Case Report: Plasmodium vivax Sporozoite Melanization in the Midgut and Salivary Gland of the Malaria Vector Anopheles darlingi.

Anopheles darlingi is the primary malaria vector in the Amazon region and is highly susceptible to both Plasmodium vivax and Plasmodium falciparum parasites. Although anopheline mosquitoes may develop melanotic encapsulation in response to Plasmodium parasites, there is no record of An. darlingi exhibiting a melanization response to P. vivax, the main malaria parasite in the Americas. Here, we report the occurrence of P. vivax sporozoite melanization in An. darlingi mosquitoes.

Profiling the antibody response of humans protected by immunization with Plasmodium vivax radiation-attenuated sporozoites.

Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates.Trial registration: ClinicalTrials.gov number: NCT01082341.

Isospora juruviarae n. sp. (Apicomplexa: Eimeriidae) from chivi vireos Vireo chivi (Vieillot, 1817) (Passeriformes: Vireonidae) in South America.

Chivi vireos Vireo chivi (Vieillot, 1817) are passerine birds widely distributed throughout Brazil, but mainly observed in the Atlantic Forest of the South and Southeast regions of the country. In this context, the current study identifies a new species of Isospora Schneider, 1881 from V. chivi captured in the Marambaia Island, on the coast of the State of Rio de Janeiro, Southeastern Brazil. The oocysts of Isospora juruviarae Andrade & Berto n. sp. are subspheroidal to ovoidal, measuring on average 26 by 24 µm. Micropyle is absent or inconspicuous. Oocyst residuum absent, but polar granules are present. Sporocysts are ellipsoidal with pointed posterior end, measuring on average 17 by × 11 µm. Stieda and Sub-Stieda bodies are present. Sporocyst residuum is present among the vermiform sporozoites, which have refractile bodies and nucleus. This morphology was different from the other Isospora spp. recorded in the same family, superfamily and parvorder as the host. Molecular identification was targeted by the amplification and sequencing of two different loci of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and one locus of the 18S small subunit ribosomal RNA (18S) gene. Phylogenetic analyses were not very efficient in forming monophyletic groups associated with host taxon, zoogeographical region or taxonomic character; however, they confirmed the identification as a new species through comparison with sequences from Isospora spp. of wild passerines. Finally, based on the morphological and molecular analyses of the oocysts recovered from the chivi vireo V. chivi in the current work, I. juruviarae is considered new to science, being the second species recorded in the host family Vireonidae and the first to have a supplementation by molecular identification.

Optimization of Plasmodium vivax infection of colonized Amazonian Anopheles darlingi.

Obtaining Plasmodium vivax sporozoites is essential for in vitro culture of liver stage parasites, not only to understand fundamental aspects of parasite biology, but also for drug and vaccine development. A major impediment to establish high-throughput in vitro P. vivax liver stage assays for drug development is obtaining sufficient numbers of sporozoites. To do so, female anopheline mosquitoes have to be fed on blood from P. vivax-infected patients through an artificial membrane-feeding system, which in turns requires a well-established Anopheles colony. In this study we established conditions to provide a robust supply of P. vivax sporozoites. Adding a combination of serum replacement and antibiotics to the membrane-feeding protocol was found to best improve sporozoite production. A simple centrifugation method appears to be a possible tool for rapidly obtaining purified sporozoites with a minimal loss of yield. However, this method needs to be better defined since sporozoite viability and hepatocyte infection were not evaluated.

Amazonian Anopheles with low numbers of oocysts transmit Plasmodium vivax sporozoites during a blood meal.

Anopheles darlingi is the main malarial vector in the Brazilian Amazon region. An. nuneztovari s.l., An. triannulatus s.l., An. evansae, and An. benarrochi s.l. do not have a defined role as malarial vectors, although they have been found to be naturally infected with Plasmodium vivax, and some develop oocysts. In this study, we evaluated the importance of low numbers of oocysts in sporozoite salivary gland invasion and transmission. Field-collected mosquitoes were experimentally infected with P. vivax. The infection rates and oocyst and sporozoite infection intensities were evaluated and compared with those of An. aquasalis. We found the highest number of oocysts in An. darlingi (mean = 39.47) and the lowest in An. nuneztovari s.l. (mean = 2). The highest number of sporozoites was observed in An. darlingi (mean = 610) and lowest in An. benarrochi s.l. (mean = 30). Plasmodium vivax DNA was detected in the saliva of all mosquito species after a blood meal. Regardless of the number of oocysts, all species transmitted sporozoites during blood meals. Considering the abundance of these mosquitoes and transmission of sporozoites, it is logical to assume that An. nuneztovari s.l. and An. triannulatus s.l. are involved in the transmission of P. vivax.

Evaluation of sustainable susceptibility to Plasmodium vivax infection among colonized Anopheles darlingi and Anopheles deaneorum.

BACKGROUND: The colonization of mosquitoes susceptible to Plasmodium vivax via direct membrane feeding assay (DMFA) has the potential to significantly advance our knowledge of P. vivax biology, vector-parasite interaction and transmission-blocking vaccine research. Anopheles darlingi and Anopheles deaneorum are important vectors of malaria in the Western Brazilian Amazon. Since 2018, well-established colonies of these species have been maintained in order to mass produce mosquitoes destined for P. vivax infection. Plasmodium susceptibility was confirmed when the colonies were established, but susceptibility needs to be maintained for these colonies to remain good models for pathogen transmission. Thus, the susceptibility was assessed of colonized mosquitoes to P. vivax isolates circulating in the Western Amazon. METHODS: Laboratory-reared mosquitoes from F10-F25 generations were fed on P. vivax blood isolates via DMFA. Susceptibility was determined by prevalence and intensity of infection as represented by oocyst load seven days after blood feeding, and sporozoite load 14 days after blood feeding. The effect of infection on mosquito survival was evaluated from initial blood feeding until sporogonic development and survival rates were compared between mosquitoes fed on infected and uninfected blood. Correlation was calculated between gametocytaemia and prevalence/intensity of infection, and between oocyst and sporozoite load. RESULTS: Significant differences were found in prevalence and intensity of infection between species. Anopheles darlingi showed a higher proportion of infected mosquitoes and higher oocyst and sporozoite intensity than An. deaneorum. Survival analysis showed that An. deaneorum survival decreased drastically until 14 days post infection (dpi). Plasmodium vivax infection decreased survival in both species relative to uninfected mosquitoes. No correlation was observed between gametocytaemia and prevalence/intensity of infection, but oocyst and sporozoite load had a moderate to strong correlation. CONCLUSIONS: Colonized An. darlingi make excellent subjects for modelling pathogen transmission. On the other hand, An. deaneorum could serve as a model for immunity studies due the low susceptibility under current colonized conditions. In the application of DMFA, gametocyte density is not a reliable parameter for predicting mosquito infection by P. vivax, but oocyst intensity should be used to schedule sporozoite experiments.

Morphometric analysis of aerobic Eimeria bovis sporogony using live cell 3D holotomographic microscopy imaging.

M onoxenous Eimeria species are widespread enteropathogenic apicomplexan protozoa with a high economic impact on livestock. In cattle, tenacious oocysts shed by E. bovis-infected animals are ubiquitously found and making infection of calves almost inevitable. To become infectious oocysts, exogenous oxygen-dependent E. bovis sporogony must occur leading to the formation of sporulated oocysts containing four sporocysts each harboring two sporozoites. Investigations on sporogony by live cell imaging techniques of ruminant Eimeria species are still absent in literature as commonly used fluorescent dyes do not penetrate resistant oocyst bi-layered wall. Sporogonial oocysts were daily analyzed by a 3D Cell Explorer Nanolive microscope to explore ongoing aerobic-dependent sporogony as close as possible to an in vivo situation. Subsequently, 3D holotomographic images of sporulating E. bovis oocysts were digitally stained based on refractive indices (RI) of oocyst bi-layered wall and sub-compartments of circumplasm using STEVE software (Nanolive), and the cellular morphometric parameters were obtained. Overall, three different E. bovis sporogony phases, each of them divided into two sub-phases, were documented: (i) sporoblast/sporont transformation into sporogonial stages, (ii) cytokinesis followed by nuclear division, and finally (iii) formation of four sporocysts with two fully developed sporozoites. Approximately 60% of sporulating E. bovis oocysts accomplished aerobic sporogony in a synchronized manner. E. bovis sporogony was delayed (i.e., 6 days) when compared to an in vivo situation where 2-3 days are required but under optimal environmental conditions. Live cell 3D holotomography analysis might facilitate the evaluation of either novel disinfectants- or anti-coccidial drug-derived effects on ruminant/avian Eimeria sporogony in vitro as discrimination of sporogony degrees based on compactness, and dry mass was here successfully achieved. Main changes were observed in the oocyst area, perimeter, compactness, extent, and granularity suggesting those parameters as an efficient tool for a fast evaluation of the sporulation degree.

Distribution, redescription, and molecular identification of Isospora striata McQuistion et al. 1997 (Eimeriidae), from woodcreepers (Dendrocolaptidae) in South America.

Woodcreepers are passerines of the family Dendrocolaptidae, which have a high forest dependency. The current work aimed to redescribe Isospora striata McQuistion et al. 1997, from two new hosts in protected areas in Brazil, revealing new localities of parasitism, in addition to providing preliminary genotypic identifications via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene from both host species. Isospora striata has oocysts that are subspheroidal to ovoidal, 19.4 × 16.8 µm with smooth wall. Oocyst residuum is absent, but micropyle and polar granules are present. Sporocysts are ovoidal, 13.6 × 8.3 µm, with both Stieda and sub-Stieda bodies. Sporocyst residuum is present and sporozoites with refractile body, nucleus, and striations. The morphological study and the 100% similarity in sequencing of the COI gene between samples of different dendrocolaptid species confirmed the identification of a single species, supporting the identification of I. striata in the Brazilian Atlantic forest and consequently the wide distribution of this coccidian species in the Neotropical Region.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy.

Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Imunização contra malária: perspectivas e desafios / Malaria immunization: prospects and challenges

O referido trabalho tem como objetivo analisar e avaliar a atual conjuntura das pesquisas científicas na busca da imunização eficaz contra a malária, destacando os principais mecanismos imunológicos e moleculares subjacentes à referida proteção, bem como, as perspectivas a curto e médio prazo. O presente estudo de revisão selecionou pesquisas nas bases de dados da Medical Literature Analysis and Retrieval System Online (Medline), National Library of Medicine (Pubmed), Scientific Electronic Library Online (SciELO), Web of Science e Scopus. Foram combinados os termos Malaria, Immunization, Vaccine and Epidemiology, com seus sinônimos remissivos e outros descritores associados, no período compreendido entre janeiro e julho de 2019. Como fator preponderante dos critérios de inclusão, foram selecionadas revisões sistemáticas com ou sem metanálise, publicadas nos últimos 5 anos, que discorressem detalhadamente sobre o tema, ou que apresentassem informações estatísticas ou históricas relevantes, relacionada ao tema. Como critérios de exclusão foram considerados: materiais literários e científicos, anteriores ao período de 2014 e que não apresentassem informações estatísticas ou histórica relevantes ao tema, ou que, não se adequassem à temática da pesquisa. Após a aplicação dos critérios de inclusão e exclusão, foi realizada a análise e seleção dos artigos. Dos 451 artigos identificados, 44 foram selecionados. As informações extraídas dos referidos trabalhos convergem no sentido de que a erradicação da malária é uma tarefa demasiadamente complexa, a qual não será alcançada com as vacinas atuais, havendo necessidade do desenvolvimento de ferramentas imunizadoras de maior eficácia. Apesar dos esforços, atualmente ainda não existe uma vacina eficaz na prevenção da infecção, mas vários estudos se encontram em andamento nessa vertente, tornando promissor o surgimento de uma vacina eficaz contra o parasita.

This study aims at analyzing and evaluating the current status of scientific research in the search for effective immunization against malaria, highlighting the key immunological and molecular mechanisms of such protection and the short- and medium-term perspectives. The search and selection of studies took place in the databases of the Medical Literature Analysis and Retrieval System Online (Medline); National Library of Medicine (Pubmed); Scientific Electronic Library Online (SciELO); Web of Science; and Scopus. The terms Malaria, Immunization, Vaccine, and Epidemiology were used, with their corresponding cross-referenced synonyms and other associated descriptors, including the period from January to July 2019. As a main factor in the inclusion criteria, systematic reviews with or without meta-analysis published in the last 5 years, presenting a detailed discourse about the topic, or relevant statistical or historical information related to the topic were selected. The following exclusion criteria were considered: literary and scientific materials, prior to 2014, and without statistical or historical information relevant to the theme, or which did not fit the research theme. After applying the inclusion and exclusion criteria, the articles were analyzed and selected. From a total of 451 identified articles, 44 were selected. The information extracted from the referred studies converge in the sense that malaria eradication is an overly complex task, which will not be achieved with the current vaccines, requiring the development of more effective immunizing tools. Despite all the efforts, there is no effective vaccine for preventing infection yet, but several studies are being developed in this area, making the emergence of an effective vaccine against the disease promising.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

The Structural and Molecular Underpinnings of Gametogenesis in Toxoplasma gondii.

Toxoplasma gondii is a widely prevalent protozoan parasite member of the phylum Apicomplexa. It causes disease in humans with clinical outcomes ranging from an asymptomatic manifestation to eye disease to reproductive failure and neurological symptoms. In farm animals, and particularly in sheep, toxoplasmosis costs the industry millions by profoundly affecting their reproductive potential. As do all the parasites in the phylum, T. gondii parasites go through sexual and asexual replication in the context of an heteroxenic life cycle involving members of the Felidae family and any warm-blooded vertebrate as definitive and intermediate hosts, respectively. During sexual replication, merozoites differentiate into female and male gametes; their combination gives rise to a zygotes which evolve into sporozoites that encyst and are shed in cat's feces as environmentally resistant oocysts. During zygote formation T. gondii parasites are diploid providing the parasite with a window of opportunity for genetic admixture making this a key step in the generation of genetic diversity. In addition, oocyst formation and shedding are central to dissemination and environmental contamination with infectious parasite forms. In this minireview we summarize the current state of the art on the process of gametogenesis. We discuss the unique structures of macro and microgametes, an insight acquired through classical techniques, as well as the more recently attained molecular understanding of the routes leading up to these life forms by in vitro and in vivo systems. We pose a number of unanswered questions and discuss these in the context of the latest findings on molecular cues mediating stage switching, and the implication for the field of newly available in vitro tools.

Toward Improving Interventions Against Toxoplasmosis by Identifying Routes of Transmission Using Sporozoite-specific Serological Tools.

BACKGROUND: Horizontal transmission of Toxoplasma gondii occurs primarily via ingestion of environmental oocysts or consumption of undercooked/raw meat containing cyst-stage bradyzoites. The relative importance of these 2 transmission routes remains unclear. Oocyst infection can be distinguished from bradyzoite infection by identification of immunoglobulin G (IgG) antibodies against T. gondii embryogenesis-related protein (TgERP). These antibodies are, however, thought to persist for only 6-8 months in human sera, limiting the use of TgERP serology to only those patients recently exposed to T. gondii. Yet recent serological survey data indicate a more sustained persistence of anti-TgERP antibodies. Elucidating the duration of anti-TgERP IgG will help to determine whether TgERP serology has epidemiological utility for quantifying the relative importance of different routes of T. gondii transmission. METHODS: We developed a serocatalytic mathematical model to capture the change in seroprevalence of non-stage-specific IgG and anti-TgERP IgG antibodies with human age. The model was fitted to published datasets collected in an endemic region of Brazil to estimate the duration of anti-TgERP IgG antibodies, accounting for variable age-force of infection profiles and uncertainty in the diagnostic performance of TgERP serology. RESULTS: We found that anti-TgERP IgG persists for substantially longer than previously recognized, with estimates ranging from 8.3 to 41.1 years. The Brazilian datasets were consistent with oocysts being the predominant transmission route in these settings. CONCLUSIONS: The longer than previously recognized duration of anti-TgERP antibodies indicates that anti-TgERP serology could be a useful tool for delineating T. gondii transmission routes in human populations. TgERP serology may therefore be an important epidemiological tool for informing the design of tailored, setting-specific public health information campaigns and interventions.

Plasmodium vivax Cell Traversal Protein for Ookinetes and Sporozoites (CelTOS) Functionally Restricted Regions Are Involved in Specific Host-Pathogen Interactions.

Following the injection of Plasmodium sporozoites by a female Anopheles mosquito into the dermis, they become engaged on a long journey to hepatic tissue where they must migrate through different types of cell to become established in parasitophorous vacuoles in hepatocytes. Studies have shown that proteins such as cell traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) play a crucial role in cell-traversal ability. Although CelTOS has been extensively studied in various species and included in pre-clinical assays it remains unknown which P. vivax CelTOS (PvCelTOS) regions are key in its interaction with traversed or target cells (Kupffer or hepatocytes) and what type of pressure, association and polymorphism these important regions could have to improve their candidacy as important vaccine antigens. This work has described producing a recombinant PvCelTOS which was recognized by ~30% P. vivax-infected individuals, thereby confirming its ability for inducing a natural immune response. PvCelTOS' genetic diversity in Colombia and its ability to interact with HeLa (traversal cell) and/or HepG2 cell (target cell) external membrane have been assessed. One region in the PvCelTOS amino-terminal region and another in its C-terminus were seen to be participating in host-pathogen interactions. These regions had important functional constraint signals (ω < 0.3 and several sites under negative selection) and were able to inhibit specific rPvCelTOS binding to HeLa cells. This led to suggesting that sequences between aa 41-60 (40833) and 141-160 (40838) represent promising candidates for an anti-P. vivax subunit-based vaccine.

Plasmodium falciparum pre-erythrocytic stage vaccine development.

Worldwide strategies between 2010 and 2017 aimed at controlling malarial parasites (mainly Plasmodium falciparum) led to a reduction of just 18% regarding disease incidence rates. Many biologically-derived anti-malarial vaccine candidates have been developed to date; this has involved using many experimental animals, an immense amount of work and the investment of millions of dollars. This review provides an overview of the current state and the main results of clinical trials for sporozoite-targeting vaccines (i.e. the parasite stage infecting the liver) carried out by research groups in areas having variable malaria transmission rates. However, none has led to promising results regarding the effective control of the disease, thereby making it necessary to complement such efforts at finding/introducing new vaccine candidates by adopting a multi-epitope, multi-stage approach, based on minimal subunits of the main sporozoite proteins involved in the invasion of the liver.

Coccidia of Columbiformes: a taxonomic review of its Eimeriidae species and Eimeria columbinae n. sp. from Columbina talpacoti (Temminck, 1809) from Brazil.

Coccidia (Chromista: Miozoa: Eimeriidae) of columbiform birds (Aves: Columbiformes) have been described since the end of the nineteenth century; however, some of these descriptions were poorly detailed or inconclusive. In this sense, the current work makes a detailed taxonomic revision reconsidering and organizing 18 Eimeria spp. and two Isospora spp. previously described or reported of Columbiformes. Along with this, a new species of Eimeria is morphologically and molecularly identified by the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and by the 18S small subunit ribosomal RNA (18S) gene from the ruddy ground-dove Columbina talpacoti (Temminck, 1809) in the Médio Paraíba region of the State of Rio de Janeiro, southeastern Brazil. Eimeria columbinae n. sp. has subspheroidal oocysts, 14.7 × 13.2 µm, with smooth, bi-layered wall, ~ 1.1 µm and length/width ratio of 1.1. Micropyle and oocyst residuum are present, but polar granule is absent. Sporocysts are ellipsoidal to slightly asymmetrical, 9.0 × 5.1 µm, with both Stieda and sub-Stieda bodies. Sporocyst residuum present and sporozoites with refractile body and nucleus. This is the 19th description of an eimerian from Columbiformes in the World, and the second to have a molecular identification of the COI and 18S genes.

Antibody Responses Against Plasmodium vivax TRAP Recombinant and Synthetic Antigens in Naturally Exposed Individuals From the Brazilian Amazon.

Thrombospondin-related adhesive protein (TRAP) is essential for sporozoite motility and the invasion of mosquitoes' salivary gland and vertebrate's hepatocyte and is, thus, considered a promising pre-erythrocytic vaccine candidate. Despite the existence of a few reports on naturally acquired immune response against Plasmodium vivax TRAP (PvTRAP), it has never been explored so far in the Amazon region, so results are conflicting. Here, we characterized the (IgG and IgG subclass) antibody reactivity against recombinant PvTRAP in a cross-sectional study of 299 individuals exposed to malaria infection in three municipalities (Cruzeiro do Sul, Mâncio Lima and Guajará) from the Acre state of the Brazilian Amazon. In addition, the full PvTRAP sequence was screened for B-cell epitopes using in silico and in vitro approaches. Firstly, we confirmed that PvTRAP is naturally immunogenic in the cohort population since 49% of the individuals were IgG-responders to it. The observed immune responses were mainly driven by cytophilic IgG1 over all other sublcasses and the IgG levels that was corelated with age and time of residence in the studied area (p < 0.05). Interestingly, only the levels of specific anti-TRAP IgG3 seemed to be associated with protection, as IgG3 responders presented a significantly higher time elapse since the last malaria episode than those recorded for IgG3 non-responders. Regarding the B-cell epitope mapping, among the 148 responders to PvTRAP, four predicted epitopes were confirmed by recognition of antibodies (PvTRAPR197-H227; PvTRAPE237-T258; PvTRAPP344-G374; and PvTRAPE439-K454). Nevertheless, the frequency of responders against these peptides were low and did not show a clear correlation with the antibody response against the corresponding antigen. Moreover, none of the linear confirmed epitopes were located in the binding regions of PvTRAP in respect to the host cell ligand. Collectively, our data confirm the PvTRAP immunogenicity among Amazon inhabitants, while suggesting that the main important B-cell epitopes are not linear.

Primaquine-quinoxaline 1,4-di-N-oxide hybrids with action on the exo-erythrocytic forms of Plasmodium induce their effect by the production of reactive oxygen species.

BACKGROUND: The challenge in anti-malarial chemotherapy is based on the emergence of resistance to drugs and the search for medicines against all stages of the life cycle of Plasmodium spp. as a therapeutic target. Nowadays, many molecules with anti-malarial activity are reported. However, few studies about the cellular and molecular mechanisms to understand their mode of action have been explored. Recently, new primaquine-based hybrids as new molecules with potential multi-acting anti-malarial activity were reported and two hybrids of primaquine linked to quinoxaline 1,4-di-N-oxide (PQ-QdNO) were identified as the most active against erythrocytic, exoerythrocytic and sporogonic stages. METHODS: To further understand the anti-malarial mode of action (MA) of these hybrids, hepg2-CD81 were infected with Plasmodium yoelii 17XNL and treated with PQ-QdNO hybrids during 48 h. After were evaluated the production of ROS, the mitochondrial depolarization, the total glutathione content, the DNA damage and proteins related to oxidative stress and death cell. RESULTS: In a preliminary analysis as tissue schizonticidals, these hybrids showed a mode of action dependent on peroxides production, but independent of the activation of transcription factor p53, mitochondrial depolarization and arrest cell cycle. CONCLUSIONS: Primaquine-quinoxaline 1,4-di-N-oxide hybrids exert their antiplasmodial activity in the exoerythrocytic phase by generating high levels of oxidative stress which promotes the increase of total glutathione levels, through oxidation stress sensor protein DJ-1. In addition, the role of HIF1a in the mode of action of quinoxaline 1,4-di-N-oxide is independent of biological activity.

Concomitant in vitro development of Eimeria zuernii- and Eimeria bovis-macromeronts in primary host endothelial cells.

Eimeria zuernii and E. bovis are host-specific apicomplexan parasites of cattle causing haemorrhagic typhlocolitis in young animals worldwide. During first merogony, both Eimeria species form giant macromeronts (>300 µm) in host endothelial cells containing >120,000 merozoites I in vivo. During the massive intracellular replication of macromeronts, large amounts of cholesterol and fatty acids are indispensable for enormous merozoite I-derived membrane production. From a metabolic perspective, host endothelial cells might be of advantage to the parasite, as transcription of several genes involved in both, cholesterol de novo biosynthesis and low density lipoprotein (LDL)-mediated uptake, are up-regulated in Eimeria macromeront-carrying host endothelial cells. In order to analyse further influence of E. zuernii/E. bovis infections on the metabolism of cholesterol, fatty acids, and glycolysis of the host endothelial cells, suitable in vitro cell culture systems are necessary. So far, in vitro cell culture systems based on primary bovine umbilical vein endothelial cells (BUVEC) are available for E. bovis-macromeront I formation, but have not been evaluated for E. zuernii. A novel E. zuernii (strain A), initially isolated from naturally infected calves in Antioquia, Colombia, was used for sporozoite isolation. Primary BUVEC monolayers were concomitantly infected with E. zuernii- and E. bovis-sporozoites, resulting in large sized macromeronts whose morphological/morphometric characteristics were compared. BUVEC carrying E. zuernii-macromeronts resulted in the release of viable and highly motile merozoites I. Overall, E. zuernii-merozoites I differed morphologically from those of E. bovis. The new E. zuernii (strain A) will allow detailed in vitro investigations not only on the modulation of cellular cholesterol processing (i. e. cholesterol-25-hydroxylase and sterol O-acyltransferase) but also on the surface expression of LDL receptors during macromeront formation.