De novo transcriptome sequencing of virulent and attenuated Histomonas meleagridis reveals basic insights about biological functions and potential pathogenic mechanisms

The flagellated protozoan Histomonas meleagridis is the causative agent of histomonosis in poultry. In turkeys the disease can coincide with high mortality whereas in chickens it is less severe but severe production losses can be noticed. Molecular data on this parasite has been scarce and genetic studies are in their infancy, which poses a challenge towards the understanding of the molecular biology of the protozoon. Majority of the genomic information in respect to biological processes and virulence factors remains unknown.In order to increase the availability of genomic information, a de novo transcriptome sequencing of H. meleagridis was performed from a virulent and an attenuated strain, cultivated in vitro as monoxenic mono-eukaryotic cultures together with Escherichia coli DH5alpha. Normalized cDNA libraries were prepared and sequenced on a Roche 454 GS FLX resulting in 1.17 million reads with an average read length of 458bp. Sequencing reads were assembled into two sets of >4500 contigs. The E. coli-specific contigs were filtered out. The remaining contigs were considered as H. meleagridis-specific and were retained for further analysis. Both sets of contigs were merged using CAP3 followed by CD-HIT EST giving rise to 3367 unique contigs, which constituted the H. meleagridis reference transcriptome database. Unique contigs from either virulent or attenuated strains were identified. In order to differentiate strain-specific contigs from the contigs present in both strains, contigs from the virulent and the attenuated strain have been assigned an extension of '_LP' and '_HP', respectively. All the contigs were searched for homologous genes to assign gene ontology (GO) terms to each, using the mapping and annotation functionality of the Blast2GO software suit. Contigs were also subjected to InterPro scans through the Blast2GO interface and any new GO term identified was assigned to the contig.