Date of Award

2010

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Animal Science

First Advisor

Worku, Mulumebet Dr.

Abstract

Escherichia coli cause mastitis upon entry into the mammary gland. Colostrum is the first milk produced by the lactating mother and provides immune nutrients for protection of the newborn. Bacterial pathogenesis may be modulated by exposure to colostral components. The objective of this study was to evaluate the effect of host immune factors in colostrum on E. coli K-12 growth, RNA transcription, and gene expression. Samples of E. coli K-12 were grown to mid-log in LB (Luria Bertani) broth and colostrum-treated LB broth. One of each culture was heat shocked to create one control and three different treatments. Growth was inhibited for the first 20 minutes in colostrum treated samples. RNA from control and treated samples was isolated using the RNeasy (Qiagen, CA) kits. The RNA was evaluated using a ND-1000 NanoDrop Spectrophotometer (NanoDrop Technologies Inc., DE) and Agilent 2100 bioanalyzer (Agilent Technologies, CA). The concentrations of samples were significantly different for each treatment (p<0.0001) and the purity was found to be not significantly affected (p>0.05). The effect of the treatments on transcription of six E. coli genes (fdoG, aceE, hslV, ldhA, rplB, and mscL) was evaluated using RT-PCR and agarose gel electrophoresis, and 93 genes were evaluated using E. coli K12 Starter V2 microarray chips (Ocimum Biosolutions, CA). Microarray data was analyzed using Imagene® 9.0 (Biodiscovery, CA). All six genes selected for PCR analysis were successfully amplified using specific primers. Colostrum, heat shock, and the combination of colostrum and xv heat shock had an effect on bands present. Analysis of all data from microarray experiments showed that both treatment and dye had an effect, however in dye swap experiment there were no dye effects on gene expression. Evaluation of gene expression was only conducted on the dye swap experiment. Colostrum treatment resulted in an upregulation of 54.1% of the genes on slide 1 and 38.0% of the genes on slide 2. In conclusion, major findings from this study indicate that colostrum and heat shock have an effect on E. coli growth, transcription, and gene expression.

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