Host defense peptides (HDPs) are a large group of small positively charged peptides that play an important role in innate immunity. Their role is more critical at early ages when other components of the immune system have not fully developed. There are three classes of avian HDPs: avian beta defensins (AvBDs), cathelicidins (Cath) and liver-expressed antimicrobial peptide 2 (LEAP-2). The objective was to compare expression of HDPs in male turkey poults at day of hatch (D0), D7, D14, D21 and D28 from the thymus, spleen, bursa, duodenum, jejunum and ileum. The expression of AvBD1, AvBD2, AvBD8, AvBD9, AvBD10, AvBD13, Cath2, Cath3 and LEAP-2 was measured using qPCR (n=6 birds/tissue/age). Data were analyzed by one-way ANOVA and Tukey's test, and significance considered at P ≤ 0.05. AvBDs and Caths exhibited greater expression in immune organs than intestinal tissues, with the greatest expression of AvBDs observed in the spleen. The intestinal tissues showed very low expression of AvBDs except for AvBD10 at D0. Similar to AvBDs, Caths expression in the immune organs was greater than the intestinal tissues with the spleen having the greatest expression among immune organs. Conversely, LEAP-2 showed greater expression in the intestinal tissues than in the immune tissues, which showed very low LEAP-2 expression unlike other HDPs. Understanding the differential expression of HDPs could reveal the innate immune status of poults, and may subsequently allow improvement of their health through appropriate mitigation strategies. / Master of Science

Avian paramyxoviruses (APMVs) consist of twelve distinct serotypes (APMV-1 to -12) isolated from a wide variety of domestic and wild birds. APMV-1/Newcastle disease virus (NDV) is the most characterized and globally important avian pathogen, because of the huge economic loss associated with the disease. However, very little information is known about the pathogenicity of APMV 2-12. APMV expresses six structural and two accessory proteins. The functions of APMV accessory proteins (V and W) are not fully established. Only the function of V protein in NDV is studied so far. V protein was found to be an IFN antgonist and a major virulent determinant of NDV. In this study, we tested for the potential functions of W protein in NDV and fuctions of V protein in other APMV serotypes. Vaccination failure is a major cause for NDV outbreak in developing and tropical countries, because of thermolabile nature of vaccine strains. Thermostable and thermolabile NDV strains exhibit difference in W protein length. In the first part of our study, we mutated the genome of a thermolabile NDV strain to express W protein of different lengths, rescued recombinant viruses by reverse genetics system and tested for thermostability. Our results showed that W protein does not confer thermostability to NDV. In the second part of study, we constructed plasmids expressing APMV -2, -3 and -6V proteins and tested for IFN antagonism by a dual luciferase reporter assay. Our results showed that APMV-3V acts as IFN antagonist by blocking IFN induction and thereby may play an important role in the evasion of innate immunity. / Master of Science

A 4.0 Kb (2.64 Mdal) plasmid was isolated from a fowl cholera strain of Pasteurella multocida (the Larsen strain) by alkaline lysis and cesium chloride purification. The plasmid, designated pLAR-1, was characterized in terms of its size and restriction sites. The restriction patterns produced by fourteen endonucleases were used to generate a restriction map. Five restriction enzymes cleaved the plasmid at multiple sites. Two enzymes, Bgl II and Sal I had unique sites on pLAR-1. Twelve of the fifty six strains of P. multocida surveyed contained plasmids of different sizes which hybridized to pLAR-1. Strains containing homologous plasmids were variable in serotype, dermonecrotoxin production, and origin (both in terms of the host and locale). pLAR-1 did not encode any of the enzymes necessary for the biochemical pathways contained within the API-20E strip or siderophore production. pLAR-1 was cloned into the BamH I site of pBR322. Resultant clones were approximately 8.363 Kb in length, ampicillin resistant and tetracycline sensitive. The pLAR-1 / Master of Science

Pasteurella multocida is an important multi-host animal and zoonotic pathogen that is capable of causing respiratory and multi-systemic diseases, bacteremia, and infections resulting from bite wounds. The glycosaminoglycan capsule (CPS) of P. multocida is an essential virulence factor, protecting the bacterium from host defenses. However, chronic infections such as bovine respiratory disease (BRD) and avian cholera may be associated with biofilm formation. Biofilm formation was inversely related to capsule production (determined by uronic acid and N-acetylglucosamine assays), and was confirmed with capsule-deficient mutants of mucoid strains. Capsule-deficient mutants formed biofilms with a larger biomass that was much thicker and smoother than encapsulated strains. Gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion demonstrated that the matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS). Therefore, CPS may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix to encase large numbers of bacterial cells. Chemical mutagenesis was performed on P. multocida strain P1059, resulting in isolation of an acapsular mutant designated as P1059-R8. A uridyltransferase encoded by gene P1059_01979 was mutated in such a way that a polar amino acid was changed to a non-polar amino acid near the active site. The protein product of P1059_01979 is important for the biosynthesis of the CPS subunit N-acetylglucosamine. CPS quantification revealed that the subunit glucuronic acid was produced in equal concentrations to the parent, but the CPS subunit N-acetylglucosamine was not detected in the chemical mutant. Biofilm formation in the chemical mutant was significantly higher than in WT P1059 and the capsule-deficient mutant. We hypothesize that P1059_01979 is essential for CPS production in P. multocida serogroup A. Histophilus somni and Pasteurella multocida cause bovine respiratory disease (BRD) and systemic infections in cattle. Following respiratory infection of calves with H. somni, P. multocida is also often isolated from the lower respiratory tract. Because H. somni normally forms a biofilm during BRD, we suspected that P. multocida may co-exist with H. somni in a polymicrobial biofilm. Interactions between the two species in the biofilm were characterized and quantified by fluorescence in situ hybridization (FISH), and the biofilm matrix of each species examined by fluorescently-tagged lectins (FTL), confocal scanning laser microscopy of in vitro biofilms and bovine pulmonary tissue following dual H. somni and P. multocida infection. FISH and FTL were used to show that P. multocida and H. somni were evenly distributed in the in vitro biofilm, and both species contributed to the polymicrobial biofilm matrix. COMSTAT z-stack image analysis revealed that the average biomass and biofilm thickness of the individual and polymicrobial biofilms were greatest when both species were present. Encapsulated P. multocida isolates not capable of forming a biofilm still formed a polymicrobial biofilm with H. somni, but only the EPS of H. somni could be detected by FTL staining of bovine tissues from which both species were isolated. Bacteria within a biofilm are more quiescent than during planktonic growth and induce less of an inflammatory response, indicating encapsulated P. multocida may take advantage of the H. somni biofilm to persist in the host during less severe, but more chronic, BRD. These results may have important implications for the management of BRD. Acute avian cholera is associated with encapsulated P. multocida, while chronic and asymptomatic cases of avian cholera are associated with acapsular P. multocida isolates. We hypothesize that biofilm formation is present and an important factor for chronic and asymptomatic avian cholera. Experimental infections of chickens with biofilm deficient P. multocida strain WT X73, proficient biofilm forming P. multocida strain X73ΔhyaD, and proficient biofilm forming clinical isolates 775 and 756 showed that virulence inversely correlated with biofilm formation. Histopathological analysis showed that biofilm forming isolates induced little inflammation in the lungs, heart, and liver, while biofilm deficient isolates induced greater inflammation. Biofilm material was located in pulmonary tissues of chickens diagnosed with chronic avian cholera using FTL staining.. Quantitative real-time PCR for expression of cytokine genes in the spleens of infected chickens indicated that P. multocida induced Th1 and Th17 immune responses during acute and chronic avian cholera. Chickens that succumbed to acute avian cholera after experimental challenge with WT X73 had high levels of INF-ƴ, IL-1β, IL-6, IL-12A, IL-22, IL-17A, and IL-17RA expression in the spleen compared to all other experimental groups. Antibody titers were low, indicating that antibodies may be less important in managing and clearing P. multocida infections. / Ph. D. / Pasteurella multocida is a zoonotic pathogen, which means it can be transferred from animals to humans as part of the normal flora of many animals including household pets such as cats and dogs, and agriculture species such as cattle. P. multocida is responsible for infected animal bites, especially those resulting from household and large cats. Additionally, P. multocida is responsible for several diseases of veterinary importance, including avian cholera and bovine respiratory disease (BRD). Capsule, composed of capsular polysaccharide (CPS), is an essential virulence factor for P. multocida. Virulence factors are genetically encoded attributes that aid the bacteria in causing an infection. Capsule covers the surface of bacterial cells, which allows P. multocida to survive within the host and avoid detection by the immune system. The P. multocida capsular serogroup A is composed of hyaluronic acid. Biofilms are communities of bacteria that survive within a hydrated matrix composed of polysaccharides, proteins, enzymes, antimicrobial compounds, extracellular DNA, and other bacterial and host components. Biofilms can be compared to multicellular organs of eukaryotes. While less complex, biofilms similarly regulate nutrients, water, composition, remove waste, and perform other processes such as DNA transfer. Biofilms protect bacterial communities by shielding them from the host immune response. Bacteria living in biofilms also grow slowly, and as a result are protected from many antibiotic treatments. While biofilm formation has been suggested for P. multocida, the biofilm has not yet been characterized. The work reported here characterizes biofilm formation by P. multocida isolates of capsular serogroup A. Biofilms formed by P. multocida were stained with fluorescently-tagged lectins, DNA stain, and other fluorescent dyes, as well as crystal violet stain. Biofilms were imaged using several microscopy techniques. Biofilm formation was prominent for serogroup A strains of P. multocida that were acapsular. However, in the presence of CPS, biofilm formation was inhibited. H. somni forms a biofilm during BRD that allows the bacterium to survive within the heart and lungs of the bovine host. BRD is often caused by several different bacterial, viral, and even parasitic microbes – resulting in a polymicrobial disease. Polymicrobial diseases are more difficult to diagnose and treat, which is a challenge when trying to control this economically important disease. Experimental infections of bovines with H. somni have resulted in polymicrobial infections with P. multocida. We hypothesize that these two bacterial species may form a mutualistic or commensalistic interaction together during BRD to improve the survival of one or both species within the host. The polymicrobial biofilm was observed using fluorescent microscopy techniques. We confirmed that H. somni and P. multocida form a polymicrobial biofilm. Avian cholera can be an acute, chronic, or asymptomatic disease that affects poultry farms and migratory flocks around the world. The spread of P. multocida and avian cholera is thought to occur through infected water, infected insects, and through other infected animals surrounding water supplies such as deer, raccoon, and even fish. We hypothesize that P. multocida can produce a biofilm and survive within the respiratory tract of birds for extended periods of time, that biofilm formation is important for the establishment of chronic and asymptomatic avian cholera, and that a biofilm assists in the spread of disease between flocks of birds. Chickens were challenged in the respiratory tract with a highly encapsulated, poor biofilm forming strain, or a prominent biofilm forming strain. After 7, 14, and 28 days chicken lungs were examined to identify bacteria, biofilm material, and inflammation. Biofilm-forming P. multocida strains were less virulent and caused less inflammation than non-biofilm forming P. multocida strains. Biofilms were visible in the airways of pulmonary tissue by scanning electron microscopy. Biofilm formation by P. multocida was observed within the pulmonary tissue of chickens with chronic and acute avian cholera.

Examination of bird assemblages along an environmental gradient which encompasses both climate and habitat change is needed if we are to better understand the potential effects of these changes for avians and the ecological process that depend upon them. Climate change is predicted to have a significant impact on deserts and desert margins, resulting in distributional shifts of entire ecosystems and new community associations. This study explores the probable responses of avian communities to increasing desertification. In general, species richness and numbers of birds in arid zones are low compared to more mesic areas. Different combinations of habitat types and the variety of patches in a landscape influence the diversity and community structures of avians in that landscape. The role of vegetation structure in avian habitat selection in semi-arid areas is dictated by horizontal habitat density as well as vertical structure. Although bird distribution is determined by habitat boundaries, most birds are flexible and can disperse across small habitat barriers. The hypothesis tested, was that bird species assemblages along an aridity gradient are affected primarily by rainfall and secondarily by habitat type. Assessing the impacts of rainfall and habitat on bird variables, such as species richness, abundance, diversity, biomass, and life history traits, were the objectives of the study. An east-west aridity gradient of 300 mm, stretching over 370 km, was chosen in central Namibia for the study area. The climate is harsh with localised rain and considerable daily fluctuations in temperature. Grasses, and trees and shrubs up to 7 m in height are the co-dominant life-forms. Surveys were conducted over three years; one winter and one summer survey in each year. Rainfall, seasons and vegetation height were recorded as environmental variables. Three structurally different habitat types were selected for stratified sampling: open areas, rivers and thickets. Open areas were dominated by grass; river refers to ephemeral dry river lines with mature trees; and thickets comprise woody shrubs and trees. At each site, the same three habitats were used for bird sampling, resulting in 15 sample units. Sampling took place on 51 discontinuous line transects of 1km in length and without a width limit. Univariate analyses included ANOVA and t-tests. Multivariate analyses consisted of cluster analysis, MRPP tests, indicator analysis, Shannon diversity index and NMS ordinations. NMS bi-plots were used to define avian community structures responding to aridity, habitat, migration and life history traits. The results showed that bird species richness, abundance, and diversity remained relatively constant across the aridity gradient, until they declined significantly once a certain aridity threshold was crossed at the most arid site. There were significantly more bird species and individual birds at the wetter sites than at the drier sites. Rivers contained more birds than thickened or open habitat types, suggesting the importance of riparian habitat types for maintaining avian diversity. The three more mesic sites included higher numbers of species from the nesting and feeding guilds, regardless of habitat type, than the two more arid sites. The aridity threshold had a significant effect on bird community structures: more migrant and nomadic species, and omnivore and insectivore species persisted in very arid conditions. From the results it was predicted that climate change will cause avian species to undergo range shifts from west to east, resulting in community composition changes and a reduction in diversity. Life history traits affect the adaptive capabilities of bird species and it is predicted that nomadism, flexibility in diet, and adaptability of nesting requirements will contribute to species persistence in the drier conditions predicted under current climate change scenarios. Dry river lines will act as refugia for avian diversity, but crucial habitat types that currently contain less diversity are also important for maintaining unique avian assemblages. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Examination of bird assemblages along an environmental gradient which encompasses both climate and habitat change is needed if we are to better understand the potential effects of these changes for avians and the ecological process that depend upon them. Climate change is predicted to have a significant impact on deserts and desert margins, resulting in distributional shifts of entire ecosystems and new community associations. This study explores the probable responses of avian communities to increasing desertification. In general, species richness and numbers of birds in arid zones are low compared to more mesic areas. Different combinations of habitat types and the variety of patches in a landscape influence the diversity and community structures of avians in that landscape. The role of vegetation structure in avian habitat selection in semi-arid areas is dictated by horizontal habitat density as well as vertical structure. Although bird distribution is determined by habitat boundaries, most birds are flexible and can disperse across small habitat barriers. The hypothesis tested, was that bird species assemblages along an aridity gradient are affected primarily by rainfall and secondarily by habitat type. Assessing the impacts of rainfall and habitat on bird variables, such as species richness, abundance, diversity, biomass, and life history traits, were the objectives of the study. An east-west aridity gradient of 300 mm, stretching over 370 km, was chosen in central Namibia for the study area. The climate is harsh with localised rain and considerable daily fluctuations in temperature. Grasses, and trees and shrubs up to 7 m in height are the co-dominant life-forms. Surveys were conducted over three years; one winter and one summer survey in each year. Rainfall, seasons and vegetation height were recorded as environmental variables. Three structurally different habitat types were selected for stratified sampling: open areas, rivers and thickets. Open areas were dominated by grass; river refers to ephemeral dry river lines with mature trees; and thickets comprise woody shrubs and trees. At each site, the same three habitats were used for bird sampling, resulting in 15 sample units. Sampling took place on 51 discontinuous line transects of 1km in length and without a width limit. Univariate analyses included ANOVA and t-tests. Multivariate analyses consisted of cluster analysis, MRPP tests, indicator analysis, Shannon diversity index and NMS ordinations. NMS bi-plots were used to define avian community structures responding to aridity, habitat, migration and life history traits. The results showed that bird species richness, abundance, and diversity remained relatively constant across the aridity gradient, until they declined significantly once a certain aridity threshold was crossed at the most arid site. There were significantly more bird species and individual birds at the wetter sites than at the drier sites. Rivers contained more birds than thickened or open habitat types, suggesting the importance of riparian habitat types for maintaining avian diversity. The three more mesic sites included higher numbers of species from the nesting and feeding guilds, regardless of habitat type, than the two more arid sites. The aridity threshold had a significant effect on bird community structures: more migrant and nomadic species, and omnivore and insectivore species persisted in very arid conditions. From the results it was predicted that climate change will cause avian species to undergo range shifts from west to east, resulting in community composition changes and a reduction in diversity. Life history traits affect the adaptive capabilities of bird species and it is predicted that nomadism, flexibility in diet, and adaptability of nesting requirements will contribute to species persistence in the drier conditions predicted under current climate change scenarios. Dry river lines will act as refugia for avian diversity, but crucial habitat types that currently contain less diversity are also important for maintaining unique avian assemblages. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The aim of this study is to improve our understanding of the ecology, spatiotemporal patterns, and risk of infectious pathogens of migratory waterfowl, using the blue-winged teal (Anas discors, BWTE), as a model. From 2007-2010, 1,869 BWTE were sampled in the prairie provinces (Alberta, Saskatchewan and Manitoba, Canada) to examine infection status and/or evidence of previous exposure to avian influenza virus (AIV), West Nile virus (WNV), and avian paramyxovirus-1 (APMV-1), in relation to host demographic variables (age, sex, body condition, exposure to other pathogens), other ecological variables such as local waterfowl breeding population density and local pond density, and year. The probability of AIV infection depended on an interaction between age and AIV antibody status. Hatch year birds with antibodies to AIV were more likely to be infected, suggesting an antibody response to an active infection. After hatch year birds with antibodies to AIV were less likely to be infected, suggesting immunity resulting from previous exposure. AIV infection was positively associated with local BWTE density, supporting the hypothesis of density dependent transmission. Exposure to WNV and APMV-1 were also associated with age and year. Furthermore, the probability of WNV exposure was positively associated with local pond density rather than host population density, likely because ponds provide suitable breeding habitat for mosquitoes, the primary vectors for transmission. We also investigated large-scale spatiotemporal trends in apparent prevalence of AIV across Canada and the United States throughout the year, using data from national avian influenza surveillance programs in Canada and the US in 2007-2010. Our analyses revealed that age, sex, year of sampling, flyway, latitude, and season (categorized by stages of the BWTE annual life cycle) were all important variables in predicting probability of AIV infection. There was an interaction between age and season. During late summer staging (August) and fall migration (September-October), hatch year birds were more likely to be infected than after hatch year birds, however there was no difference between age categories for the remainder of the year (winter, spring migration, and breeding season). Probability of infection increased non-linearly with latitude, and was highest in summer, corresponding to the beginning of fall migration when densities of birds and the proportion of susceptible hatch year birds in the population are highest. Birds in the Pacific, Central and Mississippi flyways were significantly more likely to be infected compared to those in the Atlantic flyway. Observed trends in seasonal, annual, and geographic patterns of AIV infection in BWTE across Canada and the US were primarily driven by the dynamics of AIV infection in hatch year birds. Our results demonstrate demographic as well as seasonal, latitudinal and flyway trends across Canada and the US. This research provided further evidence for the role of wild dabbling ducks, particularly BWTE, in the maintenance and ecology of AIV. This improved understanding of the role of BWTE as natural hosts, and the geographic, demographic and temporal variables that affect infection and transmission parameters, moves us closer to deciphering the overall ecology of the virus and its transmission and transportation pathways at the individual, population and continental levels. This knowledge, in turn, will permit development of better tools to predict and perhaps to prevent possible outbreaks in domestic animals as well as in humans.

published_or_final_version / abstract / toc / Microbiology / Doctoral / Doctor of Philosophy

Thesis (M.P.A.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 68-69).

Thesis (M.P.A.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 68-69). Also available in print.