Disruption of bacterial interactions and community assembly in Babesia-infected Haemaphysalis longicornis following antibiotic treatment.

BACKGROUND: A previous study highlighted the role of antibiotic-induced dysbiosis in the tick microbiota, facilitating the transstadial transmission of Babesia microti from nymph to adult in Haemaphysalis longicornis. This study builds on previous findings by analyzing sequence data from an earlier study to investigate bacterial interactions that could be linked to enhanced transstadial transmission of Babesia in ticks. The study employed antibiotic-treated (AT) and control-treated (CT) Haemaphysalis longicornis ticks to investigate shifts in microbial community assembly. Network analysis techniques were utilized to assess bacterial interactions, comparing network centrality measures between AT and CT groups, alongside studying network robustness and connectivity loss. Additionally, functional profiling was conducted to evaluate metabolic diversity in response to antibiotic treatment. RESULTS: The analysis revealed notable changes in microbial community assembly in response to antibiotic treatment. Antibiotic-treated (AT) ticks displayed a greater number of connected nodes but fewer correlations compared to control-treated (CT) ticks, indicating a less interactive yet more connected microbial community. Network centrality measures such as degree, betweenness, closeness, and eigenvector centrality, differed significantly between AT and CT groups, suggesting alterations in local network dynamics due to antibiotic intervention. Coxiella and Acinetobacter exhibited disrupted connectivity and roles, with the former showing reduced interactions in AT group and the latter displaying a loss of connected nodes, emphasizing their crucial roles in microbial network stability. Robustness tests against node removal showed decreased stability in AT networks, particularly under directed attacks, confirming a susceptibility of the microbial community to disturbances. Functional profile analysis further indicated a higher diversity and richness in metabolic capabilities in the AT group, reflecting potential shifts in microbial metabolism as a consequence of antimicrobial treatment. CONCLUSIONS: Our findings support that bacterial interaction traits boosting the transstadial transmission of Babesia could be associated with reduced colonization resistance. The disrupted microbial interactions and decreased network robustness in AT ticks suggest critical vulnerabilities that could be targeted for managing tick-borne diseases.

Microbiota-driven vaccination in soft ticks: Implications for survival, fitness and reproductive capabilities in Ornithodoros moubata.

The Ornithodoros moubata (Om) soft tick, a vector for diseases like tick-borne human relapsing fever and African swine fever, poses challenges to conventional control methods. With diminishing insecticide efficacy, harnessing the tick's microbiota through innovative approaches like microbiota-driven vaccination emerges as a promising strategy for sustainable and targeted disease control. This study investigated the intricate relationship between Pseudomonas, a keystone taxon in the Om microbiome, and its impact on tick fitness, microbiome structure and network dynamics. Utilizing in silico analyses and empirical vaccination experiments, the role of Pseudomonas within microbial networks in the tick midguts (MG) and salivary glands (SG) of Om was studied. Additionally, the consequences of anti-microbiota vaccines targeting Pseudomonas and Lactobacillus on tick fitness, microbiome diversity and community assembly were explored. The result of the study shows that in Om, Pseudomonas plays a central role in microbial networks, influencing keystone species despite being categorized as peripheral (interacting with 47 different taxa, 13 of which are keystone species). Anti-microbiota vaccination targeting Pseudomonas and Lactobacillus yields distinct effects on tick fitness, with Pseudomonas vaccination significantly impacting female tick survival, while Lactobacillus significantly reduced oviposition and fertility. Microbiome changes post-vaccination reveal diversity alterations, emphasizing the impact of vaccine choice. Community assembly dynamics and network robustness analyses highlight Pseudomonas' pivotal role, in influencing topological features and network resilience. The findings of the study provide comprehensive insights into the intricate dynamics of Om microbial networks and the potential of targeted microbiota-driven vaccines for tick control.

First data on cholesterol metabolism in Ornithodoros argasid ticks: Molecular and functional characterization of the N-terminal domain of Niemann-Pick C1 proteins.

Cholesterol is a molecule vital for tick physiology, but ticks cannot synthesize it and rely on dietary cholesterol. Therefore, tick proteins involved in cholesterol absorption and transport, such as the Niemann-Pick type C1 domain-containing (NPC1) proteins, are promising targets for anti-tick vaccine development. The aim of this study was to assess the structure, function, and protective efficacy of the NPC1 orthologues identified previously in the midgut transcriptomes of argasid ticks Ornithodoros erraticus and Ornithodoros moubata. For this purpose, their corresponding cDNA coding sequences were cloned and sequenced, their secondary and 3D structures were predicted, and their function was evaluated through RNAi-mediated gene knockdown and in vitro feeding on blood supplemented with ezetimibe, which inhibits cholesterol binding by NPC1 proteins. Subsequently, the protective efficacy of a recombinant form of NPC1 from O. moubata (rOmNPC1) was tested in a rabbit vaccine trial. While inhibiting cholesterol absorption with ezetimibe resulted in up to 77 % mortality in adult O. moubata, NPC1 gene knockdown and vaccination with rOmNPC1 decreased female reproductive performance in terms of the number and fertility of laid eggs. This study presents the initial molecular and functional insights into NPC1 proteins in soft ticks and supports the hypothesis that disrupting cholesterol metabolism diminishes tick viability and reproduction, rendering Niemann-Pick type C1 domain-containing proteins promising targets for drugs or vaccines.

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.

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.

Characterization of two Cuban colonies of Rhipicephalus microplus ticks.

Rhipicephalus microplus (Canestrini, 1888) is one of the species with medical and economic relevance that has been reported in the list of Cuban tick species. Some morphological characterizations about the R. microplus species in Cuba have been published; however, molecular studies are lacking. Molecular phylogenetic analyses have grouped R. annulatus, R. australis and three clades of R. microplus in a complex named R. microplus. The present study aimed to characterize two R. microplus tick isolates, established as colonies at the Cuban National Laboratory of Parasitology. Morphological characterization of adult specimens was carried out by using Scanning Electron Microscopy. The sequences of mitochondrial genes: 12S rRNA, 16S rRNA and the subunit I of cytochrome c oxidase (coxI) and one nuclear sequence: internal transcribed spacer 2 (its2) were used for phylogenetic analyses. The life cycle under laboratory conditions for both isolates was also characterized. Tick specimens of both colonies showed morphological characteristics comparable with those distinctive for the R. microplus species. Phylogenies based on mitochondrial gene sequences identified congruently the Cuban tick colonies within the clade A of R. microplus. The life cycle of both isolates under laboratory conditions lasted 65 ± 5 days and the reproductive performance of female ticks of each colony also were similar with approximately 2500 larvae obtained from fully engorged female ticks. This study constitutes the first molecular characterization of ticks from the R. microplus species in Cuba.