Scholarship & Research
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Item Study of diverse host immune responses to viral and bacterial pathogens(Montana State University - Bozeman, College of Agriculture, 2023) Plewa, Jack Bruno; Chairperson, Graduate Committee: Mark Jutila; This is a manuscript style paper that includes co-authored chapters.Brucella abortus is the bacterium that causes brucellosis, an infection transmitted from cattle to people, often through consumption of raw milk and contact with aborted materials. With antibiotic resistance on the rise, phage therapy for bacterial infection may become a useful approach. The direct effects of phage on mammalian cells is important to understand, yet understudied. In vivo delivery of low phage MOI to the mouse lung was more effective at diminishing Brucella burden than higher doses of phage. In an in vitro model of intracellular Brucella infection, low phage MOI was capable of minimizing human THP-1 monocyte infection, but, unexpectedly, use of higher phage MOI diminished this effect. We hypothesized that recognition of these phage preparations may induce an antiviral immune suppressive response that may counteract their anti-bacterial effects. Indeed, when the type I IFN signaling pathway was disrupted in mice, phage treatment was more effective. However, when attempting to induce type I IFN in vitro using both human monocyte and mouse macrophage cell lines, we were unable to stimulate expression of type I IFN with Brucella phage, including in response to a combination of phage and bacteria. We then examined the effect of phage treatment on macrophage cell surface markers that are indicative of activation/differentiation. Interestingly, while Brucella LPS induced expression of CD71 and CD206, the addition of phage suppressed upregulation of these markers. Our discovery of immune suppressive effects of Brucella bacteriophage is an important consideration for using phage as a treatment.Item Evaluating host plant preference and pheromone attract and kill as strategies to manage pea leaf weevil Sitona lineatus (L.) (Coleoptera: Curculionidae) in Montana(Montana State University - Bozeman, College of Agriculture, 2022) Kiju, Pragya; Chairperson, Graduate Committee: Michael A. Ivie and Kevin Wanner (co-chair); This is a manuscript style paper that includes co-authored chapters.Pea leaf weevil (Sitona lineatus) is the most common insect pest of field peas grown in Montana. Montana is the number one producer of field pea in the US. Currently, Montana producers spray insecticides at least once or twice during the spring growing season to avoid leaf damage inflicted by pea leaf weevil adults and larvae. Complete reliance on insecticides may, however, raise the risk of pea leaf weevil populations developing genetic resistance. Therefore, this project focuses on development of alternative pulse insect pest management strategies such as pheromone-based attract and kill and host plant preference. For the attract and kill strategy 6 different treatments compared combinations of aggregate pheromone (4-methyal-3,5- heptanedione) alone in two different forms; septa and pellet, pheromone with granular insecticide (Deltamethrin). To determine the effect of host variety on adult feeding preference, 10 field pea, 2 faba bean, 2 lentil and 2 chickpea varieties were assessed for feeding damage. Crescent shaped notches were counted on 10 individual plants per replicate plot. The average number of larvae within the nitrogen fixing root nodules of each variety was also recorded on 5 randomly selected individual plants per plot. Faba bean was the most preferred host while lentil and chickpea suffered almost no feeding damage. 'Delta' and 'Lifter' field pea varieties appeared to be preferred over 'DS Admiral' and 'AC Agassiz' varieties. Given the significant level of feeding on all pea and faba bean cultivars further evaluation of low-cost pheromone traps are essential to establishing an IPM control approach.Item Investigating the regulation of virulence by Sae in Staphylococcus aureus(Montana State University - Bozeman, College of Agriculture, 2020) Collins, Madison Paige Martin; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Ranjan K. Behera, Kyler B. Pallister, Tyler J. Evans, Owen Burroughs, Caralyn Flack, Fermin E. Guerra, Willis Pullman, Brock Cone, Jennifer G. Dankoff, Tyler K. Nygaard, Shaun R. Brinsmade and Jovanka M. Voyich were co-authors of the article, 'The accessory gene saeP of the saeR/S two-component gene regulatory system impacts Staphylococcus aureus virulence during neutrophil interaction' in the journal 'Frontiers in microbiology' which is contained within this dissertation.; Kyler Pallister and Jovanka M. Voyich were co-authors of the article, 'Differential analysis of host/pathogen RNA expression via next generation sequencing reveals Staphylococcus aureus utilizes saeR/S-mediated factors to inhibit human neutrophil functions following phagocytosis' which is contained within this dissertation.Staphylococcus aureus (S. aureus) is a common commensal bacterium known to colonize, at minimum, 30% of the human population. It is also capable of causing a range of diseases that span from minor skin- and soft-tissue infections to life-threatening diseases. The diversity of S. aureus infections is due to the ability of the bacteria to sense and respond to environmental change. Virulence regulation in S. aureus can be attributed to the use of two-component gene regulatory systems (TCS). TCS can sense a variety of encounters including: antibiotics, heat stress, or immune cell encounter. Neutrophils are a key leukocyte involved in bacterial clearance in the human host. It follows that S. aureus has evolved mechanisms to sense and respond to neutrophils. The Sae TCS, is immediately up-regulated after neutrophil phagocytosis and has been demonstrated to be critical in the success of S. aureus both in vitro and in vivo. SaeS, the histidine kinase, and the respective response regulator, SaeR, are established components of the Sae TCS and their importance during neutrophil evasion and pathogenesis is well established. However, little is known about two accessory genes, saeP and saeQ. Results described herein using human neutrophil and murine models of infection provide evidence that SaeP modulates the Sae-mediated response of S. aureus against human neutrophils and suggest that saeQ and saeP together impact pathogenesis in vivo. To identify additional host and pathogen factors important during neutrophil interaction, we used differential analysis of host/pathogen RNA expression via Next Generation Sequencing to define the influence of SaeR/S on the host-pathogen transcriptome following neutrophil phagocytosis. Results determined that in the early stages of S. aureus infection, SaeR/S-dependent factors significantly modulate neutrophil processes involved in several pathways including autophagy, TNF-alpha signaling, and NF-kappaB signaling. These results suggest S. aureus uses SaeR/S-regulated virulence factors to hijack human neutrophil function at the transcriptional level to inhibit proper killing by neutrophils and allow for S. aureus persistence within the host.Item Population dynamics of wheat stem sawfly, Cephus cinctus Norton, in barley in Montana(Montana State University - Bozeman, College of Agriculture, 2020) Achhami, Buddhi Bahadur; Chairperson, Graduate Committee: David K. Weaver; Gadi V. P. Reddy, Jamie D. Sherman, Robert K. D. Peterson and David K. Weaver were co-authors of the article, 'Antixenosis, antibiosis, and potential yield compensatory responses in barley cultivars exposed to wheat stem sawfly under field conditions' which is contained within this dissertation.; Gadi V. P. Reddy, Jamie D. Sherman, Robert K. D. Peterson and David K. Weaver were co-authors of the article, 'Effect of precipitation and temperature on larval survival of Cephus cinctus (Hymenoptera: Cephidae) in barley cultivars' which is contained within this dissertation.; Robert K. D. Peterson, Jamie D. Sherman, Gadi V. P. Reddy and David K. Weaver were co-authors of the article, 'Multiple decrement life tables of Cephus cinctus Norton (Hymenoptera: Cephidae) across a set of barley cultivars: the importance of plant defense versus cannibalism' which is contained within this dissertation.; Gadi V. P. Reddy, Megan L. Hofland, Robert K. D. Peterson, Jamie D. Sherman and David K. Weaver were co-authors of the article, 'Host selection and oviposition behaviors of Cephus cinctus (Hymenoptera: Cephidae) in barley' which is contained within this dissertation.Wheat stem sawfly (WSS) is an economically devastating pest of cereals grown in North America. The larva is the only feeding stage and remains confined within a host stem until it emerges as an adult the following year. This limited mobility increases larval vulnerability to mortality factors when host plant traits are hostile to survival. For instance, larval mortality is greater in barley than in solid stemmed wheat cultivars known to be resistant. Both solid stemmed wheat and barley kill neonates via host plant resistance traits. Traditionally, barley was recommended as an alternative rotational crop to prevent WSS outbreaks in wheat crops. There is limited data available regarding barley host plant resistance and questions persist. Has barley resistance changed over time? What is the impact of larval feeding injury on grain yield of barley? Do females display similar oviposition behaviors across barley cultivars that vary in susceptibility? To answer these questions. I conducted field experiments to assess resistance and possible tolerance to WSS in eight barley cultivars. Based on the number of eggs, 'Hockett' was the most attractive cultivar to WSS female (less antixenosis), while 'Craft' and 'Celebration' killed the greatest number of neonates due to antibiosis. Multiple decrement life table studies revealed that plant defense and cannibalism were two major causes of larval mortality. We measured greater yield in infested stems with dead larvae (potential tolerance) than for cut stems and both were greater than uninfested stems in all cultivars except 'Celebration'. A greenhouse study revealed that females preferred 'Hockett' over 'Craft' in frequencies of oviposition behaviors and numbers of eggs deposited. Additionally, a greater amount of the WSS attractant (Z)-3-hexenyl acetate was found in aerations from 'Hockett' plants than from 'Craft.' The amount of defensive compound linalool was greater in aerations from 'Craft' than from 'Hockett.' These results suggest that barley cultivars are equipped with varying levels of antibiosis, antixenosis, and tolerance traits against WSS. Thus, we can exploit these traits in the development of cultivars which can reduce WSS populations and decrease economic loss caused by this species.Item Quantitative assessment of Myxobolus cerebralis viability and infective success in the salmonid host(Montana State University - Bozeman, College of Agriculture, 2003) Hudson, Crystal JeanItem B cell responses in the gut and mesenteric lymph nodes of mice to infection with Eimeria falciformis(Montana State University - Bozeman, College of Agriculture, 1985) Nash, Patricia VargaItem Purification and characterization of Pseudomycin, a phytotoxin and antimycotic produced by Pseudomonas syringae(Montana State University - Bozeman, College of Agriculture, 1990) Harrison, Leslie AnnItem Proteomic and systems biology analysis of the response of monocytes to infection by Coxiella burnetii and exposure to innate immune adjuvants(Montana State University - Bozeman, College of Letters & Science, 2010) Shipman, Matthew Richard; Chairperson, Graduate Committee: Edward Dratz.Coxiella burnetii is an obligate intracellular pathogen that infects human monocytes, specifically inhabiting the phagolysosome. C. burnetii is a potential bioterror agent and is classified by the National Institute for Allergies and Infectious Diseases (NIAID) as a category B pathogen. This bacterium is remarkably infectious, requiring as little as one bacterium to cause infection. We used phase II C. burnetii, an avirulent laboratory strain that acts as a model for wild type phase I strains. Our research was directed towards a deeper understanding of the monocyte proteome in response to a) infection by phase II C. burnetii, and b) exposure to immune adjuvants known to increase monocyte resistance to infection by C. burnetii. Monomac I cells were infected with phase II C. burnetii and aliquots were taken at 24, 48, and 96 hours postinfection. Experiments with immune adjuvants that increase monocyte killing of C. burnetii, involved Monomac I cells treated with Securinine, E. coli lipopolysaccharide (LPS), and monophosphoryl lipid A (MPL). Securinine is a GABA A receptor antagonist that is being developed at Montana State University for biodefense purposes, and triggers an innate immune response that differs from classic Toll-like receptor (TLR) stimulation of innate immunity represented by LPS and MPL. We employed multiplex 2D gel electrophoresis (m2DE) using ZDyes, a new generation of covalent fluorescent protein dyes being developed at Montana State University, coupled with MS/MS analysis and bioinformatics to determine the proteome changes in Monomac I cells in response to the conditions described above, and to develop a preliminary mechanistic model using a systems biology approach to account for the observed changes and propose multiple testable hypotheses to focus downstream research efforts. We also tested the effects on Monomac I cells infected with phase II C. burnetii +/- Securinine. We observed a high proportion of cell death in the + Securinine samples, using a dosage of Securinine higher than the optimal effective dosage. The information derived from this experiment will be useful in monitoring the tendency towards cell death in Securinine treated samples both from C. burnetii infected monocytes and other cell types (e.g. neurons) that contain GABA A receptors.