Liposome-mediated DNA immunisation via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper, we have investigated the application of liposome-entrapped DNA and their cationic lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmoles dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol) and either the cationic lipid 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP) or cholesteryl 3-N-(dimethyl amino ethyl) carbamate (DC-Chol). This method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded incorporation values of 90-94% of the DNA used. Mixing or rehydration of preformed cationic liposomes with 100 μg plasmid DNA also led to similarly high complexation values (92-94%). In an attempt to establish differences in the nature of DNA association with these various liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal DNA by the dehydration-rehydration generated submicron size liposomes incorporating most of the DNA in a manner that prevents DNA displacement through anion competition. The bilayer composition of these dehydration-rehydration vesicles (DRV(DNA)) can also further influence these physicochemical characteristics with the presence of DOPE within the liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 μg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2003 Taylor & Francis Ltd.

Surfactant vesicle-mediated delivery of DNA vaccines via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper we have compared the potency of lipid-based and non-ionic surfactant based vesicle carrier systems for DNA vaccines after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into various vesicle formulations. The DRV method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded high DNA vaccine incorporation values (85-97% of the DNA used) in all formulations. Studies on vesicle size revealed lipid-based systems formed cationic submicron size vesicles whilst constructs containing a non-ionic surfactant had significantly large z-average diameters (>1500 nm). Subcutaneous vesicle-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG 1 and 1gG 2a) engendered by the plasmid encoded nucleoprotein were substantially higher after dosing twice, 28 days apart with 10 μg DRV-entrapped DNA compared to naked DNA. Comparison between the lipid and non-ionic based vesicle formulations revealed no significant difference in stimulated antibody production. These results suggest that, not only can DNA be effectively entrapped within a range of lipid and non-ionic based vesicle formulations using the DRV method but that such DRV vesicles containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2004 Elsevier B.V. All rights reserved.

Open-source mapping and services for Web-based land-cover validation

Monitoring land-cover changes on sites of conservation importance allows environmental problems to be detected, solutions to be developed and the effectiveness of actions to be assessed. However, the remoteness of many sites or a lack of resources means these data are frequently not available. Remote sensing may provide a solution, but large-scale mapping and change detection may not be appropriate, necessitating site-level assessments. These need to be easy to undertake, rapid and cheap. We present an example of a Web-based solution based on free and open-source software and standards (including PostGIS, OpenLayers, Web Map Services, Web Feature Services and GeoServer) to support assessments of land-cover change (and validation of global land-cover maps). Authorised users are provided with means to assess land-cover visually and may optionally provide uncertainty information at various levels: from a general rating of their confidence in an assessment to a quantification of the proportions of land-cover types within a reference area. Versions of this tool have been developed for the TREES-3 initiative (Simonetti, Beuchle and Eva, 2011). This monitors tropical land-cover change through ground-truthing at latitude / longitude degree confluence points, and for monitoring of change within and around Important Bird Areas (IBAs) by Birdlife International and the Royal Society for the Protection of Birds (RSPB). In this paper we present results from the second of these applications. We also present further details on the potential use of the land-cover change assessment tool on sites of recognised conservation importance, in combination with NDVI and other time series data from the eStation (a system for receiving, processing and disseminating environmental data). We show how the tool can be used to increase the usability of earth observation data by local stakeholders and experts, and assist in evaluating the impact of protection regimes on land-cover change.

Conceptual Building Information Modelling Framework for Whole‐house Refurbishment based on LCC and LCA

The UK government aims at achieving 80% CO2 emission reduction by 2050 which requires collective efforts across all the UK industry sectors. In particular, the housing sector has a large potential to contribute to achieving the aim because the housing sector alone accounts for 27% of the total UK CO2 emission, and furthermore, 87% of the housing which is responsible for current 27% CO2 emission will still stand in 2050. Therefore, it is essential to improve energy efficiency of existing housing stock built with low energy efficiency standard. In order for this, a whole‐house needs to be refurbished in a sustainable way by considering the life time financial and environmental impacts of a refurbished house. However, the current refurbishment process seems to be challenging to generate a financially and environmentally affordable refurbishment solution due to the highly fragmented nature of refurbishment practice and a lack of knowledge and skills about whole‐house refurbishment in the construction industry. In order to generate an affordable refurbishment solution, diverse information regarding costs and environmental impacts of refurbishment measures and materials should be collected and integrated in right sequences throughout the refurbishment project life cycle among key project stakeholders. Consequently, various researchers increasingly study a way of utilizing Building Information Modelling (BIM) to tackle current problems in the construction industry because BIM can support construction professionals to manage construction projects in a collaborative manner by integrating diverse information, and to determine the best refurbishment solution among various alternatives by calculating the life cycle costs and lifetime CO2 performance of a refurbishment solution. Despite the capability of BIM, the BIM adoption rate is low with 25% in the housing sector and it has been rarely studied about a way of using BIM for housing refurbishment projects. Therefore, this research aims to develop a BIM framework to formulate a financially and environmentally affordable whole‐house refurbishment solution based on the Life Cycle Costing (LCC) and Life Cycle Assessment (LCA) methods simultaneously. In order to achieve the aim, a BIM feasibility study was conducted as a pilot study to examine whether BIM is suitable for housing refurbishment, and a BIM framework was developed based on the grounded theory because there was no precedent research. After the development of a BIM framework, this framework was examined by a hypothetical case study using BIM input data collected from questionnaire survey regarding homeowners’ preferences for housing refurbishment. Finally, validation of the BIM framework was conducted among academics and professionals by providing the BIM framework and a formulated refurbishment solution based on the LCC and LCA studies through the framework. As a result, BIM was identified as suitable for housing refurbishment as a management tool, and it is timely for developing the BIM framework. The BIM framework with seven project stages was developed to formulate an affordable refurbishment solution. Through the case study, the Building Regulation is identified as the most affordable energy efficiency standard which renders the best LCC and LCA results when it is applied for whole‐house refurbishment solution. In addition, the Fabric Energy Efficiency Standard (FEES) is recommended when customers are willing to adopt high energy standard, and the maximum 60% of CO2 emissions can be reduced through whole‐house fabric refurbishment with the FEES. Furthermore, limitations and challenges to fully utilize BIM framework for housing refurbishment were revealed such as a lack of BIM objects with proper cost and environmental information, limited interoperability between different BIM software and limited information of LCC and LCA datasets in BIM system. Finally, the BIM framework was validated as suitable for housing refurbishment projects, and reviewers commented that the framework can be more practical if a specific BIM library for housing refurbishment with proper LCC and LCA datasets is developed. This research is expected to provide a systematic way of formulating a refurbishment solution using BIM, and to become a basis for further research on BIM for the housing sector to resolve the current limitations and challenges. Future research should enhance the BIM framework by developing more detailed process map and develop BIM objects with proper LCC and LCA Information.

Feature Extraction for Classification in the Data Mining Process

Dimensionality reduction is a very important step in the data mining process. In this paper, we consider feature extraction for classification tasks as a technique to overcome problems occurring because of “the curse of dimensionality”. Three different eigenvector-based feature extraction approaches are discussed and three different kinds of applications with respect to classification tasks are considered. The summary of obtained results concerning the accuracy of classification schemes is presented with the conclusion about the search for the most appropriate feature extraction method. The problem how to discover knowledge needed to integrate the feature extraction and classification processes is stated. A decision support system to aid in the integration of the feature extraction and classification processes is proposed. The goals and requirements set for the decision support system and its basic structure are defined. The means of knowledge acquisition needed to build up the proposed system are considered.

Development and application of sensitive genome-wide platforms to study the genetic and epigenetic (DNA methylation) makeup of gametes and early bovine embryos

Pour ce projet, nous avons développé une plateforme pour l’analyse pangénomique de la méthylation de l’ADN chez le bovin qui est compatible avec des échantillons de petites tailles. Cet outil est utilisé pour étudier les caractéristiques génétiques et épigénétiques (méthylation de l’ADN) des gamètes soumis aux procédures de procréation médicalement assisitée et des embryons précoces. Dans un premier temps, une plateforme d’analyse de biopuces spécifiques pour l’étude de la méthylation de l’ADN chez l’espèce bovine a été développée. Cette plateforme a ensuite été optimisée pour produire des analyses pangénomiques de méthylation de l’ADN fiables et reproductibles à partir d’échantillons de très petites tailles telle que les embryons précoces (≥ 10 ng d’ADN a été utilisé, ce qui correspond à 10 blastocystes en expansion). En outre, cet outil a permis d’évaluer de façon simultanée la méthylation de l’ADN et le transcriptome dans le même échantillon, fournissant ainsi une image complète des profils génétiques et épigénétiques (méthylation de l’ADN). Comme preuve de concept, les profils comparatifs de méthylation de l’ADN spermatique et de blastocystes bovins ont été analysés au niveau de l’ensemble du génome. Dans un deuxième temps, grâce à cette plateforme, les profils globaux de méthylation de l’ADN de taureaux jumeaux monozygotes (MZ) ont été analysés. Malgré qu’ils sont génétiquement identiques, les taureaux jumeaux MZ ont des descendants avec des performances différentes. Par conséquent, l’hypothèse que le profil de méthylation de l’ADN spermatique de taureaux jumeaux MZ est différent a été émise. Dans notre étude, des différences significatives entre les jumeaux MZ au niveau des caractéristiques de la semence ainsi que de la méthylation de l’ADN ont été trouvées, chacune pouvant contribuer à l’obtention de performances divergentes incongrues des filles engendrées par ces jumeaux MZ. Dans la troisième partie de ce projet, la même plateforme a été utilisée pour découvrir les impacts d’une supplémentation à forte concentration en donneur de méthyle universel sur les embryons précoces bovins. La supplémentation avec de grandes quantités d’acide folique (AF) a été largement utilisée et recommandée chez les femmes enceintes pour sa capacité bien établie à prévenir les malformations du tube neural chez les enfants. Cependant, plus récemment, plusieurs études ont rapporté des effets indésirables de l’AF utilisé à des concentrations élevées, non seulement sur le développement de l’embryon, mais aussi chez les adultes. Au niveau cellulaire, l’AF entre dans le métabolisme monocarboné, la seule voie de production de S-adénosyl méthionine (SAM), un donneur universel de groupements méthyles pour une grande variété de biomolécules, y compris l’ADN. Par conséquent, pour résoudre cette controverse, une forte dose de SAM a été utilisée pour traiter des embryons produits in vitro chez le bovin. Ceci a non seulement permis d’influencer le phénotype des embryons précoces, mais aussi d’avoir un impact sur le transcriptome et le méthylome de l’ADN. En somme, le projet en cours a permis le développement d’une plateforme d’analyse de la méthylation de l’ADN à l’échelle du génome entier chez le bovin à coût raisonnable et facile à utiliser qui est compatible avec les embryons précoces. De plus, puisque c’est l’une des premières études de ce genre en biologie de la reproduction bovine, ce projet avait trois objectifs qui a donné plusieurs nouveaux résultats, incluant les profils comparatifs de méthylation de l’ADN au niveau : i) blastocystes versus spermatozoïdes ; ii) semence de taureaux jumeaux MZ et iii) embryons précoces traités à de fortes doses de SAM versus des embryons précoces non traités.

Avaliação da viabilidade da aplicação de técnicas biomoleculares na autenticação de produtos alimentares

A carne continua a ser a fonte proteica mais comum no quotidiano das pessoas. Além disso, os produtos cárneos processados apresentam-se como uma mais-valia nas suas vidas agitadas. Este tipo de produto torna difícil a diferenciação das carnes utilizadas na sua confecção, sendo por isso propícios a adulteração. A Reacção em Cadeia da Polimerase (PCR) tem ganho cada vez mais importância nos laboratórios de biologia molecular, revelando-se uma técnica de análise rápida, sensível e altamente específica na identificação de espécies em produtos alimentares. No entanto, vários factores podem interferir com o processo de amplificação, pelo que alguns cuidados devem ser implementados desde a aquisição da amostra a analisar, ao seu acondicionamento e posterior extração de ADN. Existem inúmeros protocolos de extração de ADN, devendo para cada estudo avaliar-se e optar-se pelo mais adequado, considerando a finalidade estabelecida para a amostra extraída. O trabalho laboratorial apresentado nesta dissertação baseou-se em três etapas principais. Inicialmente, avaliaram-se diferentes protocolos de extração de ADN, utilizando-se amostras de carne adquiridas num talho. Entre os protocolos testados, o método de Brometo de Cetil-Trimetil-Amónio (CTAB) modificado foi o que permitiu obter amostras de ADN com maior concentração e elevado nível de pureza. Posteriormente, foram testados e optimizados diferentes protocolos de amplificação, por PCR em tempo real, para a detecção das espécies Bos taurus (vaca), Sus scrofa (porco), Equus caballus (cavalo) e Ovis aries (ovelha). Foram empregues primers específicos de espécie para a detecção de genes mitocondriais e genómicos, consoante cada protocolo. Para o caso concreto do porco, foi efectuada a avaliação de dois protocolos, singleplex com EvaGreen® e tetraplex com AllHorse, para possível aplicação dos mesmos na sua quantificação. Os resultados demonstraram elevada especificidade e sensibilidade das reacções para esta espécie, permitindo a sua detecção até um limite de 0,001 ng e 0,1%, respectivamente. Somente a primeira metodologia se mostrou adequada para quantificação. Por último, as metodologias sugeridas foram aplicadas com sucesso na análise de 4 amostras comerciais de hambúrgueres, tendo-se verificado a consistência da rotulagem em todos os casos, no que concerne a composição em termos de espécies animais. O interesse de trabalhos neste âmbito recai na importância da autenticidade dos rótulos de produtos alimentares, principalmente nos produtos cárneos, para segurança dos consumidores e salvaguarda dos produtores.

Isomérisation du lactose en lactulose en solution modèle de lactose et dans du perméat de lactosérum par électro-activation supportée par échange ionique sur résine

L’isomérisation alcaline du lactose en lactulose a été effectuée électro-chimiquement à l’aide d’un réacteur d’électro-activation en combinaison avec des résines échangeuses d’anions de polystyrène de trois types; à savoir Lewatit VP-OC-1065 faible-acide, Lewatit MP-64 moyenne-acide et Lewatit Monoplus M500 forte-acide. Les paramètres opératoires qui ont fait l’objet de cette étude ont été étudiés sur trois blocs expérimentaux pour optimiser le système. Dans le Premier bloc, les paramètres étudiés sont : (1) ratio lactose-5%(p/v) : résine échangeuse d’anions (1:0.5, 1:1 et 1:2), (2) intensité du champ électrique : 50 mA, 100 mA et 200 mA et (3) type de résines : faible, moyenne et forte. Dans le Deuxième bloc, les paramètres mis à l’étude comprenaient : (1) l’intensité du champ électrique : 300 mA, 450 mA et 550 mA, (2) le débit de la solution traitée : 25 ml / min, 50 ml/ min et 100 ml/min et (3) la surface active de la membrane adjacente au compartiment cathodique : 0.78 cm2, 7.06 cm2 et 18.1 cm2. Le Troisième bloc expérimental a été effectué sur la base de la distance entre la membrane et l’électrode : 3.1 cm, 5.6 cm et 9 cm. Le même modèle expérimental a était également réalisé avec du perméat du lactosérum d’une concentration de 7% (p/v). Les résultats obtenus ont révélé que le meilleur rendement de l’isomérisation du lactose en lactulose était obtenu après 30 minutes d’électroactivation en utilisant une solution modèle de lactose-5% avec une valeur d’environ 20.1%. Les conditions opératoires qui ont permis d’avoir ce taux de conversion sont une intensité du courant de 550 mA, un débit de la solution de 25 ml/min, une surface active de la membrane de 7.06 cm2 et une distance de 9 cm entre la cathode et la membrane qui lui y est adjacente. En utilisant le perméat de lactosérum-7%, un taux de conversion de lactose en lactulose de 8.34% a était obtenu avec une intensité du courant de 200 mA, un débit de 120 ml/min, une surface active de de 18.1cm2 et une distance de 9 cm entre la membrane et l’électrode dans le compartiment cathodique. Les analyses de variance ont indiqué un effet catalytique significatif du type de la résine. En effet, la résine-forte a permis d’avoir les plus hauts rendements de la réaction d’isomérisation par électro-activation. La résistance électrique globale du système d’électroactivation dépendait de la valeur de l’intensité du courant. Le produit final était d’une grande pureté, car il ne présentait que quelques traces de galactose (< 4%).

A importância do DNA na Medicina Dentária Forense

Com o presente trabalho pretende-se discutir a importância do DNA na resolução de casos de investigação forense. O Homem, desde os tempos mais remotos tem revelado interesse na confirmação da identidade dos seus semelhantes, pelo desenvolvimento e prática de diversas técnicas de identificação. A partir de uma revisão da literatura, fez-se o estudo dos métodos de identificação humana, sendo este mais aprofundado no que diz respeito ao DNA. Frequentemente são encontrados corpos em que a única peça disponível para o processo de identificação é o dente. As peças dentárias são as estruturas mais resistentes e estáveis do corpo humano, mantendo as suas características e propriedades por longos períodos de tempo pós-morte. A análise do DNA contribui de forma muito importante para os processos de reconhecimento humano, principalmente em casos em que outros métodos de identificação falham devido a decomposição, fragmentação, incineração ou inexistência de dados comparativos ante-morte. Para a obtenção de um perfil genético é necessário executar as seguintes etapas: extração do DNA, sua quantificação e amplificação seguida de análise de determinadas regiões do genoma. Nos dias de hoje, muitos casos de identificação necessitam de uma abordagem multidisciplinar, isto porque em algumas situações existe falta de material padrão ou, por outro lado, pode haver mais do que uma evidência a ser examinada.

Extracting DNA from ‘jaws’: high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield.

Design and implementation of an enterprise application integration solution for science and research outcomes information management using guaraná technology

This document presents an Enterprise Application Integration based proposal for research outcomes and technological information management. The proposal addresses national and international science and research outcomes information management, and corresponding information systems. Information systems interoperability problems, approaches, technologies and integration tools are presented and applied to the research outcomes information management case. A business and technological perspective is provided, including the conceptual analysis and modelling, an integration solution based in a Domain-Specific Language (DSL) and the integration platform to execute the proposed solution. For illustrative purposes, the role and information system needs of a research unit is assumed as the representative case.

Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants

Background: Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults. Results: Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week's storage compared to immediately extracted samples. After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2. The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p

Detection of Hepatitis B virus subgenotype A1 in a Quilombo community from Maranhao, Brazil

Background: The Brazilian population is mainly descendant from European colonizers, Africans and Native Americans. Some Afro-descendants lived in small isolated communities since the slavery period. The epidemiological status of HBV infection in Quilombos communities from northeast of Brazil remains unknown. The aim of this study was to characterize the HBV genotypes circulating inside a Quilombo isolated community from Maranhao State, Brazil. Methods: Seventy-two samples from Frechal Quilombo community at Maranhao were collected. All serum samples were screened by enzyme-linked immunosorbent assays for the presence of hepatitis B surface antigen ( HBsAg). HBsAg positive samples were submitted to DNA extraction and a fragment of 1306 bp partially comprising HBsAg and polymerase coding regions (S/POL) was amplified by nested PCR and its nucleotide sequence was determined. Viral isolates were genotyped by phylogenetic analysis using reference sequences from each genotype obtained from GenBank (n = 320). Sequences were aligned using Muscle software and edited in the SE-AL software. Bayesian phylogenetic analyses were conducted using Markov Chain Monte Carlo (MCMC) method to obtain the MCC tree using BEAST v.1.5.3. Results: Of the 72 individuals, 9 (12.5%) were HBsAg-positive and 4 of them were successfully sequenced for the 1306 bp fragment. All these samples were genotype A1 and grouped together with other sequences reported from Brazil. Conclusions: The present study represents the first report on the HBV genotypes characterization of this community in the Maranhao state in Brazil where a high HBsAg frequency was found. In this study, we reported a high frequency of HBV infection and the exclusive presence of subgenotype A1 in an Afro-descendent community in the Maranhao State, Brazil.

Experimental Infection of the Opossum Didelphis aurita by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense and Amblyomma dubitatum

This work evaluated the infection of opossums (Didelphis aurita) by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and their role as amplifier hosts for horizontal transmission to Amblyomma cajennense and/or Amblyomma dubitatum ticks. Infection in D. aurita was induced by intraperitoneal inoculation with R. felis (n = 4 opossums), R. bellii (n = 4), and R. parkeri (n = 2). Another group of six opossums were inoculated intraperitoneally with Leibovitz-15 sterile culture medium, representing the uninfected groups (n = 2 opossums simultaneously to each infected group). Opossum blood samples collected during the study were used for DNA extraction, followed by real-time polymerase chain reaction targeting the rickettsial gene gltA, hematology, and detection of Rickettsia spp.-reactive antibodies by indirect immunofluorescence assay. Opossums were infested with uninfected A. cajennense and/or A. dubitatum for 30 days postinoculation (DPI). Flat ticks molted from ticks fed on opossums were allowed to feed on uninfected rabbits, which were tested for seroconversion by immunofluorescence assay. Samples of flat ticks were also tested by real-time polymerase chain reaction. Inoculated opossums showed no clinical abnormalities. Antibodies to Rickettsia spp. were first detected at the second to fourth DPI, with detectable titers until the 150th DPI. Rickettsemia was detected only in one opossum inoculated with R. parkeri, at the eighth DPI. Only one A. cajennense tick (2.0%) previously fed on a R. parkeri-inoculated opossum became infected. None of the rabbits infested with opossum-derived ticks seroconverted. The study demonstrated that R. felis, R. bellii, and R. parkeri were capable to produce antibody response in opossums, however, with undetectable rickettsemia for R. felis and R. bellii, and very low rickettsemia for R. parkeri. Further studies must be done with different strains of these rickettsiae, most importantly the strains that have never gone through in vitro passages.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.