A systematic, functional genomics, and reverse vaccinology approach to the identification of vaccine candidates in the cattle tick, Rhipicephalus

In the post-genomic era, reverse vaccinology is proving promising in the development of vaccines against bacterial and viral diseases, with limited application in ectoparasite vaccine design. In this study, we present a systematic approach using a combination of functional genomics (DNA microarrays) techniques and a pipeline incorporating in silico prediction of subcellular localization and protective antigenicity using VaxiJen for the identification of novel anti-tick vaccine candidates. A total of 791 candidates were identified using this approach, of which 176 are membrane-associated and 86 secreted soluble proteins. A preliminary analysis on the antigenicity of selected membrane proteins using anti-gut antisera yielded candidates with an IgG binding capacity greater than previously identified epitopes of Bm86. Subsequent vaccination trials using recombinant proteins will not only validate this approach, but will also improve subsequent reverse vaccinology approaches for the identification of novel anti-tick vaccine candidates.The Red Meat Research Development Trust, University of Pretoria Research Development Programme, and the Technology and Human Resources for Industry Programme.http://www.elsevier.de/ttbdishb201

Comparative microarray analyses of adult female midgut tissues from feeding Rhipicephalus species

The cattle tick, Rhipicephalus microplus, has a debilitating effect on the livestock industry worldwide, owing to its being a vector of the causative agents of bovine babesiosis and anaplasmosis. In South Africa, co-infestation with R. microplus and R. decoloratus, a common vector species on local livestock, occurs widely in the northern and eastern parts of the country. An alternative to chemical control methods is sought in the form of a tick vaccine to control these tick species. However, sequence information and transcriptional data for R. decoloratus is currently lacking. Therefore, this study aimed at identifying genes that are shared between midgut tissues of feeding adult female R. microplus and R. decoloratus ticks. In this regard, a custom oligonucleotide microarray comprising of 13,477 R. microplus sequences was used for transcriptional profiling and 2476 genes were found to be shared between these Rhipicephalus species. In addition, 136 transcripts were found to be more abundantly expressed in R. decoloratus and 1084 in R. microplus. Chi-square analysis revealed that genes involved in lipid transport and metabolism are significantly overrepresented in R. microplus and R. decoloratus. This study is the first transcriptional profiling of R. decoloratus and is an additional resource that can be evaluated further in future studies for possible tick control.The Red Meat Research Development Trust Gauteng Department of Agriculture and Rural Development,University of Pretoria Research Development Program, and the National Research Foundation’s Technology and Human Resourcesfor Industry Program (Grant No. TP2010072300035).http://www.elsevier.com/locate/ttbdishb201

Gene expression profiling of adult female tissues in feeding Rhipicephalus microplus cattle ticks

The southern cattle tick, Rhipicephalus microplus, is an economically important pest, especially for resource-poor countries, both as a highly adaptive invasive species and prominent vector of disease. The increasing prevalence of resistance to chemical acaricides and variable efficacy of current tick vaccine candidates highlight the need for more effective control methods. In the absence of a fully annotated genome, the wealth of available expressed sequence tag sequence data for this species presents a unique opportunity to study the genes that are expressed in tissues involved in blood meal acquisition, digestion and reproduction during feeding. Utilising a custom oligonucleotide microarray designed from available singletons (BmiGI Version 2.1) and expressed sequence tag sequences of R. microplus, the expression profiles in feeding adult female midgut, salivary glands and ovarian tissues were compared. From 13,456 assembled transcripts, 588 genes expressed in all three tissues were identified from fed adult females 20 days post infestation. The greatest complement of genes relate to translation and protein turnover. Additionally, a number of unique transcripts were identified for each tissue that relate well to their respective physiological/biological function/role(s). These transcripts include secreted anti-hemostatics and defense proteins from the salivary glands for acquisition of a blood meal, proteases as well as enzymes and transporters for digestion and nutrient acquisition from ingested blood in the midgut, and finally proteins and associated factors involved in DNA replication and cell-cycle control for oogenesis in the ovaries. Comparative analyses of adult female tissues during feeding enabled the identification of a catalogue of transcripts that may be essential for successful feeding and reproduction in the cattle tick, R. microplus. Future studies will increase our understanding of basic tick biology, allowing the identification of shared proteins/pathways among different tissues that may offer novel targets for the development of new tick control strategies.The Red Meat Research Development Trust, University of Pretoria Research Development Programme and the Technology and Human Resources for Industry Programme, South Africa. Additional funding and student support was obtained from the National Research Foundation of South Africa.http://www.elsevier.com/locate/ijpar

A Combined functional genomics and in silico approach for the identification of anti-Rhipicephalus vaccine candidates

The cattle tick, Rhipicephalus microplus, has a debilitating effect on the livestock industry worldwide, owing to its being a vector of the causative agents of bovine babesiosis and anaplasmosis. In South Africa, co-infestation of livestock with R. microplus and R. decoloratus occurs. An alternative to chemical control methods is sought in the form of an anti-tick vaccine. Using microarray technology, this study aimed at identifying genes that are shared between midgut tissues of adult female R. microplus and R. decoloratus ticks. In addition, results from another study were used and a reverse vaccinology pipeline was devised to identify putative novel vaccine candidates. Using a custom oligonucleotide microarray comprising 13 477 R. microplus sequences, 2476 genes were found to be shared between the two abovementioned tick species. In addition, 136 were found to be more abundantly expressed in R. decoloratus and 1084 in R. microplus. Chi-square analysis revealed that genes involved in lipid transport and metabolism are significantly over-represented in R. microplus and R. decoloratus. With vaccine design in mind, considering genes that are expressed in the midgut of both tick species, 6730 genes were identified and of these, 1224 are predicted to contain membrane-spanning helices. One major limitation to anti-tick vaccine discovery in the past has been a lack of candidates to evaluate, combined with limited knowledge of the transcriptome of R. microplus. This study identified a large pool of transcripts that are expressed in the midgut of both R. microplus and R. decoloratus adult females. Of these, those that are expressed in larvae, nymphs and the midgut were identified in another study and an in silico pipeline was used to predict membrane-bound protective antigens using an alignment-free approach, which led to the identification of seven proteins that were predicted to be both glycosylphosphatidylinositol (GPI)-anchored and more likely than Bm86 to be protective antigens. Finally, epitopes were predicted and corresponding synthetic peptides were evaluated using enzyme-linked immunosorbent assay (ELISA), resulting in the identification of three epitopes that are recognized to a greater extent than previously published Bm86 epitopes, when using murine serum raised against membrane proteins from the midgut of R. microplus. These results are significant because novel R. microplus proteins that are also present in R. decoloratus were identified. Trials using recombinant protein are under way and this will ultimately validate the experimental methodology discussed in this dissertation. Finally, regardless of whether the next-generation anti-tick vaccine has been discovered, this study also led to the identification of novel reference genes that can be used for real-time PCR experiments.Dissertation (MSc)--University of Pretoria, 2013.gm2014BiochemistryUnrestricte