RNA sequencing differential gene expression analysis of isolated perfused bovine udders experimentally inoculated with Streptococcus agalactiae

Abstract

The aim of this study was to elucidate the differential gene expression in the RNA sequencing transcriptome of isolated perfused udders collected from 4 slaughtered Holstein × Zebu crossbred dairy cows experimentally inoculated with Streptococcus agalactiae. We studied 3 different statistical tools (edgeR, baySeq, and Cuffdiff 2). In summary, 2 quarters of each udder were experimentally inoculated with Strep. agalactiae and the other 2 were used as a control. Mammary tissue biopsies were collected at times 0 and 3 h after infection. The total RNA was extracted and sequenced on an Illumina HiSeq 2000 (Illumina Inc., San Diego, CA). Transcripts were assembled from the reads aligned to the bovine UMD 3.1 reference genome, and the statistical analyses were performed using the previously mentioned tools (edgeR, baySeq, and Cuffdiff 2). Finally, the identified genes were submitted to pathway enrichment analysis. A total of 1,756, 1,161, and 3,389 genes with differential gene expression were identified when using edgeR, baySeq, and Cuffdiff 2, respectively. A total of 122 genes were identified by the overlapping of the 3 methods; however, only the platelet activation presented a significantly enriched pathway. From the results, we suggest the FCER1G, GNAI2, ORAI1, and VASP genes shared among the 3 methods in this pathway for posterior biological validation.

Th17-related mammary immunity, but not a high systemic Th1 immune response is associated with protection against E. coli mastitis

“…We chose to evaluate blood cell transcriptome by RNA-seq rather than mammary tissue or milk cell gene expression as previously described by us 16,17 or other groups 18 . Indeed, this approach allowed us to do a kinetics study and to look for mechanisms over time on a tissue whose cell composition is more stable than that of milk during the mastitis course.…”

Going further post-RNA-seq: In silico functional analyses revealing candidate genes and regulatory elements related to mastitis in dairy cattle

“…RNA-sequence (RNA-seq) data were obtained from previous studies of our group (Sbardella et al ., 2019; Weller et al ., 2019). Briefly, four perfused udders were inoculated with a strain of S. agalactiae (FSL S3-026).…”

“…Expression values were calculated by counting for each gene how many aligned reads overlapped its exons using Htseq-count (Anders et al ., 2014). The significance of gene expression changes contrasts were assessed using edgeR package (Galaxy tool version 0.0.2) (Robinson et al ., 2010; Sbardella et al ., 2019; Weller et al ., 2019).…”

“…Therefore, it is necessary to develop new methodologies to control S. agalactiae , including breeding more resistant cattle through enhanced genomic selection. Genomics and transcriptomic data have elucidated gene networks and physiological cellular processes important in the response to S. agalactiae (Sbardella et al ., 2019; Weller et al ., 2019). In our laboratory, bovine extracorporeal udders have proved to be a successful tool for ex situ transcriptomic analysis of the innate response triggered by S. agalactiae in mammary tissue and display metabolic pathways associated with the inflammatory response (Pinto et al ., 2017; Sbardella et al ., 2019; Weller et al ., 2019).…”

“…Genomics and transcriptomic data have elucidated gene networks and physiological cellular processes important in the response to S. agalactiae (Sbardella et al ., 2019; Weller et al ., 2019). In our laboratory, bovine extracorporeal udders have proved to be a successful tool for ex situ transcriptomic analysis of the innate response triggered by S. agalactiae in mammary tissue and display metabolic pathways associated with the inflammatory response (Pinto et al ., 2017; Sbardella et al ., 2019; Weller et al ., 2019). In order to contribute to a better knowledge of the genes involved in mastitis development, this study aimed to investigate transcription factors and MicroRNAs related to differentially expressed genes (DEGs) previously found in these bovine extracorporeal udders infected with S. agalactiae.…”