A colorimetric assay for quantitating bovine neutrophil bactericidal activity

Area of Science:

• Veterinary Immunology
• Microbiology
• Cellular Assays

Background:

• Bovine neutrophils play a crucial role in innate immunity against bacterial infections.
• Quantifying neutrophil bactericidal activity is essential for understanding immune responses and developing treatments.
• Traditional bactericidal assays can be time-consuming and labor-intensive.

Purpose of the Study:

• To develop and validate a novel colorimetric assay for quantifying bovine neutrophil bactericidal activity against Staphylococcus aureus.
• To assess the assay's sensitivity and efficiency compared to traditional methods.
• To demonstrate the assay's utility in detecting modulations in neutrophil function.

Main Methods:

• Development of a colorimetric assay utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to detect viable Staphylococcus aureus.
• Incubation of antibody-opsonized S. aureus with bovine neutrophils, followed by lysis and MTT addition.
• Quantification of formazan production, directly proportional to the number of surviving bacteria, via spectrophotometry.
• Validation of the assay by testing the effects of various agents (colchicine, cytochalasin B, PMA, rBoIFN-gamma) on neutrophil bactericidal activity.

Main Results:

• The MTT assay demonstrated a direct relationship between formazan production and viable S. aureus numbers.
• The assay successfully detected suppressed bactericidal activity following in vitro treatment with colchicine, cytochalasin B, and phorbol 12-myristate 13-acetate.
• Recombinant bovine interferon gamma (rBoIFN-gamma) showed dose-dependent effects, enhancing activity at low levels and decreasing it at high levels.
• The assay proved sensitive, inexpensive, and completed within 3 hours, allowing for high-throughput analysis.

Conclusions:

• The developed colorimetric MTT assay is an effective and efficient tool for quantifying bovine neutrophil bactericidal activity.
• This assay provides a valuable method for studying neutrophil function and the impact of various substances on their antimicrobial capabilities.
• The MTT assay offers significant advantages in terms of speed, cost, and throughput compared to conventional bactericidal assays.