Analysis of gene expression of Babesia gibsoni cultured with diminazene aceturate using RNA sequencing

A comprehensive and quantitative method to compare gene expression may be useful for investigating the mechanisms responsible for diminazene aceturate (DA) resistance in Babesia gibsoni. Therefore, the gene expression of B. gibsoni cultured with DA was compared with those without DA using RNA sequencing (RNA-seq). Total RNA extracted from the parasites cultured with or without DA was examined using two next-generation sequencers, the 454 GS Junior and MiniSeq systems. We aimed to detect the genes differentially expressed between parasites cultured with and without DA by mapping the reads against de novo assembled contigs. The contigs, the amounts of which were more than five-fold higher in the parasite with DA than that without DA, were searched using BLAST^®, and two contigs were found as parasite genes. Real-time quantitative reverse transcription-PCR (qRT-PCR) indicated that the expression levels of both genes were significantly higher in the parasites cultured with DA than those without DA. The nucleotide sequences of two contigs established using RNA-seq were similar to those found using direct sequencing, although the 5'- and 3'-end of those sequences were different between the two sequencing methods. In conclusion, we successfully utilized RNA-seq analysis to compare gene expression between parasites cultured with and without DA. RNA-seq can be used for comprehensive and quantitative analyses of gene expression in Babesia parasites.