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Artificial tick feeding systems (ATFS) can be used to study tick biology and tick-pathogen interactions. Due to the long feeding duration of hard ticks, antibiotics are commonly added to the in vitro blood meal to prevent the blood from decaying. This may affect the ticks’ microbiome, including mutualistic bacteria that play an important role in tick biology. This effect was examined by the consecutive feeding of Ixodes ricinus larvae, nymphs, and adults in vitro with and without the supplementation of gentamicin and in parallel on calves. DNA extracted from unfed females was analyzed by 16S rRNA sequencing. The abundance of Candidatus Midichloria mitochondrii, Rickettsia helvetica and Spiroplasma spp. was measured by qPCR in unfed larvae, nymphs, and adults. Larvae and nymphs fed on calves performed significantly better compared to both in vitro groups. Adults fed on blood supplemented with gentamicin and B vitamins had a higher detachment proportion and weight compared to the group fed with B vitamins but without gentamicin. The detachment proportion and weights of females did not differ significantly between ticks fed on calves and in vitro with gentamicin, but the fecundity was significantly higher in ticks fed on calves. 16S rRNA sequencing showed a higher microbiome species richness in ticks fed on calves compared to ticks fed in vitro. A shift in microbiome composition, with Ca. Midichloria mitochondrii as dominant species in females fed as juveniles on calves and R. helvetica as the most abundant species in females previously fed in vitro was observed. Females fed in vitro without gentamicin showed significant lower loads of Ca. M. mitochondrii compared to females fed in vitro with gentamicin and ticks fed on calves. Spiroplasma spp. were exclusively detected in female ticks fed on cattle by qPCR, but 16S rRNA sequencing results also showed a low abundance in in vitro females exposed to gentamicin. In conclusion, the employed feeding method and gentamicin supplementation affected the ticks’ microbiome composition and fecundity. Since these changes may have an impact on tick biology and vector competence, they should be taken into account in studies employing ATFS. [ABSTRACT FROM AUTHOR]

Anti-tick vaccines have the potential to be an environmentally friendly and cost-effective option for tick control. In vaccine development, the identification of efficacious antigens forms the major bottleneck. In this study, the efficacy of immunization with recombinant ferritin 2 and native tick protein extracts (TPEs) against Ixodes ricinus infestations in calves was assessed in two immunization experiments. In the first experiment, each calf (n = 3) was immunized twice with recombinant ferritin 2 from I. ricinus (IrFER2), TPE consisting of soluble proteins from the internal organs of partially fed I. ricinus females, or adjuvant, respectively. In the second experiment, each calf (n = 4) was immunized with protein extracts from the midgut (ME) of partially fed females, the salivary glands (SGE) of partially fed females, a combination of ME and SGE, or adjuvant, respectively. Two weeks after the booster immunization, calves were challenged with 100 females and 200 nymphs. Blood was collected from the calves before the first and after the second immunization and fed to I. ricinus females and nymphs using an in vitro artificial tick feeding system. The two calves vaccinated with whole TPE and midgut extract (ME) showed hyperemia on tick bite sites 2 days post tick infestation and exudative blisters were observed in the ME-vaccinated animal, signs that were suggestive of a delayed type hypersensitivity (DTH) reaction. Significantly fewer ticks successfully fed on the three animals vaccinated with TPE, SGE, or ME. Adults fed on the TPE and ME vaccinated animals weighed significantly less. Tick feeding on the IrFER2 vaccinated calf was not impaired. The in vitro feeding of serum or fresh whole blood collected from the vaccinated animals did not significantly affect tick feeding success. Immunization with native I. ricinus TPEs thus conferred a strong immune response in calves and significantly reduced the feeding success of both nymphs and adults. In vitro feeding of serum or blood collected from vaccinated animals to ticks did not affect tick feeding, indicating that antibodies alone were not responsible for the observed vaccine immunity. [ABSTRACT FROM AUTHOR]

Ixodes ricinus transmits bacterial, protozoal and viral pathogens, causing disease and forming an increasing health concern in Europe. ANTIDotE is an European Commission funded consortium of seven institutes, which aims to identify and characterize tick proteins involved in feeding and pathogen transmission. The knowledge gained will be used to develop and evaluate anti-tick vaccines that may prevent multiple human tick-borne diseases. Strategies encompassing anti-tick vaccines to prevent transmission of pathogens to humans, animals or wildlife will be developed with relevant stakeholders with the ultimate aim of reducing the incidence of tick-borne diseases in humans. [ABSTRACT FROM AUTHOR]

Ixodes ricinus is the main vector of tick-borne diseases in Europe. An immunization trial of calves with soluble extracts of I. ricinus salivary glands (SGE) or midgut (ME) previously showed a strong response against subsequent tick challenge, resulting in diminished tick feeding success. Immune sera from these trials were used for the co-immunoprecipitation of tick tissue extracts, followed by LC-MS/MS analyses. This resulted in the identification of 46 immunodominant proteins that were differentially recognized by the serum of immunized calves. Some of these proteins had previously also drawn attention as potential anti-tick vaccine candidates using other approaches. Selected proteins were studied in more detail by measuring their relative expression in tick tissues and RNA interference (RNAi) studies. The strongest RNAi phenotypes were observed for MG6 (A0A147BXB7), a protein containing eight fibronectin type III domains predominantly expressed in tick midgut and ovaries of feeding females, and SG2 (A0A0K8RKT7), a glutathione-S-transferase that was found to be upregulated in all investigated tissues upon feeding. The results demonstrated that co-immunoprecipitation of tick proteins with host immune sera followed by protein identification using LC-MS/MS is a valid approach to identify antigen–antibody interactions, and could be integrated into anti-tick vaccine discovery pipelines. [ABSTRACT FROM AUTHOR]

The hard tick Ixodes ricinus is an obligate hematophagous arthropod and the main vector for several zoonotic diseases. The life cycle of this three-host tick species was completed for the first time in vitro by feeding all consecutive life stages using an artificial tick feeding system (ATFS) on heparinized bovine blood supplemented with glucose, adenosine triphosphate, and gentamicin. Relevant physiological parameters were compared to ticks fed on cattle (in vivo). All in vitro feedings lasted significantly longer and the mean engorgement weight of F0 adults and F1 larvae and nymphs was significantly lower compared to ticks fed in vivo. The proportions of engorged ticks were significantly lower for in vitro fed adults and nymphs as well, but higher for in vitro fed larvae. F1-females fed on blood supplemented with vitamin B had a higher detachment proportion and engorgement weight compared to F1-females fed on blood without vitamin B, suggesting that vitamin B supplementation is essential in the artificial feeding of I. ricinus ticks previously exposed to gentamicin. [ABSTRACT FROM AUTHOR]

Borrelia burgdorferi sensu lato , the causative agents of Lyme borreliosis, are transmitted by Ixodes ticks. Tick saliva proteins are instrumental for survival of both the vector and spirochete and have been investigated as targets for vaccine targeting the vector. In Europe, the main vector for Lyme borreliosis is Ixodes ricinus , which predominantly transmits Borrelia afzelii. We here investigated the differential production of I. ricinus tick saliva proteins in response to feeding and B. afzelii infection. Label-free Quantitative Proteomics and Progenesis QI software was used to identify, compare, and select tick salivary gland proteins differentially produced during tick feeding and in response to B. afzelii infection. Tick saliva proteins were selected for validation, recombinantly expressed and used in both mouse and guinea pig vaccination and tick-challenge studies. We identified 870 I. ricinus proteins from which 68 were overrepresented upon 24-hours of feeding and B. afzelii infection. Selected tick proteins were successfully validated by confirming their expression at the RNA and native protein level in independent tick pools. When used in a recombinant vaccine formulation, these tick proteins significantly reduced the post-engorgement weights of I. ricinus nymphs in two experimental animal models. Despite the reduced ability of ticks to feed on vaccinated animals, we observed efficient transmission of B. afzelii to the murine host. Using quantitative proteomics, we identified differential protein production in I. ricinus salivary glands in response to B. afzelii infection and different feeding conditions. These results provide novel insights into the process of I. ricinus feeding and B. afzelii transmission and revealed novel candidates for an anti-tick vaccine. [ABSTRACT FROM AUTHOR]