MS, University of Cincinnati, 2024, Arts and Sciences: Biological Sciences

Ectoparasites are ubiquitous in nature, and they can impose significant fitness costs on their hosts. Understanding the physiological and structural defense mechanisms employed by invertebrate hosts during ectoparasite infection remains limited. This lack of understanding presents an obstacle to comprehending ectoparasite resistance mechanisms in their own right and their evolutionary potential. The present study endeavors to address this knowledge gap by investigating attachment-resistance adaptations in Drosophila that mitigate the costs of ectoparasitism by mites, Gamasodes pachysetis (Parasitidae). It was found that significant variation in mite attachment duration occurred among host species of Drosophila that had been cultured in the laboratory under common environmental conditions for multiple generations. This result suggests species-level genetic differences in host mechanisms affecting mite attachment duration. Experiments with a given species, D. malerkotliana, revealed that when flies were pre-infected by mites, they exhibited reduced attachment durations compared to naive flies. This finding implies that specific protective factors were induced by mite attachment, resulting in reduced attachment duration in a subsequent infection. This response was not replicated in flies experimentally wounded to mimic mite-induced cuticle damage, suggesting that the observed reduction in attachment duration in pre-infected flies was specific to mite feeding, highlighting the importance of understanding host-parasite interactions at a molecular level. Eight (8) candidate genes were screened for their involvement in mediating mite attachment duration; the genes examined were chosen based on previous RNA-seq analyses. Gene knockdown experiments using the GAL4-UAS system identified a candidate gene of interest, HSP70Ba, which, when suppressed, significantly prolonged mite attachment duration. The results of this study support the hypothesis that Drosophila species possess (open full item for complete abstract)

Committee: Michal Polak Ph.D. (Committee Chair); Stephen Matter Ph.D. (Committee Member); Joshua Benoit Ph.D. (Committee Member) Subjects: Parasitology

Master of Science, The Ohio State University, 2024, Environment and Natural Resources

Sarcoptic mange, caused by the obligatory ectoparasite, Sarcoptes scabiei var canis, poses significant threats to wild canid populations, as it results in high levels of morbidity and mortality. Despite its impact, the disease dynamics and individual risk of infestation for coyotes (Canis latrans) in urban environments remain understudied. As human density within urban spaces continues to rise globally, understanding how disease dynamics within coyote populations is crucial for both wildlife conservation and public health. The Cook County Coyote Project, spanning from 2000 to 2023, has been examining sarcoptic mange infestations among radio-collared coyotes within the Chicago metropolitan area. In this study, we aim to (1) determine prevalence rates, identify causes of mortality, and elucidate general disease dynamics of sarcoptic mange and (2) identify specific covariates that influence an individual's likelihood of infestation within the Chicago coyote population. We found that sarcoptic mange prevalence was enzootic within our study site and was at a lower overall prevalence than studies conducted in rural areas. There was no significant increase in diagnoses between male and female coyotes; however, we were more likely to diagnosis coyotes with sarcoptic mange as adults than subadults or pups. 2% of the diagnosed coyotes fully recovered from gross lesions associated with sarcoptic mange. Coyotes that died from sarcoptic mange exposure were more common during the winter months. Individual likelihood of infestation was assessed using a generalized linear model using demographic and spatial covariates. Our best-fit model based on Akaike information criterion found that with a random effect for coyote ID, yearly home range composition and yearly age class were useful covariates in predicting individual risk of infestation; however, both fixed effects were non-significant. We hypothesize that sarcoptic mange infestations are rather influenced by inter-individua (open full item for complete abstract)

Committee: Stanley Gehrt (Advisor); Laura Pomeroy (Committee Chair); Risa Pesapane (Committee Chair) Subjects: Parasitology; Wildlife Conservation; Wildlife Management

Master of Science, The Ohio State University, 2024, Comparative Biomedical Sciences

Widespread anthelmintic resistance to the gastrointestinal nematode Haemonchus contortus has made this parasitic infection increasingly challenging to treat and prevent in small ruminants and camelids. A commercial vaccine, known as Barbervax®, contains a mixture of H11 and H-gal-GP antigens derived from gut mucosal membrane enzymes of H. contortus that play a role in digesting blood taken from the host. Antibody neutralization of these antigens causes failure of the adult H. contortus to digest blood, resulting in loss of nutrients and inability to survive and reproduce. H11 and H-gal-GP proteins are considered “hidden” antigens, meaning the host immune system does not encounter these proteins under natural infection. Therefore, repeated immunization withBarbervax® is required to maintain this protective response. One previous study has evaluated the safety of Barbervax® in camelids but efficacy was unable to be assessed due to lack of successful infection following larval inoculation. The objective of the current study was to evaluate clinical parameters of anemia, fecal egg counts, and humoral immune responses of healthy young alpacas after immunizing with Barbervax® in comparison to non-vaccinated controls, all having the same natural, environmental exposure to existing parasite-laden pastures. We hypothesized vaccination would demonstrate a decrease in trichostrongyle (H. contortus) egg production compared to non-vaccinated alpacas. A crossover-like study was performed where twenty alpacas (298 ± 66 days of age) were assigned to be initially vaccinated with Barbervax® (n=10) or no treatment (n=10). Three doses of Barbervax® were administered at three-week intervals to the vaccinate group. Fecal egg counts (FECs) and blood were collected ten days prior the start of the study, Day 0, and every 21 days thereafter to evaluate packed cell volume and antibody titers. Each study group was kept on separate pastures for natural exposure to parasites. Tracer sheep (n=2 p (open full item for complete abstract)

Committee: Antoinette Marsh (Advisor); Jeffrey Lakritz (Committee Member); Andrew Niehaus (Committee Member); Joseph Lozier (Committee Member) Subjects: Parasitology

Master of Sciences, Case Western Reserve University, 2024, Pathology

Malaria caused by the Plasmodium vivax parasite is a leading cause of disease burden in many tropical and subtropical parts of the world. Infection leads to the generation of antibodies specific to parasite proteins, such as apical membrane antigen 1 (AMA1). This ligand plays a role in invasion of reticulocytes by binding of the rhoptry neck (RON) complex, which is composed of RON proteins. This complex is inserted by the parasite into the cell surface. In malaria infections, IgG1 and IgG3 isotype frequencies are increased. These isotypes can activate complement or bind Fc receptors to initiate effector functions. As part of a project that isolates cDNA from individual PvAMA1- specific IgG expressing B cells, we aim to develop a rapid method to identify the IgG subclass from these B cells to provide a more comprehensive view of immunoglobulin generation in these individuals.

Committee: Neena Singh (Committee Chair); Jürgen Bosch (Committee Member); Christopher King (Committee Member) Subjects: Immunology; Molecular Biology; Parasitology; Pathology

Doctor of Philosophy (PhD), University of Toledo, 2023, Biomedical Sciences (Medical Microbiology and Immunology)

Chagas disease is a chronic disabling neglected parasitic disease caused by the protozoa Trypanosoma cruzi (T. cruzi) for which there is no standardized treatment for patients with chronic Chagas disease nor preventative vaccine. The infective form of T. cruzi (i.e., trypomastigotes) are highly resistant to killing by complement. The complement system is an integral part of the immune system that comprises of 3 pathways: classical, lectin and alternative pathway. The alternative pathway (AP) is an essential immune effector mechanism resulting in various consequences including pathogen clearance. Factor H (FH), an important negative regulator of the AP on cell surfaces and in blood, contains 20 short consensus repeat domains, and binds to complement activation products (i.e., C3b/C3d) and polyanionic host cell markers such as sialic acid or glycosaminoglycans on cell surfaces. FH uses its 4 N-terminal domains to inactivate the AP, while using other domains for interacting with C3b/d in combination with polyanionic markers on cell surfaces. Various pathogens hijack FH to inactivate the AP as an immune evasion strategy. T. cruzi uses a trans-sialidase enzyme to hijack host sialic acids to the parasite surface that are hypothesized to bind FH. Previous studies by others show that FH binds to complement-opsonized T. cruzi and that removal of sialic acid from the parasite surface iv increases complement-mediated lysis of T. cruzi trypomastigotes. However, whether FH binds directly to T. cruzi in the absence of complement opsonization, the FH domains involved, and the functional consequences of FH binding are not known. We investigated whether FH bound directly to the parasite in the absence of complement activation and the FH domains involved in this interaction. Only trypomastigotes, but not epimastigotes (non-infective, complement susceptible) bound FH directly in a dose-dependent manner. Although domain 20 was hypothesized as important for FH binding given it is the (open full item for complete abstract)

Committee: Viviana Ferreira (Committee Chair); Kevin Pan (Committee Member); Guillermo Vazquez (Committee Member); Stanislaw Stepkowski (Committee Member); Ritu Chakravarti (Committee Member) Subjects: Immunology; Parasitology

Doctor of Philosophy, Case Western Reserve University, 2023, Genetics

Whole genome sequencing (WGS) is an ever expanding tool in the field of genetics, and is widely used to characterize human genetic variation. There are multiple large-scale sequencing studies being conducted today worldwide, like All of Us, Three Million African Genomes, and GenomeAsia 100k. The addition of these diverse datasets alone can be transformative to our understanding of genetics, but the increase in the diversity of populations sampled also has the potential to reveal additional and novel information relevant to health and disease. Specifically, whole genome sequence (WGS) analyses of DNA from human whole blood may be able to capture genetic variation in other species that can affect both individual and public health. The research detailed in this dissertation aims to illustrate the utility of human WGS data for infectious disease, by showing that the malaria causing parasite Plasmodium can be sensitively detected from unmapped reads (UMRs) from WGS data. Malaria has a significant global health burden, and elimination of the disease has been a goal since the 1950s. Recently, there have been roadblocks in the progress of malaria elimination that can only be resolved through additional research efforts. Development of this detection methodology could be the tool required to better define the parasite population, identify problematic populations, and solve the roadblocks limiting elimination success.

Committee: Thomas LaFramboise Ph.D. (Committee Chair); Scott Williams Ph.D. (Advisor); Peter Zimmerman Ph.D. (Advisor); Dana Crawford Ph.D. (Committee Member); Arlene Dent MD, Ph.D. (Committee Member) Subjects: Biomedical Research; Epidemiology; Genetics; Parasitology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2022, College of Sciences and Health Professions

Telomeres are a nucleoprotein structure at the end of the chromosome and are essential for genome integrity and chromosome stability. Telomere lengths are primarily maintained by a telomerase-mediated pathway but can be maintained by a homologous recombination-mediated pathway. However, detailed mechanisms of telomere maintenance are still unclear in many eukaryotes, including an important human pathogen, Trypanosoma brucei. Telomeres can be elongated by telomerase in T. brucei, a causative agent of fatal sleeping sickness in humans and nagana in cattle. T. brucei evades host immune response by regularly switching its major surface antigen, variant surface glycoprotein (VSG), a process known as antigenic variation. The telomere structure and telomere proteins play critical roles in T. brucei pathogenesis. In mammalian, yeast, and plant cells, ssDNA binding proteins with OB-fold domains play important roles in coordinating telomere G- and C-strand syntheses. However, no such protein has been described in T. brucei to be specifically associated with the telomere. We identified POLIE, an A-type DNA polymerase, as a crucial telomere complex component in T. brucei and essential in maintaining telomere integrity in T. brucei. Depletion of POLIE in T. brucei leads to an increased amount of DNA damage at telomere/subtelomere, increased frequency of gene conversion-mediated VSG switching, and an increased amount of the telomeric circles (T-circles), suggesting a potential role of POLIE in suppressing DNA recombination at the telomere and the subtelomere. However, I find that telomeric and subtelomeric DNA recombination is unlikely to be mediated by the increased telomeric R-loop level as the telomeric repeat-containing RNA (TERRA) level is significantly lower in POLIE-depleted cells. The telomere G-rich 3'overhangs are dramatically elongated in POLIE-depleted cells, indicating a potential role of POLIE to coordinate telomere G- and C-strand syntheses and suggesting that the (open full item for complete abstract)

Committee: Bibo Li (Advisor); Girish Shukla (Committee Member); Aaron Severson (Committee Member); Keith McCrae (Committee Member); Valentin Börner (Other); Kurt Runge (Other) Subjects: Biochemistry; Genetics; Molecular Biology; Parasitology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2022, College of Sciences and Health Professions

Trypanosoma brucei (T. brucei) causes sleeping sickness in humans and regularly switches its major surface antigen, variant surface glycoprotein (VSG), to evade the host immune response. TbRAP1, a telomere protein, is essential for VSG silencing, suppresses the level of telomeric repeat containing RNA (TERRA) and the telomeric R-loop, and suppresses DNA recombination-mediated VSG switching events. However, the mechanism of TbRAP1-mediated VSG silencing is still poorly understood. Like other RAP1 orthologs, TbRAP1 has conserved functional domains. We have identified that the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal (NLS) that is required for its interaction with importin-alpha and its nuclear localization. However, how is TbRAP1 localized to the telomere was unclear. Known RAP1 homologs either bind telomere DNA directly through their Myb and Myblike domains or are recruited to the telomere through their interaction with duplex telomere DNA binding factors. We have identified both a double-stranded and a single-stranded DNA binding activities in TbRAP1. Both these activities depend on a short, highly positively charged peptide that resides in the MybLike domain and overlaps with the TbRAP1 NLS. Surprisingly, we have also identified an RNA Recognition Motif (RRM)-mediated RNA binding activity in TbRAP1, a novel finding in RAP1 homologs. TbRAP1 RRM recognizes consensus sequences of VSG 3'UTRs in vitro and binds the active VSG RNA in vivo. Concentration-dependent competition between TbRAP1's RNA and dsDNA binding activities suggests a novel mechanism where the active VSG RNA can antagonize the local TbRAP1 dsDNA binding-mediated VSG silencing to sustain VSG monoallelic expression. Recently we also have identified TbTRF-Interacting Factor 3 (TbTIF3) in a yeast 2- (open full item for complete abstract)

Committee: Bibo Li Ph.D. (Advisor); Aaron Severson Ph.D. (Committee Member); Donny D. Licatalosi (Committee Member); Zihua Gong M.D., Ph.D. (Committee Member); Valentin Börner Ph.D. (Committee Member); Merlin Nithya Gnanapragasam Ph.D. (Committee Member) Subjects: Biology; Genetics; Molecular Biology; Parasitology

Doctor of Philosophy, The Ohio State University, 2022, Microbiology

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally. Cutaneous leishmaniasis (CL) is the most common form, characterized by chronic skin lesions. Currently, there are no approved vaccines for human use. We have generated centrin knock out Leishmania (L.) mexicana (LmexCen-/-) mutants using CRISPR/Cas9. Centrin is a cytoskeletal protein required only for intracellular amastigote replication in Leishmania. Here, we investigated the safety, immunogenicity, and efficacy of LmexCen-/- parasites in vitro and in vivo. Our data shows that LmexCen-/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected macrophages and dendritic cells, compared to LmexWT. Furthermore, LmexCen-/- parasites are safe in susceptible mouse models and efficacious against challenge with LmexWT in genetically different BALB/c and C57BL/6 mice. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens compared to the controls. Overall, we demonstrate that LmexCen-/- parasites are a promising candidate vaccine against CL in pre-clinical models. Next, we explored the metabolic drivers of these vaccine-mediated immunological profiles. Metabolomics are emerging as a useful tool to uncover unknown networks that govern immune regulation and determine functional specialization. We analyzed the metabolic changes occurring after immunization with LmexCen-/- and compared them with LmexWT infection. Our results show enriched aspartate metabolism and pentose phosphate pathway (PPP) in ears immunized with LmexCen-/- parasites. These pathways are both known to promote M1 polarization in macrophages, and PPP in particular induces nitric oxide production in macrophages cultured with LmexCen-/-, suggesting a shift to a pro-inflammatory phenotype following immunization. Furthermore, immunized mice showed enriched t (open full item for complete abstract)

Committee: Abhay Satoskar (Advisor); Pravin Kaumaya (Committee Member); Steve Oghumu (Committee Member); Jesse Kwiek (Committee Member) Subjects: Immunology; Microbiology; Neurosciences; Parasitology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2022, Biological Sciences

Though parasites are a ubiquitous in both terrestrial and aquatic ecosystems, the important role parasites play in community ecology is often overlooked. Thus, the goal of my dissertation was to determine how parasites impact ecological communities by altering the personality, foraging behavior, and community interactions of a host. Crayfish Faxonius rustucis are the second intermediate host of the digenetic trematode Microphallus spp. Microphallid load impacted crayfish personality along a bold shy spectrum, causing crayfish to become bolder in the presence of a threatening odor and less exploratory in novel environments, whether or not a food source was present. Additionally, crayfish infected with Microphallus spp. consumed significantly less macrophyte tissue that as parasite load increase. This decrease in consumption was species-specific, with parasite load causing the sharpest decrease in the consumption of Chara sp., followed by Elodea canadensis, Ceratophyllum demersum, and Potamotgeton richardsonii. Additionally, females were more effected by parasite load than males, resulting in a sharper decrease in foraging for the female crayfish. Crayfish were placed in larger community mesocosms with both macrophytes (Chara sp., Myriophallum exalbescens, C. demersum, and P. richardsonii) and animal prey (Campeloma decisum, Dreissena polymorpha, and Dicosmoecus sp.) to determine how microphallid load affected consumption of both plant and animal prey as well as community interactions between the competing animal prey. Macrophyte consumption significantly decreased with increasing parasite load, but the number of animal prey consumed was not affected by parasite load. However, animal weight change was slightly impacted by parasite load, such that D. polymorpha weight remained constant, Dicosmoecus sp. gained about 5% of their body weight, and C. decisum lost approximately 5% of their body weight, which could indicate shifts in the competition between these species. Ov (open full item for complete abstract)

Committee: Paul Moore Ph.D. (Advisor); Steven Cady Ph.D. (Other); Curtis Blankespoor Ph.D. (Committee Member); Robert Huber Ph.D. (Committee Member); Daniel Pavuk Ph.D. (Committee Member) Subjects: Behavioral Sciences; Biology; Ecology; Parasitology

Doctor of Philosophy (PhD), Ohio University, 2022, Translational Biomedical Sciences

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. It affects 7 to 8 million people worldwide and leads to approximately 50,000 deaths per year. In vitro and in vivo studies had demonstrated that T. cruzi infection causes an imbalance in the hypothalamic-pituitary-adrenal (HPA) axis that is accompanied by a progressive decrease in growth hormone (GH) and prolactin (PRL) production. Inactivating mutations in the GH receptor gene cause Laron Syndrome (LS) in humans. The main characteristics of LS subjects are increased serum GH levels and decreased insulin-like growth factor-I (IGF-I); they are highly resistant to diabetes and cancer. Remarkably, CD incidence in these individuals is diminished despite living in highly endemic areas. No study to date has examined the effect of GH/IGF-I axis imbalance during T. cruzi infection, neither has anyone exclusively examined the absence of T. cruzi infection in LS subjects, presenting with high GH and low IGF-I serum levels. The long-term goal of this dissertation was to understand the association of clinically diminished T. cruzi infection in LS subjects. The studies presented in this dissertation aimed to investigate the effect of GH on T. cruzi infection. We first analyzed previous experimental data on T. cruzi infection that shows an HPA axis imbalance with increasing glucocorticoids (GC) and decreasing GH and PRL secretion from the pituitary. Moreover, decreased GH levels in vitro and in vivo conditions are often associated with increased susceptibility to T. cruzi infection, in contrast, treatment with GH grants protection in the host. Next, we characterized the effect of elevated GH levels on T. cruzi infection in vitro. We treated T. cruzi with different hormones including GH, IGF-I, PRL, and EGF. Results showed that in vitro GH treatment significantly reduces T. cruzi infection. Importantly, the combination of high GH and low IGF-I, as seen in LS individuals, decreased T. cruzi infection. This stud (open full item for complete abstract)

Committee: Mario J. Grijalva PhD (Advisor); John J. Kopchick PhD (Advisor) Subjects: Biology; Endocrinology; Molecular Biology; Parasitology

Honors Theses, Ohio Dominican University, 2021, Honors Theses

Leishmaniasis is a neglected tropical disease which affects millions of people every year across 6 continents. Current antileishmanial drugs have become less effective due to resistance, have a high toxicity, and are too expensive for many lower socioeconomic countries. New drug candidates can be explored and synthesized by natural drug synthesis and high throughput screening (HTS). This paper aimed to explore both methods in search of novel antileishmanial compounds. For the natural product approach, the ABC ring of berberine will be optimized to improve its antileishmanial properties. For the HTS approach, a piperazine based small molecule has been synthesized and will be evaluated for its antileishmanial properties.

Committee: Janet Antwi (Advisor); Blake Mathys (Other); Arlene Ramkissoon (Committee Member); John Marazita (Committee Chair) Subjects: Biochemistry; Biology; Biomedical Research; Chemistry; Epidemiology; Organic Chemistry; Parasitology; Pharmaceuticals; Pharmacology

Bachelor of Arts, Wittenberg University, 2019, Biochemistry/Molecular Biology

Trypanosoma brucei, a parasitic protozoan, is the etiologic agent of African Sleeping Sickness (Trypanosomiasis), a disease that threatens the lives of thousands in sub-Saharan Africa, with a 100% mortality rate in untreated cases. Transfer RNA (tRNA) is an adaptor molecule which acts as the intermediary between nucleotide and amino acid sequences, linking the information encoded in the genome to protein synthesis. Naturally occurring nucleotide modifications of tRNA are critical for translation; and therefore, essential for the viability of the organism. RNA editing involves the chemical modification of one nucleotide to another; in T. brucei, some tRNAs undergo essential editing events from adenosine to inosine. The heterodimeric enzymes, called adeonsine deaminases acting on tRNA (ADAT2/3), catalyzes the deamination of the first position of the anticodon in tRNA. Inosine in the wobble base position allows a single tRNA to decode multiple codons, implicating ADAT2/3 as a potential anti-parasitic therapeutic target. This enzyme consists of two structurally unique subunits, ADAT2 and ADAT3, both of which bind Zn2+. Given its essentiality in parasitic viability, it is important to understand the enzyme's structure and function. Since the structure of ADAT2/3 has been difficult to elucidate, a Tripartite Split-Green Fluorescent Protein (GFP) Association Assay was used to determine the correct orientation of each subunit in vitro. This assay uses GFP split into three fragments, which fluoresce when they are all in close proximity to one another. By strategically tagging the termini of the enzyme with the two small linker fragments, and incubating with the larger reporter, the correct orientation of the ADAT2/3 heterodimer can be determined on the basis of fluorescence of a reassembled GFP. To validate the results of the assay, a general cross linking experiment, followed by mass spectrometry, should also be conducted. Ultimately, elucidation of ADAT2/3 structure is imp (open full item for complete abstract)

Committee: Margaret Goodman (Advisor); Michelle McWhorter (Committee Member); Daniel Marous (Committee Member) Subjects: Biochemistry; Microbiology; Molecular Biology; Parasitology

Master of Science in Biology, Cleveland State University, 2020, College of Sciences and Health Professions

Colpodella species are free living biflagellated protists that prey on algae and bodonids in a process known as myzocytosis. Colpodella species are phylogenetically closely related to Apicomplexa. Cyst and trophozoite stages have been identified in Colpodella species, however the mechanisms of encystation and excystation are unknown. The timing and transition of the life cycle stages are unknown. In the present study I investigated the life cycle of Colpodella sp. (ATCC 50594). Colpodella sp. were grown in diprotist cultures in tissue culture flasks containing Hay medium bacterized with Enterobacter aerogenes and containing Bodo caudatus as prey. I wanted to know the timing and duration of the life cycle and the transition stages of Colpodella sp. Six time course experiments were performed to determine the duration of the life cycle stages, to investigate the transition of stages within the life cycle, and to identify the predominant cyst stage in resting cultures. In all time course experiments cells were formalin-fixed and stained with Sam-Yellowe's trichrome for visualization by light microscopy. Confocal and differential interference contrast (DIC) microscopy was performed to determine cross reactivity of Plasmodium species and Toxoplasma gondii specific antibodies against Colpodella sp. proteins and to identify cell morphology, respectively. Transmission electron microscopy (TEM) was performed to visualize the ultrastructure of Colpodella sp. Data obtained shows the duration of Colpodella sp. life cycle is thirty-six hours. Colpodella sp. were most active between 20-28 hours. Trophozoites actively swimming, feeding, forming transient cysts, and excysting were observed. Bodo caudatus excysted and encysted earlier in the culture. Previously undocumented life cycle stages identified include precyst stages, multinucleate cysts, egress of trophozoites with incomplete cytokinesis, and predominant single nuclei mature resting cysts. Multinucleate cysts of Colpodella s (open full item for complete abstract)

Committee: Tobili Sam-Yellowe Ph.D. (Advisor); Girish Shukla Ph.D. (Committee Member); Michael Walton Ph.D. (Committee Member) Subjects: Microbiology; Parasitology

Doctor of Philosophy, Case Western Reserve University, 2019, Pathology

Toxoplasma gondii is an obligate intracellular protozoan parasite that can cause disease in the brain and the eye. It is estimated that 1/3 of the population worldwide is chronically infected. After host cell invasion, T. gondii resides in a specialized compartment known as the parasitophorous vacuole (PV) that protects the parasite from being targeted for lysosomal degradation. T. gondii, additionally, manages to avoid autophagy by inducing EGFR signaling in host cells early, during host cell invasion. The brain and eye are the most clinically relevant organs in infection with T. gondii. The parasite reaches these organs hematogeneously after overcoming the blood-brain and blood-retinal barriers. Therefore, we set out to determine the role of EGFR expression in modulating T. gondii invasion to the brain and the eye. Transgenic mice with conditional expression of a dominant negative form of EGFR (DN EGFR) were infected with T. gondii. Deficiency in EGFR activation resulted in diminished parasite load and histopathology in the brain and eye. This was accompanied by reduction of parasite foci in brain endothelial cells, accumulation of LC3 structures around the parasite and parasite killing that was dependent on autophagy proteins and lysosomal enzymes. Thus, expression of EGFR in endothelial cells is an important regulator of T. gondii invasion to the brain and eye that promotes survival of the parasite. The previously described mechanisms by which T. gondii engages EGFR signaling occur early during invasion. Therefore, we hypothesized that the parasite must have an alternative mechanism to maintain EGFR signaling at later timepoints. We found that there is phosphorylation of EGFR that persists for up to 18h and is dependent on PKC/-Src signaling. Since EGFR signaling is relevant to parasite survival throughout the cycle of infection, we set out to determine whether in vivo inhibition of EGFR after pre-established infection with T. gondii led to control (open full item for complete abstract)

Committee: Carlos Subauste MD (Advisor); Donald Anthony (Committee Chair); David Canaday (Committee Member); W Henry Boom (Committee Member); Cathleen Carlin (Committee Member); Clive Hamlin (Committee Member) Subjects: Biomedical Research; Immunology; Microbiology; Parasitology; Pathology

Master of Science, The Ohio State University, 2018, Comparative Veterinary Medicine

Haemonchosis in camelids remains a challenging disease to treat, and prevention has become increasingly problematic due to widespread anthelmintic resistance. Barbervax® is an adjuvanted vaccine containing natural H-11, H-gal-GP antigens obtained from Haemonchus contortus adults via a proprietary process and solubilized in Quil A. This vaccine is approved for use in Australia, after demonstrating its safety and efficacy in sheep and goats. There are no published studies evaluating Barbervax in other ruminants/pseudoruminants such as camelids which can be parasitized with H. contortus. The vaccine utilizes a mixture of the native parasite gut mucosal membrane enzymes including H-gal-GP and H11 (in a Quil A adjuvant), involved in digesting a blood meal from the host. This study monitored the safety profile of the Barbervax® vaccine in a group of adolescent alpacas. Although designed into the original study of vaccine efficacy, the experimental infection with viable H. contortus third stage larvae could not be completed due to lack of detectable significant variation of infection following experimental challenge. Twelve alpacas (158 + 15 days) were randomized to vaccination with Barbervax® or no treatment. Three doses of Barbervax® were administered at 3 week intervals and investigators involved in animal monitoring and sample collection were blinded to the groupings. Clinical pathologic parameters were evaluated 7 days before vaccination, and 1 and 2 months post-vaccination. Daily clinical observations were made and specific observations regarding the injection site and rectal temperatures were monitored in each alpaca twice daily for 1 week following vaccination. Fecal egg counts, packed cell volume, and total protein were monitored following challenge with 1500 H. contortus larvae each on days 42, 46, and 50. An increase in rectal temperature for a duration of 2 days (range 2-4 days) was observed post-vaccination. Vaccinated alpacas were lethargic for 2-3 days follo (open full item for complete abstract)

Committee: Antoinette Marsh (Advisor); Jeffrey Lakritz (Committee Member); Andrew Niehaus (Committee Member); Greg Habing (Committee Member) Subjects: Animal Diseases; Parasitology; Veterinary Services

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2018, College of Sciences and Health Professions

Plasmodium falciparum virulence proteins and molecules required for assembly of the knobs used for cytoadherence are exported through the Maurer's clefts which are formed in the erythrocyte cytoplasm immediately following merozoite invasion into the erythrocyte host cell. Proteins participating in knob formation, cytoadherence or immune evasion include variant-surface antigens (VSAs) that are trafficked alone or chaperoned by other parasite proteins through the Maurer's clefts. In this study, recombinant PfMC-2TM was expressed using HeLa based in vitro human cell free expression system. Further, the stage specific expression, trafficking, solubility and topology of endogenous PfMC-2TM in the parasite and erythrocyte cytoplasm was investigated in 3D7 and FC-27 strains of P. falciparum. Detailed cell biological and biochemical characterizations were performed to identify the mode of PfMC-2TM export from the parasite to erythrocyte cytoplasm. Following Brefeldin A (BFA) treatment of parasites, PfMC-2TM traffic was evaluated using immunofluorescence and western blotting with antibodies reactive with PfMC-2TM. Our data shows that PfMC-2TM is sensitive to BFA treatment in the ring and trophozoite stage. Permeabilization of infected erythrocytes with streptolysin O (SLO) and saponin, showed that the N and C-termini of PfMC-2TM are exposed to the erythrocyte cytoplasm with the central portion of the protein protected in the MC membranes. PfMC-2TM was expressed as early as 4 h post invasion (hpi), was tightly colocalized with Maurer's cleft resident protein REX-1 and trafficked to the erythrocyte membrane without a change in solubility. PfMC-2TM associates with the infected erythrocyte membrane as an integral membrane protein and is seen to be transiently exposed on the infected red blood cell surface. PfMC-2TM remains insoluble upon association with the MC and erythrocyte membrane, suggestive of protein-lipid interactions with membranes of the MC and erythrocyte. PfMC-2TM i (open full item for complete abstract)

Committee: Tobili Sam-Yellowe (Advisor); Girish Shukla (Committee Member); Roman Kondratov (Committee Member); Bin Zhang (Committee Member); Severson Aaron (Other); Ronald Blanton (Other) Subjects: Biochemistry; Biology; Parasitology

Master of Sciences, Case Western Reserve University, 2018, Biology

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) forms the basis of many current malaria rapid diagnostic tests (RDTs). However, parasites lacking the pfhrp2 gene do not express the PfHRP2 protein and are, therefore, not identifiable by PfHRP2-detecting RDTs. In this study, the performance of the SD Bioline Malaria Ag P.f/Pan RDT together with pfhrp2 variation in Madagascar was evaluated. The study also evaluated pfhrp2 gene variation in PNG. Genomic DNA isolated from patient blood samples from Madagascar (n = 260) and PNG (n = 169) were subjected to molecular detection (18S rRNA PCR, followed by post-PCR LDR-FMA) for the identification of Plasmodium spp. infections. PCR amplification of the pfhrp2 gene, sequencing and gene analysis enabled studying of gene variation. PCR diagnosis showed that 28.8% (75/260) in Madagascar and 81.1% (137/169) in PNG had Plasmodium infections. 94.6% (71/75) and 91.2% (125/137), of the infections were P. falciparum in Madagascar and PNG, respectively. Compared to molecular detection, the sensitivity and specificity of the RDT (in Madagascar) for P. falciparum detection were 87% and 89%, respectively. From randomly selected pfhrp2 gene-positive samples, 16 pfhrp2 gene sequences from Madagascar and 18 pfhrp2 gene sequences from PNG were generated. Although extensive variations of the pfhrp2 gene were observed in both countries, this study showed that there was no indication of pfhrp2 deletion. The study also did not observe a clear correlation between pfhrp2 sequence structure and RDT detection rates. Although the absence of pfhrp2 deletion from the samples screened here is encouraging, continued monitoring of the efficacy of RDTs currently used in Madagascar and PNG is warranted.

Committee: Peter Zimmerman A. (Advisor); Daniel Tisch J. (Committee Member); Emmitt Jolly R. (Committee Member); Hillel Chiel (Committee Chair) Subjects: Biology; Epidemiology; Genetics; Molecular Biology; Parasitology

Doctor of Philosophy, Case Western Reserve University, 2017, Biology

Schistosomiasis is a disease of global scale with more than 240 million people infected and more than 700 million people at risk of infection. Historically, treatment of this disease has been with one primary drug, praziquantel. Due to this, concern for resistance developing in the parasitic worms that cause this disease has risen. The three species of blood flukes responsible for the majority of the schistosomiasis disease burden are Schistosoma mansoni (S. mansoni), S. japonicum, and S. haematobium. These trematodes have a complex life cycle in which they undergo distinct morphological changes during the transitions from free-swimming to invertebrate and vertebrate host stages. The molecular factors and mechanisms which regulate their development during these metamorphoses are widely unknown. A better understanding of the basic biology of these worms and the factors which drive their development will be critical in the process of identifying new treatment targets or developing preventative measures. Forkhead Box (Fox) proteins, are a family of transcription factors that play key roles in a variety of biological processes including development, metabolism, stress tolerance, and lifespan and have been identified in a number of organisms. The major goal of this thesis is to identify and characterize the complement of forkhead box genes found in the S. mansoni genome. Here, we have identified 15 Fox genes, two with multiple isoforms, which were classified into 11 classes (SmFoxA, SmFoxC, SmFoxD, SmFoxF, SmFoxG, SmFoxJ, SmFoxK, SmFoxL, SmFoxN, SmFoxO, and SmFoxP) using bioinformatics and a phylogenetic comparison to mouse, fly, nematode, and yeast forkhead proteins. Additionally, the expression pattern during the sporocyst, cercarial, 4h schistosomula, and adult stages was determined for each gene using absolute quantitative PCR. To test functionality, SmFox-GAL4 DBD fusion proteins were tested in a modified yeast one-hybrid to determine their ability to activ (open full item for complete abstract)

Committee: Emmitt Jolly (Advisor) Subjects: Biology; Parasitology

Doctor of Philosophy, The Ohio State University, 2016, Comparative and Veterinary Medicine

Surveillance for wildlife diseases is critical to our understanding of the emergence, transmission, persistence and control of infectious diseases at the interface of humans, domestic animals, and wildlife populations. Neospora caninum is a protozoan parasite capable of infecting a wide range of canid and ungulate species. The importance of the disease relates to economic losses, mainly derived from endemic or epidemic abortions in cattle. In the United States, coyotes and dogs are believed to be the main definitive hosts and white-tailed deer and cows are the main intermediate hosts. Our overall aim was to better understand the wildlife-livestock interface of N. caninum in natural settings. First, we estimated the true prevalence of N. caninum in three ruminant species by using Bayesian inference. We identified and discussed differences between apparent and true prevalence (TP). Differences in TP for some species suggest differences in the epidemiology of N. caninum for these co-located populations. Second, we evaluated the environmental phase of N. caninum shed in wild canid scats. Results suggested that the role of this environmental phase in the transmission to ruminants is likely minor. Finally, we evaluated the role of host species heterogeneity in the epidemiology of N. caninum circulating in a community. We identified differences in the patterns of immunity, age structure, and maternal and/or fetal antibody duration in three intermediate (ruminant) host species. Also, we estimated the species-specific contributions to the persistence of this pathogen in a community. This research was approached from the One Health perspective and provided a better understanding of N. caninum dynamics at the wildlife-livestock interface in an ecosystem.

Committee: Rebecca Garabed (Advisor); Mark Moritz (Committee Member); Barbara Wolfe (Committee Member); William Saville (Committee Member) Subjects: Animal Diseases; Applied Mathematics; Biology; Biostatistics; Computer Science; Conservation; Cultural Anthropology; Ecology; Environmental Health; Epidemiology; Geographic Information Science; Health Sciences; Livestock; Parasitology; Veterinary Services; Wildlife Conservation; Zoology