A Fixed Cohort Field Study of Gene Expression in Circulating Leukocytes From Dairy Cows With and Without Mastitis

Abstract

Specifically designed gene expression studies can be used to prioritize candidate genes and identify novel biomarkers affecting resilience against mastitis and other diseases in dairy cattle. The primary goal of this study was to assess whether specific peripheral leukocyte genes expressed differentially in a previous study of dairy cattle with postpartum disease, also would be expressed differentially in peripheral leukocytes from a diverse set of different dairy cattle with moderate to severe clinical mastitis. Four genes were selected for this study due to their differential expression in a previous transcriptomic analysis of circulating leukocytes from dairy cows with and without evidence of early postpartum disease. An additional 15 genes were included based on their cellular, immunologic, and inflammatory functions associated with resistance and tolerance to mastitis. This fixed cohort study was conducted on a conventional dairy in Washington state. Cows >50 days in milk (DIM) with mastitis (n = 12) were enrolled along with healthy cows (n = 8) selected to match the DIM and lactation numbers of mastitic cows. Blood was collected for a complete blood count (CBC), serum biochemistry, leukocyte isolation, and RNA extraction on the day of enrollment and twice more at 6 to 8-days intervals. Latent class analysis was performed to discriminate healthy vs. mastitic cows and to describe disease resolution. RNA samples were processed by the Primate Diagnostic Services Laboratory (University of Washington, Seattle, WA). Gene expression analysis was performed using the Nanostring System (Nanostring Technologies, Seattle, Washington, USA). Of the four genes (C5AR1, CATHL6, LCN2, and PGLYRP1) with evidence of upregulation in cows with mastitis, three of those genes (CATHL6, LCN2, and PGLYRP1) were investigated due to their previously identified association with postpartum disease. These genes are responsible for immunomodulatory molecules that selectively enhance or alter host innate immune defense mechanisms and modulate pathogen-induced inflammatory responses. Although further research is warranted to explain their functional mechanisms and bioactivity in cattle, our findings suggest that these conserved elements of innate immunity have the potential to bridge disease states and target tissues in diverse dairy populations.

Differential gene expression in peripheral leukocytes of pre-weaned Holstein heifer calves with respiratory disease

“…These differences in leukocyte profiles between calves and mature cattle are also the likely reason that three of our genes of interest ( LIF , IFNG , and TNF) had consistently low copy numbers in our samples and were unable to be reliably included in DGE analysis. Although these genes are well documented inflammatory mediators in older cattle [ 14 , 17 , 29 , 30 ], they are evidently not prolifically expressed by peripheral leukocytes of young calves.…”

“…Genes were prioritized that have strong evidence for their role in amplification or attenuation of inflammatory processes, the antibacterial or antiviral actions of their protein products, or their involvement in disease resistance and tolerance mechanisms. Five internal control genes ( ACTB , GOLGA5 , OSBPL2 , SMUG1 , and YWHAZ ) were also selected based on their previously demonstrated suitability as controls in bovine peripheral leukocytes [ 29 ].…”

“…RNA samples were processed by the NanoString Technologies Proof of Principle Services in Seattle, WA. Gene expression analysis of the 19 target genes and five housekeeping genes was performed using the Nanostring System (Nanostring Technologies, Seattle, Washington, USA), as previously described [ 29 ]. Gene expression was measured using a custom CodeSet for the selected genes in the Nanostring nCounter Analysis System 2.0 (NanoString Technologies, Seattle, WA).…”

Regulatory network of miRNA, lncRNA, transcription factor and target immune response genes in bovine mastitis

“…Bovine mastitis, caused by a variety of organisms, including Staphylococci, Streptococci, and Enterobacteria, is a mammary gland infection posing a great threat to the dairy industry, and is the costliest disease affecting cattle, due to its impact on the quality and quantity of milk production 1 , 2 . The immune response to pathogens in the mammary gland is highly complex, having to first identify the pathogen, recruit the appropriate immune cells, remove the pathogen, and then terminate the inflammatory response 3 . Recent studies have shown that mammary epithelial cells (MEC) initiate this process by changing the expression of various genes which are responsible for a wide range of immunological and inflammatory responses 4 .…”

Regulatory Network of miRNA, lncRNA, Transcription Factor and Target Immune Response Genes in Bovine Mastitis

“…Bovine mastitis, caused by a variety of organisms, including Staphylococci, Streptococci, and Enterobacteria, is a mammary gland infection posing a great threat to the dairy industry, and is the costliest disease affecting cattle, due to its impact on the quality and quantity of milk production 1,2 . The immune response to pathogens in the mammary gland is highly complex, having to rst identify the pathogen, recruit the appropriate immune cells, remove the pathogen, and then terminate the in ammatory response 3 . Recent studies have shown that mammary epithelial cells (MEC) initiate this process by changing the expression of various genes, responsible for a wide range of immunological and in ammatory responses 4 .…”