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- Antileishmanial activity of meroditerpenoids from the macroalgae Cystoseira baccataPublication . Sousa, Carolina Bruno de; Gangadhar, Katkam N.; Morais, Thiago R.; Conserva, Geanne A. A.; Vizetto-Duarte, C; Pereira, H.; Laurenti, Marcia D.; Campino, Lenea; Levy, Debora; Uemi, Miriam; Barreira, Luísa; Custódio, L.; Passero, Luiz Felipe D.; Lago, Joao Henrique G.; Varela, JoãoThe development of novel drugs for the treatment of leishmaniases continues to be crucial to overcome the severe impacts of these diseases on human and animal health. Several bioactivities have been described in extracts from macroalgae belonging to the Cystoseira genus. However, none of the studies has reported the chemical compounds responsible for the antileishmanial activity observed upon incubation of the parasite with the aforementioned extracts. Thus, this work aimed to isolate and characterize the molecules present in a hexane extract of Cystoseira baccata that was found to be bioactive against Leishmania infantum in a previous screening effort. A bioactivity-guided fractionation of the C. baccata extract was carried out and the inhibitory potential of the isolated compounds was evaluated via the MIT assay against promastigotes and murine macrophages as well as direct counting against intracellular amastigotes. Moreover, the promastigote ultrastructure, DNA fragmentation and changes in the mitochondrial potential were assessed to unravel their mechanism of action. In this process, two antileishmanial meroditerpenoids, (3R)- and (3S)-tetraprenyltoluquinol (1a/1b) and (3R)- and (3S)-tetraprenyltoluquinone (2a/2b), were isolated. Compounds 1 and 2 inhibited the growth of the L. infantum promastigotes (IC50 = 44.9 +/- 4.3 and 94.4 +/- 10.1 mu M, respectively), inducing cytoplasmic vacuolization and the presence of coiled multilamellar structures in mitochondria as well as an intense disruption of the mitochondrial membrane potential. Compound 1 decreased the intracellular infection index (IC50 = 25.0 +/- 4.1 mu M), while compound 2 eliminated 50% of the intracellular amastigotes at a concentration > 88.0 mu M. This work identified compound 2 as a novel metabolite and compound 1 as a biochemical isolated from Cystoseira algae displaying antileishmanial activity. Compound 1 can thus be an interesting scaffold for the development of novel chemotherapeutic molecules for canine and human visceral leishmaniases studies. This work reinforces the evidence of the marine environment as source of novel molecules. (C) 2017 Elsevier Inc. All rights reserved.
- Molecular detection of Leishmania DNA and identification of blood meals in wild caught phlebotomine sand flies (Diptera: Psychodidae) from southern PortugalPublication . Maia, Carla; Parreira, Ricardo; Cristovao, Jose Manuel; Freitas, Ferdinando Bernardino; Afonso, Maria Odete; Campino, LeneaBackground: Zoonotic visceral leishmaniasis caused by Leishmania infantum which is transmitted by phlebotomine sand flies (Diptera, Psychodidae) is endemic in the Mediterranean basin. The main objectives of this study were to (i) detect Leishmania DNA and (ii) identify blood meal sources in wild caught female sand flies in the zoonotic leishmaniasis region of Algarve, Portugal/Southwestern Europe. Methods: Phlebotomine sand flies were collected using CDC miniature light traps and sticky papers. Sand flies were identified morphologically and tested for Leishmania sp. by PCR using ITS-1 as the target sequence. The source of blood meal of the engorged females was determined using the cyt-b sequence. Results: Out of the 4,971 (2,584 males and 2,387 females) collected sand flies, Leishmania DNA was detected by PCR in three females (0.13%), specifically in two specimens identified on the basis of morphological features as Sergentomyia minuta and one as Phlebotomus perniciosus. Haematic preferences, as defined by the analysis of cyt-b DNA amplified from the blood-meals detected in the engorged female specimens, showed that P. perniciosus fed on a wide range of domestic animals while human and lizard DNA was detected in engorged S. minuta. Conclusions: The anthropophilic behavior of S. minuta together with the detection of Leishmania DNA highlights the need to determine the role played by this species in the transmission of Leishmania parasites to humans. In addition, on-going surveillance on Leishmania vectors is crucial as the increased migration and travelling flow elevate the risk of introduction and spread of infections by Leishmania species which are non-endemic.
- The first detection of Leishmania major in naturally infected Sergentomyia minuta in PortugalPublication . Campino, Lenea; Cortes, Sofia; Dionisio, Lidia; Neto, Luis; Afonso, Maria Odete; Maia, CarlaPhlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.
- Neutralization-based seroprevalence of Toscana virus and sandfly fever Sicilian virus in dogs and cats from PortugalPublication . Alwassouf, Sulaf; Maia, Carla; Ayhan, Nazli; Coimbra, Mónica; Cristovão, José Manuel; Richet, Herve; Bichaud, Laurence; Campino, Lenea; Charrel, Remi N.Sandfly-borne phleboviruses are endemic in the Mediterranean basin. However, levels of exposure of human and animal populations are inadequately researched. Toscana virus (TOSV) is present in Portugal where it causes human infection and disease; in contrast there are few data for sandfly fever Sicilian virus (SFSV) which has neither been isolated nor detected by molecular tests and for which there are only limited serological data. The sera collected from 1160 dogs and 189 cats in southern Portugal were tested for the presence of neutralizing antibodies against TOSV and SFSV, two viruses recognized as distinct serocomplexes in the Mediterranean region. Our data showed (i) seropositivity to TOSV and SFSV in dogs at a rate of 6.8 and 50.8 %, respectively, and (ii) that 3.7 % of cats were seropositive for TOSV. TOSV findings are in line with previous results obtained with less stringent serological assays. Our results for SFSV in dogs clearly indicate that the virus is circulating widely and that humans may be exposed to infection via the dogs. Although the presence of SFSV was suggested by haemagglutination inhibition in 4/1690 human sera in 1974, this is the first time, as far as we know, that SFSV has been shown to circulate so widely in dogs in Portugal. Future studies should be directed at isolating strains of SFSV in Portugal from dogs, humans and sandflies collected in high prevalence regions. As dogs appear to be good sentinels for SFSV, their role as a possible reservoir in the natural cycle should also be considered.
- Prevalence and correlates of antibodies to Neospora caninum in dogs in PortugalPublication . Maia, Carla; Cortes, Helder; Brancal, Hugo; Lopes, Ana Patricia; Pimenta, Paulo; Campino, Lenea; Cardoso, LuisNeosporosis, caused by Neospora caninum, is an important cause of abortion in cattle and of neurological disease in dogs. This study investigated the prevalence and correlates of antibodies to N. caninum in 441 dogs from the five regions of mainland Portugal. A commercial competitive enzyme-linked immunosorbent assay (cELISA) was used and specific antibodies were detected in 35 (7.9%) dogs. Seroprevalence levels were significantly different among some of the studied regions, as well as between stray dogs (13.6%) and hunting dogs (1.7%). The difference between seropositivity in dogs presenting musculoskeletal or neurological signs (21.4%) and that in animals without clinical signs compatible with neosporosis (5.6%) was close to statistical significance. This is the first report on the seroprevalence of N. caninum in dogs in Portugal. Neosporosis should be considered in the differential diagnosis of neurological disorders of dogs.
- Bacterial and protozoal agents of feline vector-borne diseases in domestic and stray cats from southern PortugalPublication . Maia, Carla; Ramos, Claudia; Coimbra, Monica; Bastos, Filipa; Martins, Angela; Pinto, Pedro; Nunes, Monica; Vieira, Maria Luisa; Cardoso, Luis; Campino, LeneaBackground: Feline vector-borne diseases (FVBD) have emerged in recent years, showing a wider geographic distribution and increased global prevalence. In addition to their veterinary importance, domestic cats play a central role in the transmission cycles of some FVBD agents by acting as reservoirs and sentinels, a circumstance that requires a One Health approach. The aim of the present work was to molecularly detect feline vector-borne bacteria and protozoa with veterinary and zoonotic importance, and to assess associated risk factors in cats from southern Portugal. Methods: Six hundred and forty-nine cats (320 domestic and 329 stray), from veterinary medical centres and animal shelters in southern Portugal, were studied. Anaplasma spp./Ehrlichia spp., Babesia spp., Bartonella spp., Borrelia burgdorferi sensu lato, Hepatozoon spp. and Leishmania spp. infections were evaluated by polymerase chain reaction (PCR) in blood samples. Results: One hundred and ninety-four (29.9%) cats were PCR-positive to at least one of the tested genera or complex of FVBD agents. Sixty-four (9.9%) cats were positive to Leishmania spp., 56 (8.6%) to Hepatozoon spp., 43 (6.6%) to Babesia spp., 35 (5.4%) to Anaplasma spp./Ehrlichia spp., 19 (2.9%) to Bartonella spp. and 14 (2.2%) to B. burgdorferi s.l. Thirty-three (5.1%) cats were positive to two (n = 29) or three (n = 4) genera/complex. Babesia vogeli, Bartonella clarridgeiae, Bartonella henselae, Ehrlichia canis, Hepatozoon felis and Leishmania infantum were identified by DNA sequencing. Conclusions: The occurrence of FVBD agents in southern Portugal, some of them with zoonotic character, emphasizes the need to alert the veterinary community, owners and public health authorities for the risk of infection. Control measures should be implemented to prevent the infection of cats, other vertebrate hosts and people.
- Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in PortugalPublication . Maia, Carla; Dionisio, Lidia; Afonso, Maria Odete; Neto, Luis; Cristovao, Jose Manuel; Campino, LeneaThe Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.
- The recombinant protein rSP03B is a valid antigen for screening dog exposure to Phlebotomus perniciosus across foci of canine leishmaniasisPublication . Kostalova, T.; Lestinova, T.; Maia, C.; Sumova, P.; Vlkova, M.; Willen, L.; Polanska, N.; Fiorentino, E.; Scalone, A.; Oliva, G.; Veronesi, F.; Cristovao, J. M.; Courtenay, O.; Campino, Lenea; Gradoni, L.; Gramiccia, M.; Volf, P.The frequency of sandfly-host contacts can be measured by host antibody levels against sandfly salivary proteins. Recombinant salivary proteins are suggested to represent a valid replacement for salivary gland homogenate (SGH); however, it is necessary to prove that such antigens are recognized by antibodies against various populations of the same species. Phlebotomus perniciosus (Diptera: Psychodidae) is the main vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in southwest Europe and is widespread from Portugal to Italy. In this study, sera were sampled from naturally exposed dogs from distant regions, including Campania (southern Italy), Umbria (central Italy) and the metropolitan Lisbon region (Portugal), where P. perniciosus is the unique or principal vector species. Sera were screened for anti-P. perniciosus antibodies using SGH and 43-kDa yellow-related recombinant protein (rSP03B). Arobust correlation between antibodies recognizing SGH and rSP03B was detected in all regions, suggesting substantial antigenic cross-reactivity among different P. perniciosus populations. No significant differences in this relationship were detected between regions. Moreover, rSP03B and the native yellow-related protein were shown to share similar antigenic epitopes, as canine immunoglobulin G (IgG) binding to the native protein was inhibited by pre-incubation with the recombinant form. These findings suggest that rSP03B should be regarded as a universal marker of sandfly exposure throughout the geographical distribution of P. perniciosus.