Klebsiella spp. are coliform bacteria that cause mastitis in dairy cattle and account for high mortality rates in infected cows leading to a significant financial loss. Recent outbreaks indicate that within farms a single strain can be responsible for clinical signs in multiple animals. Identification of the virulence of factors enabling Klebsiella spp. survival in the mammary glands of multiple animals may provide insight into host adaptation. In this study, Klebsiella spp. strains were evaluated for their ability to evade neutrophil killing, the primary immune defense in the bovine mammary gland. Our research focused on capsule and biofilm production by Klebsiella spp. when strains were grown in Luria Broth or skim milk to examine the effects on evasion of neutrophil killing. Biofilm production was not significantly related to the ability to resist neutrophil killing nor was capsule (P = 0.29). Farm (P < 0.001), media type (P < 0.005), and strain type by cow (P < 0.001) were found to play significant roles in neutrophil evasion. This suggests farm of origin, media type used, and cow all may play a role in evasion of neutrophils by Klebsiella spp. Further evaluation of virulence factor expression in different media types and the role of individual cow immune responses may provide insight into ability of Klebsiella spp. to cause outbreaks of mastitis in multiple animals. / Master of Science

Thesis (doctoral)--Göteborg University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Göteborg University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Tissue homeostasis is necessary for optimal organ functioning. The onset of tissue trauma compromises the homeostatic environment resulting in widespread cell death with the likelihood of exposure to invading micro-organisms. Early stage elimination of microbes and immunomodulation is co-ordinated by leukocytes of the innate immune system of which neutrophils and macrophages play a pivotal role. Leukocyte-released pro-inflammatory factors are vital in the containment of infection but bring with it a degree of collateral tissue destruction. Thus cascading stages during inflammation must be tightly regulated to bring about timely tissue regeneration and regained homeostasis. However, chronic inflammatory diseases e.g. rheumatoid arthritis highlights the existence of defective regulation at numerous stages during this transition, often leading to debilitating disease progression. Recently published findings by our research group identified the anti-inflammatory properties of α-defensin - an anti-microbial peptide released from dying human neutrophils - on stimulated macrophages. Thus the main objective of my research was to gain an understanding into the molecular actions of α-defensins which inhibit the macrophage inflammatory potential. Strong evidence supported the propensity of α-defensins to inhibit both intracellular and secreted protein synthesis, as assessed by de novo 35S-radiolabeled Methionine incorporation. Inhibition was not attributed to endoplasmic reticulum stress events, a common diagnosis in the regulation of global translation. Supporting evidence using cell-free systems identified a fundamental block in translation with the inclusion of α-defensin. Biochemical studies linked the ability of α-defensin to bind non-specifically to oligonucleotide sequences. This binding potential was also demonstrated on ribosomal RNA (rRNA), impeding its migration through electrophoretic gels. Immunocytochemical assays proposed an emerging suggestion of α-defensins in macrophages concentrated in close proximity to ribosomes around the perinuclear region. Evidence of suggested defensin/ribosome accumulation after 24hrs after treatment were attempted but to date remained unconfirmed. Attempts to determine the fate of these proposed accumulations were inconclusive, assessed by autophagy assays and ribosome semiquantitation. This thesis describes for the first time an enhanced understanding into the intracellular inhibitory mechanisms of α-defensins on macrophages and possibly other cell types. Understanding the molecular impact of α-defensins provide key insights into this novel inflammatory regulator, with the potential to be utilized in future immunotherapies.

published_or_final_version / Biochemistry / Master / Master of Philosophy

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