Veterinary Research Communications

, Volume 38, Issue 3, pp 209–219 | Cite as

Formation of NET, phagocytic activity, surface architecture, apoptosis and expression of toll like receptors 2 and 4 (TLR2 and TLR4) in neutrophils of mastitic cows

  • Dilip K. Swain
  • Mohar Singh Kushwah
  • Mandheer Kaur
  • Tapas K. Patbandha
  • Ashok K. Mohanty
  • Ajay K. Dang
Original Article


Neutrophils employ both oxidative and non oxidative mechanisms to destroy pathogens. Function of neutrophils coming in milk during mammary invasion is not clearly understood in dairy animals. Therefore, the present study was designed in 36 Sahiwal cows to see the changes occurring in the neutrophil activity of cows suffering from subclinical (SCM) and clinical mastitis (CM). Cows were divided into three groups as healthy (n = 12), SCM (n = 12) and CM (n = 12) groups on the basis of CMT scoring, gross morphological changes in milk, bacteriological examination of milk and by counting milk SCC. Significantly (P < 0.05) higher milk SCC, neutrophil percent and significantly (P < 0.05) lower viability of both blood and milk neutrophils were observed in CM group of cows as compared to SCM and control group of cows. Phagocytic activity (PA) was significantly (P < 0.05) higher in milk neutrophils of SCM and CM cows as compared to control cows. Toll like receptors 2 and 4 in blood and milk neutrophils were found to be significantly (P < 0.05) higher, whereas, apoptosis in terms of altered mitochondrial transmembrane potential, Caspase 3 and 7 activities were found to be significantly (P < 0.05) lower in CM cows. Alterations in surface architecture of neutrophils in terms of formation of pseudopods was observed by scanning electron microscope (SEM) and found to be higher in CM cows. Blood neutrophils were found to be spherical as compared to milk neutrophils. Formation of neutrophil extracellular trap (NETs) were found milk neutrophils of CM cows, whereas, SCM and healthy cows did not exhibit NET formation. The study indicated a positive correlation between lower neutrophil apoptosis and higher expression of TLR2 and TLR4 with the formation of NETs and change in surface architecture. Formation of NET like structures seemed to be an effective mode of defense employed by neutrophils of cows suffering from clinical mastitis.


Neutrophils Apoptosis TLR2 TLR4 NET Mastitis Cows 



The authors are thankful to Dr. V.V. Ramamurthy, Dept of Entomology, IARI, New Delhi for helping to carry out SEM of blood and milk neutrophils. We are also thankful to Dr. Sandhya Toki, PGI Chandigarh, for assisting in carrying out the flow cytometry. We are highly thankful to Department of Biotechnology, Ministry of Science and Technology, India (BT/PR13016/AAQ/01/411/2009) for providing financial support to carry out this study.

Conflict of Interest Statement

The authors declare that they have no conflict of interest


  1. Alluwaimi AM (2004) The cytokines of bovine mammary gland: prospects for diagnosis and therapy. Res Vet Sci 77(3):211–22PubMedCrossRefGoogle Scholar
  2. Bannerman DD (2009) Pathogen-dependent induction of cytokines and other soluble inflammatory mediators during intramammary infection of dairy cows. J Anim Sci 87:10–25PubMedCrossRefGoogle Scholar
  3. Bannerman DD, Paape MJ, Lee JW, Zhao X, Hope JC, Rainard P (2004) Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intra mammary infection. Clin Diagn Lab Immunol 11:463–472PubMedCentralPubMedGoogle Scholar
  4. Brinkmann VU, Reichard C, Goosmann B, Fauler Y, Uhlemann DS, Weiss YW, Zychlinsky A (2004) Neutrophil extracellular traps kill bacteria. Science 303:1532–1535PubMedCrossRefGoogle Scholar
  5. Burton JL, Erskine RJ (2003) Immunity and mastitis. Some new ideas for an old disease. Vet Clin N Am Food Anim Pract 19:1–45CrossRefGoogle Scholar
  6. Burvenich C, Bannerman DD, Lippolis JD, Peelman L, Nonnecke BJ, Kehrli MEJ, Paape MJ (2007) Cumulative physiological events influence the inflammatory response of the bovine udder to Escherichia coli infections during the transition period. J Dairy Sci 90:E39–E54PubMedCrossRefGoogle Scholar
  7. Dang AK, Kapila S, Tomar P, Singh C (2007) Relationship of blood and milk cell counts with mastitic pathogens in Murrah buffaloes. Ital J Anim Sci 6(2):821–824Google Scholar
  8. Dang AK, Mukherjee J, Kapila S, Mohanty AK, Kapila R, Prasad S (2010) In vitro phagocytic activity of milk neutrophils during lactation cycle in Murrah buffaloes of different parity. J Anim Physiol Nut 94:706–711CrossRefGoogle Scholar
  9. Dang AK, Prasad S, De K, Mukherjee J, Sandeep IVR, Mutoni G, Pathan MM, Jamwal M, Kapila S, Kapila R, Kaur H, Dixit S, Mohanty AK, Prakash BS (2012) Effect of supplementation of Vitamin E, copper and zinc on the in vitro phagocytic activity and lymphocyte proliferation index of peripartum Sahiwal (Bos indicus) cows. J Anim Physiol Nut. doi: 10.1111/j.1439-0396.2011.01272.x Google Scholar
  10. de Souza FN, Sanchez EMR, Gidlund MA, Heinemann MB, Reis LC, Libera AMMPD, Cerqueira MMOP (2012) The innate immunity in bovine mastitis: the role of pattern-recognition receptors. Am J Immunol 8(4):166–178CrossRefGoogle Scholar
  11. DeLeo FR (2004) Modulation of phagocyte apoptosis by bacterial pathogens. Apoptosis 9:399–413PubMedCrossRefGoogle Scholar
  12. Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, Weinrauch Y, Brinkmann V, Zychlinsky A (2007) Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 176:231–241PubMedCentralPubMedCrossRefGoogle Scholar
  13. Harmon RJ (2001) Somatic cell counts: A primer. Proc. National Mastitis Council Annual Meeting, In, pp 3–9Google Scholar
  14. Hogeveen HK, Hujips K, Lam TJ (2011) Economic aspects of mastitis: New developments. NZ Vet J 59:16–23CrossRefGoogle Scholar
  15. Kawai T, Akira S (2011) Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34:637–650PubMedCrossRefGoogle Scholar
  16. Kebir DEI, Filep JG (2013) Targeting neutrophil apoptosis for enhancing the resolution of inflammation. Cells 2:330–348PubMedCentralPubMedCrossRefGoogle Scholar
  17. Kennedy AD, DeLeo FR (2009) Neutrophil apoptosis and the resolution of infection. Immunol Res 43:25–61PubMedCrossRefGoogle Scholar
  18. Kobayashi SD, Voyich JM, Burlak C, DeLeo FR (2005) Neutrophils in the innate immune response. Arch Immunol Ther Exp 53:505–517Google Scholar
  19. Lippolis JD, Reinhardt TA, Goff JP, Horst RL (2006) Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk. Vet Immunol Immunopathol 15,113(1–2):248–55CrossRefGoogle Scholar
  20. Mehrzad J, Duchateau L, Pyorola S, Burvenich C (2002) Blood and milk neutrophil chemiluminescence and viability in primiparous and pluriparous dairy cows during late pregnancy, around parturition and early lactation. J Dairy Sci 85:3268–3276PubMedCrossRefGoogle Scholar
  21. Mehrzad J, Duchateau L, Burvenich C (2004) Viability of milk neutrophils and severity of bovine coliform mastitis. J Dairy Sci 87:4150–4162PubMedCrossRefGoogle Scholar
  22. Nathan C (2006) Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 6:173–182PubMedGoogle Scholar
  23. Paape MJ, Mehrzad J, Zhao X, Detileux J, Burvenich C (2002) Defense of the bovine mammary gland by polymorphonuclear neutrophil leukocytes. J Mammary Gland Biol Neoplasia 7:109–121PubMedCrossRefGoogle Scholar
  24. Paape MJ, Bannerman DD, Zhao X, Lee JW (2003) The bovine neutrophil: structure and function in blood and milk. Vet Res 34(5):597–627PubMedCrossRefGoogle Scholar
  25. Papayannopoulos V, Zychlinsky A (2009) NETs: a new strategy for using old weapons. Trends Immunol 30:513–521PubMedCrossRefGoogle Scholar
  26. Petzl W, Zerbe HJ, Guntler J, Yang W, Seyfert HM (2008) Escherichia coli, but not Staphylococcus aureus triggers an early increased expression of factors contributing to the innate defense in the udder of the cow. Vet Res 39:18PubMedCrossRefGoogle Scholar
  27. Pyorala S (2003) Indicators of inflammation in the diagnosis of mastitis. Vet Res 34:565–578PubMedCrossRefGoogle Scholar
  28. Sordillo LM, Streicher KL (2002) Mammary gland immunity and mastitis susceptibility. J Mammary Gland Biol Neoplasia 7(2):135–146PubMedCrossRefGoogle Scholar
  29. Tian SZ, Chang CJ, Chiang CC, Peh HC, Huang MC, Lee JW, Zhao X (2005) Comparison of morphology, viability and function between blood and milk neutrophils from peak lactating goats. Canad J Vet Res 69:39–45Google Scholar
  30. Van Oostveldt K, Paape MJ, Dosogne H, Burvenich C (2002) Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils. Domest Anim Endocrinol 22:37–50.Google Scholar
  31. Zhao X, Lacasse P (2008) Mammary tissue damage during bovine mastitis: Causes and control. J Anim Sci 86(1):57–65CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Dilip K. Swain
    • 1
  • Mohar Singh Kushwah
    • 1
  • Mandheer Kaur
    • 1
  • Tapas K. Patbandha
    • 2
  • Ashok K. Mohanty
    • 3
  • Ajay K. Dang
    • 1
  1. 1.Lactation and Immuno-Physiology LaboratoryNational Dairy Research InstituteKarnalIndia
  2. 2.Dept. of Livestock Production ManagementCollege of Vet. Sc. and A.H. Junagadh Agri. Uni.JunagadhIndia
  3. 3.Principal Scientist Animal Biotechnology Centre National Dairy Research InstituteKarnalIndia

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