This study compared peripheral blood mononuclear cell (PBMC) transcriptional responses to Mycoplasma bovis using in vitro infection followed by RNA sequencing. The manuscript reports that seven lactating, primiparous contemporary and unselected Holstein cows were used (as reported) and five approximately two-year-old female bison were included. PBMC were isolated from whole blood by density gradient centrifugation, red blood cells were lysed with ACK buffer, and cells were resuspended in complete RPMI.
For infection experiments, PBMC were plated at 1 × 10^6 cells/well and infected with the M. bovis isolate NADC1 at a multiplicity of infection (MOI) of approximately 0.5. Mock-infected controls received culture medium. Cells were incubated for 24 hours at 37°C, 5% CO2 before RNA was extracted using TRIzol and a column-based purification kit.
Libraries were prepared using an Illumina Stranded mRNA kit and sequenced on a NextSeq 1000 to generate 100-bp paired-end reads, with an average of 22.06 million reads per sample. Reads were quality-checked and trimmed, then aligned to the Bos taurus ARS-UCD 2.0 or Bison UMD 1.0 genome assemblies. Raw counts were generated and differential gene expression (DEG) was calculated with DESeq2. DEG were considered significant with log2 fold change > |1| and Benjamini–Hochberg false discovery rate (FDR) < 0.05. Ingenuity Pathway Analysis (IPA) was used for canonical pathway enrichment. Raw sequence data are available on GenBank (PRJNA1439942).
At 24 hours post-infection, the numbers of significantly differentially expressed genes (DEG) reported were: contemporary Holsteins (CH) 3,904 DEGs (1,651 upregulated, 2,253 downregulated); unselected Holsteins (UH) 4,871 DEGs (2,018 upregulated, 2,853 downregulated); and bison 2,714 DEGs (1,212 upregulated, 1,502 downregulated). The UH group exhibited the greatest number of DEG, while bison had the fewest, indicating host genotype and species differences in PBMC transcriptional responsiveness to M. bovis.
Comparison across genotypes revealed that CH and UH shared 2,291 DEGs, reflecting greater similarity between Holstein lines. Only 129 CH DEGs and 296 UH DEGs overlapped with bison, and 382 DEGs were common to all three host types. Supplementary tables in the source list the full sets of unique and shared DEGs by genotype.
IPA identified 529, 589, and 266 significantly enriched pathways (P < 0.05) for CH, UH, and bison PBMC, respectively. In CH, enriched activated pathways were primarily associated with innate immune cell function such as neutrophil degranulation; pathways with decreased expression included wound healing, IL-17A signaling, and osteoarthritis-related pathways. UH showed many similar enriched pathways but with higher expression in pathways linked to adaptive responses to intracellular pathogens (class I MHC antigen processing and presentation), pattern recognition receptor signaling, and cytokine signaling including interferon pathways and the pathogen-induced cytokine storm signaling pathway (PICSS).
Bison PBMC pathway enrichment differed from both Holstein genotypes. Bison responses were characterized by upregulation of pathways involved in cytokine and chemokine signaling, classical macrophage activation, and strong type I and II interferon pathway activity, along with activation of IL-17 and cachexia-related pathways. In contrast to Holsteins, the bison signature showed a relative decrease in the neutrophil degranulation pathway.
Within the pathogen induced cytokine storm signaling pathway (PICSS), species-specific differences were pronounced. Bison PBMC demonstrated broad and strong upregulation of interferons, multiple cytokines, and chemokines. Reported log2 fold changes for bison included IFNB1 (1.95) and IFNG (4.23); only IFNG was upregulated in UH (3.86). TNF was increased in bison (3.28), consistent with activation of the cachexia pathway in that host.
Several interleukins showed markedly greater induction in bison than in Holsteins: IL1A (5.87), IL2 (2.03), IL6 (7.90), IL10 (2.42), IL17A (6.51), IL21 (3.93), and IL23A (3.04). IL12A and IL22 were upregulated in both UH and bison but to a greater extent in bison (IL12A 3.48 and IL22 6.27 in bison vs IL12A 2.04 and IL22 2.10 in UH). Chemokine changes were also genotype- and species-specific: CCL2 (2.23) and CXCL10 (1.94) were upregulated in UH, whereas CCL5 (3.08) was upregulated only in bison.
The most significant DEGs called in each host type included genes implicated in immune regulation and cellular processes. In CH, the top significant DEGs included SLA (Src-like adaptor protein involved in negative regulation of T-cell receptor signaling) and KCNN4 (a potassium-calcium-activated channel important for multinucleate giant cell formation). UH top DEG hits included CROCC, FRMD8, ABTB2, and LENG8 (reported as downregulated). In bison PBMC, highly significant DEGs included ABCC3 and SLC44A3 among others. Full gene lists and supplementary data are provided in the source article.
The source interprets these findings as evidence that PBMC transcriptional responses to M. bovis differ both by species (Holstein cattle versus bison) and by Holstein genotype (contemporary selected line versus an unselected control line). Holstein PBMC—particularly UH—showed greater overall transcriptional responsiveness by DEG count, while bison exhibited a more focused proinflammatory and interferon-rich program. The authors suggest these species- and genotype-specific immune signatures may underlie observed differences in clinical disease: M. bovis acts as a primary, often severe pathogen in bison with strong proinflammatory responses, whereas in cattle it is commonly associated with the multifactorial bovine respiratory disease complex. The source notes that selection for increased milk production in contemporary Holsteins is associated with genomic changes affecting immunity, which may influence responses to M. bovis.
Note: all numerical results, methods, and interpretations above are taken directly from the cited source article; any additional experimental details or follow-up outcomes not reported in that source are not included here.