The bovine teat canal: Its role in pathogen recognition and defence of the mammary gland

The bovine mammary gland is remarkable in that it produces over three litres of milk daily as an exocrine secretion, which passes through the teat into the external environment. Despite this substantial volume of fluid flow and that milk is one of the most nutritional mediums in which microorganisms can thrive, for the most part, this process occurs without colonisation of pathogens in the interior of the gland. Nevertheless, mastitis, when it does occur, is a significant dairy management problem. The major factor restricting ingress of pathogens into the gland has been assumed by most researchers in the field to be the length and diameter of the teat canal and the tightness of the teat sphincter. However, it has become increasingly clear in recent years that epithelial tissues have considerable innate immune functionality that contributes significantly to host defence. The presence of innate immune components within the teat end tissues has been only very superficially described to date. The presence of antimicrobial activity in the protein fraction of the teat canal lining has been previously demonstrated, as well as the presence of immune cells within some teat-end tissues, however, a detailed investigation into the identity of these proteins and cell types has not been undertaken. The aims of this thesis are to firstly, characterise the protein component of the teat canal lining (Chapter 3) and secondly, identify key immune-related cells in healthy lactating dairy cows (Chapter 4).0 A third aim is to assess changes in the cell population and immune-related factors that occur within teat-end tissues during mammary involution (Chapter 5). The fourth and final aim is to begin to characterise the immediate localised inflammatory response within teat-end tissues to the presence of mastitis-causing pathogens (Chapter 6), a question that has never been previously addressed. The approach used to address these aims were to (i) apply proteomics methodologies to the characterisation of the proteins within the teat canal lining, and (ii) examination of teat-end tissues by fluorescence immunohistochemistry using antibodies directed against known immune cell markers. The results revealed that aside from the major keratin proteins, the teat canal lining contained several other proteins and protein families (e.g. the S100 and serpin families) that differed from that of the cornified layer of the surrounding teat skin (Chapter 3). Immunofluorescent analysis revealed that there were substantial differences in the density and distribution of immune cells between the teat-end tissues. The Fürstenberg’s rosette is especially rich in antigen-presenting cells (Chapter 4). Drying-off of the udder (mammary involution) was associated with an increase in some immune cell types in the teat sinus, and an increased abundance of some antimicrobial proteins in the teat canal lining, but relatively few and minor additional changes (Chapter 5). The introduction of pathogens into the teat canal resulted in similar changes in the abundance of teat canal lining proteins, but no obvious changes in localised immune cell abundance or distribution after 24 hours exposure (Chapter 6). The presence of specific antimicrobial proteins in the teat canal lining, and the presence of immune cells within the Fürstenberg’s rosette and teat sinus epithelia demonstrate that these tissues play an active role in host defence. Thus, the research presented in this thesis has laid the foundation for a more complete understanding of host-defence functionality in the teat. This has implications for devising novel approaches to reducing susceptibility to mastitis in dairy cows

Keratin and S100 calcium-binding proteins are major constituents of the bovine teat canal lining

The bovine teat canal provides the first-line of defence against pathogenic bacteria infecting the mammary gland, yet the protein composition and host-defence functionality of the teat canal lining (TCL) are not well characterised. In this study, TCL collected from six healthy lactating dairy cows was subjected to two-dimensional electrophoresis (2-DE) and mass spectrometry. The abundance and location of selected identified proteins were determined by western blotting and fluorescence immunohistochemistry. The variability of abundance among individual cows was also investigated. Two dominant clusters of proteins were detected in the TCL, comprising members of the keratin and S100 families of proteins. The S100 proteins were localised to the teat canal keratinocytes and were particularly predominant in the cornified outermost layer of the teat canal epithelium. Significant between-animal variation in the abundance of the S100 proteins in the TCL was demonstrated. Four of the six identified S100 proteins have been reported to have antimicrobial activity, suggesting that the TCL has additional functionality beyond being a physical barrier to invading microorganisms. These findings provide new insights into understanding host-defence of the teat canal and resistance of cows to mastitis

Application of ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (Orbitrap™) for the determination of beta-casein phenotypes in cow milk

The objective of this study was to determine the β-CN phenotypes in cow milk collected from HF and cross-bred HF dairy cattle in Phu Dong, Vietnam. In total, 85 samples of raw milk were collected from 85 individual cows. Beta-casein (β-CN) phenotypes in cow milk were determined using ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). The results showed that three β-CN variants A1, A2 and I were detected and identified in the milk samples. Five β-CN phenotypes A1A1, A1A2, A1I, A2A2 and A2I were found with percentages of 0.035, 0.400, 0.059, 0.482 and 0.024, respectively. The higher proportion of β-CN phenotype A2A2 compared to other phenotypes was expected because of changes in dairy cow breeding in Phu Dong, Vietnam

Proteomics data in support of the quantification of the changes of bovine milk proteins during mammary gland involution

Here we provide data from three proteomics techniques; two-dimensional electrophoresis (2-DE) followed by identification of selected spots using PSD MALDI-TOF MS/MS, one-dimensional gel electrophoresis followed by LC-MS/MS analysis of gel slices (GeLC) and dimethyl isotopic labelling of tryptic peptides followed by Orbitrap MS/MS (DML), to quantify the changes in the repertoire of bovine milk proteins that occurs after drying off. We analysed skim milk and whey sampled at day 0 and either day 3 or day 8 after drying off. These analyses identified 45 spots by MALDI-TOF, 51 proteins by GeLC and 161 proteins by DML, for which the detailed data work-up is presented as three Excel files. The data supplied in this article supports the accompanying publication “Changes in the repertoire of bovine milk proteins during mammary involution” (Boggs et al., 2015) [1]. Data are available via ProteomeXchange with identifiers ProteomeXchange: PXD003110 and ProteomeXchange: PXD003011. Keywords: Host defence, Dairy, Drying off, Proteomic

Release of beta-casomorphins during in-vitro gastrointestinal digestion of reconstituted milk after heat treatment

© 2020 Elsevier Ltd This study assessed the impact of heat treatment on beta-casomorphin 5 (β-CM5) and beta-casomorphin 7 (β-CM7) after in-vitro gastrointestinal digestion of milk representing beta-casein (β-CN) A1A1, A2A2 and A2I phenotype. After heat treatment at 73 °C/20 s, 85 °C/5 min and 121 °C/12 min, milk samples were subjected to in-vitro gastrointestinal digestion. β-CM5/7 were analysed using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and further confirmed using ultra high-performance liquid chromatography-high resolution accurate mass Orbitrap™ mass spectrometry (UHPL-HRMS). β-CM5 was not released after in-vitro gastrointestinal digestion of all heated milk. Similarly, β-CM7 was not released in all milk with β-CN A2A2 phenotype. However, this peptide level ranged from 127.25 to 198.10 ng/mL (4.94–7.70 ng/mg protein) in heated milk with β-CN A1A1 phenotype, whereas it was released at much lower levels ranging from 19.35 to 24.50 ng/mL (0.71–0.91 ng/mg protein) in heated milk with β-CN A2I phenotype

Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep

Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health