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Cell free mitochondrial DNA in serum and milk associated with bovine mastitis: a pilot study

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Abstract

Mastitis is inflammation of mammary gland affecting all the species of domestic animals. Fragments of the mitochondrial genome released from dying cells are considered surrogate markers of mitochondrial injury. We hypothesized that bovine mastitis would be associated with increased cell free mitochondrial DNA (mtDNA) content in serum and milk. Milk and serum samples were collected from sub-clinical mastitic and normal animals. Mastitis was confirmed by California mastitis test and bacterial isolation. Oxidative stress, nitric oxide and inflammatory cytokines were also estimated. Real time polymerase chain reaction was conducted in serum and milk from sub-clinical mastitic animals and compared with healthy animals targeting the mtDNA genes cytochrome b. Mastitis animals showed higher oxidative stress markers and nitric oxide along with higher level of inflammatory cytokines. Cell free mtDNA was significantly higher in serum and milk of mastitic animals comparing to that of healthy control. The higher cell free relative mtDNA content in mastitis animals indicates injury to the mammary epithelial cells and thereby releasing the mtDNA in the milk and blood. This mtDNA may be a bio-marker of oxidative stress and tissue injury in bovine mastitis.

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References

  • Anderson S, de Bruijn MH, Coulson AR, Eperon IC, Sanger F, Young IG (1982) Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian mitochondrial genome. J Mol Biol 156(4):683–717

    Article  CAS  Google Scholar 

  • Barbalat R, Ewald SE, Mouchess ML, Barton GM (2011) Nucleic acid recognition by the innate immune system. Annu Rev Immunol 29:185–214

    Article  CAS  Google Scholar 

  • Berezin AE (2015) Circulating cell-free mitochondrial DNA as biomarker of cardiovascular risk: New challenges of old findings. Angiol 3:161.

  • Blum JW, Dosogne H, Hoeben D, Vangroenweghe F, Hammon HM, Bruckmaier RM, Burvenich C (2000) Tumor necrosis factor-alpha and nitrite/nitrate responses during acute mastitis induced by Escherichia coli infection and endotoxin in dairy cows. Domest Anim Endocrinol 19(4):223–235

    Article  CAS  Google Scholar 

  • Bochniarz M, Zdzisińska B, Wawron W, Szczubiał M, Dąbrowski R (2017) Milk and serum IL-4, IL-6, IL-10, and amyloid A concentrations in cows with subclinical mastitis caused by coagulase-negative staphylococci. J Dairy Sci 100(12):9674–9680

    Article  CAS  Google Scholar 

  • Bouchard L, Blais S, Desrosiers C, Zhao X, Lacasse P (1999) Nitric oxide production during endotoxin-induced mastitis in the cow. J Dairy Sci 82(12):2574–2581

    Article  CAS  Google Scholar 

  • Boyapati RK, Tamborska A, Dorward DA, Ho GT (2017) Advances in the understanding of mitochondrial DNA as a pathogenic factor in inflammatory diseases. F1000Research 6:169. 

    Article  Google Scholar 

  • Braun JS, Hoffmann O, Schickhaus M, Freyer D, Dagand E, Bermpohl D, Mitchell TJ, Bechmann I, Weber JR (2007) Pneumolysin causes neuronal cell death through mitochondrial damage. Infect Immun 75(9):4245–4254

    Article  CAS  Google Scholar 

  • Brenaut P, Lefèvre L, Rau A, Laloë D, Pisoni G, Moroni P, Bevilacqua C, Martin P (2014) Contribution of mammary epithelial cells to the immune response during early stages of a bacterial infection to Staphylococcus aureus. Vet Res 45:16

    Article  Google Scholar 

  • Cesa S (2004) Malondialdehyde contents in infant milk formulas. J Agric Food Chem 52(7):2119–2122

    Article  CAS  Google Scholar 

  • Cossarizza A, Pinti M, Nasi M, Gibellini L, Manzini S, Roat E, De Biasi S, Bertoncelli L, Montagna JP, Bisi L, Manzini L, Trenti T, Borghi V, Mussini C (2011) Increased plasma levels of extracellular mitochondrial DNA during HIV infection: a new role for mitochondrial damage-associated molecular patterns during inflammation. Mitochondrion 11(5):750–755

    Article  CAS  Google Scholar 

  • Ellis TN, Beaman BL (2004) Interferon-gamma activation of polymorphonuclear neutrophil function. Immunology 112(1):2–12

    Article  CAS  Google Scholar 

  • Elmore S (2007) Apoptosis. A review of programmed cell death. Toxicol Pathol 35(4):495–516

    Article  CAS  Google Scholar 

  • Finkel T (2012) Signal transduction by mitochondrial oxidants. J Biol Chem 287(7):4434–4440

    Article  CAS  Google Scholar 

  • Genestier AL, Michallet MC, Prévost G, Bellot G, Chalabreysse L, Peyrol S, Thivolet F, Etienne J, Lina G, Vallette FM, Vandenesch F, Genestier L (2005) Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils. J Clin Invest 115(11):3117–3127

    Article  CAS  Google Scholar 

  • Góth L (1991) A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta 196(2–3):143–151

    Article  Google Scholar 

  • Gu B, Zhu Y, Zhu W, Miao J, Deng Y, Zou S (2009) Retinoid protects rats against neutrophil-induced oxidative stress in acute experimental mastitis. Int Immunopharmacol 9(2):223–229

    Article  CAS  Google Scholar 

  • Haslinger B, Strangfeld K, Peters G, Schulze-Osthoff K, Sinha B (2003) Staphylococcus aureus alpha-toxin induces apoptosis in peripheral blood mononuclear cells: role of endogenous tumour necrosis factor-alpha and the mitochondrial death pathway. Cell Microbiol 5(10):729–741

    Article  CAS  Google Scholar 

  • Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, Knippers R (2001) DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 61(4):1659–1665

    CAS  PubMed  Google Scholar 

  • Johannsen DL, Ravussin E (2009) The role of mitochondria in health and disease. Curr Opin Pharmacol 9(6):780–786

    Article  CAS  Google Scholar 

  • Komine K, Kuroishi T, Komine Y, Watanabe K, Kobayashi J, Yamaguchi T, Kamata S, Kumagai K (2004) Induction of nitric oxide production mediated by tumor necrosis factor alpha on staphylococcal enterotoxin C-stimulated bovine mammary gland cells. Clin Diagn Lab Immunol 11(1):203–210

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar M, Kumar R, Sharma A, Jain VK (2016) Investigations on prevalence and oxidative stress aspects of mastitis in buffaloes. Ital J Anim Sci 6(sup2):978–979

    Article  Google Scholar 

  • Lamkanfi V, Dixit M (2010) Manipulation of host cell death pathways during microbial infections. Cell Host Microbe 8:44–54

    Article  CAS  Google Scholar 

  • Lobet E, Letesson JJ, Arnould T (2015) Mitochondria: a target for bacteria. Biochem Pharmacol 94(3):173–185

    Article  CAS  Google Scholar 

  • Long E, Capuco AV, Wood DL, Sonstegard T, Tomita G, Paape MJ, Zhao X (2001) Escherichia coli induces apoptosis and proliferation of mammary cells. Cell Death Differ 8:808–816

    Article  CAS  Google Scholar 

  • Madesh M, Balasubramanian KA (1998) Microtitre plate assay for superoxide dismutase using MTT reduction by superoxide. Indian J Biochem Biophys 35:184–188

    CAS  PubMed  Google Scholar 

  • Malik AN, Czajka A (2013) Is mitochondrial DNA content a potential biomarker of mitochondrial dysfunction? Mitochondrion 13(5):481–492

    Article  CAS  Google Scholar 

  • Middleton JR, Saeman A, Fox LK, Lombard J, Hogan JS, Smith KL (2014) The National Mastitis Council: a global organization for mastitis control and milk quality, 50 years and beyond. J Mammary Gland Biol Neoplasia 19(3–4):241–251

    Article  Google Scholar 

  • Mishra P, Chan DC (2014) Mitochondrial dynamics and inheritance during cell division, development and disease. Nat Rev Mol Cell Biol 15(10):634–646

    Article  CAS  Google Scholar 

  • Mosovsky K, Silva E, Troyer R, Propst-Graham K, Dow S (2014) Interaction of interferon gamma-induced reactive oxygen species with ceftazidime leads to synergistic killing of intracellular Burkholderia pseudomallei. Antimicrob Agents Chemother 58(10):5954–5963

    Article  Google Scholar 

  • Nagasawa Y, Yoshio K, Kazue S, Fuyuko T, Tomohito H (2018) Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load. Anim Sci J 89:259–266

    Article  CAS  Google Scholar 

  • Nakahira K, Hisata S, Choi AM (2015) The roles of mitochondrial damage-associated molecular patterns in diseases. Antioxid Redox Signal 23(17):1329–1350

    Article  CAS  Google Scholar 

  • Patrushev M, Kasymov V, Patrusheva V, Ushakova T, Gogvadze V, Gaziev A (2004) Mitochondrial permeability transition triggers the release of mtDNA fragments. Cell Mol Life Sci 61(24):3100–3103

    Article  CAS  Google Scholar 

  • Radostits OM, Gay CC, Hinchcliff K, Constable PD (2000) Veterinary medicine. A textbook of the diseases of cattle, horses, sheep, pigs, and goats, 9th edn. Saunders Elsevier, Philadelphia, pp 179–183, 250–255

  • Ryman VE, Packiriswamy N, Sordillo LM (2015) Role of endothelial cells in bovine mammary gland health and disease. Anim Health Res Rev 16:135–149

    Article  Google Scholar 

  • Ryter SW, Kim HP, Hoetzel A, Park JW, Nakahira K, Wang X, Choi AMK (2007) Mechanisms of cell death in oxidative stress. Antioxid Redox Signal 9:49–89

    Article  CAS  Google Scholar 

  • Shafiq-Ur-Rehman (1984) Lead-induced regional lipid peroxidation in brain. Toxicol Lett 21(3):333–337

    Article  CAS  Google Scholar 

  • Sharma N, Jeong DK (2013) Stem cell research: a novel boulevard towards improved bovine mastitis management. Int J Biol Sci 9(8):818–829

    Article  Google Scholar 

  • Sharma L, Verma AK, Rahal A, Kumar A, Nigam R (2016) Relationship between serum biomarkers and oxidative stress in dairy cattle and buffaloes with clinical and sub-clinical mastitis. Biotechnology 15:96–100

    Article  CAS  Google Scholar 

  • Skulachev VP (1999) Mitochondrial physiology and pathology; concepts of programmed death of organelles, cells and organisms. Mol Asp Med 20(3):139–184

    Article  CAS  Google Scholar 

  • Sordillo LM, Streicher KL (2002) Mammary gland immunity and mastitis susceptibility. J Mammary Gland Biol Neoplasia 7(2):135–146

    Article  Google Scholar 

  • Su WJ, Chang CJ, Peh HC, Lee SL, Huang MC, Zhao X (2002) Apoptosis and oxidative stress of infiltrated neutrophils obtained from mammary glands of goats during various stages of lactation. Am J Vet Res 63:241–246

    Article  CAS  Google Scholar 

  • Suriyasathaporn W, Vinitketkumnuen U, Chewonarin T, Boonyayatra S, Kreausukon K, Schukken YH (2006) Higher somatic cell counts resulted in higher malondialdehyde concentrations in raw cows’ milk. Int Dairy J 16:1088–1091

    Article  CAS  Google Scholar 

  • Sursal T, Stearns-Kurosawa DJ, Itagaki K, Oh SY, Sun S, Kurosawa S, Hauser CJ (2013) Plasma bacterial and mitochondrial DNA distinguish bacterial sepsis from sterile systemic inflammatory response syndrome and quantify inflammatory tissue injury in nonhuman primates. Shock 39(1):55–62

    PubMed  PubMed Central  Google Scholar 

  • Thannickal VJ, Fanburg BL (2000) Reactive oxygen species in cell signaling. Am J Phys Lung Cell Mol Phys 279(6):L1005–L1028

    CAS  Google Scholar 

  • Turk R, Piras C, Kovačić M, Samardžija M, Ahmed H, De Canio M, Urbani A, Meštrić ZF, Soggiu A, Bonizzi L, Roncada P (2012) Proteomics of inflammatory and oxidative stress response in cows with subclinical and clinical mastitis. J Proteome 75(14):4412–4428

    Article  CAS  Google Scholar 

  • Turk R, Koledić M, Maćešić N, Benić M, Dobranić V, Đuričić D, Cvetnić, Samardžija M (2017) The role of oxidative stress and inflammatory response in the pathogenesis of mastitis in dairy cows. Mljekarstvo 67(2):91–101

    Article  Google Scholar 

  • Wagner SA, Jones DE, Apley MD (2009) Effect of endotoxic mastitis on epithelial cell numbers in the milk of dairy cows. Am J Vet Res 70:796–799

    Article  Google Scholar 

  • Wenceslau CF, McCarthy CG, Szasz T et al (2014) Mitochondrial damage-associated molecular patterns and vascular function. Eur Heart J 35(18):1172–1177

    Article  CAS  Google Scholar 

  • Wilde CJ,  Addey C V,  Peaker M (1996) Effects of immunization against an autocrine inhibitor of milk secretion in lactating goats. J Physiol 491 (2):465–469

    Article  CAS  Google Scholar 

  • Woollard DC, Indyk HE (2014) Colorimetric determination of nitrate and nitrite in milk and milk powders - use of vanadium (III) reduction. Int Dairy J 35(1):88–94

    Article  CAS  Google Scholar 

  • Zhao X, Lacasse P (2008) Mammary tissue damage during bovine mastitis: causes and control. J Anim Sci 86:57–65

    Article  CAS  Google Scholar 

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Acknowledgements

The first author acknowledged Department of Science and Technology, Government of India for providing the necessary financial support to carry out this work through Women Scientist Scheme, DST-WOS-A vide Ref. No. SR/WOS - A/LS-1026/2015.

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Authors and Affiliations

  1. Animal Disease Research Centre, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab, 141004, India

    Geeta Devi Leishangthem & Gursimran Filia

  2. School of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab, 141004, India

    Niraj Kumar Singh

  3. Department of Veterinary Pathology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab, 141004, India

    Nittin Dev Singh & Amarjit Singh

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  1. Geeta Devi Leishangthem
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Correspondence to Geeta Devi Leishangthem.

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Leishangthem, G.D., Singh, N.K., Singh, N.D. et al. Cell free mitochondrial DNA in serum and milk associated with bovine mastitis: a pilot study. Vet Res Commun 42, 275–282 (2018). https://doi.org/10.1007/s11259-018-9735-z

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