Hostname: page-component-848d4c4894-r5zm4 Total loading time: 0 Render date: 2024-07-03T01:16:59.059Z Has data issue: false hasContentIssue false
  • English
  • Français

A two-step immunomagnetic separation of somatic cell subpopulations for a gene expression profile study in bovine milk

Published online by Cambridge University Press:  08 August 2018

Sara Divari ,
Laura Starvaggi Cucuzza ,
Fulvio Riondato ,
Paola Pregel ,
Paola Sacchi ,
Roberto Rasero ,
Bartolomeo Biolatti  and
Francesca Tiziana Cannizzo
Sara Divari*
Affiliation:
Department of Veterinary Science, University of Turin, Largo Braccini 2, 10095 Grugliasco, Turin, Italy
Laura Starvaggi Cucuzza
Affiliation:
Department of Veterinary Science, University of Turin, Largo Braccini 2, 10095 Grugliasco, Turin, Italy
Fulvio Riondato
Affiliation:
Department of Veterinary Science, University of Turin, Largo Braccini 2, 10095 Grugliasco, Turin, Italy
Paola Pregel
Affiliation:
Department of Veterinary Science, University of Turin, Largo Braccini 2, 10095 Grugliasco, Turin, Italy
Paola Sacchi
Affiliation:
Department of Veterinary Science, University of Turin, Largo Braccini 2, 10095 Grugliasco, Turin, Italy
Roberto Rasero
Affiliation:
Department of Veterinary Science, University of Turin, Largo Braccini 2, 10095 Grugliasco, Turin, Italy
Bartolomeo Biolatti
Affiliation:
Department of Veterinary Science, University of Turin, Largo Braccini 2, 10095 Grugliasco, Turin, Italy
Francesca Tiziana Cannizzo
Affiliation:
Department of Veterinary Science, University of Turin, Largo Braccini 2, 10095 Grugliasco, Turin, Italy
*
*For correspondence; e-mail: sara.divari@unito.it
Get access Rights & Permissions [Opens in a new window]

Abstract

The objective of this study was to demonstrate the usefulness of an immunomagnetic method to purify subpopulations of milk somatic cells. The experiment was conducted on milk samples collected from healthy cows (n = 17) and from cows with clinical mastitis (n = 24) due to a Staphylococcus aureus natural infection. A two-step immunomagnetic purification was applied to simultaneously separate three somatic cell subpopulations from the same milk sample. Total RNA was extracted and qPCR was performed to determinate mRNA levels of innate immunity target genes in purified somatic cell subpopulations. Good quality and quantity of RNA allowed the reference gene analysis in each cell subpopulation. An up-regulation of the main genes involved in innate immune defence was detected in separated polymorphonuclear neutrophilic leucocytes-monocytes and lymphocytes of mastitic milk. These results and flow cytometric analysis suggest that the immunomagnetic purification is an efficient method for the isolation of the three populations from milk, allowing the cells to be studied separately.

Keywords

Mastitisinnate immunitygene expressionmilk somatic cellsmagnetic separation
Type
Research Article
Information
Journal of Dairy Research , Volume 85 , Issue 3 , August 2018 , pp. 281 - 287
Copyright
Copyright © Hannah Dairy Research Foundation 2018 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Albenzio, M & Caroprese, M 2011 Differential leucocyte count for ewe milk with low and high somatic cell count. Journal of Dairy Research 78 43–48Google Scholar
Albenzio, M, Santillo, A, Caroprese, M, D'Angelo, F, Marino, R & Sevi, A 2009 Role of endogenous enzymes in proteolysis of sheep milk. Journal of Dairy Science 92 79–86Google Scholar
Baumert, A, Bruckmaier, RM & Wellnitz, O 2009 Cell population, viability, and some key immunomodulatory molecules in different milk somatic cell samples in dairy cows. Journal of Dairy Research 76 356Google Scholar
Boutinaud, M, Herve, L & Lollivier, V 2015 Mammary epithelial cells isolated from milk are a valuable, non-invasive source of mammary transcripts. Frontiers in Genetics 6 1–12Google Scholar
Bustin, S, Benes, V, Garson, J, Hellemans, J, Huggett, J, Kubista, M, Mueller, R, Nolan, T, Pfaffl, MW, Shipley, GL, Vandesompele, J & Wittwer, CT 2009 The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55 611–622Google Scholar
Cannizzo, FT, Pegolo, S, Starvaggi Cucuzza, L, Bargelloni, L, Divari, S, Franch, R, Castagnaro, M & Biolatti, B 2013 Gene expression profiling of thymus in beef cattle treated with prednisolone. Research in Veterinary Science 95 540–547Google Scholar
Caroprese, M, Marzano, A, Schena, L & Sevi, A 2008 Technical note: immunomagnetic procedure for positive selection of macrophages in ovine milk. Journal of Dairy Science 91 1908–1912Google Scholar
De Maria, R, Divari, S, Spada, F, Oggero, C, Mulasso, C, Maniscalco, L, Cannizzo, FT, Bianchi, M, Barbarino, G, Brina, N & Biolatti, B 2010 Progesterone receptor gene expression in the accessory sex glands of veal calves. Veterinary Research 167 291–296Google Scholar
Fonseca, I, Antunes, GR, Paiva, DS, Lange, CC, Guimarães, SEF & Martins, MF 2011 Differential expression of genes during mastitis in Holstein-Zebu crossbreed dairy cows. Genetics and Molecular Research 10 1295–1303Google Scholar
Griesbeck-Zilch, B, Meyer, HHD, Kühn, CH, Schwerin, M & Wellnitz, O 2008 Staphylococcus aureus and Escherichia coli cause deviating expression profiles of cytokines and lactoferrin messenger ribonucleic acid in mammary epithelial cells. Journal of Dairy Science 91 2215–2224Google Scholar
Koess, C & Hamann, J 2008 Detection of mastitis in the bovine mammary gland by flow cytometry at early stages. Journal of Dairy Research 75 225–232Google Scholar
Lewandowska-Sabat, AM, Boman, GM, Downing, A, Talbot, R, Storset, AK & Olsaker, I 2013 The early phase transcriptome of bovine monocyte-derived macrophages infected with Staphylococcus aureus in vitro. BMC Genomics 14 891Google Scholar
O'Sullivan, CA, Joyce, PJ, Sloan, T & Shattock, AG 1992 Capture immunoassay for the diagnosis of bovine mastitis using a monoclonal antibody to polymorphonuclear granulocytes. Journal of Dairy Research 59 122–133Google Scholar
Oviedo-Boyso, J, Valdez-Alarcón, JJ, Cajero-Juárez, M, Ochoa-Zarzosa, A, López-Meza, JE, Bravo-Patiño, A & Baizabal-Aguirre, VM 2007 Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis. Journal of Infection 54 399–409Google Scholar
Pfaffl, MW 2001 A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29 e45Google Scholar
Pilla, R, Malvisi, M, Snel, GGM, Schwarz, D, Konig, S, Czerny, CP & Piccinini, R 2013 Differential cell count as an alternative method to diagnose dairy cow mastitis. Journal of Dairy Science 96 1653–1660Google Scholar
Pietrocola, G, Arciola, CR, Rindi, S, Di Poto, A, Missineo, A, Montanaro, L & Speziale, P 2011 Toll-like receptors (TLRs) in innate immune defense against Staphylococcus aureus. The International Journal of Artificial Organs 34 799–810Google Scholar
Sánchez-Macías, D, Morales-dela Nuez, A, Torres, A, Hernández-Castellano, LE, Jiménez-Flores, R, Castro, N & Argüello, A 2013 Effects of addition of somatic cells to caprine milk on cheese quality. International Dairy Journal 29 61–67Google Scholar
Si-Tahar, M, Touqui, L & Chignard, M 2009 Innate immunity and inflammation-two facets of the same anti-infectious reaction. Clinical and Experimental Immunology 156 194–198Google Scholar
Sorg, D, Danowski, K, Korenkova, V, Rusnakova, V, Küffner, R, Zimmer, R, Meyer, HHD & Kliem, H 2013 Microfluidic high-throughput RT-qPCR measurements of the immune response of primary bovine mammary epithelial cells cultured from milk to mastitis pathogens. Animal 7 799–805Google Scholar
Spalenza, V, Girolami, F, Bevilacqua, C, Riondato, F, Rasero, R, Nebbia, C, Sacchi, P & Martin, P 2011 Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes. Veterinary Journal 189 278–283Google Scholar
Vandesompele, J, De Preter, K, Pattyn, F, Poppe, B, Van Roy, N, De Paepe, A & Speleman, F 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3 research0034.1- research0034.11Google Scholar
Yang, Z, Fu, Y, Liu, B, Zhou, E, Liu, Z, Song, X, Li, D & Zhang, N 2013 Farrerol regulates antimicrobial peptide expression and reduces Staphylococcus aureus internalization into bovine mammary epithelial cells. Microbial Pathogenesis 65 1–6Google Scholar
Ye, J, Coulouris, G, Zaretskaya, I, Cutcutache, I, Rozen, S & Madden, TL 2012 Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13 134Google Scholar
Supplementary material: PDF

Divari et al. supplementary material

Figure S1

Download Divari et al. supplementary material(PDF)
PDF 223 KB