Background Beyond conventional transcriptome profiling, RNA-sequencing (RNA-Seq) enables the discovery of variants within expressed genes linked to complex traits such as mastitis resistance or susceptibility. In this study, RNA-Seq was performed on milk somatic cells from uninfected Holstein cows with no history of mastitis (Neg, n = 9) or with subclinical intramammary infections (sIMI) caused by Prototheca spp. (P+ , n = 11) or Streptococcus agalactiae (Sa+ , n = 11). The objective was to identify transcriptome-derived sequence variants detectable under specific microbiological conditions that may contribute to the modulation of host transcriptional responses. By integrating these transcript-derived variants with quantitative trait locus (QTL) annotations and enrichment analyses, we aimed to highlight genomic regions functionally associated with mastitis susceptibility or resilience. Results Using the CLC Genomic Workbench, a total of 306,440, 264,132, and 246,777 unique variants were detected in Neg, P+ , and Sa+ groups, respectively, with SNPs being more abundant than INDELs. High-impact variants were identified in immune-related genes (TNIP1, TNIP3, IL10RB, IL2RA, IL15RA), suggesting post-transcriptional modulation of immune and inflammatory responses under different microbiological conditions. Variants in non-coding regulatory regions indicate that transcriptional control may also contribute to host susceptibility. QTL enrichment using GALLO showed consistent associations with milk production traits, while clinical mastitis QTLs were specifically enriched in P+ , reflecting context-dependent regulation of immune responses. Conclusions These findings provide new insights into the molecular basis of host responses to intramammary infections and support the value of a transcriptome-driven approach for variant discovery. Variants identified in immune-related genes and regulatory regions suggest that both immune gene modulation and post-transcriptional regulation may contribute to susceptibility to sIMI. Upon further functional validation, these findings may inform precision breeding strategies to enhance mastitis resilience in dairy cattle.
Transcriptome-derived variants in milk reveal host response signatures to subclinical intramammary infection in Holstein cattle
Vanzin A.;Bisutti V.;Cecchinato A.;Gallo L.;Giannuzzi D.;Pegolo S.
2026
Abstract
Background Beyond conventional transcriptome profiling, RNA-sequencing (RNA-Seq) enables the discovery of variants within expressed genes linked to complex traits such as mastitis resistance or susceptibility. In this study, RNA-Seq was performed on milk somatic cells from uninfected Holstein cows with no history of mastitis (Neg, n = 9) or with subclinical intramammary infections (sIMI) caused by Prototheca spp. (P+ , n = 11) or Streptococcus agalactiae (Sa+ , n = 11). The objective was to identify transcriptome-derived sequence variants detectable under specific microbiological conditions that may contribute to the modulation of host transcriptional responses. By integrating these transcript-derived variants with quantitative trait locus (QTL) annotations and enrichment analyses, we aimed to highlight genomic regions functionally associated with mastitis susceptibility or resilience. Results Using the CLC Genomic Workbench, a total of 306,440, 264,132, and 246,777 unique variants were detected in Neg, P+ , and Sa+ groups, respectively, with SNPs being more abundant than INDELs. High-impact variants were identified in immune-related genes (TNIP1, TNIP3, IL10RB, IL2RA, IL15RA), suggesting post-transcriptional modulation of immune and inflammatory responses under different microbiological conditions. Variants in non-coding regulatory regions indicate that transcriptional control may also contribute to host susceptibility. QTL enrichment using GALLO showed consistent associations with milk production traits, while clinical mastitis QTLs were specifically enriched in P+ , reflecting context-dependent regulation of immune responses. Conclusions These findings provide new insights into the molecular basis of host responses to intramammary infections and support the value of a transcriptome-driven approach for variant discovery. Variants identified in immune-related genes and regulatory regions suggest that both immune gene modulation and post-transcriptional regulation may contribute to susceptibility to sIMI. Upon further functional validation, these findings may inform precision breeding strategies to enhance mastitis resilience in dairy cattle.
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