Master of Science / Division of Biology / David A. Haukos / The lesser prairie-chicken is a prairie grouse native to the southwestern Great Plains that has experienced significant population and habitat declines since European settlement. Ongoing declines prompted the U.S. Fish and Wildlife Service to list lesser prairie-chickens as threatened under the Endangered Species Act in spring of 2014. In fall of 2015, the listing was vacated on procedural grounds and the lesser prairie-chicken was removed from listing in summer 2016. Despite the legislative change, considerable conservation efforts emerged with the initial listing and have continued following the removal of the species from the threatened and endangered species list. Understanding how lesser prairie-chickens use landscapes and how management actions can influence their space use is important for long-term strategies to meet conservation goals. I modeled lesser prairie-chicken habitat selection relative to landscape mosaics of vegetation patches generated through patch-burn grazing, microclimate, and vegetation characteristics across their range. I captured, attached GPS satellite or VHF radio transmitters to, tracked, and measured vegetation characteristics used by and available to female lesser prairie-chickens across the northern portion of their range in Kansas and Colorado. Female lesser prairie-chickens use all patch types created in a patch-burn grazing mosaic, with female selecting greater time-since-fire patches (>2-years post-fire) for nesting, 2-year post-fire patches during the spring lekking season, 1- and 2-year post-fire patches during the summer brooding period, and 1-year post-fire units during the nonbreeding season. Available vegetation structure and composition in selected patches during each life-cycle stage was similar to the needs of female lesser prairie-chickens during that life-cycle stage. To assess their selected microclimate conditions, I deployed Maxim Integrated Semiconductor data loggers (iButtons) at female flush locations and across a landscape inhabited by lesser prairie-chickens. Females selected locations that minimized thermal stress at microsite, patch, and landscape scales during peak midday temperatures during summer. Females selected midday locations based on vegetation characteristics; where selected sites had >60% forb cover and <25% grass cover, or >75% grass cover and <10% forb cover. In addition, females selected sites with greater visual obstruction. I measured vegetation composition and structure at use and available sites at four study areas located along the precipitation gradient characterizing the full extent of the lesser prairie-chicken range. Vegetation structure use by females varied in relation to long-term precipitation patterns. Females used sites with lower visual obstruction than available during the fall and spring. However, they used vegetation composition that was similar to available within each study area. Overall, my findings indicate that lesser prairie-chickens require structural and compositional heterogeneity to support a suite of habitat needs throughout the year. Therefore, management should focus on providing structural and compositional heterogeneity across landscapes. Greater heterogeneity in vegetation conditions can be achieved through management practices that allow domestic grazers to select grazing locations, such as patch-burn grazing or increased pasture area.

The cytochromes P450 (P450s) are important enzymes involved in metabolic pathways, which use exogenous and endogenous substances as their substrate for various enzymatic reactions. These enzymes can also use precarcinogens as their substrate and activate them into carcinogens, which leads to a cancer development. If the P450s are induced, the cancer risk increases. Some chemopreventive compounds may induce the P450s and thus be harmful to the human body. Therefore it is necessary to pay enough attention to a study of the mechanism of action of P450s and the influence of the chemopreventive compounds on the activity of cytochromes P450. mRNA expression of most of the P450s isoforms is detected in a number of healthy (nontransformed) tissues, viz. liver, brain, heart, colon, kidney or placenta. Nevertheless there are a few P450s isoforms which mRNAs are expressed at relatively low levels in the nontransformed tissues, whereas the expression in the transformed tissues is significantly higher. One of these P450s is CYP2W1, which can be used as a prognostic marker for colorectal cancer - therefore it is useful to be able to detect a presence of this enzyme in various tissues. A detection of P450s can be accomplished by using a method Western blot. In this method, the immunodetection is achieved by using...

Glyphosate based herbicides are among the most widely used herbicides in the world. The purpose of this study was to determine developmental toxicity of glyphosate, the active ingredient in the common herbicide Roundup, on developing chicken embryos. Few studies have examined toxic effects of glyphosate alone versus the full compound formulations of Roundup, which include adjuvants and surfactants. Adjutants and surfactants are added to aid in solubility and absorption of glyphosate. In this study chicken embryos were exposed at the air cell on embryonic day 6 to 19.8 or 9.9 mg / Kg egg mass of glyphosate in Roundup or glyphosate only. Chickens treated with 19.8 and 9.9 mg / Kg glyphosate in Roundup showed significant reduction in survivability compared to glyphosate alone treatments and controls. On embryonic day 18, embryos were sacrificed for evaluation of developmental toxicity using wet embryo mass, dry embryo mass, and yolk mass as indicators. Morphology measurements were taken on liver mass, heart mass, tibiotarsus length and beak length. Embryos treated with 19.8 mg / Kg glyphosate and 9.9 mg / Kg glyphosate in Roundup showed significant reductions in wet and dry embryo mass and yolk mass. Tibiotarsus length in 9.9 mg / Kg glyphosate in Roundup treatments were significantly reduced compared to 9.9 mg / Kg glyphosate treatments. Beak length was significantly reduced in 9.9 mg /Kg glyphosate in Roundup treatments compared to all other groups.

Master of Science / Food Science / Randall K. Phebus / Campylobacter is recognized as a leading cause of bacterial gastroenteritis. In the United States, Campylobacter causes an estimated 600,000 illnesses and 55 deaths each year at a cost of over $1.3 billion. It is estimated that 80 percent of Campylobacter infections are foodborne with almost 50 percent of these cases attributed to poultry. Based on these statistics, Campylobacter and poultry is considered by some to be the riskiest pathogen-food combination. Campylobacter illness is usually self-limiting but serious illness and complications can occur. Serious illness requires treatment with antibiotics, but with emerging antibiotic resistance observed in Campylobacter isolates, treatment options might be limited. Therefore, it is of importance to reduce significantly the consumer’s exposure to Campylobacter through poultry consumption. In July 2011, USDA FSIS’s new performance standard for Campylobacter in chicken and turkey slaughter establishments went into effect. For chicken, the standard allows no more than eight Campylobacter-positive samples out of a fifty-one sample set. Methods for Campylobacter detection and enumeration include direct plating using a medium such as Campy-Cefex, MPN techniques, ELISA, and PCR. To meet the new performance standard the industry will need to consider improvements in poultry production. Improvements likely will not be limited to processing interventions such as scalding, picking, evisceration, and chilling. Improvements may include on-farm interventions such as enhanced biosecurity, use of competitive exclusion or vaccinations, good hygiene practices, and improved staging at introduction to processing. Post-processing interventions that might be considered include freezing or further processing (i.e. cooking) of poultry products from Campylobacter-positive flocks. Significant improvements in establishments’ food safety programs are expected to occur to meet the standard and are predicted to result in an estimated reduction of 5,000 Campylobacter illnesses per year.

Call number: LD2668 .T4 1950 W55 / Master of Science

Photocopy of typescript. / Digitized by Kansas Correctional Industries

The poultry industry has successfully applied breeding and production programmes to meet growing consumer demands for chicken meat and eggs. Over the last four decades, poultry breeders have selected birds not only for productivity, but also for improved health, welfare, fitness and environmental robustness. Intensive production settings contribute to faster spread of diseases and greater losses in production due to increased morbidity and mortality of the flock. Traditional methods of disease treatment and prevention have played a critical role in control of disease. However, growing resistance of pathogens to therapeutic measures and consumer concerns led to the withdrawal of antibiotics as growth promoting additives in chicken feed. In addition, some vaccines have been overcome by increasing variation and virulence of pathogens and are no longer successful in disease prevention. The emergence of virulent and drug resistant pathogens have emphasised the need to focus on other solutions to disease, particularly natural genetic resistance. Genetic loci or gene expression patterns associated with the differential resistance of lines to specific pathogens have been identified, providing valuable markers for selective breeding. However, to date relatively few of these have been successfully incorporated into commercial lines. An ability to suppress or resist multiple pathogens, by selection for improved innate immune robustness has also been studied but it has not been introduced in commercial production, partly as the phenotype is ill-defined. Previous studies that focused on pro-inflammatory cytokines and their mRNA levels expressed by innate immune effector cells (heterophils and macrophages) identified differences between resistant and susceptible chicken lines, with the former producing stronger responses, supporting efforts to select poultry with an efficient early innate response. Here, small-scale qPCR screening and cellular techniques were evaluated with the conclusion that a more rapid, cheaper and reproducible method needs to be applied. The main objective of this project was therefore to design and validate a diagnostic tool that could be used to phenotype the immune responses of chickens at the level of innate immunity. For this purpose, a panel of 89 genes was selected based on previously published infection studies and on RNA-seq results obtained from stimulation of heterophils, macrophages and dendritic cells with lipopolysaccharide (LPS). Target genes were cloned and sequenced to optimise the design of qPCR reactions and primers. A multiplex qPCR platform, the Fluidigm 96.96 Dynamic Array, was selected as the tool of choice with the capacity to measure transcription of 96 genes of interest in 96 samples simultaneously. The preamplification reaction was optimised and the platform validated using a commercial line of chickens housed in clean or pathogen-challenged environments. Lymphoid tissues, including bursa of Fabricius, spleen, ileum with Peyer’s patches, caecal tonsils, and blood leukocytes were isolated and transcript levels for immune-related genes defined between organs, birds and farms. For qPCR analysis, a panel of reference genes was normalised and TBP, ACTB and GAPDH genes were selected and validated as the most stable. The high-throughput qPCR analysis identified peripheral blood leukocytes as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially expressed between birds housed in varying hygienic environments. The research described here could potentially aid the selection of poultry for improved immune robustness. The technical optimisation and validation of a new tool to simultaneously quantify expression of tens of relevant immune-related genes will prime research in many areas of avian biology, especially to define baseline immune gene expression for selection, the basis of differential resistance, and host responses to infection, vaccination or immuno-modulatory substances.

Macrophages are present in every tissue, and have a central role in immune responses, development and homeostasis. Typically recognised as scavenger cells phagocytising pathogens and dead cells, macrophages also regulate the innate and adaptive immune responses via the secretion of cytokines. In mammals, the differentiation, proliferation, and survival of macrophages are controlled by macrophage colony-stimulating factor, or CSF1, which acts through the CSF1 receptor (CSF1R), a ligand-dependent protein tyrosine kinase. IL34, a more recently discovered cytokine with a differential expression, shares the CSF1R. Natural or artificial knock out of these genes in mice and rats depletes macrophage populations with consequent pleiotropic effects on development of multiple organs. The mammalian CSF1R is exclusively expressed on the cells of the macrophage lineage, and their progenitors. For this reason, the CSF1R promoter has been used to generate fluorescent reporter transgenic mice, to permit analysis of macrophage function in vivo. Macrophages are present in very large numbers from midgestation in mice, but dynamic studies of their biology are difficult in a mammal. The chick has been used extensively as a developmental biology model because of the ease of visualisation and manipulation in ovo. It has the added advantage of being economically important. At the start of this project, the factors that control avian myelopoiesis had not been identified. Indeed, CSF1 was not identified in the chicken genome. The primary objective of this research was to characterise the chicken mononuclear phagocyte system. To this end, the CSF1, IL34, and CSF1R genes in chicken and zebra finch were identified from respective genomic/cDNA sequence resources. Comparative analysis of the avian CSF1R loci revealed likely orthologs of mammalian macrophage-specific promoters and enhancers, and the CSF1R gene was shown to be expressed specifically in macrophages of the developing chick embryo. These observations formed the basis of the generation of a chicken CSF1R reporter transgenic by a colleague in the laboratory. Structure-based modelling, comparative amino acid sequence analysis and co-evolution study across all vertebrates demonstrated the conservation of the IL34/CSF1/CSF1R complex in birds. Modelling also suggested that IL34 was a four helix bundle factor, structurally related to CSF1, which was subsequently confirmed by published crystal structure. To show that these factors were active in birds, chicken CSF1 and IL34 were expressed in HEK293 cells. Although chicken CSF1 lacked the interchain disulphide present in the mammalian protein, it formed a dimer. Both factors were able to promote the generation of pure macrophage cultures when added to chicken bone marrow. The specificity of action of chCSF1 and chIL34 on chCSF1R was assessed using murine myeloid IL-3 dependent Ba/F3 cells stably transfected with chCSF1R. Either chCSF1 or chIL34 alone could substitute for IL3 in receptor-expressing cells and caused them to differentiate further into the monocytic lineage pathway and to undergo growth arrest. The avian factors were not active on mammalian CSF1R. The observed species specificity and inactivity of the CSF1R inhibitor GW2580 in chicken were linked to the dissimilarities between the avian and mammalian CSF1/IL34/CSF1R proteins. To enable functional studies in vivo, a project was initiated to produce a monoclonal antibody against chicken CSF1R. Binding of the monoclonal to cells demonstrated that CSF1R was, indeed, monocyte-macrophage restricted. Chicken CSF1 was expressed as a fusion protein with the domains 3 and 4 of the chicken immunoglobulin. This increased the half-life of the recombinant chCSF1 without impairing its activity. Injection of chCSF1-Fc in the neural tube of stage HH21 chick embryos stimulated the proliferation of embryonic macrophages. Similarly, four consecutive daily injections of chCSF1-Fc in chicken hatchlings resulted in an increase in tissue macrophage number, notably in the spleen, liver and lung. To investigate the pathway of development of macrophages during embryogenesis, bone marrow from chicken ubiquitously expressing EGFP was transplanted into the circulation of stage HH16-17 embryos. The results demonstrated effective colonisation of the hematopoietic organs, and highlighted the presence of large numbers of macrophages in embryonic tissues, similar to those seen in MacGreen mice. The results are discussed in the context of the proposed yolk sac origin of some macrophage subpopulations, such as microglia cells and Langerhans cells, and the presence of a clonogenic macrophage-committed progenitor in the bone marrow that is distinct from the pluripotent stem cell. Bone marrow-derived macrophages (BMDMs) grown in CSF1 have been used extensively as a model to understand gene regulation in mice. The cloning and expression of chicken CSF1 permitted the production of large numbers of BMDMs from chicken bone marrow. To enable the characterisation of chicken macrophages and comparison to mammalian BMDMs, the gene expression profile of these cells was examined using RNAseq. For comparison, mid incubation embryos and a fibroblast line were also profiled. These data could identify several novel chicken macrophage-specific transcripts that may assist in further dissection of macrophage differentiation in birds and contribute to chicken genome annotation. Overall, this project has demonstrated that the CSF1/IL34/CSF1R system is conserved in birds, and controls the generation of monocytes and tissue macrophages. It has provided the tools to enable detailed analysis of the function of this system in embryogenesis and immunity.

Inclusion body hepatitis (IBH) has been occurring as an economically important, emerging disease of broiler chickens in several countries. Historically, IBH has been identified as a secondary disease, often associated with common immunosuppressive diseases. However, few studies have identified IBH as a primary disease with no apparent association with immunosuppressive diseases. The objectives of this study were to develop an animal model of IBH in commercial broilers, to demonstrate vertical transmission of fowl adenoviruses (FAdVs) in broiler breeders and to control IBH in broilers by vaccinating their parents with an inactivated FAdV vaccine. In order to develop an animal model of IBH in commercial broilers, fourteen-day old broilers were inoculated intramuscularly with 1x104 1x107 CCID50 of either FAdV x11a-like virus, two strains of FAdV-8a (FAdV-8a strain TR-59 and FAdV-8a strain T8-A) or FAdV-11strain 1047. Four days following FAdV inoculation, 5% - 15% mortality was observed and dead birds showed histologic lesions of hemorrhagic necrotizing hepatitis. This animal model reproduced the clinical disease, and pathological lesions of IBH that have been described in commercial broilers. In order to demonstrate vertical transmission of the FAdV, 35-week-old broiler breeders were inoculated with 1x106 CCID50 of either FAdV x11a-like virus, FAdV-8a strain TR-59, FAdV-8a strain T8-A or FAdV-11 strain 1047. Eggs from infected breeders were collected and hatched seven days post-inoculation. Clinical signs or mortality were not observed in parents; however broiler progeny derived from broiler breeders inoculated with FAdV-8a strain T8-A had 30% IBH mortality by seven days of age. The hexon gene loop 1 sequence of the virus isolated from affected broiler progeny showed 100% identity to FAdV-8a strain T8-A. In order to demonstrate protection of broilers against IBH by vaccination of their parents, four groups of broiler breeders were vaccinated with either FAdV-8a strain T8-A (2x107 or 2x104 CCID50) formulated with 20% oil-in-water emulsion, or FAdV x11a-like virus (2x107 or 2x104 CCID50) formulated with 20% oil-in-water emulsion at the age of 12 and 15 weeks. The control group was inoculated with 20% oil-in-water emulsion. Broiler progeny were challenged with FAdV-8a strain T8-A to study the immunoprotective effect of the vaccine. Although, survival of broilers following FAdV-8a strain T8-A challenge was not significantly different among vaccinated and non-vaccinated groups (P>0.05), immunoprotective effect was enhanced by the increase dose of FAdV antigens (P>0.05). Further studies are necessary to improve the vaccine efficacy to protect broilers against IBH.<p> In conclusion, the results of this study support the hypothesis that IBH in broilers in Canada is a vertically-transmitted primary disease with no known immunosuppressive involvement. The results also demonstrated that inactivated antigens of FAdV are able to partially protect broilers against IBH by vaccinating their parents. Further studies with different formulations, and priming the immune system of broiler breeders with live FAdV prior to vaccination with inactivated FAdV vaccines are necessary to improve the efficacy of inactivated IBH vaccine.

It has been shown in many species that individuals exhibit consistent differences in behaviour over time and/or across situations. These differences in behaviour are called personality. One way to categorise personality types typically used for rodents, is along a proactive-reactive gradient, which describes how individuals cope with stressful challenges. Proactive individuals pay less attention to their environment, form routines easily and take longer to adapt when routines are broken compared to reactive individuals. Avian species have to date rarely been described along this gradient, thus the generality of this description across species is unclear. The present study has investigated variation in proactivity-reactivity in red junglefowl chicks (Gallus gallus). To observe the chicks’ coping styles, a proactive-reactive test was conducted where the chicks were trained to form a routine, which was then broken. Their behavioural response to this was recorded and used as a measure for proactivity-reactivity. The behavioural response was then linked to individual behavioural variation in additional personality assays. Individuals that were more vigilant in the proactive-reactive test often uttered stress calls and took longer to complete the test. In contrast, individuals that walked more and did not utter stress calls had a shorter time to complete the test. These findings can be used to describe proactive red junglefowl chicks; those that are more stressed when routines are broken, compared to calmer reactive individuals. I found no difference in routine formation between proactive and reactive red junglefowl chicks, suggesting that what describes proactive and reactive individuals may vary across species.