Browsing by Author "Antunes, S."
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- An insight into the transcriptome of Rhipicephalus bursa: differential gene expression upon feeding and in response to salivary glands infection with Babesia ovisPublication . Couto, J.; Antunes, S.; Ferrolho, J.; Santos, A.S.; Santos-Silva, M.M.; de la Fuente, J.; Domingos, A.Understanding the paradigm of the pathogen-tick-host triad remains an intricate issue. Basic research focusing on each one of these interplays is necessary to better comprehend this dynamic system. One of such complex association is the Babesia ovis, transmitted by the Rhipicephalus bursa ticks. B. ovis is an intra-erythrocytic protozoan parasite highly pathogenic parasite, causing severe infection in sheep. Pathogen and vector co-evolved and during infection a substantial molecular dialogue/interchanges takes place. The pathogen is adapted to both its hosts, and remarkably transovarial and transstadial transmissions are present in the vector. In ticks, the salivary glands (SG) play important role in both, feeding and pathogen interaction, being a vehicle for pathogen transmission and a barrier that they need to surpass.
- Anaplasma marginale and Theileria annulata in questing ticks from PortugalPublication . Antunes, S.; Ferrolho, J.; Domingues, N.; Santos, A.S.; Santos-Silva, M.M.; Domingos, A.Ticks are ubiquitous arthropods and vectors of several pathogenic agents in animals and humans. Monitoring questing ticks is of great importance to ascertain the occurrence of pathogens and the potential vector species, offering an insight into the risk of disease transmission in a given area. In this study 428 host-seeking ticks, belonging to nine species of Ixodidae and collected from 17 of the 23 Portuguese mainland subregions, were screened for several tick-borne agents with veterinary relevance: Anaplasma marginale, Anaplasma ovis, Anaplasma centrale, Babesia spp., Coxiella burnetii and Theileria spp. Prevalence was assessed by PCR and amplified amplicons sequenced for validation of results. Twenty ticks, in a total of 428, were found positive: one Ixodes ventalloi for Theileria annulata and four Dermacentor marginatus, one Haemaphysalis punctata, five Ixodes ricinus, five I. ventalloi, and four Rhipicephalus sanguineus sensu lato for A. marginale. According to the reviewed literature, this is the first report of A. marginale and T. annulata detection in I. ventalloi. Furthermore, the amplification of A. marginale DNA in several tick species suggests a broad range for this agent in Portugal that might include other uncommon species as R. sanguineus s.l. This work provides new data towards a better understanding of tick-pathogen associations and also contributes to the surveillance of tick-borne agents in geographic areas with limited information.
- Detection and phylogenetic characterization of Theileria spp. and Anaplasma marginale in Rhipicephalus bursa in PortugalPublication . Ferrolho, A.; Antunes, S.; Santos, A.S.; Velez, R.; Padre, L.; Cabezas-Cruz, A.; Santos-Silva, M.M.; Domingos, A.Ticks are obligatory blood-sucking arthropod (Acari:Ixodida) ectoparasites of domestic and wild animals as well as humans. The incidence of tick-borne diseases is rising worldwide, challenging our approach toward diagnosis, treatment and control options. Rhipicephalus bursa Canestrini and Fanzago, 1877, a two-host tick widely distributed in the Palearctic Mediterranean region, is considered a multi-host tick that can be commonly found on sheep, goats and cattle, and occasionally on horses, dogs, deer and humans. R. bursa is a species involved in the transmission of several tick-borne pathogens with a known impact on animal health and production. The aim of this study was to estimate R. bursa prevalence in Portugal Mainland and circulating pathogens in order to contribute to a better knowledge of the impact of this tick species. Anaplasma marginale and Theileria spp. were detected and classified using phylogenetic analysis. This is the first report of Theileria annulata and Theileria equi detection in R. bursa ticks feeding on cattle and horses, respectively, in Portugal. This study contributes toward the identification of currently circulating pathogens in this tick species as a prerequisite for developing future effective anti-tick control measures.
- Efficacy of the Vaccine Candidate Based on the P0 Peptide against Dermacentor nitens and Ixodes ricinus TicksPublication . Rodríguez Mallon, A.; Encinosa Guzmán, P.E.; Bello, Y.; Domingos, A.; Antunes, S.; Kopacek, P.; Santos, A.S.; Velez, R.; Perner, J.; Ledesma Bravo, F.L.; Frantova, H.; Erhart, J.; Rodríguez, R.; Fuentes, A.; Diago, D.; Joglar, M.; Méndez, L.; Estrada, M.P.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.
- Molecular heterogeneity of Rhipicephalus sanguineus sensu lato and screening for Ehrlichia canis in mainland PortugalPublication . Santos, G.S.; Couto, J.; Silva-Perdosa, R.; Ferrolho, J.; Santos, A.S.; Santos-Silva, M.M.; Antunes, S.; Domingos, A.The present study aimed to expand knowledge regarding the molecular characterization of R. sanguineus s.l. in Portugal and to screen for ticks naturally infected with E. canis. A total of 113 R. sanguineus s.l. were collected questing or attached to domestic and wild animals from the 18 administrative regions of mainland Portugal. All the ticks were analyzed for the 16S rRNA and the partial sequences obtained showed high genetic similarities with specimens belonging to the temperate lineage. These sequences revealed eight haplotypes (H1‒H8), with a genetic distance ranging from 0.3% to 1.4%. A convenience sample representing approximately 75% of all the R. sanguineus s.l. ticks collected was tested for the presence of E. canis by qPCR for the dsb gene. No ticks were found to be infected with this pathogen. Accordingly, further studies are required to determine the role of the R. sanguineus s.l. temperate lineage in E. canis maintenance and transmission, as well as to elucidate if a different R. sanguineus s.l. lineage or other tick species act as E. canis vectors for dogs in Portugal.
- PCR screening of tick-borne agents in sensitive conservation areas, Southeast PortugalPublication . Santos-Silva, M.M.; Melo, P.; Santos, N.; Antunes, S.; Duarte, L.R.; Ferrolho, J.; Milhano, N.; Santos, P.T.; Domingos, A.; Santos, A.S.The Southeast region of Portugal, particularly the Guadiana valley, is currently the reintroduction territory of Lynx pardinus (Iberian lynx), one of the most endangered felids in the world that is only found in the Iberian Peninsula. Over the last century, populations have declined, placing L. pardinus at extremely high risk of extinction in the wild and relying on reintroduction projects. Among the aspects taken into account in the establishment of new populations is the sanitary status of the selected habitats, especially concerning infectious diseases, including tick-borne pathogens (TBPs). This study presents the results of TBPs survey on ticks collected at sensitive conservation areas of Southeast Portugal. From 2012 to 2014, 231 ticks obtained from vegetation, sympatric domestic and wild animals were submitted for analysis. The presence of Babesia spp., Cytauxzoon spp., Theileria spp., Hepatozoon spp., Anaplasma spp., Ehrlichia spp., Candidatus Neoehrlichia mikurensis, among other Anaplasmataceae, and Coxiella burnetii were investigated by PCR. Six tick species were recorded, Dermacentor marginatus (n = 13/5.6%), Hyalomma lusitanicum (n = 175/75.8%), Ixodes ricinus (n = 4/1.7%), Rhipicephalus bursa (n = 7/3.0%), R. pusillus (n = 21/9.1%) and R. sanguineus sensu lato (n = 11/4.8%). The molecular screening confirmed the presence of two tick-borne pathogens, C. burnetii (N = 34) and Anaplasma platys (N = 1), and one tick-endosymbiont, Candidatus Midichloria mitochondrii (N = 45). The results obtained provide new information on the circulation of ticks and TBPs with potential veterinary importance in Iberian lynx habitat.
- Rhipicephalus bursa Sialotranscriptomic Response to Blood Feeding and Babesia ovis Infection: Identification of Candidate Protective AntigensPublication . Antunes, S.; Couto, J.; Ferrolho, J.; Rodrigues, F.; Nobre, J.; Santos, A.S.; Santos-Silva, M.M.; de la Fuente, J.; Domingos, A.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.
- Vitellogenin and Lachesin knockdown in Rhipicephalus bursa influence in feeding and pathogen acquisitionPublication . Antunes, S.; Ferrolho, J.; Nobre, J.; Couto, J.; Santos, A.S.; Santos-Silva, M.M.; de la Fuente, J.; Domingos, A.Rhipicephalus bursa is considered as a multi-host tick being the primary hosts of this species, cattle, sheep and goats. R. bursa is widely distributed in the Mediterranean region and has been described as being involved in the transmission of agents of the genus Anaplasma, Babesia, Theileria, Rickettsia among others, with a known impact on animal health. Control of ticks by vaccination has the advantages of being costeffective, reducing environment contamination and preventing the selection of drugresistant ticks. The major constrain in the development of new anti-tick vaccines is still the identification of effective antigens. The present work studies the influence of tick feeding in gene expression as well as interactions between R. bursa tick and the protozoa B. ovis using silencing of genes by RNA interference (RNAi).