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Lates Calcarifer) Peripheral Blood Against Immune defence mechanisms of barramundi (Lates calcarifer) peripheral blood against Streptococci Kelly-Anne Masterman BSc. (hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 The School of Biological Science 1 Abstract IMMUNE DEFENCE MECHANISMS OF BARRAMUNDI (Lates calcarifer) PERIPHERAL BLOOD AGAINST STREPTOCOCCI Aquaculture now supplies more than half of the fish used for human consumption and is a major contributor to global food security. As aquaculture growth must be sustained to continue to supply increasing population-driven demand for food fish, efficiency must be increased. In spite of major inroads in disease control through mass vaccination of farm fish, bacterial diseases continue to cause substantial losses, particularly in tropical and warm- temperate regions where aquaculture is expanding fastest. To develop new vaccines, and improve existing vaccines for effective disease control in these regions, an improved understanding of the mechanisms of pathogen immune evasion and dissemination within the host may identify new vaccine targets. Streptococcus agalactiae and Streptococcus iniae are significant pathogens of warm and temperate farmed and wild fish. Both have a broad host range, express a polysaccharide capsule as a major virulence determinant and cause similar pathologies characterized by rapid sepsis, followed by meningitis and death. S. iniae causes significant mortality in barramundi, Lates calcarifer. Interestingly, S. agalactiae ST261, although causing mortality in wild fish along the Queensland coast, does not appear to cause mortality in barramundi farmed in close proximity. This difference provides a basis for comparison, in order to explore the requirements for blood colonization and sepsis by S. iniae in barramundi, and to increase our understanding of sepsis in fish. S. iniae grew rapidly in barramundi blood, doubling in less than 30 minutes in a whole blood bactericidal assay. In contrast, S. agalactiae was unable to multiply. Moreover, E. coli DH5 was completely killed in barramundi blood during the same incubation period, suggesting 2 that antibacterial humoral and cellular immune defences were functional within the blood bactericidal model. A capsular defective strain of S. iniae also survived in the blood- bactericidal assay, however the rapid proliferation was reduced, suggesting that while the capsule is important for bacterial colonisation it is not the only means used to evade blood- borne defences. As gram-positive pathogens are particularly susceptible to lysozyme due to the dominance of muramic acids in the cell wall, the role of lysozyme was investigated. Lysozyme levels in barramundi serum and plasma were undetectable in a lysis assay using Micrococcus lysodeitikus, indicating very low levels of circulating lysozyme in healthy barramundi. The first responder immune cells in blood are neutrophils and these are critical in the prevention of sepsis; for example, granulocytopenia (circulating granulocyte deficiency) is associated with increased susceptibility to Escherichia coli K1 and Klebsiella pneumoniae sepsis in neonates, and this can be partially repaired by injection of granulocyte colony stimulating factor (G-CSF) to increase neutrophil numbers (Deshmukh et al., 2014). This critical role in the prevention of sepsis implies that pathogens that cause sepsis must therefore be capable of evading or subverting neutrophilic response. As neutrophils are short-lived and previous work on fish neutrophils has focused on populations derived from hematopoietic tissues (head-kidney), cells that are likely to be immature in their development, a novel procedure for effective enrichment of mature neutrophils from barramundi peripheral blood was devised. Use of a dextran layer during Percoll density gradient separation effectively removed reticulocytes and erythrocytes from barramundi blood and enriched neutrophils to around 40-50% in the resulting cell suspensions. These mature enriched neutrophils did not release reactive oxygen species (ROS) or 3 myeloperoxidase (MPO) in response either Streptococcus species tested at an MOI of 1 but showed functionality by response to PMA and CaI positive control. Production of neutrophil extracellular traps (NETs) was investigated in plate-based DNA release assays. There was a low level of DNA release in response to both capsulated and acapsular strains of S. iniae and S. agalactiae, but microbial nucleases did not appear to play a role in evasion of NETs or neutrophils. Moreover, S. iniae and S. agalactiae did not kill or induce apoptosis in barramundi peripheral blood neutrophils. However, both capsular and acapsular strains of S. iniae, but not S. agalactiae, bound to mature peripheral blood neutrophils in an actin- independent manner but were not phagocytosed. This suggests that S. iniae may recruit neutrophils, but block activation and subsequent phagocytosis. It may be that S. iniae co- opts the innate ability of neutrophils to pass through tight junctions in order to cross the blood brain barrier and cause meningitis. Future work should focus on the mechanisms by which this key bactericidal process in peripheral blood neutrophils is blocked by S. iniae, as the factors involved in neutrophil interaction are potentially targets for vaccination against S. iniae in farmed fish. 4 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. 5 Publications during candidature A reliable method for enrichment of neutrophils from peripheral blood in barramundi (Lates calcarifer). Masterman KA, Barnes AC. Fish Shellfish Immunol. 2016 Sep 15;58:174-176. doi: 10.1016/j.fsi.2016.09.028 PMID: 27640156 Publications included in this thesis No publications included. 6 Contributions by others to the thesis No contributions by others. Statement of parts of the thesis submitted to qualify for the award of another degree None. 7 Acknowledgements Thank you to the Australian Postgraduate Association for my scholarship, which made it financially possible for me to do this PhD at all. Thank you to Andy and the Barnes lab for the help, support, coffees and comradery. Special big thanks to Emma, a great office buddy and Jerome, always happy to help, all the time. Much appreciation to Emma and Sheridan for thesis editing support. In memorial to Jared Purton, I wish I could have shared this bumpy journey and all its travails with you, sure could have done with bouncing ideas off you. Biggest hugs and thanks of all to Andrew, and my parents, for all the support and encouragement along the way. Couldn’t have done it with Andrew, who has just been a fabulous dive buddy, sounding board and who is great at talking me down when I’m going loopy. You make my life happier and easier and I appreciate it all. Between submitting my thesis for assessment and receiving the comments back, Andrew died on a tragic scuba diving accident. My loss is enormous, and I dedicate this thesis to him as it simply wouldn’t have been completed without him. 8 Keywords Cell biology, innate immunity, neutrophil, aquaculture, Streptococci, teleost, vaccines Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 060601, Biochemistry and Cell Biology, 80% ANZSRC code: 060605, Microbiology, 20% Fields of Research (FoR) Classification FoR code: 0601 Biochemistry and Cell Biology, 80% FoR code: 0605 Microbiology, 20% 9 Table of contents Streptococcal pathogenesis in barramundi (Lates calcarifer): the host innate immune response and how Streptococci circumvent them. CHAPTER ONE: General background introduction 1.1 Aquaculture, food security and sustainability .......................................................................... 18 1.2 Fish pathogenic Streptococci ................................................................................................... 23 1.2.1 Streptococcus iniae ..................................................................................................... 25 1.2.2 Streptococcus agalactiae ...........................................................................................
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