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

Achieving equity through 'gender autonomy': the challenges for VET policy and practice

This paper is based on research carried out in an EU Fifth Framework project on 'Gender and Qualification'. The research partners from five European countries investigated the impact of gender segregation in European labour markets on vocational education and training, with particular regard to competences and qualifications. The research explored the part played by gender in the vocational education and training experiences of (i) young adults entering specific occupations in child care, electrical engineering and food preparation/service (ii) adults changing occupations

Unique Mitochondrial Single Nucleotide Polymorphisms Demonstrate Resolution Potential to Discriminate Theileria parva Vaccine and Buffalo-Derived Strains

Distinct pathogenic and epidemiological features underlie different Theileria parva strains resulting in different clinical manifestations of East Coast Fever and Corridor Disease in susceptible cattle. Unclear delineation of these strains limits the control of these diseases in endemic areas. Hence, an accurate characterization of strains can improve the treatment and prevention approaches as well as investigate their origin. Here, we describe a set of single nucleotide polymorphisms (SNPs) based on 13 near-complete mitogenomes of T. parva strains originating from East and Southern Africa, including the live vaccine stock strains. We identified 11 SNPs that are non-preferentially distributed within the coding and non-coding regions, all of which are synonymous except for two within the cytochrome b gene of buffalo-derived strains. Our analysis ascertains haplotype-specific mutations that segregate the different vaccine and the buffalo-derived strains except T. parva-Muguga and Serengeti-transformed strains suggesting a shared lineage between the latter two vaccine strains. Phylogenetic analyses including the mitogenomes of other Theileria species: T. annulata, T. taurotragi, and T. lestoquardi, with the latter two sequenced in this study for the first time, were congruent with nuclear-encoded genes. Importantly, we describe seven T. parva haplotypes characterized by synonymous SNPs and parsimony-informative characters with the other three transforming species mitogenomes. We anticipate that tracking T. parva mitochondrial haplotypes from this study will provide insight into the parasite’s epidemiological dynamics and underpin current control efforts

Evaluating Transmission Paths for Three Different Bartonella spp. in Ixodes ricinus Ticks Using Artificial Feeding

Bartonellae are facultative intracellular alpha-proteobacteria often transmitted by arthropods. Ixodes ricinus is the most important vector for arthropod-borne pathogens in Europe. However, its vector competence for Bartonella spp. is still unclear. This study aimed to experimentally compare its vector competence for three Bartonella species: B. henselae, B. grahamii, and B. schoenbuchensis. A total of 1333 ticks (1021 nymphs and 312 adults) were separated into four groups, one for each pathogen and a negative control group. Ticks were fed artificially with bovine blood spiked with the respective Bartonella species. DNA was extracted from selected ticks to verify Bartonella-infection by PCR. DNA of Bartonella spp. was detected in 34% of nymphs and females after feeding. The best engorgement results were obtained by ticks fed with B. henselae-spiked blood (65.3%) and B. schoenbuchensis (61.6%). Significantly more nymphs fed on infected blood (37.3%) molted into adults compared to the control group (11.4%). Bartonella DNA was found in 22% of eggs laid by previously infected females and in 8.6% of adults molted from infected nymphs. The transovarial and transstadial transmission of bartonellae suggest that I. ricinus could be a potential vector for three bacteria

On the boundary of the dispersion-managed soliton existence

A breathing soliton-like structure in dispersion-managed (DM) optical fiber system is studied. It is proven that for negative average dispersion the breathing soliton is forbidden provided that a modulus of average dispersion exceed a threshold which depends on the soliton amplitude.Comment: LaTeX, 8 pages, to appear in JETP Lett. 72, #3 (2000

The Sasa-Satsuma higher order nonlinear Schrodinger equation and its bilinearization and multi-soliton solutions

Higher order and multicomponent generalizations of the nonlinear Schrodinger equation are important in various applications, e.g., in optics. One of these equations, the integrable Sasa-Satsuma equation, has particularly interesting soliton solutions. Unfortunately the construction of multi-soliton solutions to this equation presents difficulties due to its complicated bilinearization. We discuss briefly some previous attempts and then give the correct bilinearization based on the interpretation of the Sasa-Satsuma equation as a reduction of the three-component Kadomtsev-Petvishvili hierarchy. In the process we also get bilinearizations and multi-soliton formulae for a two component generalization of the Sasa-Satsuma equation (the Yajima-Oikawa-Tasgal-Potasek model), and for a (2+1)-dimensional generalization.Comment: 13 pages in RevTex, added reference

Leading Order Temporal Asymptotics of the Modified Non-Linear Schrodinger Equation: Solitonless Sector

Using the matrix Riemann-Hilbert factorisation approach for non-linear evolution equations (NLEEs) integrable in the sense of the inverse scattering method, we obtain, in the solitonless sector, the leading-order asymptotics as $t$ tends to plus and minus infinity of the solution to the Cauchy initial-value problem for the modified non-linear Schrodinger equation: also obtained are analogous results for two gauge-equivalent NLEEs; in particular, the derivative non-linear Schrodinger equation.Comment: 29 pages, 5 figures, LaTeX, revised version of the original submission, to be published in Inverse Problem

Potential role of ticks as vectors of bluetongue virus

When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not to survive the northern European winter, and transovarial transmission in Culicoides is not recorded, we examined the potential vector role of ixodid and argasid ticks for bluetongue virus. Four species of ixodid ticks (Ixodes ricinus, Ixodes hexagonus, Dermacentor reticulatus and Rhipicephalus bursa) and one soft tick species, Ornithodoros savignyi, ingested BTV8-containing blood either through capillary feeding or by feeding on artificial membranes. The virus was taken up by the ticks and was found to pass through the gut barrier and spread via the haemolymph into the salivary glands, ovaries and testes, as demonstrated by real-time reverse transcriptase PCR (PCR-test). BTV8 was detected in various tissues of ixodid ticks for up to 21 days post feeding and in Ornithodoros ticks for up to 26 days. It was found after moulting in adult Ixodes hexagonus and was also able to pass through the ovaries into the eggs of an Ornithodoros savignyi tick. This study demonstrates that ticks can become infected with bluetongue virus serotype 8. The transstadial passage in hard ticks and transovarial passage in soft ticks suggest that ticks have potential vectorial capacity for bluetongue virus. Further studies are required to investigate transmission from infected ticks to domestic livestock. This route of transmission could provide an additional clue in the unresolved mystery of the epidemiology of Bluetongue in Europe by considering ticks as a potential overwintering mechanism for bluetongue virus