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Item Microbiomes and zoonoses: dynamics of the black flying fox (Pteropus alecto) gastrointestinal microbiome(Montana State University - Bozeman, College of Agriculture, 2022) Jones, Devin Nicole; Chairperson, Graduate Committee: Raina K. Plowright; This is a manuscript style paper that includes co-authored chapters.Land-use change is increasingly recognized as a driver of spillover of zoonotic pathogens. Australian black flying foxes (Pteropus alecto) are experiencing extensive loss of habitat which reduces available food, particularly in winter. Hendra virus (HeV, family: Paramyxoviridae) was isolated from horses and humans in 1994 and P. alecto was later identified as the reservoir host. As habitat loss threatens these bat populations, and Hendra virus continues to spill over to horses annually, it is important to understand factors that influence bat health and viral shedding. Because gastrointestinal tract (GIT) microbiomes are important for host health and are understudied in flying foxes, the goal of this research was to understand the natural dynamics of the P. alecto GIT microbiome and its associations with diet, body composition, markers of inflammation, and viral shedding. We sampled Pteropus alecto near Brisbane from 2018-2020. We captured bats returning from foraging and collected rectal swabs to determine the GIT microbiome using 16S rRNA amplicon sequencing. In addition to feces for dietary analysis, we also collected samples to measure health and infection, including blood to measure neutrophil-to-lymphocyte ratios, urine to detect Hendra virus, and bioelectrical impedance analysis to measure body fat. These data enabled us to determine how the P. alecto GIT microbiome varied within individuals over time and in the context of physiological, ecological, and dietary shifts. Lastly, we asked if we could predict health outcomes using the GIT microbiome. We found that P. alecto GIT microbiomes are highly dynamic over time, through different life stages, between foraging strategies, and that the type of diet is associated with GIT microbiome diversity. Bats consuming native foods had lower GIT microbiome diversity compared to those consuming introduced and cultivated foods. Despite associations between body fat and HeV infection, the GIT microbiome was not able to predict these health outcomes. These results suggest that P. alecto GIT microbiomes are highly dynamic and may not contribute significantly to host health. Future research should incorporate more health metrics or other approaches to microbiome profiling to determine if the GIT microbiome could be used as a biomarker of health.Item Using gastrointestestinal organoids to study infectious diseases in humans and bats(Montana State University - Bozeman, College of Agriculture, 2021) Hashimi, Marziah; Chairperson, Graduate Committee: Diane Bimczok; This is a manuscript style paper that includes co-authored chapters.The gastrointestinal epithelium plays a critical role in protecting the gastrointestinal mucosa from invading microorganism such as bacteria or a viruses. Helicobacter pylori (H. pylori) infection of human gastric epithelium causes gastric cancer, which is the third leading cause of cancer-related mortality worldwide. Dendritic cells (DCs)--which are antigen presenting cells--are responsible for the activation of T cells. However, the mechanism by which DCs are recruited to the gastric epithelium is still unknown. We hypothesized that the DCs are recruited to the gastric epithelium in a chemokine- dependent manner. For my thesis work, I utilized human primary gastric epithelial organoids cells to test this hypothesis and evaluate the recruitment of DCs to the epithelium under normal conditions and upon H. pylori infection. Using monocyte-derived DCs in a chemotaxis assay, I showed that these cells are recruited to H. pylori-infected organoid supernatant. I showed that this recruitment is chemokine- dependent, as it was significantly decreased when a chemokine receptor inhibitor was included in the chemotaxis assay. COVID-19 is caused by severe respiratory syndrome coronavirus-2 (SARS-CoV-2). In addition to respiratory symptoms, COVID-19 patients can also have diarrhea and vomiting, indicating that SARS-CoV-2 may infect the gastrointestinal tract. Bats are thought to be the natural reservoirs for SARS-CoV-2, however there is no known bat gastrointestinal model to study SARS-CoV-2 infection. In the second part of my thesis, I developed Jamaican fruit bat (JFB), Artibeus jamaicensis) gastrointestinal organoids (JFB organoids). I successfully developed organoids from JFB stomach, proximal and distal intestine. I showed via histology and gene expression that developed organoids do indeed recapitulate their corresponding tissues from which they were derived. I also tested whether the JFB distal intestinal organoids were susceptible to SARS-CoV-2 infection. While they do not support the active replication of SARS-CoV-2 infection, they did show antiviral and pro-inflammatory responses. My results also showed that SARS-CoV-2 does not induce programmed cell death in the organoids.Item Effect of landscape fragmentation on bat population dynamics and disease persistence in Uruguay(Montana State University - Bozeman, College of Agriculture, 2021) Botto Nunez, German; Chairperson, Graduate Committee: Raina K. Plowright; This is a manuscript style paper that includes co-authored chapters.The transmission of pathogens into novel host species, a process known as spillover, requires a series of conditions to align in space and time. A series of imperfect barriers prevent the jump of pathogens from one species to others. These may include the distribution and abundance of the primary host, the survival of the pathogen in the environment and the susceptibility of the recipient host to the pathogen. Only when permissive conditions align in time and space can the spillover occur. Spillovers may be relatively rare events and the understanding of the dynamics of the barriers is constrained by the ability of detecting and analyzing such events. Systems where spillover does not occur, despite apparent presence of all required conditions, provide an opportunity to understand barriers preventing inter-species transmission. Vampire bat-borne rabies in Uruguay provides such an opportunity. Despite large and stable livestock density, presence of vampire bats, and circulation of the virus in close proximity, the country did not experience livestock rabies outbreaks until 2007. Here we combined historical review, field sampling, and statistical and mathematical modeling to understand the factors driving the emergence of rabies in Uruguay in 2007 and the previous absence of the disease. Our results suggest that rabies outbreaks in the country are spatially and temporally associated with fragmentation of grasslands. We showed that proposed increased connectivity among colonies, in response to fragmentation, is sufficient to explain longer persistence of the virus in the bat colonies, allowing more opportunities for virus transmission to livestock. We showed that connectivity has a strong effect on rabies persistence and that reproductive seasonality and population turnover have only marginal effects compared to connectivity. As connectivity driven by shared feeding areas might not be detectable by genetic analyses of the bats, we proposed the use of a widespread virus persistently infecting bats as a marker to trace connectivity across colonies. Combined, the results presented here provide tools that can be applied to intervene and apply countermeasures to prevent spillover.