Date of Award

2012

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Animal Science

First Advisor

Worku, Mulumebet Dr.

Abstract

Concerns for animal welfare, food safety, and security fuel interest in understanding modulators of innate immunity. The accumulation of neutrophils and activation of toll-like receptors (TLRs) at infection sites are critical events of innate host defense. The objectives of this study were to evaluate the effects of Lipopolysaccaride (LPS), Peptidoglycan (PGN), Nystatin (NYS), and Quebracho (Q) extract on bovine neutrophils activation in relation to expression of genes encoding TLR2, TLR4, natural resistance-associated macrophage protein 1 (Nramp1), and the cytokines TNF-α and IL-10. Genes in genomic DNA were detected from blood samples, except IL-10. Neutrophils were isolated from four Holstein Friesian cows and cultured with LPS (100ng), PGN (1μg), NYS (2500U), or ethanol (62%); Q (1.5X) or maintained in PBS at the following time intervals: 0, 15, 30 or 60 minutes. Total RNA was extracted and concentration, purity and time of incubation, had an effect on RNA concentration, producing highly significant results (p<0.0001). Specific primers were used to amplify Nramp1, TLR2, TLR4, TNF-α and IL10 mRNA using a reverse transcriptase polymerase chain reaction (RT-PCR). Neutrophils suspended in PBS showed basal expression of TLR 2, 4, and TNF-α. Exposure of neutrophils to LPS resulted in induction of Nramp1. Peptidoglycan induced gene expression of TLR 2 at 60 minutes. Nystatin treatment induced TLR 2 expression but suppressed Nramp1 at 60 minutes. Gene modulation with ethanol was seen with TLR 2 and Nramp1. Quebracho suppressed NRAMP1 gene expression after 30 and 60 minutes. ELISA results did not show a treatment effect, but demonstrated a significant decrease in TNF-α secretion compared to PBS and/or ethanol (p <0.05), after 30 minutes of incubation. This study demonstrated modulation of genes expressed by treatment with bacterial components and immunomodulators in bovine neutrophils in vitro.

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