A ticking time bomb hidden in plain sight.

The deer tick transmits nearly half of the known tick-borne pathogens in the United States, and its expanding geographic range increases the risk of human infection. To decrease the abundance of and infection risk from deer ticks, approaches that include vaccines for human use and for animal hosts are desired.

Tick salivary glycans - a sugar-coated tick bite.

Ticks are hematophagous arthropods that transmit disease-causing pathogens worldwide. Tick saliva deposited into the tick-bite site is composed of an array of immunomodulatory proteins that ensure successful feeding and pathogen transmission. These salivary proteins are often glycosylated, and glycosylation is potentially critical for the function of these proteins. Some salivary glycans are linked to the phenomenon of red meat allergy - an allergic response to red meat consumption in humans exposed to certain tick species. Tick salivary glycans are also invoked in the phenomenon of acquired tick resistance wherein non-natural host species exposed to tick bites develop an immune response that thwarts subsequent tick feeding. This review dwells on our current knowledge of these two phenomena, thematically linked by salivary glycans.

Specific mRNA lipid nanoparticles and acquired resistance to ticks.

Acquired resistance to ticks can develop when animals are repeatedly exposed to ticks. Recently, acquired resistance to Ixodes scapularis was induced in guinea pigs immunized with an mRNA-lipid nanoparticle vaccine (19ISP) encoding 19 I. scapularis proteins. Here, we evaluated specific mRNAs present in 19ISP to identify critical components associated with resistance to ticks. A lipid nanoparticle containing 12 mRNAs which included all the targets within 19ISP that elicited strong humoral responses in guinea pigs, was sufficient to induce robust resistance to ticks. Lipid nanoparticles containing fewer mRNAs or a single mRNA were not able to generate strong resistance to ticks. All lipid nanoparticles containing salp14 mRNA, however, were associated with increased redness at the tick bite site - which is the first manifestation of acquired resistance to ticks. This study demonstrates that more than one I. scapularis target within 19ISP is required for resistance to ticks, and that additional targets may also play a role in this process.

Croquemort elicits activation of the immune deficiency pathway in ticks.

The immune deficiency (IMD) pathway directs host defense in arthropods upon bacterial infection. In Pancrustacea, peptidoglycan recognition proteins sense microbial moieties and initiate nuclear factor-κB-driven immune responses. Proteins that elicit the IMD pathway in non-insect arthropods remain elusive. Here, we show that an Ixodes scapularis homolog of croquemort (Crq), a CD36-like protein, promotes activation of the tick IMD pathway. Crq exhibits plasma membrane localization and binds the lipid agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Crq regulates the IMD and jun N-terminal kinase signaling cascades and limits the acquisition of the Lyme disease spirochete B. burgdorferi. Additionally, nymphs silenced for crq display impaired feeding and delayed molting to adulthood due to a deficiency in ecdysteroid synthesis. Collectively, we establish a distinct mechanism for arthropod immunity outside of insects and crustaceans.

Dome1-JAK-STAT signaling between parasite and host integrates vector immunity and development.

Ancestral signaling pathways serve critical roles in metazoan development, physiology, and immunity. We report an evolutionary interspecies communication pathway involving a central Ixodes scapularis tick receptor termed Dome1, which acquired a mammalian cytokine receptor motif exhibiting high affinity for interferon-gamma (IFN-γ). Host-derived IFN-γ facilitates Dome1-mediated activation of the Ixodes JAK-STAT pathway. This accelerates tick blood meal acquisition and development while upregulating antimicrobial components. The Dome1-JAK-STAT pathway, which exists in most Ixodid tick genomes, regulates the regeneration and proliferation of gut cells-including stem cells-and dictates metamorphosis through the Hedgehog and Notch-Delta networks, ultimately affecting Ixodes vectorial competence. We highlight the evolutionary dependence of I. scapularis on mammalian hosts through cross-species signaling mechanisms that dually influence arthropod immunity and development.

Repeated Tick Infestations Impair Borrelia burgdorferi Transmission in a Non-Human Primate Model of Tick Feeding.

The blacklegged tick, Ixodes scapularis, is the predominant vector of Borrelia burgdorferi, the agent of Lyme disease in the USA. Natural hosts of I. scapularis such as Peromyscus leucopus are repeatedly infested by these ticks without acquiring tick resistance. However, upon repeated tick infestations, non-natural hosts such as guinea pigs, mount a robust immune response against critical tick salivary antigens and acquire tick resistance able to thwart tick feeding and Borrelia burgdorferi transmission. The salivary targets of acquired tick resistance could serve as vaccine targets to prevent tick feeding and the tick transmission of human pathogens. Currently, there is no animal model able to demonstrate both tick resistance and diverse clinical manifestations of Lyme disease. Non-human primates serve as robust models of human Lyme disease. By evaluating the responses to repeated tick infestation, this animal model could accelerate our ability to define the tick salivary targets of acquired resistance that may serve as vaccines to prevent the tick transmission of human pathogens. Towards this goal, we assessed the development of acquired tick resistance in non-human primates upon repeated tick infestations. We report that following repeated tick infestations, non-human primates do not develop the hallmarks of acquired tick resistance observed in guinea pigs. However, repeated tick infestations elicit immune responses able to impair the tick transmission of B. burgdorferi. A mechanistic understanding of the protective immune responses will provide insights into B. burgdorferi-tick-host interactions and additionally contribute to anti-tick vaccine discovery.

The environment, the tick, and the pathogen - It is an ensemble.

Ixodes scapularis is one of the predominant vectors of Borrelia burgdorferi, the agent of Lyme disease in the USA. The geographic distribution of I. scapularis, endemic to the northeastern and northcentral USA, is expanding as far south as Georgia and Texas, and northwards into Canada and poses an impending public health problem. The prevalence and spread of tick-borne diseases are influenced by the interplay of multiple factors including microbiological, ecological, and environmental. Molecular studies have focused on interactions between the tick-host and pathogen/s that determine the success of pathogen acquisition by the tick and transmission to the mammalian host. In this review we draw attention to additional critical environmental factors that impact tick biology and tick-pathogen interactions. With a focus on B. burgdorferi we highlight the interplay of abiotic factors such as temperature and humidity as well as biotic factors such as environmental microbiota that ticks are exposed to during their on- and off-host phases on tick, and infection prevalence. A molecular understanding of this ensemble of interactions will be essential to gain new insights into the biology of tick-pathogen interactions and to develop new approaches to control ticks and tick transmission of B. burgdorferi, the agent of Lyme disease.

Identification of novel conserved Ixodes vaccine candidates; a promising role for non-secreted salivary gland proteins.

Ixodes ricinus and Ixodes scapularis are the main vectors for the causative agents of Lyme borreliosis and a wide range of other pathogens. Repeated tick-bites are known to lead to tick rejection; a phenomenon designated as tick immunity. Tick immunity is mainly directed against tick salivary gland proteins (TSGPs) and has been shown to partially protect against experimental Lyme borreliosis. TSGPs recognized by antibodies from tick immune animals could therefore be interesting candidates for an anti-tick vaccine, which might also block pathogen transmission. To identify conserved Ixodes TSGPs that could serve as a universal anti-tick vaccine in both Europe and the US, a Yeast Surface Display containing salivary gland genes of nymphal I. ricinus expressed at 24, 48 and 72 h into tick feeding was probed with either sera from rabbits repeatedly exposed for 24 h to I. ricinus nymphal ticks and/or sera from rabbits immune to I. scapularis. Thus, we identified thirteen TSGP vaccine candidates, of which ten were secreted. For vaccination studies in rabbits, we selected six secreted TSGPs, five full length and one conserved peptide. None of these proteins hampered tick feeding. In contrast, vaccination of guinea pigs with four non-secreted TSGPs - two from the current and two from a previous human immunoscreening - did significantly reduce tick attachment and feeding. Therefore, non-secreted TSGPs appear to be involved in the development of tick immunity and are interesting candidates for an anti-tick vaccine.

Tick transmission of Borrelia burgdorferi to the murine host is not influenced by environmentally acquired midgut microbiota.

BACKGROUND: Ixodes scapularis is the predominant tick vector of Borrelia burgdorferi, the agent of Lyme disease, in the USA. Molecular interactions between the tick and B. burgdorferi orchestrate the migration of spirochetes from the midgut to the salivary glands-critical steps that precede transmission to the vertebrate host. Over the last decade, research efforts have invoked a potential role for the tick microbiome in modulating tick-pathogen interactions. RESULTS: Using multiple strategies to perturb the microbiome composition of B. burgdorferi-infected nymphal ticks, we observe that changes in the microbiome composition do not significantly influence B. burgdorferi migration from the midgut, invasion of salivary glands, or transmission to the murine host. We also show that within 24 and 48 h of the onset of tick feeding, B. burgdorferi spirochetes are within the peritrophic matrix and epithelial cells of the midgut in preparation for exit from the midgut. CONCLUSIONS: This study highlights two aspects of tick-spirochete interactions: (1) environmental bacteria associated with the tick do not influence spirochete transmission to the mammalian host and (2) the spirochete may utilize an intracellular exit route during migration from the midgut to the salivary glands, a strategy that may allow the spirochete to distance itself from microbiota in the midgut lumen effectively. This may explain in part, the inability of environment-acquired midgut microbiota to significantly influence spirochete transmission. Unraveling a molecular understanding of this exit strategy will be critical to gain new insights into the biology of the spirochete and the tick. Video Abstract.

Immunization of guinea pigs with cement extract induces resistance against Ixodes scapularis ticks.

As hematophagous parasites, many tick species are important vectors of medical and veterinary disease agents. Proteins found in tick saliva and midgut have been used with some success in immunizations of animal hosts against feeding ticks, and whole saliva has been used effectively in this capacity against Ixodes scapularis, the primary vector of tickborne pathogens in the United States. Tick saliva is a complex substance containing hundreds of proteins, and the identification of specific protective antigens is ongoing. We performed a series of experiments immunizing guinea pigs with extracts prepared from midgut or attachment cement collected from adult female I. scapularis followed by challenge with nymphs of the same species. Midgut extract did not induce protective immunity, while immunization with cement extract resulted in partial protection of hosts as evidenced by premature tick detachment and 34-41% reduction in tick engorgement weights. Proteomic characterization of I. scapularis cement was performed, demonstrating that the cement extract was compositionally different from tick saliva, and vitellogenin-like lipoproteins were the most abundant proteins in cement extract (>40%). Cement was also heavily enriched with lysozymes and defensins, including those originating from both the mammalian host as well as ticks. These results demonstrate that I. scapularis cement contains immunogenic components capable of stimulating host resistance against tick feeding. Because the cement is present at the tick-host interface for an extended period of time during the feeding process, these antigens present auspicious candidates for further evaluation and potential inclusion in an anti-tick vaccine.

Borrelia miyamotoi Meningoencephalitis in an Immunocompetent Patient.

Borrelia miyamotoi is an underdiagnosed cause of tick-borne illness in endemic regions and, in rare cases, causes neurological disease in immunocompetent patients. Here, we present a case of serologically confirmed Borrelia miyamotoi meningoencephalitis in an otherwise healthy patient who rapidly improved following initiation of antibiotic therapy.

Cytochrome b Drug Resistance Mutation Decreases Babesia Fitness in the Tick Stages But Not the Mammalian Erythrocytic Cycle.

Human babesiosis is an emerging tick-borne malaria-like illness caused by Babesia parasites following their development in erythrocytes. Here, we show that a mutation in the Babesia microti mitochondrial cytochrome b (Cytb) that confers resistance to the antibabesial drug ELQ-502 decreases parasite fitness in the arthropod vector. Interestingly, whereas the mutant allele does not affect B. microti fitness during the mammalian blood phase of the parasite life cycle and is genetically stable as parasite burden increases, ELQ-502-resistant mutant parasites developing in the tick vector are genetically unstable with a high rate of the wild-type allele emerging during the nymphal stage. Furthermore, we show that B. microti parasites with this mutation are transmitted from the tick to the host, raising the possibility that the frequency of Cytb resistance mutations may be decreased by passage through the tick vector, but could persist in the environment if present when ticks feed.

Tick immunity using mRNA, DNA and protein-based Salp14 delivery strategies.

Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms. salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays. This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.

Hygromycin A in the Lymelight.

Lyme disease, which is caused by the spirochete Borrelia burgdorferi, is on the rise. Current treatment relies on broad-spectrum antibiotics that perturb the gut microbiome. In a recent paper in Cell, Leimer et al. demonstrate the utility of a long-forgotten antibiotic, Hygromycin A, as a spirochete-specific antibacterial that is conducive to gut health.

mRNA vaccination induces tick resistance and prevents transmission of the Lyme disease agent.

Ixodes scapularis ticks transmit many pathogens that cause human disease, including Borrelia burgdorferi. Acquired resistance to I. scapularis due to repeated tick exposure has the potential to prevent tick-borne infectious diseases, and salivary proteins have been postulated to contribute to this process. We examined the ability of lipid nanoparticle­containing nucleoside-modified mRNAs encoding 19 I. scapularis salivary proteins (19ISP) to enhance the recognition of a tick bite and diminish I. scapularis engorgement on a host and thereby prevent B. burgdorferi infection. Guinea pigs were immunized with a 19ISP mRNA vaccine and subsequently challenged with I. scapularis. Animals administered 19ISP developed erythema at the bite site shortly after ticks began to attach, and these ticks fed poorly, marked by early detachment and decreased engorgement weights. 19ISP immunization also impeded B. burgdorferi transmission in the guinea pigs. The effective induction of local redness early after I. scapularis attachment and the inability of the ticks to take a normal blood meal suggest that 19ISP may be used either alone or in conjunction with traditional pathogen-based vaccines for the prevention of Lyme disease, and potentially other tick-borne infections.

The Lyme disease agent co-opts adiponectin receptor-mediated signaling in its arthropod vector.

Adiponectin-mediated pathways contribute to mammalian homeostasis; however, little is known about adiponectin and adiponectin receptor signaling in arthropods. In this study, we demonstrate that Ixodes scapularis ticks have an adiponectin receptor-like protein (ISARL) but lack adiponectin, suggesting activation by alternative pathways. ISARL expression is significantly upregulated in the tick gut after Borrelia burgdorferi infection, suggesting that ISARL signaling may be co-opted by the Lyme disease agent. Consistent with this, RNA interference (RNAi)-mediated silencing of ISARL significantly reduced the B. burgdorferi burden in the tick. RNA-seq-based transcriptomics and RNAi assays demonstrate that ISARL-mediated phospholipid metabolism by phosphatidylserine synthase I is associated with B. burgdorferi survival. Furthermore, the tick complement C1q-like protein 3 interacts with ISARL, and B. burgdorferi facilitates this process. This study identifies a new tick metabolic pathway that is connected to the life cycle of the Lyme disease spirochete.

Many countries around the world are seeing an increase in the number of patients diagnosed with Lyme disease, with often serious joint, heart, and neurologic complications. This illness is caused by species of 'spirochete' bacteria that live and multiply inside black-legged ticks, and get injected into mammals upon a bite. Ticks are not simply 'syringes' however, and a complex relationship is established between spirochetes and their host. This is particularly true since Lyme disease-causing bacteria such as Borrelia burgdorferi rely on ticks to obtain energy and nutrients. Tang, Cao et al. delved into these complex interactions by focusing on the molecular cascades (or pathways) involving adiponectin, a hormone essential for regulating sugar levels and processing fats. Analyses of gene and protein databases highlighted that ticks carry a receptor-like protein for adiponectin but not the hormone itself, suggesting that an alternative pathway is at play. This may involve B. burgdorferi, which gets its fats and sugars from its host. And indeed, experiments showed that ticks produced more of the adiponectin receptor-like protein when they carried B. burgdorferi; conversely, silencing the receptor reduced the number of surviving spirochetes inside the tick. Further exploration showed that the receptor mediates molecular cascades that help to process fat molecules; these are associated with spirochete survival. In addition, the receptor-like protein was activated by C1QL3, a 'complement 1q domain-contained' molecule which might be part of the tick energy-making or immune systems. Larger quantities of C1QL3 were found in ticks upon B. burgdorferi infection, suggesting that the spirochete facilitates an interaction that boosts activity of the adiponectin receptor-like protein. Overall, the work by Tang and Cao et al. revealed a new pathway which B. burgdorferi takes advantage of to infect their host and multiply. Targeting this molecular cascade could help to interfere with the life cycle of the spirochete, as well as fight Lyme disease and other insect-borne conditions.

Probing an Ixodes ricinus salivary gland yeast surface display with tick-exposed human sera to identify novel candidates for an anti-tick vaccine.

In Europe, Ixodes ricinus is the most important vector of human infectious diseases, most notably Lyme borreliosis and tick-borne encephalitis virus. Multiple non-natural hosts of I. ricinus have shown to develop immunity after repeated tick bites. Tick immunity has also been shown to impair B. burgdorferi transmission. Most interestingly, multiple tick bites reduced the likelihood of contracting Lyme borreliosis in humans. A vaccine that mimics tick immunity could therefore potentially prevent Lyme borreliosis in humans. A yeast surface display library (YSD) of nymphal I. ricinus salivary gland genes expressed at 24, 48 and 72 h into tick feeding was constructed and probed with antibodies from humans repeatedly bitten by ticks, identifying twelve immunoreactive tick salivary gland proteins (TSGPs). From these, three proteins were selected for vaccination studies. An exploratory vaccination study in cattle showed an anti-tick effect when all three antigens were combined. However, immunization of rabbits did not provide equivalent levels of protection. Our results show that YSD is a powerful tool to identify immunodominant antigens in humans exposed to tick bites, yet vaccination with the three selected TSGPs did not provide protection in the present form. Future efforts will focus on exploring the biological functions of these proteins, consider alternative systems for recombinant protein generation and vaccination platforms and assess the potential of the other identified immunogenic TSGPs.

Grappling with the tick microbiome.

Ixodes scapularis and Ixodes pacificus are the predominant vectors of multiple human pathogens, including Borrelia burgdorferi, one of the causative agents of Lyme disease in North America. Differences in the habitats and host preferences of these closely related tick species present an opportunity to examine key aspects of the tick microbiome. While advances in sequencing technologies have accelerated a descriptive understanding of the tick microbiome, molecular and mechanistic insights into the tick microbiome are only beginning to emerge. Progress is stymied by technical difficulties in manipulating the microbiome and by biological variables related to the life cycle of Ixodid ticks. This review highlights these challenges and examines avenues to understand the significance of the tick microbiome in tick biology.