Defining immune function and identifying correlates of protection against Chlamydia abortus in sheep

Chlamydia abortus is an obligate intracellular bacterial pathogen and is one of the most common causes of infectious abortion in sheep world-wide. The organism is responsible for the disease known as ovine enzootic abortion (OEA), which manifests in late pregnancy. Development of a safe and effective vaccine deployed alongside existing molecular diagnostic tests remains a long-term ambition. This approach is dependent on improving our knowledge of disease pathogenesis that leads to tissue damage (pathogen and immune cell-mediated) in the sheep placenta. Immune responses that control disease and those responses that facilitate pregnancy are not always compatible or completely understood. Interferon-gamma (IFN-ɣ), the signature cytokine of T-helper (Th)-1 cells, is known to control C. abortus growth. Little is known about other cytokines co-expressed during OEA that might contribute to control or disease progression. Incidentally, knowledge of the existence of other cluster of differentiation (CD)4+ve T cells that produce interleukin-(IL)-4, IL-10 or IL-17A in sheep are limited or non-existent. These capability gaps need to be addressed first, to enable measurement of the quality of immune responses during pregnancy or OEA. This thesis addressed the hypothesis: Systemic immune responses of sheep uniquely correlate with pregnancy outcome following OEA. Three major research aims were undertaken: 1. To expand the range of assays to characterise CD4+ve T cell immune responses in sheep. 2. To develop and apply tools to investigate local immune regulation in the ovine placenta. 3. To investigate immunological correlates of protection against OEA. Specific immunological assays developed in this project enabled single cell identification, multi-parameter cytokine assessment and quantification of specific immunomodulatory molecule transcripts. Studies revealed that IFN-ɣ and IL-10 were initiated upon antigen or mitogen stimulation and their kinetics are as reported for other mammals. Presumptive T-regulatory cells were identified in peripheral blood mononuclear cells (PBMC). IFN-ɣ and IL-17A-expressing cells include Th-1 and Th-17 cells which are differentially represented in mitogen-activated PBMC. Analysis of placental immunity involved collection of placental tissues and associated lymph nodes at full term of pregnancy. Selective predominance of classical major histocompatibility complex Class I transcripts were identified but no indoleamine-2, 3 dioxygenase-1 transcripts within the placental tissues were found and no natural killer cell staining observed. Collectively, this expression pattern of ‘immunological parameters’ are unique to date to placental mammals. Refined and new assays were fundamental to the experimental design to investigate immune responses to OEA. New patterns of Chlamydia-specific responses were identified. Selective elevation of IFN-ɣ and IL-10 during the latent stage of disease prior to pregnancy was observed. These cytokines remained elevated during and after active phases of OEA. Measurement of targeted immunological parameters failed to identify unique responses correlated with protection and do therefore not support the PhD hypothesis. This work has significantly extended the knowledge of placental immunity and cellular immunity during OEA utilizing species-validated immunological assays. Several new areas of investigation have been identified. Concurrent progress in antigen discovery, transcriptomic analysis and tissue organoid cultures have now provided an environment to accelerate innovative research for reproductive disease vaccinology. Defined protective immune signatures will inform on the design of new safe vaccines to OEA, by refinement of protective antigen selection and mechanisms of vaccine delivery. Placental tissue-associated organoids may facilitate antigen discovery for other sheep reproductive pathogens. Together, these proposed areas of further investigation may enable development of novel multi-pathogen vaccines to prevent reproductive diseases of small ruminants.