O vírus respiratório sincicial humano (HRSV) é o principal agente viral causador de doença respiratória em bebês e crianças em idade pré-escolar. A fim de estudar a variabilidade genética de HRSV, grupo B, com inserção de 60 nucleotídeos no gene G, selecionamos amostras de aspirado de nasofaringe de crianças menores de 5 anos de idade, com doença respiratória aguda, admitidas no hospital universitário da Universidade de São Paulo. Testamos 521 amostras, das quais 35,3% foram positivas para HRSV. A região G2 da glicoproteína G foi utilizada para genotipar essas amostras. Todas as amostras do grupo B apresentaram a inserção de 60 nucleotídeos no gene da proteína G, como descrito anteriormente em Buenos Aires, em 1999. As modificações de aminoácidos e nucleotídeos dessas amostras foram comparadas com outras amostras com inserção de 2001-2005. A seqüência de nucleotídeos duplicados foi a cópia exata dos 60 nucleotídeos precedentes em vírus mais antigos, mas as cópias do segmento duplicado acumularam substituições de nucleotídeos em vírus mais recentes. / Human respiratory syncytial virus (HRSV) is the leading viral cause of respiratory illness in infants and young children. In order to study the genetic variability of HRSV group B, with 60-nucleotide duplication in the gene G, we selected nasopharyngeal aspirates samples of children less than five years of age, with acute respiratory illness admitted in the university hospital of São Paulo (USP). We tested 521 samples and the HRSV-detection test positivity rate was 35.3%. The G2 region of glycoprotein G was used as genotyping default. All type B HRSV had a 60-nucleotide duplication in the attachment protein gene like previously described in Buenos Aires, in 1999. Changes in aminoacids and nucleotides in these samples were compaired with other samples with duplication from 2001-2005. The duplicated nucleotide sequence was an exact copy of the preceding 60 nucleotides in early viruses, but copies of the duplicated segment accumulated nucleotide substituions in more recent viruses.

This thesis discusses the issue of predicting of the effect of amino acid substitutions on protein funkcion, based on phylogenetic analysis method, inspired by tool MAPP. Significant number of genetic diseases is caused by nonsynonymous SNPs manifested as single point mutations on the protein level. The ability to identify deleterious substitutions could be useful for protein engineering to test whether the proposed mutations do not damage protein function same as for targeting disease causing harmful mutations. However the experimental validation is costly and the need of predictive computation methods has risen. This thesis describes desing and implementation of a new in silico predictor based on the principles of evolutionary analysis and dissimilarity between original and substituting amino acid physico-chemical properties. Developed algorithm was tested on four datasets with 74,192 mutations from 16,256 sequences in total. The predictor yields up to 72 % accuracy and in the comparison with the most existing tools, it is substantially less time consuming. In order to achieve the highest possible efficiency, the optimization process was focused on selection of the most suitable (a) third-party software for calculation of a multiple sequence alignment, (b) overall decision threshold and (c) a set of physico-chemical properties.

No description available.

Lumnitzera littorea and Lumnitzera racemosa are mangrove species distributed widely along the Indonesian coasts. Besides their ecological importance, both are of interest owing to their wealth of natural products, some of which constitute potential sources for medicinal applications. We aimed to discover and characterize new anti-infective compounds, based on population-level sampling of both species from across the Indonesian Archipelago. Root metabolites were investigated by TLC, hyphenated LC-MS/MS and isolation, the internal transcribed spacer (ITS) region of rDNA was used for genetic characterization. Phytochemical characterization of both species revealed an unusual diversity in sulfated constituents with 3,3’,4’-tri-O-methyl-ellagic acid 4-sulfate representing the major compound in most samples. None of these compounds was previously reported for mangroves. Chemophenetic comparison of L. racemosa populations from different localities provided evolutionary information, as supported by molecular phylogenetic evidence. Samples of both species from particular locations exhibited anti-bacterial potential (Southern Nias Island and East Java against Gram-negative bacteria, Halmahera and Ternate Island against Gram-positive bacteria). In conclusion, Lumnitzera roots from natural mangrove stands represent a promising source for sulfated ellagic acid derivatives and further sulfur containing plant metabolites with potential human health benefits.

Female mating rates vary widely, even among closely related species, but the reasons for this variation are not fully understood. Across Drosophila species, female mating frequencies are positively associated with sperm length. This association may be due in part to sperm limitation, with longer-spermed species transferring fewer sperm, or to cryptic female choice. However, a previously overlooked factor is sperm metabolic rate, which may correlate with sperm length. If faster-metabolizing sperm accumulate agerelated cellular damage more quickly, then females should remate sooner to obtain fresh sperm. Alternatively, frequent female mating may select for increased sperm competitiveness via increased metabolism. Here, we measure sperm metabolism across 13 Drosophila species and compare these measures to published data on female mating rate and on sperm length. Using fluorescent lifetime imaging microscopy, we quantify NAD(P)H metabolism ex vivo, in intact organs. Phylogenetically controlled regression reveals that sperm metabolic rate is positively associated with sperm length and with female mating frequency. Path analysis shows sperm length driving sperm metabolism and sperm metabolism either driving or being driven by female mating rate. While the causal directionality of these relationships remains to be fully resolved, and the effect of sperm metabolism on sperm aging and/or sperm competitiveness remains to be established, our results demonstrate the importance of sperm metabolism in sexual selection.

Bdellovibrionaceae sind außergewöhnliche Bakterien, da sie als die kleinsten bekannten räuberischen Organismen gelten. Seit ihrer Entdeckung in Bodenproben im Jahr 1962 durch Stolp und Starr konnte eine weite Verbreitung in der Natur nachgewiesen werden. Besonderes Interesse erlangten Bdellovibrionaceae durch ihre Fähigkeit bakterielle Erreger wie Escherichia coli, Salmonella und Yersinia zu attackieren. Der bestuntersuchte Vertreter der Familie Bdellovibrionaceae ist Bdellovibrio bacteriovorus. Er durchläuft einen komplexen Lebenszyklus, der nur partiell verstanden ist. In der Attack-Phase werden durch einen noch nicht aufgeklärten Mechanismus potentielle Beutebakterien erkannt und der interzelluläre Kontakt hergestellt. Nachdem eine Pore in der Zellwand der ausschließlich Gram-negativen Beutebakterien erzeugt wurde, dringt B. bacteriovorus in den periplasmatischen Raum ein. Nach etwa 30 Minuten ist der Invasionsprozess abgeschlossen und man kann die Ausbildung sphärischer Bdelloblasten beobachten. Im Beutebakterium verdaut B. bacteriovorus makromolekulare Bestandteile des Wirtes und wächst zu einem langen spiralförmigen Stäbchen aus. Es setzt schließlich die Zellteilung ein bei der 5 bis 30 Tochterzellen in Abhängigkeit zur Größe des Beutebakteriums freiwerden. Mit der Reproduktion ist der parasitäre Lebenszyklus nach etwa 3 Stunden beendet. Die komplexe Regulation der Expression von Proteinen konnte für die einzelnen Wachstumsphasen durch ein- sowie zweidimensionale Gelelektrophorese nachgewiesen werden. In der Vergangenheit haben verschiedene Studien die Komplexität der Wechselwirkung mit den Beutebakterien belegt. Hierbei wurde, neben dem offensichtlichen Abbau makromolekularer Bestandteile der Beutebakterien, ein Recycling und Einbau von Membranbestandteilen wie Lipopolysacchariden (LPS) und Outer Membrane Proteine (OmP) in das Membransystem von B. bacteriovorus diskutiert. Die biologische Interpretation dieses Phänomens ist eine erhöhte Effizienz in der Reproduktion von B. bacteriovorus wenn es Bestandteile des Wirtsbakteriums wiederverwertet im Gegensatz zur Eigensynthese. Trotz der morphologischen Ähnlichkeit und des ungewöhnlichen Lebensstils wurde festgestellt, dass die Familie der Bdellovibrionaceae phylogenetisch sehr heterogen ist. Es werden zur Zeit zwei Gattungen unterschieden Bdellovibrio und Bacteriovorax. Aufgrund von 16S rRNA Untersuchungen konnten auch innerhalb der Gattungen eine Vielzahl von phylogenetisch distinkten Arten nachgewiesen werden. In der Literatur wird eine regulierende Wirkung auf pathogene Gram-negative Bakterien für aquatische Systeme durch Bdellovibrionaceae beschrieben. Weiterhin konnten sie bei verschiedenen Nutztieren im Verdauungstrakt mikrobiologisch nachgewiesen werden. Ein positiver Einfluss auf die Gesundheit der Tiere wurde bei Vorkommen von B. bacteriovorus festgestellt. Für eine Charakterisierung von Umweltisolaten werden in der vorliegenden Arbeit genetische Methoden vorgestellt. Hierfür wurden Hybridisierungsmethoden und PCR-methoden entwickelt, die es ermöglichen aus der Umwelt isolierte Bdellovibrionaceae phylogenetisch einzuordnen. Es konnte gezeigt werden, das sowohl B. bacteriovorus als auch Bacteriovorax stolpii im Verdauungstrakt von Nutztieren vorkommen. Es ist weiterhin gelungen eine PCR-methode für Kotproben zu entwickeln, die einen direkten Nachweis ermöglicht ohne mikrobiologische Anzucht. B. bacteriovorus ist ein Gram-negatives Bakterium, allein dieser Sachverhalt spiegelt die Schwierigkeit wider, wenn der enzymattische Abbau von Zellwandbestandteilen der Beutebakterien Ziel der Untersuchung ist, da auch die Beutebakterien Gram-negativ sind. Es ergibt sich aufgrund eines ähnlichen Zellwandaufbaus ein immenses analytisches Problem die makromolekularen Bestandteile von Wirtsbakterium und Jäger zu trennen. Um dem Lebensstil angepasst Modifikationen von B. bacteriovorus zu untersuchen, wurde in dieser Arbeit LPS, charakteristischer Bestandteil der Äußeren Membran, von Wildtypstamm B. bacteriovorus HD100 und seiner wirtsunabhängigen Mutante HI100 isoliert und das Lipid A strukturell aufgeklärt. Die Isolation gelang durch die Ausnutzung unterschiedlicher Fällungseigenschaften des LPS von B. bacteriovorus HD100 gegenüber des E. coli K-12 LPS aus dem Extraktionsmittel. Weiterhin konnte nachgewiesen werden, das B. bacteriovorus S-Form LPS besitzt. Die außergewöhnliche Struktur des Lipid A von B. bacteriovorus wurde im Detail mit massenspektrometrischen Methoden, ein- und zweidimensionalen NMR Methoden sowie mikrochemischer Methoden in Kombination mit der GC/MS charakterisiert. Der Fettsäureanker besteht aus a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. Dies stellt eine neuartige Struktur dar, da an allen bisher bekannten Lipid A´s sich Brönstedtsäuren am hydrophilen Fettsäureanker befinden, die in physiologischer Lösung durch Protonenabgabe negative Ladungen tragen. Im Lipid A von B. bacteriovorus sind diese Substituenten durch den Neutralzucker Mannose ersetzt. Weiterhin wurden als Fettsäuren nur 3-Hydroxyfettsäuren nachgewiesen, wobei sich auf die 6 gebunden Fettsäuren etwa 1,5 Doppelbindungen verteilen. Dieses Lipid A zeigt eine wesentlich reduzierte endotoxische Aktivität im Vergleich zu E. coli Lipid A und weist als biophysikalische Besonderheit eine erhöhte Fluidität über einen weiten Temperaturbereich auf. Die vorgestellte 16S rRNA Analyse und die Strukturanalyse des Lipid A von B. bacteriovorus belegen seine besondere Stellung in der Welt der Bakterien. / Bdellovibrionaceae are extraordinary bacteria known as the smallest predatory organism so far. Since their discovery in soil samples by Stolp and Starr 1963 they have been detected in a wide range of other natural habitats. Bdellovibrionaceae became the focus of attention concerning their ability to attack pathogens like Escherichia coli, Salmonella and Yersinia. For Bdellovibrio bacteriovorus the most detailed studies are available. The up to now only partially understood lifecycle consists of several complex phases. In the attack phase B. bacteriovorus is motile possessing flagella and the preys are recognized by an unknown mechanism. After attachment on the cell wall within 15 to 30 minutes a pore is formed which is used as entrance to the periplasmatic space of the Gram-negative prey bacteria. The completion of the invasion process can be observed by the change of the prey s shape to spherical bdelloblasts. Inside of the prey B. bacteriovorus degrades macromolecular compounds and transforms into a long spirally shaped rod. At the end of the lifecycle the rod divides yielding 5 up to 30 daughter cells depending on the size of the prey bacteria. This reproduction phase is completed within 3 hours. The complex regulation of expression of a certain number of proteins was observed by one and two dimensional gelelectrophoresis. The complexity of the interaction between predator and prey were examined in several studies. Besides the obvious degradation of macromolecular compounds of the prey, reutilising of lipopolysaccharides (LPS) and Outer Membrane Proteins (OmP) into the membrane system of B. bacteriovorus was discussed. The biological interpretation of such behaviour was that it is more efficient for reproduction to recycle components of the prey than to perform de novo synthesis. Unexpectedly, despite the unique predatory lifecycle and common morphological features, Bdellovibrionaceae show a great phylogenetical diversity based on 16S rRNA analyses. Bdellovibrionaceae are divided into the three species Bdellovibrio bacteriovorus, Bacteriovorax stolpii, Bacteriovorax starrii and some strains yet to be assigned. In two studies Bdellovibrionaceae were found as part of regulation processes for decreasing the number of pathogenic Gram-negative bacteria in aquatic systems. Furthermore, B. bacteriovorus were found in the intestinal tract of several domestic animals showing a positive influence on the state of health. For the phylogenetic characterization of environmental isolates techniques were developed based on hybridisation methods and the PCR. In this work we detected B. bacteriovorus and B. stolpii strains in the gut of animals. Furthermore, a PCR method for direct detection of Bdellovibrionaceae in fecal samples was developed. B. bacteriovorus are Gram-negative bacteria. This fact complicates the study of the degradation of the prey s cell wall as it possesses the architecture of Gram-negative bacteria also. Furthermore, the search for important modifications of the cell wall of B. bacteriovorus concerning the predatory lifestyle becomes an analytical problem. In this study we isolated LPS of the wild type strain B. bacteriovorus HD100 and its host independent mutant strain HI100. For the isolation of pure B. bacteriovorus HD100 S-form LPS we took advantage of different precipitation properties in the extraction solvent of E. coli K-12 LPS and the predator s LPS. The structure of the lipid A was examined in detail by mass spectrometric methods, one- and two-dimensional NMR and chemical analytical techniques. The novel structure consists of backbone built of a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. This is the first known natural lipid A without negatively charged substituents in physiological solution. The lipid A of B. bacteriovorus carries the neutral sugar mannose instead of Brönstedt acids. Furthermore, the lipid A exclusively consists of 3-hydroxy fatty acids with approximately 1.5 double bounds distributed on six bounded fatty acids. This lipid A shows a significant decreased endotoxic activity in comparison to E. coli lipid A and revealed increased fluidity over broad temperature range as further remarkable biophysical property. The 16S rRNA analysis and the structural analysis of the lipid A of B. bacteriovorus document the unique position in the world of bacteria.

Parareptilien stellen ein artenreiches Monophylum paläozoischer und früh-mesozoischer Amnioten dar; sie bilden innerhalb der Reptilien die Schwestergruppe zu den Eureptilien. Pareiasaurier, eine Untergruppe der Parareptilien, sind eine bedeutende Komponente spät-permischer …kosysteme. Jüngst wurde ein Schwestergruppenverhältnis zu den Nycyeroletern entdeckt, mit welchen sie die "Pareiasauromorpha" bilden. Diese Arbeit befasst sich mit der Beschreibung einiger dieser bisher schlecht untersuchten Parareptilien und deren Verwandtschaftsverhältnisse.Der Pareiasaurier Deltavjatia vjatkensis und der Nycteroleter Emeroleter levis aus der russischen Lokalität Kotel''nich werden mithilfe neu entdeckten Materials im Detail beschrieben. Das gut erhaltene Deltavjatja Materials umfasst mehrere Grössenstadien und erlaubt eine Analyse der Wachstumsraten. Eine geometrisch-morphometrische Analyse des Schädeldachs lässt eine allometrische Zunahme von Schnauzenlänge und Postorbitalregion erkennen. Auch wird der historisch erste Pareiasaurier, Parasaurus geinitzi, aus dem oberpermischen Kupferschiefer von Deutschland neu beschrieben. Zudem ergibt eine Neuanalyse der russischen Nycteroleter eine Synonymie von Tokosaurus perforatus und Macroleter poezicus. Eine phylogenetische Analyse unter Verwendung von Parsimonie und bayesischen Methoden ergibt eine basale Stellung von Parasaurus und eine Monophylie der Nycteroleter; letztere wird aber nur durch die Parsimonie unterstützt. Alle phylogenetischen Methoden ergeben jedoch eine Monophylie der Pareiasauromorpha. Eine strato-kladistische Analyse zeigt zudem eine ähnliche Topologie. Eine biogeographische Analyse der Pareiasauromorpha ergibt mehrfache Verbreitungsereignisse nach Russland und China. Diese stimmen überein mit denen anderer oberpermischer Gruppen, jedoch werden nur wenige dieser Ereignisse komplett bei allen Taxa gefunden. / Parareptilia is a diverse, yet enigmatic clade of fossil amniotes, sister-group to Eureptilia within Reptilia. Pareiasauria, a widely distributed and speciose parareptile group, was a prominent part of Late Permian ecosystems, yet few taxa have been examined in detail. This work describes some poorly known parareptiles, using this data to assess interrelationships and evaluate evolutionary trends within the clade. A reassessment of a group of derived parareptiles, the Russian ''nycteroleters'', results in the synonymy of Tokosaurus perforatus with Macroleter poezicus. A phylogenetic analysis of parareptilian relationships recovers a ''nycteroleter'' monophyly in the parsimony analysis, but not with Bayesian, with the genus Bashkyroleter paraphyletic in both. A monophyletic clade consisting of the nycteroleters and pareiasaurs, termed Pareiasauromorpha, is supported by all methods. Parasaurus geinitzi von Mayer, 1857 is redescribed and confirmed to be a pareiasaur based on the anatomy of the type specimens and fragmentary material that has subsequently been assigned to the taxon. Well-preserved cranial and postcranial material of the pareiasaur Deltavjatia vjatkensis from Kotel''nich, Russia, is described in detail for the first time, including specimens of radically different size classes. The first geometric morphometric analysis of a parareptile reveals an allometric increase in snout length along with an increase in postorbital length. A reassessment of the relationships of Pareiasauromorpha using different methods of phylogenetic inference recovers similar results. The topologies are not well supported, but are relatively consistent with the stratigraphic record. Biogeographic analysis of pareiasauromorph taxa recovers complex and reticulated patterns of dispersal and vicariance, including multiple dispersal events into both Russia and China; patterns that are consistent with those seen in other Late Permian groups, though few events are shared by all clades.

The present study aimed to evaluate bacterial isolates and the microbiome of dairies. The specific aims were: to characterize Staphylococcus spp. isolated from mastitic milk, to evaluate the presence of Lactococcus in mastitic milk as a potential causative agent of mastitis, to evaluate the association between microbiome and milk quality parameters, and to characterize Staphylococcus spp. isolated from production lines of Minas Frescal cheese. The detection of genes encoding virulence factors (enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, and selx), hemolysins (hla, hlb, hld, hlg, and hlgv), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin (tst)), genes encoding antibiotic resistance (resistance to tetracycline (tetK, tetL, and tetM), erythromycin (ermA, ermB, and ermC), methicillin (mecA and mecC), and tobramycin (ant(4\')-Ia)), molecular typing (spa, SCCmec, and agr types), and phenotyping regarding antibiotic resistance were performed in staphylococci isolates from mastitic milk, and from cheese processing plant samples. Staphylococcus aureus was identified in the majority of isolates from both origins. Several virulence factor genes were detected. The distribution of genes encoding staphylococcal enterotoxins (85.0% - 85.7% of isolates were positive for one or more enterotoxin gene) was highlighted and the gene related to H toxin was the most prevalent. Methicillin-resistant Staphylococcus aureus were identified in isolates from mastitic milk (4.1%) and cheese processing (6.0%); the genotyping and phenotyping of these isolates were described. t605 had the highest frequency in the S. aureus population studied. In mastitic milk, Lactococcus was suggested as the causative agent of an outbreak of mastitis in a dairy farm. Using next generation sequencing, the abundance of Lactococcus was observed in microbiome samples. Bacterial isolation and DNA sequencing confirmed the presence of Lactococcus lactis and Lactococcus garvieae. The microbiome of environmental samples and bulk tank milk from the dairy farm showed the Lactococcus genus among the most common bacterial taxa, suggesting other sources of this genus. Regarding milk quality parameters, the microbiome of bulk tank milk from several dairy farms was associated with somatic cell count and bacterial count. The core microbiome was described and many genera of importance were identified. Among the associations performed between microbiome and milk quality parameters, the identification of Streptococcus in samples classified with high somatic cell count and high bacterial count was highlighted. Several bacterial taxa with relative abundance significantly higher in samples classified as high and low cell count and bacterial count were shown. Real-time polymerase chain reaction was also performed associated with bacterial diversity, bacterial taxa, and bacterial count. These findings highlight the need to control and prevent bacterial contamination in the dairy industry, from herd to consumers. / O presente estudo apresentou como objetivo avaliar isolados bacterianos e microbioma de lácteos. Os objetivos específicos foram: caracterizar Staphylococcus spp. isolados de leite de vacas com mastite, avaliar a presença de Lactococcus em leite de vacas com mastite como um potencial agente causador de mastite, avaliar a associação entre microbioma de leite de tanque e parâmetros da qualidade de leite, e caracterizar Staphylococcus spp. isolados de linhas de processamento de queijo Minas frescal. A detecção de genes codificadores de fatores de virulência (enterotoxinas (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, e selx), hemolisinas (hla, hlb, hld, hlg, e hlgv), toxinas exfoliativas (eta, etb e etd), leucocidina de Panton-Valentine (pvl), toxina da síndrome do choque tóxico (tst)), genes codificadores de resistência a antibióticos (resistência a tetraciclina (tetK, tetL e tetM), eritromicina (ermA, ermB e ermC), meticilina (mecA e mecC) e tobramicina (ant(4\')-Ia)), tipagem molecular (spa, SCCmec e agr types), e fenotipagem quanto à resistência a antibióticos foram realizadas em estafilococos isolados de leite de vacas com mastite e de amostras de planta de processamento de queijo. Staphylococcus aureus foi identificado na maioria dos isolados de ambas as origens. Diversos genes de fatores de virulência foram detectados, com destaque para a distribuição de genes codificadores de enterotoxinas estafilocócicas (85,0%-85,7% dos isolados foram positivos para um ou mais genes codificadores de enterotoxinas), sendo o gene relacionado com a toxina H o mais frequente. Staphylococcus aureus meticilina resistente foram identificados em isolados de leite de vacas com mastite (4.1%) e em processamento de queijo (6.0%); o perfil genotípico e fenotípico destes isolados foram descritos. t605 foi o mais freqüente na população de S. aureus estudada. Em leite de vacas com mastite, Lactococcus foi sugerido como o agente causador de um surto de mastite numa fazenda leiteira. Usando sequenciamento de nova geração, a abundância de Lactococcus foi observada no microbioma das amostras. O isolamento e sequenciamento de DNA confirmaram a presença de Lactococcus lactis e Lactococcus garvieae. O microbioma de amostras ambientais e de leite de tanque da fazenda mostrou o gênero Lactococcus entre os mais comuns, sugerindo outras fontes deste gênero. Contemplando parâmetros da qualidade de leite, o microbioma de leite de tanque de várias fazendas leiteiras foi relacionado com contagem de células somáticas e contagem bacteriana. O core microbiome foi descrito e muitos gêneros bacterianos de importância foram identificados. Dentre as análises realizadas associando microbioma com parâmetros da qualidade de leite, foi destacada a identificação de Streptococcus em amostras classificadas com alta contagem de células somáticas e alta contagem bacteriana. Diversos táxons bacterianos com abundância relativa significativamente maior em amostras classificadas com alta e baixa contagem de células somáticas e contagem bacteriana foram mostrados. Reação em cadeia da polimerase em tempo real também foi realizada e associada com diversidade bacteriana, táxons bacterianos e contagem bacteriana. Estes levantamentos confirmam a necessidade de controlar e prevenir a contaminação bacteriana na indústria de lácteos, do rebanho leiteiro até os consumidores.

O coronavírus de perus (TCoV) é o agente etiológico associado a síndrome de mortalidade entérica das aves (PEMS). PEMS é uma enfermidade entérica, aguda e altamente contagiosa dos perus caracterizada por depressão, anorexia, diarréia e alta mortalidade em lotes de perus comerciais. A presença do coronavírus de perus (TCov) foi pesquisada em 29 amostras de conteúdo intestinal de perus entre 10 e 104 dias de idade que apresentaram enterite severa no período de 2004 a 2006. A detecção do TcoV foi realizada realizada através da técnica da transcriptase reversa e da reação em cadeia pela polimerase (RT-PCR), mediante a amplificação da região 3 UTR, seguida pela amplificação dos genes 3 e 5. A caracterização molecular dos vírus foi realizada mediante a amplificação dos genes 3 e 5, que mostrou similaridade genética entre as amostras, mas diferenças com as sequencias dos outros TCoVs publicados previamente. Em relação ao gene 3, as amostras apresentaram maior relação com o vírus da bronquite infecciosa das aves (IBV), enquanto que com o gene 5 houve maior identidade com os cronavírus de faisão (PhCoV). Nossos resultados sugerem que a estratégia de amplificação da região 3 UTR provou ser uma estratégia eficaz para a detecção do TcoV em conteúdo intestinal. / Turkey coronavirus (TCoV) is causative agent associated to Poult Enteritis and Mortality Syndrome (PEMS) in turkeys wideworld. The disease is characterized by an acute highly contagious enteric disease of turkeys characterized by depression, anorexia, diarrhea and high mortality in co mMercial turkey flocks. The presence of turkey coronavirus (TCoV) in 29 intestinal content samples from turkey flocks aged between 10 and 104 days with severe enteritis was monitored in the period of 2004 to 2006. TCoV detection was accomplished by the reverse transcriptase-polymerase chain reaction (RT-PCR), through amplification of the 3´UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5, and showed evidence of genetic similarity between them, although they differed of sequences of other TCoVs described in the literature. In relation to gene 3, samples showed greater relationship with chicken infectious bronchitis virus (IBV), and while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3´UTR region has proved to an effective means of detection of TCoV in intestinal contents.

Made available in DSpace on 2016-01-26T12:51:28Z (GMT). No. of bitstreams: 1 adrianomondini_tese.pdf: 12162182 bytes, checksum: fe0a4d238020f614624084ebb47e1011 (MD5) Previous issue date: 2010-03-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Dengue belongs to the Flavivirus genus and is the most common arboviral infection worldwide. It can be caused by four antigenically different serotypes (DENV 1-4). These serotypes are transmitted mainly by the bite of Aedes aegypti mosquitoes. The vector is widely associated with human activity and the influence of organized social space favors the interaction among vector, virus and man, making populated areas sources of dengue dispersion. In this study, we performed a molecular, spatial and temporal study of DENV transmission through positive samples of blood and infected mosquitoes captured in São José do Rio Preto/SP in a period of four years. Material and Methods: Serum samples of patients presenting dengue like symptoms and pools of mosquitoes had their viral RNA extracted and were tested by Multiplex- RT-PCR with Flavivirus generic primers based on non-structural protein (NS5) in the first round, followed by Nested assays with species-specific primers for the identification of DENV 1-3, yellow fever virus, Saint Louis encephalitis virus (SLEV) among others. Positive samples were analyzed spatially and phylogenetically. Results and Discussion: We analyzed 613 blood samples for four years: 199 in 2006, 94 in 2007, 313 in 2008 and 10 in 2009. The positivity was high in 2006 and 2007, with 106 and 51 infected patients, respectively. The major dengue serotype circulating during the 2006 and 2007 epidemics was DENV-3 and few cases of DENV-2, which is an indication of its recent introduction in the municipality. We also reported the first outbreak of SLEV in Brazil in 2006. Among DENV patients in 2008, only seven were infected by DENV-3 and 90 were infected by DENV-2, suggesting the reemergence of this serotype. We detected the circulation of DENV-1 in two Abstract xxv patients in 2008 and in four patients in 2009. Nearly 1200 mosquitoes were captured from December 2007 to March 2008. We have captured 814 Aedes aegypti mosquitoes, which were divided in 463 pools. Only 3.67% of them were positive for DENV-3 and DENV-2. Pools containing only male mosquitoes were positive for DENV, indicating the presence of transovarial transmission. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000 2001. Sixty DENV-3 from São José do Rio Preto formed a monophyletic group (lineage 1), closely related to the remaining 22 isolates (lineage 2). We assumed that these lineages appeared before 2006 in different occasions. The possibility of inferring the spatio-temporal dynamics from genetic data has been generally little explored, and it may shed light on DENV circulation. The use of both geographic and temporally structured phylogenetic data provided a detailed view on the spread of at least two dengue viral strains in a populated urban area. / Dengue pertence ao gênero Flavivirus e é a infecção por arbovírus mais comum no mundo todo. Pode ser causada por quatro sorotipos antigenicamente distintos (DENV 1-4). Estes sorotipos são transmitidos pela picada do mosquito Aedes aegypti. O vetor está amplamente associado a atividade humana e a influencia do espaço urbano favorece a interação entre o vetor, o vírus e o homem, tornando áreas populosas, grandes centros de dispersão do dengue. Neste estudo, foi realizada um estudo molecular, espacial e temporal da transmissão de DENV através de amostras positivas de sangue e de mosquitos infectados capturados em São José do Rio Preto/SP, num período de quatro anos. Materiais e métodos: Soro de pacientes apresentando sintomas de dengue e pools de mosquitos tiveram seu RNA viral extraído e foram testados por Multiplex-RT-PCR, com primers genéricos de Flavivirus baseados na proteína não estrutural 5 (NS5) numa primeiro ciclo,seguida por ensaios Nested com primers específicos para DENV, para o vírus da febre amarela, para o vírus da encefalite de Saint Louis, entre outros. As amostras positivas foram analisadas espacial e filogeneticamente. Resultados e discussão: Analisamos 613 amostras de soro durante 4 anos: 199 em 2006; 94 em 2007; 313 em 2008 e 10 em 2009. A positividade foi alta em 2006 e 2007, com 106 e 51 pacientes infectados, respectivamente. O principal sorotipo circulante durante as epidemias de 2006-2007 foi DENV-3 e poucos casos de DENV-2, o que pode ser a indicação de sua recente introdução no município. Nós também descrevemos a primeira epidemia de SLEV no Brasil em 2006. Dentre os pacientes com DENV em 2008, apenas sete estavam infectados com DENV-3 e 90 com DENV-2, sugerindo a reemergência do sorotipo. Nós Resumo xxiii detectamos a circulação de DENV-1 em dois pacientes em 2009 e em quatro pacientes em 2009. Aproximadamente 1200 mosquitos foram capturados entre Dezembro 2007 e Março de 2008. Capturamos 814 mosquitos Aedes aegypti, que foram divididos em 463 pools. Apenas 3,67% deles foram positivos para DENV-2 e DENV-3. Pools contendo apenas machos foram positivos para DENV, indicando a presença de transmissão transovariana. Nós obtivemos sequências de 82 pacientes dentre 174 amostras de sangue. Nós fomos capazes de geocodificar 46 sequências. O alinhamento gerou gerou nucleotídeos com 399 bp com 134 taxa. A análise filogenética indicou que todas as amostras foram de DENV-3 e estavam relacionadas às cepas circulantes na ilha da Martinica em 2000-2001. Sessenta pacientes com DENV- 3 de São José do Rio Preto formaram um grupo monofilético (linhagem 1), intimamente relacionado com os outros 22 isolados (linhagem 2). Nós assumimos que estas linhagens apareceram antes de 2006 em ocasiões diferentes. A possibilidade de inferir a dinâmica espaço-temporal através de dados genéticos é relativamente pouco explorada e pode esclarecer acirculação de DENV. O uso de dados filogenéticos estruturadosgeograficamente e temporalmente forneceu uma visão detalhada na dispersão de, pelo menos, duas cepas virais distintas numa área urbana.