African swine fever virus transmission cycles in Central Europe: Evaluation of wild boar-soft tick contacts through detection of antibodies against Ornithodoros erraticus saliva antigen.

BACKGROUND: African swine fever (ASF) is one of the most complex viral diseases affecting both domestic and wild pigs. It is caused by ASF virus (ASFV), the only DNA virus which can be efficiently transmitted by an arthropod vector, soft ticks of the genus Ornithodoros. These ticks can be part of ASFV-transmission cycles, and in Europe, O. erraticus was shown to be responsible for long-term maintenance of ASFV in Spain and Portugal. In 2014, the disease has been reintroduced into the European Union, affecting domestic pigs and, importantly, also the Eurasian wild boar population. In a first attempt to assess the risk of a tick-wild boar transmission cycle in Central Europe that would further complicate eradication of the disease, over 700 pre-existing serum samples from wild boar hunted in four representative German Federal States were investigated for the presence of antibodies directed against salivary antigen of Ornithodoros erraticus ticks using an indirect ELISA format. RESULTS: Out of these samples, 16 reacted with moderate to high optical densities that could be indicative of tick bites in sampled wild boar. However, these samples did not show a spatial clustering (they were collected from distant geographical regions) and were of bad quality (hemolysis/impurities). Furthermore, all positive samples came from areas with suboptimal climate for soft ticks. For this reason, false positive reactions are likely. CONCLUSION: In conclusion, the study did not provide stringent evidence for soft tick-wild boar contact in the investigated German Federal States and thus, a relevant involvement in the epidemiology of ASF in German wild boar is unlikely. This fact would facilitate the eradication of ASF in the area, although other complex relations (wild boar biology and interactions with domestic pigs) need to be considered.

A microRNA profile of the saliva in the argasid ticks Ornithodoros erraticus and Ornithodoros moubata and prediction of specific target genes.

Ornithodoros erraticus and Ornithodoros moubata ticks are the main vectors of the agents of human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin and Africa, respectively. Tick ​​saliva is crucial for complete tick feeding and pathogen transmission, as it contains numerous molecules such as proteins, lipids, and non-coding RNAs (ncRNA) including microRNAs (miRNA). MiRNAs are small ncRNAs capable of regulating the expression of their target messenger RNA (mRNA) leading to degradation or inhibition of its translation into protein. Research on miRNAs from ixodid ticks has revealed that miRNAs are involved in the regulation of different physiological processes of ticks, as well as in the modulation of host gene expression, immune response to tick bite and pathogen transmission. Regarding argasid ticks, there is not information about their miRNAs or their potential involvement in tick physiology and/or in the regulation of the tick-host-pathogen interactions. The aim of this work was to profile the miRNAs expressed in the saliva of O. erraticus and O. moubata, and the in silico prediction and functional analysis of their target genes in the swine host. As a whole, up to 72 conserved miRNAs families were identified in both species: 35 of them were shared and 23 and 14 families were unique to O. erraticus and O. moubata, respectively. The most abundant miRNAs families were mir-1, mir-10 and let-7 in O. erraticus and let-7, mir-252, mir-10 in O. moubata. Four miRNAs sequences of each species were validated by RT-qPCR confirming their presence in the saliva. Target gene prediction in the host (Sus scrofa) and functional analysis showed that the selected miRNAs are mainly involved in processes related to signal transduction, regulation of mRNA transcription and gene expression, synapse regulation, immune response, angiogenesis and vascular development. These results suggest that miRNAs could play an important role at the tick-host interface, providing new insights into this complex relationship that may contribute to a more precise selection of tick molecules for the development of therapeutic and immune strategies to control tick infestations and tick-borne pathogens.

Function-guided selection of salivary antigens from Ornithodoros erraticus argasid ticks and assessment of their protective efficacy in rabbits.

The identification of new protective antigens for the development of tick vaccines may be approached by selecting antigen candidates that have key biological functions. Bioactive proteins playing key functions for tick feeding and pathogen transmission are secreted into the host via tick saliva. Adult argasid ticks must resynthesise and replace these proteins after each feeding to be able to repeat new trophogonic cycles. Therefore, these proteins are considered interesting antigen targets for tick vaccine development. In this study, the salivary gland transcriptome and saliva proteome of Ornithodoros erraticus females were inspected to select and test new vaccine candidate antigens. For this, we focused on transcripts overexpressed after feeding that encoded secretory proteins predicted to be immunogenic and annotated with functions related to blood ingestion and modulation of the host defensive response. Completeness of the transcript sequence, as well as a high expression level and a high fold-change after feeding were also scored resulting in the selection of four candidates, an acid tail salivary protein (OeATSP), a multiple coagulation factor deficiency protein 2 homolog (OeMCFD2), a Cu/Zn-superoxide dismutase (OeSOD) and a sulfotransferase (OeSULT), which were later produced as recombinant proteins. Vaccination of rabbits with each individual recombinant antigen induced strong humoral responses that reduced blood feeding and female reproduction, providing, respectively, 46.8%, 45.7%, 54.3% and 31.9% protection against O. erraticus infestations and 0.7%, 3.9%, 3.1% and 8.7% cross-protection against infestations by the African tick, Ornithodoros moubata. The joint protective efficacy of these antigens was tested in a second vaccine trial reaching 58.3% protection against O. erraticus and 18.6% cross-protection against O. moubata. These results (i) provide four new protective salivary antigens from argasid ticks that might be included in multi-antigenic vaccines designed for the control of multiple tick species; (ii) reveal four functional protein families never tested before as a source of protective antigens in ticks; and (iii) show that multi-antigenic vaccines increase vaccine efficacy compared with individual antigens. Finally, our data add value to the salivary glands as a protective antigen source in argasids for the control of tick infestations.

Comparison of salivary gland and midgut microbiome in the soft ticks Ornithodoros erraticus and Ornithodoros moubata.

Introduction: Ornithodoros erraticus and Ornithodoros moubata are the main vectors of African swine fever virus (ASFV) and the human relapsing fever spirochetes Borrelia hispanica and Borrelia crocidurae in the Mediterranean region and Borrelia duttoni in continental Africa. Manipulation of the tick microbiome has been shown to reduce vector fitness and competence in tick vectors, suggesting that the identification of key microbial players associated with tick tissues can inform interventions such as anti-microbiota vaccines to block pathogen development in the midgut and/or salivary glands. Methods: In this study, we analyzed and compared the microbiome of the salivary glands and midgut of O. erraticus and O. moubata. For the taxonomic and functional characterization of the tissue-specific microbiome, we used 16S rRNA amplicon sequencing and prediction of metabolic profiles using PICRUSt2. Co-occurrence networks were built to characterize the community assembly and identify keystone taxa in each tick species. Results: Our results revealed differences in the composition, diversity, and assembly of the bacterial microbiome of salivary glands and midgut within each tick species, but differences were more noticeable in O. moubata. Differences were also found in the microbiome of each tissue, salivary gland and midgut, between species. However, the 'Core Association Networks (CAN)' analysis revealed conserved patterns of interacting taxa in tissues within and between tick species. Different keystone taxa were identified in O. erraticus and O. moubata tissues, but Muribaculaceae and Alistipes were found as keystone taxa in the salivary glands of both tick species which justifies their use as anti-microbiota vaccine candidates to alter the microbiome and reduce tick fitness and/or block pathogen transmission. The high similarity of predicted metabolic pathways profiles between tissues of the two tick species suggests that taxonomic variability of the microbiome is not associated with significant changes in microbial functional profiles. Conclusion: We conclude that the taxonomic structure of the microbiome in O. erraticus and O. moubata is tissue-specific, suggesting niche partitioning of bacterial communities associated to these soft ticks. However, shared keystone taxa and conserved patterns of interacting taxa between tissues and tick species suggest the presence of key microbial players that could be used as anti-microbiota vaccine candidates to affect tick physiology and/or pathogen colonization.

Self-assembled protein arrays from an Ornithodoros moubata salivary gland expression library.

Protein interactions play a critical role in the regulation of many biological events and their study in a high-throughput format has become a key area of proteomic research. Nucleid Acid Programmable Protein Arrays (NAPPA) technology allows the construction of protein arrays from cDNA expression libraries in high-throughput cell-free systems to study protein interaction and functions. Tick saliva contains antihemostatic, anti-inflammatory, and immunosuppressive proteins that counteract the host hemostatic, immune, and inflammatory responses allowing the ingestion of host blood and facilitating its infection by the tick-borne pathogens. Identification of such proteins and their functions could help in the selection of antigenic targets for the development of antitick and transmission-blocking vaccines. With that aim, we have prepared a cDNA expression library from the salivary glands of Ornithodoros moubata and subsequently produced a self-assembled protein microarray using 480 randomly selected clones from that library. The reproducibility of the array, its representativeness of the tick salivary protein repertoire, and the functionality of the in situ expressed proteins have been checked, demonstrating that it is a suitable tool for the identification and functional characterization of soft tick salivary molecules that interact with host proteins. Several clones in the array were shown to bind to human recombinant P-selectin. One of them was a likely secreted tick phospholipase A2, which may represent a potential new ligand for P-selectin. As these salivary molecules are likely involved in blood meal acquisition through the modulation of the host immune and hemostatic responses, this new high-throughput tool could open new avenues for development of new therapeutic agents and control strategies against ticks and tick-borne pathogens.

First molecular and functional characterisation of ferritin 2 proteins from Ornithodoros argasid ticks.

Ferritins are iron-binding proteins that play critical functions in iron metabolism. Tick ferritins are essential in blood feeding, reproduction, iron transport, and protection of ticks from the iron-mediated oxidative stress during blood feeding and digestion. In ixodids, ferritin 2 (Fer2) is responsible for iron transport into peripheral tissues, it is critically involved in tick reproduction and has been identified as a good candidate antigen to be included in anti-tick vaccines. In argasids, information on the molecular and functional characteristics of ferritins is almost nonexistent. Given the potential of ixodid Fer2 as a vaccine target, the aim of the current study was to characterise the Fer2 orthologues in Ornithodoros erraticus (OEFer2) and O. moubata (OMFer2), including functional analyses by RNAi gene knockdown and the assessment of the protective efficacy of recombinant Fer2 protein in an animal vaccination trials. Characterisation and analysis of the OMFer2 and OEFer2 amino acid sequences showed high similarity to each other, and high similarity to the Fer2 sequences of ixodid species as well, confirming that Fer2 is highly conserved between both tick families and suggesting a similar function in the physiology of both argasid and ixodid ticks. Fer2 gene knockdown in O. moubata reduced egg hatchability rate and the subsequent number of emerging nymphs-1 up to 71%. Conversely, Fer2 gene knockdown in O. erraticus did not affect the treated ticks even though the Fer2 mRNA expression level was reduced by 90%. The recombinant form of O. moubata Fer2 (tOMFer2) was highly immunogenic and induced strong humoral responses when administered to rabbits formulated with Montanide adjuvant. The protective effect of the anti-tOMFer2 response was limited. While in O. erraticus, we did not observe any protective effect, in O. moubata it induced a significant reduction in oviposition without affecting the other parameters analysed. Accordingly, Fer2 seems to be involved in O. moubata embryogenesis. This study provides the first data on the molecular and functional characterisation of Fer2 in soft tick species and paves the way for further studies aimed at unveiling the functional aspects of Fer2 in soft ticks and confirming its potential as a vaccine candidate antigen.

A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva.

BACKGROUND: The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. The prevention and control of these diseases would greatly benefit from the elimination of O. erraticus populations, and anti-tick vaccines are envisaged as an effective and sustainable alternative to chemical acaricide usage for tick control. Ornithodoros erraticus saliva contains bioactive proteins that play essential functions in tick feeding and host defence modulation, which may contribute to host infection by tick-borne pathogens. Hence, these proteins could be candidate antigen targets for the development of vaccines aimed at the control and prevention of O. erraticus infestations and the diseases this tick transmits. The objective of the present work was to obtain and characterise the proteome of the saliva of O. erraticus adult ticks as a means to identify and select novel salivary antigen targets. METHODS: A proteomics informed by transcriptomics (PIT) approach was applied to analyse samples of female and male saliva separately using the previously obtained O. erraticus sialotranscriptome as a reference database and two different mass spectrometry techniques, namely liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition mode and sequential window acquisition of all theoretical fragment ion spectra MS (SWATH-MS). RESULTS: Up to 264 and 263 proteins were identified by LC-MS/MS in the saliva of O. erraticus female and male ticks, respectively, totalling 387 non-redundant proteins. Of these, 224 were further quantified by SWATH-MS in the saliva of both male and female ticks. Quantified proteins were classified into 23 functional categories and their abundance compared between sexes. Heme/iron-binding proteins, protease inhibitors, proteases, lipocalins and immune-related proteins were the categories most abundantly expressed in females, while glycolytic enzymes, protease inhibitors and lipocalins were the most abundantly expressed in males. Ninety-seven proteins were differentially expressed between the sexes, of which 37 and 60 were overexpressed in females and males, respectively. CONCLUSIONS: The PIT approach demonstrated its usefulness for proteomics studies of O. erraticus, a non-model organism without genomic sequences available, allowing the publication of the first comprehensive proteome of the saliva of O. erraticus reported to date. These findings confirm important quantitative differences between sexes in the O. erraticus saliva proteome, unveil novel salivary proteins and functions at the tick-host feeding interface and improve our understanding of the physiology of feeding in O. erraticus ticks. The integration of O. erraticus sialoproteomic and sialotranscriptomic data will drive a more rational selection of salivary candidates as antigen targets for the development of vaccines aimed at the control of O. erraticus infestations and the diseases it transmits.

First Data on Ornithodoros moubata Aquaporins: Structural, Phylogenetic and Immunogenic Characterisation as Vaccine Targets.

Ornithodoros moubata transmits African swine fever and human relapsing fever in Africa. The elimination of O. moubata populations from anthropic environments is expected to improve the prevention and control of these diseases. Tick vaccines have emerged as a sustainable method for tick control, and tick aquaporins (AQPs) are promising targets for tick vaccines due to their vital functions, immunogenicity and ease of access by neutralising host antibodies. This study aimed at the systematic identification of the AQPs expressed by O. moubata (OmAQPs) and their characterisation as vaccine targets. Therefore, AQP coding sequences were recovered from available transcriptomic datasets, followed by PCR amplification, cloning, sequence verification and the analysis of the AQP protein structure and epitope exposure. Seven OmAQPs were identified and characterised: six were aquaglyceroporins, and one was a water-specific aquaporin. All of these were expressed in the salivary glands and midgut and only three in the coxal glands. Epitope exposure analysis identified three extracellular domains in each AQP, which concentrate overlapping B and T cell epitopes, making them interesting vaccine targets. Based on these domain sequences, a set of ten antigenic peptides was designed, which showed adequate properties to be produced and tested in pilot vaccine trials.

Sialotranscriptomics of the argasid tick Ornithodoros moubata along the trophogonic cycle.

The argasid tick Ornithodoros moubata is the main vector of human relapsing fever (HRF) and African swine fever (ASF) in Africa. Salivary proteins are part of the host-tick interface and play vital roles in the tick feeding process and the host infection by tick-borne pathogens; they represent interesting targets for immune interventions aimed at tick control. The present work describes the transcriptome profile of salivary glands of O. moubata and assesses the gene expression dynamics along the trophogonic cycle using Illumina sequencing. De novo transcriptome assembling resulted in 71,194 transcript clusters and 41,011 annotated transcripts, which represent 57.6% of the annotation success. Most salivary gene expression takes place during the first 7 days after feeding (6,287 upregulated transcripts), while a minority of genes (203 upregulated transcripts) are differentially expressed between 7 and 14 days after feeding. The functional protein groups more abundantly overrepresented after blood feeding were lipocalins, proteases (especially metalloproteases), protease inhibitors including the Kunitz/BPTI-family, proteins with phospholipase A2 activity, acid tail proteins, basic tail proteins, vitellogenins, the 7DB family and proteins involved in tick immunity and defence. The complexity and functional redundancy observed in the sialotranscriptome of O. moubata are comparable to those of the sialomes of other argasid and ixodid ticks. This transcriptome provides a valuable reference database for ongoing proteomics studies of the salivary glands and saliva of O. moubata aimed at confirming and expanding previous data on the O. moubata sialoproteome.

Proteomics informed by transcriptomics for a qualitative and quantitative analysis of the sialoproteome of adult Ornithodoros moubata ticks.

BACKGROUND: The argasid tick Ornithodoros moubata is the main vector in mainland Africa of African swine fever virus and the spirochete Borrelia duttoni, which causes human relapsing fever. The elimination of populations of O. moubata would contribute to the prevention and control of these two serious diseases. Anti-tick vaccines are an eco-friendly and sustainable means of eliminating tick populations. Tick saliva forms part of the tick-host interface, and knowledge of its composition is key to the identification and selection of vaccine candidate antigens. The aim of the present work is to increase the body of data on the composition of the saliva proteome of adult O. moubata ticks, particularly of females, since in-depth knowledge of the O. moubata sialome will allow the identification and selection of novel salivary antigens as targets for tick vaccines. METHODS: We analysed samples of female and male saliva using two different mass spectrometry (MS) approaches: data-dependent acquisition liquid chromatography-tandem MS (LC-MS/MS) and sequential window acquisition of all theoretical fragment ion spectra-MS (SWATH-MS). To maximise the number of proteins identified, a proteomics informed by transcriptomics analysis was applied using the O. moubata salivary transcriptomic dataset previously obtained by RNA-Seq. RESULTS: SWATH-MS proved to be superior to LC-MS/MS for the study of female saliva, since it identified 61.2% more proteins than the latter, the reproducibility of results was enhanced with its use, and it provided a quantitative picture of salivary components. In total, we identified 299 non-redundant proteins in the saliva of O. moubata, and quantified the expression of 165 of these in both male and female saliva, among which 13 were significantly overexpressed in females and 40 in males. These results indicate important quantitative differences in the saliva proteome between the sexes. CONCLUSIONS: This work expands our knowledge of the O. moubata sialome, particularly that of females, by increasing the number of identified novel salivary proteins, which have different functions at the tick-host feeding interface. This new knowledge taken together with information on the O. moubata sialotranscriptome will allow a more rational selection of salivary candidates as antigen targets for tick vaccine development.

RNA-seq analysis and gene expression dynamics in the salivary glands of the argasid tick Ornithodoros erraticus along the trophogonic cycle.

BACKGROUND: The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. Tick salivary proteins secreted to the host at the feeding interface play critical roles for tick feeding and may contribute to host infection by tick-borne pathogens; accordingly, these proteins represent interesting antigen targets for the development of vaccines aimed at the control and prevention of tick infestations and tick-borne diseases. METHODS: To identify these proteins, the transcriptome of the salivary glands of O. erraticus was de novo assembled and the salivary gene expression dynamics assessed throughout the trophogonic cycle using Illumina sequencing. The genes differentially upregulated after feeding were selected and discussed as potential antigen candidates for tick vaccines. RESULTS: Transcriptome assembly resulted in 22,007 transcripts and 18,961 annotated transcripts, which represent 86.15% of annotation success. Most salivary gene expression took place during the first 7 days after feeding (2088 upregulated transcripts), while only a few genes (122 upregulated transcripts) were differentially expressed from day 7 post-feeding onwards. The protein families more abundantly overrepresented after feeding were lipocalins, acid and basic tail proteins, proteases (particularly metalloproteases), protease inhibitors, secreted phospholipases A2, 5'-nucleotidases/apyrases and heme-binding vitellogenin-like proteins. All of them are functionally related to blood ingestion and regulation of host defensive responses, so they can be interesting candidate protective antigens for vaccines. CONCLUSIONS: The O. erraticus sialotranscriptome contains thousands of protein coding sequences-many of them belonging to large conserved multigene protein families-and shows a complexity and functional redundancy similar to those observed in the sialomes of other argasid and ixodid tick species. This high functional redundancy emphasises the need for developing multiantigenic tick vaccines to reach full protection. This research provides a set of promising candidate antigens for the development of vaccines for the control of O. erraticus infestations and prevention of tick-borne diseases of public and veterinary health relevance, such as TBRF and ASF. Additionally, this transcriptome constitutes a valuable reference database for proteomics studies of the saliva and salivary glands of O. erraticus.

In silico selection of functionally important proteins from the mialome of Ornithodoros erraticus ticks and assessment of their protective efficacy as vaccine targets.

BACKGROUND: New candidate protective antigens for tick vaccine development may be identified by selecting and testing antigen candidates that play key biological functions. After blood-feeding, tick midgut overexpresses proteins that play essential functions in tick survival and disease transmission. Herein, Ornithodoros erraticus midgut transcriptomic and proteomic data were examined in order to select functionally significant antigens upregulated after feeding to be tested as vaccine candidate antigens. METHODS: Transcripts annotated as chitinases, tetraspanins, ribosomal protein P0 and secreted proteins/peptides were mined from the recently published O. erraticus midgut transcriptome and filtered in a second selection step using criteria based on upregulation after feeding, predicted antigenicity and expression in the midgut proteome. Five theoretical candidate antigens were selected, obtained as recombinant proteins and used to immunise rabbits: one chitinase (CHI), two tetraspanins (TSPs), the ribosomal protein P0 (RPP0) and one secreted protein PK-4 (PK4). RESULTS: Rabbit vaccination with individual recombinant candidates induced strong humoral responses that mainly reduced nymph moulting and female reproduction, providing 30.2% (CHI), 56% (TSPs), 57.5% (RPP0) and 57.8% (PK4) protection to O. erraticus infestations and 19.6% (CHI), 11.1% (TSPs), 0% (RPP0) and 8.1% (PK4) cross-protection to infestations by the African tick Ornithodoros moubata. The joint vaccine efficacy of the candidates was assessed in a second vaccine trial reaching 66.3% protection to O. erraticus and 25.6% cross-protection to O. moubata. CONCLUSIONS: These results (i) indicate that argasid chitinases and RPP0 are promising protective antigens, as has already been demonstrated for ixodid chitinases and RPP0, and could be included in vaccines targeting multiple tick species; (ii) reveal novel protective antigens tetraspanins and secreted protein PK-4, never tested before as protective antigens in ticks; and (iii) demonstrate that multi-antigenic vaccines increased vaccine efficacy compared with individual antigens. Lastly, our data emphasize the value of the tick midgut as a source of protective candidate antigens in argasids for tick control.

Function-guided selection of midgut antigens from Ornithodoros erraticus ticks and an evaluation of their protective efficacy in rabbits.

The identification of candidate protective antigens for the development of tick vaccines may be approached by selecting antigen candidates that play key biological functions. Tick midgut proteins that play essential functions in tick survival and disease transmission are upregulated in response to blood feeding and digestion. In this study, Ornithodoros erraticus midgut transcriptomic and proteomic data upon feeding were inspected to select functionally relevant antigens to be assessed as vaccine candidate antigens. For this, we primarily focused on proteins with relevant biological functions in key physiological processes for ticks and tick-host-pathogen interactions. Later, we used additional criteria based on overexpression after feeding, predicted antigenicity and cellular localisation, resulting in the selection of four theoretical candidates, two aquaporins (OeAQP, OeAQP1), one ABC transporter (OeABC) and one selenoprotein T (OeSEL). Rabbit vaccination with synthetic immunogenic peptides designed from the extracellular antigenic regions of the selected candidates induced humoral responses that reduced tick feeding and reproduction performance. Both AQPs and OeSEL demonstrated significant protection efficacy against the homologous species O. erraticus, but lower non-significant cross-species protection against Ornithodoros moubata. Conversely, OeABC showed no protection against the homologous species O. erraticus, but significant cross-species protection against O. moubata. These results are the first demonstration of the protective potential of argasid aquaporins, suggesting that they might be included in vaccines for the control of multiple tick species. Additionally, these results also unveiled two novel protective antigens from argasid ticks, OeABC and OeSEL, belonging to functional protein families that have never been explored as a source of vaccine candidates and are deserving of further studies. Finally, our data add value to the midgut as a protective candidate antigen source in argasids for the control of tick infestations.

Epidemiological surveillance of schistosomiasis outbreak in Corsica (France): Are animal reservoir hosts implicated in local transmission?

Environmental and anthropogenic changes are expected to promote emergence and spread of pathogens worldwide. Since 2013, human urogenital schistosomiasis is established in Corsica island (France). Schistosomiasis is a parasitic disease affecting both humans and animals. The parasite involved in the Corsican outbreak is a hybrid form between Schistosoma haematobium, a human parasite, and Schistosoma bovis, a livestock parasite. S. bovis has been detected in Corsican livestock few decades ago raising the questions whether hybridization occurred in Corsica and if animals could behave as a reservoir for the recently established parasite lineage. The latter hypothesis has huge epidemiological outcomes since the emergence of a zoonotic lineage of schistosomes would be considerably harder to control and eradicate the disease locally and definitively needs to be verified. In this study we combined a sero-epidemiological survey on ruminants and a rodent trapping campaign to check whether schistosomes could shift on vertebrate hosts other than humans. A total of 3,519 domesticated animals (1,147 cattle; 671 goats and 1,701 sheep) from 160 farms established in 14 municipalities were sampled. From these 3,519 screened animals, 17 were found to be serologically positive but were ultimately considered as false positive after complementary analyses. Additionally, our 7-day extensive rodent trapping (i.e. 1,949 traps placed) resulted in the capture of a total of 34 rats (Rattus rattus) and 4 mice (Mus musculus). Despite the low number of rodents captured, molecular diagnostic tests showed that two of them have been found to be infected by schistosomes. Given the low abundance of rodents and the low parasitic prevalence and intensity among rodents, it is unlikely that neither rats nor ruminants play a significant role in the maintenance of schistosomiasis outbreak in Corsica. Finally, the most likely hypothesis is that local people initially infected in 2013 re-contaminated the river during subsequent summers, however we cannot definitively rule out the possibility of an animal species acting as reservoir host.

A proteomic approach to the identification of tegumental proteins of male and female Schistosoma bovis worms.

Schistosoma bovis, a parasite of ruminants, can live for years in the bloodstream in spite of the immune response of its host. The parasite tegument covers the entire surface of the worm and plays a key role in the host-parasite relationship. The parasite molecules involved in host immune response evasion mechanisms must be expressed on the tegument surface and are potential targets for immune or drug intervention. The purpose of the present work was to identify the tegumental proteomes of male and female S. bovis worms, in particular the proteins expressed on the outermost layers of the tegument structure. Adult worms of each sex were treated separately with trypsin in order to digest their tegumental proteins, after which the peptides released were analysed by LC-MS/MS for identification. This experimental approach afforded valuable information about the protein composition of the tegument of adult S. bovis worms. A range of tegumental proteins was identified, most of which had not been identified previously in this species. Although an absolute purification of the proteins expressed on the outermost layers of the tegument structure was not achieved, it is likely that present among the proteins identified are some of the molecules most closely associated with the tegument surface. Our study also suggests that there may be differences in the protein composition of the tegument of male and female schistosomes. Finally, the presence of actin and GAPDH on the surface of male and female worms and the presence of enolase exclusively on the surface of male worms were verified by confocal microscopy.

Pathogenicity of endophytic entomopathogenic fungi to Ornithodoros erraticus and Ornithodoros moubata (Acari: Argasidae).

The argasid ticks O. erraticus and O. moubata are of great medical and veterinary importance because they are vectors of the African swine fever virus and several species of human relapsing fever borreliae. Biocontrol of these ticks using entomopathogenic fungi has not been previously reported. We examined the pathogenicity to different developmental stages of these two argasids of six strains of the fungal species Beauveria bassiana (strains Bb1764 and Bb2157), Lecanicillium lecanii (strains Ll586, Ll618 and Ll3047) and Tolypocladium cylindrosporum (strain Tc3398). Three strains, Bb1764, Bb2157, and Tc3398, caused in Spanish O. erraticus mean mortality rates between 34.4% and 62% in 14-28 days post-inoculation. Additionally, Bb2157 also induced in African O. moubata mean mortality rates of 31.9%. The remaining strains caused lower mortality rates and were not considered effective. This is the first study in which some strains of entomopathogenic fungi are found to be effective against argasid ticks of the genus Ornithodoros, and its results might justify further efforts towards the application of entomopathogenic fungal strains as anti-argasid biocontrol agents.

De novo assembly and analysis of midgut transcriptome of the argasid tick Ornithodoros erraticus and identification of genes differentially expressed after blood feeding.

Ticks are hematophagous vectors of great medical and veterinary importance because they transmit numerous pathogenic microorganisms to humans and animals. The argasid Ornithodoros erraticus is the main vector of tick-borne human relapsing fever and African swine fever in the Mediterranean Basin. Tick enterocytes express bioactive molecules that perform key functions in blood digestion, feeding, toxic waste processing and pathogen transmission. To explore new strategies for tick control, in this work we have obtained and compared the midgut transcriptomes of O. erraticus female ticks before and after a blood meal and identified the genes whose expression is differentially regulated after feeding. The transcript sequences were annotated, functionally and structurally characterised and their expression levels compared between both physiological conditions (unfed females and fed females at 2 days post-engorgement). Up to 29,025 transcripts were assembled, and 9290 of them corresponded to differentially expressed genes (DEGs) after feeding. Of these, 4656 genes were upregulated and nearly the same number of genes was downregulated in fed females compared to unfed females. BLASTN and BLASTX analyses of the 29,025 transcripts allowed the annotation of 9072 transcripts/proteins. Among them, the most numerous were those with catalytic and binding activities and those involved in diverse metabolic pathways and cellular processes. The analyses of functional groups of upregulated DEGs potentially related to the digestion of proteins, carbohydrates and lipids, and the genes involved in the defence response and response to oxidative stress, confirm that these processes are narrowly regulated in ticks, highlighting their complexity and importance in tick biology. The expression patterns of six genes throughout the blood digestion period revealed significant differences between these patterns, strongly suggesting that the transcriptome composition is highly dynamic and subjected to important variation along the trophogonic cycle. This may guide future studies aimed at improving the understanding of the molecular physiology of tick digestion and digestion-related processes. The current work provides a more robust and comprehensive understanding of the argasid tick digestive system.

Evaluation of the protective efficacy of Ornithodoros moubata midgut membrane antigens selected using omics and in silico prediction algorithms.

The African argasid tick Ornithodoros moubata transmits two important pathogens, the African swine fever virus and the spirochete Borrelia duttoni, the cause of human relapsing fever. To date, only conventional control measures such as widespread application of acaricides, strict control measures, and animal movement restrictions have been implemented to confine these diseases. Vaccines against tick infestations have the potential to be among the most efficacious interventions for the management of these diseases. Plasma membrane-associated proteins upregulated in tick midgut cells in response to blood feeding and digestion are thought to play vital functions in tick physiology and in the transmission of tick-borne pathogens. In addition, their antigenic extracellular regions are easily accessible to antibodies synthesised by immunised hosts, which makes them interesting targets for tick vaccine design. The mialomes (midgut transcriptomes and proteomes) of unfed O. moubata females and of engorged females at 48 h post-feeding have recently been obtained, providing a wealth of predicted midgut protein sequences. In the current study, these mialomes were screened using in silico tools to select predicted antigenic transmembrane proteins that were upregulated after feeding (516 proteins). The functionally annotatable proteins from this list (396 proteins) were then manually inspected following additional criteria in order to select a finite and easy-manageable number of candidate antigens for tick vaccine design. The extracellular antigenic regions of five of these candidates were obtained either as truncated recombinant proteins or as KLH-conjugated synthetic peptides, formulated in Freund's adjuvant, and individually administered to rabbits to assess their immunogenicity and protective potential against infestations by O. moubata and the Iberian species Ornithodoros erraticus. All candidates were highly immunogenic, but provided low protection against the O. moubata infestations (ranging from 7% to 39%). Interestingly, all candidates except one also protected against infestations by O. erraticus, achieving higher efficacies against this species (from 20% to 66%). According to their protective potential, three of the five antigens tested (Om17, Om86 and OM99) were considered little suitable for use in tick vaccines, while the other two (OM85 and OM03) were considered useful antigens for tick vaccine development, deserving further studies.

A proteomic approach to the identification of salivary proteins from the argasid ticks Ornithodoros moubata and Ornithodoros erraticus.

The saliva of ticks contains anti-haemostatic, anti-inflammatory and immunomodulatory molecules that allow these parasites to obtain a blood meal from the host and help tick-borne pathogens to infect the vertebrate host more efficiently. This makes the salivary molecules attractive targets to control ticks and tick-borne pathogens. Although Ornithodoros moubata and O. erraticus are important argasid ticks that transmit severe diseases, to date only a few of their salivary proteins have been identified. Here we report our initial studies using proteomic approaches to characterize the protein profiles of salivary gland extracts (SGE) from these two argasids. The present work describes the proteome of the SGEs of both tick species, their antigenic spots, and the identification of several of their proteins. The whole number of identifications was low despite the good general quality of the peptide mass maps obtained. In the O. moubata SGE, 18 isoforms of a protein similar to O. savignyi TSGP1 were identified. In the O. erraticus SGE we identified 6 novel proteins similar to unknown secreted protein DS-1 precursor, NADPH dehydrogenase subunit 5, proteasome alpha subunit, ATP synthase F0 subunit 6, lipocalin and alpha tubulin. Finally, the current drawbacks of proteomics when applied to the identification of acarine peptides and proteins are discussed.

Purification and characterization of a 45-kDa concealed antigen from the midgut membranes of Ornithodoros erraticus that induces lethal anti-tick immune responses in pigs.

Ornithodoros erraticus is an argasid tick that can transmit severe diseases such as human relapsing fever and African swine fever. In the search for a vaccine against this parasite, a crude extract of tick midgut membranes (GME) was obtained that in pigs and mice induced a protective response able to kill up to 80% of the nymphs in the first 72 h post-feeding and to reduce the fecundity of females by more than 50%. To identify the protective antigens, the GME was subjected to successive biochemical fractionations and the resulting simpler protein fractions were inoculated in pigs. A 45-kDa antigen, the so-called Oe45, was detected, purified and demonstrated to be responsible for the protection induced by the GME. Oe45 seems to be a membrane protein that is presumably expressed on the luminal membrane of midgut epithelial cells. Oe45 consists of at least two differently charged bands (cationic and neutral), which show antigenic cross-reactivity. The possibility that these bands might be different isoforms of the same protein is discussed. Although Oe45 is constitutively expressed at low levels throughout the trophogonic cycle, its expression is up-regulated by the ingestion of blood, as suggested by the higher levels observed between 6 and 72 h post-feeding.