Browsing by Subject "Parasitology"

Background: Recent epidemiological studies demonstrate that the prevalence of the fifth major human malaria parasite, Plasmodium knowlesi (monkey malaria), is often underestimated and misdiagnosed with standard microscopy blood film. We sought to adapt and compare a new simple molecular diagnostic method for P. knowlesi with the gold standard nested molecular assay and microscopy blood film in P. knowlesi hotspot areas in Sarawak, Malaysia. In addition, we analyzed the statistical association between P. knowlesi positive test results and demographic and behavioral/occupational risk factors.

Methods: The study was conducted at Sibu, Kapit and Sarikei Hospitals in Sarawak, Malaysia. 115 blood samples were collected from malaria suspected patients seeking treatment at these hospitals. Samples were analyzed by microscopy, Nested polymerase chain reaction (PCR) and single-step PCR. Sensitivity, specificity, and practical value of the new single-step PCR assay was calculated. Bivariate and multivariate regression was conducted to test the possible risk factors for the detection of P. knowlesi.

Results: Single-step PCR showed low sensitivity (51.92%, 95%CI 37.63 - 65.99%) compared to nested PCR and 46.03% (95%CI 33.39 - 59.06%) compared to microscopy. When compared to nested PCR, microscopy had a false positive rate of 20.6%. However, it only missed 2 cases of P. knowlesi. The mean age in the study population was 40.35. Patients enrolled at Kapit hospital had higher odds ratio for positive P. knowlesi PCR results (adjusted OR = 4.46, 95%CI 1.16 – 11.51). Age above 21 years (adjusted OR = 6.28, 95%CI 1.53 – 25.64), male gender (adjusted OR = 2.46, 95%CI 0.91 – 6.65) and living near a vegetation (Plantation, forest, fruit trees or wet rice paddy) (adjusted OR = 5.96, 95%CI 1.11 – 31.83) were associated with increased risk for P. knowlesi infection.

Conclusions: Data from this study showed that single-step PCR has a low sensitivity and thus, it is not a suitable alternative for accurate detection of P. knowlesi. Further studies are required for assessment and development of other diagnostic assays or new primer sets. Multivariate analysis revealed that adult men over the age of 21 who live near agricultural areas have the highest risk for P. knowlesi malaria infection. Large- scale descriptive studies of both non-human hosts and vectors would greatly influence prevention and control strategies of this zoonotic disease.

Increased attention has recently been placed on understanding the natural variation of the malaria parasite Plasmodium vivax across the globe, as in 2020 alone, P. vivax caused an estimated 4.5 million malaria cases and lead to over 600,000 deaths around the world. P. vivax infections in central Africa have been of particular interest, as humans in Sub-Saharan Africa frequently possess a P. vivax resistance allele known as the Duffy-negative phenotype that is believed to prevent infection in these individuals. However, new reports of asymptomatic and symptomatic infections in Duffy-negative individuals in Africa raise the possibility that P. vivax is evolving to evade host resistance.Whole genome sequencing has become more common as a means of understanding the population diversity of P. vivax. However, there is still a scarcity of information about P. vivax in central Africa. In this dissertation, I analyze whole genome sequencing data from a new P. vivax sample collected from the Democratic Republic of the Congo in central Africa. By studying P. vivax from central Africa, we can begin to understand the evolutionary history of the pathogen in this part of the world as it relates to the global context of this pathogen. I also investigate the relationship of P. vivax in the DRC with a potential animal reservoir of a closely related species, P. vivax-like, in non-human primates in this region. Due to the scarcity of P. vivax samples in central Africa, I also investigated methods with which to best make use of whole genome sequencing data, particularly in generating phylogenetic trees. While many studies of P. vivax genetic diversity employ whole genome variation data in order to study evolutionary relationships of P. vivax populations, in this dissertation I make use of the P. vivax apicoplast, a non-photosynthetic plastid organelle genome. The apicoplast genome is five times longer than the mitochondrial genome and does not undergo recombination, making it a valuable locus for studying P. vivax evolutionary history using phylogenetic trees.

Multiple biological processes occur in a time-dependent fashion. One of the most prevalent ways of regulating rhythmic biology is by controlling the timing and dosage of gene expression. Gene regulatory networks are groups of regulators that control target genes through multiple mechanisms, including direct DNA binding to alter expression dynamics, post-transcriptional activation, and protein degradation. Networks that control oscillating processes, such as circadian rhythms and cell cycles, have several hallmark properties, such as high interconnectedness and the disproportionate presence of certain patterns of regulator-target wiring (network motifs.) They are also self-sustaining and can control large suites of repeating, periodic gene expression.While circadian rhythms and cell cycles are fairly well-studied, there are still some prominent gaps in knowledge. Multiple parasites exhibit remarkably rhythmic life cycles, particularly the Plasmodium genus that causes malaria, but it is unknown whether these rhythms are driven by an oscillator within the parasite or are imposed by pre-existing host rhythms. In addition, the principles which guide oscillator gene regulatory network (oGRN) evolution are only partially understood, and lack examples of network-wide analysis on a finer scale. In this dissertation, I approach both questions primarily by examining the dynamics of periodic gene expression on a whole-transcriptome level. In Chapter 2, I investigate evidence for an innate oscillator in P. falciparum. I found multiple known hallmarks of oGRN-controlled gene expression—independent control of gene expression, conservation of ordering between strains, and genetic control of period length—and conclude that the parasite drives its own asexual reproduction cycle. In Chapter 3, I examine the dynamics of cell cycle-controlled transcription in two closely related species of the budding yeast Saccharomyces cerevisiae, S. paradoxus and S. uvarum. S. cerevisiae is a model organism for cell cycle research, providing a tractable system for studying oGRN evolution principles. I found intriguing differences between the species, including marked changes in the periodicity and gene expression levels of several known members of the core cell cycle oscillation machinery, warranting further study and validation. I conclude by discussing how studying oGRNs of both distantly- and closely-related species to model organisms furthers our understanding of governing evolutionary principles and our ability to form gene network hypotheses.

Abstract

Introduction: Mongolia is a country known for its rich nomadic and pastoral culture, with populations of people who work in environments that are densely populated with ticks and TBP animal reservoirs. TBPs typically undergo transstadial transmission, but transovarial transmission may also occur. Transovarial transmission events have been demonstrated in laboratory settings, but few studies have evaluated transovarial transmission of TBPs in field settings within the host-vector ecosystem. Tick borne pathogens of most concern in Mongolia are Rickettsia spp., Anaplasma spp., Borrelia spp., Babesia spp., Ehrlichia spp., and tick-borne encephalitis. In this study, specific aims were: 1) To determine the prevalence of tick borne pathogens, particularly Rickettsia spp., Anaplasma spp., and Ehrlichia spp. among various tick species at different developmental life stages; and 2) To understand the role of animal reservoirs and vertical transmission of TBPs among feeding ticks at different life stages using larval and nymph ticks collected in the wild from small mammal reservoirs, as well as eggs laid by engorged wild-caught adult female ticks and reared in the laboratory.

Methods: In this cross-sectional study, ticks in their larvae and nymph life stages were collected off of captured rodents across seven soums (districts) in three aimags (provinces) situated in the Northern region of Mongolia from June 20th to July 23rd, 2016. Engorged adult ticks were collected from livestock located in three soums within three aimags from May 6th to 22nd, 2016. Tick eggs were collected from engorged ticks from May 9th to June 1st, 2016. Ticks were tested by PCR to detect the presence of Rickettsia spp., Anaplasma spp., and Ehrlichia spp.

Results: There were 546 (88%) larval and 72 (12%) nymphal Dermacentor spp. ticks collected. There were 588 (95%) of 618 ticks allocated into 42 larvae and 18 nymph pools (60 pools total). All tick pools were PCR-positive for Rickettsia spp. and no tick pools were PCR-positive for Anaplasma/Ehrlichia spp. minimum infection rate (MIR) for R. raoultii ranged from 6.7% to 28.6%. Of the 60 tick pools, 50 (522 ticks total) were matched with rodent rickettsial infection history status. There were 31 (62%) tick pools or 362 (69%) of individual ticks found on rodents with no history of rickettsial infection. The majority of ticks discovered to have no association with rodents with rickettsial infection history were larvae (352/362 individual ticks). There were 38 adult fed female ticks collected. All adult fed ticks were PCR-positive for Rickettsia spp. and 2 (5%) were PCR-positive for Anaplasma/Ehrlichia spp. There were 33 ticks that laid eggs. PCR testing of eggs showed a 91% (30/33) positivity for Rickettsia spp. and one pool of eggs was PCR-positive for Anaplasma/Ehrlichia spp. All sequenced Rickettsia spp. products were identified to be R. raoultii and all sequenced Anaplasma/Ehrlichia spp. were An. ovis.

Conclusions: This study identified transovarial transmission of Rickettsia spp. and Anaplasma spp. among D. nuttalli ticks. This study also found a low association between rodents with history of Rickettsia spp. infection and infection status of biting ticks. Additional study is needed to further assess the proportion of transovarial transmission found in nature. Specifically, testing of individual tick eggs and larvae should be conducted. A better understanding of the ecology of TBPs in nature can provide public health and human and veterinary medicine with a greater awareness of the burden of TBPs in Mongolia.

While the Madre de Dios department of Peru has the nation's highest leishmaniasis incidence, limited research on the disease and its sand fly vector exists in the region. Considering the diverse and adaptive nature of sand flies, understanding vector ecology is essential to directing leishmaniasis control efforts. This study addresses this need by investigating the following aims: 1) describe sand fly abundance and biodiversity in communities along the Madre de Dios River; 2) identify environmental and socio-demographic factors associated with sand fly abundance and biodiversity in these communities; and 3) examine ecological risk factors associated with leishmaniasis in the communities. In order to accomplish these aims, the research team collected sand flies, obtained leishmaniasis case data, conducted community and household surveys, measured soil samples and studied land cover data in communities along the Madre de Dios River. We compared sand fly abundance, sand fly biodiversity and leishmaniasis incidence with soil chemistry, socio-demographic and land cover measures. We ran negative binomial regressions and Poisson regressions to investigate bivariate relationships between dependent and independent variables. We developed a multivariate model that predicts greater potential vector and non-vector abundance in areas with greater forest coverage, lower infrastructure indices and lower soil pH. This study provides an important overview of endemic sand fly fauna in Madre de Dios and presents an initial description of factors associated with sand fly populations in the region.

Parasitism is integral to primate evolution, contributing to major life history tradeoffs with other processes critical to reproductive success and survival. I investigate how infection with the tapeworm Taenia serialis affects geladas (Theropithecus gelada) in the Simien Mountains National Park, Ethiopia. After identifying T. serialis as the parasite behind the protuberant cysts regularly observed in geladas with the use of molecular tools, I described an overall cyst prevalence of 4.8% in the study population (Chapter 2). To identify infections that do not present as visible cysts, I adapted a non-invasive monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) to detect circulating Taenia spp. antigen in gelada urine (Chapter 4). This assay detected Taenia antigen with high accuracy (98.4% specificity, 98.5% sensitivity, and an AUC (area under the curve) of 0.99). Implementing this assay in the study population, I found that infection is substantially more widespread than would be predicted based on the occurrence of visible T. serialis cysts (16.5% of individuals of unknown status tested positive for antigen presence at least once). Contrary to the female-bias observed in many Taenia-host systems, I found no significant sex bias in either cyst presence or antigen presence. Age, on the other hand, predicted cyst presence (older individuals were more likely to show cysts) but not antigen presence (Chapter 3). This indicates that T. serialis may infect individuals early in life but result in visible disease only later in life. I found that cysts were strongly associated with decreased survival and reproductive success in adult geladas (Chapter 4). Counter to expectations, T. serialis cysts were not strongly associated with decreased fecal testosterone metabolite concentrations (Chapter 5). This suggests that the mechanisms underlying the wild T. serialis-gelada relationship differ from those observed in experimental systems. Together, the analyses contained in this dissertation offer novel insights into the predictors, costs, and consequences of a trophically transmitted larval parasite in wild primates.

The sickle-cell trait hemoglobin genotype (HbAS) is known to protect against severe malaria caused by Plasmodium falciparum. However, the biological mechanisms behind this protection are not well understood. Cyclophilin 19B (PfCyP-19B) is a parasitic gene that produces the protein cyclophilin 19B, a member of the unfolded protein response that is important in parasitic protein folding and trafficking. We set out to measure the transcript expression level of PfCyP-19B to investigate its potential role in the mechanisms that confer protection for HbAS individuals. RNA was extracted from both in vivo samples collected from Malian children as well as in vitro samples harvested throughout a 48-hour incubation period. RNA extracts were reverse transcribed and transcript expression was measured via qPCR. Wilcoxon rank sum and bootstrapping methods were used to analyze transcript units between parasites grown in normal (HbAA) and HbAS red blood cells. The results from our cross-sectional in vivo data revealed under expression of PfCyP-19B among individuals with the HbAS genotype compared to those with the HbAA genotype (Wilcoxon rank sum p=0.006). In vitro time series results showed no significant difference in PfCyP-19B transcript expression levels between genotypes but did display a 24-hour pattern of peak expression for both HbAA and HbAS genotypes. The under expression of PfCyP-19B among HbAS individuals could be linked to impaired protein trafficking, interfering with the parasite’s ability to display surface proteins vital for cytoadherence and severe disease manifestation. The 24-hour peak transcript expression displayed in vitro roughly aligns with the P. falciparum parasite stage transition states, suggesting cyclophilin 19B may aid in parasitic transitions.

This dissertation is an investigation of the systematics, phylogeography, and ecology of a globally distributed fungal family, the Elaphomycetaceae. In Chapter 1, we assess the literature on fungal phylogeography, reviewing large-scale phylogenetics studies and performing a meta-data analysis of fungal population genetics. In particular, we examined the possible effects of asexuality, trophic niche, dispersal method, and ocean barriers on population structure. In Chapter 2, we examine the systematics and phylogeography of the Elaphomycetaceae, a family consisting of the truffle genus Elaphomyces and the stalked genus Pseudotulostoma, hypothesizing that the mammal-dispersed truffle would show evidence of dispersal limitation. Using DNA sequence data, we determined that Pseudotulostoma is derived from a lineage of Elaphomyces, indicating that Elaphomyces as currently defined is paraphyletic. The distribution of each subgenus of Elaphomyces is nearly global; representative species have been found on every continent save Africa and Antarctica. This biogeographic pattern does not follow the pattern expected by a scenario of continental vicariance. Dating analysis in BEAST confirmed that broadly distributed clades are, in most cases, too young for this pattern to be explained by continental vicariance, indicating that occasional long-distance dispersal has been a significant component in the biogeographic history of the Elaphomycetaceae. This finding contradicts our initial hypothesis that the mammal-dispersed truffles would be dispersal- limited. In Chapter 3, we investigate the role of Elaphomyces as a host for the fungal parasite Elaphocordyceps, a parasite derived from insect pathogens that attacks both insect larvae and Elaphomyces, its only fungal host. We examined the biogeography of Elaphocordyceps isolated from Elaphomyces specimens in order to test whether it, like its host, showed recent connections between the Southern and Northern Hemispheres. We also evaluated the pathogenicity of infection as determined by a visual rubric for the truffle gleba, the phylogenetic distribution of Elaphocordyceps species on its host, testing for seasonal, climate, and host-parasite effects. In Chapter 4, based on the phylogeographic pattern seen in Elaphomyces that resembles that of some air-dispersed fungi, we used theoretical and experimental methods to test whether Elaphomyces could be dispersed by air. We tested the capacity for air dispersal with an experimental test of passive air dispersal on the powdery spores of Elaphomyces morettii and found that these large spores could disperse over a short distance (10 m) in comparable numbers with the spores of the giant puffball Calvatia cyathiformis, which is known to be air-dispersed. The major findings of this thesis are that 1) fungi in general show high dispersal ability, but that trophic niche and dispersal mode may affect population structure, 2) that Pseudotulostoma, a stalked genus, is derived from the truffle Elaphomyces, 3) that the Elaphomycetaceae have experienced frequent long-distance dispersal despite 4) that the fitness of Elaphomyces as indicated by glebal development varies with host-parasite interactions based on species identity, but not with climate or season, and that 5) Elaphomyces spores, should they be released into the air, can remain in the air long enough to be dispersed long distances by the wind. The overall conclusion of this thesis is that, while Elaphomyces is clearly reliant on animal vectors for excavation and dispersal, its past history of long-distance dispersal and current spore trajectories indicate it can be passively air-dispersed as well.

Recent experiments and comparative surveys in Southern US salt marshes revealed that a common larval trematode parasite, Parorchis acanthus, generated a trophic cascade that protected foundational marsh plants (Spartina alterniflora) from drought-associated overgrazing by suppressing the per capita grazing impacts of its host, the marsh periwinkle (Littoraria irrorata). While it is clear that parasites can play a positive role in mediating marsh ecosystem response to disturbance, there is still little known about the context dependency of this interaction, the role of definitive avian hosts in regulating parasite prevalence, and whether other commonly-occurring parasites may also modify processes that underpin ecosystem stability. The purpose of this project was to extend the current understanding of the roles played by parasites and their hosts in mediating marsh ecosystem stability. A field manipulation of Littoraria density in which infection prevalence with Parorchis acanthus was held at a constant value revealed that these parasites yielded positive impacts on Spartina aboveground biomass at middling densities of snails, but the positive effects of parasites were negligible at both low, and high snail densities. Surveys of drought-impacted marshes revealed that birds – the definitive hosts for trematode that infect Littoraria – congregated within die-off areas and that increased bird usage of die-off areas was associated with increased trematode parasitism in snails within grazer fronts, decreased per capita grazing rates of snails, and proportionate decreases in ecosystem die-off rate. Multi-site bird exclusion and mechanistic field studies experimentally confirmed that birds increased ecosystem resistance to drought-driven die-off by acting as the dispersive vectors for parasites that suppress Littoraria grazing. Finally, we explored how the trematode Cercaria opaca in ribbed mussels (Geukensia demissa) influenced the facultative mutualism between Guekensia and Spartina – an interaction that underlies marsh ecosystem resilience to drought-associated die-off. A field manipulation using experimentally infected mussels revealed that mutualistic benefits to Spartina decreased with increasing infection intensity in mussels. Subsequent mechanistic experiments demonstrated that increasing infection with C. opaca decreased mussel biodeposit production, the functional trait underlying mutualistic benefits to Spartina. Additionally, increasing parasite load was associated with decreased strength of both shells and byssal attachments, potentially explaining the relatively higher predation on heavily infected mussels in our field study. A survey of five North Carolina salt marshes revealed that infection intensity in mussels increased with proximity to die-off areas, indicating that C. opaca could influence marsh recovery following die-off events. Taken together, these results underscore the importance of parasitism’s influence on Southern salt marsh ecosystem stability and more generally show that parasites can be major arbiters of community structure and functioning.

Malaria, caused by the apicomplexan parasite Plasmodium, is a disease which affects up to 500 million people each year. Historically, malaria infection has been combated both through the control of its vector, the Anopheles mosquito, and use of a variety of drugs, such as quinine (1800s) and chloroquine (1900s). However, with the evolution of resistance to the majority of available anti-malarial drugs, current approaches have settled upon combinatorial therapies. The most effective of these currently are ACTs (Artemisinin Combination Therapies - Artemisinin derivatives combined with a number of other drugs). However reports of Artemisinin resistance are continuing to emerge, suggesting that new approaches and increased understanding of the Plasmodium parasite is required.

Beginning with the complete sequencing of Plasmodium falciparum genome and continuing with comprehensive profiling of both the parasite's proteome and transcriptome, various genomic approaches applied in the study of malaria have led to significant new insights into the underlying biology of this parasite. While these new findings have greatly increased our understanding of genetic regulation within the malaria parasite, they largely have not yet translated into new therapeutic approaches. For this reason, considerable attention has been paid to the study of human genetic disorders which convey resistance to malaria, in the hopes that elucidating the mechanisms behind these resistances might lead to increased understanding of the parasite's biology and thus novel therapeutic approaches.

Sickle cell (HbS) erythrocytes are well known to resist malaria infection. However, the molecular basis of this resistance, long been recognized as multifactorial, contains elements which remain poorly understood. Here we show that the dysregulated erythrocytic microRNA composition, present in both HbAS and HbSS erythrocytes, is a significant determinant of resistance against the malaria parasite Plasmodium falciparum. During the intraerythrocytic lifecycle of P. falciparum, a subset of erythrocyte microRNAs translocate into the parasite. Two microRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite mRNAs and, via impaired ribosomal loading, resulted in translational inhibition of the target mRNA. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical microRNA activity which may present a novel host defense strategy against complex eukaryotic pathogens. In addition, the formation of these chimeric transcripts even in normal host erythrocytes illustrates a unique parasitic post-transcriptional adaptation to the host-cell environment.

Malaria persists as a global health problem, with 212 million cases and 429,000 deaths worldwide in 2015 alone. It is caused by the apicomplexan parasite Plasmodium, which follows a complex life cycle that consists of multiple stages spanning from the human host to the mosquito vector. Among the Plasmodium parasites causing human malaria, the deadliest species is Plasmodium falciparum. Most P. falciparum parasites follow an asexual cycle in human erythrocytes that is characterized by a tightly synchronized continuous cascade of gene expression, although a small proportion commits to a sexual fate. Parasites committed to the sexual stage develop into male and female gametocytes over 9-12 days, with mature gametocytes being the only form of the parasite transmissible to the mosquito vector.

This commitment to a sexual fate is rare, and little is known about the transcriptional programs related to sexual commitment and mating-type determination. Furthermore, discrete changes that occur in these cells are largely undetectable in traditional bulk-cell analyses. Bulk-cell analyses were used to establish models for synchronous stage-specific transcriptional programming during the asexual intraerythrocytic developmental cycle (IDC) but left little resolved in terms of cellular heterogeneity and cell-fate decisions. Due to these limitations, we developed unbiased single-cell approaches on a microfluidic platform to analyze single parasites during late asexual and sexual stages. This work was divided into two main parts. The first focused on single-cell gene expression in male and female mid-to-late stage gametocytes. We captured 90 single parasites and compared the expression of 91 genes, including 87 gametocyte-specific genes. Our analysis identified distinct gene clusters whose expression associated with male, female, or all gametocytes. In addition, a small number of male gametocytes clustered separately from female gametocytes based on sex-specific expression independent of stage. RNA fluorescent in situ hybridization (RNA-FISH) validated the mutually exclusive expression pattern of male and female transcripts in gametocytes. These analyses uncovered novel male and female markers that are expressed as early as stage III gametocytogenesis, providing further insight into Plasmodium sex-specific differentiation previously masked in population analyses.

The second part of this work centered on single-cell RNA sequencing (scRNA-seq) of P. falciparum late asexual and sexual stages. First, we uncovered a large number of previously undefined gametocyte-specific genes. 46 asexual cells were then segregated into three separate clusters based on the differential expression of SERAs, rhoptries, and EXP2 plus transporters. RNA-FISH of cluster-specific genes validated this distinct stage-specific expression during the IDC and defined the highly variable transcriptional pattern of EXP2. Additionally, these analyses indicated huge variations in the stage-specific transcript levels among parasites. Overall, scRNA-seq and RNA-FISH of P. falciparum revealed distinct stage transitions and unexpected degrees of heterogeneity with potential impact on transcriptional regulation during the IDC and adaptive responses to the host.