Mechanisms of vaccine protection in chickens against challenge with virulent Mycoplasma synoviae

Abstract

The Vaxsafe MS vaccine is used globally to control infections with Mycoplasma synoviae (MS) in commercial poultry. It provides long-term protective immunity against airsacculitis and tracheitis caused by M. synoviae. However, the mechanisms involved in the protection afforded by the vaccine are not well understood. This study aimed to investigate and compare tracheal mucosal responses to challenge with virulent M. synoviae in chickens vaccinated with Vaxsafe MS and in unvaccinated chickens. The tracheal mucosal transcriptional profiles were obtained using messenger RNA sequencing. Compared to the unvaccinated-unchallenged chickens, 64 genes were differentially transcribed in the vaccinated-challenged chickens and 321 genes were differentially transcribed in the unvaccinated-challenged chickens. In vaccinated-challenged chickens, functional categories enriched with up-regulated genes included IL4 production, TCR signalling (without T-cell co-stimulation), T-cell activation/proliferation/differentiation and B-cell activation/proliferation. In the unvaccinated-challenged chickens, many other functional categories were also enriched with upregulated genes, including Positive regulation of the MAPK/ERK cascades, Positive regulation of IFN gamma production, T-cell co-stimulation, Negative regulation of IL6 production, IL10 production, Toll-like receptor signalling, Neutrophil activation/degranulation, DAP12 signalling, Chemotaxis, Phagocytosis, Cell killing, PD-1 signalling, Negative regulation of non-canonical NF-κB signal transduction, Isotype switching and Somatic diversification of immune receptors. The transcriptional changes in the unvaccinated-challenged chickens were indicative of T-helper-1-cell-mediated inflammation and dysregulated primary T- and B-cell responses, which were absent in the vaccinated-challenged chickens. In the vaccinated-challenged chickens the changes were indicative of a memory T-follicular-helper-cell-dependent secondary B-cell response, suggesting that this response was protective against the dysregulated inflammatory response seen in unvaccinated chickens.

Keywords: Mycoplasma synoviae; RNA sequencing; Vaxsafe® MS; chicken tracheal mucosae; vaccine protection.

Figure 1

Haematoxylin and eosin-stained upper tracheal cross sections. A Unvaccinated-challenged group. B Vaccinated-challenged group. C Unvaccinated-unchallenged group. M, mucosa; T, tracheal cartilage; G, mucus glands; double-headed black arrows indicate the mucosal thickness; single-headed black arrows indicate ciliated pseudostratified columnar epithelia; black arrowheads indicate immune cell infiltration. The scale bars indicate 50 µm.

Figure 2

Mean-difference plots of log fold change versus log CPM. A Unvaccinated-challenged chickens compared to unvaccinated-unchallenged chickens. B Vaccinated-challenged chickens compared to unvaccinated-unchallenged chickens. N indicates the number of genes, red dots and numbers indicate up-regulated genes, blue dots and numbers indicate down-regulated genes, grey dots and numbers indicate the genes with no significant difference in transcription. C Venn diagram showing the differentially expressed genes that are unique to or shared by the two challenged groups of chickens. The blue circle represents the unvaccinated-challenged group (compared to unvaccinated-unchallenged birds) and the red circle represents the vaccinated-challenged group (compared to unvaccinated-unchallenged birds).

Figure 3

Biological pathways enriched with up-regulated genes. C: the unvaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. VC: the vaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. The size of the bubble indicates the proportion of the genes in the pathway that were up-regulated.

Figure 4

Immune response associated gene ontology terms (biological processes) enriched with up-regulated genes. C: the unvaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. VC: the vaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. The size of the bubble indicates the proportion of the genes in the GO term that were up-regulated.

Figure 5

Leukocyte mediated immunity associated gene ontology terms (biological processes) enriched with up-regulated genes. C: the unvaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. VC: the vaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. The size of the bubble indicates the proportion of the genes in the GO term that were up-regulated.

Figure 6

Lymphocyte mediated immunity associated gene ontology terms (biological processes) enriched with up-regulated genes. C: the unvaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. VC: the vaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. The size of the bubble indicates the proportion of the genes in the GO term that were up-regulated.

Figure 7

T-cell mediated immunity associated gene ontology terms (biological processes) enriched with up-regulated genes. C: the unvaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. VC: the vaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. The size of the bubble indicates the proportion of the genes in the GO term that were up-regulated.

Figure 8

B-cell mediated immunity associated gene ontology terms (biological processes) enriched with up-regulated genes. C: the unvaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. VC: the vaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. The size of the bubble indicates the proportion of the genes in the GO term that were up-regulated.

Figure 9

Cytokine response associated gene ontology terms (biological processes) enriched with up-regulated genes. C: the unvaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. VC: the vaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. The size of the bubble indicates the proportion of the genes in the GO term that were up-regulated.

Figure 10

Signal transduction associated gene ontology terms (biological processes) enriched with up-regulated genes. C: the unvaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. VC: the vaccinated-challenged chickens compared to the unvaccinated-unchallenged chickens. The size of the bubble indicates the proportion of the genes in the GO term that were up-regulated.