Veterinary World

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Research (Published online: 16-03-2015)

14. Neutrophil dynamics in the blood and milk of crossbred cows naturally infected with Staphylococcus aureus - Dilip K. Swain, Mohar Singh Kushwah, Mandheer Kaur and Ajay K. Dang

Veterinary World, 8(3): 336-345



   doi: 10.14202/vetworld.2015.336-345


Dilip K. Swain: Division of Dairy Cattle Physiology, Lactation and Immunophysiology Laboratory, National Dairy Research Institute, Karnal - 132 001, Haryana, India;

Mohar Singh Kushwah: Division of Dairy Cattle Physiology, Lactation and Immunophysiology Laboratory, National Dairy Research Institute, Karnal - 132 001, Haryana, India;

Mandheer Kaur: Division of Dairy Cattle Physiology, Lactation and Immunophysiology Laboratory, National Dairy Research Institute, Karnal - 132 001, Haryana, India;

Ajay K. Dang: Division of Dairy Cattle Physiology, Lactation and Immunophysiology Laboratory, National Dairy Research Institute, Karnal - 132 001, Haryana, India;


Received: 02-11-2014, Revised: 31-01-2015, Accepted: 09-02-2015, Published online: 16-03-2015


Corresponding author: Parveen Kumar, e-mail:

Citation: Swain DK, Kushwah MS, Kaur M, Dang AK (2015) Neutrophil dynamics in the blood and milk of crossbred cows naturally infected with Staphylococcus aureus, Veterinary World 8(3):336-345.

Aim: The present study was designed to evaluate the neutrophil dynamics in terms of the functional competence during subclinical mastitis (SCM) and clinical mastitis (CM).

Materials and Methods: A total of 146 Karan fries cows were screened and were divided into three groups as control (n=12), SCM, n=12 and CM, n=12 groups on the basis of California mastitis test scoring, bacteriological evaluation, gross and morphological changes in milk and by counting milk somatic cell count (SCC). Both blood and milk polymorphonuclear neutrophils (PMNs) were isolated in the study. Phagocytic activity (PA) was studied by spectrophotometrically; neutrophil extracelluar traps (NETs) were studied by scanning electron microscopy (SEM); CD44 was quantified by flow cytometry and apoptosis was studied by fluorescent microscopy.

Results: Significantly (p<0.05) higher SCC, PA was found in milk of CM cows as compared to SCM and control cows. Significantly lower (p<0.05) apoptosis was observed in PMNs isolated from both blood and milk of CM group of cows when compared to control and SCM group. The milk neutrophils of CM group of cows formed NETs as evidenced from the SEM images. Surface expression of CD44 revealed a significantly (p<0.05) lower expression in milk neutrophils of CM group of cows when compared to SCM and control group of cows.

Conclusion: The study indicated a positive correlation between delayed neutrophil apoptosis, persistent staying of neutrophils at the site of infection along with formation of NETs as the strategies to fight against the pathogens in the udder during Staphylococcal mastitis. The study forms a strong base for future molecular research in terms of neutrophil recruitment and neutrophil removal from the site of infection.

Keywords: apoptosis, CD44, mastitis, neutrophil, neutrophil extracelluar traps, Staphylococcus aureus.

1. Paape, M.J., Bannerman, D.D., Zhao, X. and Lee, J.W. (2003) The bovine neutrophil: Structure and function in blood and milk. Vet. Res., 34(5): 597-627.
2. Borregaard, N. (2010) Neutrophils, from marrow to microbes. Immunity, 33(5): 657-670.
3. Michael, A.R. and Stephen, C.N. (2011) Mastitis and its impact on structure and function in the ruminant mammary gland. J Mammary Gland Biol. Neoplasia., 16(4), 275-289.
4. Rainard, P. and Riollet, C. (2006) Innate immunity of the bovine mammary gland. Vet. Res., 37(3): 369-400.
5. Petzl, W., Zerbe, H.J., Guntler, J., Yang, W. and Seyfert, H.M. (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(2): 18-26.
6. Bratton, D.L. and Henson, P.M. (2011) Neutrophil clearance: When the party is over, clean-up begins. Trends Immunol., 32(8): 350-357.
PMid:21782511 PMCid:PMC3151332
7. Sadik, C.D., Kim, N.D. and Luster, A.D. (2011) Neutrophils cascading their way to inflammation. Trends Immunol., 32(10): 452-460.
PMid:21839682 PMCid:PMC3470857
8. Kennedy, A.D. and DeLeo, F.R. (2009) Neutrophil apoptosis and the resolution of infection. Immunol. Res., 43: 25-61.
9. Rainard, P., Fromageau, A., Cunha, P. and Gilbert, F.B. (2008) Staphylococcus aureus lipoteichoic acid triggers inflammation in the lactating bovine mammary gland. Vet. Res., 39(5): 52-58.
10. Brenaut, P., Lefèvre, L., Rau, A., Laloë, D., Pisoni, G., Moroni, P., Bevilacqua, C., and 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.
PMid:24521038 PMCid:PMC3937043
11. Leitner, G., Lubashevsky, E., Glickman, A., Winkler, M. and Trainin, Z. (2003) Development of a Staphylococcus aureus vaccine against mastitis in dairy cows- challenge trials. Vet. Immunol. Immunopathol., 93(1-2): 31-38.
12. Swain, D.K., Kushwah, M.S., Kaur, M., Patbandha, T.K., Mohanty, A.K. and Dang, A.K. (2014) 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. Vet. Res. Commun., 38: 209-219.
13. Mehrzad, J., Duchateau, L. and Burvenich, C. (2004) Viability of milk neutrophils and severity of bovine coliform mastitis. J. Dairy Sci., 87(12): 4150-4162.
14. Dang, A.K., Prasad, S., De, K., Mukherjee, J., Sandeep, I.V.R., Mutoni, G., Pathan, M.M., Jamwal, M., Kapila, S., Kapila, R., Kaur, H., Dixit, S., Mohanty, A.K. and Prakash, B.S. (2013) 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. Anim. Nutr., 97(2), 315-321.
15. Pyorala, S. (2003) Indicators of inflammation in the diagnosis of mastitis. Vet. Res., 34(5): 565-578.
16. Aitken, S.L., Corl, C.M. and Sordillo, L.M. (2011) Immunopathology of mastitis: Insights into disease recognition and resolution. J. Mammary Gland Biol. Neoplasia., 16(4): 291-304.
17. Sharma, N., Singh, N.K. and Bhadwal, M.S. (2011) Relationship of somatic cell count and matitis; an overview. Asian Aust. J. Anim. Sci., 24(3): 429-438.
18. Kebir, D.E.I. and Filep, J.G. (2013) Targeting neutrophil apoptosis for enhancing the resolution of inflammation. Cell, 2(2): 330-348.
PMid:24709704 PMCid:PMC3972676
19. Boutet, P., Boulanger, D., Gillet, L. and Bureau, F.L. (2004) Delayed neutrophil apoptosis in bovine subclinical mastitis. J. Dairy Sci., 87(12): 4104-4114.
20. Lotz, S., Aga, S., Wilde, I., Zandbergen, G., Hartung, T., Solbach, W. and Laskay, T. (2004) Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2. J. Leukocyte Biol. 75(3): 467-477.
21. Sladek, Z., Rysanek, D. and Faldyna, M. (2005a) Effect of Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine mammary gland tissue pool neutrophils in vitro. Vet. Med., 50: 11-23.
22. Sladek, Z., Rysanek, D., Ryznarova, H. and Faldyna, M. (2005b) Neutrophil apoptosis during experimentally induced Staphylococcus aureus mastitis. Vet. Res., 36(4): 629-643.
23. Yamamoto, A., Taniuchi, S., Tsuji, S., Mausi, M. and Kobayashi, Y. (2002) Role of reactive oxygen species in neutrophil apoptosis following ingestion of heat-killed Staphylococcus aureus. Clin. Exp. Immunol., 129(3): 479-484.
PMid:12197889 PMCid:PMC1906464
24. Nathan, C. (2006) Neutrophils and immunity: Challenges and opportunities. Nat. Rev. Immunol., 6(3): 173-182.
25. Brinkmann, V., Laube, B, Abu Abed, U., Goosmann, C. and Zychlinsky, A. (2010) Neutrophil extracellular traps: how to generate and visualize them. J. Vis. Exp., 36: 1724.
26. Papayannopoulos, V. Zychlinsky, A. (2009) NETs: A new strategy for using old weapons. Trends Immunol., 30(11): 513-521.