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

Published online by Cambridge University Press:  11 December 2012

D. Sorg ,
K. Danowski ,
V. Korenkova ,
V. Rusnakova ,
R. Küffner ,
R. Zimmer ,
H. H. D. Meyer  and
H. Kliem
D. Sorg
Affiliation:
Physiology Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany ZIEL – Research Center for Life and Food Sciences, Weihenstephaner Berg 1, 85350 Freising, Germany
K. Danowski
Affiliation:
Physiology Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany ZIEL – Research Center for Life and Food Sciences, Weihenstephaner Berg 1, 85350 Freising, Germany
V. Korenkova
Affiliation:
Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic
V. Rusnakova
Affiliation:
Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic
R. Küffner
Affiliation:
Institute for Bioinformatics, Ludwigs-Maximilians-Universität München, Amalienstraße 17, 80333 München, Germany
R. Zimmer
Affiliation:
Institute for Bioinformatics, Ludwigs-Maximilians-Universität München, Amalienstraße 17, 80333 München, Germany
H. H. D. Meyer
Affiliation:
Physiology Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany ZIEL – Research Center for Life and Food Sciences, Weihenstephaner Berg 1, 85350 Freising, Germany
H. Kliem*
Affiliation:
Physiology Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany ZIEL – Research Center for Life and Food Sciences, Weihenstephaner Berg 1, 85350 Freising, Germany
*
†E-mail: [email protected]
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Abstract

Bovine mastitis, the inflammation of the udder, is a major problem for the dairy industry and for the welfare of the animals. To better understand this disease, and to implement two special techniques for studying mammary gland immunity in vitro, we measured the innate immune response of primary bovine mammary epithelial cells (pbMEC) from six Brown Swiss cows after stimulation with the heat-inactivated mastitis pathogens, Escherichia coli 1303 and Staphylococcus aureus 1027. The cells were extracted and cultivated from milk instead of udder tissue, which is usually done. The advantages of this technique are non-invasiveness and less contamination by fibroblasts. For the first time, pbMEC gene expression (GE) was measured with a microfluidic high-throughput real-time reverse transcription-quantitative PCR platform, the BioMark HD™ system from Fluidigm. In addition to the physiological analysis, the precision and suitability of this method was evaluated in a large data set. The mean coefficient of variance (± s.e.) between repeated chips was 4.3 ± 0.4% for highly expressed and 3.3 ± 0.4% for lowly expressed genes. Quantitative PCR (qPCR) replicate deviations were smaller than the cell culture replicate deviations, indicating that biological and cell culture differences could be distinguished from the background noise. Twenty-two genes (complement system, chemokines, inflammatory cytokines, antimicrobial peptides, acute phase response and toll-like receptor signalling) were differentially expressed (P < 0.05) with E. coli. The most upregulated gene was the acute phase protein serum amyloid A3 with 618-time fold. S. aureus slightly induced CCL5, IL10, TLR4 and S100A12 expression and failed to elicit a distinct overall innate immune response. We showed that, with this milk-derived pbMEC culture and the high-throughput qPCR technique, it is possible to obtain similar results in pbMEC expression as with conventional PCR and with satisfactory precision so that it can be applied in future GE studies in pbMEC.

Keywords

bovine mastitisgene expression profilingmicrofluidic qPCRprimary bovine mammary epithelial cellsinnate immune response
Type
Physiology and functional biology of systems
Information
animal , Volume 7 , Issue 5 , May 2013 , pp. 799 - 805
Copyright
Copyright © The Animal Consortium 2012

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