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The project has two broad aims: <OL> <LI> To identify the intrinsic differences in the way that macrophages from asymptomatic, paucibacillary and multibacillary forms of paratuberculosis respond to infection by Mycobacterium avium subspecies paratuberculosis [Map]. <LI> To compare immuno-inflammatory gene expression in gut-associated lymphoid tissue and relate these differences to the different pathological forms of paratuberculosis. </ol> These aims will be addressed by analysing immuno-inflammatory gene expression of infected macrophages and gut-associated lymphoid tissues using functional genomics [microarray] technology. Consequently, a major aim will be the construction, verification and validation of an oligonucleotide microarray of ruminant immuno-inflammatory genes.
Progress: <BR> Scientific highlights: Two oligonucleotide microarrays have been designed based on a 2-generation format. The first generation oligonucleotide array, the Ruminant Immuno-inflammatory Gene Reference Array (RIGRA) was designed with 12 cytokines, 17 positive control genes (including 4 PCR products) and 7 negative control genes. The RIGRA chip has been used for verification and validation experiments. The chip has been used to optimise and standardise hybridisation experiments. The expression of the cytokine and housekeeping genes has been quantified and the accuracy verified by comparison with our established RNAse Protection Assay (RPA). Oligonucleotides from two sources have been compared and this has resulted in finding a cheaper supplier enabling the production of a second-generation chip containing more genes and hence a more comprehensive gene array. A second version of the RIGRA chip (RIGRA-2) has been designed to include “landing lights”, spiked controls using the SpotReport Oligo Array Validation System (Strategene) and olignonucleotides representing 13 cytokine genes (the additional gene represented on the array is IL-18). The chip will be used to examine the normal variation of gene expression in unaffected sheep. RNA has been extracted and quantified for six asymptomatic sheep and the hybridisations to the array are currently underway. <BR> <BR>
The second generation array; the Ruminant Immuno-inflammatory Gene Universal Array (RIGUA) has been designed to represent 516 immuno-inflammatory genes including all sequenced ruminant genes associated with the immune system and immuno-inflammatory genes from other species whose sequence is not publicly available for a ruminant species. Sequences of known ruminant control genes, house keeping genes and immuno-inflammatory genes were extracted from the public database GenBank. Optimised oligonucleotides were designed in collaboration with Sigma-Genosys Limited (London Road, Pampisford, Cambridge, UK). Gene sequences of known immuno-inflammatory genes, but not publicly available for a ruminant species, were extracted from the public database GenBank. Conserved regions within genes represented by other species (including human, rodent and pig) were selected using between species gene sequence comparisons. Oligonucleotide probes were designed from conserved regions with a 3 prime gene sequence bias. Using the Oligo 6 programme software (Medprobe, PO Box 2640, St.Hanshaugen, N-0131, Oslo, Norway) two non-overlapping 75 mer oligonucleotides were designed. All oligonucleotides probes were compared with the complete NCBI GenBank database using the BLAST search similarity algorithm. The purpose of using BLAST was to check the polarity of the probe, to ensure that it had 100% homology to the intended target sequence and to eliminate possible similarities to irrelevant genes that potentially could produce false positive signals on the microarrays. <BR> <BR>
The manufacture of both these chips has required significant bioinformatics and data mining. A Microsoft Access database has been developed to record all stages of this procedure. The database is subdivided into 3 categories:
<OL> <LI> Clinical Database: describes the gross and histopathological condition of an individual animal and selected tissues. This category also records bacteriology data, parasitic load and final diagnosis.
<LI> Microarray gene information database: describes the genes represented on the chip, including the complete sequence, the oligonucleotides designed to represent the gene, and the parameters that characterise the oligonucleotide sequence.
<LI> LIMS (Laboratory Information Management System): describes the experiments that were conducted and the RNA quality and concentration.
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To date the number of clinical cases (from 4 farms) examined by pathology are as follows:
<UL> <LI> Asymptomatic 12
<LI> Paucibacilliary 11
<LI> Multibacilliary 4 </uL>
Alveolar macrophages were collected from selected sheep and infected with the lab strain of Map. Rna has been extracted from these infected cells in preparation for hybridisation onto the RIGUA chip.
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