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The vector competence of blood-feeding arthropods is influenced by the interaction between pathogens and the immune system of the vector. The Toll and IMD (immune deficiency) signaling pathways play a key role in the regulation of innate immunity in both the Drosophila model and blood-feeding insects. However, in ticks (chelicerates), immune determination for pathogen acquisition and transmission has not yet been fully explored. Here, we have mapped homologs of insect Toll and IMD pathways in the European tick Ixodes ricinus, an important vector of human and animal diseases. We show that most genes of the Toll pathway are well conserved, whereas the IMD pathway has been greatly reduced. We therefore investigated the functions of the individual components of the tick Toll pathway and found that, unlike in Drosophila, it was specifically activated by Gram-negative bacteria. The activation of pathway induced the expression of defensin (defIR), the first identified downstream effector gene of the tick Toll pathway. Borrelia, an atypical bacterium and causative agent of Lyme borreliosis, bypassed Toll-mediated recognition in I. ricinus and also resisted systemic effector molecules when the Toll pathway was activated by silencing its repressor cactus via RNA interference. Babesia, an apicomplexan parasite, also avoided Toll-mediated recognition. Strikingly, unlike Borrelia, the number of Babesia parasites reaching the salivary glands during tick infection was significantly reduced by knocking down cactus. The simultaneous silencing of cactus and dorsal resulted in greater infections and underscored the importance of tick immunity in regulating parasite infections in these important disease vectors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jalovecka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Babesia divergens is an emerging tick-borne pathogen considered as the principal causative agent of bovine babesiosis in Europe with a notable zoonotic risk to human health. Despite its increasing impact, considerable gaps persist in our understanding of the molecular interactions between this parasite and its hosts. In this study, we address the current limitation of functional genomic tools in B. divergens and introduce a stable transfection system specific to this parasite. We define the parameters for a drug selection system hdhfr -WR99210 and evaluate different transfection protocols for highly efficient generation of transgenic parasites expressing GFP. We proved that plasmid delivery into bovine erythrocytes prior to their infection is the most optimal transfection approach for B. divergens , providing novel evidence of Babesia parasites' ability to spontaneously uptake external DNA from erythrocytes cytoplasm. Furthermore, we validated the bidirectional and symmetrical activity of ef-tgtp promoter, enabling simultaneous expression of external genes. Lastly, we generated a B. divergens knockout line by targeting a 6-cys-e gene locus. The observed dispensability of this gene in intraerythrocytic parasite development makes it a suitable recipient locus for further transgenic application. The platform for genetic manipulations presented herein serves as the initial step towards developing advanced functional genomic tools enabling the discovery of B. divergens molecules involved in host-vector-pathogen interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cubillos, Snebergerova, Borsodi, Reichensdorferova, Levytska, Asada, Sojka and Jalovecka.)

Hematophagous arthropods are responsible for the transmission of a variety of pathogens that cause disease in humans and animals. Ticks of the Ixodes ricinus complex are vectors for some of the most frequently occurring human tick-borne diseases, particularly Lyme borreliosis and tick-borne encephalitis virus (TBEV). The search for vaccines against these diseases is ongoing. Efforts during the last few decades have primarily focused on understanding the biology of the transmitted viruses, bacteria and protozoans, with the goal of identifying targets for intervention. Successful vaccines have been developed against TBEV and Lyme borreliosis, although the latter is no longer available for humans. More recently, the focus of intervention has shifted back to where it was initially being studied which is the vector. State of the art technologies are being used for the identification of potential vaccine candidates for anti-tick vaccines that could be used either in humans or animals. The study of the interrelationship between ticks and the pathogens they transmit, including mechanisms of acquisition, persistence and transmission have come to the fore, as this knowledge may lead to the identification of critical elements of the pathogens' life-cycle that could be targeted by vaccines. Here, we review the status of our current knowledge on the triangular relationships between ticks, the pathogens they carry and the mammalian hosts, as well as methods that are being used to identify anti-tick vaccine candidates that can prevent the transmission of tick-borne pathogens.

Babesiosis is a tick-transmitted zoonosis caused by apicomplexan parasites of the genus Babesia. Treatment of this emerging malaria-related disease has relied on antimalarial drugs and antibiotics. The proteasome of Plasmodium, the causative agent of malaria, has recently been validated as a target for anti-malarial drug development and therefore, in this study, we investigated the effect of epoxyketone (carfilzomib, ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome inhibitors on the growth and survival of Babesia. Testing the compounds against Babesia divergens ex vivo revealed suppressive effects on parasite growth with activity that was higher than the cytotoxic effects on a non-transformed mouse macrophage cell line. Furthermore, we showed that the most-effective compound, carfilzomib, significantly reduces parasite multiplication in a Babesia microti infected mouse model without noticeable adverse effects. In addition, treatment with carfilzomib lead to an ex vivo and in vivo decrease in proteasome activity and accumulation of polyubiquitinated proteins compared to untreated control. Overall, our results demonstrate that the Babesia proteasome is a valid target for drug development and warrants the design of potent and selective B. divergens proteasome inhibitors for the treatment of babesiosis., (Copyright © 2018. Published by Elsevier Ltd.)

Although apicomplexan parasites of the group Piroplasmida represent commonly identified global risks to both animals and humans, detailed knowledge of their life cycles is surprisingly limited. Such a discrepancy results from incomplete literature reports, nomenclature disunity and recently, from large numbers of newly described species. This review intends to collate and summarize current knowledge with respect to piroplasm phylogeny. Moreover, it provides a comprehensive view of developmental events of Babesia, Theileria , and Cytauxzoon representative species, focusing on uniform consensus of three consecutive phases: (i) schizogony and merogony, asexual multiplication in blood cells of the vertebrate host; (ii) gamogony, sexual reproduction inside the tick midgut, later followed by invasion of kinetes into the tick internal tissues; and (iii) sporogony, asexual proliferation in tick salivary glands resulting in the formation of sporozoites. However, many fundamental differences in this general consensus occur and this review identifies variables that should be analyzed prior to further development of specific anti-piroplasm strategies, including the attractive targeting of life cycle stages of Babesia or Theileria tick vectors.

Background: Babesia divergens is the most common blood parasite in Europe causing babesiosis, a tick-borne malaria-like disease. Despite an increasing focus on B. divergens, especially regarding veterinary and human medicine, the sexual development of Babesia is poorly understood. Development of Babesia sexual stages in the host blood (gametocytes) plays a decisive role in parasite acquisition by the tick vector. However, the exact mechanism of gametocytogenesis is still unexplained., Methods: Babesia divergens gametocytes are characterized by expression of bdccp1, bdccp2 and bdccp3 genes. Using previously described sequences of bdccp1, bdccp2 and bdccp3, we have established a quantitative real-time PCR (qRT-PCR) assay for detection and assessment of the efficiency of B. divergens gametocytes production in bovine blood. We analysed fluctuations in expression of bdccp genes during cultivation in vitro, as well as in cultures treated with different drugs and stimuli., Results: We demonstrated that all B. divergens clonal lines tested, originally derived from naturally infected cows, exhibited sexual stages. Furthermore, sexual commitment was stimulated during continuous growth of the cultures, by addition of specific stress-inducing drugs or by alternating cultivation conditions. Expression of bdccp genes was greatly reduced or even lost after long-term cultivation, suggesting possible problems in the artificial infections of ticks in feeding assays in vitro., Conclusions: Our research provides insight into sexual development of B. divergens and may facilitate the development of transmission models in vitro, enabling a more detailed understanding of Babesia-tick interactions.