Exploring splice variants in milk leukocytes of dairy cows with subclinical intramammary infection due to Prototheca spp. and Streptococcus agalactiae

Alternative splicing events lead to different mRNA isoforms, potentially translated into proteins with altered structures or impaired functions, or both. This phenomenon may influence the resistance or susceptibility to diseases, such as mastitis. To explore this aspect, this study aims to analyze transcript expression, focusing on cases where multiple mRNA isoforms are present, in milk somatic cells from Holstein cattle affected by subclinical intramammary infection caused by Prototheca spp. (P+, n = 11) or Streptococcus agalactiae (Sa+, n = 11), compared with uninfected animals (Neg, n = 9). The RNA-sequencing data were analyzed using the CLC Genomics Workbench (23.0.5, Qiagen) with a large gap read mapping approach and Bos taurus ARS-UCD1.3 reference genome to identify the differentially expressed transcripts (DET) among the groups. In addition, a functional analysis of the identified DET, combined with the identification of functional variants within the expressed regions, was performed. The comparison P+ versus Neg revealed 27 annotated DET, 11 annotated DET with novel length, and 7 novel DET with no previously annotated associated gene or length. These DET mainly originated from immune-related genes involved in pathways strictly linked to the immune and inflammatory responses (i.e., antigen presentation pathway, MHC class II antigen presentation pathway, macrophage classical activation signaling pathway). In contrast, the Sa+ versus Neg comparison revealed a total of 26 DET, including 17 annotated transcripts, 8 annotated transcripts with a novel length, and 1 novel transcript from a nonannotated gene. In this case, a predominance of enriched pathways related to metabolism and detoxification processes was observed (i.e., FXR/RXR activation, xenobiotic metabolism general signaling pathway, glutathione-mediated detoxification). Functional variants were identified in regions overlapping DET encoded by KRT78, CSN1S1, and MYBPC1, which were downregulated in P+ when compared with Neg group and potentially related to mastitis resistance/susceptibility traits. The transcripts and associated functional variants identified in this study may contribute to a better understanding of bovine mastitis pathogenesis and development, providing useful insights for improving animal health and management strategies.