Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86

<p>Abstract</p> <p>Background</p> <p>For 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 <it>Rhipicephalus appendiculatus </it>and <it>Rhipicephalus (Boophilus) microplus </it>ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against <it>R. microplus </it>were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against <it>R. appendiculatus</it>. 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 <it>R. microplus </it>and <it>R. appendiculatus</it>. 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.</p> <p>Results</p> <p>The 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 <it>R. microplus </it>and <it>R. appendiculatus</it>. 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 <it>R. appendiculatus </it>and <it>R. microplus </it>revealed a more continuous Bm86 antigen abundance in <it>R. microplus </it>throughout its one-host life cycle compared to the three-host tick <it>R. appendiculatus </it>where large variations were observed between different life stages.</p> <p>Conclusion</p> <p>Based on these results, ELF1A can be proposed as an initial reference gene for normalization of quantitative RT-PCR data in whole <it>R. microplus </it>and <it>R. appendiculatus </it>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 <it>R. appendiculatus</it>.</p

De novo assembly and annotation of the Amblyomma hebraeum tick midgut transcriptome response to Ehrlichia ruminantium infection

The South African bont tick Amblyomma hebraeum is a hematophagous vector for the heartwater disease pathogen Ehrlichia ruminantium in southern Africa. During feeding, the tick’s enterocytes express proteins that perform vital functions in blood digestion, including proteins that may be involved in E. ruminantium acquisition, colonization or immunity. To delineate the molecular mechanism of midgut response to E. ruminantium infection, we performed comparative analyses of midgut transcriptomes of E. ruminantium infected engorged A. hebraeum nymphs, and infected adult male and female ticks with their corresponding matched uninfected controls, before and during feeding. A total of 102,036 unigenes were annotated in public databases and their expression levels analyzed for engorged nymphs as well as unfed and partly-fed adult ticks. There were 2,025 differentially expressed genes (DEGs) in midguts, of which 1,225 unigenes were up-regulated and 800 unigenes were down-regulated in the midguts of infected ticks. Annotation of DEGs revealed an increase in metabolic and cellular processes among E. ruminantium infected ticks. Notably, among the infected ticks, there was up-regulation in the expression of genes involved in tick immunity, histone proteins and oxidative stress responses. We also observed up-regulation of glycoproteins that E. ruminantium could potentially use as docking sites for host cell entry. Insights uncovered in this study offer a platform for further investigations into the molecular interaction between E. ruminantium and A. hebraeum

Experimental transmission of Anaplasma marginale by male Dermacentor reticulatus

[Background] Bovine anaplasmosis has been reported in several European countries, but the vector competency of tick species for Anaplasma marginale from these localities has not been determined. Because of the wide distributional range of Dermacentor reticulatus within Europe and the major role of Dermacentor spp. as a vector of A. marginale in the United States, we tested the vector competency of D. reticulatus for A. marginale.[Results] Male D. reticulatus were allowed to feed for 7 days on a calf persistently infected with a Zaria isolate of A. marginale, after which they were removed and held off-host for 7 days. The ticks were then allowed to feed a second time for 7 days on a susceptible tick-naïve calf. Infection of calf No. 4291 was detected 20 days post exposure (p.i.) and confirmed by msp4 PCR. Thirty percent of the dissected acquisition fed ticks was infected. In addition, A. marginale colonies were detected by light microscopy in the salivary glands of the acquisition fed ticks. Transmission of A. marginale to calf No. 9191 was confirmed by examination of Giemsa-stained blood smears and msp4 PCR. Ticks were dissected after transmission feeding and presence of A. marginale was confirmed in 18.5% of the dissected ticks.[Conclusion] This study demonstrates that D. reticulatus males are competent vectors of A. marginale. Further studies are needed to confirm the vector competency of D. reticulatus for other A. marginale strains from geographic areas in Europe.This research was supported by grants from the European Community, INCO-DEV program (project No. 003713), entitled 'Epidemiology and new generation vaccines for Ehrlichia and Anaplasma infections of ruminants', the Junta de Comunidades de Castilla-La Mancha, Spain (project 06036-00 ICSJCCM), entitled "Epidemiología de zoonosis transmitidas por garrapatas en Castilla–La Mancha" and was facilitated through the Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3), financed by the International Cooperation Program of the European Union, coordination action project No. 510561.Peer reviewe