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

Distribution and severity of placental lesions caused by the Chlamydia abortus 1B vaccine strain in vaccinated ewes

Chlamydia abortus infects livestock species worldwide and is the cause of enzootic abortion of ewes (EAE). In Europe, control of the disease is achieved using a live vaccine based on C. abortus 1B strain. Although the vaccine has been useful for controlling disease outbreaks, abortion events due to the vaccine have been reported. Recently, placental pathology resulting from a vaccine type strain (vt) infection has been reported and shown to be similar to that resulting from a natural wild-type (wt) infection. The aim of this study was to extend these observations by comparing the distribution and severity of the lesions, the composition of the predominating cell infiltrate, the amount of bacteria present and the role of the blood supply in infection. A novel system for grading the histological and pathological features present was developed and the resulting multi-parameter data were statistically transformed for exploration and visualisation through a tailored principal component analysis (PCA) to evaluate the difference between them. The analysis provided no evidence of meaningful differences between vt and wt strains in terms of the measured pathological parameters. The study also contributes a novel methodology for analysing the progression of infection in the placenta for other abortifacient pathogens.EEA MercedesFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Caspe, Sergio Gaston. Moredun Research Institute; Reino UnidoFil: Caspe, Sergio Gaston. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Palarea-Albaladejo, Javier. Biomathematics & Statistics Scotland; Reino UnidoFil: Underwood, Clare. Moredun Research Institute; Reino UnidoFil: Livingstone, Morag. Moredun Research Institute; Reino UnidoFil: Wattegedera, Sean Ranjan. Moredun Research Institute; Reino UnidoFil: Milne, Elspeth. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Sargison, Neil Donald. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Chianini, Francesca. Moredun Research Institute; Reino UnidoFil: Longbottom, David. Moredun Research Institute; Reino Unid

The extent of placental pathology is negatively correlated to birth weight in ewes infected with the wild-type strain of Chlamydia abortus

The placenta is the organ that allows the exchange of oxygen and nutrients between maternal and foetal blood, supplying the requirements of the growing foetus. Consequently, any factor that alters placental integrity may affect foetal nutrition, viability and lamb birth weight. Reproductive diseases, such as ovine enzootic abortion (OEA), impact on foetal viability due to the reduction in the functional area for maternofoetal exchange. This study aimed to consider the impact of pathological features of OEA placental lesions on lamb birth weight and indirectly on foetal viability. To investigate the relationship between birth weight and various OEA-related parameters, data from 562 lambs/foetuses from animals experimentally challenged with Chlamydia abortus strain S26/3 and from uninfected animals were analysed. The parameters investigated included the number of foetuses/lambs delivered (single/multiple), foetus/lamb sex, length of gestation, the proportion of placentas affected by lesions (percentage of gross placental pathology), foetal viability (live/aborted) and the number of C. abortus organisms shed in post-parturition vaginal excretions. The results suggest that the length of gestation and the proportion of placentas affected by lesions are the main contributors to birth weight variability, whereas the other factors, including foetal viability (live or aborted outcomes), were found to be less relevant co-variables. The study determined the strongest positive and negative correlations between birth weight were with the length of gestation and the extent of placental pathology, respectively. These results may indicate that economic losses associated with OEA infections result not only from aborted foetuses but also from the surviving lambs that are born weaker and consequently are more susceptible to diseases.EEA MercedesFil: Caspe, Sergio Gaston. Moredun Research Institute; Reino UnidoFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Caspe, Sergio Gaston. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Palarea-Albaladejo, Javier. Biomathematics & Statistics Scotland; Reino UnidoFil: Palarea-Albaladejo, Javier. University of Girona. Deparment of Computer Sciences, Applied Mathematics and Statistics; EspañaFil: Livingstone, Morag. Moredun Research Institute; Reino UnidoFil: Wattegedera, Sean Ranjan. Moredun Research Institute; Reino UnidoFil: Milne, Elspeth. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Sargison, Neil Donald. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Longbottom, David. Moredun Research Institute; Reino Unid

The 1B vaccine strain of Chlamydia abortus produces placental pathology indistinguishable from a wild type infection

Chlamydia abortus is one of the most commonly diagnosed causes of infectious abortion in small ruminants worldwide. Control of the disease (Enzootic Abortion of Ewes or EAE) is achieved using the commercial live, attenuated C. abortus 1B vaccine strain, which can be distinguished from virulent wild-type (wt) strains by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Published studies applying this typing method and whole-genome sequence analyses to cases of EAE in vaccinated and non-vaccinated animals have provided strong evidence that the 1B strain is not attenuated and can infect the placenta causing disease in some ewes. Therefore, the objective of this study was to characterise the lesions found in the placentas of ewes vaccinated with the 1B strain and to compare these to those resulting from a wt infection. A C. abortus-free flock of multiparous adult ewes was vaccinated twice, over three breeding seasons, each before mating, with the commercial C. abortus 1B vaccine strain (Cevac® Chlamydia, Ceva Animal Health Ltd.). In the second lambing season following vaccination, placentas (n = 117) were collected at parturition and analysed by C. abortus-specific real-time quantitative PCR (qPCR). Two placentas, from a single ewe, which gave birth to live twin lambs, were found to be positive by qPCR and viable organisms were recovered and identified as vaccine type (vt) by PCR-RFLP, with no evidence of any wt strain being present. All cotyledons from the vt-infected placentas were analysed by histopathology and immunohistochemistry and compared to those from wt-infected placentas. Both vt-infected placentas showed lesions typical of those found in a wt infection in terms of their severity, distribution, and associated intensity of antigen labelling. These results conclusively demonstrate that the 1B strain can infect the placenta, producing typical EAE placental lesions that are indistinguishable from those found in wt infected animals

Antibody responses to recombinant protein fragments of the major outer membrane protein and polymorphic outer membrane protein POMP90 in Chlamydophila abortus-infected pregnant sheep

Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans

A Trifecta of New Insights into Ovine Footrot for Infection Drivers, Immune Response and Host Pathogen Interactions.

Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industry’s biggest welfare problems. The complex aetiology of footrot makes in-situ or in-vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved, how they may interact with the host and ultimately providing a way to identify targets for future hypotheses driven investigative work. Here we present the first combined global analysis of the bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intra tissue and surface bacterial populations and the most abundant bacterial transcriptome were analysed, demonstrating footrot affected skin has a reduced diversity and increased abundances of, not only the causative bacteria Dichelobacter nodosus, but other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica. Host transcriptomics reveals a suppression of biological processes relating to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot affected interdigital skin comparted to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome which could lead to the identification of future therapeutic targets