Browsing by Author "Bosco-Lauth, Angela, committee member"
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Item Embargo Furthering the development of the Jamaican fruit bat as an animal model for immunology(Colorado State University. Libraries, 2023) Burke, Bradly E., author; Schountz, Tony, advisor; Henao-Tamayo, Marcela, advisor; Zabel, Mark, committee member; Bosco-Lauth, Angela, committee member; Hansen, Tod, committee memberBats are the only flying mammals capable of powered sustained flight and encompass over 1,400 species. Bats interconnect important ecological services, agricultural crop services, agricultural husbandry health, and human health. The order of chiroptera is largely understudied for immunological research even though bats are reservoirs for viruses that are transmissible to livestock and humans including, henipaviruses, filoviruses, coronaviruses, and lyssaviruses. In 2006 Colorado State University established a breeding colony of Jamaican fruit bats (Artibeus jamaicensis) for use in the study of bat-borne viral infections. Establishment of a bat colony is only the first step to the development of an animal model. The next step to develop an animal model is to elucidate the immunological systems and functionality of them. To elucidate the immunological systems, bat specific reagents are needed to characterize and perform hypothesis driven research. However, the paucity of bat specific reagents largely limits hypothesis driven research. Antibodies and cell lines are foundational reagents to immunology and virology. Antibodies are integral reagents used for many immunological and biochemical assays: ELISAs, ELISPOTs, western blots, cytometric bead assays, magnetic bead pull-down, surface plasmon resonance, microscopy, in vivo cellular depletion, and flow cytometry. Cell lines allow for in vitro assays and the propagation of viruses to be performed. The production and validation of Jamaican fruit bat specific antibodies targeting CD3γ, CD4, and CD8α epitopes for the investigation of cytotoxic CD8 T cells, and CD4 T helper cells are described in this study. Furthermore, the identification and validation of commercially available cross-reactive antibodies targeted to epitopes for various proteins and glycolipids: asialo GM1, CD3ε, CD8α, CD19, CD34, CD40, CD44, CD45, CD80, CD104, CD154, CD161, and MHC-II. This work has described their use in highly quantitative flow cytometric analysis, enrichment of cell populations by fluorescently activated cell sorting, and highly qualitative anatomical data by microscopy. This study has identified a novel CD19+CD3+ T cell population under homeostatic conditions of the Jamaican fruit bat immune system that has not been identified in humans, mice, or other bat species under a homeostatic state. The use of anti-asialo GM1 in vivo treatment of Jamaican fruit bats to target natural killer cells is also described in this work. Lastly the construction of plasmids for the production of Jamaican fruit bat growth factors: epidermal growth factor, wnt3a, R-Spondin-2, noggin, and gastrin are described for their use in culturing Jamaican fruit bat primary cells – especially gastrointestinal crypt stem cells. Validation of cellular markers and reagents is a crucial first step in the investigative process that allows for the generation of informed conclusions – in any study. Furthermore, validation of cellular markers holds a higher level of imminent need and accuracy in underdeveloped animal models. This work provides a framework for other researchers in the advancement of underdeveloped animal models for immunology to more rigorously test antibody cross-reactivity. Furthermore, this work highlights the need to build a robust body of literature of cross-reactive antibodies for underdeveloped animal models.Item Embargo SARS-CoV-2 evolution and within-host variation in nonhuman animals(Colorado State University. Libraries, 2024) Bashor, Laura, author; VandeWoude, Sue, advisor; Stenglein, Mark, committee member; Bosco-Lauth, Angela, committee member; Sloan, Dan, committee member; Gagne, Roderick B., committee memberThe COVID-19 pandemic originated following spillover of SARS-CoV-2 from non-human animals into humans. Despite concentrated efforts before and after the pandemic, current research is constrained by the impracticality of witnessing initial host shift events and transmission dynamics that shape infectious disease emergence. SARS-CoV-2 transmission from humans to a range of domestic and wild species has been well documented; furthermore, spillback into humans from white-tailed deer, mink, hamsters, domestic cats, and lions has also been reported. SARS-CoV-2, like other RNA viruses, has the ability to adapt rapidly following host shifts. These cross-species transmission events can accelerate novel variant emergence through selection for genetic variation that improves virus fitness in a novel host environment. To evaluate the possibility that cross-species transmission accelerates SARS-CoV-2 evolution and variant emergence, we employed next-generation sequencing of viral genomes recovered from experimentally and naturally infected animals to characterize within-host virus populations. We demonstrated the use of experimental exposure studies as a controlled system to test hypotheses surrounding SARS-CoV-2 adaptation in cats (Felis catus), dogs (Canis lupus familiaris), hamsters (Mesocricetus auratus), ferrets (Mustela putorius furo), deer mice (Peromyscus maniculatus), bushy-tailed woodrats (Neotoma cinerea), Brazilian free-tailed bats (Tadarida brasiliensis), striped skunks (Mephitis mephitis), red foxes (Vulpes vulpes) and mule deer (Odocoileus hemionus). We also evaluated publicly available sequencing data from infected felids, and investigated within-host dynamics in natural infections of Amur tigers (Panthera tigris altaica), African lions (Panthera leo), and spotted hyenas (Crocuta crocuta) in a zoo environment. Our initial work investigated SARS-CoV-2 evolution across three passages in Vero cells and experimentally infected cats (n = 6), dogs (n = 3), hamsters (n = 3), and a ferret (n = 1). We observed the rapid selection and fixation of five SARS-CoV-2 mutations in Vero cells, followed by their reversion in dogs, cats and hamsters 1-3 days post-infection. We noted 14 emergent variants across the SARS-CoV-2 genome, including increased variation in the SARS-CoV-2 spike protein. Emergent variants included mutations not detected in the original virus stocks used for inoculation, and several defining mutations of variant lineages of concern in humans. Finally, we noted increased signs of adaptation in dogs, which did not shed infectious virus, including six nonsynonymous mutations in the SARS-CoV-2 open-reading frames (ORFs) encoding proteins for virus replication. In particular, this work underscored the potential for accelerated viral evolution in cell culture systems used commonly in virological research. This work has been published and represents Chapter 2 of this dissertation. Our next study built upon this work by investigating SARS-CoV-2 evolution in three experimental cohorts of domestic cats (n=23) infected through direct inoculation and cat-to-cat contact transmission. We observed high numbers of within-host variants in SARS-CoV-2 genomes recovered from cats compared to what is documented in humans, over half of which were nonsynonymous changes. The number of variants detected was positively correlated with the experimental dose of virus inoculum, and fewer variants were observed in contact cats. Similar to the previous study, mutations occurring at the same positions as defining VOC mutations, and signatures of positive selection in the viral spike (S) gene were observed. Our concurrent analysis of publicly available SARS-CoV-2 sequences showed no evidence for independent evolutionary trajectories associated with natural infections of domestic cats or other felids, and confirmed susceptibility of felids to the breadth of variants circulating in human populations. This work has also been published and represents Chapter 3 of this dissertation. We subsequently investigated SARS-CoV-2 evolution in longitudinal samples collected from Amur tigers (n=2), African lions (n=11), and spotted hyenas (n=4) infected during an outbreak at the Denver Zoo. Longitudinal nasal swabs were collected from infected individuals over an approximately three-month sampling period. We determined that the outbreak was caused by a single introduction of the Delta sublineage AY.20, which was a rare variant circulating in human populations at the time. We inferred a transmission chain from tigers to lions to hyenas, which was consistent with the appearance of clinical signs in infected animals. We observed expansion and diversification of within-host virus populations, and signatures of both purifying and positive selection. The strongest signs of positive selection were evident in the viral nucleocapsid (N) gene, and in viruses recovered from hyenas. Four candidate species-specific adaptive mutations, two of which are in the N gene, were identified in lions and hyenas (N A254V) and hyenas alone (ORF1ab E1724D, S T274I, and N P326). This work is presented in Chapter 4 of this dissertation. In Chapter 5, we evaluated a large dataset of peridomestic wildlife species experimentally infected with two SARS-CoV-2 variants, WA01 and Delta. Study species included deer mice (n=3), bushy-tailed woodrats (n=3), Brazilian free-tailed bats (n=4), striped skunks (n=5), red foxes (n=9), and mule deer (n=6). Distinct dynamics were observed in within-host virus populations recovered from WA01- and Delta- infected animals. This included increased within-host variation, relative effective population size, and genomic signatures of positive selection in WA01 animals. In contrast to our first study in domestic dogs, Brazilian free-tailed bats, which also did not shed infectious virus, did not show increased signs of adaptation. We also observed a potential host barrier to infection in skunks and one fox, followed by the emergence of potential de novo mutations. Six novel mutations were also detected in contact-exposed mule deer. Our findings suggest that mule deer populations, similar to what has been documented in closely related white-tailed deer, should be investigated for accelerated SARS-CoV-2 evolution. Collectively, our work reveals the unique dynamics of SARS-CoV-2 evolution and transmission in both naturally- and experimentally- infected felids. We observed rapid viral adaptation both in vitro and in vivo, highlighting advantages and limitations of experimental animal infections for studies of viral evolution. In each study, we used publicly available data to contextualize our experimental data and identify broader patterns. Furthermore, we identified specific SARS-CoV-2 mutations and genomic regions under selective pressures across a range of animal species, setting the groundwork for future mechanistic studies. Our findings underscore the importance of a One Health approach to understanding SARS-CoV-2 evolution, and the need for surveillance in animal populations.Item Open Access Strategies to maintain market access for pork and enhance functionality of beef proteins(Colorado State University. Libraries, 2023) Cochran, Hannah, author; Martin, Jennifer, advisor; Bosco-Lauth, Angela, committee member; Garry, Franklyn, committee member; Roman-Muniz, Noa, committee memberAfrican swine fever is a high-consequence foreign animal disease endemic to sub-Saharan Africa and the island of Sardinia. The U.S. is the world's third largest pork producer, and ASF introduction would severely disrupt the pork supply chain, emphasizing a need to protect market access for U.S. proteins. However, niche producers raising swine intended for exhibition may not follow stringent biosecurity protocols and livestock show circuits may promote untracked animal movement across the country, potentially exacerbating virus spread in the event of ASF incursion into the U.S. Two Qualtrics surveys designed to evaluate knowledge, understanding, and perceptions of ASF and biosecurity principles of youth swine exhibitors and adults involved in the exhibition swine industry were distributed via flyers, emails, and canvassing at livestock shows. Youth exhibitors (age 21 and under) answered questions assessing their knowledge and provided basic demographic information, including their home state and states to which they traveled for exhibitions. Adult respondents answered the same questions assessing their knowledge and provided information on their time involved in the swine industry and number of shows attended by the youth they advise (if any). Youth respondents (n = 127) lived in 14 states and exhibited in 23 states, with 35% and 28% holding membership in state and national swine organizations, respectively. When provided with a list of ASF clinical signs, 34 individuals (26.9%) correctly identified all symptoms. Twenty-nine individuals (23%) incorrectly responded that ASF has been found in the U.S., and ten (7.9%) believed the virus cannot spread between pigs. Increased biosecurity understanding in youth exhibitors showed a significant relationship with an increase in years involved (p<0.05). Adult respondents (n = 211) had been involved in the swine industry for an average of 21 years, and the youth they advised attended 14 exhibitions in an average year. Nearly all adults (90.5%) identified direct contact with infected animals as a method of ASF transmission, while far fewer (36.39%) identified animal feed as a possible mechanism of transmission. These responses indicate highly varied knowledge of symptoms, routes of transmission, and biosecurity recommendations. Youth membership in state or national swine organizations offers a route for outreach and educational activities to enhance foreign animal disease preparedness, and adult presence at swine exhibitions allows for a wide variety of programming for all ages to better serve all levels of understanding. Fluctuations in the beef supply chain due to COVID-19 triggered discussions on methods to fully utilize edible proteins from beef carcasses, such as collagen. One potential method is the addition of collagen powder to beef frankfurters to replace a fraction of lean grind. The inclusion of NOVAPRO® collagen powder to beef franks at three hydration levels resulted in no significant differences (p>0.05) in water activity, pH, or shear force values between the treatment groups. Additionally, trained sensory panelists did not discern differences between treatment or control samples when asked to rate attributes that included beef flavor intensity, seasoning intensity, springiness, and mouth coating, indicating that NOVAPRO® powder could be added to processed meat products to reduce costs without compromising product quality.Item Open Access Studying the impact of air pollution and pesticide mixtures on respiratory health in Fresno and Tulare counties of central California(Colorado State University. Libraries, 2022) Hughes, Matthew Lawrence, author; Magzamen, Sheryl, advisor; Anderson, Brooke, committee member; Schaeffer, Joshua, committee member; Bosco-Lauth, Angela, committee memberResidents of California's Central Valley are exposed to some of the worst air quality in the United States, as well as high levels of pesticides owing to the region's large agricultural economy. There is ample evidence that exposure to air pollution is associated with adverse respiratory health outcomes, and some evidence from occupational and community-based studies that exposure to pesticides has negative impacts on respiratory health as well. Epidemiologic research on air pollution and pesticides often considers these exposures one at a time in relation to health outcomes, but humans are exposed to pollutants simultaneously in mixtures. In this study we used multiple linear regression models to look at linear relationships of three criteria air pollutants and biomarkers of organophosphates (dialkyl-phosphates or DAPs) with urinary leukotriene E4 (LTE4), a biomarker of respiratory inflammation, in participants in four Central California communities (n=80). We then used Bayesian Kernel Machine Regression models to study these criteria air pollutants and DAPs as a mixture and determine if this mixture had a relationship with respiratory health in this population. We also studied these relationships at two different times of the year (January and June) to study whether and how this relationship between an air pollution-pesticide mixture and the respiratory health outcome changed seasonally. Our multiple linear regression models revealed that dimethyl-phosphates had a statistically significant association with respiratory health in January, which suggests that LTE4 can be used as a biomarker for respiratory inflammation in populations with low asthma prevalence. The results of our BKMR analysis were not statistically significant but did suggest interactions between the exposures in our air pollution-pesticide mixture. Despite a small sample size, this study adds to the limited research on environmental mixtures, and the effects of pesticide exposure on respiratory health.