Abstract
BackgroundFor accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus) microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility.ResultsThe transcription levels of nine potential reference genes: β-actin (ACTB), β-tubulin (BTUB), elongation factor 1α (ELF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione S-transferase (GST), H3 histone family 3A (H3F3A), cyclophilin (PPIA), ribosomal protein L4 (RPL4) and TATA box binding protein (TBP) were measured in all life stages of R. microplus and R. appendiculatus. ELF1A was found to be the most stable expressed gene in both species following analysis by both geNorm and Normfinder software applications, GST showed the least stability. The expression profile of Bm86 in R. appendiculatus and R. microplus revealed a more continuous Bm86 antigen abundance in R. microplus throughout its one-host life cycle compared to the three-host tick R. appendiculatus where large variations were observed between different life stages.ConclusionBased on these results, ELF1A can be proposed as an initial reference gene for normalization of quantitative RT-PCR data in whole R. microplus and R. appendiculatus ticks. The observed differences in Bm86 expression profile between the two species alone can not adequately explain the lack of a Bm86 vaccination effect in R. appendiculatus.
Highlights
For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential
Quantitative room temperature (RT)-PCR The efficiencies of the quantitative RT-PCR's were uniformly high and ranged from 91% to 103%, making all assays suitable for quantitative analysis (Table 1)
Nine candidate reference genes from different functional classes were identified in the expressed sequence tag (EST) databases of R. microplus and R. appendiculatus and their expression stability throughout the life cycle of these two tick species was evaluated
Summary
For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. Besides causing direct production losses and leather damage due to their blood-feeding habit, both ticks are able to transmit a wide variety of pathogens Both tick species overlap in their distribution, but R. microplus is more widespread and occurs in subtropical and tropical areas of the world whereas the distribution of R. appendiculatus, known as the brown ear tick, is limited to areas with a humid climate from southern Sudan to the southeastern coast of South Africa. Control of ticks worldwide relies principally on the use of acaricides, but two vaccines targeting R. microplus were commercialized in the 1990s: TickGARD Plus® in Australia and Gavac® in Cuba Both are based on the same recombinant antigen named Bm86, a glycoprotein of unknown function which is located predominantly on the surface of midgut digest cells [2]. Bm86-based vaccines showed cross-protection against various other tick species, e.g. Rhipicephalus (Boophilus) annulatus [3], Rhipicephalus (Boophilus) decoloratus, Hyalomma anatolicum and Hyalomma dromedarii [4], they were not effective against Amblyomma variegatum and R. appendiculatus [4,5]
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