Host-pathogen interactions during Mycoplasma gallisepticum infection and vaccination in chickens

© 2021 Sathya Nadeeshani Kulappu ArachchigeMycoplasma gallisepticum is an important respiratory pathogen of chickens with a worldwide distribution. The molecular basis of chronic pathological changes in the trachea, caused by M. gallisepticum and the immune responses induced after infection or vaccination with M. gallisepticum are poorly understood, particularly during the chronic stages of disease. Live attenuated vaccines are commonly used and effective against infection with M. gallisepticum, but currently available commercial live-attenuated vaccines have limitations that prevent their optimal application in the field. M. gallisepticum ts-304 is a novel live-attenuated vaccine strain that has been shown to be safe and more effective than the current commercial vaccine strain ts-11, from which it was derived. Tools for investigating host gene transcription using RNA-seq were developed with the aim of identifying transcriptional changes underlying the chronic pathological changes, caused by M. gallisepticum in the trachea and assessing the duration of immunity and the effect of immunosuppression on the efficacy of the ts-304 vaccine against those transcriptional changes. The findings led to identification of genes that are involved in inflammation, cytokine production and signalling, cell proliferation, and impaired formation of the cilia, cytoskeleton and intercellular junctions in unvaccinated-challenged birds compared to vaccinated-unchallenged, vaccinated-challenged, and unvaccinated-unchallenged birds. In addition, the absence of transcriptional changes associated with impaired formation of intercellular junctions and several of those involved in inflammation in older birds, compared to young birds, suggested age-dependent differences in the susceptibility of chickens to pathological changes associated with infection with M. gallisepticum. The ts-304 vaccine was able to protect chickens against the transcriptional changes caused by M. gallisepticum in the trachea until at least 57 weeks after vaccination. In order to investigate the immune responses mounted in the trachea after infection with M. gallisepticum in vaccinated and unvaccinated chickens, pathological changes were assessed by histopathological examination and the immune response cells accumulating in tracheal mucosa were identified by indirect immunofluorescent staining. The levels of transcription of a panel of cytokine and chemokine genes in the trachea were quantified using RT-qPCR. After infection, there was a significant increase in the tracheal mucosal thickness and in transcription of the genes for IFN-gamma, IL-17, RANTES and CXCL-14, and significant downregulation of transcription of the gene for IL-2. B cells, CD4+ cells, CD3+ cells and macrophages (KUL01+) were detected in the tracheal mucosa of unvaccinated/challenged birds, but not CD8+ cells. Paradoxical transcription of the genes for IFN-gamma and IL-2 and depletion of CD8+ cells suggested that M. gallisepticum caused immune dysregulation. In addition, the presence of CD4+ cells, and the upregulation of IFN-gamma and IL-17 in the absence of a significant difference in the level of transcription of IL-13, in the challenged-only group compared to the other groups were suggestive of suppression of Th2 responses, as has been seen in infections with other mycoplasmas in other animal species. Upregulation of IFN-gamma, and the presence of CD4+ cells, B cells and macrophages, also suggested enhanced antigen processing and presentation by antigen presenting cells, possibly leading to macrophage-driven phagocytosis in unvaccinated chickens. Vaccinated birds appeared to be protected against challenge and this protection afforded by the ts-304 vaccine appeared to be mediated by mucosal humoral memory, resulting in a strong anamnestic mucosal antibody response. Four immunosuppression models were developed using infection with chicken anaemia virus (CAV) or infectious bursal disease virus (IBDV) to suppress the cell-mediated or humoral immune responses at vaccination or at challenge to enable the effects of immunosuppression on the protection induced by ts-304 vaccine to be assessed. These studies showed that the efficacy of the ts-304 vaccine was reduced by immunosuppression, resulting in exacerbation of tracheal lesions, a higher level of colonisation of the trachea by virulent M. gallisepticum and reduced systemic serological responses against M. gallisepticum. Investigations of the genome-wide transcriptional profiles of these chickens found that infection with CAV and IBDV adversely affected activation, proliferation and function of immune cells, including T and B lymphocytes and macrophages, with impacts on phagocytosis, immunoglobulin class switching and several immune-signalling pathways in the tracheal mucosa, suggesting the mechanisms underlying the reduced efficacy of the ts-304 vaccine in immunosuppressed chickens