Transcriptome Analysis of Paramphistomum cervi in Sheep (Ovis aries) Using Next Generation Sequencing
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Date
2013
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AAU, Anand
Abstract
Rumen fluke, Paramphistomum cervi, is responsible for production losses to the livestock (mainly sheep, goat and buffalo) and meat industries due to clinical disease, reduced weight gain and milk production, and deaths. Surprisingly, despite the massive economic loss, knowledge of the fiindamental molecular biology of Paramphistomum cervi is scanty. Currently, there is a need to focus on the development of new approaches for the prevention and control of Paramphistomosis in livestock. Paramphistomum cervi were found mainly in the rumen and were light pink in colour with a sucker at the tip of the cone and another sucker ventrally at the posterior end. The body of P. cervi was pear-shaped, slightly concave ventrally (conical) and convex dorsally. Caeca were wide, pursued a serpentine course and reached anterior margin of posterior sucker (acetabulum) with blind ends more dorsal than lateral. Genital pore was situated behind intestinal bifiircation. Histological peculiarities of the muscular organs such as a liorchis type pharynx, gracile type genital opening, Paramphistomum type acetabulum in median sagittal section and distinctly lobed testes, situated a little obliquely tandem were also supported the histological diagnosis. The present study explores, for the first time, the transcriptome and proteome of the adult stage of the rumen fluke, Paramphistomum cenn of sheep using Ion Torrent PGM sequencing and in silico analysis, and also provides a basis for applied outcomes such as the development of drug and vaccines against this neglected parasite of veterinary importance. Transcriptome analysis oi Paramphistomum cenn of sheep was carried out to study identification of differentially expressed genes. Relative levels of transcription determined for individual molecules, were also characterized on basis of homology, gene ontology and pathway mapping. Total of 2,944,061 reads were generated and after removing low quality reads 2,178,136 (73.98%) sequences remained. These 2,178,136 reads fi-om the cDNA library of sheep Paramphistomes were then mapped with reference file data. Out of 11,593 sequences, 11,588 sequences were mapped and classified based on homology searches, protein motifs, gene ontology and biological pathway mapping. BLASTx manual annotation revealed 11,582 sequences displaying significant similarity to known genes in NCBI (99.94% of total P. cervi sequences). Species distribution of BLAST hits based on the BLASTx analysis showed that most of sequences matched to species of Trematode parasites, mainly to Clonorchis sinensis. Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Fasciola hepatica. A significant proportion (56.6%) of the transcriptome of Paramphistomum cervi was inferred to encode 6,562 conserved protein domains or family signatures. Amongst the InterPro domains identified 'protein kinase-like domain' (130, 2.9%) 'Protein kinase, catalytic domain' (109, 2.4%), 'WD40/YVTN repeat-like-containing domain' (91. 2.0%), 'armadillo-like helical' (74. 1.6%), 'armadillo-type fold' (72.1.6%)) and WD40-repeat-containing domain' (69, 1.5%)) were most abundant. KEGG annotated a total of 1,926 (16.6%) sequences were fiirther classified into 108 pathways by using the pathway identification tool in Blast2G0. Total 14 parent pathways were identified and categorized into four types, with metabolic pathways being the most abundant, followed by genetic information processing, environmental processing and organismal systems. Based on the annotation, based on conserved domains and protein families, total 6,527 sequences (56.32%)) were assigned to GO term. GO annotation of the Paramphistomum cervi predicted peptides revealed 4,473 different GO teniis, which were assigned to three main GO categories of biological process (2,645 GO terms; 59.1%), cellular component (644 GO terms; 14.4%) and molecular function (1,184 GO terms; 26.5%). The integration of transcriptomic and proteomic datasets generated herein sets the scene for fiiture studies, aimed at exploring the potential roles of molecules which may play at the host-parasite interface and for estabhshing novel strategies for the treatment or control of parasitic fluke infections. Recent transcriptome analysis of Paramphistomum cervi has identified novel genes and genes not previously reported for Paramphistomes, as well as the identification of the molecular mechanisms for host-dependent maturation, immune evasion, development and signaling.
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VETERINARY PARASITOLOGY, AN ANALYSIS