Manuel Rodriguez Valle

Surface display libraries (SDL) have predominantly been utilized for the screening of peptides, and single-chain variable IgG fragments, however, the use of SDL for the expression and purification of proteins is gaining interest. Prokaryote SDL express proteins within the periplasm, limiting the application of common screening techniques, such as ELISA and FACS, to assess the viability of recombinant toxin before purification. A previous attempt to express a functional holocyclotoxin-1 (HT1) from the Australian paralysis tick (Ixodes holocyclus) using a prokaryotic system was unsuccessful. In this study, the coding sequence (CDS) of HT1 was cloned into the pYD1 plasmid and transformed by electroporation into IMTV014 and EBY100 yeast cell lines. Post induction, recombinant HT1 was identified on the cell surface of IMTV014/ht1 and EBY100/ht1 transformants by FACS, Western blot, and ELISA utilizing dog anti-paralysis tick IgG. The recombinant HT1 was purified, and functionality confirmed by an in vitro synaptosome-binding assay. This research reports for the first time the extracellular expression and display of a functional HT1 on the surface of the S. cerevisiae. It also provides evidence that yeast display libraries provide a viable technology to produce recombinant toxins, and their screening using high throughput methodologies such as FACS.

36 The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that induce host 37 paralysis. Salivary glands and viscera were dissected from fully engorged female I. holocyclus ticks col-38 lected from dogs and cats with paralysis symptoms. cDNA from both tissue samples were sequenced 39 using Illumina HiSeq 100 bp pair end read technologies. Unique and non-redundant holocyclotoxin 40 sequences were designated as HT2–HT19, as none were identical to the previously described HT1. 41 Specific binding to rat synaptosomes was determined for synthetic HTs, and their neurotoxic capacity 42 was determined by neonatal mouse assay. They induced a powerful paralysis in neonatal mice, particu-43 larly HT4 which produced rapid and strong respiratory distress in all animals tested. This is the first 44 known genomic database developed for the Australian paralysis tick. The database contributed to the 45 identification and subsequent characterization of the holocyclotoxin family that will inform the develop-46 ment of novel anti-paralysis control methods.

Background: Rhipicephalus (Boophilus) microplus is an obligate blood feeder which is host specific to cattle. Existing knowledge pertaining to the host or host breed effects on tick transcript expression profiles during the tick-host interaction is poor. Results: Global analysis of gene expression changes in whole R. microplus ticks during larval, pre-attachment and early adult stages feeding on Bos indicus and Bos taurus cattle were compared using gene expression microarray analysis. Among the 13,601 R. microplus transcripts from BmiGI Version 2 we identified 297 high and 17 low expressed transcripts that were significantly differentially expressed between R. microplus feeding on tick resistant cattle [Bos indicus (Brahman)] compared to R. microplus feeding on tick susceptible cattle [Bos taurus (Holstein-Friesian)] (p ≤ 0.001). These include genes encoding enzymes involved in primary metabolism, and genes related to stress, defence, cell wall modification, cellular signaling, receptor, and cuticle formation. Microarrays were validated by qRT-PCR analysis of selected transcripts using three housekeeping genes as normalization controls. Conclusion: The analysis of all tick stages under survey suggested a coordinated regulation of defence proteins, proteases and protease inhibitors to achieve successful attachment and survival of R. microplus on different host breeds, particularly Bos indicus cattle. R. microplus ticks demonstrate different transcript expression patterns when they encounter tick resistant and susceptible breeds of cattle. In this study we provide the first transcriptome evidence demonstrating the influence of tick resistant and susceptible cattle breeds on transcript expression patterns and the molecular physiology of ticks during host attachment and feeding. The microarray data used in this analysis have been submitted to NCBI GEO database under accession number GSE20605 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20605.

Rhipicephalus microplus-the cattle tick-is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis. Video Link The video component of this article can be found at https://www.jove.com/video/55747/

The Rhipicephalus microplus genome is large and complex in structure, making it difficult to assemble a genome sequence and costly to resource the required bioinformatics. In light of this, a consortium of international collaborators was formed to pool resources to begin sequencing this genome. We have acquired and assembled genomic DNA into contigs that represent over 1.8 Gigabase pairs of DNA from gene-enriched regions of the R. microplus genome. We also have several datasets containing transcript sequences from a number of gene expression experiments conducted by the consortium. A web-based resource was developed to enable the scientific community to access our datasets and conduct analysis through a web-based bioinformatics environment called YABI. The collective bioinformatics resource is termed CattleTickBase. Our consortium has acquired genomic and transcriptomic sequence data at approximately 0.9X coverage of the gene-coding regions of the R. microplus genome. The YABI tool will facilitate access and manipulation of cattle tick genome sequence data as the genome sequencing of R. microplus proceeds. During this process the CattleTickBase resource will continue to be updated.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of $50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments ($100–200 bp) and the usefulness of microarrays to study knock-down phenotypes.

Ticks are important vectors of pathogens and secreted neurotoxins with approximately 69 out of 692 tick species having the ability to induce severe toxicoses in their hosts. The Australian paralysis tick (Ixodes holocyclus) is known to be one of the most virulent tick species producing a flaccid paralysis and fatalities caused by a family of neurotoxins known as holocyclotoxins (HTs). The paralysis mechanism of these toxins is temperature dependent and is thought to involve inhibition of acetylcholine levels at the neuromuscular junction. However, the target and mechanism of this inhibition remain uncharacterised. Here, we report that three members of the holocyclotoxin family; HT-1 (GenBank AY766147), HT-3 (GenBank KP096303) and HT-12 (GenBank KP963967) induce muscle paralysis by inhibiting the dependence of transmitter release on extracellular calcium. Previous study was conducted using extracts from tick salivary glands, while the present study is the first to use pure toxins from I. holocyclus. Our findings provide greater insight into the mechanisms by which these toxins act to induce paralysis. Ticks (order Acarina) are arthropods members of the class Arachnida, which includes spiders (order Araneae) and scorpions (order Scorpionida). These ectoparasites need to feed on the host's blood to complete their life cycle and secrete a complex mixture of proteins in the saliva to counter host defenses 1–5. A significant number of ticks, in particular from the genus Ixodes cause paralysis and toxicoses to the host 1,6,7. Neurotoxins secreted in the saliva of the Australia paralysis tick (Ixodes holocyclus) caused an ascending flaccid paralysis and respiratory failure that can be fatal particularly in cats and dogs. The clinical symptoms of the paralysis appear in the host between three and four days after the attachment of an adult tick 8–10. Other ticks that cause paralysis with major implication on human and animal health are Dermacentor andersoni and Dermacentor variabilis in North America, and Ixodes rubicundus and Rhipicephalus evertis in Africa 8. Previous study conducted by Cooper and co-worker indicated that I. holocyclus (Iho) toxins inhibit the release of acetylcholine from neuromuscular junction to cause paralysis 11. However, the target and mechanism of inhibition by these holocyclotoxins have not been determined. All former studies related with Iho toxins had been conducted using crude or partially purified salivary gland extract obtained from I. holocyclus 11. The present study investigated the effects of the synthetic HT1, HT3 and HT12 on the skeletal neuromuscular junction using electrophysiological recordings. The current study reports that three members of the holocyclotoxin family; HT-1 (GenBank AY766147), HT-3 (GenBank KP096303) and HT-12 (GenBank KP963967) induce muscle paralysis by inhibiting the dependence of transmitter release on extracel-lular calcium. Results In this study, both native and synthesised holocyclotoxins were tested by electrophysiological analysis of neu-rotransmission using the mouse neuromuscular junction. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded before, during and after HT exposure on the neuromuscular junctions. No change in the rise and decay times was noted for MEPPs at all concentrations tested for each HT peptide (Fig. 1A). MEPP frequency and amplitude were also unaffected, suggesting that the HT peptides were not targeting the calcium independent release of vesicles nor the postsynaptic acetylcholine receptors to induce paralysis. Each peptide reduced evoked EPP amplitudes in a dose dependent manner (Fig. 1B,C). No significance was found between individual HTs at each concentration tested. Incubation of preparations with native HTs resulted in a significant dose dependent decrease in mean EPP amplitude (Fig. 1D). The decrease in EPP amplitudes with maintained amplitude of MEPPs indicates the HTs act via a presynaptic mechanism. The holocyclotoxins likely

Current strategies for the control of the cattle tick Boophilus microplus include the use of chemicals as the principal control method. These methods, however, have met with partially successful results. The recent development of immunological methods for the control of the cattle tick has opened new possibilities for the design of control strategies. Employing the results obtained by us in experiments testing the effect of vaccination with the recombinant vaccine, Gavac TM (Heber Biotec S.A.), on tick populations, we have developed a model to evaluate, through a computer program, the efficacy of the vaccine as a control method. The action of the vaccine on the control of tick populations was simulated and the specific serum antibody titers required to decrease the tick population in the field were calculated. The specific serum antibody titer required to decrease the tick population in the field after the first vaccination scheme was found to be > 57 200 and the antibody titer required to maintain this effect when the vaccine is already acting and after successive revaccinations was found to be > 27 500, Considerations about revaccination schemes and combination between vaccination and acaricide treatments as possible control strategies are discussed.

Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein (SwissProt Accession No. P20736), which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS. The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N-glycans, from Man 5 GlcNAc 2 to Man 9 GlcNAc 2 of which, Man 8-9 GlcNAc 2 were the most abundant. O-Glycopeptides are distributed mostly towards the protein N-terminus. While the first N-glycosylated site (Asn 122) is located between EGF-like domains 2 and 3, where the O-glycopeptides were found, two other N-glycosylated sites (Asn 329 and Asn 363) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr.

The control of tick populations by using conventional
strategies possess several problems, including the appearance
of organophosphate resistant strains, among others.
The possibility of using alternative strategies such as
vaccination with tick antigens has been suggested by
several authors. One particular antigen ( Bm86) has been
described and shown to be able to induce a protective
immunity against the cattle tick Boophilus microplus. In
this paper, we demonstrate by means of immunohistochemical
staining that this antigen is conserved among several
strains of this species. These results correlate with those
showing that animals vaccinated with a preparation of
recombinant Bm86 were protected against challenge with
the four different strains tested, including one resistant to
organophosphates. These results favour the immunization
with recombinant Bm86 for the control of the cattle tick
B. microplus.

Abstract
Background: Rhipicephalus (Boophilus) microplus (Rmi) a major cattle ectoparasite and tick borne disease vector,
impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete
Chelicerate genome, which includes ticks, mites, spiders, scorpions and crustaceans. Model arthropod genomes
such as Drosophila and Anopheles are too taxonomically distant for a reference in tick genomic sequence analysis.
This study focuses on the de-novo assembly of two R. microplus BAC sequences from the understudied R microplus
genome. Based on available R. microplus sequenced resources and comparative analysis, tick genomic structure
and functional predictions identify complex gene structures and genomic targets expressed during tick-cattle
interaction.
Results: In our BAC analyses we have assembled, using the correct positioning of BAC end sequences and
transcript sequences, two challenging genomic regions. Cot DNA fractions compared to the BAC sequences
confirmed a highly repetitive BAC sequence BM-012-E08 and a low repetitive BAC sequence BM-005-G14 which
was gene rich and contained short interspersed elements (SINEs). Based directly on the BAC and Cot data
comparisons, the genome wide frequency of the SINE Ruka element was estimated. Using a conservative approach
to the assembly of the highly repetitive BM-012-E08, the sequence was de-convoluted into three repeat units, each
unit containing an 18S, 5.8S and 28S ribosomal RNA (rRNA) encoding gene sequence (rDNA), related internal
transcribed spacer and complex intergenic region.
In the low repetitive BM-005-G14, a novel gene complex was found between to 2 genes on the same strand.
Nested in the second intron of a large 9 Kb papilin gene was a helicase gene. This helicase overlapped in two
exonic regions with the papilin. Both these genes were shown expressed in different tick life stage important in
ectoparasite interaction with the host. Tick specific sequence differences were also determined for the papilin gene
and the protein binding sites of the 18S subunit in a comparison to Bos taurus.
Conclusion: In the absence of a sequenced reference genome we have assembled two complex BAC sequences,
characterised novel gene structure that was confirmed by gene expression and sequencing analyses. This is the
first report to provide evidence for 2 eukaryotic genes with exon regions that overlap on the same strand, the first
to describe Rhipicephalinae papilin, and the first to report the complete ribosomal DNA repeated unit sequence
structure for ticks. The Cot data estimation of genome wide sequence frequency means this research will underpin
future efforts for genome sequencing and assembly of the R. microplus genome.

Ixodes holocyclus is the paralysis tick commonly found in Australia. I. holocyclus does not cause paralysis in the primary host – bandicoots, but markedly affects secondary hosts such as companion animals, livestock and humans. Holocyclotoxins are the neurotoxin molecules in I. holocyclus responsible for paralysis symptoms. There is a limited understanding of holocyclotoxins due to the difficulties in purifying and expressing these toxins in vitro. Next-generation sequencing technologies were utilised for the first time to generate transcriptome data from two cDNA samples –salivary glands samples collected from female adult ticks engorged on paralysed companion animals and on bandicoots. Contig-encoded proteins in each library were annotated according to their best BLAST match against several databases and functionally assigned into six protein categories: housekeeping, transposable elements, pathogen-related, hypothetical, secreted and novel. The " secreted protein " category is comprised of ten protein families: enzymes, protease inhibitors, antigens, mucins, immunity-related, lipocalins, glycine-rich, putative secreted, salivary and toxin-like. Comparisons of contig representation between the two libraries reveal the differential expression of tick proteins collected from different hosts. This study provides a preliminary description of the I. holocyclus tick salivary gland transcriptome.

The field of reverse vaccinology developed as an outcome of the genome sequence revolution. Following the introduction of live vaccinations in the western world by Edward Jenner in 1798 and the coining of the phrase 'vaccine', in 1881 Pasteur developed a rational design for vaccines. Pasteur proposed that in order to make a vaccine that one should 'isolate, inactivate and inject the microorganism' and these basic rules of vaccinology were largely followed for the next 100 years leading to the elimination of several highly infectious diseases. However, new technologies were needed to conquer many pathogens which could not be eliminated using these traditional technologies. Thus increasingly, computers were used to mine genome sequences to rationally design recombinant vaccines. Several vaccines for bacterial and viral diseases (i.e. meningococcus and HIV) have been developed, however the ongoing challenge for parasite vaccines has been due to their comparatively larger genomes. Understanding the immune response is important in reverse vaccinology studies as this knowledge will influence how the genome mining is to be conducted. Vaccine candidates for anaplasmosis, cowdriosis, theileriosis, leishmaniasis, malaria, schistosomiasis, and the cattle tick have been identified using reverse vaccinology approaches. Some challenges for parasite vaccine development include the ability to address antigenic variability as well the understanding of the complex interplay between antibody, mucosal and/or T cell immune responses. To understand the complex parasite interactions with the livestock host, there is the limitation where algorithms for epitope mining using the human genome cannot directly be adapted for bovine, for example the prediction of peptide binding to major histocompatibility complex motifs. As the number of genomes for both hosts and parasites increase, the development of new algorithms for pan-genomic mining will continue to impact the future of parasite and ricketsial (and other tick borne pathogens) disease vaccine development.

The cattle tick (Rhipicephalus microplus) affects cattle industries in tropical and subtropical countries because it is the vector of babesiosis and anaplasmosis which constitutes a threat to the health of cattle. During blooding feeding, ticks secrete saliva containing a complex of bioactive molecules into the injured site to evade host's defensive responses. Serine protease inhibitors (serpins) are important anti-haemostatic molecules present in tick saliva that are necessary for a successful blood feeding. Several serpin sequences have been reported in R. microplus but there is a gap of information about their functions during host–parasite interactions. In this study, the RmS-15 expressed in the yeast Pichia pastoris was characterised using kinetic assays and in vitro analysis. The inhibitory enzymatic assays conducted showed that RmS-15 is a physiological inhibitor of thrombin with a stoichiometric inhibition (SI) of 1.5 and high inhibition affinity with ka = 9.3 ± 0.5 × 104 M −1 s −1. RmS-15 delayed the clotting of plasma in a dose-dependent manner as determined in a recalcification time assay. Significant elevated ELISA titres were observed in tick resistant and susceptible cattle on day 28 after the tick infestation (p < 0.001). This data suggests direct contact of RmS-15 with the immune system of the host at the tick-feeding site. The present study contributed to the understanding of the biological functions of R. microplus serpins during host–parasite interactions which contributes to the design of future innovative methods for tick control.

The recombinant Bm86-based tick vaccines have shown their efficacy for the control of cattle ticks, Rhipicephalus (Boophilus) microplus and R. annulatus infestations. However, cattle ticks often co-exist with multi-host ticks such as Hyalomma and Amblyomma species, thus requiring the control of multiple tick infestations for cattle and other hosts. Vaccination trials using a R. microplus recombinant Bm86-based vaccine were conducted in cattle and camels against Hyalomma dromedarii and in cattle against Amblyomma cajennense immature and adult ticks. The results showed an 89% reduction in the number of H. dromedarii nymphs engorging on vaccinated cattle, and a further 32% reduction in the weight of the surviving adult ticks. In vaccinated camels, a reduction of 27% and 31% of tick engorgement and egg mass weight, respectively was shown, while egg hatching was reduced by 39%. However, cattle vaccination with Bm86 did not have an effect on A. cajennense tick infestations. These results showed that Bm86 vaccines are effective against R. microplus and other tick species but improved vaccines containing new antigens are required to control multiple tick infestations.

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5–6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated(CPDA) and EDTA tubes. Although >30 mg semi-engorged ticks fed more reliably, ticks as small as 15 mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10 mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA.This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

A comprehensive study of eight paintings of the Cuban artist Amelia Peláez were analysed by physic - chemical
methods. Four paintings belong to her Paris and Modernism period (1927 to 1934) and the others to Cuba,
development and esthetic definition period (1934 to 1968). Samples were taken to study each paint layer
and its painting sequence with polarized light microscopy. Pigments characterization was carried out by energy
dispersive x ray of scanning electron microscope. Organic materials present in each sample were analyzed with a
Fourier transform infrared spectrometer. This research shows new information about Amelia’s paint technique. In
her first painting period she used zinc white to prime while commercial priming was present in her second period
paintings (1934 to 1968). Most of the layers examined show complex mixtures of different kind of pigments, for
example, application of an opaque color (chrome green) mixed with a bright transparent one (viridian green). This
scientific study is a contribution to Cuban art and an aid to guarantee authenticity of Cuban paintings.