Date of Award

2010

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Animal Science

First Advisor

Worku, Mulumebet Dr.

Abstract

An animal's ability to fight off infections with Escherichia coli can be impacted by polymorphisms in the TLR-4 gene. In goats, the effects of polymorphisms of the TLR4 gene are not known and need to be researched to improve goat herd production. The objectives of this study were to evaluate gene polymorphism and transcription of TLR-4 and to assess the effects of LPS exposure on gene expression and signaling in neutrophils from Spanish and Boer goats. To study genetic polymorphisms, gene segments using primers specific for TLR-4 were amplified. PCR products were then examined on a 1% agarose gel to determine amplification. Single Stranded Conformational Polymorphism (SSCP) Gels of PCR products were used to determine any polymorphisms between samples. To evaluate TLR-4 and CD-14 expression and the effects of LPS in caprine neutrophils, blood neutrophils were isolated from Spanish and Boer goats. Neutrophils were treated with and without 0111 B4 serotype E. coli LPS. Cell surface expression of TLR-4 and CD-14 were determined by Flow Cytometry. Transcription of TLR-4, CD14, and TNF-α after stimulation with LPS was determined by Reverse transcriptase PCR. Secretion of the cytokine IL-8 was determined by ELISA. A 494 base pair fragment of the TLR-4 gene was amplified from genomic DNA. Gene alleles run on SSCP Gels were found to be similar in all six goats used. Both TLR-4 and TNFα were transcribed in goat blood neutrophils. Treatment with LPS did not have an effect on transcription of TLR-4, xiii TNFα, CD-14, or GAPDH mRNA. Treatment with LPS resulted in increased secretion of IL-8. These studies provide evidence for polymorphisms in the TLR-4 gene and transcription and translation of TLR-4 associated genes in neutrophils from Boer and Spanish goats. Further studies are warranted.

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