Frankenbacteriosis targeting interactions between pathogen and symbiont to control infection in the tick vector.

Tick microbiota can be targeted for the control of tick-borne diseases such as human granulocytic anaplasmosis (HGA) caused by model pathogen, Anaplasma phagocytophilum. Frankenbacteriosis is inspired by Frankenstein and defined here as paratransgenesis of tick symbiotic/commensal bacteria to mimic and compete with tick-borne pathogens. Interactions between A. phagocytophilum and symbiotic Sphingomonas identified by metaproteomics analysis in Ixodes scapularis midgut showed competition between both bacteria. Consequently, Sphingomonas was selected for frankenbacteriosis for the control of A. phagocytophilum infection and transmission. The results showed that Franken Sphingomonas producing A. phagocytophilum major surface protein 4 (MSP4) mimic pathogen and reduce infection in ticks by competition and interaction with cell receptor components of infection. Franken Sphingomonas-MSP4 transovarial and trans-stadial transmission suggests that tick larvae with genetically modified Franken Sphingomonas-MSP4 could be produced in the laboratory and released in the field to compete and replace the wildtype populations with associated reduction in pathogen infection/transmission and HGA disease risks.

An Update of Evidence for Pathogen Transmission by Ticks of the Genus Hyalomma.

Current and likely future changes in the geographic distribution of ticks belonging to the genus Hyalomma are of concern, as these ticks are believed to be vectors of many pathogens responsible for human and animal diseases. However, we have observed that for many pathogens there are no vector competence experiments, and that the level of evidence provided by the scientific literature is often not sufficient to validate the transmission of a specific pathogen by a specific Hyalomma species. We therefore carried out a bibliographical study to collate the validation evidence for the transmission of parasitic, viral, or bacterial pathogens by Hyalomma spp. ticks. Our results show that there are very few validated cases of pathogen transmission by Hyalomma tick species.

First Expert Elicitation of Knowledge on Possible Drivers of Observed Increasing Human Cases of Tick-Borne Encephalitis in Europe.

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

Dermacentor reticulatus - a tick on its way from glacial refugia to a panmictic Eurasian population.

The ornate dog tick (Dermacentor reticulatus) shows a recently expanding geographic distribution. Knowledge on its intraspecific variability, population structure, rate of genetic diversity and divergence, including its evolution and geographic distribution, is crucial to understand its dispersal capacity. All such information would help to evaluate the potential risk of future spread of associated pathogens of medical and veterinary concern. A set of 865 D. reticulatus ticks was collected from 65 localities across 21 countries, from Portugal in the west to Kazakhstan and southern Russia in the east. Cluster analyses of 16 microsatellite loci were combined with nuclear (ITS2, 18S) and mitochondrial (12S, 16S, COI) sequence data to uncover the ticks' population structures and geographical patterns. Approximate Bayesian computation was applied to model evolutionary relationships among the found clusters. Low variability and a weak phylogenetic signal showing an east-west cline were detected both for mitochondrial and nuclear sequence markers. Microsatellite analyses revealed three genetic clusters, where the eastern and western cluster gradient was supplemented by a third, northern cluster. Alternative scenarios could explain such a tripartite population structure by independent formation of clusters in separate refugia, limited gene flow connected with isolation by distance causing a "bipolar pattern", and the northern cluster deriving from admixture between the eastern and western populations. The best supported demographic scenario of this tick species indicates that the northern cluster derived from admixture between the eastern and western populations 441 (median) to 224 (mode) generations ago, suggesting a possible link with the end of the Little Ice Age in Europe.

The control of Hyalomma ticks, vectors of the Crimean-Congo hemorrhagic fever virus: Where are we now and where are we going?

At a time of major global, societal, and environmental changes, the shifting distribution of pathogen vectors represents a real danger in certain regions of the world as generating opportunities for emergency. For example, the recent arrival of the Hyalomma marginatum ticks in southern France and the concurrent appearance of cases of Crimean-Congo hemorrhagic fever (CCHF)-a disease vectored by this tick species-in neighboring Spain raises many concerns about the associated risks for the European continent. This context has created an urgent need for effective methods for control, surveillance, and risk assessment for ticks and tick-borne diseases with a particular concern regarding Hyalomma sp. Here, we then review the current body of knowledge on different methods of tick control-including chemical, biological, genetical, immunological, and ecological methods-and the latest developments in the field, with a focus on those that have been tested against ticks from the genus Hyalomma. In the absence of a fully and unique efficient approach, we demonstrated that integrated pest management combining several approaches adapted to the local context and species is currently the best strategy for tick control together with a rational use of acaricide. Continued efforts are needed to develop and implement new and innovative methods of tick control.

Spatial and Temporal Circulation of Babesia caballi and Theileria equi in France Based on Seven Years of Serological Data.

Caused by two blood parasites, Babesia caballi and Theileria equi, equine piroplasmosis is a tick-borne disease that poses major health and economic issues for the equine industry. Our objective was to gain insight into the spatio-temporal variations of parasite circulation in France, where the disease is known to be enzootic, but has been the subject of few studies. Seroprevalence was assessed for each parasite thanks to 16,127 equine sera obtained between 1997 and 2003 from all over France and analysed through complement fixation tests. Results indicated that 13.2% (5-27% depending on the region) of horses were seropositive for T. equi and 9.5% (3-25%) for B. caballi. Regardless of the year, horses from the southern regions of France were the most affected by B. caballi or T. equi infection, while the proportion of horses having antibodies against T. equi increased over time. These results highlight the heterogeneity of the circulation of both piroplasms, which may be linked with ecological diversity and vector distribution. Our data provide baseline information regarding the sero-epidemiology of B. caballi and T. equi infection in horses in France, making it now possible to select regions for future studies on risk factors, and design and implement effective targeted measures against equine piroplasms.

Eco-epidemiology of equine piroplasmosis and its associated tick vectors in Europe: A systematic literature review and a meta-analysis of prevalence.

When studying a vector-borne disease, an eco-epidemiological approach is vital for a comprehensive understanding of how the pathogen circulates amongst populations. Equine piroplasmosis (EP), a tick-borne disease caused by the protozoans Babesia caballi and Theileria equi, is endemic in the Mediterranean basin of Europe and causes both animal health and economic issues for the equine sector. With no vaccine available, defining the episystem of the disease can help to identify which components of the host-pathogen-vector-environment system to target to improve preventive measures. In this systematic literature review, we collected relevant data on the eco-epidemiology of EP in Europe. The 62 studies remaining after the selection procedure explored potential vectors, indicators of parasite circulation and putative risk factors of EP. Eight hard tick species were identified as potential vectors of one or both piroplasm species. Meta-analyses were then conducted on prevalence and seroprevalence data in equids in European countries, demonstrating an estimated seroprevalence of 30% and 8% and prevalence of 25% and 2% for T. equi and B. caballi, respectively. Finally, herd management practices and environmental risk factors analysed in studies showed no real consensus between studies, but revealed a general trend highlighting age and exposure to ticks as risk factors, and vaccination as a protective factor. Through this study, we point out that only a few studies have focused on disease management practices and even fewer have studied the effect of environmental parameters on equid infections. Further investigation in these areas is required to better characterize the eco-epidemiology of EP and risk factors associated with this disease.

Investigation of Transovarial Transmission of Bartonella henselae in Rhipicephalus sanguineus sensu lato Ticks Using Artificial Feeding.

Bartonella henselae is a slow-growing, Gram-negative bacterium that causes cat scratch disease in humans. A transstadial transmission of the bacteria from larvae to nymphs of Rhipicephalus sanguineus sensu lato (s.l.) ticks, suspected to be a potential vector of the bacteria, has been previously demonstrated. The present study aims to investigate transovarial transmission of B. henselae from R. sanguineus s.l. adults to their instars. Adult ticks (25 males and 25 females) were fed through an artificial feeding system on B. henselae-infected goat blood for 14 days, and 300 larvae derived from the experimentally B. henselae-infected females were fed on noninfected goat blood for 7 days. Nested PCR and culture were used to detect and isolate B. henselae in ticks and blood samples. Bartonella henselae DNA was detected in midguts, salivary glands, and carcasses of the semi-engorged adults and pooled tick feces (during feeding and post-feeding periods). After the oviposition period, B. henselae DNA was detected in salivary glands of females (33.3%), but not in pooled eggs or larvae derived from the infected females. However, B. henselae DNA was detected by nested PCR from the blood sample during larval feeding, while no viable B. henselae was isolated by culture. According to our findings, following infected blood meal, B. henselae could remain in the tick midguts, move to other tissues including salivary glands, and then be shed through tick feces with limited persistency. The presence of bacterial DNA in the blood during larval feeding shows the possibility of transovarial transmission of B. henselae in R. sanguineus s.l. ticks.

Experimental Infection of Ticks: An Essential Tool for the Analysis of Babesia Species Biology and Transmission.

Babesiosis is one of the most important tick-borne diseases in veterinary health, impacting mainly cattle, equidae, and canidae, and limiting the development of livestock industries worldwide. In humans, babesiosis is considered to be an emerging disease mostly due to Babesia divergens in Europe and Babesia microti in America. Despite this importance, our knowledge of Babesia sp. transmission by ticks is incomplete. The complexity of vectorial systems involving the vector, vertebrate host, and pathogen, as well as the complex feeding biology of ticks, may be part of the reason for the existing gaps in our knowledge. Indeed, this complexity renders the implementation of experimental systems that are as close as possible to natural conditions and allowing the study of tick-host-parasite interactions, quite difficult. However, it is unlikely that the development of more effective and sustainable control measures against babesiosis will emerge unless significant progress can be made in understanding this tripartite relationship. The various methods used to date to achieve tick transmission of Babesia spp. of medical and veterinary importance under experimental conditions are reviewed and discussed here.

Of fungi and ticks: Morphological and molecular characterization of fungal contaminants of a laboratory-reared Ixodes ricinus colony.

Establishing and maintaining tick colonies in the laboratory is essential for studying their biology and pathogen transmission, or for the development of new tick control methods. Due to their requirement for very high humidity, these laboratory-bred colonies are frequently subject to fungal contamination. In the present study, we aimed to identify the fungal species that contaminated a laboratory-reared colony of Ixodes ricinus through microscopic observation and molecular identification. We identified three different taxa isolated from the ticks: Aspergillus parasiticus, Penicillium steckii, and Scopulariopsis brevicaulis. These three species are usually regarded as environmental saprophytic molds but both direct and indirect evidence suggest that they could also be considered as entomopathogenic fungi. Although we do not have any direct evidence that the fungi isolated from I. ricinus in this study could cause lethal infections in ticks, we observed that once infected, heavy fungal growth coupled with very high mortality rates suggest that studying the entomopathogenic potential of these fungi could be relevant to biological tick control.

Transmission of Bartonella henselae within Rhipicephalus sanguineus: Data on the Potential Vector Role of the Tick.

Bartonella henselae is a fastidious intraerythrocytic, gram-negative bacteria that causes cat scratch disease in humans. Ixodes ricinus has been confirmed to be a competent vector of B. henselae, and some indirect evidences from clinical cases and epidemiological studies also suggested that some other tick species, including Rhipicephalus sanguineus, may transmit the bacteria. B. henselae has been detected in R. sanguineus but no experimental investigations have been performed to evaluate the vector competency of this tick species regarding B. henselae transmission. To this end, this work aimed to assess the transstadial transmission of B. henselae between larvae and nymphs of R. sanguineus as well as transmission by nymphs infected at the larval stage. Four hundred B. henselae negative larvae were fed with B. henselae-infected blood by using an artificial membrane feeding system. After five days of feeding, B. henselae was detected by PCR in 57.1% (8/14) of engorged larval pools, 66.7% (4/6) of semi-engorged larval pools, and 66.7% (2/3) of larval feces pools. After molting, B. henselae DNA was also detected in 10% (1/10) of nymph pools, but not in tick feces. After a pre-fed step of nymphs infected at the larval stage on non-infected blood meal, B. henselae was detected by PCR in blood sample from the feeder, but no Bartonella colonies could be obtained from culture. These findings showed that B. henselae could be transstadial transmitted from R. sanguineus larvae to nymphs, and also suggest that these nymphs may retransmitted the bacteria through the saliva during their blood meal. This is the first study that validated the artificial membrane feeding system for maintaining R. sanguineus tick colony. It shows the possibility of transstadial transmission of B. henselae from R. sanguineus larvae to nymphs.

Multiple Antigenic Peptide-Based Vaccines Targeting Ixodes ricinus Neuropeptides Induce a Specific Antibody Response but Do Not Impact Tick Infestation.

Synthetic peptide vaccines were designed to target the neuropeptides innervating Ixodes ricinus salivary glands and hindgut and they were tested for their capacity to afford protective immunity against nymphs or larvae and Anaplasma phagocytophilum-infected nymph infestation, in mice and sheep, respectively. In both models, the assembly of SIFamide (SIFa) or myoinhibitory peptide (MIP) neuropeptides into multiple antigenic peptide constructs (MAPs) elicited a robust IgG antibody response following immunization. Nevertheless, no observable detrimental impact on nymphs was evidenced in mice, and, unfortunately, the number of engorged nymphs on sheep was insufficient for firm conclusions to be drawn, including for bacterial transmission. Regarding larvae, while vaccination of the sheep did not globally diminish tick feeding success or development, analyses of animals at the individual level revealed a negative correlation between anti-SIFa and MIP antibody levels and larva-to-nymph molting success for both antigens. Our results provide a proof of principle and precedent for the use of MAPs for the induction of immunity against tick peptide molecules. Although the present study did not provide the expected level of protection, it inaugurates a new strategy for protection against ticks based on the immunological targeting of key components of their nervous system.

Failed Disruption of Tick Feeding, Viability, and Molting after Immunization of Mice and Sheep with Recombinant Ixodes ricinus Salivary Proteins IrSPI and IrLip1.

To identify potential vaccine candidates against Ixodes ricinus and tick-borne pathogen transmission, we have previously sequenced the salivary gland transcriptomes of female ticks infected or not with Bartonella henselae. The hypothesized potential of both IrSPI (I. ricinus serine protease inhibitor) and IrLip1 (I. ricinus lipocalin 1) as protective antigens decreasing tick feeding and/or the transmission of tick-borne pathogens was based on their presumed involvement in dampening the host immune response to tick feeding. Vaccine endpoints included tick larval and nymphal mortality, feeding, and molting in mice and sheep. Whether the antigens were administered individually or in combination, the vaccination of mice or sheep elicited a potent antigen-specific antibody response. However, and contrary to our expectations, vaccination failed to afford protection against the infestation of mice and sheep by I. ricinus nymphs and larvae, respectively. Rather, vaccination with IrSPI and IrLip1 appeared to enhance tick engorgement and molting and decrease tick mortality. To the best of our knowledge, these observations represent the first report of induction of vaccine-mediated enhancement in relation to anti-tick vaccination.

Putative Role of Arthropod Vectors in African Swine Fever Virus Transmission in Relation to Their Bio-Ecological Properties.

African swine fever (ASF) is one of the most important diseases in Suidae due to its significant health and socioeconomic consequences and represents a major threat to the European pig industry, especially in the absence of any available treatment or vaccine. In fact, with its high mortality rate and the subsequent trade restrictions imposed on affected countries, ASF can dramatically disrupt the pig industry in afflicted countries. In September 2018, ASF was unexpectedly identified in wild boars from southern Belgium in the province of Luxembourg, not far from the Franco-Belgian border. The French authorities rapidly commissioned an expert opinion on the risk of ASF introduction and dissemination into metropolitan France. In Europe, the main transmission routes of the virus comprise direct contact between infected and susceptible animals and indirect transmission through contaminated material or feed. However, the seasonality of the disease in some pig farms in Baltic countries, including outbreaks in farms with high biosecurity levels, have led to questions on the possible involvement of arthropods in the transmission of the virus. This review explores the current body of knowledge on the most common arthropod families present in metropolitan France. We examine their potential role in spreading ASF-by active biological or mechanical transmission or by passive transport or ingestion-in relation to their bio-ecological properties. It also highlights the existence of significant gaps in our knowledge on vector ecology in domestic and wild boar environments and in vector competence for ASFV transmission. Filling these gaps is essential to further understanding ASF transmission in order to thus implement appropriate management measures.

Experimental Ixodes ricinus-Sheep Cycle of Anaplasma phagocytophilum NV2Os Propagated in Tick Cell Cultures.

The causative agent of tick-borne fever and human granulocytic anaplasmosis, Anaplasma phagocytophilum, is transmitted by Ixodes ricinus, and is currently considered an emerging disease throughout Europe. In this study, we established a model of A. phagocytophilum sheep infection and I. ricinus transmission using the European Norway variant 2 ovine strain (NV2Os) propagated in both IDE8 and ISE6 tick cells. Two sheep were inoculated with IDE8 tick cells infected with NV2Os. Both sheep developed A. phagocytophilum infection as determined by qPCR and PCR, the presence of fever 4 days post inoculation (dpi), the observation of morulae in granulocytes at 6 dpi, and the detection of A. phagocytophilum antibodies at 14 dpi. A. phagocytophilum was detected by PCR in skin, lung, small intestine, liver, spleen, uterus, bone marrow, and mesenteric lymph node from necropsies performed at 14 and 15 dpi. One sheep was infested during the acute phase of infection with I. ricinus nymphs from a pathogen-free colony. After molting, A. phagocytophilum transstadial transmission in ticks was validated with qPCR positive bacterial detection in 80% of salivary glands and 90% of midguts from female adults. Infected sheep blood collected at 14 dpi was demonstrated to be able to infect ISE6 tick cells, thus enabling the infection of two additional naive sheep, which then went on to develop similar clinical signs to the sheep infected previously. One of the sheep remained persistently infected until 115 dpi when it was euthanized, and transmitted bacteria to 70 and 2.7% of nymphs engorged as larvae during the acute and persistent infection stages, respectively. We then demonstrated that these infected nymphs were able to transmit the bacteria to one of two other naive infested sheep. As expected, when I. ricinus females were engorged during the acute phase of infection, no A. phagocytophilum transovarial transmission was detected. The development of this new experimental model will facilitate future research on this tick-borne bacterium of increasing importance, and enable the evaluation of any new tick/transmission control strategies.

The Immunomodulatory Effect of IrSPI, a Tick Salivary Gland Serine Protease Inhibitor Involved in Ixodes ricinus Tick Feeding.

Ticks are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Hard tick feeding is a slow process-taking up to several days-and necessitates extended control over the host response. The success of the feeding process depends upon injection of tick saliva, which not only controls host hemostasis and wound healing, but also subverts the host immune response to avoid tick rejection that creates a favorable niche for the survival and propagation of diverse tick-borne pathogens. Here, we report on the molecular and biochemical features and functions of an Ixodes ricinus serine protease inhibitor (IrSPI). We characterize IrSPI as a Kunitz elastase inhibitor that is overexpressed in several tick organs-especially salivary glands-during blood-feeding. We also demonstrated that when IrSPI is injected into the host through saliva, it had no impact on tissue factor pathway-induced coagulation, fibrinolysis, endothelial cell angiogenesis or apoptosis, but the protein exhibits immunomodulatory activity. In particular, IrSPI represses proliferation of CD4+ T lymphocytes and proinflammatory cytokine secretion from both splenocytes and macrophages. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in I. ricinus feeding.

The Essential Role of Tick Salivary Glands and Saliva in Tick Feeding and Pathogen Transmission.

As long-term pool feeders, ticks have developed myriad strategies to remain discreetly but solidly attached to their hosts for the duration of their blood meal. The critical biological material that dampens host defenses and facilitates the flow of blood-thus assuring adequate feeding-is tick saliva. Saliva exhibits cytolytic, vasodilator, anticoagulant, anti-inflammatory, and immunosuppressive activity. This essential fluid is secreted by the salivary glands, which also mediate several other biological functions, including secretion of cement and hygroscopic components, as well as the watery component of blood as regards hard ticks. When salivary glands are invaded by tick-borne pathogens, pathogens may be transmitted via saliva, which is injected alternately with blood uptake during the tick bite. Both salivary glands and saliva thus play a key role in transmission of pathogenic microorganisms to vertebrate hosts. During their long co-evolution with ticks and vertebrate hosts, microorganisms have indeed developed various strategies to exploit tick salivary molecules to ensure both acquisition by ticks and transmission, local infection and systemic dissemination within the vertebrate host.

Serine Protease Inhibitors in Ticks: An Overview of Their Role in Tick Biology and Tick-Borne Pathogen Transmission.

New tick and tick-borne pathogen control approaches that are both environmentally sustainable and which provide broad protection are urgently needed. Their development, however, will rely on a greater understanding of tick biology, tick-pathogen, and tick-host interactions. The recent advances in new generation technologies to study genomes, transcriptomes, and proteomes has resulted in a plethora of tick biomacromolecular studies. Among these, many enzyme inhibitors have been described, notably serine protease inhibitors (SPIs), whose importance in various tick biological processes is only just beginning to be fully appreciated. Among the multiple active substances secreted during tick feeding, SPIs have been shown to be directly involved in regulation of inflammation, blood clotting, wound healing, vasoconstriction and the modulation of host defense mechanisms. In light of these activities, several SPIs were examined and were experimentally confirmed to facilitate tick pathogen transmission. In addition, to prevent coagulation of the ingested blood meal within the tick alimentary canal, SPIs are also involved in blood digestion and nutrient extraction from the meal. The presence of SPIs in tick hemocytes and their involvement in tick innate immune defenses have also been demonstrated, as well as their implication in hemolymph coagulation and egg development. Considering the involvement of SPIs in multiple crucial aspects of tick-host-pathogen interactions, as well as in various aspects of the tick parasitic lifestyle, these molecules represent highly suitable and attractive targets for the development of effective tick control strategies. Here we review the current knowledge regarding this class of inhibitors in tick biology and tick-borne pathogen transmission, and their potential as targets for future tick control trials.

The Tick Microbiome: Why Non-pathogenic Microorganisms Matter in Tick Biology and Pathogen Transmission.

Ticks are among the most important vectors of pathogens affecting humans and other animals worldwide. They do not only carry pathogens however, as a diverse group of commensal and symbiotic microorganisms are also present in ticks. Unlike pathogens, their biology and their effect on ticks remain largely unexplored, and are in fact often neglected. Nonetheless, they can confer multiple detrimental, neutral, or beneficial effects to their tick hosts, and can play various roles in fitness, nutritional adaptation, development, reproduction, defense against environmental stress, and immunity. Non-pathogenic microorganisms may also play a role in driving transmission of tick-borne pathogens (TBP), with many potential implications for both human and animal health. In addition, the genetic proximity of some pathogens to mutualistic symbionts hosted by ticks is evident when studying phylogenies of several bacterial genera. The best examples are found within members of the Rickettsia, Francisella, and Coxiella genera: while in medical and veterinary research these bacteria are traditionally recognized as highly virulent vertebrate pathogens, it is now clear to evolutionary ecologists that many (if not most) Coxiella, Francisella, and Rickettsia bacteria are actually non-pathogenic microorganisms exhibiting alternative lifestyles as mutualistic ticks symbionts. Consequently, ticks represent a compelling yet challenging system in which to study microbiomes and microbial interactions, and to investigate the composition, functional, and ecological implications of bacterial communities. Ultimately, deciphering the relationships between tick microorganisms as well as tick symbiont interactions will garner invaluable information, which may aid in the future development of arthropod pest and vector-borne pathogen transmission control strategies.