Abstract The age of an adult female mosquito is a critical determinant of its ability to transmit a range of human pathogens such as the dengue viruses. Since most pathogens require a relatively long period of development in their mosquito vector before they can be transmitted to a new host, only older insects are of epidemiological importance. It has been proposed that life-shortening strains of the inherited bacterial symbiont Wolbachia may be potentially useful to shift mosquito population age structure towards younger individuals, thereby reducing pathogen transmission without eradicating the mosquito population. However, life-shortening Wolbachia strains do not occur in mosquitoes naturally. To facilitate the evaluation of this potential strategy, the major aim of this project was to transfer a life-shortening Wolbachia strain, wMelPop, from the fruit fly, Drosophila melanogaster, to the dengue vector, Aedes aegypti. After successful transinfection, I then characterized the wMelPop infection in this mosquito species, and the associated phenotypic effects of this Wolbachia strain on several aspects of Ae. aegypti life-history. The horizontal transfer of the obligate intracellular bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. Previous attempts to directly transfer wMelPop from D. melanogaster to Ae. aegypti have been unsuccessful, presumably due to mal-adaptation of wMelPop to this novel mosquito host. In an attempt to adapt wMelPop to the mosquito intracellular environment to facilitate transfer, we serially passaged this infection for 3 years in Aedes cell culture. After long-term serial passage in mosquito cell lines, wMelPop was then initially reintroduced into its native host, D. melanogaster, by embryonic microinjection to examine the phenotypic outcome of this process. The cell line-adapted wMelPop strains were characterized by a loss of infectivity when reintroduced into Drosophila, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility (CI), when compared to the original wMelPop strain. These results were suggestive of adaptation of this Wolbachia strain to the mosquito intracellular environment. Subsequently, we successfully established wMelPop in Ae. aegypti using embryonic microinjection. Two independently transinfected Ae. aegypti lines, PGYP1 and PGYP2, were generated. Analogous to infection in Drosophila, the presence of wMelPop in both of these lines halved adult mosquito lifespan under a range of laboratory conditions. The new association is stable, and wMelPop is maternally inherited with high fidelity across Ae. aegypti life span. It is capable of inducing near perfect CI that does not diminish as male Ae. aegypti age, which should facilitate its invasion into natural field populations and persistence over time. Fecundity assays using young PGYP1 females revealed that wMelPop infection induced minimal costs for reproductive fitness for females during their first gonotrophic cycle. The phenotypic effects of wMelPop on several aspects of Ae. aegypti life-history were additionally characterized. Overall, minor costs of wMelPop infection for pre-imaginal survivorship, development and adult size were observed. However, wMelPop infection decreased the viability of quiescent Ae. aegypti eggs over time. Similarly, the reproductive fitness of wMelPop-infected Ae. aegypti females declined with age. These results revealed a general pattern associated with wMelPop pathogenesis in Ae. aegypti, where host fitness costs incurred by this infection increase during aging of both immature and adult life-history stages. We also discovered that wMelPop-infected Ae. aegypti have a reduced ability to utilise blood from non-human hosts for egg development. Blood feeding by wMelPop-infected females on mouse, guinea pig or chicken hosts resulted in a near complete abolishment of reproductive output associated with both a decline in the numbers of eggs oviposited as well as the hatching rate of successfully laid eggs. In contrast, the reproductive output of wMelPop-infected females fed human blood was normal, with intermediate effects observed with other vertebrate blood sources. Removal of Wolbachia from mosquitoes using antibiotic treatment restored egg development to standard levels on all blood sources. Further blood feeding assays over two reproductive cycles definitively illustrated a nutritional interaction between host blood source and egg development in wMelPop-infected Ae. aegypti. This dramatic phenotype may provide new insights into the nutritional basis of mosquito anthropophily. The results presented in this thesis highlight several important parameters required to theoretically model the infection dynamics of wMelPop, and its potential impacts on Ae. aegypti populations. Moreover, the successful establishment of wMelPop in Ae. aegypti forms the primary basis for further field-based evaluations, which will ultimately determine the viability of this Wolbachia-based strategy as an applied tool to reduce dengue transmission.

Chagas disease is caused by the parasite Trypanosoma cruzi and transmitted by multiple triatomine vectors across the Americas. In Central America, the predominant vector is Triatoma dimidiata, a highly adaptable and genetically diverse Hemiptera. In this research, we used a novel reduced-representation DNA sequencing approach to discover community dynamics among multiple biotic factors associated with Chagas disease in Central America, and assess the infestation patterns of T. dimidiata after seasonal and chemical disturbances in Jutiapa, Guatemala. For our first study, we used a hierarchical sampling design to obtain multi-species DNA data found in the abdomens of 32 T. dimidiata specimens from Central America. We aimed to understand (1) the prevalence of T. cruzi infection, (2) the population genetics of the vector and parasite, (3) the blood meal history of the vector, and (4) gut microbial diversity. Our results indicated the presence of nine infected vectors harboring two distinct DTUs: TcI and possibly TcIV. We found significant clusters among T. dimidiata populations in countrywide and within-country levels associated with sylvatic ecotopes and diverse domestic genotypes. There was significantly higher bacteria species richness in infected T. dimidiata abdomens than those that were not infected, with further analysis suggesting that gut bacteria diversity relates to both T. cruzi infection and the local environment. We identified vertebrate blood meals from five T. dimidiata abdomens including chicken, dog, duck and human; however, additional detection methods are necessary to confidently identify blood meal sources. In our second study, we analyzed the GBS genotypes of 440 T. dimidiata specimens collected in two towns of Jutiapa, Guatemala. Our aim was to assess (1) the domestic population patterns that aid the recovery of T. dimidiata after an insecticide treatment in El Carrizal and (2) the seasonal changes that regulate the dispersal of the vector in the untreated communities of El Chaperno. Results showed that the insecticide application was effective at reducing the population abundance immediately after the application in El Carrizal; nevertheless, 18-month post-treatment the town-wide infestation and genetic diversity were recovering. Within-house relatedness among specimens recovered 18 months post-treatment, suggesting that the insecticide treatment failed to fully eliminate domiciliated colonies. In contrast, lack of change in abundance or genetic diversity in El Chaperno implied absence of dispersers from sources beyond the town periphery, while evidence of a decrease of relatedness among individuals implied dispersal among houses. After the insecticide treatment in El Carrizal, population reduction led to lack of genetic spatial autocorrelation; nevertheless, rapid dispersal into neighboring houses lead to autocorrelation 18 months after the insecticide treatment. This pattern was also observed in El Chaperno, where an increase in spatial autocorrelation during seasonal dispersal suggests spillover to close-by households. The creation of a novel genomics pipeline allowed us to understand community and dispersal patterns of T. dimidiata and other biotic factors important for the prevalence and transmission of Chagas disease at local and regional levels. Future studies should include complementary approaches for taxa verification (e.g. bacteria 16S barcoding, PCR-base detection), as well as expand the scope of local population analyses to peridomestic and sylvatic genotypes that could suggest a broader range of vector sources and region-wide patterns of temporal and spatial dispersion.

Globally emphasis has been given to identify emerging and re-emerging pathogens. Rapid urban expansion creates a problem which is two-fold. Firstly, increasing slum living conditions due to inadequate rate of infrastructure development results in an increased reliance on natural resources, including the capture and consumption of surrounding wildlife to subsist, thereby facilitating the transfer of emergent zoonotic pathogens. Secondly, through activities such as pollution or alien species introductions, the rapid transformation of once pristine environments, alters natural systems, potentially exposing these environments to new bacterial pathogens. Therefore, the main aim of this study was to assess overlooked bacterial species harboured by four host species (Bathyergus suillus, Georychus capensis, Cryptomys hottentotus hottentotus and Fukomys damarensis) belonging to the subterranean rodent family Bathyergidae, which inhabit an environment well-suited for an array of bacterial species, and which varied in their exposure to human settlements. Bacterial prevalence and diversity was initially evaluated using broad-range PCR techniques in combination with nucleotide sequencing. This revealed high levels of bacterial prevalence (82.91%) and mixed infections (22.60%) in bathyergid species. Two bacterial groups, the Bacillus cereus complex (a group of soil-dwelling bacterial strains with pathogenic potential with an overall prevalence of 8.55%) and haemotropic Mycoplasma strains (vector-borne bacterial strains of zoonotic potential with an overall prevalence of 1.28%) were subsequently selected for further genetic analysis with genus and species-specific PCRs. Bacillus molecular screening and phylogenetic analyses was achieved by targeting four gene regions with seven published primer assays and two novel PCR assays. This enabled identification of two B. cereus complex strains in bathyergid lungs and revealed an overall B. cereus complex prevalence of 17.95% for the 234 bathyergid lung samples screened. Bacillus genome prevalence was significantly higher in B. suillus individuals (45.35%), sampled in a peri-urban environment, compared to the other bathyergid species sampled from pristine habitats (ranging from 0% - 4.44%). Anthropogenic activities in the area where B. suillus was sampled could, at least partially, attribute to the perceived difference between urban and naturally sampled bathyergid species, highlighting the role of B. suillus to act as both a reservoir of potentially zoonotic pathogens and as a sentinel for anthropogenic soiling. Mycoplasma molecular screening using three different PCR assays, all targeting the 16S rRNA gene region, confirmed an overall haemotropic Mycoplasma prevalence of 24.13% in the 286 bathyergid organs (lung, spleen and liver) screened. A significantly higher prevalence and diversity of haemotropic Mycoplasma strains was found in B. suillus lungs (41.86%) compared to its naturally occurring relatives (ranging from 0%-36%). Phylogenetic analyses identified six novel haemotropic Mycoplasma strains, all grouping within a discrete monophyletic cluster, sister to Mycoplasma coccoides, and comprising two well-supported sub-clusters. The human introduction of commensal rodents harbouring Mycoplasma strains transferred through cosmopolitan arthropod vectors to indigenous bathyergids, likely underlies the higher prevalence in urban areas, although other biotic and abiotic factors affecting ectoparasite load also merit consideration. The data generated by the current study indicate the need to identify largely overlooked and potentially zoonotic bacterial pathogens in subterranean mammals and emphasises the importance of monitoring anthropogenically-introduced, opportunistic pathogens and the threats they pose to vulnerable communities and co-occurring, free-living animal species / Dissertation (MSc)--University of Pretoria, 2017. / Airports Company of South Africa (ACSA) / National Research Foundation (NRF) / Zoology and Entomology / MSc / Unrestricted

Climate change will likely have impacts on disease vector distribution. Posing a significant health threat in the 21st century, risk of tick-borne diseases may increase with higher annual mean temperatures and changes in precipitation. We modeled the current and future potential distribution of the Ixodes ricinus tick species in Europe. The Genetic Algorithm for Rule-set Prediction (GARP) was utilized to predict potential distributions of I. ricinus based on current (1990-2010 averages) and future (2040-2060 averages) environmental variables. A ten model best subset was created out of a possible 200 models based on omission and commission criteria. Our results show that under the A2 climate change scenario the potential habitat range for the I. ricinus tick in Europe will expand into higher elevations and latitudes (e.g., Scandinavia, the Baltics, and Belarus), while contracting in other areas (e.g., Alps, Pyrenees, interior Italy, and northwestern Poland). Overall, a potential habitat expansion of 3.8% in all of Europe is possible. Our results may be used to inform climate change adaptation efforts in Europe.

Rodents have been reported to play a significant role as reservoirs of over 22 rodent-associated Bartonella species. In this study, we contrast prevalence and diversity of Bartonella infections in 377 small mammals, representative of three terrestrial rodent genera, namely Aethomys, Gerbilliscus and Rhabdomys and one subterranean mole-rat species (Bathyergus suillus). The latter was sampled in close proximity to an informal human settlement, whereas the afore-mentioned murid rodent genera were sampled across a range of landscapes inclusive of natural, agricultural, urban, peri-urban and rural settings, from three provinces (Free State, Gauteng and Western Cape) in South Africa. Molecular estimates of Bartonella infection rates were determined through multi-gene screening of DNA extracted from clinical samples, primarily heart and spleen. PCR assays targeting the citrate synthase (gltA) and NADH dehydrogenase gamma subunit (nuoG) and/or beta subunit of bacterial RNA polymerase (rpoB) genes were used to ensure enhanced molecular estimates of Bartonella prevalence. Aethomys had the highest infection rate (86.7%), whereas Rhabdomys had the lowest (15%). Nucleotide sequencing and phylogenetic analyses revealed that the different primers sets used for Bartonella screening have different affinities to the different strains present in rodents from South Africa. Furthermore, the presence of Bartonella co-infections, confirmed through the presence of multiple peaks at 15% of the nucleotide sequences sites, ranged from 33.8% (in Aethomys) to 42.9% (in Gerbilliscus species) for the gltA gene region. For Aethomys ineptus, of the discrete Bartonella lineages recovered, one was closely related to zoonotic B. elizabethae. The latter species, which is associated with Rattus hosts worldwide and has been linked to cases of human endocarditis, suggests spillover from invasive to indigenous rodents. This is supported by previous studies indicating that indigenous Micaelamys namaquensis, a highly adaptable species, which like Aethomys is capable of utilizing natural and modified landscapes also hosts B. elizabethae-related lineages. Of potential public health importance, Bathyergus suillus were shown to be infected with a zoonotic Bartonella species, B. rochalimae. Our results further indicate that the level of anthropogenic transformation is significantly correlated with Bartonella prevalence, with Rhabdomys sampled from rural settings in the Western Cape Province having infection rates of 36% versus 0% in a nearby urban setting. This study also uncovered high levels of strain diversity in members of the Gerbilliscus cryptic species complex, sampled from an agricultural setting. The overall Bartonella PCR-positivity rate was 67.5 % and the gltA gene phylogeny confirmed the presence of six discrete Bartonella gerbil-specific lineages (I-VI). Lineages I and II clustered with Bartonella strains identified previously in G. leucogaster sampled from Sandveld nature reserves in the Free State Province South Africa, whereas lineages III-VI comprised of lineages that were restricted to either G. leucogaster or to G. brantsii, indicative of host-specificity. From the findings of this study, it is clear that the public health importance of the Bartonella species present in indigenous rodents warrants further investigation as at least two species, with known zoonotic potential (B. elizabethae and B. rochalimae) were shown to be present in rodents sampled in close proximity to human settlements. / Thesis (PhD (Zoology))--University of Pretoria, 2019. / This research was made possible through the financial support from the (AZD-IRT), CDC Co-Ag 5 NU2GGH001874-02-00 and through the NRF incentive and SARChI PI funding awarded to ADSB. / Zoology and Entomology / PhD (Zoology) / Unrestricted

Aedes aegypti mosquitoes, vectors for many human diseases, begin life as larvae developing in water, potentially exposed to runoff with herbicides and pesticides. This study serves as a novel investigation into the transstadial effects of exposure to Roundup on A. aegypti life history, immunity, and stress response and aims to account for these effects in an R0 model for vectorborne disease transmission. Prior work has shown that Roundup negatively affects mosquito life history. I hypothesized that larval exposure to the maximum sublethal dose of Roundup (7189µg/L) would negatively impact A. aegypti life history, immunity (candidate gene approach), and stress response (heat shock protein expression and fluctuating asymmetry). No significant differences were found for survival from the larval to adult stages, body size, size or shape fluctuating asymmetry, or sex ratio. However, the Roundup treatment group developed significantly slower for both time to pupation and to adult eclosion (both p < 0.0001). Adult immune gene expression showed no difference between groups, but the larval immune genes Dome (JAK-STAT pathway) and Spatzle (TOLL pathway) were downregulated in the Roundup treatment (p=0.0383 and p=0.0035, respectively), suggesting the larvae have reduced immunity. This study suggests that Roundup may have off-target effects on A. aegypti mosquitoes that are unaccounted for by current models, and these effects may potentially alter disease transmission to human hosts.

The recent discovery of Amblyomma maculatum sensu lato (s. l.) ticks in southern Arizona has renewed discussions around species designations for members of the Amblyomma maculatum tick group. Amblyomma maculatum s. l. from Arizona appear to be morphologically intermediate between A. maculatum sensu stricto (s. s.) and A. triste s. s. At present there is no conclusive species designation for the ticks from Arizona. My research focused on analyzing the systematics of both A. maculatum s. l. and Rickettsia parkeri, a common bacterial pathogen transmitted by these ticks. In the laboratory, A. maculatum s. l. from Arizona and A. maculatum s. s. from Georgia readily mated on experimental animals to produce F1 hybrid ticks; there was no difference in fertility with these two populations when compared with homologous populations. However, the F1 hybrids produced during these experiments exhibited diminished fitness and did not produce a viable F2 generation. These results suggest that A. maculatum s. l. and A. maculatum s. s. represent separate biological species. Results of the crossbreeding experiment conflict with recent genetic analyses of A. maculatum s. l. and A. maculatum s. s. suggesting they are a single species. Thus, I developed and optimized 14 microsatellite loci that amplify both A. maculatum s. s. and A. maculatum s. l. These novel microsatellite markers can be used in future analyses of A. maculatum s. l. and A. maculatum s. s. to further test for conspecificity between the two. I also investigated the genetic relationships within geographically distinct R. parkeri strains through development and implementation of a multi-locus sequence typing analysis. I showed that while there is no consistent genetic delineation of strains isolated from A. maculatum s. l. versus A. maculatum s. s., there is a subset of R. parkeri strains from A. maculatum s. l. that appear to represent an intermediate genotype between the North and South American strains. While the biological causes for these results are not immediately clear, coevolution of R. parkeri and A. maculatum s. l. may account for the detection of the intermediate genotype only found in association with A. maculatum s. l.

Emerging infectious diseases continue to threaten human health and healthcare resources across the world, and with the sustained emergence of some like Lyme disease, this trend is only expected to get worse. Even though some research has examined the spread of Lyme disease in different parts of the United States, focusing on its origin, spread, surveillance, and reporting, there has been minimal research on the recent spread of Lyme disease into and within Central and Southern Appalachia. This is an important gap, considering that this region is at the edge of the disease's expanding incidence and range. Considering this factor and the consequences that this disease may have on the residents of Appalachia who already face an array of underlying problems like chronic conditions, decreased access to health care, and exposure through outdoor work like resource extraction, addressing this gap will be important in understanding the current and future spatial range and impacts of the continued emergence of Lyme disease in Appalachia. This study thus sought to examine the extent of the emergence of Lyme disease in Appalachia between 2000 and 2019 using spatial scan statistic. This type of analysis uses spatiotemporal information to quantify disease emergence and diffusion using retrospective case and location data within a GIS environment; hence it helped quantify the spatial distribution and diffusion of Lyme disease into and within Appalachia and identify additional areas that can be targeted with public health prevention measures. We found five statistically significant clusters of Lyme disease across the contiguous US, indicating that the disease expanded spatially over the study period. Lyme disease has more than tripled across the US, with the number of counties with RR>1 increasing from 130 in 2000 to 453 by 2019. Lyme disease also increased more than eightfold in Appalachia, with spread southwards into and within Appalachia. The number of Appalachian counties with RR>1 increased from 16 in 2000 to 127 in 2019. These findings are important in understanding the current and future spatial range and the impacts of the continued emergence of Lyme disease in Appalachia. With this understanding, we can minimize the misdiagnosis of Lyme disease and inform public health action to reduce public vulnerability. / Master of Science / Emerging infectious diseases are diseases that have not been present, the persistence of diseases that were previously unknown, or those diseases that are rapidly increasing in a given location. These diseases continue to threaten human health and healthcare resources across the world, and with the sustained emergence of some like Lyme disease, this trend is only expected to get worse. Even though some research has investigated the spread of Lyme disease in different parts of the United States, focusing on its origin, spread, monitoring, and reporting, there has been minimal research on the recent spread of Lyme disease in Central and Southern Appalachia. This is an important gap, considering that this region is next to areas where the disease has recently spread, thereby making it vulnerable to continued spread. Considering this factor and the consequences that this disease may have on the residents of Appalachia who already face many underlying problems like long-term conditions, decreased access to health care, and exposure through outdoor work like resource extraction, addressing this gap will be important in understanding the current and future geographic occurrence and impacts of the continued spread of Lyme disease in Appalachia. This study thus sought to investigate the extent of the spread of Lyme disease in Appalachia between 2000 and 2019 using statistics and identification of places with a higher than expected number of cases. This type of analysis uses location information to quantify disease occurrence and spread using past case and location data using GIS software; hence it helped identify the distribution and spread of Lyme disease into and within Appalachia and identify additional areas that can be targeted with public health prevention measures. We found five areas with higher than expected number of Lyme disease cases across the contiguous US, indicating that the disease increased over the study period. Lyme disease has more than tripled across the US, with the number of counties with higher cases than expected increasing from 130 in 2000 to 453 by 2019. Lyme disease also increased more than eightfold in Appalachia, with spread southwards into and within Appalachia. The number of Appalachian counties with higher than expected Lyme disease cases increased from 16 in 2000 to 127 in 2019. These findings are important in understanding the current and future spread and the impacts of this continued spread of Lyme disease in Appalachia. With this understanding, we can properly detect Lyme disease and inform public health action to reduce public vulnerability.

This study evaluates the roles of temperature variability and disease awareness in the emergence of Chagas disease (American trypanosomiasis). Chagas disease is endemic in Latin America and primarily spreads to humans directly via the triatomine vector. Hosts for most triatomine species are mainly rodents and occasionally dogs. The disease itself is caused by a parasitic protozoan, Trypanosoma cruzi (T. cruzi) which is found in the triatomine's feces and is often spread while the triatomine is consuming a blood meal. T. cruzi from feces enters the body via an abrasion on the skin, the mucous membranes, conjunctivae, or through consumption. To determine the risk of Chagas disease transmission one must define qualities that make the triatomine an effective disease vector as well as investigate the level of disease awareness among physicians and the population within the vector's range. This thesis maps triatomine species within the U.S. that harbor T. cruzi naturally and that exhibit qualities of domesticity. These qualities are defined by whether the species bites humans and dogs as well as reports that the species has been found in the domestic setting. Ranges illustrating temperature thresholds for increased triatomine activity for 2000 and 2030 are also depicted. Additionally, outcomes of a physician survey are presented to gauge the status of Chagas disease awareness in areas at higher risk for disease transmission. Results reveal limited consideration of Chagas disease in physician diagnosis despite the higher risk range which extends through the southern U.S. and is predicted to expand significantly by 2030. / Master of Science

A large epizootic of the vector-borne disease bluetongue occurred in northern Europe from 2006-2009, costing the economies of the infected countries several hundreds of millions of euros. During this time, the United Kingdom (UK) was exposed to the risk of bluetongue by windborne incursions of infected Culicoides biting midges from the northern coast of mainland Europe. The first outbreaks which occurred in the UK in 2007 were attributed to this cause. Although bluetongue virus (BTV) no longer appears to be circulating in northern Europe, it is widely suggested that it and other midge-borne diseases may emerge again in the future, particularly under a changing climate. Spread of BTV is strongly influenced by the weather and climate however limited use has been made of meteorologically based models to generate predictions of its spread to the UK. The extent to which windborne BTV spread can be modelled at timescales from days to decades ahead, to inform tactical and strategic decisions taken to limit its transmission, is therefore examined here. An early warning system has been developed to predict possible incursion events on a daily timescale, based on an atmospheric dispersion model adapted to incorporate flight characteristics of the Culicoides vectors. The system’s warning of the first UK outbreak in September 2007 was found to be greatly beneficial to the UK livestock industry. The dispersion model is also shown to be a useful post-outbreak epidemiological analysis tool. A novel approach has been developed to predict BTV spread into the UK on climate-change timescales as dispersion modelling is not practical over extended periods of time. Using a combination of principal component and cluster analyses the synoptic scale atmospheric circulations which control when local weather conditions are suitable for midge incursions were determined. Changes in the frequency and timing of these large scale circulations over the period 2000 to 2050 were then examined using an ensemble of regional climate model simulations. The results suggest areas of UK under the influence of easterly winds may face a slight increase in risk and the length of the season where temperatures are suitable for BTV replication is likely to increase by around 20 days by 2050. However a high level of uncertainty is associated with these predictions so a flexible decision making approach should be adopted to accommodate better information as it becomes available in the future.