Chapter 1: Introduction and Literature Review; Chapter 2: A retrospective study of anthrax on the Ghaap plateau, Northern Cape Province of South Africa, with special reference to the 2007 - 2008 outbreaks; Chapter 3: Insights gained from sample diagnostics during anthrax outbreaks in the Kruger National Park, South Africa; Chapter 4: Through the lens: a microscopic and molecular evaluation of archival blood smears from the 2010 anthrax outbreaks in Kruger National Park, South Africa; Chapter 5: A distribution snapshot of anthrax in South Africa: multiple locus variable number of tandem repeats analyses of Bacillus anthracis isolates from epizootics spanning 4 decades across southern Africa; Chapter 6: Isolation and Whole Genome Analysis of a Lytic Bacteriophage Infected Bacillus anthracis Isolate from Pafuri, South Africa; Chapter 7: Characterisation of temperate bacteriophages infecting Bacillus cereus sensu stricto group in the anthrax endemic regions of South Africa; Chapter 8: General discussion, conclusions and recommendations. / Thesis (PhD)--University of Pretoria, 2016. / German Research Foundation (DFG) / National Research Foundation (NRF) of South Africa. / Veterinary Tropical Diseases / PhD / Unrestricted

Striped skunks (Mephitis mephitis) are distributed across much of North America with variable habitat preferences and behaviors recorded throughout their range. Striped skunks also readily adapt to human activity and act as hosts to many common pathogens and parasites that infect other wildlife, domestic animals, and humans. Despite how common striped skunks are in both anthropogenic and natural landscapes, few studies have investigated the influence of human activity on striped skunk ecology, and regional studies of the species in the lower Midwest are uncommon. I captured, radiocollared, and tracked striped skunks in southern Illinois during April 2018-August 2021. I used these data along with camera trap photos, necropsies, and spatial data layers in a GIS to quantify winter denning behavior, pathogen prevalence, habitat selection, and survival. Individual striped skunks used 3-21 unique dens during a single winter and denned consecutively in 1 location for 2-59 days. Three striped skunks participated in communal denning, and ≤3 striped skunks were observed at a den concurrently. Eleven mammalian species were observed at striped skunk dens, and the presence of a striped skunk at a den was positively associated with the presence of Virginia opossums (Didelphis virginiana). Human modification had no significant effect on the number of dens used by a striped skunk, but human modification, distance to stream/shoreline, and mean daily temperature had significant negative effects on striped skunk denning duration. Winter denning behavior of striped skunks in southern Illinois followed the latitudinal gradient of behavior across North America, and dens are a shared resource where direct and indirect intraspecific and interspecific interactions occur. No striped skunks tested positive for canine parvovirus (CPV) or Toxoplasma gondii, 55 striped skunks (83.33%) tested positive for Babesia microti, 24 striped skunks (28.6%) tested positive for Leptospira spp., and 5 striped skunks (6%) tested positive for canine distemper virus (CDV). As distance to permanent water increased, so did probability of infection with Leptospira spp. and CDV, which may be due to pathogen persistence in temporary water sources. No other spatial or temporal covariates affected pathogen presence indicating that pathogen transmission via striped skunks is equally likely across the urban-rural gradient. However, the high prevalence of B. microti indicates further study of vectors is needed in the area. I radiotracked 41 (20 F, 21 M) striped skunks and estimated 3,255 locations (x ̅ per individual =79 ± 43 locations; SD) for analyses of home ranges and habitat selection. Annual home ranges varied in size from 14.2-1196.0 ha (x ̅ =270.5 ±257.1 ha) and annual core areas ranged from 2.7-201.1 ha (x ̅ =55.0 ±48.5 ha). Male home ranges and core areas were larger than those of females (Home range: W =86, P <0.001, Core area: W =85, P <0.001) but did not differ by season (Home range: F3,43 =1.2, P =0.317, Core area: F3,43 =1.3, P =0.276). At the second order of habitat selection, striped skunks preferred developed, grassland/pasture, and forest cover types, areas with less canopy cover, areas with moderate levels of human modification, and spaces closer to permanent water and roads. At the third order of habitat selection, selection by individuals was significant but was so variable that trends were difficult to identify. Although striped skunk preferences are expected to differ across their geographic range, my study indicates striped skunk home ranges and habitat selection within one region can vary drastically, making it difficult to elucidate trends and further reinforcing striped skunks as a quintessential generalist species. I radiocollared 63 striped skunks and tracked them for 6,636 radiodays (x ̅ per individual =105 ± 11 days; SE) for survival analysis. Fifty-seven percent of individuals in my study had unknown fates and 43% were found deceased. I attributed 8% of mortalities to predation, 25% to vehicle collisions, 33% to disease or poor body condition, and 33% to unknown causes. Disease or poor body condition and vehicle collisions are top causes of mortality for striped skunks in other populations. I used the null model to estimate a monthly survival rate of 0.91 (95% CI: 0.87-0.94) and annual survival rate of 0.32 (95% CI: 0.20-0.48). This estimated annual survival rate is similar to reports from other stable striped skunk populations, so I expect it represents a stable striped skunk population in southern Illinois. Overall, my study highlighted variability in striped skunk preferences and behavior across an urban-rural gradient and discussed pathogen transmission implications of this variability.

Emerging wildlife diseases represent a serious threat to conservation efforts. Impacts of emerging multi-host pathogens can vary greatly among species as well as geographically, and understanding which populations will be at greatest risk is essential for conserving biodiversity. Snake fungal disease (SFD), caused by the fungal pathogen Ophidiomyces ophidiicola, is responsible for lethal infections in snakes and has contributed to the decline of multiple North American snake populations. However, which species are most affected by this disease and how infections vary regionally remains unknown. Here we sampled 44 different species across 14 sites throughout the Southeastern and Mid-Atlantic United States. We found a strong effect of latitude on both pathogen prevalence and severity, with more severe infections at more northern latitudes. We also found high variability in pathogen prevalence and infection severity among species. There was a strong positive relationship between pathogen prevalence and disease severity, suggesting that SFD is not just highly prevalent in some populations but also highly virulent. More broadly, our results support that SFD likely has continued impacts on snake populations with some species experiencing greater disease than others attributed to spatial and host variation. / Master of Science / Conserving biodiversity is a significant challenge. Wildlife species are under multiple threats including habitat loss, changing climate, species introductions, pollution, and infectious diseases. Emerging wildlife diseases can pose a major problem for wildlife as they often go undetected until they cause substantial declines for the affected species, sometimes leading to population extirpations and extinction events. Snake fungal disease (SFD) is an emerging disease caused by the fungal pathogen Ophidiomyces ophidiicola, which has contributed to the decline of some North American snake populations. However, little is known about differences in infection, transmission, and host responses to SFD in a broader community context. To investigate the dynamics of this pathogen, we collected swab samples from 44 species from a total of 14 sites in New Jersey, Virginia, North Carolina, South Carolina, Georgia, Florida, and Louisiana. We sampled individual snakes to examine variation over a geographic gradient and among species. We found high variability among sites with more severe disease at northern sites. There was also high variability among species and some populations experienced both high pathogen prevalence and disease severity. Our results show that SFD is highly variable within snake communities and may still be causing population level effects.

Emerging infectious diseases of wildlife have threatened host populations of diverse taxa in recent history, which is largely attributable to anthropogenic global change. In three data chapters, this dissertation examines the drivers of individual- to population-level variation in how host populations respond to novel and emerging pathogens. Each chapter explores these processes in bat populations of North America, predominantly the Northeast and Midwest regions of the United States, impacted by the emerging fungal pathogen that causes white-nose syndrome, Pseudogymnoascus destructans. In Chapter 2, I disentangle the effects of adaptive host traits and environmental influences in driving host population stabilization of the little brown bat (Myotis lucifugus), finding that host-pathogen coexistence in this system is the product of their complex interaction. In Chapter 3, I characterize the range-wide variation in white-nose syndrome impacts on a federally endangered and poorly studied species, the Indiana bat (Myotis sodalis), as well as environmental and demographic determinants of its declines over epidemic time. In Chapter 4, I explore the role of individual variation in roosting microclimate selection of little brown bats in driving their infection severity, yielding important insights into the pathophysiology and environmental dependence of white-nose syndrome. Ultimately, this dissertation characterizes complex drivers of variation in host responses to emerging and invading pathogens, yielding insights essential to the successful mitigation of their impacts. / Doctor of Philosophy / In the same way that Covid-19 swept through our global human population in the year 2020, novel infectious diseases have threatened wildlife populations, sometimes to the point of extinction. Often, however, the processes driving the impacts of novel infectious diseases in wildlife are unknown, despite being important information to protect susceptible populations. In this dissertation, I explore how North American bat populations have been impacted by a recently emerged disease, white-nose syndrome, and what processes cause variation in how individual bats and bat colonies have responded to the disease. In Chapter 2, I explore how the little brown bat (Myotis lucifugus) has evolved to co-exist with its new pathogen and how this coexistence is affected by environmental conditions like temperature and humidity. In Chapter 3, I characterize variation in how populations of the Indiana bat (Myotis sodalis) have responded to white-nose syndrome and how environmental and demographic conditions have affected declines since the disease first emerged. In Chapter 4, I explore how the temperatures used by little brown bats during hibernation affect the severity of their infection, giving us important information on how bats survive with white-nose syndrome and the role of temperature. Altogether, the research in this dissertation describes complex interactions between hosts, pathogens, and their environment in driving the patterns we observe after the emergence of novel infectious diseases.

We live in an era of global change, where emerging infectious diseases such as Ebola, Zika, bird flu, and white nose syndrome are affecting humans, wildlife, and domesticated species at an increasing rate. To understand and predict the dynamic spread of these infectious agents and other symbionts through host populations and communities, we need dynamic mathematical models that accurately portray host-symbiont transmission. But transmission is an inherently difficult process to measure or study, because it is actually a series of interacting processes influenced by abiotic and biotic factors at multiple scales, and thus empirical tests of the transmission function within epidemiological models are rare. Therefore, in this dissertation, I explore factors at the individual, population, and community-levels that influence host contact rates or symbiont transmission success in a common snail-symbiont system, providing a detailed description of the multi-faceted nature of symbiont transmission. From a review of the ecological literature, I found that most models assume that transmission is a linear function of host population density, whereas most empirical studies describe transmission as a nonlinear function of density. I then quantified the net nonlinear transmission-density relationship in a system where ectosymbiotic oligochaetes are directly transmitted among snail hosts, and I explored the ecological mechanisms underlying the nonlinear transmission-density relationship observed in the field via intraspecific transmission success and contact rate experiments in the laboratory. I found that the field results could be explained by heterogeneity in transmission success among snails with different characteristics and nonlinear contact-density relationships caused by non-instantaneous handling times. After I 'unpacked'population-level transmission dynamics into those individual-level mechanistic processes, I used this same approach to examine higher-level ecological organization by describing the mechanistic underpinnings of interspecific or community-level transmission in the same snail-symbiont system. I found that low interspecific transmission rates in the field were the product of opposing interactions between high population densities, high prevalences of infection, and very low interspecific transmission success caused by strong symbiont preferences for their current host species. Unpacking transmission in this way resulted in one of the most detailed empirical studies of transmission dynamics in a wildlife system, and yielded many surprising new insights in symbiont ecology that would not have been discovered with a purely phenomenological or holistic view of transmission. Though simple, linear, and holistic epidemiological models will always be important tools in disease ecology, 'unpacking'transmission rates and adding heterogeneity and nonlinearity to models, as I have done here, will become increasingly important as we work to maximize model prediction accuracy in this era of increased disease emergence. / Ph. D.

Lyme borreliosis (LB) is the most frequent vectorborne disease in the Northern Hemisphere. It is a complex bacterial zoonosis involving vertebrate hosts and hard ticks of the genus Ixodes. The causative agents, bacteria of the LB group of spirochaetes, form a species complex comprising 17 named species. As is the case for most microbial pathogens, epidemiological and ecological studies require appropriate genotyping. Although the use of single loci may provide rapid results, there are serious disadvantages, in particular when inferring evolutionary relationships or geographic population structure. A novel multilocus sequence analysis (MLSA) system of the LB group spirochaetes has been developed based on housekeeping genes to overcome these problems. Here, the system is optimized and tested using extracted spirochaetal DNA directly from ticks and then utilized to obtain insights into the migration and spread of individual species as well as to investigate the evolutionary origins of the species complex. Species belonging to the LB group of spirochetes display different patterns and levels of host specialisation which makes this an ideal system to study the impact of host associations on spread of zoonotic tickborne diseases. For example, Borrelia valaisiana and B. garinii are transmitted exclusively by birds while B. afzelii is transmitted by rodents. I demonstrate that the migration of the LB species is dependent on, and limited by, the migration of their respective hosts. I also show the presence of B. afzelii strains in England and, through the use of the MLSA scheme, demonstrate that the strains are highly structured. A close evolutionary relationship between B. afzelii and its rodent host species is shown. Furthermore, through phylogenetic analyses, some evidence of a coevolutionary relationship between the LB group species and their major group of vector species, the Ixodes persulcatus species complex, is presented and a Eurasian origin for the species group is suggested.

The inadvertent spread of exotic pests and pathogens has resulted in devastating losses for bees. The vast majority of bee disease research has focused on a single species of managed bee, the European honey bee (Apis mellifera). More recently, pathogen spillover from managed bees is implicated in the decline of several bumble bee species (Bombus spp.) demonstrating a need to better understand the mechanisms driving disease prevalence in bees, transmission routes, and spillover events. RNA viruses, once considered specific to honey bees, are suspected of spilling over from managed honey bees into wild bumble bee populations. To test this, I collected bees and flowers in the field from areas with and without honey bee apiaries nearby. Prevalence of deformed wing virus (DWV) and black queen cell virus (BQCV) as well as replicating DWV infections in Bombus vagans and B. bimaculatus were highest in bumble bees collected near honey bee apiaries (χ 12 < 6.531, P < 0.05). My results suggest that honey bees are significant contributors of viruses to bumble bees. Flowers have been suspected as bridges in virus transmission among bees. I detected bee viruses on 18% of the flowers collected within honey bee apiaries and detected no virus on flowers in areas without apiaries, thus providing evidence that viruses are transmitted at flowers from infected honey bees. In controlled experiments using captive colonies in flight cages, I found that honey bees leave viruses on flowers but not equally across plant species. My results suggest that there are differences in virus ecology mediated by floral morphology and/or pollinator behavior. No bumble bees became infected in controlled experiments, indicating that virus transmission through plants is a rare event that is likely to require repeated exposure. The few studies examining viruses in bumble bees are generally limited to virus detection, resulting in little understanding of the conditions affecting virus titers. In honeybees, infections may remain latent, capable of replicating under certain conditions, such as immunosuppression induced by pesticide exposure. I tested whether exposure to imidacloprid, a neonicotinoid pesticide, affects virus titers in bumble bees. In previous honey bee studies, imidacloprid exposure increased virus titers. In contrast, I found that bumble bee exposure to imidacloprid decreased BQCV and DWV titers (χ42 < 20.873, p < 0.02). My findings suggest that virus-pesticide interactions are species-specific and results from honey bee studies should not be generalized across other bee species. Having found that honey bees are significant contributors of viruses to wild bees and flowers, I investigated how honey bee management practices affect disease spread and developed recommendations and tools to lesson the risk of spillover events. Honey bee disease may be exacerbated by migratory beekeeping which increases stress and opportunities for disease transmission. I experimentally tested whether migratory conditions contribute to disease spread in honey bees and found negative yet varying effects on bees suggesting that the effects of migratory practices may be ameliorated with rest time between pollination events. State apiary inspection programs are critical to controlling disease spread and reducing the risk of spillover. However, these programs are often resource constrained. I developed and deployed a toolkit that enables state programs to prioritize inspections and provide a platform for beekeeper education. Using novel data collected in Vermont, I discovered several promising avenues for future research and provided realistic recommendations to improve bee health.

In this dissertation I evaluated the ecological and evolutionary mechanisms that promote the spread of non-native parasites infecting novel hosts under contemporary and future climate conditions. Thorough assessment of the impact of introduced parasites and an understanding of the potential effects of climate change on parasite distributions and densities will promote effective conservation of native aquatic biodiversity. The spread of an introduced nematode parasite, Camallanus cotti, infecting the native Hawaiian stream fish, Awaous stamineus, across the Hawaiian Islands provided an opportunity to examine how biotic (densities of introduced & native hosts, individual host traits, genetic diversity) and environmental (land-use, water chemistry) factors promote novel host-parasite interactions. In addition to completing archipelago-wide surveys of parasite distributions and densities in native fish hosts, I characterized geographic patterns of genetic variation in C. cotti to assess gene flow, identify likely conduits of introduction and spread of the parasite across the archipelago. Finally, I utilized a natural precipitation gradient across the Hamakua coast on the island of Hawai`i, as a natural analog to conditions predicted by climate change, to assess the relationship between precipitation and infection of A. stamineus by C. cotti. I found the distribution C. cotti has become decoupled from that of the non-native hosts and that the parasite infects native fishes in remote, relatively pristine watersheds. The abundance, intensity, and prevalence of C. cotti infecting A. stamineus are influenced by a suite of factors, but notably parasitism increases with decreasing precipitation. This finding suggest that infection of native Hawaiian fishes by introduced parasites will increase if climate conditions change as expected. Genetic analysis indicates that C. cotti has spread across the archipelago following an initial introduction on O'ahu with subsequent dispersal to Maui and then underwent stepwise dispersal to other islands in the archipelago. Significant genetic structure also was detected across islands, suggesting that dispersal potential is constrained, which in turn suggests that remediation efforts focusing on invasion hotspots or areas of concern could be effective at reducing parasites loads in native fishes. / acase@tulane.edu

World-wide amphibian declines sparked concern and encouraged investigation into potential causes beginning in the 1980’s. Infectious disease has been identified as one of the major potential contributors to amphibian declines. For example, Ranavirus has caused amphibian die-offs throughout the United States. Investigators isolated Ranavirus from dead or moribund amphibians during large-scale die-offs of amphibians in the Cades Cove area of Great Smoky Mountains National Park in 1999-2001. In 2009, after nearly a decade without follow-up monitoring, I undertook an investigation to determine if the virus persisted in the area, and if so, to assess spatial, temporal, and taxonomic patterns in prevalence. Three amphibian breeding ponds, including Gourley Pond, the site of these earlier mortality events, were monitored for Ranavirus during the 2009 amphibian breeding season. A peak in prevalence occurred at Gourley Pond corresponding to a massive amphibian die-off. Prevalence varied among three different taxonomic groups during this mortality event with the highest prevalence, 84%, detected in larval Ambystomatids, 44.4% prevalence in adult Newts, and no virus detected in adult Plethodontids. I did not detect virus at either of the other two breeding ponds despite equivalent sampling effort, similar community composition, and close proximity to Gourley Pond. These results suggest that the severity and spatial extent of Ranavirus in Cades Cove remains unchanged since its initial detection a decade ago. Also, despite the observed massive die-offs there is no evidence of local amphibian extinction at Gourley Pond.

<p>Fungal pathogens likely play an important role in regulating populations of tree seedlings and preserving forest diversity, due to their ubiquitous presence and differential effects on survival. Host-specific mortality from natural enemies is one of the most widely tested hypotheses in community ecology to explain the high biodiversity of forests. The effects of fungal pathogens on seedling survival are usually discussed under the framework of the Janzen-Connell (JC) hypothesis, which posits that seedlings are more likely to survive when dispersed far from the parent tree or at low densities due to pressure from host-specific pathogens (Janzen 1970, Connell 1971). One of the key challenges to assessing the importance of JC effects has been to identify and quantify the effects of the large numbers of potential pathogens required to maintain host diversity. The primary objectives of this research were to (1) characterize the fungi associated with seedling disease and mortality for a number of important southeastern US forest tree species; and (2) determine if these associations are consistent with the Janzen-Connell hypothesis in terms of differential effects on seedling survival.<br></p><p>Culture-based methods and ribosomal DNA (rDNA) sequencing were used to characterize the fungal community in recently dead and live seedlings of thirteen common tree species in a temperate mixed hardwood forest (North Carolina, USA), with the goal of identifying putative seedling pathogens. Cultures were initially classified and grouped into 130 operational taxonomic units (OTUs) using 96% internal transcribed spacer (ITS) sequence similarity; 46% of all OTUs were found only once. Using rarefaction, it was concluded that the richness of the system was not fully sampled and likely included over 200 taxa (based on non-parametric richness estimators). Species richness did not differ between sampling sites or among the five most common hosts sampled. The large ribosomal subunit (LSU) region of rDNA was then sequenced for representative samples of common OTUs and refined identifications using a constrained maximum likelihood phylogenetic analysis. Phylogenetic placement verified strong BLAST classifications, and allowed for placement of unknown taxa to the order level, with many of these unknowns placed in the Leotiomycetes and Xylariales (Sordariomycetes).<br> </p><p>Next, a hierarchical Bayesian model was developed to predict the effects of multiple putative fungal pathogens on individual seedling survival, without forcing the effects of multiple fungi to be additive. The process of disease was partitioned into a chain of events including incidence, infection, detection, and survival, and conditional probabilities were used to quantify each component individually, but in the context of one another. The use of this modeling approach was illustrated by examining the effects of two putative fungal pathogens, <italics>Colletotrichum acutatum</italics> and <italics>Cylindrocarpon</italics> sp. A, an undescribed species of <italics>Cylindrocarpon</italics>, on the survival of five seedling hosts in both a maximum likelihood and Bayesian framework.<br> </p><p>Finally, the model was used to assess the impacts of these fungi on seedling survival, alone and in combination, using data on five potential fungal pathogens and five hosts. Multi-host fungi had differential effects on seedling survival depending on host identity, and multiple infections may impact survival even when single infections do not. Evaluating these interactions among multiple plant and fungal species generates a set of targeted hypotheses of specific plant-fungal combinations that could help us better understand pathogen-driven diversity maintenance at larger scales than previously possible. Building on these results, some recommendations are provided as to how the Janzen-Connell hypothesis can be re-evaluated with respect to host specificity, pathogen distribution, and environmental context.</p> / Dissertation