Immunopathogenesis of Chlamydia abortus infections in vaccinated and non-vaccinated ewes
Item statusRestricted Access
Embargo end date16/11/2023
Caspe, Sergio Gastón
Enzootic abortion of ewes (EAE) is caused by the obligate intracellular Gram-negative bacterium Chlamydia abortus. EAE is considered one of the most important causes of infectious abortion in sheep in many parts of the world. A necrosuppurative placentitis is typically associated with EAE. The primary function of the placenta is to provide fetal nutrition, therefore, anything that affects organ integrity may indirectly affect the viability of the fetus. The severity of placental lesions giving rise to abortion is a consequence of multiple interacting factors, in particular chlamydial growth, host immune responses and hormonal balance. A better understanding of these interactions may help to explain why EAE results in different pregnancy outcomes spanning: abortions, the birth of weak lambs that may die during the first days of their life or the birth of healthy lambs. Effective control of the disease is achieved through a combination of diagnosis, antibiotic therapy, flock management and vaccination. In the UK, vaccination is carried out using the live C. abortus 1B vaccine (vt) strain, which has been associated with abortion events in sheep flocks. The research hypothesis for this PhD thesis is that the distribution of C. abortus in the ovine placenta, pathological lesions and clinical outcomes of infection are not homogeneous within and between infected ewes and ewes vaccinated with the live 1B vaccine. This study aimed to increase knowledge of the pathogenesis of C. abortus in the placenta and uterus and its relationship with pregnancy outcome, thereby informing on improved diagnosis and control of the disease. The objectives were to: 1. Determine the relationship between phenotypical patterns of immune cell infiltration and the different outcomes of chlamydial infection in multifetal pregnancies. 2. Investigate the distribution and severity of lesions in placentas from wild-type (wt) C. abortus infected ewes and their relationship with pregnancy outcome. 3. Compare the distribution and severity of the lesions caused by the commercial C. abortus 1B vaccine strain with those resulting from a wt C. abortus infection. The uteri and placentas from experimental wt C. abortus -challenged twin-bearing ewes with different pregnancy outcomes (dead/dead, dead/live and live/live) were collected after parturition to address objective 1. Tissues were also collected from non-infected and EAE-free ewes as negative controls. All samples were analysed using a broad range of immune cell features, including cell surface antigens, T-cell transcription factors and cytokines. Elevated lymphocytes (cluster of differentiation (CD)4+, CD8+, gamma-delta T cells and natural killer cells), interleukin (IL)-10, tumour necrosis factor-alpha (TNF-α) and Forkhead box P3 (Foxp3) responses were observed by immunohistochemistry (IHC). Interferon-gamma and IL-17A increases were measured by in situ hybridisation. Additionally, a C. abortus major outer membrane protein (MOMP) antibody was used to detect Chlamydiae in the tissues to determine the relationship between pregnancy outcome, pathological lesion, and presence of immune cells. The level of T-helper (Th) and T-regulatory cell (Treg) features revealed statistically significant group effects, showing the important role that the balance of lymphocyte subsets may play in the different pregnancy outcomes in ewes. In addressing objective 2, several features associated with EAE, including placental lesions, length of gestation, lambing outcome, modified Ziehl-Neelsen scores, and the number of copies of C. abortus genomes were analysed, using the co-variables sex of the lamb (male or female) and type of gestation (single, multiple). One of the most significant parameters that was statistically associated with pregnancy outcome was the severity of placental lesions. For that reason, placentas presenting differences in the severity and distribution of lesions were examined and compared (specifically 0, 10, 25, 40, 50, 60, 85, 90 and 100% of the placental area affected). Where placentas exhibited gross lesions of between 0 and 20% of the placental surface, the difference was statistically significant between pregnancy and lamb birth weight. There were some variations in severity when the percentage of lesions increased from 25 % to 100%, but the statistical analysis of those differences was less significant. Vaccination with commercial vaccines containing the vt strain is a key strategy for controlling EAE in sheep flocks, but this strain has in some circumstances been associated with abortion. A pathological comparison was, therefore, made between placentas infected with the vt strain and EAE lesions caused by a wt strain, to address objective 3. Placentas collected from an EAE-free commercial sheep flock were assessed for gross pathological lesions and analysed for the presence of chlamydial DNA by real-time quantitative polymerase chain reaction (qPCR). Two placentas were observed to exhibit lesions consistent with EAE and were also the only two found positive by qPCR. Following isolation of the strain, PCR-restriction fragment length polymorphism (RFLP) and whole-genome sequence analyses, to distinguish vt from wt infections, confirmed the presence of only the vt strain. Comparative analyses were performed by histology and IHC for chlamydial labelling, to evaluate the difference between vt and wt strain placentas. The lesions caused by both, vt and wt strains were found to be indistinguishable. The results obtained from this study demonstrate that the distribution and severity of the placental lesions and chlamydial load are not uniform in both vt and wt infected ewes, confirming the research hypothesis. This study contributes relevant information about the pathogenesis of EAE that will help to inform the iterative improvement of control strategies. It is also a new starting point for future research into the rate of vertical transmission in vt and wt infected ewes, and the impact of this transmission on the reproductive performance of these intrauterine infected lambs in their adult life.