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International audience; BackgroundRhipicephalus sanguineus sensu lato (s.l.) is the most widely distributed ixodid tick and is a vector of major canine and human pathogens. High-throughput technologies have revealed that individual ticks carry a high diversity of pathogens, including bacteria, protozoa and viruses. Currently, it is accepted that co-infections (multiple pathogen species within an individual) are very common in ticks and influence pathogen acquisition and transmission as well as host infection risk. However, little is known on the impact of the genetic diversity of pathogens on the incidence of co-infections. Herein, we studied the frequency of co-infections in R. sanguineus (s.l.) and their association with the genetic diversity of Ehrlichia canis.MethodsRhipicephalus sanguineus (s.l.) female ticks (n = 235) were collected from healthy farm dogs in three districts of Pakistan. Microfluidic real-time PCR, a powerful nanotechnology for high-throughput molecular detection of pathogens, was used to test the presence of 25 bacterial and seven parasitic species in individual ticks. The genetic diversity of E. canis was evaluated by characterizing the trp36 gene.ResultsA total of 204 ticks were infected with at least one pathogen and 109 co-infected with two (80%) or three (20%) pathogens. Rickettsia massiliae (human pathogen) and E. canis (zoonotic dog pathogen) were the most common pathogens co-infecting (30.4%) ticks. Furthermore, all identified co-infections included R. massiliae and/or E. canis. Multiple correspondence analysis (MCA) revealed that single infections did not show clear regional association whereas some co-infections were restricted to certain geographical regions. The sequence analysis of trp36 in representative samples allowed the identification of three E. canis strains with low genetic diversity, and the strain found in Muzaffargarh district appeared to be more adapted to co-infection with R. massiliae.ConclusionsRhipicephalus sanguineus (s.l.) harbors multiple co-infections with human and dog pathogens of zoonotic potential. Findings of this study suggest that genetic diversity of E. canis may favor co-infections with different pathogens.

Tick populations are controlled through the application of chemical pesticides. However, the rise in chemical resistance has prompted the investigation of other control methods such as the use of tick vaccines. Proteomic analysis provides valuable information about the possible function and localization of proteins, as candidate vaccine proteins are often either secreted or localized on the cell-surface membrane. Progress in the utilization of proteomics for the identification of novel treatment targets has been significant. However, their use in tick-specific investigations is still quite novel, with the continual development of tick-specific methodologies essential. In this study, an innovative sample preparation method was utilized to isolate epithelial cells from tick midguts to identify the membrane-bound proteins. Proteomic analysis was conducted comparing crude and innovative sample preparation methods with 692 and 1242 tick-specific proteins, 108 and 314 surface proteins respectively, isolated from the midguts of semi-engorged Rhipicephalus microplus adult female ticks. This research reports a novel preparation protocol for the analysis of tick midgut proteins which reduces host protein contamination.

Ixodes holocyclus, the eastern paralysis tick, is a significant parasite in Australia in terms of animal and human health. However, very little is known about its virome. In this study, next-generation sequencing of I. holocyclus salivary glands yielded a full-length genome sequence which phylogenetically groups with viruses classified in the Iflaviridae family and shares 45% amino acid similarity with its closest relative Bole hyalomma asiaticum virus 1. The sequence of this virus, provisionally named Ixodes holocyclus iflavirus (IhIV) has been identified in tick populations from northern New South Wales and Queensland, Australia and represents the first virus sequence reported from I. holocyclus.

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

Tick innate immunity involves humoral and cellular responses. Among the humoral effector molecules in ticks are the defensins which are a family of small peptides with a conserved γ-core motif that is crucial for their antimicrobial activity. Defensin families have been identified in several hard and soft tick species. However, little is known about the presence and antimicrobial activity of defensins from the Australian paralysis tick Ixodes holocyclus. In this study the I. holocyclus transcriptome was searched for the presence of defensins. Unique and non-redundant defensin sequences were identified and designated as holosins 1 – 5. The antimicrobial activity of holosins 2 and 3 and of the predicted γ-cores of holosins 1–4 (HoloTickCores 1–4), was assessed using Gram-negative and Gram-positive bacteria as well as the fungus Fusarium graminearum and the yeast Candida albicans. All holosins had molecular features that are conserved in other tick defensins. Furthermore holosins 2 and 3 were very active against the Gram-positive bacteria Staphylococcus aureus and Listeria grayi. Holosins 2 and 3 were also active against F. graminearum and C. albicans and 5 μM of peptide abrogate the growth of these microorganisms. The activity of the synthetic γ-cores was lower than that of the mature defensins apart from HoloTickCore 2 which had activity comparable to mature holosin 2 against the Gram-negative bacterium Escherichia coli. This study reveals the presence of a multigene defensin family in I. holocyclus with wide antimicrobial activity., JJV is supported by Project FIT (Pharmacology, Immunotherapy, nanoToxicology), which was funded by the European Regional Development Fund and acknowledges a grant from the research organization RVO: RO0516. Transcriptome analysis of I. holocyclus viscera and salivary glands was funded by the Australian Research Council linkage project LP120200836 and Elanco Animal Health. MT and AV would like to acknowledge generous funding by the Hessen State Ministry of Higher Education, Research and the Arts (HMWK) via the LOEWE Centre for Translational Biodiversity Genomics (LOEWE-TBG) and the LOEWE Center for Insect Biotechnology and Bioresources.

The attachment to host skin by Rhipicephalus microplus larvae induces a series of physiological events at the attachment site. The host-parasite interaction might induce a rejection of the larvae, as is frequently observed in Bos taurus indicus cattle, and under certain conditions in Bos taurus taurus cattle. Ticks deactivate the host rejection response by secreting specific proteins and lipids that play an essential role in manipulation of the host immune response. The available genomic information on the R. microplus tick was mined using bioinformatics approaches to identify R. microplus lipocalins (LRMs). This in silico examination revealed a total of 12 different putative R. microplus LRMs (LRM1-LRM12). The identity of the LRM family showed high sequence variability: from 6% between LRM7 and LRM8 to 55.9% between LRM2 and LRM6. However, the three-dimensional structure of the lipocalin family was conserved in the LRMs. The B and T cell epitopes in these lipocalins were then predicted, and six of the LRMs (5, 6, 9, 10, 11 and 12) were used to examine the host immune interactions with sera and peripheral blood mononuclear cells (PBMCs) collected from tick-susceptible and tick-resistant cattle challenged with R. microplus. On days 28-60 after tick infestation, the anti-LRM titres were higher in the resistant group compared with the susceptible cattle. After 60 day, the anti-LRM titres (except LRM9 and LRM11) decreased to zero in the sera of both the tick-resistant and tick-susceptible cattle. Using cell proliferation assays, the PBMCs challenged with some of the predicted T cell epitopes (LRM1_T1, T2; LRM_T1, T2 and LRM12_T) exhibited a significantly higher number of IFN-γ-secreting cells (Th1) in tick-susceptible Holstein-Friesians compared with tick-resistant Brahman cattle. In contrast, expression of the Th2 cytokine (IL-4) was lower in Holstein-Friesians cattle compared with Brahman cattle. Moreover, this study found that LRM6, LRM9 and LRM11 play important roles in the mechanism by which R. microplus interferes with the host's haemostasis mechanisms.

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.

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)., This meeting was supported by the Bill & Melinda Gates Foundation.

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 similar to 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 (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Background: Rhipicephalus (Boophilus) microplus evades the host's haemostatic system through a complex protein array secreted into tick saliva. Serine protease inhibitors (serpins) conform an important component of saliva which are represented by a large protease inhibitor family in Ixodidae. These secreted and non-secreted inhibitors modulate diverse and essential proteases involved in different physiological processes. Methods: The identification of R. microplus serpin sequences was performed through a web-based bioinformatics environment called Yabi. The database search was conducted on BmiGi V1, BmiGi V2.1, five SSH libraries, Australian tick transcriptome libraries and RmiTR V1 using bioinformatics methods. Semi quantitative PCR was carried out using different adult tissues and tick development stages. The cDNA of four identified R. microplus serpins were cloned and expressed in Pichia pastoris in order to determine biological targets of these serpins utilising protease inhibition assays. Results: A total of four out of twenty-two serpins identified in our analysis are new R. microplus serpins which were named as RmS-19 to RmS-22. The analyses of DNA and predicted amino acid sequences showed high conservation of the R. microplus serpin sequences. The expression data suggested ubiquitous expression of RmS except for RmS-6 and RmS-14 that were expressed only in nymphs and adult female ovaries, respectively. RmS-19, and -20 were expressed in all tissues samples analysed showing their important role in both parasitic and non-parasitic stages of R. microplus development. RmS-21 was not detected in ovaries and RmS-22 was not identified in ovary and nymph samples but were expressed in the rest of the samples analysed. A total of four expressed recombinant serpins showed protease specific inhibition for Chymotrypsin (RmS-1 and RmS-6), Chymotrypsin / Elastase (RmS-3) and Thrombin (RmS-15). Conclusion: This study constitutes an important contribution and improvement to the knowledge about the physiologic role of R. microplus serpins during the host-tick interaction.

Manuel Rodriguez-Valle,1 Leonardo Canan-Hadden,2 Olivia Niebla2 1Animal Biotechnology Division, 2Analytical Division, Centre for Genetic Engineering and Biotechnology, Havana, Cuba Abstract: The ectoparasite Rhipicephalus (Boophilus) microplus causes severe economic losses to the cattle industry in tropical and subtropical regions, and transmits endoparasites, such as Babesia bovis. The glycoprotein Bm95 is homologous to Bm86, a surface membrane protein of gut epithelial cells in R. microplus, and has been shown to efficiently control this ectoparasite in regions of the Americas. The immunostimulant properties of Bm86 have already been demonstrated after its coinjection with hepatitis B surface antigen (HBsAg) and the infectious bovine rhinotracheitis virus. This study evaluated the carrier and immunostimulant properties of Bm95 using low immunogenic Neisseria meningitidis capsular C polysaccharide (Men CpS) and HBsAg. We produced two polysaccharide-Bm95 conjugates by carbodiimide (MenCpSBm-c) and reductive amination (MenCpSBm-ra) methods. These conjugates were characterized and evaluated in mice. Antibody titers against Men CpS were significantly higher in mice immunized with MenCpSBm-ra (2,350±250, P

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.