Project description:Sheep (N=6) infested with Psoroptes ovis mites were bled weekly and circulating leukocytes isolated from their whole blood samples. RNA was extracted and processed onto Agilent Bovine Microarrays and differentially expressed genes identified between the following time points (0 (Baseline (pre-infestation)), 1 week, 3 weeks and 6 weeks post-infestation).
Project description:The presence of components of the RNA interference (RNAi) pathway in Psoroptes ovis, an ectoparasitic mite responsible for psoroptic mange, was investigated through interrogation of the P. ovis genome. Homologues of transcripts representing critical elements for achieving effective RNAi in the mite, Tetranychus urticae and the model organisms Caenorhabditis elegans and Drosophila melanogaster were identified and, following the development of a non-invasive immersion method of double stranded RNA delivery, gene silencing by RNAi was successfully demonstrated in P. ovis. Significant reductions in transcript levels were achieved for three target genes which encode the Group 2 allergen (Pso o 2), mu-class glutathione S-transferase (PoGST-mu1) and beta-tubulin (Po?tub). This is the first demonstration of RNAi in P. ovis and provides a mechanism for mining transcriptomic and genomic datasets for novel control targets against this economically important ectoparasite.
Project description:Transcriptional analysis of ovine skin response to infection with the parasitic mite Psoroptes ovis using the Agilent ovine transcriptome microarray platform. The results of statistical analysis (differential gene expression across the time course of infection was determined using a one way-analysis of variance (ANOVA) with a Student-Newman-Keuls (SNK) post-hoc test in Genespring GX 11.0 (Agilent Technologies, UK) comparing each of the 5 time points, non-infected and 1, 3, 6 and 24 hours post infection. Multiple test correction was performed using the Benjamini & Hochberg False Discovery Rate (FDR) procedure with an FDR corrected p-value cut-off set at 0.05) and fold change analysis (Fold change analysis was performed on the one-way anova dataset, probes with a fold change greater than 2 between any of the conditions were carried forward) are in archive E-TABM-1012.additional.zip. Quality control: Six biological replicates (sheep, n=6), multiple biopsies pooled from each animal at each time point of infection. Pooled samples from each sheep at each time point hybridised in single-dye (Cy3) format to Agilent ovine arrays. Positive and negative control probes utilised to assess array QC and real time qRT-PCR validation of selected array probes
Project description:Comparative expression profiles between unstimulated ovine keratinocytes and keratinocytes stimulated with whole mite antigen and whole mite wash in vitro, over a time course of 1, to 48 hours, using the RIGUA custom array (Watkins et al., 2008) and real-time RT-PCR
Project description:Sheep scab, caused by infestation with Psoroptes ovis, is highly contagious, results in intense pruritus, and represents a major welfare and economic concern. Here, we report the first draft genome assembly and gene prediction of P. ovis based on PacBio de novo sequencing. The ∼63.2-Mb genome encodes 12,041 protein-coding genes.
Project description:BACKGROUND: Sheep scab, caused by infestation with the ectoparasitic mite Psoroptes ovis, results in the rapid development of cutaneous inflammation and leads to the crusted skin lesions characteristic of the disease. We described previously the global host transcriptional response to infestation with P. ovis, elucidating elements of the inflammatory processes which lead to the development of a rapid and profound immune response. However, the mechanisms by which this response is instigated remain unclear. To identify novel methods of intervention a better understanding of the early events involved in triggering the immune response is essential. The objective of this study was to gain a clearer understanding of the mechanisms and signaling pathways involved in the instigation of the immediate pro-inflammatory response. RESULTS: Through a combination of transcription factor binding site enrichment and pathway analysis we identified key roles for a number of transcription factors in the instigation of cutaneous inflammation. In particular, defined roles were elucidated for the transcription factors NF-kB and AP-1 in the orchestration of the early pro-inflammatory response, with these factors being implicated in the activation of a suite of inflammatory mediators. CONCLUSIONS: Interrogation of the host temporal response to P. ovis infestation has enabled the further identification of the mechanisms underlying the development of the immediate host pro-inflammatory response. This response involves key regulatory roles for the transcription factors NF-kB and AP-1. Pathway analysis demonstrated that the activation of these transcription factors may be triggered following a host LPS-type response, potentially involving TLR4-signalling and also lead to the intriguing possibility that this could be triggered by a P. ovis allergen.