Host transcriptome response to Mycoplasma bovis and bovine viral diarrhea virus in bovine tissues

Abstract

Background: Mycoplasma bovis is a prominent pathogen associated with respiratory disease in livestock. Respiratory disease in cattle often involves co-infection, where a primary viral infection can weaken the host immune system and thus enhance subsequent bacterial infection. The objective of this study was to investigate changes in the host (cattle) transcriptome during bacterial-viral co-infection. RNA sequencing was done in whole blood cells (WBC), liver, mesenteric lymph node (MLN), tracheal-bronchial lymph node (TBLN), spleen, and thymus collected from Control animals (n = 2), animals infected with M. bovis (MB; n = 3), and animals infected with M. bovis and bovine viral diarrhea virus (BVDV) (Dual; n = 3).

Results: Thymus and spleen had the greatest number of differentially expressed genes (DEGs) out of all tissues analyzed. In spleen, genes involved in maintenance of the extracellular matrix (ECM) including collagen type XV alpha 1 chain (COL15A1), collagen type IV alpha 2 chain (COL4A2), and heparan sulfate proteoglycan 2 (HSPG2) were the most significantly downregulated in Dual compared to Control and MB. In thymus, complement 3 (C3) was a highly significant DEG and upregulated in Dual compared to Control and MB. Interferon alpha inducible protein 6 (IFI6) and interferon-induced transmembrane proteins (IFITM1 and IFITM3), were significantly associated with infection status and upregulated in spleen and thymus of Dual compared to Control and MB.

Conclusion: Downregulation of ECM components may cause degradation of the ECM and contribute to increased viral spread due to co-infection. Hyperactivation of complement pathway genes may contribute to damage to the thymus and influence severity of co-infection. Co-expression of IFI6, IFITM1 and IFITM3 across lymphoid tissues may be connected to enhanced pathogenesis in co-infection. These findings suggest co-infection exacerbates disease severity through modulation of ECM components in spleen and complement and coagulation cascades in the thymus. These impacted pathways may underlie thymic atrophy and impaired pathogen clearance due to BVDV and M. bovis co-infection.

Keywords: Bovine; Bovine viral diarrhea virus; Gene expression; Mycoplasma bovis.

Fig. 1

Principal component analysis (PCA) and correlation heatmap plots across all samples and tissues. (A) PCA plots for all samples analyzed. (B) PCA plots for all samples, excluding liver and WBC. (C) Correlation heatmap for all samples. Red and blue colors represent the highest and lowest correlation values, respectively. Tissues are highlighted in different colors and samples labeled on x- and y-axis. Samples belonging to Control, M. bovis (MB), and Dual groups as well as those derived from liver, mesenteric lymph node (MLN), serum, spleen, tracheal-bronchial lymph node (TBLN), thymus, and whole blood cells (WBC) are shown in different colors

Fig. 2

Volcano plots of differentially expressed genes (DEGs) between Control and M. bovis (MB) in liver, whole blood cell (WBC), mesenteric lymph node (MLN), spleen, and thymus. The x-axis indicates log2 foldchange and the y-axis indicates -log10 adjusted p-value for each DEG. DEGs with an adjusted p-value < 0.05 were deemed significant. Upregulated DEGs are shown in blue. Downregulated DEGs are shown in red. Non-significant genes are shown in grey. PTPRO = protein tyrosine phosphatase receptor type O; DGAT2 = diacylglycerol O-acyltransferase 2; ATP2C2 = ATPase secretory pathway Ca2 + transporting 2; LOC100298428 = uncharacterized LOC100298428

Fig. 3

Volcano plots of differentially expressed genes (DEGs) between (A) Control vs Dual and (B) M. bovis (MB) vs Dual in liver, whole blood cell (WBC), mesenteric lymph node (MLN), tracheal-bronchial lymph node (TBLN), spleen, and thymus. The x-axis indicates log2 foldchange and the y-axis indicates -log10 adjusted p-value for each DEG. DEGs with an adjusted p-value < 0.05 were deemed significant. Upregulated DEGs are shown in blue. Downregulated DEGs are shown in red. Non-significant genes are shown in grey. SLC45A3 = solute carrier family 45 member 3; ISM1 = isthmin 1; PTPRO = protein tyrosine phosphatase receptor type O; IFI6 = interferon alpha inducible protein 6; FADS1 = fatty acid desaturase 1; ARSG = arylsulfatase G; TMEM119 = transmembrane protein 119; SCD = stearoyl-CoA desaturase; DGAT2 = diacylglycerol O-acyltransferase 2; OAS2 = 2’-5’-oligoadenylate synthetase 2; IFI27 = interferon alpha inducible protein 27; AICDA = activation induced cytidine deaminase; ARL5C = ARF like GTPase 5 C; STMN3 = stathmin 3; CCL14/16 = chemokine ligand 14/16; COL15A1 = collagen type XV alpha 1 chain; HSPG2 = heparan sulfate proteoglycan 2; COL4A2 = collagen type IV alpha 2 chain; C3 = complement C3

Fig. 4

Intersection plots of unique and shared differentially expressed genes (DEGs) across tissues. Intersection plots in (A) Control vs. M. bovis (MB), (B) Control vs. Dual, and (C) MB vs. Dual for DEGs found in liver, spleen, thymus, mesenteric lymph node (MLN), tracheal-bronchial lymph node (TBLN), and whole blood cell (WBC). Individual dots under the bar graph indicate DEGs unique to a tissue. More than one dot under a bar indicates that the DEG is shared by the dotted tissues

Fig. 5

Pathways significantly enriched for differentially expressed genes. A subset of impacted biological processes associated with DEGs in Control vs. Dual and M. bovis (MB) vs. Dual comparisons in (A) Thymus and (B) Spleen. Gene ontology (GO) enrichment analysis was performed with DAVID and pathways with a p-value < 0.05 (dashed line) were considered significant. Pathways are shown in the y-axis and the -log10 adjusted p-value on the x-axis

Fig. 6

Heatmaps of shared differentially expressed genes (DEGs) in Control vs. Dual and M. bovis (MB) vs. Dual comparisons within impacted pathways in (A) spleen and (B) thymus. The color scale indicates the magnitude of expression (log counts per million (logCPM)) of the respective gene across samples

Fig. 7

Weighted gene co-expression network analysis (WGCNA) in lymphoid tissues for Control, M. bovis (MB), and Dual groups. (A) Module-treatment relationship graph where each row represents the module eigen value and each column represents infection status. The cells within the matrix show the correlation coefficient and p-value. Modules were found in mesenteric lymph node (MLN), tracheal-bronchial lymph node (TBLN), thymus, and spleen. (B) Correlation between module membership (MM) and gene significance (GS) of Module III genes, where MM represents the correlation between gene expression and the module eigen values and GS represents the correlation between gene expression and co-infection status. (C) Biological processes, pathways, and molecular functions enriched with co-expressed genes in Module III. The y-axis indicates the gene ontology (GO) term and the x-axis indicates the -log10 p-value. The size of the dot indicates the number of co-expressed genes enriched in the GO term. Enriched GO terms with a p-value < 0.05 (dashed line) were considered significant. IFI6 = interferon alpha inducible protein 6; BST2 = bone marrow stromal cell antigen 2; HERC6 = HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase Family Member 6; ISG15 = interferon-stimulated gene 15; IFITM1/3 = interferon-induced transmembrane proteins 1/3