Selection of Biomarkers from Differentially Expressed Genes in Leukocytes of Buffalos Treated with Recombinant Bovine Somatotropin: The Importance of Sample Size for Reliable Discriminating Systems

Authors

  • Lorenzo Castigliego Department of Veterinary Sciences, University of Pisa, Via delle Piagge 2, 56124 Pisa, Italy
  • Filippo Jodi Carrieri Department of Veterinary Sciences, University of Pisa, Via delle Piagge 2, 56124 Pisa, Italy
  • Andrea Armani Department of Veterinary Sciences, University of Pisa, Via delle Piagge 2, 56124 Pisa, Italy
  • Marco Mazzi Animal Production Research Centre (PCM), Agriculture Research Council (CRA), Via Salaria, 31, I-00015 Monterotondo, Rome, Italy
  • Carlo Boselli Experimental Zooprophylactic Institute of Lazio and Tuscany , Via Appia Nuova 1411, I-00178 Rome, Italy
  • Goffredo Grifoni Experimental Zooprophylactic Institute of Lazio and Tuscany , Via Appia Nuova 1411, I-00178 Rome, Italy
  • Daniela Gianfaldoni Department of Veterinary Sciences, University of Pisa, Via delle Piagge 2, 56124 Pisa, Italy
  • Alessandra Guidi Department of Veterinary Sciences, University of Pisa

DOI:

https://doi.org/10.6000/1927-520X.2016.05.01.1

Keywords:

Recombinant bovine somatotropin, Buffalos, Biomarkers, Screening tests, Sample size

Abstract

The research on biomarkers to detect livestock treated with recombinant bovine somatotropin (rbST) is still an open issue. In fact, beyond undertaking confirmation methods, there is the need to develop simple and inexpensive screening tests. In this direction, some proposals have been forwarded, mostly involving the measurement of circulating molecules, whereas the possibility of using biomarkers related to gene expression is a field under investigation. The present study was carried out on sixteen buffalos, eight of which treated with rbST. Blood samples were collected six times during the treatment to investigate on the presence of differentially expressed genes in leukocytes. Analysis with the microarray technique was performed on two sampling moments, in order to obtain a first selection of genes. Further analysis was carried out by real time RT-PCR, in order to create a discriminating linear system. A study on the variation of the error related to the number of samples included in statistics was also performed. Results showed that, including an increasing number of samples to build the discriminating algorithm, the b-error grows and tends to stabilize on 6.5%. This study clearly shows the paramount importance of including a proper number of samples to obtain reliable algorithms.

References

Cannizzo FT, Pegolo S, Starvaggi Cucuzza L, et al. Gene expression profiling of thymus in beef cattle treated with prednisolone. Res Vet Sci 2013; 95: 540-7. http://dx.doi.org/10.1016/j.rvsc.2013.03.025 DOI: https://doi.org/10.1016/j.rvsc.2013.03.025

De Jager N, Hudson NJ, Reverter A, et al. Chronic exposure to anabolic steroids induces the muscle expression of oxytocin and a more than fiftyfold increase in circulating oxytocin in cattle. Physiol Genom 2011; 43: 467-78. http://dx.doi.org/10.1152/physiolgenomics.00226.2010 DOI: https://doi.org/10.1152/physiolgenomics.00226.2010

Kinkead RA, Elliott CT, Cannizzo FT, Biolatti B, Mooney MH. Proteomic identification of plasma proteins as markers of growth promoter abuse in cattle. Anal Bioanal Chem 2015; 407: 4495-507. http://dx.doi.org/10.1007/s00216-015-8651-0 DOI: https://doi.org/10.1007/s00216-015-8651-0

Ludwig SKJ, Smits NGE, Cannizzo FT, Nielen MWF. Potential of treatment-specific protein biomarker profiles for detection of hormone abuse in cattle. J Agric Food Chem 2013; 61: 4514-9. http://dx.doi.org/10.1021/jf4004972 DOI: https://doi.org/10.1021/jf4004972

Pinel G, Weigel S, Antignac JP, et al. Targeted and untargeted profiling of biological fluids to screen for anabolic practices in cattle. Trends Anal Chem 2010; 29: 1269-80. http://dx.doi.org/10.1016/j.trac.2010.06.010 DOI: https://doi.org/10.1016/j.trac.2010.06.010

Commission Decision 2002/657/EC of 12 August 2002 im-plementing Council Directive 96/23/EC concerning the per-formance of analytical methods and the interpretation of res-ults Official Journal of the European Community L 221: 8-36.

Council Decision 1999/879/EC of 17 December 1999 concerningthe Placing on the Market and Administration of bovineSomatotrophin(BST) and repealing Decision 90/218/EEC Official Journal of the European Community L331: 71-72.

Le Breton MH, Rochereau-Roulet S, Pinel G, Cesbron N, Le Bizec B. Elimination kinetic of recombinant somatotropin in bovine. Anal Chim Acta 2009; 637: 121-7. http://dx.doi.org/10.1016/j.aca.2008.09.003 DOI: https://doi.org/10.1016/j.aca.2008.09.003

Le Breton MH, Rochereau-Roulet S, Chéreau S Pinel G, Delatour T, Le Bizec B. Identification of cows treated with recombinant bovine somatotropin. J Agric Food Chem 2010; 58: 729-33. http://dx.doi.org/10.1021/jf903032q DOI: https://doi.org/10.1021/jf903032q

Smits NG, Blokland MH, Wubs KL, Nessen MA, van Ginkel LA, Nielen MW. Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum. Anal Bioanal Chem 2015; 407: 6041-50. http://dx.doi.org/10.1007/s00216-015-8775-2 DOI: https://doi.org/10.1007/s00216-015-8775-2

Castigliego L, Iannone G, Grifoni G, et al. Natural and recombinant bovine somatotropin: immunodetection with a sandwich ELISA. J Dairy Res 2007; 74: 79-85. http://dx.doi.org/10.1017/S0022029906002159 DOI: https://doi.org/10.1017/S0022029906002159

Suárez-Pantaleón C, Huet AC, Kavanagh O, et al. Production of polyclonal antibodies directed to recombinant methionyl bovine somatotropin. Anal Chim Acta 2013; 761: 186-93. http://dx.doi.org/10.1016/j.aca.2012.11.041 DOI: https://doi.org/10.1016/j.aca.2012.11.041

Zhao X, McBride BW, Trouten-Radford LM, Golfman L, Burton JH. Somatotropin and insulin-like growth factor-I concentrations in plasma and milk after daily or sustained-release exogenous somatotropin administrations. Domest Anim Endocrinol 1994; 11: 209-16. http://dx.doi.org/10.1016/0739-7240(94)90028-0 DOI: https://doi.org/10.1016/0739-7240(94)90028-0

Doué M, Dervilly-Pinel G, Cesbron N, et al. Clinical biochemical and hormonal profiling in plasma: a promising strategy to predict growth hormone abuse in cattle. Anal Bioanal Chem 2015; 407: 4343-9. http://dx.doi.org/10.1007/s00216-015-8548-y DOI: https://doi.org/10.1007/s00216-015-8548-y

Ludwig SKJ, Smits MGE, Bremer MGEG, Nielen MWF. Monitoring milk for antibodies against recombinant bovine somatotropin using a microsphere immunoassay-based biomarker approach. Food Control 2012; 26: 68-72. http://dx.doi.org/10.1016/j.foodcont.2011.12.011 DOI: https://doi.org/10.1016/j.foodcont.2011.12.011

Pinel G, Buon R, Aviat F, et al. Recombinant bovine somatotropin misuse in cattle Evaluation of Western blotting and 2D electrophoresis methods on biological samples for the demonstration of its administration. Anal Chim Acta 2005; 529: 41-6. http://dx.doi.org/10.1016/j.aca.2004.07.063 DOI: https://doi.org/10.1016/j.aca.2004.07.063

Rochereau-Roulet S, Gaudin I, Chéreau S, et al. Develop-ment and validation of an enzyme-linked immunosorbent assay for the detection of circulating antibodies raised against growth hormone as a consequence of rbST treatment in cows. Anal Chim Acta 2011; 700: 189-93. http://dx.doi.org/10.1016/j.aca.2011.01.035 DOI: https://doi.org/10.1016/j.aca.2011.01.035

Smits NG, Bremer MG, Ludwig SK, Nielen MW. Development of a flow cytometric immunoassay for recombinant bovine somatotropin-induced antibodies in serum of dairy cows. Drug Testing and Anal 2012; 4: 362-7. http://dx.doi.org/10.1002/dta.336 DOI: https://doi.org/10.1002/dta.336

Castigliego L, Armani A, Grifoni G, et al. Effects of growth hormone treatment on the expression of somatotropic axis genes in the skeletal muscle of lactating Holstein cows. Dom Anim Endocrinol 2010; 39: 40-53. http://dx.doi.org/10.1016/j.domaniend.2010.02.001 DOI: https://doi.org/10.1016/j.domaniend.2010.02.001

Castigliego L, Li XN, Armani A, et al. Somatotropic gene response to recombinant growth hormone treatment in buffalo leucocytes. Biol Chem 2011; 392: 1145-54. http://dx.doi.org/10.1515/BC.2011.187 DOI: https://doi.org/10.1515/BC.2011.187

Smyth GK. Limma: linear models for microarray data. In: Gentleman R, Carey V, Dudoit S, Irizarry R, Huber W, editors. Bioinformatics and Computational Biology Solutions using R and Bioconductor. New York: Springer 2005; pp. 397-420. http://dx.doi.org/10.1007/0-387-29362-0_23 DOI: https://doi.org/10.1007/0-387-29362-0_23

Jeay S, Sonenshein GE, Postel-Vinay MC, Kelly PA, Baixeras E. Growth hormone can act as a cytokine controlling survival and proliferation of immune cells: new insights into signaling pathways. Mol Cell endocrinol 2002; 188: 1-7. http://dx.doi.org/10.1016/S0303-7207(02)00014-X DOI: https://doi.org/10.1016/S0303-7207(02)00014-X

Welniak LA, Sun R, Murphy WJ. The role of growth hormone in T-cell development and reconstitution. J Leukocyte Biol 2002; 71: 381-7. DOI: https://doi.org/10.1189/jlb.71.3.381

Velkeniers B, Dogusan Z, Naessens F, Hooghe R, Hooghe-Peters EL. Prolactin growth hormone and the immune system in humans. Cell Mol Life Sci 1998; 54: 1102-8. http://dx.doi.org/10.1007/s000180050239 DOI: https://doi.org/10.1007/s000180050239

Elvinger F, Hansen PJ, Head HH, Natzke RP. Actions of bovine somatotropin on polymorphonuclear leukocytes and lymphocytes in cattle. J Dairy Sci 1991; 74: 2145-2152. http://dx.doi.org/10.3168/jds.S0022-0302(91)78387-2 DOI: https://doi.org/10.3168/jds.S0022-0302(91)78387-2

Burton JL, McBride BW, Kennedy BW, Burton JH, Elsasser TH, Woodward B. Hematological profiles in dairy cows treated with recombinant bovine somatotropin. J Anim Sci 1992; 70: 1488-95. DOI: https://doi.org/10.2527/1992.7051488x

Khaliq T, Rahman ZU. Haematological Studies of Nili-Ravi Buffaloes Injected with Recombinant Bovine Somatotropin. Pak Vet J 2010; 30: 53-7.

Canales RD, Luo Y, Willey JC, et al. Evaluation of DNA microarray results with quantitative gene expression platforms. Nat Biotechnol 2006; 24: 1115-22. http://dx.doi.org/10.1038/nbt1236 DOI: https://doi.org/10.1038/nbt1236

Carraro L, Ferraresso S, Cardazzo B, et al. Expression profiling of skeletalmuscle in young bulls treated with steroidal growth promoters. Physiol Genom 2009; 38: 138-48. http://dx.doi.org/10.1152/physiolgenomics.00014.2009 DOI: https://doi.org/10.1152/physiolgenomics.00014.2009

Pegolo S, Cannizzo FT, Biolatti B, Castagnaro M, Bargelloni L. Transcriptomic profiling as a screening tool to detect trenbolone treatment in beef cattle. Res Vet Sci 2014; 96: 472-81. http://dx.doi.org/10.1016/j.rvsc.2014.03.020 DOI: https://doi.org/10.1016/j.rvsc.2014.03.020

Reiter M, Walf VM, Christians A, Pfaffl MW, Meyer HH. Modification of mRNA expression after treatment with anabolic agents and the usefulness for gene expression-biomarkers. Anal Chim Acta 2007; 586: 73- 81. http://dx.doi.org/10.1016/j.aca.2006.10.049 DOI: https://doi.org/10.1016/j.aca.2006.10.049

Toffolatti L, Rosa Gastaldo L, Patarnello T, et al. Expression analysis of androgen-responsive genes in the prostate of vealcalvestreated with anabolichormones. Domest Anim Endocrinol 2006; 30: 38-55. http://dx.doi.org/10.1016/j.domaniend.2005.05.008 DOI: https://doi.org/10.1016/j.domaniend.2005.05.008

Chuaqui RF, Bonner RF, Best CJM, et al. Post-analysis follow-up and validation of microarray experiments. Nat Genet 2002; 32: 509-14. http://dx.doi.org/10.1038/ng1034 DOI: https://doi.org/10.1038/ng1034

Wurmbach E, Yuen T, Sealfon SC. Focused microarray analysis. Methods 2003; 31: 306-16. http://dx.doi.org/10.1016/S1046-2023(03)00161-0 DOI: https://doi.org/10.1016/S1046-2023(03)00161-0

Etienne W, Meyer MH, Peppers J, Meyer RA Jr. Comparison of mRNA gene expression by RT-PCR and DNA microarray. BioTechniques 2004; 36: 618-21. DOI: https://doi.org/10.2144/04364ST02

Rajeevan MS, Vernon SD, Taysavang N, Unger ER. Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR. J Mol Diagn 2001; 3: 26-31. http://dx.doi.org/10.1016/S1525-1578(10)60646-0 DOI: https://doi.org/10.1016/S1525-1578(10)60646-0

Downloads

Published

2016-04-06

How to Cite

Castigliego, L., Jodi Carrieri, F., Armani, A., Mazzi, M., Boselli, C., Grifoni, G., Gianfaldoni, D., & Guidi, A. (2016). Selection of Biomarkers from Differentially Expressed Genes in Leukocytes of Buffalos Treated with Recombinant Bovine Somatotropin: The Importance of Sample Size for Reliable Discriminating Systems. Journal of Buffalo Science, 5(1), 1–13. https://doi.org/10.6000/1927-520X.2016.05.01.1

Issue

Section

Articles