Transcriptome profiling of Streptococcus uberis-induced mastitis reveals fundamental differences between immune gene expression in the mammary gland and in a primary cell culture model

Abstract

Streptococcus uberis is a prevalent causative organism of mastitis and resides naturally in the environment of the dairy cow making prevention of the disease difficult. A bovine cDNA microarray comprising approximately 22,000 expressed sequence tags was used to evaluate the transcriptional changes that occur in the mammary gland after the onset of clinical Strep. uberis mastitis. Five lactating Friesian heifers were intramammary infused in an uninfected quarter with approximately 1,000 to 1,500 cfu of a wild-type strain of Strep. uberis. Microarray results showed that Strep. uberis mastitis led to the differential expression of more than 2,200 genes by greater than 1.5-fold compared with noninfected control quarters. The most highly upregulated genes were associated with the immune response, programmed cell death, and oxidative stress. Quantitative real-time reverse transcription PCR analysis confirmed the increase in mRNA expression of immune-related genes complement component 3, clusterin, IL-8, calgranulin C, IFN-gamma , IL-10, IL-1beta, IL-6, toll-like receptor-2, tumor necrosis factor-alpha, serum amyloid A3, lactoferrin, LPS-bonding protein, and oxidative stress-related genes metallothionein 1A and superoxide dimutase 2. In contrast, a decrease of mRNA levels was observed for the major milk protein genes. Bovine mammary epithelial cells in culture challenged with the same Strep. uberis strain used to induce clinical mastitis in the in vivo animal experiment did not cause a change in the mRNA levels of the immune-related genes. This suggests that the expression of immune-related genes by mammary epithelial cells may be initiated by host factors and not Strep. uberis. However, challenging epithelial cells with different Strep. uberis strains and Staphylococcus aureus resulted in an increase in the mRNA expression of a subset of the immune-related genes measured. In comparison, an Escherichia coli challenge caused an increase in the majority of immune-related genes measured. Results demonstrate the complexity of the bovine mammary gland immune response to an infecting pathogen and indicate that a coordinated response exists between the resident, recruited, and inducible immune factors.

Microfluidic high-throughput RT-qPCR measurements of the immune response of primary bovine mammary epithelial cells cultured from milk to mastitis pathogens

“…However, two important innate immune genes, TNFa and CD14, were identified as differentially expressed by the RT-qPCR, but not on the microarray. Swanson et al (2009) infected heifers with Streptococcus uberis and measured the transcriptional changes in the mammary tissue on a DNA microarray. Of the regulated genes, they validated 11 innate immune genes with RT-qPCR.…”

“…Possibly, the dose of inoculation was insufficient. It could have been too low, taking into account that there is a dose-dependent immune response of pbMEC to lipopolysaccharide (LPS) and S. aureus (Wellnitz and Kerr, 2004) and a study by Swanson et al (2009) with pbMEC from tissue showed an upregulation in four of nine measured immune genes to S. aureus with a much higher MOI of 1000. Another possibility is that we missed the proper time frame of the immune response: one study showed an early immune response of MECs to S. aureus that decreased to resting levels after 8 to 16 h (Strandberg et al, 2005).…”

Neurotoxicity of cerebro-spinal fluid from patients with Parkinson’s disease on mesencephalic primary cultures as an in vitro model of dopaminergic neurons

“…Following digestion, DMEM/F-12 (Gibco-BRL, Invitrogen Life Technologies, Carlsbad, CA, USA) containing 0.04 mg/ml DNase (Sigma-Aldrich) was added and the mixture was incubated at 37˚C for 8-10 min, as described in detail previously (8)(9)(10). The resulting single cell suspension seeded into a 24-well cell culture plate pre-coated coated with polylysine and then cultured under standard conditions (37˚C, 5% CO 2 ).…”

Cell-based models to test the effects of milk-derived bioactives

“…This study shows the importance of developing models with polarised mammary epithelial cells and that BME-UV1 may be a good model for studying, for example, developmental processes of the bovine mammary gland in vitro. Primary cells of the bovine mammary gland have also been used as models including both organoids, explants and epithelial cells for studies of effects of growth factors, steroids, lactoferrin, retinoids, cytokines, biogenic amines, mammary extracts, and endpoint parameters such as mitogenic activity and gene expression have been measured Bauman, 1999 andWeber et al, 1999;Akers et al, 2000;Ellis et al, 2000;Purup et al, 2000 andWeng et al, 2005;Thorn et al, 2006 andErnens et al, 2007;Fusi et al, 2008;Swanson et al, 2009;Wang and Baumrucker, 2010). Both primary mammary cells and cell lines of human and rodent origin have been used to study effects of milk-derived bioactives such as CLA and phytoestrogens.…”