BUBALINE NEUTROPHILS FUNCTIONAL ACTIVITY AND LYMPHOCYTES BLASTOGENESIS RESPONSES IN Staphylococcus aureus INFECTION, in vitro: EFFECT OF IMMUNOMODULATION
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Date
2019-11
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA
Abstract
Buffaloes are important dairy animals of Andhra Pradesh (64.33 lakhs population) that contribute 69% of the total milk produced in the state. Staphylococcus aureus is one of the the most important Gram-positive bacterial pathogen that causes pneumonia, mastitis, phlebitis, meningitis, urinary tract infections, osteomyelitis, endocarditis and superficial skin lesions such as furunculosis etc in animals. Immune response against invading pathogens is crucial for keeping up the host’s health status. The desired optimal immune response in a host may be achieved through immunomodulation. Many synthetic, microbial and natural products are reported to act as immunomodulators. Phytochemicals are naturally occurring plant derived compounds with bioactive potentials and are used as immunomodulators in the treatment and management of infections. Phagocytic activity of neutrophils and lymphocytes proliferation assay or lymphocyte transformation test with PBMC which are considered as indicators of innate and adaptive immune responses, respectively, are included in this study to investigate the immunomodulatory activity of phytochemicals curcumin and quercetin. In this study blood samples were collected from apparently clinically healthy she buffaloes (4 to 5 years of age with similar physiological status) and infected in vitro with S. aureus isolated from clinical mastitic milk sample. Phagocytic activity of the neutrophils was measured by semi quantitative nitroblue tetrazolium assay (NBT). The reduction of NBT to formazan by the superoxide anion generated in the respiratory burst is measured at 540 nm. Bubaline neutrophils are treated with different concentrations of curcumin and quercetin, separately and incubated with live S. aureus / opsonized S. aureus / zymosan. The phagocytic activity of bubaline neutrophils treated with 25 μM curcumin and incubated with opsonized S. aureus is significantly different (p<0.05) with phagocytic activity of neutrophils treated with 50 μM curcumin and incubated with opsonized S. aureus. The phagocytic activity of bubaline neutrophils treated with 50 μM curcumin and incubated with opsonized S. aureus is significantly different (p<0.05) with phagocytic activity of neutrophils treated with 50 μM of quercetin and incubated with opsonized S. aureus.
The in vitro lymphocyte proliferation assay is routinely used as a measure to assess the functional status of lymphocytes. Though initially radioactive isotopes were used in cell proliferation assays due to radiation hazards associated with radio isotope assays the tetrazolium salt-based assays are employed for the proliferation assay. In the present study XTT {sodium3'-[1-[(phenylamino)-carbonyl]-3, 4-tetrazolium]-bis (4-methoxy-6- nitro) benzene-sulfonic acid hydrate} assay was used in cell proliferation assays. The stimulation index (S.I) of PBMC stimulated with Con A is significantly different (p<0.05) with the S.I of PBMC treated with 25 μM & 50 μM of quercetin and stimulated with Con A. The S.I of PBMC stimulated with Con A is significantly different (p<0.05) with the S.I of PBMC treated with 25 μM & 50 μM of quercetin and stimulated with heat inactivated S. aureus. Similarly, the S.I of PBMC treated with different concentrations of immunomodulators is significantly different (p<0.05) with other related groups. Therefore it is concluded that the phytochemicals curcumin and quercetin exhibited immunomodulatory activity on innate and adaptive immune response of buffaloes. Further research is required to establish the mechanisms of action underlying it.
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