The physiology of the keratin plug formation in the teat canal of dairy cattle and its interaction with current and novel methods for prevention of intramammary infections : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science, Massey University, Turitea, Palmerston North, New Zealand

Chapter 6 was published as an open access article in the Journal of Dairy Science under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 (CC BY-NC-ND) license. Chapter 7 was published as an open access article in Veterinary Sciences under a Creative Commons Attribution (CC BY) license. Both articles are attached.The incidence of intramammary infections (IMI) in dairy cows in the early dry period is the highest of the lactation cycle when methods to prevent IMI are not applied. This high incidence is comparable only with that observed near calving. At the end of lactation, the teat is sealed by a plug formed mainly by keratinised cells, detritus and proteinaceous material. Research suggests that the keratin plug acts as a physicochemical barrier throughout the dry period that impedes the entrance of bacteria. However, the physiological mechanism of keratin plug formation is still uncertain. The main objectives of this thesis were to characterise the physiological functions of the teat canal (TC) during the early dry period and assess how they associate with the presence of IMI. A further objective was to evaluate the modes of action of a current mastitis preventative containing bismuth subnitrate and a novel formulation of micronized keratin that is under investigation as a teat seal for preventing IMI during the early dry period. To address these objectives a novel biopsy method was developed to allow investigation of the physiological characteristics of the epithelial tissues of the TC. A transcriptomic analysis of the TC epithelium after drying off showed that epithelial cells decreased expression of mitotic and immune-response related genes. A Streptococcus uberis strain was used in a challenge study aiming to examine mechanisms of colonization in the TC and the response of the epithelial tissue to progressing infection. This Streptococcus uberis challenge did not result in colonization of the TC nor in IMI with S. uberis. Nevertheless, a reduction in the thickness of the stratum granulosum and the keratin layer of the TC epithelium was observed. This coincided with an increase in TC colonization by non-pathogenic bacteria and a decline in the concentration of certain cytokines after drying off. These changes observed in the TC epithelium support previous reports showing increased incidence of IMI by non-pathogenic bacteria during the early dry period. Antimicrobial effects and neutrophil cell responses were evaluated in vitro in two studies to test previously hypothesised action mechanisms for bismuth subnitrate and a novel keratin-based internal teat sealant (ITS) formulation. Bismuth subnitrate showed an inhibitory effect on bacterial growth, contrary to the current description of ITS as non-pharmacological, inert physical barriers. No activation of a cellular response was observed for keratin or bismuth formulations in vitro. Bismuth subnitrate and keratin were also tested in vivo for their effect on the formation of the keratin plug. The hypothesis of this study was that these treatments induce expression of mitogenic genes that induce a faster sealing of the teat canal. There was no modification of gene expression after treating cows with bismuth subnitrate or the novel keratin-based ITS formulation during the formation of the natural keratin plug, and no modification of the closure status of the teat canal lumen, suggesting that neither of the two treatments induced an improved sealing of the teat canal after drying off through increased keratin production. These findings contribute to the knowledge of keratin plug formation and physiological characteristics of the TC during involution. They align with and partially explain some of the literature-reported events observed during the early dry period. The knowledge gained provides support for future product development aimed to increase protection of the mammary gland during the dry period