Microarray-based gene expression profiling of peripheral blood mononuclear cells in dairy cows with experimental hypocalcemia and milk fever

Abstract

Although a molecular diagnostic assay using clinically accessible tissue, such as blood, would facilitate evaluation of disease conditions in humans and animals, little information exists on microarray-based gene expression profiling of circulating leukocytes from clinically hypocalcemic cows. Therefore, peripheral blood mononuclear cells from dairy cows with experimentally induced hypocalcemia or spontaneous milk fever were subjected to oligo-microarray analysis to identify specific biomarker genes. In experimental hypocalcemia induced by a 4-h infusion of 10% disodium EDTA (n=4), 32 genes were significantly up- or downregulated compared with control treatment (4-h infusion of 11% calcium EDTA; n=4). In cows with milk fever (n=8), 98 genes were expressed differentially (either up- or downregulated) compared with healthy parturient cows (n=5). From these data, the following 5 genes were selected as being strongly related to both experimental hypocalcemia and milk fever: protein kinase (cAMP-dependent, catalytic) inhibitor β (PKIB); DNA-damage-inducible transcript 4 (DDIT4); period homolog 1 (PER1); NUAK family, SNF1-like kinase, 1 (NUAK1); and expressed sequence tag (BI537947). Another gene (neuroendocrine secretory protein 55, NESP55) was also determined to be specific for milk fever, independently of hypocalcemia. The mRNA expression of these 6 genes in milk fever cases was verified by quantitative real-time reverse-transcription PCR and was significantly different compared with their expression in healthy parturient cows. In the present study, the selected genes appeared to be candidate biomarkers of milk fever because the continuous interactions between blood cells and the entire body suggest that subtle intracellular changes occur in association with disease. However, before any genomic biomarkers are incorporated into clinical evaluation of the disease, the effect of hypocalcemia on the mRNA expression of these genes in the tissues that regulate calcium homeostasis in dairy cows should be determined.

Plasma fluctuation in estradiol-17β and bone resorption markers around parturition in dairy cows

“…The plasma intact parathyroid hormone (iPTH) concentration was measured via an electrochemiluminescence immunoassay (Elecsys PTH Kit; Roche Diagnostics, Mannheim, Germany). As a check of the assay validity, plasma samples from cows with experimentally induced hypocalcemia (n=4) [ 19 ] were preliminary subjected to measuring iPTH concentrations. Plasma iPTH concentration (median, minimum‒maximum) increased to the maximum level during experimental hypocalcemia (1,228 p g/m l , 755‒1,587 p g/m l ) compared to the level before the experiment (60.8 p g/m l , 19.7‒128.3 p g/m l ).…”

“…Plasma iPTH concentration (median, minimum‒maximum) increased to the maximum level during experimental hypocalcemia (1,228 p g/m l , 755‒1,587 p g/m l ) compared to the level before the experiment (60.8 p g/m l , 19.7‒128.3 p g/m l ). Plasma iPTH concentrations in clinical healthy parturient cows and spontaneous milk fever cases using this assay have been described in our previous study [ 19 ]. Plasma tartrate-resistant acid phosphatase isoform 5b (TRAP5b) activity was measured fluorometrically as described previously [ 13 , 22 ].…”

Gene Mapping and Gene-Set Analysis for Milk Fever Incidence in Holstein Dairy Cattle

“…Moreover, terms related to protein phosphorylation, regulation of apoptotic process, gene expression regulation, and intracellular signal transduction were also enriched with candidate genes for milk fever incidence. Of particular interest, Sasaki et al (2014) investigated gene expression of peripheral blood mononuclear cells in milk fever vs. healthy cows. Notably, upregulated genes were involved in regulation of protein amino acid phosphorylation, intracellular signaling cascade, apoptosis, and cell death, while downregulated genes were implicated in cation-binding, transcription regulation, and DNA-binding ( Sasaki et al, 2014 ; Dervishi and Ametaj, 2017 ).…”

“…Of particular interest, Sasaki et al (2014) investigated gene expression of peripheral blood mononuclear cells in milk fever vs. healthy cows. Notably, upregulated genes were involved in regulation of protein amino acid phosphorylation, intracellular signaling cascade, apoptosis, and cell death, while downregulated genes were implicated in cation-binding, transcription regulation, and DNA-binding ( Sasaki et al, 2014 ; Dervishi and Ametaj, 2017 ). Therefore, our results provide further evidence that processes such as protein phosphorylation and regulation of apoptosis, among others, could be implicated in the development of periparturient hypocalcemia in dairy cows.…”

“…Using a multiple-marker mapping approach, Elo et al (1999) identified a genomic region on chromosome 23 significantly associated with veterinary treatment, including milk fever, in Finnish Ayrshire dairy cattle. Furthermore, Sasaki et al (2014) , using a microarray-based gene expression study of peripheral blood mononuclear cells, suggested genes PKIB, DDIT4, PER1 , and NUAK1 as potential biomarkers for milk fever predisposition.…”