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Disposal of greywater is a neglected challenge facing rapidly growing human populations. Here, we define greywater as wastewater that originates from household activities (e.g., washing dishes, bathing, and laundry) but excludes inputs from the toilet. Pollutants in greywater can include both chemical and biological contaminates that can significantly impact human, animal, and environmental health under certain conditions. We evaluate greywater disposal practices in nonsewered, low-income residential areas in Kasane (264 dwellings/ha), Kazungula (100 du/ha), and Lesoma (99 du/ha) villages in Northern Botswana through household surveys (n = 30 per village). Traditional pit latrines were the dominant form of sanitation (69%, n = 90, 95% CI, 59%–79%) while 14% of households did not have access to onsite sanitation (95% CI 0%–22%). While greywater disposal practices varied across villages, respondents in all sites reported dumping greywater into the pit latrine. Frequency varied significantly across villages with the highest level reported in Kasane, where residential density was greatest (p &lt, 0.014, χ2 = 9.13, 61% (n = 23, 95% CI 41%–81%), Kazungula 41% (n = 22, 95% CI 20%–62%), Lesoma 13% (95% CI 0%–29%). Disposal of greywater in this manner was reported to limit contamination of the household compound and reduce odors, as well as pit latrine fecal levels. Some respondents reported being directed by local health authorities to dispose of greywater in this manner. Environmentally hazardous chemicals were also dumped directly into the pit latrine to reduce odors. With high household to pit latrine ratios particularly in rental properties (4.2 households, SD = 3.32, range = 15 units, average household size 5.3, SD = 4.4), these greywater and pit latrine management approaches can significantly alter hydraulic loading and leaching of chemicals, microorganisms, and parasites. This can dramatically expand the environmental footprint of pit latrines and greywater, increasing pollution of soil, ground, and surface water resources. Challenges in greywater disposal and pit latrines must be addressed with urgency as health behaviors directed at minimizing negative aspects may amplify the environmental impacts of both greywater and pit latrine excreta.

Rift Valley fever (RVF) is endemic in many countries in Sub-Saharan Africa and is responsible for severe outbreaks in livestock characterized by a sudden onset of abortions and high neonatal mortality. During the last decade, several outbreaks have occurred in Southern Africa, with a very limited number of cases reported in Botswana. To date, published information on the occurrence of RVF in wild and domestic animals from Botswana is very scarce and outdated, despite being critical to national and regional disease control. To address this gap, 863 cattle and 150 buffalo sampled at the interface between livestock areas and the Chobe National Park (CNP) and the Okavango Delta (OD) were screened for the presence of RVF virus (RVFV) neutralizing antibodies. Antibodies were detected in 5.7% (n=863), 95% confidence intervals (CI) (4.3–7.5%) of cattle and 12.7% (n=150), 95% CI (7.8–19.5%) of buffalo samples. The overall prevalence was significantly higher (p=0.0016) for buffalo [12.7%] than for cattle [5.7%]. Equally, when comparing RVF seroprevalence in both wildlife areas for all pooled bovid species, it was significantly higher in CNP than in OD (9.5 vs. 4%, respectively; p=0.0004). Our data provide the first evidence of wide circulation of RVFV in both buffalo and cattle populations in Northern Botswana and highlight the need for further epidemiological and ecological investigations on RVF at the wildlife–livestock–human interface in this region. This study was funded by the Food and Agriculture Organization, EMPRES Animal Health Unit, and the Wildlife Health and Ecology Unit, through two Letters of Agreement with CIRAD (PR 43231 and PR 45371).

Disposal of greywater is a neglected challenge facing rapidly growing human populations. Here, we define greywater as wastewater that originates from household activities (e.g., washing dishes, bathing, and laundry) but excludes inputs from the toilet. Pollutants in greywater can include both chemical and biological contaminates that can significantly impact human, animal, and environmental health under certain conditions. We evaluate greywater disposal practices in nonsewered, low-income residential areas in Kasane (264 dwellings/ha), Kazungula (100 du/ha), and Lesoma (99 du/ha) villages in Northern Botswana through household surveys (n = 30 per village). Traditional pit latrines were the dominant form of sanitation (69%, n = 90, 95% CI, 59%–79%) while 14% of households did not have access to onsite sanitation (95% CI 0%–22%). While greywater disposal practices varied across villages, respondents in all sites reported dumping greywater into the pit latrine. Frequency varied significantly across villages with the highest level reported in Kasane, where residential density was greatest (p < 0.014, χ2 = 9.13, 61% (n = 23, 95% CI 41%–81%), Kazungula 41% (n = 22, 95% CI 20%–62%), Lesoma 13% (95% CI 0%–29%). Disposal of greywater in this manner was reported to limit contamination of the household compound and reduce odors, as well as pit latrine fecal levels. Some respondents reported being directed by local health authorities to dispose of greywater in this manner. Environmentally hazardous chemicals were also dumped directly into the pit latrine to reduce odors. With high household to pit latrine ratios particularly in rental properties (4.2 households, SD = 3.32, range = 15 units, average household size 5.3, SD = 4.4), these greywater and pit latrine management approaches can significantly alter hydraulic loading and leaching of chemicals, microorganisms, and parasites. This can dramatically expand the environmental footprint of pit latrines and greywater, increasing pollution of soil, ground, and surface water resources. Challenges in greywater disposal and pit latrines must be addressed with urgency as health behaviors directed at minimizing negative aspects may amplify the environmental impacts of both greywater and pit latrine excreta. Published version

Sustainable management of dryland river systems is often complicated by extreme variability of precipitation in time and space, especially across large catchment areas. Understanding regional water quality changes in southern African dryland rivers and wetland systems is especially important because of their high subsistence value and provision of ecosystem services essential to both public and animal health. We quantified seasonal variation of Escherichia coli (E. coli) and Total Suspended Solids (TSS) in the Chobe River using spatiotemporal and geostatistical modeling of water quality time series data collected along a transect spanning a mosaic of protected, urban, and developing urban land use. We found significant relationships in the dry season between E. coli concentrations and protected land use (p = 0.0009), floodplain habitat (p = 0.016), and fecal counts from elephant (p = 0.017) and other wildlife (p = 0.001). Dry season fecal loading by both elephant (p = 0.029) and other wildlife (p = 0.006) was also an important predictor of early wet season E. coli concentrations. Locations of high E. coli concentrations likewise showed close spatial agreement with estimates of wildlife biomass derived from aerial survey data. In contrast to the dry season, wet season bacterial water quality patterns were associated only with TSS (p

Diarrheal disease is an important health challenge, accounting for the majority of childhood deaths globally. Climate change is expected to increase the global burden of diarrheal disease but little is known regarding climate drivers, particularly in Africa. Using health data from Botswana spanning a 30-year period (1974–2003), we evaluated monthly reports of diarrheal disease among patients presenting to Botswana health facilities and compared this to climatic variables. Diarrheal case incidence presents with a bimodal cyclical pattern with peaks in March (ANOVA p < 0.001) and October (ANOVA p < 0.001) in the wet and dry season, respectively. There is a strong positive autocorrelation (p < 0.001) in the number of reported diarrhea cases at the one-month lag level. Climatic variables (rainfall, minimum temperature, and vapor pressure) predicted seasonal diarrheal with a one-month lag in variables (p < 0.001). Diarrheal case incidence was highest in the dry season after accounting for other variables, exhibiting on average a 20% increase over the yearly mean (p < 0.001). Our analysis suggests that forecasted climate change increases in temperature and decreases in precipitation may increase dry season diarrheal disease incidence with hot, dry conditions starting earlier and lasting longer. Diarrheal disease incidence in the wet season is likely to decline. Our results identify significant health-climate interactions, highlighting the need for an escalated public health focus on controlling diarrheal disease in Botswana. Study findings have application to other arid countries in Africa where diarrheal disease is a persistent public health problem. Published version

Introduction and Justification: Diarrheal disease is a leading cause of death in children in low- and moderate-income countries. Food, food waste, and water are all vehicles that can promote the spread of diarrheal disease-causing bacteria like Campylobacter, Salmonella enterica, and E. coli. Resistance to commonly used antibiotics is on the rise, making them difficult to manage. This study aimed to determine prevalence and antibiotic resistance profiles of Campylobacter, S. enterica, and E. coli isolated from food, food waste, and water samples obtained from the Chobe Region of Botswana. In addition, the survival of two common pathogens, E. coli and C. jejuni, on kale, a type of leafy green commonly consumed raw, was determined. Methods: Samples were collected from the Chobe region of Botswana in 2022 including water from the local river, food (produce, beef, pork, and poultry) from local vendors, and food scraps from the landfill. Food samples were enriched in the appropriate selective media: Brilliant Green Bile Broth for E. coli, Bolton Broth for Campylobacter, and Rappaport Vassiliadis Broth for S. enterica. Water samples were collected using modified USEPA methods1103.1 and 1604, E.coli isolation was performed by plating on RAPID E.coli2 agar and incubation at 37°C for 2h and 44°C for 16-22h. Campylobacter, S. enterica, and E. coli were isolated from meat, poultry, and water samples before being sent to Virginia Tech, while enriched bacterial pellets from the produce were shipped for screening and isolation at Virginia Tech. E.coli were confirmed by PCR detecting the phoA gene (all E. coli), and classified as pathogenic through screening for the eae (present in enterohemorrhagic and enteropathogenic E.coli), stx1 and stx2 (present in enterohemorrhagic E. coli) and est1b ( present in Enterotoxigenic E.coli) genes. Campylobacter isolates were confirmed using a genera-specific PCR while S. enterica isolates were confirmed using invA primers. These enrichment and primer sets were tested as part of a study to determine the survival of E. coli O157:H7 and C. jejuni on kale during a 21-day shelf life. E. coli and S. enterica isolates were subjected to antibiotic resistance testing using the Kirby-Bauer Disk Diffusion method. Results: Methods for detection of inoculated E. coli O157:H7 on kale indicated survival for the majority of the shelf-life (up to 19 d), in comparison, C. jejuni was undetectable by day 13 using enrichment and PCR or plating. From the Botswanan samples, E. coli was isolated from 20% of produce, 49% of meat, and 84.7% of water. Salmonella was only isolated from produce samples (2.4%, 7/294). Resistance was uncommon among the Salmonella isolates with only one isolate being resistant to chloramphenicol. No Campylobacter were isolated from the screened produce, meat, or food waste. E. coli resistant to 3 or more classes of antibiotics (MCR) were identified in 15.5% of produce, and 22.2% of meat isolates. Isolation of E. coli or Salmonella from meat was not associated with a particular food type. In contrast, isolation of E. coli was more common from certain types of vegetables and fruits. Antibiotic-resistant E. coli were isolated more commonly from beef, poultry, and pork than from produce. Multi-class resistant E. coli were isolated from fruits, greens, soil associated, and above ground associated vegetables, beef, and poultry. Water samples were collected from the same time period as the food samples. E. coli isolation, especially pathogens (based on eae presence) was more frequent from environmental water samples collected during the wet season compared to the dry season. Water samples collected during periods of increased rainfall were more likely to contain E. coli isolates, especially pathogens. S. enterica and Diarrheagenic E. coli isolates, especially MCR isolates, pose a significant risk of illness to consumers. Strategies to reduce the circulation of these pathogens in foods and water sources are needed. Master of Science in Life Sciences People can get sick with diarrheal diseases after consuming contaminated food and water. These illnesses are difficult to treat and control when the bacteria causing them are resistant to antibiotics. Campylobacter, Salmonella, and diarrheagenic E. coli are three types of bacteria that can cause illness from food and water. These illnesses disproportionately affect people, especially children, in low-and moderate-income countries like Botswana. Little is known about the prevalence of Campylobacter, Salmonella, and diarrheagenic Escherichia coli in Botswana. This study aimed to determine the prevalence of these microorganisms as well as how resistant they are to different types of antibiotics. Samples from produce, beef, poultry, pork, and recreational water sources were collected in the Chobe region of Botswana over the course of 2022. Food samples were collected from different vendors and food scraps were obtained from the landfill. E. coli and Salmonella were isolated out of meat and water samples in Botswana while produce samples were shipped as mixed cultures to Virginia Tech where E. coli, Salmonella, and Campylobacter isolation or confirmation was done. Once the target bacteria were isolated, their resistance to certain antibiotics was tested. Salmonella was only found in produce from samples collected during October-December. No Campylobacter was found from produce, meat, or food waste. More E. coli was isolated from fruit or vegetable food waste collected from the landfill than from produce bought at local vendors. E. coli was obtained from meat purchased from local vendors more often than samples from the landfill. E. coli was found more often in water when there was more rainfall. E. coli that causes illness was also more likely to be obtained during the wet seasons. Resistant E. coli that could not be killed by the screened antibiotics, were classified as multi-drug resistant when it was resistant to more than three antibiotics. Rainfall, season, and the food source influenced if E. coli isolates were likely to be multi-drug resistant. While more research is needed to determine how these bacteria are moving in the environment and gaining resistance to antibiotics, the findings of this study show they are present in the environment and require further research.

Water is a primary shared resource that connects all species across the landscape and can facilitate shared exposure to a community of waterborne pathogens. Despite remarkable global progress in sanitation and hygiene development in the past two decades, infectious diarrhea remains a prominent public health threat in sub-Saharan Africa. This thesis identifies and discusses persistent challenges limiting the success of current waterborne disease management strategies and several existing research hurdles that continue to impede characterization of microbial transmission and transport. In this work, the Chobe River watershed in Northern Botswana serves as a target study site for the application of hydrological modeling tools to quantify emergent water quality and health challenges in Southern Africa. A watershed model with extensive data requirements, the Hydrological Simulation Program – Fortran (HSPF), is used to identify primary data gaps and model assumptions that limit the progress of model development, and guide opportunities for data collection, tool development, and research direction. Environmental pathogen exposure risk and epidemiological outbreak dynamics are best described by interactions between the coupled human and environmental processes within a system. The challenge of reducing diarrheal disease incidence strengthens a call for research studies and management plans that join multiple disciplines and consider a range of spatiotemporal scales. Master of Science

Free-ranging banded mongooses (Mungos mungo) in northeastern Botswana are infected by a novel Mycobacterium tuberculosis complex pathogen, M. mungi, which putatively infects mongooses through lesions in the skin (often the planum nasale) from an environmental reservoir. To understand the epidemiology of the yearly and highly seasonal outbreaks of M. mungi in this population of banded mongooses, researchers need to understand what factors influence banded mongoose exposure to M. mungi and banded mongoose susceptibility to M. mungi infection. Researchers have no baseline data on the behavioral ecology of this population of banded mongooses - such as home range dynamics, denning ecology, movement ecology, and foraging ecology, all of which may play a role in banded mongoose exposure to M. mungi. Further, researchers have highlighted the potential role of prolonged elevations of glucocorticoids in impairing cell-mediated immunity, which would play a significant role in determining susceptibility to a mycobacterium such as M. mungi, however, researchers have no data on the endocrinology of banded mongooses. Finally, researchers have not detected M. mungi infection in any other population of banded mongooses. Our study population has a gradient of troops (social groups) that vary from troops with extremely close association with humans in a town, to troops associated with humans at tourist lodges within the Chobe National Park, to troops with no discernible association with humans within the national park and surrounding forest reserve. Researchers have few data on how synanthropy (living with humans) affects banded mongoose behavioral ecology and no data on how synanthropy affects banded mongoose endocrinology. Researchers do not know whether or how the high level of synanthropy in this population of banded mongooses plays a role in the epidemiology of M. mungi outbreaks. Thus, we document here some aspects of banded mongoose home range dynamics, movement metrics, denning ecology and foraging behavior for our study population in northeastern Botswana. We present a novel method for screening data from global positioning system (GPS) collars for large measurement error and we present a detailed home range study. We also document the spatio-temporal dynamics of glucocorticoid production among several banded mongoose study troops across our study site, using a non-invasive assay for fecal glucocorticoid metabolites, which we validated and also present here. We tested to see which factors, including nutritional limitation, predation risk, and reproduction (and associated competition, agonistic encounters, and predation), best explained the variation in glucocorticoid production among our study troops over several years. We found that the metrics traditionally used to screen data from GPS collars, horizontal dilution of precision (HDOP) or fix dimension (2-D or 3-D), performed poorly relative to a new screening metric that we propose, the estimated elevation error (EEE). We propose that researchers use our screening method, which combines test data and a model-averaging information-theoretic framework that uses a priori candidate models of telemetry measurement error. Although we recommend including EEE in a priori candidate models, it may not describe telemetry error in other systems as well as it did in our own. Banded mongooses in our study population formed troops of a median of 13 adults (IQR: 11 to 21 adults) and these troops used home ranges of a median of 68 ha (IQR: 39 to 134 ha) with core areas of a median of 15 ha (IQR: 9 to 28 ha). These cores (statistically-clumped space use) occurred at a median volume contour of 66 % (IQR: 58 to 71 %). Synanthropic troops showed more clumped area use than apoanthropic troops (those living away from humans). Synanthropic troops also used man-made structures for den sites in SI{81}{percent} of nights, fed from refuse sites in 13 % of foraging observations, and drank from anthropogenic water sources in 78 % of drinking observations. From our conducted adrenocorticotropic hormone challenge, we detected valid increases in fecal glucocorticoid metabolite concentrations in mongoose feces using our four tested enzyme-immunoassays. An 11-oxoetiocholanolone assay detecting 11,17-dioxoandrostanes (11,17-DOA) performed best. Using this assay, we detected expected decreases in fecal glucocorticoid metabolite concentrations 48 h after administering dexamethasone sodium phosphate. We also validated this assay using biological events as challenges, in which captive mongooses showed higher fecal glucocorticoid metabolite concentrations during reproductive activity, agonistic encounters, and depredation events. The time delay of fecal glucocorticoid metabolite excretion approximately corresponded with food transit time, at a minimum of approximately 24 h. Fecal glucocorticoid metabolite metabolism was minimal up to 8 h post-defecation. Reproduction and its associated challenges dramatically increased glucocorticoid production, which otherwise remained low and stable in a captive troop with a constant food supply and lowered predation risk. Variation in glucocorticoid production in free-ranging banded mongooses was best explained by food limitation as described by current nutritional limitation (proportion of fecal organic matter), recent rainfall (which increases soil macrofauna availability), and access to concentrated anthropogenic food resources. Habitat differences in soil macrofauna density and reproductive events also explained variation in glucocorticoid production in free-ranging mongooses, but to a much lower degree. Predation risk, as measured by canopy cover (escape from aerial predators) and group size (decreased per capita vigilance) explained very little of the variation in glucocorticoid production. In the late dry season, banded mongooses in our population may face a "perfect storm" of nutritional limitation, agonistic encounters at concentrated food resources, aggressive evictions, estrus, competition for mates, parturition, and predation pressure on pups. We suspect that this prefect storm may push glucocorticoid responses into homeostatic overload and may impair cell-mediated immunity in banded mongooses. Ph. D.