between snails found in river water (blue) versus sugar refineryeffluent (red) for Bulinus globosus (left) and Lymnaea natalensis (right). Differences are significant(p<0.05, t test).

Disease PrevalenceDuring year one Dr. Mwansa’s team at the UTH will coordinate human schistosomiasis monitoringin and around the Nakambala Sugar Estates 4 times per year (dry season, wet season, and transitionseasons) as part of an ongoing national monitoring program to determine baseline schistosomiasisinfection patterns and intensities. Infection prevalences, egg counts, and observed morbidity datawill be collected for both S. mansoni and S. haematobium. Surveys will also be administered aspart of the standard protocol to determine water use habits, existing knowledge regarding schisto-somiasis, and perceived health effects of infection. Similarly, baseline cattle Fasciola sp. infectiondata will be collected 4 times per year during year one through Mr. Andrew Phiri’s existing net-work of abattoirs throughout the Kafue Flats region (Phiri et al., 2007, 2005). Prevalence data andliver condition scores are already collected by these abattoirs as a standard government-enforcedpractice and the provenance of cattle are noted. Using this information collected from abattoirs tofocus our work, we will determine local Fasciola sp. infection prevalence and intensity at select siteswhere sugar estate effluent enters the Kafue Flats (Figure 1 UR) as well as hydrologically similar

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adjacent sites without effluent impacts. We will test individual cattle in targeted areas alreadyfrequented by Mr. Phiri using coprological examination for Fasciola sp. eggs to determine localprevalence and intensity patterns.

Ecological MonitoringSnail diversity/density/infection/stoichiometry, periphyton biomass/diversity/stoichiometry, andwater nutrients will be measured in water bodies across and surrounding the Nakambala SugarEstates. Mr. Lilanda and I will conduct baseline studies on all relevant ecological factors every twomonths during years one, two, and three. All drainage ditches carrying mixtures of runoff waterfrom both the fertilized fields and effluent from the sugar refinery will be sampled strategicallyto achieve maximum variability in water C, N, and P concentrations. While field runoff occursuniformly all along the ditch length, refinery effluent is injected into the canals at discrete points.These injection points are crucial areas where large nutrient gradients occur. Sampling points willbe located proximally upstream and downstream of these injection points to evaluate the effectsof the addition of refinery effluent to canal ecosystems. Additional sampling points will be placedprogressively further downstream from these injection points as the effluent is diluted with fieldrunoff. In addition, sampling points will be placed in irrigation feeder ditches carrying Kafue Riverwater (no fertilizer or effluent impacts) as an endmember representing a relatively pristine base statefor these canals. The effects of exported sugar estate nutrients on disease risk in the surroundingKafue Flats will also be evaluated by comparing sampling points within waterways carrying effluent-impacted water out of the sugar estates to the Kafue Flats (Figure 1 UL) and sampling points withinadjacent waterways with no effluent impacts. Water samples will be collected from all samplingpoints, filtered, and frozen for nutrient analyses at CU Boulder.

Snail density and diversity will be determined by examining three 0.5 m2 quadrats persampling point using proper protective equipment (gloves, waders, etc.). All snails within thesequadrats will be hand collected, identified to species, and will be examined for trematode infections.If less than 50 snails are collected from the quadrats then snails from the immediatly surroundingareas will be picked by hand until at least 50 snails are collected per sampling point. These snailswill then be individually measured via calipers, crushed, and examined for mature and prepatentinfections using a dissecting microscope. All schistosomes and fasciolids will be collected and placedin 70% ethanol for later molecular identification in the United States. Snail tissues will be frozenand returned to the United States for C, N, and P analyses to evaluate the effects of infection andsource water on snail nutrient stoichiometry.

Periphyton/detritus samples will be aspirated from a standardized 0.2 m2 area from sedi-ments and macrophytes. Six periphyton samples will be collected per sample point. Three sampleswill be combusted at 500◦C to determine periphyton ash free dry mass per unit area while theother three samples will by analyzed for C, N, and P. A representative periphyton sample willbe preserved in Lugol’s (IKI) solution and returned to the United States to determine periphytonspecies diversity. We will examine these data for links between algal species and water nutrients.

2.3.5 Lab Study

Question: Does added protein and/or added carbohydrates increase snail sizes and cercarial shed-ding rates?Rationale: Physiologically when snails are infected with schistosomes they become drained ofcarbohydrates and proteins. Because these are in high demand in infected snails, supplementationwith these components should increase snail size and cercarial shedding rates.

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Hypothesis: We hypothesize that infected snails supplemented with protein will exhibit increasedgrowth, survival, and cercarial shedding rates while those supplemented with carbohydrates willsee even larger increases in growth, survival, and cercarial shedding rates due to extremely highcarbohydrate demand by the snail/parasite system. We also expect to see cercarial shedding ratespeak when equal portions of proteins and carbohydrates are present.

During year one, lab work will be conducted at CU Boulder in parallel with the Zambiafield work. Feeding trials will be used to determine the effects of varying amounts of proteinsand carbohydrates on snail survival, snail growth, and cercariae production. Uninfected snailsand snails infected with human schistosomes will be obtained from the Biomedical Research Insti-tute (http://www.afbr-bri.com/). Feeding experiments will be conducted using both Biomphalariaglabrata snails infected with S. mansoni and Bulinus truncatus truncatus snails infected with S.haematobium. Food carbohydrate (as rice starch) will be varied to mimic particulate contributionsfrom sugar refinery effluent and food protein will be varied to simulate variations of periphytonprotein through changes in N supply (Austin et al., 2009). Both rice starch and a high protein fishfood will be combined within an agar matrix to produce five different relative proportions: 0:100,25:75, 50:50, 75:25, and 100:0.

These five different food treatments will be fed to both an uninfected snail group and aninfected snail group in a 5 X 2 factorial design with 10 replicates for each treatment with bothspecies (B. glabrata/S. mansoni – 100 snails; B. truncatus/S. haematobium – 100 snails). Eachsnail will be maintained individually in spring water within 1.6 L plastic containers in a randomizedconfiguration. Water will be changed weekly and 10 g food pellets will be supplied weekly. Snailshell lengths will be measured weekly using calipers for both the infected and uninfected groups todetermine the effect of diet on snail growth and egg masses will be removed from the water, dried,and massed as they appear to measure reproductive output. Relative changes in cercarial sheddingrates will be measured in the infected group weekly, with the first measurement taken just beforethe initiation of the experiment. Each snail will be placed in a separate 60 ml centrifuge tube with30 ml of spring water for three hours (8 AM to 12 PM) where it will shed cercariae. At the end ofthis interval snails will be removed and placed back in their holding containers and the number ofcercariae shed will be counted under a dissecting microscope. Uninfected snails will also undergothe same weekly regimen to avoid confounding effects. Feeding trials will continue for 20 weeksat which point all snails will be dissected, parasite masses weighed, and snail tissues sampled forC/N/P analyses. Results from this work will inform both the field component and the mesocosmstudy.

2.3.6 Mesocosm Study

Question: What are the mechanisms through which fertilizer and refinery effluent influences snailsizes, distributions, and infection?Rationale: Adding fertilizer to a system will increase the protein content of the periphyton andthus the nutritional quality of the periphyton. Added particulate carbohydrates will settle onto theperiphyton as fine detritus and be directly grazed by snails.Hypothesis: We hypothesize that snail densities and sizes will increase in the high fertilizertreatments and cercarial shedding rates will increase marginally while in the high carbohydratetreatments snail shedding rates will drastically increase. The treatment causing the highest diseaserisk will be the high fertilizer and high carbohydrate treatment.

During year two, 1,200 L mesocosms (plastic stock tanks) will be constructed to link mech-

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anistic results from the lab feeding trials to ecosystem-level infection patterns determined in thefield studies. We will conduct a 3 X 2 X 2 factorial design mesocosm experiment manipulatingnutrient (N and P) concentrations (low and high) and carbohydrate (rice starch) concentrations(low and high) outdoors at the 30th Street greenhouse facility at the UC Boulder. Nutrient andcarbohydrate concentrations will be based on the range of concentrations encountered in the field.Each treatment will be replicated 6 times for both infected and uninfected snails for a total of 48mesocosms. The bottom of each mesocosm will be covered with 2 cm of sand and nutrient poorwell water will be added to a uniform depth. Each mesocosm will be seeded with planktonic andperiphytic algae and zooplankton from a range of ponds. In addition, each mesocosm will receive20 g damp weight of common macrophytes and their associated epiphytes (for a total of 60 g ofmacrophytes per mesocosm).

N and P concentrations will be manipulated by adding NaH2PO4 and NaNO3 in concert at aconstant molar ratio (N:P = 16) within the range of values observed from the field survey to producehigh, medium, and low treatments. Similarly, carbohydrate concentrations will be manipulated byadding rice starch to each mesocosm to produce three different concentrations whose actual valueswill be determined based on the field study. N, P, and TDOC will be measured in the watercolumn weekly and additional nutrients will be added to keep mesocosm water nutrient valuesstable to mimic a constant supply from fertilizer runoff and sugar refinery effluent. UninfectedB. glabrata snails from BRI will be placed in each mesocosm (50 per mesocosm). Snails will bemeasured before placement so that each mesocosm has approximately the same proportion of sizeclasses. For the infected group, S. mansoni eggs from BRI will be placed into 1-liter porous plasticchambers suspended at the mesocosm surface (fully submerged but within clear view of sunlight)and allowed to hatch so that the parasites can seek out and infect their snail hosts. We will add∼800 eggs to each mesocosm in the infected group twice a week for the duration of the experiment.All mesocosms will be covered with 1-mm mesh lids to minimize colonization of unintended floraand fauna and the entire site will be securely fenced.

Mesocosms will be monitored biweekly from May until October, at which point colderweather will hinder any further study. Algal growth, snail density/size/reproduction/infection,and shedding rates of schistosome cercariae will be measured biweekly throughout the study. Phy-toplankton productivity will be measured by filtering phytoplankton from 200 ml water columnsamples and measuring chlorophyll a. Periphyton productivity will be measured by removing at-tached periphyton from standard length (∼5 cm) strips of flagging tape attached to the side ofeach mesocosm, suspending in 30 ml DI water, and measuring chlorophyll a. Periphyton biomasswill be quantified by scraping attached periphyton from 75 by 25 mm glass slides and computingAFDM. Slides and flagging tape will be surrounded by screen enclosures to prevent grazing bysnails. Additional glass slides will be collected to measure periphyton C, N, and P.

Snail density will be quantified by visually counting the number of snails within each meso-cosm; snail size will be measured by recording the number of snails that can be visually placedwithin each of a set of predetermined size classes (1 to 5 mm, 5.1 to 10 mm, 10.1 to 15 mm, andgreater than 15 mm). Random snail egg masses will be chosen from those deposited on plexiglasssheets (∼11cm2) placed within each mesocosm and the number of eggs per randomly chosen eggmass will be counted (up to a maximum of 10 egg masses). Once S. mansoni infections have hadtime to develop within the snails (between 1 and 2 months after exposure) snail infection prevalenceand cercarial production rates will be measured by randomly selecting adult snails from each meso-cosm (number of snails selected = half of the starting number of snails per mesocosm) and placingeach snail into an individual 60 ml centrifuge tube with 30 ml of spring water and allowing themto float for one day within each snail’s respective mesocosm. Snails will then be released back intotheir respective mesocosms and the number of cercariae released per snail during that day will be

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Table 2: Work ScheduleYear 1 2 3

Recruit Undergraduate Students for the Project

Human Schistosomiasis Data Collection

Cattle Fasciolosis Data Collection

Field Sampling

Laboratory Feeding Trials

Mesocosm Experiment

Implementation of Nutrient Remediation Mechanisms

Prepare Manuscripts for Publication

Mentor CU Boulder Undergraduate Conducting Science Research

Mentor UNZA MS Student Conducting Science Research

Design Zambia Disease Ecology Course

Teach Zambia Disease Ecology Course

counted to estimate both snail infection prevalence and daily per capita production of schistosomecercariae. When temperatures become too low to accurately conduct this experiment, the meso-cosms will be destructively sampled and all snails will be dissected to determine the prevalence ofmature and prepatent infections and snail tissue will be analyzed for C, N, and P.

Nutrient RemediationThe third year of this project will be spent developing an on-site nutrient remediation pilot projectfor the Nakambala Sugar Estates. Emphasis will be placed on cost-effective and low-maintenancesystems which achieve focused nutrient reductions at levels deemed appropriate based on our workduring previous portions of this project. Solutions will avoid the most severe impacts of nutrient-induced disease risk in and around the sugar estates. Nutrient remediation work will include: (i)adjustments to current infrastructure to reduce nutrient leakage into surrounding ecosystems, (ii)improvements to current nutrient management schedules to reduce nutrient runoff, and (iii) con-struction of wetlands to remove nutrients from effluent for discharge into the surrounding KafueFlats. Results from the field work portion of this study will guide the types of remediation tech-niques pursued and at what locations around the sugar estates. Leaks in the current system willbe addressed so that nutrient-rich waters will be discharged at very specific locations to allow forfocused remediation.

Nutrient runoff is higher when either (1) fertilizer is applied shortly before a rain eventor (2) when nutrients are applied in excess of plant demand (Chichester, 1977). Currently atNakambala Sugar Estates, in the 12 month growing cycle of sugarcane, two fertilizer applicationsare carried out (i.e basal and top dressing). Basal fertilizers are applied at the time of planting fornewly planted crops or just before the first irrigation for crops sprouting from previously harvestedsugarcane. Top dressing is applied, “as a rule of thumb”, when the crop growth reaches knee height(2 to 3 months of age depending on the season). Fertilizer application rates vary depending onplant type (newly planted or sprouting) or soil type, but on average the total application rate perseason (1 year) is 100 g per m2 (N:P = 2) split between the basal and top dressing. Previouslycollected field data will be used to determine what conditions cause pulses of nutrients to enterthe irrigation ditches. Using these data we will offer recommendations regarding proper temporalspacing between fertilizer applications and irrigation. We will also test field soils for N and P todetermine if N and/or P are being applied in excess of sugarcane stoichiometric demand, with

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the excess nutrients leaching from the soils into the waterways. Based on these results we willrecommend altered stoichiometries for applied nutrients to both reduce nutrient runoff and savemoney otherwise spent on wasted nutrients.

A pilot constructed wetland will be placed at a focal area of high nutrient outflow. Intercep-tion of diffuse agricultural runoff using constructed and restored wetlands can complement improvedsource control measures to reduce nutrient losses from agricultural landscapes and buffer impactson receiving waters (Dinnes et al., 2002; Mitsch et al., 2001; amd D. A. Kovaic, 1993; Petersen et al.,1992). In addition, constructed wetlands need little or no maintenance and can be build with exist-ing local materials. The effectiveness of different processes that affect removal and retention of N(NH3 volatilization, nitrification, denitrification, nitrogen fixation, plant and microbial uptake, min-eralization (ammonification), nitrate reduction to ammonium (nitrate-ammonification), anaerobicammonia oxidation (ANAMMOX)) and P (fragmentation, sorption, desorption, burial, leaching)vary depending on the type of wetland constructed (Figure 6 A1-A4) (Vymazal, 2007). For exam-ple, constructed wetlands with vertical sub-surface flow (VSSF) successfully remove ammonia-Nbut denitrification is limited. On the other hand, constructed wetlands with horizontal sub-surfaceflow (HSSF) provide good conditions for denitrification but the ability of these system to nitrifyammonia is very limited.

For this reason we will develop a multi-tiered wetland mixing multiple wetland types toachieve sufficient nutrient removal. The fist component in our nutrient removal system will be a free-floating plant (FFP) wetland. FFP wetlands remove both N and P moderately well through plantuptake and removal (Figure 6B and 6C). Specifically, the water hyacinth (Eichhornia crassipes),which is present throughout the region of the sugar estates (Figure 6D), is extremely effective atsequestering both N and P (Boyd, 1976). Phosphorus standing stock within water hyacinth canamount to 45 g of P per m2 and the annual amount of phosphorus taken up by water hyacinthcould be as high as 126 g of P per m2 (Vymazal, 1995). This FFP can be constructed quickly andcheaply and is the simplest wetland in the system to build. A depression will be excavated 1 to 1.5m below the existing ground surface into the low-permeability clay subsoils present at the site. Aportion of the topsoil will be turned into the base of the wetland as a growth medium and source oforganic matter for denitrifying bacteria. Water depth will be maintained at ∼1 m and the wetlandwill be supplied with water hyacinth cuttings from plants already growing within the sugar estates.Water hyacinth will be harvested periodically to remove nutrients from the system and the entireconstructed wetland will be fenced to avoid human-contact with this extremely nutrient-enrichedsystem. Nutrient content of the water flowing out of this FFP wetland will be measured and ifadditional nutrient remediation is needed to meet the targets to mitigate disease risk determined inthe lab and mesocosm studies, then a HSSF and/or VSSF wetland will be constructed downstreamdepending on the nutrient species in need of removal.

2.3.7 Management and Timeline

Table 2 shows the overall project timeline. John Mischler (CU Boulder) will be the PI of the project,with the overall responsibility for moving it forward and will be involved in all aspects of the project.In addition John Mischler will develop and teach a disease ecology course in Zambia through CUBoulder. Pieter Johnson (CU Boulder), the host mentor, will lend his expertise in the designingand conducting of the lab and mesocosm work at CU Boulder as well as advise regarding fieldwork in Zambia. James Mwansa (partner mentor - UTH) will lead efforts in Zambia to quantifyschistosomiasis prevalence in the human populations in and around the sugar estates as well asoffer drug treatments for the disease. In addition he will develop community-based programs toraise awareness of schistosomiasis transmission and risks. Vincent Lilanda (Public Health Officer -

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Nakambala Sugar Estates) will provide access to the Nakambala Sugar Estates and assist in mappingcanal systems within the sugar estates as well as implementing nutrient remediation mechanisms.Andrew Phiri (UNZA) will lead efforts to quantify fasciolosis prevalence in cattle grazing in theKafue Flats at sites surrounding the sugar estates. Charles Michelo (UNZA) will support the projectby contributing his disease mapping expertise as well as providing a Zambian MS student to aidin all aspects of the project. John Mischler will mentor this MS student in Zambia as well as twoundergraduate students at CU Boulder. The team assembled here is well-equipped to manage thismultidisciplinary project involving disease monitoring, ecological field surveys, lab and mesocosmwork, and coordination with industrial partners for nutrient remediation.

2.3.8 Expected Outcomes

This project takes a multi-pronged approach involving field, mesocosm, and lab work to understandmechanistic links between nutrients and disease while also attempting to mitigate some of thesenutrient-enhanced risks through nutrient remediation. Ultimately the success of this project willbe measured in the effectiveness of nutrient remediation strategies in reducing host snail densitiesas well as the effects of our coupled nutrient management/drug treatment approach to reducingdisease burdens as measured in humans and cattle. Our goal is to reduce the currently unregulatedflows of nutrients from the Nakambala Sugar Estates into the surrounding aquatic ecosystemsto levels that do not encourage the development of health risks to humans and wildlife throughenhanced transmission of schistosomiasis and fasciolosis. Our nutrient remediation methods will becost-effective and simple to maintain with local resources thus increasing the chances of continuedfunctioning and long-term sustainability. While initial measures may indicate the success of thisapproach, long term monitoring of water nutrients and disease prevalence is needed to understandthe sustainability and efficacy of nutrient management in reducing disease risk.

2.4 Broader Impacts

The work we propose here is vital within the context of the current anthropogenic acceleration ofnutrient cycling. Developing countries like Zambia have seen recent drastic increases in develop-ment and fertilizer use (200% increase since 1970) (Naseem and Kelly, 1999). Care must be takento ensure that this development can progress without potentially damaging changes in ecosystemgoods and services. Specifically, the effects of nutrient enrichment on ecosystems may carry risksfor human health and well-being through increases in the risk of contracting schistosomiasis andfasciolosis. Our work will forge partnerships between ecologists, epidemiologists, health care pro-fessionals, and industry that have never been attempted in Zambia before. Through this integratedapproach we hope to increase connectivity between various stakeholders and provide a catalyst forcooperation in developing sustainable solutions to human health problems with ecological dimen-sions.

Professional Development Additionally, John Mischler will receive training in sustainability throughboth this research project and the interactions inherent therein as well as developing and teachingan international disease ecology course in Zambia. John Mischler’s PhD training has bridgedthe areas of biogeochemical cycling and disease ecology. This proposed work will put him incontact with both industrial, veterinarian, and health care partners. John Mischler will be exposedto the economic concerns and constraints inherent within industry while also getting experiencewith disease monitoring and the social and community aspects of disease prevention. These newperspectives will expand his understanding of coupled human-environmental systems and help him

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to target his science to where it will do its most good within real-world frameworks.John Mischler will also design a disease ecology course (through CU Boulder) during year

one that he will then teach in Zambia during year two and year three. He already has experiencedesigning and teaching field courses in Zambia (http://www.colorado.edu/ebio/gradstudents/

mischler/WildlandsCourseInformation.html) and would develop this course in a similar way.The focus of this course will be on the disease ecology of humans, livestock, and wildlife. Fieldlocations will include the Nakambala Sugar Estates, the Kafue Flats, the Bagweulu floodplains, etc.We will focus on schistosomiasis, fasciolosis, diseases of lechwe, and interactions between diseaseand conservation (lion reintroductions, etc.). He will work with his collaborator Charles Michelo topair American students with Zambian students from UNZA to foster a true international learningenvironment. This interaction will have benefits for both the Zambians and the Americans and willpromote cross-cultural collaboration on interdisciplinary topics.

Figure 6: A1-A4: Constructed wetlands for wastewater treatment (from top to bottom): constructedwetland with free-floating plants (FFP), constructed wetland with free water surface and emergent

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(http://www.colorado.edu/ebio/gradstudents/mischler/WildlandsCourseInformation.html)

(http://www.colorado.edu/ebio/gradstudents/mischler/WildlandsCourseInformation.html)


macrophytes (FWS), constructed wetland with horizontal sub-surface flow (HSSF, HF), constructedwetland with vertical sub-surface flow (VSSF, VF) (reproduced from Vymazal (2007)). B: Effective-ness of different wetland types at various nitrogen removal processes. C: Effectiveness of differentwetland types at various phosphorus removal processes. D: Water hyacinth growing at the Nakam-bala Sugar Estates.

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Data Management Plan

This project will generate ecological data detailing the impacts of the Nakambala SugarEstates on surrounding water bodies. Seasonal data will be collected on water column nutrientsand specific components of the aquatic systems as relevant to the transmission of schistosomiasisand fasciolosis. These data will include periphyton nutrient stoichioimetry, intermediate host snailtissue nutrient stoichiometry, snail biodiversity, snail densities, and snail infection data. Thisproject will also generate cattle infection data for fasciolosis. This data will be paired with humanschistosomiasis infection data from the area collected by Dr. James Mwansa at the UniversityTeaching Hospital in Luaska. It is vital to (i) synthesize these data into a useful data product fordistribution, (ii) make these data available to other researchers, and (iii) voucher raw materials(snail tissue, periphyon, etc.) for alternative uses by other researchers.

John Mischler will coordinate data management activities at CU Boulder. We will use onlinedatabases to facilitate data sharing, public availability and international collaboration. All datacollected will be made publicly available. All data will be stored in the generally accessible ASCIIcsv format. Already, interest has been shown by international organizations to gain access toour data (CONTRAST - http://www.eu-contrast.eu/, SCI - http://www3.imperial.ac.uk/

schisto). Also, Dr. Mwansa has expressed interest in the generated data products for use withthe National Neglected Tropical Diseases Programme in Zambia. Vincent Lilanda, the chief publichealth officer at Nakambala Sugar Estates, has additionally expressed interest in using these datato better manage human health on the sugar estates. Once the work surrounding this project ispublished, all data will be placed on a specially designed website hosted by CU Boulder and designedby John Mischler to enhance data availability. This website will contain data with various levels ofprocessing. Data will be provided in a raw form but will also be used to generate infection mapsand figures for distribution to project partners and all others who are interested. This website willcontain disclaimers and conditions regarding the use of these data in other publications or productsbased on agreements between project partners. Small amounts of vouchered raw material (water,soil, tissue, etc.) will be archived in a secure freezer at CU Boulder for distribution to any interestedresearchers who wish to run additional analyses on these samples.

The educational curriculum developed in conjunction with this project will be shared on thecourse website. This website will contain case studies that other educators will be able to use outsideof Zambia. Detailed lesson plans in conjunction with real-time picture, videos, and stories fromthe field will enrich this curriculum and serve as a potent illustration of the connections betweenunsustainable development, environmental degradation, ecosystem goods and services, and diseaserisk.

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http://www.eu-contrast.eu/

http://www3.imperial.ac.uk/schisto

http://www3.imperial.ac.uk/schisto

Facilities, Equipment and Other Resources

This research will be carried out in several laboratories at the University of Colorado Boulder.Alan Townsend manages a biogeochemistry laboratory at CU Boulder that includes a walk-infreezer, space for sample preparation and digestion, autoanalyzers and other analytical equipment,hoods, and other standard laboratory equipment for analysis of nutrients in solid and aqueoussamples. In addition, we will make use of Pieter Johnson’s laboratory equipped with temperatureand light controlled controlled rooms with racks for aquaria and a suite of dissecting and compoundmicroscopes and cameras for the feeding trials experiments and to maintain and dissect snails. The30th Street greenhouse facility at CU Boulder is already in use for various mesocosm studies andis ideally suited for our mesocosm work. In Zambia we will be able to rent a 4-wheel drive vehicleand driver for transport between field sites and field equipment can be brought from the U.S. orbuilt in Zambia. The University of Zambia can provide us with microscopes that will be set up inlab space dedicated to us by the Nakambala Sugar Estates. Samples will be held in chest freezersin Zambia until they are ready for transport to the U.S.

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Budget Justification

A. Salaries and Wages – Senior Personnel: The Principal Investigator, John Mischler, willwork full time (100% effort) on the project for the entire 3 calendar years of this 3 year project. Hiscompensation ($50,000 per year; $150,000 total) is calculated based on the precedent of postdoctoralsalary within the Department of Ecology and Evolutionary Biology at the University of ColoradoBoulder. He will be responsible for overall project direction and coordination, for assuring suc-cessful project completion including submission of progress reports as required. John Mischler willsupervise the undergraduate student, oversee all field work and experimental work both in Zambiaand CU Boulder, coordinate work with international partners in Zambia, and prepare manuscriptsfor publication. He will use his expertise in biogeochemical cycling and disease ecology as well ashis contacts in Zambia for all aspects of this project.

B. Salaries and Wages Other Personnel: A total of $6000 is requested to fund an undergrad-uate student during years 1 and 2 of this project. Support is requested for 10 hours of work perweek during the summer and fall semester for a total of 300 hours per year, 600 ours total, at theprevailing rate of $10 per hour. The undergraduate student will be involved with the laboratoryfeeding trial, the mesocosm study, and various laboratory analyses.

C. Fringe Benefits: Fringe Benefits are calculated at $32.2% for the postdoctoral PI and $1.2%for the undergraduate research assistant. We calculate the total fringe for the project by multi-plying the PI salary per calendar year by $32.2% ($50,000*0.322=$16,100) and the undergraduatewages per calendar year by 1.2% ($3000*0.012=$36). We request 3 calendar years of support for thePI and 2 calendar years of support for the undergraduate research assistant ($16,100*3 + $36*2)for a total of $48,372 during the three years of the project.

D. Permanent Equipment: No permanent equipment is requested for this work as all permanentequipment required is supplied by the host institution (the University of Colorado Boulder) andthe partner institution (the University Teaching Hospital and the University of Zambia).

E. Travel1. Domestic Travel: Funds ($1,825 per year) are requested to attend the annual Ecological Societyof America conference once per year for each of the 3 years of the project to present findings andnetwork with other scientists. These funds will be used for round trip airfare, 3 days of per diem,ground transport, and registration fees. In addition $950 per year is requested for years 2 and3 of the project to attend the NSF Fellows meeting in accordance with SEES guidelines. Thesefunds will be used for round trip airfare, 2 days per diem, and ground transport. The total fundsrequested for domestic travel over the 3 year project period are $7,375.1. Foreign Travel: The field portion of this work will take place entirely in Zambia and the partnerinstitution (University of Zambia, University Teaching Hospital) is also located in Zambia. Funds($15,000 per year) are requested to travel from Denver, USA to Lusaka, Zambia to conduct field-work and meet with collaborators. For the entire 3 calendar years of the project, trips will be takenapproximately every 8 weeks and each trip will last 10 days (7 trips per year). One round trip costsapproximately $2,000 in airfare (7 trips = $14,000) and lodging is estimated at $50 per night (10nights = $500). The remaining $500 will pay for food and ground transportation to and from theairport.

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John Mischler Budget Justification November 26, 2013

F. Participant Support Costs: Not Applicable1. Stipends: Not Applicable2. Travel: Not Applicable3. Subsistence: Not Applicable4. Other: Not Applicable

G. Other Direct Costs1. Materials and Supplies: Funds ($15,000 per year; $45,000 total) are requested to cover directcosts for this work. To conduct fieldwork in Zambia a four-wheel-drive vehicle and driver will needto be rented for each 10 day field trip. Rental prices for the vehicle are estimated at $50 per dayand the cost of hiring a driver is estimated at the cost of $30 per day. Vehicle/driver rental willbe for 10 days which calculates to $800 per trip ($16,800 total over 3 years). Fuel for this vehicleis estimated at $200 per trip ($4,200 total over 3 years). Materials for molecular identification ofparasites will cost $2,100 total. Additionally, water NO3 will be analyzed in labs at CU Boulderat $3 per sample. There will be an estimated 60 water samples collected per field trip and 7 fieldtrips per year ($3,780 total over 3 years). The mesocosm experiment requires 48 three hundred fiftygallon stock tanks ($180 each; $8,640 total) and the feeding trial requires 200 1.6 L containers ($3each; $600 total). Approximately $2,500 per year ($7,500 total over 3 years) is needed for reagentsand materials to measure all other nutrient species in the water as well as nutrients within solidsamples (snails and periphyton). An additional $500 is needed for other supplies such as springwater, rice starch, fish food, etc.2. Publication Costs/Documentation/Dissemination: A total of $880 is requested for printing,copying, and dissemination.3. Consultant Services: Not Applicable4. Computer Services: Not Applicable5. Subawards: Not Applicable6. Other: Not Applicable

H. Total Direct Costs: $302,747.I. Indirect Costs: At CU Boulder indirect costs are calculated at 26% of the total direct costs.For the 3 year period indirect costs total $78,714.J. Total Direct and Indirect Costs: $381,461.K. Residual funds: NoneL. Amount of this Request: $381,461.M. Cost Sharing Proposed Level: Not required

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Biographical Sketch:John A Mischler: Project Principal Investigator

(a) Professional Preparation

University of Colorado Boulder Ecology May 2014 (expected)The Pennsylvania State University Geosciences May 2009Augustana College Geology and Physics May 2005

(b) Appointments

2014 Graduate Part Time Instructor, University of Colorado Boulder2012 - 2013 Instructor, Wildlands Studies, Zambia2012 Teaching Assistant, University of Colorado Boulder2011 - 2012 Instructor, McNeil Academic Program, University of Colorado Boulder2006 Graduate Teaching Assistant, the Pennsylvania State University, State College, PA2006 Graduate Teaching Assistant, the Nyanza Project, Kigoma, Tanzania2003-2005 Leaning Assistant, Augustana College, Rock Island, IL

(c) ProductsMcGlue, M.M., M.J. Soreghan, E. Michel, J.A. Todd, A.S. Cohen, J. A. Mischler, C.S.O’connell, O.S. Castaneda, R.J. Hartwell, H.H. Nkotagu, K.E. Lezzar. (2010). Environmentalcontrols on rift lake shell carbonates: A view from Lake Tanganyika”s littoral, Palaios, v. 25, no.7, 426-438, doi: 10.2110/palo.2009.p09-160r

Mischler, J. A., T. A. Sowers, R. B. Alley, M. Battle, J. R. McConnell, L. Mitchell, T. Popp, E.Sofen, and M. K. Spencer (2009), Carbon and hydrogen isotopic composition of methane over thelast 1000 years, Global Biogeochem. Cycles, 23, GB4024, doi:10.1029/2009GB003460.

Walker, R.T., A. Bayasgalan, R. Carson, R. Hazelett, L. McCarthy, J. Mischler, E. Molor, P.Sarantsetseg, L. Smith, B. Tsogtbadrakh, G. Tsolmon, (2006), Geomorphology and structure ofthe Jid right-lateral strike-slip fault in the Mongolian Altay mountains. Journal of StructuralGeology. 28 (9), 1607-1622

(d) Synergistic Activities

� Departmental Sponsor, Ducks Unlimited, 2013 to present

� Development of curriculum for teaching conservation ecology in Zambia in the field

� NSF GK-12 fellow, Creekside Elementary and Casey Middle School, 2010-2012

� Steering Committee, EPA STAR 2011 conference, “Communicating Science to a BroaderAudience”, 2010-2011

� Science Advisor, Kasanka Eco-Expeditions, Kasanka, Zambia, 2012 to present

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John Mischler Biographical Sketch November 26, 2013

(e) Collaborators and Other Affiliations(1) Collaborators and Co-EditorsWaleed Abdalati, Ph.D., University of Colorado BoulderKhalid Hussein, Ph.D., University of Colorado BoulderPieter Johnson, Ph.D., University of Colorado BoulderVincent Lilanda, M.S., Nakambala Sugar EstatesValerie McKensie, Ph.D., University of Colorado BoulderCharles Michelo, Ph.D., University of ZambiaJames Mwansa, Ph. D., University Teaching Hospital, Lusaka, ZambiaAndrew Phiri, M.S., University of ZambiaPhillip Taylor, Ph.D., University of Colorado BoulderAlan Townsend, Ph.D., University of Colorado BoulderPeter Walker, M.S., Colorado Division of Wildlife

(2) Graduate Advisors and Postdoctoral SponsorsM.S. advisor, Richard Alley, Ph.D., the Pennsylvania State UniversityM.S. advisor, Todd Sowers, Ph.D., the Pennsylvania State UniversityPh.D. advisor, Alan Townsend, Ph.D., University of Colorado BoulderPostdoctoral Sponsor, Pieter Johnson, Ph.D., University of Colorado Boulder

(3) Thesis Advisor and Postgraduate-Scholar SponsorStephanie Hayden (undergraduate), University of Colorado Boulder

(4) Undergraduate Research Assistants (last 5 years)Maggie Kriz, University of Colorado BoulderKelly Miller, University of Colorado BoulderEmily Oliver, University of Colorado BoulderMelissa Woodring, University of Colorado BoulderRebecca Woythal, University of Colorado Boulder

Total Undergraduate Advisees = 6

(f) Identification of U.S. CitizenshipJohn Mischler is a U.S. citizen

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Dr. Pieter Johnson Department of Ecology and Evolutionary Biology

Ramaley N122, Campus Box 334 Boulder, Colorado 80309-0334 303-492-8981, Fax: 303-492-8699 www.colorado.edu/eeb/facultysites/pieter www.aquaticparasites.org

November 25, 2013 NSF Science, Engineering and Education for Sustainability Fellows (SEES Fellows) Program Dear Members of the Selection Committee: I am writing to confirm my strong commitment to serve as the host mentor for John Mischler's SEES postdoctoral project (The effects of anthropogenic nutrients on parasitic diseases). I have collaborated with John on his Ph.D. research and am excited to form an even more direct partnership. By way of this letter, I pledge my support for the proposed research, which will focus on understanding how nutrient pollution influences infections by important human and wildlife parasites such as human blood flukes (Schistosoma spp.) and cattle liver flukes (Fasciola spp.) in Zambia. Based on our past work together, I believe strongly in John’s ability to complete this ambitious project, which has far-reaching implications for managing disease and understand the ecosystem drivers of infection. I am extremely enthusiastic about the work John has proposed both here at CU Boulder and at the Nakambala Sugar Estates in Zambia. This research is a logical next step in light of both the remarkable connections John has single-handedly developed in Zambia and the state of knowledge concerning trematodes, nutrients, and disease. Over the past four years, John has built a strong research program in Zambia and has extensive experience with the local ecology and culture. Devastating diseases such as schistosomiasis, which have complex life cycles involving humans and freshwater snails, are critically influenced by local human practices that affect snails, freshwater ecosystems, and human interactions with water. Agricultural areas, for example, have enormous potential to exacerbate this disease by both creating more standing water habitats and through runoff of fertilizers, which can promote snails by enhancing algal growth and altering host stoichiometry. As one can imagine, this is a difficult project both scientifically but especially logistically. John's field sites are appropriately-constrained and ideally suited for an investigation of the ecological connections between disease emergence and nutrient enrichment within a human-impacted landscape. It is rare to find such a convenient and opportune field site in sub-Saharan Africa, a region where schistosomiasis is endemic. The lab and mesocosm work John has planned in cooperation with my lab will provide valuable information regarding the mechanistic relationships between nutrients and disease – something so often lacking from correlative epidemiological studies. John's connections with Nakambala allow for the possibility of not only studying the effects of nutrients on schistosomiasis, but of applying management protocols based on his work to actually minimize disease risk, thus providing tangible and essential results for all stakeholders involved. I plan to support John’s work both as an advisor on project design and by providing essential equipment and facilities here in Colorado. I am a disease ecologist with broad experience working on aquatic pathogens both in the field and in the lab, including those found in humans, fishes, amphibians, crayfish and snails. I intend to work closely with John in designing the field survey program in Zambia and in implementing experimental research here in Colorado. Our lab has worked directly with Schistosoma mansoni, and we are currently renovating one of our lab spaces into a Biosafety-Level 2 facility to work

Dr. Pieter Johnson Department of Ecology and Evolutionary Biology

Ramaley N122, Campus Box 334 Boulder, Colorado 80309-0334 303-492-8981, Fax: 303-492-8699 www.colorado.edu/eeb/facultysites/pieter www.aquaticparasites.org with parasites such as schistosomes. In addition, our primary laboratory is well equipped to work with parasitological samples, including 3 Olympus SZX10 stereodissecting scopes and 2 compound microscopes with fluorescence and DIC capacity (Olympus BX51 and BX41). We also have four temperature control rooms and a mesocosm facility that will be ideally suited for the proposed experiments evaluating the influence of nutrient chemistry on snail infections. I will also provide mentoring activities designed to enhance John’s career development. The University of Colorado at Boulder provides a rich atmosphere for mentoring post-doctoral scholars in research and educational activities. To accommodate the John’s specific career goals, an adaptive mentoring strategy will be implemented with the goal of strengthening his skills in ecology, data analysis, experimentation, manuscript preparation, grantsmanship, teaching and outreach. Building from materials developed through my Professional Development course, I will also meet regularly with John to cover career-building topics, including authorship and research ethics, developing successful grant proposals, career pathway alternatives, the interview process, and work-life balance. Outside of the laboratory, John will be given extensive opportunities to build his teaching portfolio, including guest lectures in undergraduate and graduate courses, and development of a 3-credit disease ecology course, and the mentoring of undergraduate and graduate students. John has already excelled in this area and I strongly support his interest “teaching as research” community with CU's Teaching Institute for Graduate Education Research (TIGER). Beyond the classroom, John and I will also work to develop disease-research educational modules that emphasize the influential and cost-effective role of ecology in disease management. I am excited to add my knowledge of disease ecology to the already assembled expertise John has brought to this proposal, including Dr. James Mwansa (Zambia's governmental schistosomiasis treatment program) and Vincent Lilanda (chief public health office – Nakambala Sugar Estates). This project carries a unique combination of ecology, human health, and industry-related expertise that makes it extremely applicable to developing countries such as Zambia. This work will be a timely addition to emerging knowledge that will help ensure sustainable development in rapidly developing countries where schistosomiasis is endemic.

Sincerely yours,

Pieter Johnson, PhD Associate Professor David and Lucile Packard Fellow University of Colorado

Pieter T. J. Johnson Ecology and Evolutionary Biology 303.492.5623 (phone)Ramaley N122, Campus Box 334 303.492.8699 (fax)University of Colorado [email protected], Colorado 80309-0334 www.colorado.edu/eeb/facultysites/pieter (a) Professional Preparation 1998 Stanford University: B.S. with Honors and Distinction, Biological Sciences2006 University of Wisconsin, Madison: Ph.D. in Zoology with minor in Ecological Statistics

(b) Appointments 2007-2013 Associate Professor, University of Colorado2008-2013 Packard Fellowship, David and Lucile Packard Foundation2007-2013 Assistant Professor, University of Colorado2005-2006 Postdoctoral Fellow, Center for Limnology2002-2005 National Science Foundation Graduate Research Fellowship2000-2002 University Prize Fellowship, University of Wisconsin, Madison1998-2000 Research Associate, Claremont McKenna College, 1998-2000

(c) Products Five Publications Related to Current Proposal (*with student coauthors)(PDFs and full list of publications available at: http://www.colorado.edu/eeb/facultysites/pieter)

Johnson, P. T. J., Lund*, P. J., Hartson*, R. B., and T. P. Yoshino (2009). Community diversity reduces Schistosoma mansoni transmission and human infection risk. Proceedings of the Royal Society of London, Series B 276: 1657-1663.

Johnson, P. T. J., Townsend, A. R., Cleveland, C. C., Glibert, P. M., Howarth, R. W., McKenzie, V. J., Rejmankova, E. and M. Ward (2010). Linking environmental nutrient enrichment and disease emergence in humans and wildlife. Ecol. Appl. 20: 16-29.

Johnson, P. T. J. and S. Paull (2011). The ecology of disease emergence in fresh waters. Freshwater Biology 56: 638-657.

Johnson, P. T. J., Chase, J. M., Dosch, K. L., Gross, J., Hartson, R. B., Larson, D., Sutherland, D. R. and S. R. Carpenter (2007). Aquatic eutrophication promotes pathogenic infection in amphibians. Proc. Nat. Acad. Sci. 104: 15781-15786.

Joseph*, M. B., Mihaljevic*, J. R., Arellano*, A. L., Kueneman*, J. G., Preston*, D. L., Cross, P. C. and P. T. J. Johnson (2013). Taming wildlife disease: bridging the gap between science and management. Journal of Applied Ecology 50: 702-712.

Five Additional PapersJohnson, P. T. J., Preston*, D. L., Hoverman, J. T., and K. L. D. Richgels* (2013). Biodiversity

reduces disease through predictable changes in host community competence. Nature 494: 230-234.

Johnson, P. T. J. and J. T. Hoverman (2012). Parasite diversity and coinfection drive pathogen infection success and host fitness. Proceedings of the National Academy of Sciences 109: 9006-9011.

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http://www.colorado.edu/eeb/facultysites/pieter

http://www.colorado.edu/eeb/facultysites/pieter

Johnson, P. T. J., Rohr, J. R., Hoverman, J. T., Kellermanns, E., Bowerman, J. and K. B. Lunde* (2012). Living fast and dying of infection: host life history drives interspecific variation in infection and disease risk. Ecology Letters 15: 235-242.

Johnson, P. T. J., Dobson, A., Lafferty, K. D., Marcogliese, D., Memmott, J., Orlofske, S., Poulin, R., and D. W. Thieltges (2010). When parasites become prey: ecological and epidemiological significance. Trends in Ecology & Evolution 25: 362-371.

Hoverman, J. T., Mihaljevic*, J. R., Richgels*, K. L. D., Kerby, J. L. and P. T. J. Johnson (2012). Widespread co-occurrence of virulent pathogens within California amphibian communities. EcoHealth 9: 288-292.

(d) Synergistic Activities • Organizer, “Disease emergence and amphibian decline: using ecology to understand

patterns and promote restoration”, Ecological Society of America Annual Meeting, 2007• Co-Organizer, “Towards a general theory for how climate change will affect infectious

disease”, Ecological Society of America Annual Meeting, 2010; Organizer of symposium for annual American Society of Parasitologists meeting 2010, Colorado Springs, Colorado: “Ecology of Amphibian Helminths” involving 15 participants.

• Institutional Animal Use and Care Committee, University of Colorado• Mentor, Undergraduate Research Programs (BURST, UROP, SURE, SMART, MASP,

REU), University of Colorado (35 students since 2006)

(e) Collaborators and other Affiliations Collaborators in the past 48 months (other than those listed in above publications): Jay Bowerman (Sunriver Nature Center), Andrew Blaustein (Oregon State University), Cherie Briggs (UC Santa Barbara), Cory Cleveland (Montana State University), Rob Dillon Jr. (College of Charleston), Andrew Dobson (Princeton University), Ken Forshay (Environmental Protection Agency), Frank Gleason (University of Sydney), Pat Glibert (University of Maryland), Jackson Gross (Louisiana State University), Robert Howarth (Cornell University), Anthony Ives (University of Wisconsin), Lee Kats (Pepperdine College), Jake Kerby (University of South Dakota), Marm Kilpatrick (UC Santa Cruz), Armand Kuris (UC Santa Barbara), Kevin Lafferty (USGS), David Marcogliese (Environment Canada), Valerie McKenzie (University of Colorado), Julian Olden (University of Washington), Robert Poulin (University of Otago), Thomas Raffel (University of South Florida), Jason Rohr (University of South Florida), Jeff Shields (VIMS), Chris Solomon (University of Wisconsin), David Thieltges (University of Otago), Alan Townsend (University of Colorado).

Graduate Advisor: Stephen R. Carpenter, University of WisconsinPostdoctoral Advisor: M. Jake Vander Zanden, University of Wisconsin

Thesis Advisor and Postgraduate Sponsor: Sara Paull, Daniel Preston, Sarah Orlofske, Katherine Richgels, Max Joseph, Joseph Mihaljevic, Ian Buller, Travis McDevitt-Giles, Brett Goodman, Jason Hoverman, Chelsea Wood, Bethany Hoye, Kim Medley, Sarah Haas

TOTAL = 14

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BIOGRAPHICAL SKETCH : Dr. James C.L.Mwansa

(a) Professional Preparations-PPD Laboratory Services CDC/NIH (USA) Lusaka, Zambia; Certificate – 2010; Good Clinical Laboratory Practice-WHO Geneva; Certificate of Attendance - 1987; Immunology and Biotechnology of Infectious Diseases-Manchester University (UK); PhD. 1986, Medical Microbiology, (Antibiotic Resistance in Salmonella and Shigella

in Manchester area)-Manchester University (UK); Dp. Bact. (Post Graduate Diploma in Bacteriology) 1982; Medical Bacteriology-University of Zambia; BSc (Human Biology) 1976; Medical Microbiology, Pathology, Parasitology, Anatomy,

Physiology, Clinical Chemistry

(b) Appointments1990 -2013 Honorary Lecturer, University of Zambia and have supervised undergraduate and post graduate research projects in Infectious diseases at BSc., Masters and PhD levels.1990 –2013 consultant Medical Microbiologist and Honorary Lecturer University Teaching Hospital 1987- 1989 Medical Microbiologist and Head of Laboratories, Tropical Diseases Research Centre, Ndola- Zambia1980-1981 Elective Warwick Pathology Laboratory-UK Medical Microbiology1978-1980 Laboratory scientist University Teaching Hospital ,Lusaka-Zambia.

(c) ProductsLodh N, Mwansa JC, Mutengo MM, Shiff CJ. Diagnosis of Schistosoma mansoni without the stool: comparison of three diagnostic tests to detect Schiostosoma mansoni infection from filtered urine in Zambia. Am J Trop Med Hyg. 2013 Jul;89(1):46-50. doi: 10.4269/ajtmh.13-0104. Epub 2013 May 28.

Ngandwe Kalungwana, David Mwakazanga, James Mwansa, Mable Mwale Mutengo, Seter Siziya (2012) Pervalence and factors associated with Schistosomiasis in Ng’ombe Township of Lusaka urban district. Journal of Agricultural and Biomedical Sciences 1(1):7-11 ISSN 2226-6410

Victor Mwanakasale, Seter Siziya, James Mwansa, Artemis Koukounari, Alan Fenwick (2009), Impact of iron supplementation on schistosomiasis control in Zambian school children in a highly endemic area, Malawi Medical Journal; 21(1):12 - 18 March 2009

A.Fenwick, J.P. Webster, E.Bosque-Olivia, L.Blair, F.M.Fleming, Y. Zhang, A. Ghaba, J.R. Stothard, A.F. Gabrielli, A.C.A. Clements, N.B. Kabateriene, S.Toure, R. Dembele, U Nyandindi, J Mwansa, and A Koukounari (2009) The Schistosomiasis Control Initiative (SCI) ; Rationale, development and Implementation from 2002 -2008 Parasitology (2009), 136, 1719-1730

Mable M. Mutengo, Victor Mudenda, James C. Mwansa, Kennedy Kaonga, Sandie Sianongo, Helen I. Wamulume and Cecilia J. Shinondo. (2009)Presence of Schistosomiasis in Genital Biopsies from patients at the University Teaching Hospital in Lusaka, Zambia. Medical Journal of Zambia, 3:114-118

Kachimba J.S. and Mwansa J.C.L. (2008). Dealing with Zambia's bilharzia burden. Medical Journal of Zambia. 34(4); 139 - 140.

Narcis B. Kabatereine, Fiona Fleming, Ursuline Nyandindi, James Mwansa and Lynsney Blair (2006). The control of Schistosomiasis and soil transmitted helminths in East Africa. Trends in Parasitology 22 (7): 332-339

(d) Synergistic ActivitiesPresently working closely in collaboration with the School of Veterinary Medicine to define the epidemiology and biology of the epidemic infectious and zoonotic disease.

In 2010 to 2011 I have been WHO consultant for developing Neglected Tropical Disease National Master Plans for Kenya, Malawi, Zimbabwe, and Zambia.

2000-2009 : National Schistosomiasis and Soil Transmitted Helminths Control Programme Chairperson

In 2009 trained as WHO consultant on International Health Regulations (IHR) Capacity Country Assessment. Has been a WHO advisor to develop Core capacities for IHR in Eritrea, and Zambia

2005: I was instrumental in introducing the NTD control programme in Zambia in particular Mass drug administration (MDA) for Schistosomiasis and Soil transmitted helminths in conjunction with School Health and Nutrition Programme in Zambia including training of trainers.

(e) Collaborators and Other AffiliationsCollaborators and Co-EditorsJames Chipeta, University of ZambiaAlan Fenwick, Imperial College LondonJohn S Kachimba, University Teaching Hospital ZambiaNgandwe Kalungwana, University of ZambiaDavid Mwakazanga, Tropical Diseases Research Center, Ndola, ZambiaVictor Mwanakasale, Ndola Central HospitalSeter Siziya, the Copperbelt University

Graduate Advisors and Postdoctoral Sponsorsnone listed

Thesis Advisor and Postgraduate-Scholar SponsorMable Mwale-Mutengo, Ph.D., University of Zambia

RESEARCH Schistosomiasis in Zambia

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