Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is on the increase, resulting in renewed attentiveness to this potentially life threatening emerging zoonotic disease. The molecular mechanisms underlying the pathophysiology and intra-erythrocytic development of these parasites are poorly understood. This impedes concerted efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell biological and molecular functional genomics tools, we describe the intra-erythrocytic development cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic Babesia spp. This is further correlated for the first time to nuclear content increases during intra-erythrocytic development progression, providing insight into the part of the life cycle that occurs during human infection. High-content temporal evaluation elucidated the contribution of the different stages to life cycle progression. Moreover, molecular descriptors indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation. Additionally, differential expression is observed as the parasite equilibrates its developmental stages during its life cycle. Together, this information provides the first temporal evaluation of the functional transcriptome of B. divergens parasites, information that could be useful in identifying biological processes essential to parasite survival for future anti-babesiacidal discoveries.

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 (EST) 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. Utilizing a custom oligonucleotide microarray designed from available singletons (BmiGI Version 2.1) and EST 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.

In the wake of the ‘omics’ explosion of data, reverse vaccinology approaches are being applied more readily as an alternative for the discovery of candidates for next generation diagnostics and vaccines. Promising protective antigens for the control of ticks and tick-borne diseases can be discovered by mining available omics data for immunogenic epitopes. The present study aims to explore the previously obtained Rhipicephalus bursa sialotranscriptome during both feeding and Babesia infection, to select antigenic targets that are either membrane-associated or a secreted protein, as well as unique to the ectoparasite and not present in the mammalian host. Further, they should be capable of stimulating T and B cells for a potential robust immune response, and be non-allergenic or toxic to the host. From the R. bursa transcriptome, 5706 and 3025 proteins were identified as belonging to the surfaceome and secretome, respectively. Following a reverse genetics immunoinformatics pipeline, nine preferred candidates, consisting of one transmembrane-related and eight secreted proteins, were identified. These candidates showed a higher predicted antigenicity than the Bm86 antigen, with no homology to mammalian hosts and exposed regions. Only four were functionally annotated and selected for further in silico analysis, which examined their protein structure, surface accessibility, flexibility, hydrophobicity, and putative linear B and T-cell epitopes. Regions with overlapping coincident epitopes groups (CEGs) were evaluated to select peptides that were further analyzed for their physicochemical characteristics, potential allergenicity, toxicity, solubility, and potential propensity for crystallization. Following these procedures, a set of three peptides from the three R. bursa proteins were selected. In silico results indicate that the designed epitopes could stimulate a protective and long-lasting immune response against those tick proteins, reflecting its potential as anti-tick vaccines. The immunogenicity of these peptides was evaluated in a pilot immunization study followed by tick feeding to evaluate its impact on tick behavior and pathogen transmission. Combining in silico methods with in vivo immunogenicity evaluation enabled the screening of vaccine candidates prior to expensive infestation studies on the definitive ovine host animals.

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 2,476 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 1,084 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.2

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