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:BackgroundSheep scab is caused by Psoroptes ovis and is arguably the most important ectoparasitic disease affecting sheep in the UK. The disease is highly contagious and causes and considerable pruritis and irritation and is therefore a major welfare concern. Current methods of treatment are unsustainable and in order to elucidate novel methods of disease control a more comprehensive understanding of the parasite is required. To date, no full genomic DNA sequence or large scale transcript datasets are available and prior to this study only 484 P. ovis expressed sequence tags (ESTs) were accessible in public databases.ResultsIn order to further expand upon the transcriptomic coverage of P. ovis thus facilitating novel insights into the mite biology we undertook a larger scale EST approach, incorporating newly generated and previously described P. ovis transcript data and representing the largest collection of P. ovis ESTs to date. We sequenced 1,574 ESTs and assembled these along with 484 previously generated P. ovis ESTs, which resulted in the identification of 1,545 unique P. ovis sequences. BLASTX searches identified 961 ESTs with significant hits (E-value < 1E-04) and 584 novel P. ovis ESTs. Gene Ontology (GO) analysis allowed the functional annotation of 880 ESTs and included predictions of signal peptide and transmembrane domains; allowing the identification of potential P. ovis excreted/secreted factors, and mapping of metabolic pathways.ConclusionsThis dataset currently represents the largest collection of P. ovis ESTs, all of which are publicly available in the GenBank EST database (dbEST) (accession numbers FR748230 - FR749648). Functional analysis of this dataset identified important homologues, including house dust mite allergens and tick salivary factors. These findings offer new insights into the underlying biology of P. ovis, facilitating further investigations into mite biology and the identification of novel methods of intervention.