Christine Maritz-Olivier

Evidence of the role of tick subolesin in gene expression.

BackgroundSubolesin is an evolutionary conserved protein that was discovered recently in Ixodes scapularis as a tick protective antigen and has a role in tick blood digestion, reproduction and development. In other organisms, subolesin orthologs may be involved in the control of developmental processes. Because of the profound effect of subolesin knockdown in ticks and other organisms, we hypothesized that subolesin plays a role in gene expression, and therefore affects multiple cellular processes. The objective of this study was to provide evidence for the role of subolesin in gene expression.ResultsTwo subolesin-interacting proteins were identified and characterized by yeast two-hybrid screen, co-affinity purification and RNA interference (RNAi). The effect of subolesin knockdown on the tick gene expression pattern was characterized by microarray analysis and demonstrated that subolesin RNAi affects the expression of genes involved in multiple cellular pathways. The analysis of subolesin and interacting protein sequences identified regulatory motifs and predicted the presence of conserved protein kinase C (PKC) phosphorylation sites.ConclusionCollectively, these results provide evidence that subolesin plays a role in gene expression in ticks.

Strategies for new and improved vaccines against ticks and tick-borne diseases

Ticks infest a variety of animal species and transmit pathogens causing disease in both humans and animals worldwide. Tick–host–pathogen interactions have evolved through dynamic processes that accommodated the genetic traits of the hosts, pathogens transmitted and the vector tick species that mediate their development and survival. New approaches for tick control are dependent on defining molecular interactions between hosts, ticks and pathogens to allow for discovery of key molecules that could be tested in vaccines or new generation therapeutics for intervention of tick–pathogen cycles. Currently, tick vaccines constitute an effective and environmentally sound approach for the control of ticks and the transmission of the associated tick‐borne diseases. New candidate protective antigens will most likely be identified by focusing on proteins with relevant biological function in the feeding, reproduction, development, immune response, subversion of host immunity of the tick vector and/or molecules vital for pathogen infection and transmission. This review addresses different approaches and strategies used for the discovery of protective antigens, including focusing on relevant tick biological functions and proteins, reverse genetics, vaccinomics and tick protein evolution and interactomics. New and improved tick vaccines will most likely contain multiple antigens to control tick infestations and pathogen infection and transmission.

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

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.

Cattle tick vaccine researchers join forces in CATVAC

A meeting sponsored by the Bill & Melinda Gates Foundation was held at the Avanti Hotel, Mohammedia, Morocco, July 14–15, 2015. The meeting resulted in the formation of the Cattle Tick Vaccine Consortium (CATVAC).

Evolution of the tissue factor pathway inhibitor-like Kunitz domain-containing protein family in Rhipicephalus microplus

One of the principle mechanisms utilised by ticks to obtain a blood meal is the subversion of the hosts haemostatic response. This is achieved through the secretion of saliva containing anti-haemostatic proteins into the feeding lesion. Lineage-specific expansion of predicted secretory protein families have been observed in all previously studied ticks and occurred in response to adaptation to a blood-feeding environment. Of these, the predominant families are common between both hard and soft ticks. One of these families, namely the Kunitz domain-containing protein family, includes proven tissue factor pathway inhibitor-like (TFPI-like) anti-haemostatics such as ixolaris and penthalaris that play a crucial role during tick feeding. Although Kunitz-type proteins have been found in Rhipicephalus microplus, the TFPI-like Kunitz protein family has not yet been studied. We report a comprehensive search for TFPI-like Kunitz domain-containing proteins in R. microplus expressed sequence tag libraries, resulting in the identification of 42 homologues. The homologues were bioinformatically and phylogenetically studied, including the application of an intensive Bayesian Markov Chain Monte Carlo (MCMC) analysis of the individual Kunitz domain nucleotide sequences. We show that the R. microplus TFPI-like Kunitz protein family groups into two main clades that presumably underwent ancient duplication, which indicates that a whole genome duplication event occurred at least 150 million years ago. Evidence for recent and ancient gene and domain duplication events was also found. Furthermore, the divergence times of the various tick lineages estimated in this paper correspond with those presented in previous studies. The elucidation of this large protein familys evolution within R. microplus adds to current knowledge of this economically important tick.

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.

RNAi in Arthropods: Insight into the Machinery and Applications for Understanding the Pathogen-Vector Interface

The availability of genome sequencing data in combination with knowledge of expressed genes via transcriptome and proteome data has greatly advanced our understanding of arthropod vectors of disease. Not only have we gained insight into vector biology, but also into their respective vector-pathogen interactions. By combining the strengths of postgenomic databases and reverse genetic approaches such as RNAi, the numbers of available drug and vaccine targets, as well as number of transgenes for subsequent transgenic or paratransgenic approaches, have expanded. These are now paving the way for in-field control strategies of vectors and their pathogens. Basic scientific questions, such as understanding the basic components of the vector RNAi machinery, is vital, as this allows for the transfer of basic RNAi machinery components into RNAi-deficient vectors, thereby expanding the genetic toolbox of these RNAi-deficient vectors and pathogens. In this review, we focus on the current knowledge of arthropod vector RNAi machinery and the impact of RNAi on understanding vector biology and vector-pathogen interactions for which vector genomic data is available on VectorBase.

Transmembrane proteins - mining the cattle tick transcriptome

Managing the spread and load of pathogen-transmitting ticks is an important task worldwide. The cattle tick, Rhipicephalus microplus, not only impacts the economy through losses in dairy and meat production, but also raises concerns for human health in regards to the potential of certain transmitted pathogens becoming zoonotic. However, novel strategies to control R. microplus are hindered by lack of understanding tick biology and the discovery of suitable vaccine or acaricide targets. The importance of transmembrane proteins as vaccine targets are well known, as is the case in tick vaccines with Bm86 as antigen. In this study, we describe the localization and functional annotation of 878 putative transmembrane proteins. Thirty proteins could be confirmed in the R. microplus gut using LC-MS/MS analysis and their roles in tick biology are discussed. To the best of our knowledge, 19 targets have not been reported before in any proteomics study in various tick species and the possibility of using the identified proteins as targets for tick control are discussed. Although tissue expression of identified putative proteins through expansive proteomics is necessary, this study demonstrates the possibility of using bioinformatics for the identification of targets for further evaluation in tick control strategies.

SNP Analysis Infers that Recombination Is Involved in the Evolution of Amitraz Resistance in Rhipicephalus microplus

Rhipicephalus microplus, better known as the Asiatic cattle tick, is a largely invasive ectoparasite of great economic importance due to the negative effect it has on agricultural livestock on a global scale, particularly cattle. Tick-borne diseases (babesiosis and anaplasmosis) transmitted by R. microplus are alarming as they decrease the quality of livestock health and production. In sub-Saharan Africa, cattle represent a major source of meat and milk, but this region of the world is severely affected by the Rhipicephalus microplus tick. The principal method for tick control is the use of chemical acaricides, notably amitraz, which was implemented in the 1990’s after resistance to other acaricides surfaced. However, the efficiency of chemical control is hindered by an increase in the frequency of mutant resistance alleles to amitraz in tick populations. Presently, the only way to assess amitraz resistance is by means of larval packet tests, but this technique is time-consuming and not particularly cost effective. The main aims of this study were three-fold. First, we attempted to correlate two known SNPs in the octopamine/tyramine (OCT/Tyr) receptor with amitraz resistance in South African field samples of R. microplus. Second, we calculated gametic disequilibrium for these SNPs to determine whether they are randomly associated. Lastly, we conducted a study to assess the evolutionary effects of recombination within the OCT/Tyr receptor. Our results confirmed that the two SNPs are associated with amitraz resistance in the South African tick strain, and that they are in gametic disequilibrium. Additionally, recombination was detected in the OCT/Tyr receptor generating two recombinant haplotypes. These results are of concern to farmers in sub-Saharan Africa, and the emergence of amitraz resistance should be closely monitored in future. Therefore, we present a quick and affordable RFLP based diagnostic technique to assess amitraz resistance in field samples of R. microplus.

Genetic diversity, acaricide resistance status and evolutionary potential of a Rhipicephalus microplus population from a disease-controlled cattle farming area in South Africa

The Southern cattle tick, Rhipicephalus microplus is a hematophagous ectoparasite of great veterinary and economic importance. Along with its adaptability, reproductive success and vectoring capacity, R. microplus has been reported to develop resistance to the major chemical classes of acaricides currently in use. In South Africa, the Mnisi community in the Mpumalanga region offers a unique opportunity to study the adaptive potential of R. microplus. The aims of this study therefore included characterising acaricide resistance and determining the level and pattern of genetic diversity for R. microplus in this region from one primary population consisting of 12 communal dip-stations. The level of acaricide resistance was evaluated using single nucleotide polymorphisms (SNPs) in genes that contribute to acaricide insensitivity. Additionally, the ribosomal internal transcribed spacer 2 (ITS2) gene fragments of collected individuals were sequenced and a haplotype network was constructed. A high prevalence of alleles attributed to resistance against formamidines (amitraz) in the octopamine/tyramine (OCT/Tyr) receptor (frequency of 0.55) and pyrethroids in the carboxylesterase (frequency of 0.81) genes were observed. Overall, the sampled tick population was homozygous resistant to pyrethroid-based acaricides in the voltage-gated sodium channel (VGS) gene. A total of 11 haplotypes were identified in the Mnisi R. microplus population from ITS2 analysis with no clear population structure. From these allele frequencies it appears that formamidine resistance in the Mnisi community is on the rise, as the R. microplus populations is acquiring or generating these resistance alleles. Apart from rearing multi-resistant ticks to commonly used acaricides in this community these ticks may pose future problems to its surrounding areas.