Browsing by Author "Gilleard, John"

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    An Extract of the Tapeworm, Hymenolepis diminuta, Inhibits Neutrophil Migration in vitro
    (2017) Graves, Nicholas; McKay, Derek; Gilleard, John; Yipp, Bryan; Finney, Constance
    Neutrophils are not typically the first cell that comes to mind when the immune response to infection with helminths is considered. However, data are emerging showing that these cells may play an overlooked role in response to helminths perhaps early in the infection and particularly those species that cause tissue damage (e.g. Nippostrongylus brasiliensis - a gut nematode (roundworm)). Additionally, different helminth products have been shown to directly activate neutrophils in vitro (e.g. crude extracts of the porcine cestode Cysticercus cellulosae) and directly induce their chemotaxis (e.g. excretory/secretory products of the sheep nematode Haemonchus contortus). Hymenolepis diminuta crude extract (HdE) has been shown to inhibit dinitrobenzene sulfonic acid-induced colitis. Intraperitoneal injection of this crude extract recruited a population of peritoneal exudate cells dominated by neutrophils at 4h post-injection. Remarkably, these neutrophil-predominant PECs evoked increased production of the anti-inflammatory cytokine IL-10 from co-cultures of activated murine splenic CD4+ T cells. HdE did not directly induce murine neutrophil chemotaxis in vitro and direct application of HdE to neutrophils was not preferentially cytotoxic as assessed by trypan blue staining and LDH release. HdE did however induce Ca2+ mobilization and respiratory burst by murine neutrophils in vitro. In contrast to its ability to recruit neutrophils to the peritoneal cavity, HdE inhibited neutrophil chemotaxis in response to WKYMVm and keratinocyte derived chemokine (KC). HdE was unable to modulate neutrophil migration towards leukotriene B4. Biochemical characterizations (boiling, trypsinization, acidification, and sodium metaperiodate treatment) suggested that an acid-stable glycoprotein was responsible for its ability to block migration towards WKYMVm. The ability of HdE to block migration towards WKYMVm appears to be independent of the phosphorylation of the mitogen-activated protein kinase, p38. HdE did not directly induce production of the cytokines, IL-10, TNF-α, and KC. It also did not modulate production of these cytokines induced by other activators. In conclusion: (1) HdE activated neutrophils to mobilize Ca2+ into the cytoplasm and produce ROS; and, (2) an acid-stable glycoprotein component of the HdE inhibited neutrophil migration towards WKYMVm.
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    Biological insights into the parasitic nematode Heligmosomoides bakeri from bulk and single-cell transcriptomics
    (2023-12-05) Pollo, Stephen Michael Jerome; Wasmuth, James; Gilleard, John; Lynch, Tarah; Niu, Dongyan
    Heligmosomoides bakeri, a parasitic nematode (roundworm) of mice, is closely related to economically important parasites of livestock and the hookworm parasites of humans. As a murine parasite it is more amenable to being maintained and manipulated in a controlled laboratory environment than its relatives. The worm enters its host during its third larval stage and develops through another larval stage into adults that reside in the lumen of the small intestine to mate and lay their eggs. Unravelling these processes and other processes critical to H. bakeri survival could reveal new targets to use for drug discovery for controlling this group of parasites as well as refine previous predictions of immunomodulatory molecules, which have therapeutic potential. My thesis aims to describe the gene expression of H. bakeri during the parasitic phase of its lifecycle. I first used RNA sequencing to investigate how the (whole worm) expression of genes varies across the parasite’s infection. Using differential gene expression analyses I identified genes important for development and genes important for the males vs the females. I also uncovered evidence of an increased importance for anaerobic respiration in the adults compared to the larvae and hypothesize that aerobic conditions are important for the critical developmental processes of molting and cuticle synthesis. I then analyzed single-cell RNA-seq data from young adult male and female worms. Leveraging cell type markers from the model nematode C. elegans allowed me to putatively identify gamete, embryo, intestine, hypodermis, neuron, and muscle cells. The resulting cluster expression profiles can guide investigations into tissue-specific aspects of the adult worms. Putative intestinal transcription profiles suggest compartmentalization of function along the anterior-posterior axis of the worms, with an emphasis on protein synthesis in the anterior portion, as has been observed in other worms. Embryonic profiles are noticeably different from C. elegans embryogenesis, particularly with respect to paternal contributions to the early embryo. Overall, these datasets extend our understanding of how H. bakeri lives as a parasite and provide a public resource for further investigation into genes of interest. They also lay the groundwork for more comprehensive comparisons among the phylum Nematoda.
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    Creation of Bioinformatics Approaches to Understand Host-Pathogen Molecular Mimicry
    (2024-09-10) Rich, Kaylee Dawn; Wasmuth, James D.; Gilleard, John; Lewis, Ian; Peters, Nathan
    Imitation is not the sincerest form of flattery; for some it is the key to survival. Across phyla, pathogens imitating aspects of their host is crucial to their continued survival and proliferation. Also called molecular mimicry, imitation of host biomolecules has been observed to target a wide range of processes, including host signaling pathways and the immune system. Identification of molecular mimicry can provide insights into pathogen virulence and pathogenicity. Additionally, in silico detection methods can predict currently unknown host-pathogen interactions. Here, I describe my development of a bioinformatic method for mimicry detection using protein sequence data, with the ultimate aim to further our collective understanding of molecular mimicry. In Chapter 2, I resurrected and improved upon a bioinformatic k-mer based mimicry detection method, which I assessed on 32 human-infecting pathogens. During my assessment, I exposed potential concerns with the method and recommended avenues for further improvement. Chapter 3 describes my critical evaluation of each step in the pipeline and subsequent development of mimicDetector, a new pipeline for molecular mimicry identification. During my evaluation, I assessed the performance of five protein search tools with two amino acid substitution scoring matrices on varying query lengths. From these results, I determined the combination best suited to mimicry detection and implemented a new filtering method which better assesses similarity between pathogen and host protein sequences. Lastly, in Chapter 4, I applied mimicDetector to a focused investigation of the causative agent of human tuberculosis, Mycobacterium tuberculosis (Mtb). Among my predicted mimics were potentially novel Mtb-human protein interactions as well as a protein frequently implicated in Mtb molecular mimicry. My investigation highlights strategies used by Mtb to subvert host defenses and provides insight into the role mimics play in pathogenicity and virulence.
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    Design of Novel Algorithms for Comparative Analysis of Complex Gene Families and their Application to Nematode Detoxification Pathways
    (2017) Curran, David Michael; Wasmuth, James; Gilleard, John; Van Marle, Guido; Chua, Gordon; Cribb, Alastair
    Parasitic nematodes present a current and devastating global problem, infecting billions of people, and causing huge production losses in both crops and livestock. There are a limited number of anthelmintic drugs available to treat these infections, and resistance has arisen quickly and spread across the globe. Xenobiotic metabolism is a well-known mechanism of drug resistance in insects, and evidence suggests it may also play a role in the development of drug resistance in parasitic nematodes. Identifying candidate enzymes in the free-living nematodes may help to understand or combat the rising resistance crisis in the parasites. However, identifying many of the protein sequences that may be involved in xenobiotic metabolism can prove challenging due to high sequence divergence and draft quality genome assemblies. This work focuses on novel software to detect hard-to-find genes, as well as methods of performing comparative phylogenetic analyses, both between species and within a population. In the absence of specific selective pressures, the phylogeny of a multi-species gene family will tend to agree with the underlying species tree. Conversely, adaptive evolution tends to manifest as incongruence in the gene tree as well as lineage-specific expansions and contractions. These properties, collectively termed phylogenetic instability, have been found to be good predictors of proteins that directly interact with the environment. I have developed an algorithm to quantify phylogenetic instability in a gene tree, and show that it correlates exceptionally well with the known substrate specificity of human cytochrome P450 enzymes. I then apply this technique to five detoxification gene families (cyp, fmo, sdr, ugt, gst) from five free-living nematode species (Caenorhabditis elegans, C. briggsae, C. brenneri, C. remanei, and Pristionchus pacificus). These gene families are known to act on both endogenous and xenobiotic molecules, and these new methods allow me to predict which are likely involved in xenobiotic metabolism. This will aid in the study of these enzymes, including their orthologs in the parasitic species.
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    Developing long-read Oxford Nanopore nemabiome metabarcoding for ovine gastrointestinal nematode community analysis and diagnostics
    (2024-02-02) Charrier, Eleonore; Gilleard, John; Wasmuth, James; Poissant, Jocelyn; Levy, Michel; McVeigh, Paul; Summers, Mindi
    Parasitic gastrointestinal nematodes (GIN) are major causes of disease in animals and humans and the routine use of anthelmintic drugs in their management is increasingly ineffective due to widespread anthelmintic drug resistance. Consequently, there is a growing need for improved diagnostic tools to enable more evidence-based approaches to control including more targeted and sustainable drug use. ITS-2 nemabiome metabarcoding is a powerful method to determine the relative abundance of GIN species isolated from fecal samples from a number of animal species. This thesis presents research aiming to improve this method to increase its flexibility, widen its applicability and make it more suitable for more routine diagnostics and surveillance. Sheep are an excellent system to develop GIN molecular diagnostic methods; GIN are arguably better studied in sheep than in any other host due to their major impact on sheep production. The research in this thesis has three main aims: (i) To transition nemabiome metabarcoding away from the short-read Illumina platform to the long-read Oxford Nanopore Technologies (ONT) platform. (ii) To apply nemabiome metabarcoding directly to DNA from fecal material rather than harvested parasite populations. (iii) To develop nemabiome metabarcoding to determine the absolute abundance of GIN in fecal samples rather than just their relative abundance. Chapter 2 presents a set of four new non-redundant nematode rRNA databases and a WebApp, ‘PrimerTC’, to assess the taxonomic conservation of primers targeting different regions of the rRNA cistron across the nematode phylum. These new resources should enable more targeted and more comprehensive metabarcoding for both parasitic and free-living nematodes. Chapter 3 introduces ITS-1/5.8S/ITS-2 rRNA long-read ONT metabarcoding for GIN species relative quantitation. This chapter also pioneers the application of ITS-2 and ITS-1/5.8S/ITS-2 metabarcoding directly to DNA extracted from ovine fecal samples. Chapter 4 explores a synthetic DNA spike-in approach for absolute quantitation of GIN species in ovine fecal samples using both ITS-2 rDNA Illumina short-read and ITS-1/5.8S/ITS-2 long-read nemabiome metabarcoding. The new tools and methodologies presented in this thesis should pave the way for more widescale adoption of nemabiome metabarcoding as a tool in research, surveillance, and routine diagnostics.
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    Exploring the ‘Nemabiome’: Deep Amplicon Sequencing to Investigate Community Structure and Drug Resistance in Parasitic Gastrointestinal Nematodes of Livestock
    (2018-01-12) Avramenko, Russell; Gilleard, John; Colwell, Douglas; De Buck, Jeroen; Wasmuth, James
    Parasitic gastrointestinal nematodes are one of the most important pathogen groups impacting livestock health, welfare and production worldwide. Control in both animals and humans is heavily dependent on the prophylactic administration of anthelmintic drugs. However, there are widespread and increasing reports of anthelmintic drug resistance in many parasitic nematode species making current approaches to control unsustainable. Consequently, there is an urgent need for better tools for diagnosis, surveillance and research to support the development of sustainable approaches to parasite control. Although co-infection with multiple nematode species within a single host is common, there are few tools with which to study the composition of these complex parasite communities. Nematode species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. This thesis introduces the concept of the ‘nemabiome’ as the community of mixed nematode species inhabiting the gastrointestinal tract of the host. Chapter 2 presents the development of a deep-amplicon (metabarcoding) sequencing approach, targeting the ITS-2 rDNA locus, to define the species composition of the nemabiome. This approach is analogous to deep-amplicon sequencing methodologies used to study the bacterial ‘microbiome’. Chapter 3 uses this methodology to assess the species composition of nematode communities in cattle from Canada, the United States and Brazil. It also describes changes in the nemabiome before and after anthelmintic treatment in Canadian cattle. Chapter 4 illustrates the ability of this approach to describe the nemabiome of commercial and conservation bison herds in western Canada, which previously had little information regarding species prevalence. Chapter 5 introduces another deep-amplicon sequencing application; screening for DNA sequence polymorphisms associated with benzimidazole drug resistance in mixed nematode infections. The isotype-1 β-tubulin locus is screened for three polymorphisms associated with benzimidazole resistance. Chapter 6 describes the application of this approach to screen for benzimidazole resistance associated polymorphisms in the cattle and bison parasite populations characterised in Chapters 3 and 4. These approaches represent vital tools needed to conduct epidemiological studies, surveillance of parasite species, improve the sensitivity of drug efficacy testing and assess anthelmintic resistance on a large scale.
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    The Genomic Basis of Adaptive Response to Anthelmintic Treatment in Two Species of Parasitic Nematodes
    (2022-06-08) Jasper, Russell; Yeaman, Sam; Wasmuth, James; Gilleard, John
    Despite rapid increases in next-generation sequencing studies, relatively little is known about how often species evolve convergently at the genotypic level, using the same loci to respond to a similar selection pressure (hereafter, genotypic convergence). Here we investigate similar adaptive responses in two species of parasitic nematodes, H. contortus and T. circumcincta, and test for possible genotypic convergence between species. After treating populations on farms across Western Canada with antiparasitic drugs, either fenbendazole or ivermectin, we use whole genome shotgun sequencing to identify loci putatively involved in drug resistance in each species and test for genotypic convergence. Despite both species facing similar environments and selection pressures and showing similar resistance statuses, we found no evidence for polygenic genotypic convergence for resistance. Although the response to selection was not strongly repeated between species, we did identify six candidate genes for resistance which were repeated between species.
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    The invasion process of the European strain of Echinococcus multilocularis in Western Canada
    (2022-02) Santa, Maria Alejandra; Musiani, Marco; Ruckstuhl, Kathreen; Gilleard, John; Massolo, Alessandro
    Genetic studies on Echinococcus multilocularis (Em), the causative agent of human alveolar echinococcosis (AE), are essential to understand the ecological and evolutionary processes underlying changes in the geographic distribution of this parasite, and to assess the potential impact on public health. The recent detection of European-like haplotypes circulating in wild hosts in Western Canada, and the unprecedented detection of human AE cases in Alberta, make it a priority to assess the extent of the distribution of this strain and its origin(s) into Canada. The overall aim of my thesis was to investigate the invasion process of the European strain of Em in Western Canada. I reviewed the invasions by parasites with complex-life cycles using the invasion of the European strain as a model to develop an ecological framework for investigating the invasion of parasites transmitted in predator-prey systems. Then, I developed a real-time PCR assay to detect co-infections of Em and E. canadensis in fecal and worm-mixed samples, providing the first evidence of co-infections of these two parasites in red foxes and coyotes, and the first molecularly confirmed record of E. canadensis (G10) in coyotes. When used along with an automated magnetic bead-based DNA extraction method, the real-time PCR test proved to have a high sensitivity to detect Em in fecal samples, increasing efficiency and throughput. The genetic analysis using deep amplicon sequencing and multiple mitochondrial and nuclear markers showed a lower intraspecific diversity and a higher abundance of the European-type haplotypes. Moreover, most genetic variants were closely related to the original European clade, with the dominance of one variant in each province and low genetic diversity of Em, supporting the hypothesis of a relatively recent introduction. The spatial analysis showed a genetic discontinuity between western provinces, suggesting different founder events and isolation by distance of some genetic variants. Finally, although the prevalence of Em infection was higher in red foxes than coyotes, there was a higher proportion of coyotes with high intensity of infection, even at lower population densities than red foxes. Therefore, coyotes might be considered the main spreaders of the European strain in Alberta.
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    Molecular detection and distribution of Fascioloides magna and its snail host in Elk Island National Park
    (2024-09-20) Meyers, Olivia; Pruvot, Mathieu; Kutz, Susan; Gilleard, John; Maraj, Ramona
    Giant liver fluke (Fascioloides magna) is a parasitic worm found in wild and domestic ruminants in North America. Infection with F. magna, known as fascioloidiasis, causes liver damage, fibrosis, and inflammation that can negatively impact the fitness of hosts. In Elk Island National Park (EINP), Alberta, Canada, this parasitic disease is suspected to contribute to the ongoing moose population decline. To manage the park’s moose population, fluke control measures, including anthelmintic treatment and environmental control strategies, can reduce the prevalence of fascioloidiasis. However, efficient, targeted interventions require knowledge on the fine-scale distribution of the parasite. In this thesis, I investigated the epidemiology of F. magna to guide an effective and minimally invasive fluke control program in EINP. First, I developed and validated a molecular assay to detect F. magna in freshwater snails. A multiplex qPCR assay was created to specifically target F. magna as well as an internal amplification control endogenous to freshwater snails. I used this assay, along with its constituent singleplex assay, to diagnose F. magna infections in lymnaeid snails from EINP. The singleplex assay proved to be more sensitive than the multiplex, making it a more accurate tool for epidemiological surveys. Stagnicola elodes was identified as an intermediate host species of F. magna in EINP, with an estimated prevalence of 5.8%. Second, I determined the spatial distribution of F. magna and its snail host in EINP and assessed whether environmental factors could predict their occurrence among water bodies. I found that snail hosts for F. magna were generally widespread across the park and were associated with physiochemical and plant-related environmental indicators at water bodies. Snail infections with F. magna were relatively rare, observed at only 12% of sites. Using remote-sensing environmental data, I created fine-scale predictive models to forecast the occurrence of snail hosts and F. magna-infected snails across water bodies. However, these predictive occupancy models demonstrated poor predictive accuracy. This work has contributed a molecular tool for the surveillance of F. magna, explored the utility of ecological niche modeling for the epidemiological control of the parasite, and furthered our understanding of fascioloidiasis in EINP.
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    Molecular Diagnostics and Epidemiology of Anthelmintic Efficacy in Hookworms and Whipworms
    (2023-05-10) Venkatesan, Abhinaya; Gilleard, John; Wasmuth, James; Mains, Paul; Van Marle, Guido
    Broad-spectrum anthelmintics are widely used for treating parasitic nematode infections in humans and animals. The frequent and extensive use of these drugs has resulted in widespread anthelmintic resistance in nematodes of livestock, which is increasingly well documented. In contrast, while anecdotal and clinical data often suggest poor efficacy, the extent and molecular basis of anthelmintic resistance in parasitic nematodes of humans and companion animals is still poorly understood. This thesis uses molecular genetic approaches to investigate two significant and contemporary examples of poor anthelmintic efficacy in parasitic nematodes. Firstly, a parasite of dogs, Ancylostoma caninum, for which there have been many clinical and anecdotal reports of poor efficacy for multiple drug classes for some time. Secondly, an important parasite of humans, Trichuris trichiura, where a population which is refractory to albendazole-ivermectin combination therapy has recently been identified. Ancylostoma caninum (canine hookworm) is an important gastrointestinal parasitic nematode of dogs. The parasite prevalence in the USA has seen a dramatic increase in the past decade with anecdotal reports of reduced drug efficacy and suspected cases of anthelmintic resistance. Chapter 2 of the thesis identifies and functionally characterizes a novel benzimidazole resistance mutation at codon 134 of the A. caninum isotype-1 β-tubulin gene. A deep amplicon sequencing approach was utilized for the large-scale screening of this novel and other previously described benzimidazole resistance mutations, revealing that benzimidazole resistance is now widespread in A. caninum from pet dogs across the USA. Chapter 3 examines the molecular epidemiology of the benzimidazole resistance alleles and the genetic diversity of A. caninum in pet dogs to investigate the emergence and spread of anthelmintic resistance in the parasite populations. The results support the hypothesis that at least some benzimidazole resistance alleles originated in racing greyhound kennels due to intense drug selection pressure and subsequently spread into pet dog populations via the rehoming of retired racing greyhounds. Chapter 4 focuses on the second parasitic nematode, Trichuris trichiura (human whipworm). This soil transmitted helminth (STH) species of global health importance has been particularly problematic due to its poor sensitivity to benzimidazoles, which are widely administered anthelmintics in Mass Drug Administration (MDA) programs. This thesis chapter investigates genetic differences in Trichuris populations from clinical efficacy trials in three different countries: Côte d’Ivoire, Lao PDR, and Tanzania. Genetic analysis suggests that the parasite populations in Côte d’Ivoire, which is non-responsive to albendazole-ivermectin treatment, is a separate Trichuris species, distinct from T. trichiura. The results from this thesis illustrate the power of molecular genetic approaches to elucidate the basis of poor anthelmintic efficacy and provide a framework for molecular epidemiology and surveillance studies.
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    Molecular Genetic Analysis of Benzimidazole Resistance in the Parasitic Nematodes Haemonchus contortus and Haemonchus placei
    (2015-06-18) Chaudhry, Umer; Gilleard, John
    The benzimidazoles are one of the most important anthelmintic drug classes used to control parasitic nematodes in domestic animals and humans. They have been intensively used in the livestock sector for over 30 years resulting in widespread resistance in small ruminant parasite species such as Haemonchus contortus. The work presented in this thesis investigates the molecular genetics of benzimidazole resistance in H. contortus and the closely related species Haemonchus placei. The major focus is on India and Pakistan, two regions that were considered to be potentially informative as to how resistance mutations arise and spread due to resistance being at a relatively early stage. Chapter II, explores parasite populations in small ruminants in the Punjab region of Pakistan showing that co-infections with the two species were relatively common and that interspecies F1 hybrids were present in several populations. This raises the possibility of introgression of anthelmintic resistance genes from one species to another. Chapter III, presents a study in southern India which provides phylogenetic evidence that the F200Y (TAC) mutation has arisen multiple independent times in the region. In contrast, the E198A (GCA) mutation appears to have arisen and spread from a single origin. This is the first genetic evidence of the spread of an anthelmintic resistance mutation between locations for any parasitic nematode species to date. Chapter IV compares the molecular genetics of benzimidazole resistance in H. contortus populations under intense drug selection pressure on small ruminant government farms in Pakistan with parasite populations that are under minimal selection pressure in neighbouring rural areas. Overall, the results of this work are consistent with the hypothesis that the F200Y (TAC) isotype-1 -tubulin mutation appears by recurrent mutation during selection as well as being present in the standing genetic variation present prior to the onset of selection. Finally, Chapter V, presents evidence that benzimidazole resistance is at an early stage of emergence in H. placei from cattle in the southern and mid-west USA. This raises a significant concern that clinical resistance could emerge quite quickly as more benzimidazoles are used in cattle as is the current trend.
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    Protocol for the systematic review of strategies used in beef cattle to prevent or control internal or external parasites
    (2021-08) Agbese, Samuel; Sanguinetti, V. Margarita; Gilleard, John; Ganshorn, Heather; Windeyer, M. Claire
    This paper describes the protocol for a systematic review that will evaluate the following question: What strategies are most effective for the prevention or control of internal or external parasites in beef cattle herds? The overall objective of this systematic review is to critically assess and synthesize the current published knowledge on the aforementioned review question.
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    Structural Variation in the Caenorhabditis elegans Genome: Challenges and Quality Assurance Strategies for Reliable Variant Calling
    (2023-08) Lesack, Kyle James; Wasmuth, James; Van Marle, Guido; MacCallum, Justin; Gilleard, John
    Obtaining an accurate and comprehensive representation of structural variation is crucial for understanding how large alterations in chromosome structure contribute to phenotype diversity and drive genome evolution. Despite continuous efforts into improving methods for identifying structural variation from whole genome sequencing data, accurate variant calling remains challenging. The barriers to progress in this area are complex and multifactorial but the technical limitations of short-read sequencing technologies and limited availability of suitable benchmarking resources for non-human species feature prominently. This thesis includes an in-depth evaluation of several commonly used tools for identifying structural variants from short- and long-read DNA sequencing data from natural Caenorhabditis elegans strains. The results of these comparisons revealed that popular tools yield considerably different results, which are described in detail in Chapter 2. A major aim of this project was to identify sources of error and variability that tool developers could address in the future. Surprisingly, the order of reads in PacBio FASTQ files were revealed to affect the predicted structural variants. Chapter 3 describes these results and demonstrates how alignment sorting algorithms contribute to the problem. In Chapter 4, an analysis of structural variation in 14 natural C. elegans strains is described. Importantly, this work demonstrates how long-read DNA sequencing data can be successfully used to identify structural variants at the population level.
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    The Role of Quaternary Structure Organization in Prion Strain Selection and Adaptation
    (2023-08) Chang, Sheng Chun; Gilch, Sabine; Braun, Janice; Gilleard, John; Tsutsui, Shigeki; Booth, Stephanie
    Prion diseases are fatal and infectious neurodegenerative diseases caused by the misfolded, protease-resistant, and aggregated prion protein isoform, PrPSc. Unlike conventional pathogens, the propagation of prions does not depend on the replication of genetic material, but different cases of infection can lead to PrPSc adopting specific conformations resulting in different and stably transmissible phenotypic properties that are, by definition, the determinants of different strains. The distribution of PrPSc quaternary structure has previously been determined to vary across prion strains, and different PrPSc aggregate assemblies in turn harbour varying levels of infectivity. We were interested in determining how PrPSc aggregate size modulate strain properties and how these properties are affected by different inoculation routes, specifically in comparing peripheral versus intracerebral infection. Using in vivo murine models, we explored prion disease pathogenesis and strain selection upon inoculation of different PrPSc protein quaternary structures or prion strains. Transgenic mice overexpressing cervid prion protein inoculated with different elk chronic wasting disease PrPSc fractions exhibited strikingly different survival times and disease phenotypes, including variations in the prion biochemical, neuropathological, and biophysical profiles; interestingly, these properties converged upon passaging, though not the differing clinical signs. Gene-targeted mice expressing the cervid prion protein at physiological levels infected with CWD prions either directly into the brain or from the periphery resulted in different biochemical and neuropathological properties, notably with a converging conformational profile among the peripherally inoculated group. Similar observations were recorded where animals peripherally inoculated with CWD prion aggregates exhibited markedly reduced variations in survival times compared to infection directly into the brain. These results indicate that the direct selection of PrPSc conformers, or PrPSc conformational mutation, is likely to occur in the presence of a barrier interfering with the ideal replication setting as adaptation under a more restrictive set of circumstances takes place. This research demonstrates the intricacies of prion strain selection and adaptation that would provide novel insight into future investigations of the evolution and emergence of prion strains.
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    The use of natural genetic variation in Caenorhabditis elegans to identify novel polymorphisms that can confer benzimidazole resistance in nematodes
    (2014-06-12) Latheef, Sharmilah Luthfia Jezmine; Gilleard, John
    Benzimidazoles are important drugs for parasite control. Benzimidazole resistance is widespread in parasites of domestic animals, and an emerging problem in human parasites. Caenorhabditis elegans is a powerful model system to study biology of drug resistance. Using natural genetic variation in wild populations of the non-parasitic nematode C. elegans, three novel amino acid substitutions and a deletion in the β-tubulin drug target have been identified that confer varying levels of benzimidazole resistance. These residues are different to those previously reported in other organisms including nematode parasites or fungi and provide new candidate polymorphisms to be investigated in parasitic nematode species including human parasites where resistance is poorly understood. They may also represent new residues important for drug binding. In addition, presence of these resistance conferring polymorphisms in wild populations of a free-living nematode may indicate benzimidazole drug residues in the environment having a significant impact on natural fauna. Finally, the presence of null ben-1 β-tubulin alleles in wild C. elegans populations indicates the functional redundancy of this gene in nature.