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Browsing by Author "Lappin, Michael R., advisor"

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    Assessment of novel strategies for the prevention and treatment of feline upper respiratory tract infections in shelters and feline herpesvirus-1 in laboratory settings
    (Colorado State University. Libraries, 2019) Contreras, Elena T., author; Lappin, Michael R., advisor; Olea-Popelka, Francisco J., committee member; Dow, Steven W., committee member; Mayo, Christie E., committee member; Veir, Julia K., committee member
    Feline upper respiratory tract infection (URI) and its pathogens are ubiquitous in the feline population. Most URI cases are due to viral infections with feline herpesvirus-1 (FHV-1) and/or feline calicivirus (FCV) with secondary bacterial infections. After acute exposure to FHV-1, most cats develop persistent, latent infections with reactivation particularly during times of stress and immune suppression. Clinical signs including ocular and nasal discharge, sneezing, conjunctivitis, anorexia, lethargy, and pyrexia can vary in severity from mild and transient to severe and life-threatening. Preventive measures such as vaccination, stress reduction, environmental modifications, and infection control have lessened illness, recurrence, and spread, and many successful therapies such as antibiotics for secondary bacterial components, systemic and ocular antivirals for FHV-1, supportive care, and non-specific immune stimulation have helped to reduce the severity of illness and decrease mortality in cats. Despite these advancements in management strategies, many cats and kittens continue to suffer from URI, and those in crowded environments continue to become severely ill and either die or are euthanized. Furthermore, many animal shelters still lack information and resources regarding successful implementation of URI prevention and treatment protocols, and thus URI remains one of the most common medical reasons for euthanasia in shelters. This syndrome results in poor quality of life, and extended lengths of stay in shelters can lead to high financial burdens. Further work is needed to better understand the pathogenesis of the syndrome as well as improved preventives and treatments. The goals of the work described in this dissertation were to evaluate novel preventive and treatment strategies to decrease the incidence and severity of URI in shelters and with an emphasis on FHV-1 in experimental studies. This body of work was conducted in both the controlled research environment as well as in the animal shelter environment. Chapter 1 provides an overview of URI with a specific focus on FHV-1 and FCV and Chapter 2 presents the brief research objectives for each of the studies in this body of work. Three of the studies (Chapters 4, 5, and 7) in this body of work evaluated novel immune stimulants and preventive measures for primary FHV-1 infection and recrudescent FHV-1 in purpose-bred, experimentally infected cats in a controlled research setting. Chapter 4 evaluated a plant-based nutraceutical, Carnivora™, with anti-inflammatory and immune modulating components and its effects on recrudescence of clinical signs and viral shedding in young adult cats upon repeat challenge of FHV-1. Our study found that cats that were administered Carnivora™ had significantly less clinical manifestations of FHV-1 disease when compared to the control group. Chapter 5 assessed a new mucosal formulation of a liposomal toll-like receptor immune stimulant (LTC) as both a preventive and treatment for FHV-1 in purpose-bred kittens. This study found that administration of LTC as a preventive 24 hours prior to FHV-1 challenge resulted in some positive clinical effects and decreased shedding of FHV-1 DNA, whereas administration of LTC as a treatment during illness with FHV-1 did not influence clinical course of FHV-1 illness. Chapter 7 explored the use of a pheromone product in these same purpose-bred kittens and its effects on stress reduction, relaxation, and recrudescence of FHV-1 clinical signs. Results indicated that the pheromone product decreased stress, increased relaxation, and decreased some of the clinical signs of FHV-1 recrudescence in the kittens. Two of the studies (Chapters 3 and 6) evaluated novel immune stimulants and preventives in open-admission shelter environments. Chapter 3 explored whether the addition of an inactivated, broader spectrum FCV vaccine to a standard vaccination protocol at a shelter, would result in decreased incidence, duration, and severity of URI and oral ulceration in cats. The study did not find evidence that the additional vaccine protected cats from developing URI, severe URI, or oral ulceration indicative of calicivirus. Chapter 6 evaluated administration of the LTC discussed in Chapter 5 to cats in an open-admission shelter. Cats were administered the LTC upon admission to determine whether it would result in decreased incidence and severity of URI. The study did not find significant evidence that the LTC protected cats from developing URI or severe URI in the shelter, nor did it significantly impact clinical course of illness. Although neither of the shelter experiments had significant findings regarding the preventive product being tested, results provided additional important information regarding immune compromise and potential for immunomodulatory therapeutics and stimulation in the shelter environment, risk factors contributing to URI onset, timing, prevalence, severity, and outcomes in shelter environments. The work described in this dissertation has increased our knowledge of FHV-1 preventive and treatment options and mitigating and risk factors that might contribute to URI occurrence, recrudescence, and resolution. We hope that the findings in this work will help to decrease prevalence and severity of URI and improve the outcome for cats and kittens with URI, especially in the shelter environment.
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    Comprehensive investigation of chronic enteropathy in dogs through a prospective clinical trial, immunoassays, and RNA-sequencing
    (Colorado State University. Libraries, 2024) Manchester, Alison C., author; Dow, Steven, advisor; Lappin, Michael R., advisor; Avery, Anne, committee member; Webb, Craig, committee member
    Chronic enteropathy is a common condition in dogs causing recurrent or persistent gastrointestinal clinical signs. Pathogenesis is thought to involve intestinal mucosal inflammatory infiltrates, but histopathological evaluation does not predict treatment response, inform prognosis, or correlate with clinical remission. Many dogs may improve clinically with dietary intervention, but between 15 to 40% of dogs are refractory to all therapies. This negatively impacts quality of life for dogs and their families and can lead to euthanasia. Better understanding of the cellular and molecular differences between CE and health is necessary to improve outcomes for these dogs, and to enable use of the dog as a translational model for study of inflammatory intestinal conditions across species. The goal of this work was to critically evaluate the pathogenesis of CE in dogs through use of in vitro assays, a prospective clinical trial, and next-generation sequencing based approaches. Preliminary studies have highlighted an important role for intestinal bile acids in the pathogenesis of canine and human chronic enteropathies. Fecal bile acid populations differ between healthy dogs and dogs with CE. However, there has been little work to evaluate potential consequences of these metabolic shifts in dogs. We therefore investigated potential immunomodulatory roles of primary and secondary bile acids through in vitro experiments with canine macrophages. Both the primary bile acid cholic acid (CA) and the secondary bile acid lithocholic acid (LCA) influenced LPS-induced cytokine production via canine monocyte-derived macrophages similarly, with suppression of TNF-α secretion and enhancement of IL-10 secretion. Neither BA altered the expression of the BA receptor TGR5. Transcriptomic analysis revealed that CA activated inflammatory signaling pathways in macrophages involving type II interferon signaling and the aryl hydrocarbon receptor, whereas LCA activated pathways related to nitric oxide signaling and cell cycle regulation. Thus, we concluded that both primary and secondary BAs are active modulators of macrophage responses in dogs, with differential and shared effects evident with sequencing analysis. Diet is the most effective management strategy for dogs with CE, enabling two-thirds of patients to achieve clinical remission from their disease. Various dietary strategies may be beneficial. Nutritional formulae sourcing protein from amino acids have been used for the induction of remission in human Crohn's disease patients for decades. We conducted a prospective clinical trial involving exclusive feeding of the first diet sourcing protein from individual amino acids to 23 client-owned dogs with CE to determine its ability to induce clinical remission and begin to tease apart mechanisms of action. After 2 weeks of EL, 68% of dogs consuming the diet were classified as responders. At the conclusion of the 8 week feeding trial, 16/23 dogs (70%) were considered clinical responders. Feeding EL caused shifts in fecal bacterial communities, which differed between responders and non-responders, suggesting that diet's ability to modulate gut bacterial populations may predict its efficacy. Serum biomarker concentrations were unchanged throughout the study apart from serum alkaline phosphatase activity. Results of this study indicate that an amino acid based diet is another option to treat dogs with CE and implicates the intestinal microbiota in achievement of remission in these patients. Most studies comparing healthy and CE dogs completed to date have been limited in scope, evaluating individual or a small collection of biomarkers or cell types. This has hampered advancement of the understanding of CE pathogenesis in dogs. Ultimately, this results in generic treatment strategies for dogs and leaves a substantial proportion unable to achieve clinical remission from their disease. To this end, we applied next-generation transcriptomic sequencing to mRNA from duodenal biopsies from CE dogs and healthy beagle dogs. Results of this analysis highlighted important roles for epithelial cell gene signatures in differentiating CE tissues from healthy ones. Commonly implicated cytokines like TNF-α, IL-12, or IL-10 were not differentially expressed, but pathway analysis highlighted a potential role for upregulation of anti-viral pathways in CE dogs. This preliminary study underscores the power of RNA sequencing to provide a broad overview of cellular activities in tissues of interest, and question widely accepted theories regarding dysfunction present in the gut of dogs with CE. Single-cell RNA sequencing offers a high-resolution molecular technique enabling characterization of gene expression on an individual cell basis. This approach overcomes traditional barriers to disease investigation (e.g., species-specific reagents) and allows for definition of cell subtypes within heterogeneous samples. We thus employed single-cell RNA sequencing to catalog and compare the diversity of cells present in duodenal mucosal endoscopic biopsies from 3 healthy dogs and 4 dogs with CE. We identified populations of epithelial cells, T cells, myeloid cells, and plasma cells, with contributions from both the healthy and CIE samples. Neutrophils from CE samples exhibited a more inflammatory transcriptional program. T cells were broadly divided into non-resident and tissue resident subtypes, though minimal transcriptomic differences were appreciated within this class of cells. One subset of epithelial cells from CE dogs showed differential expression of a gene encoding a 2-pore potassium channel (KCNK16). Our results reveal a previously unappreciated cellular heterogeneity in canine duodenal mucosa and provides insights into molecular mechanisms underlying CE in dogs. The cell type gene signatures determined through this work will enable better understand the subtleties of canine intestinal physiology to allow more accessible interrogation of cellular activities in health and disease. The results of the studies described add further nuance and detail to understanding of the pathogenesis and management of canine CE. We have documented the power of transcriptomic analysis for differentiation of intestinal mucosal molecular programs in health and CE. Further investigation into intestinal bile acids, duodenal mucosal T cell subtypes and neutrophils, and intestinal epithelial cell activities are indicated.