Mycobacterium paratuberculosis causes a chronic progressive wasting disease in sheep. Most of the data on the molecular pathogenesis of the disease has come from studies in other species in the advanced stages of the disease or from macrophage infection models. Molecular techniques used in these studies include microarray analysis, selective capture of transcribed sequences (SCOTS) and RNA arbitrarily primed-PCR (RAP-PCR). The objective of this study was to examine host gene expression during the initial stages of M. ptb. infection and during the clinical stage of infection to identify known or novel genes regulating the response to infection, and to define genes that can be used to identify M. ptb. infected animals earlier than currently possible. Long distance differential display PCR (DD-PCR) was employed to identify differentially expressed genes in lymph node and ileal tissues collected from uninfected, early stage infected or clinically infected Merino sheep. The advantages of this technique include the ability to look at regulated transcripts using small amounts of starting material. RNA was extracted using the Qiagen mini RNA extraction kit. RNA was DNase treated and reverse transcribed to cDNA using oligo(dT). DD-PCR was performed according to Clontech's Delta Differential Display Kit with some modifications. Preliminary DD-PCR studies using two different primer sets revealed amplification of subsets of genes, although none appeared to be differentially expressed between uninfected and early stage infected animals. Additional primer combinations are being evaluated.