Ana Sofia Santos

Over the years, tick-borne pathogens (TBPs) have garnered significant interest due to their medical, veterinary and economic importance. Additionally, TBPs have drawn attention to how these microorganisms interact with their own vectors, increasing the risk to human and animal infection of emerging and reemerging zoonoses. In this sense, ticks, which are obligate hematophagous ectoparasites, have a key role in maintaining and transmitting TBPs among humans and animals. The aim of this study was to assess the prevalence of neglected TBPs in mainland Portugal, namely Anaplasma spp., Babesia spp., Ehrlichia spp. and Neoehrlichia mikurensis. DNA fragments were detected in questing ticks collected from five different ecological areas under investigation. To the best of the authors’ knowledge, this study reports new worldwide findings, including B. bigemina infecting Ixodes frontalis, Ixodes ricinus and Rhipicephalus sanguineus sensu lato. Additionally, it presents new findings in Portugal of N. mikurensis infecting I. ricinus and of presumably Wolbachia endosymbionts being detected in I. ricinus. Overall, there were 208 tick samples that were negative for all screened TBPs. The results herein obtained raise concerns about the circulation of neglected TBPs in mainland Portugal, especially in anthropophilic ticks, highlighting the importance of adopting a One Health perspective.

The control of ticks through vaccination offers a sustainable alternative to the use of chemicals that cause contamination and the selection of resistant tick strains. However, only a limited number of anti-tick vaccines have reached commercial realization. In this sense, an antigen effective against different tick species is a desirable target for developing such vaccines. A peptide derived from the tick P0 protein (pP0) conjugated to a carrier protein has been demonstrated to be effective against the Rhipicephalus microplus, Rhipicephalus sanguineus, and Amblyomma mixtum tick species. The aim of this work was to assess the efficacy of this peptide when conjugated to the Bm86 protein against Dermacentor nitens and Ixodes ricinus ticks. An RNAi experiment using P0 dsRNA from I. ricinus showed a dramatic reduction in the feeding of injected female ticks on guinea pigs. In the follow-up vaccination experiments, rabbits were immunized with the pP0-Bm86 conjugate and challenged simultaneously with larvae, nymphs, and the adults of I. ricinus ticks. In the same way, horses were immunized with the pP0-Bm86 conjugate and challenged with D. nitens larva. The pP0-Bm86 conjugate showed efficacies of 63% and 55% against I. ricinus and D. nitens ticks, respectively. These results, combined with previous reports of efficacy for this conjugate, show the promising potential for its development as a broad-spectrum anti-tick vaccine.

Ixodes ricinus is an important vector of several human and veterinary infectious agents. Its wide geographical distribution and permissive feeding behaviour have prompted earlier studies on its population genetics. Results were, nevertheless, not conclusive. Furthermore, no research has fully focused on the south-western distribution range of I. ricinus, where exchanges between European and North African populations are more likely to occur. The presence of an additional species, Ixodes inopinatus, in the area further confuses the topic, as the two species are hard to differentiate morphologically. The present work describes the testing of microsatellite markers previously described for I. ricinus using Portuguese and Tunisian tick populations of both species. In addition, new microsatellite loci were developed to complement the available marker toolbox. Loci showed different amplification successes across subpopulations, with Tunisian DNA less readily amplified. Altogether, 15 loci were considered suitable for genetic analyses of Portuguese subpopulations, 10 for Tunisian samples, and seven, common to both populations, were considered to be informative at the inter-continental level. A preliminary analysis of both datasets revealed two isolated populations, which can correspond to two different species. Furthermore, Tunisian specimens identified by sequencing of 16S rDNA as having I. ricinus or I. inopinatus sequence profiles all clustered together in one single population using the proposed microsatellites. This confirms that taxonomic decisions based only on 16S rRNA gene sequencing can be misleading. The application of the proposed set of microsatellite markers to a larger sample, representative of the south-western Ixodes’ distribution range, will be crucial to clarify the distribution of both species.

The negative impact of ticks and tick-borne diseases on animals and human health is driving research to discover novel targets affecting both vectors and pathogens. The salivary glands are involved in feeding and pathogen transmission, thus are considered as a compelling target to focus research. In this study, proteomics approach was used to characterize Rhipicephalus bursa sialoproteome in response to Babesia ovis infection and blood feeding. Two potential tick protective antigens were identified and its influence in tick biological parameters and pathogen infection was evaluated. Results demonstrate that the R. bursa sialoproteome is highly affected by feeding but infection is well tolerated by tick cells. The combination of both stimuli shifts the previous scenario and a more evident pathogen manipulation can be suggested. Knockdown of ub2n led to a significative increase of infection in tick salivary glands but a brusque decrease in the progeny, revealing its importance in the cellular response to pathogen infection, which is worth pursuing in future studies. Additionally, an impact in the recovery rate of adults (62%), the egg production efficiency (45.75%), and the hatching rate (88.57 %) was detected. Building knowledge on vector and/or pathogen interplay bridges the identification of protective antigens and the development of novel control strategies.

Bartonella quintana is a facultative intracellular bacterium and the causative agent of trench fever. The disease was reported during the World Wars in pre-antibiotic era and is associated with louse infestation and poor hygiene conditions. Bartonella bacteraemia may result in endocarditis mostly in people with existing heart valve abnormalities. We report a case of endocarditis caused by B. quintana in a 77-year-old woman with previous valvulopathy. This active endocarditis case was characterized by aortic root involvement 5 years after surgical aortic valve replacement. Although the initial serological tests had induced to a presumptive diagnosis of Q fever, B. quintana infection was confirmed by PCR and sequencing. Detection of Bartonella DNA in valvular and abscess specimens was determinant to confirm Bartonella infection in the absence of other associated risk factors. Bartonella infection should be considered in patients with pre-existing valvular disease and with a blood culture-negative endocarditis.

Ixodes Latreille, 1795 is the largest and broadest distributed genus of the family Ixodidae Murray, 1877. Its members are present in all zoogeographic regions, remote islands, and territories close to the poles. Plus, 63 species out of the 244 described have been recorded to feed on humans. Some are mega vectors, as those belonging to Ixodes ricinus-I. persulcatus complex, but others are so poorly studied that their vector role is difficult to access. This is the case of Ixodes ventalloi Gil Collado, 1936. This species is recorded in Northern Africa and Western Europe, mostly in Mediterranean basin countries, occurring along with other moisture-demanding ticks, as Haemaphysalis spp., I. frontalis, and I. ricinus. In fact, I. ventalloi not only shares vertebrate hosts (including humans) with the latter but may as well play a role in the enzootic cycle of some Ixodes-borne agents. This chapter updates information regarding this poorly studied tick, revising the available systematic, ecological, and microbiological data, discussing the potential public health relevance.

Q fever, caused by Coxiella burnetii, is a zoonosis that presents a worldwide distribution and affects both humans and animals. The route of dispersal of the pathogen by ruminants into the environment usually involves stages of abortion and parturition, nevertheless the agent can, also, be detected in other animal samples. Therefore it is considered as important in terms of proper diagnosis, as well as, for epidemiology and surveillance purposes, to genotype the pathogen. The aim of the current study was to investigate the presence of different genotypes of the agent in animals that had suffered from abortion during a two-year survey in Greece. Sixty nine tissue samples (37 stomach contents, 11 liver samples, 21 cotyledons) were collected from 59 abortion cases in sheep (N = 45) and goats (N = 14) from 65 farms at eight different areas of Greece. Samples were screened by qPCR and positive ones were further genotyped using a 10-locus multiple loci (ms 1, 3, 7, 12, 20, 21, 22, 26, 30 and 36) variable number of tandem repeat analysis (MLVA) method. Three genotypes were identified in sheep (A, B, C). Samples representing each of the obtained MLVA profile were further used for MST genotyping. Ten spacers (Cox 2, 5, 6, 18, 20, 22, 37, 51, 56 and 57) were amplified. A close relatedness among the identified MLVA genotypes was confirmed since they all belonged to MST group 32. The current study introduces into the aspect of genotyping of C. burnetii in Greece. Further studies are needed to explore the presence of more genotypes, to associate the genotypes circulating in the animal and tick population with those causing human disease in order to further expand on the epidemiological aspects of the pathogen.

Ticks are among the most prevalent blood-feeding arthropods, and they act as vectors and reservoirs for numerous pathogens. Sialotranscriptomic characterizations of tick responses to blood feeding and pathogen infections can offer new insights into the molecular interplay occurring at the tick-host-pathogen interface. In the present study, we aimed to identify and characterize Rhipicephalus bursa salivary gland (SG) genes that were differentially expressed in response to blood feeding and Babesia ovis infection. Our experimental approach consisted of RNA sequencing of SG from three different tick samples, fed-infected, fed-uninfected, and unfed-uninfected, for characterization and inter-comparison. Overall, 7,272 expressed sequence tags (ESTs) were constructed from unfed-uninfected, 13,819 ESTs from fed-uninfected, and 15,292 ESTs from fed-infected ticks. Two catalogs of transcripts that were differentially expressed in response to blood feeding and B. ovis infection were produced. Four genes coding for a putative vitellogenin-3, lachesin, a glycine rich protein, and a secreted cement protein were selected for RNA interference functional studies. A reduction of 92, 65, and 51% was observed in vitellogenin-3, secreted cement, and lachesin mRNA levels in SG, respectively. The vitellogenin-3 knockdown led to increased tick mortality, with 77% of ticks dying post-infestation. The reduction of the secreted cement protein-mRNA levels resulted in 46% of ticks being incapable of correctly attaching to the host and significantly lower female weights post-feeding in comparison to the control group. The lachesin knockdown resulted in a 70% reduction of the levels associated with B. ovis infection in R. bursa SG and 70% mortality. These results improved our understanding of the role of tick SG genes in Babesia infection/proliferation and tick feeding. Moreover, lachesin, vitellogenin-3, and secreted cement proteins were validated as candidate protective antigens for the development of novel tick and tick-borne disease control measures.