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

Abstract

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.