Die Kryptosporidiose stellt aufgrund ihres zoonotischen Charakters und der Entwicklung chronischer Durchfälle bei Immunsupprimierten ein hohes Gesundheitsrisiko für den Menschen, aber ebenso für Tiere dar. Derzeit verfügbare Therapeutika ermöglichen keine zuverlässige Bekämpfung klinischer Symptome oder eine Erregerelimination, daher ist die Erforschung neuer Therapieansätze dringend notwendig. CDPK stellen in diesem Zusammenhang interessante Zielmoleküle dar, da sie zwar in Pflanzen und Protisten einschließlich Apikomplexa, jedoch nicht in Pilzen und Säugetieren vorkommen. Trotz der Entdeckung vielversprechender neuer Wirkstoffe gegen CpCDPK1 in den letzten Jahren ist zur Lokalisation und Funktion von CDPK in C. parvum wenig bekannt.Diese Arbeit belegt die Transkription von sechs CpCDPK in vitro und beschreibt erstmals die Länge der 3’UTR von CpCDPK. Die Translation wurde durch den Nachweis spezifischen Proteins in Sporozoiten im Immunoblot sowie die Lokalisation von CpCDPK1 mit Hilfe der Immunfluoreszenz belegt. Möglicherweise wird die CpCDPK1 durch N-Myristoylierung an Membranen gebunden, an die Oberfläche von Zoiten gebracht und sezerniert. Eine Rolle des Enzyms im Invasions- und Egressmechanismus des Parasiten wird diskutiert.

Die Rindertuberkulose ist eine chronische Erkrankung, die von Mycobacterium (M.) bovis und M. caprae, Mitgliedern des Mycobacterium-tuberculosis-Komplex (MTC), ausgelöst wird. Tuberkulose-Erregern werden sowohl mittels kultureller als auch molekulare Untersuchungsmethoden nachgewiesen. Ziel der vorliegenden Studie war es, die Sensitivität des DNA-Nachweises von Tuberkulose-Erregern zu steigern. Dafür wurden drei Fragestellung im Bereich der molekularen Mykobakterien-Diagnostik bearbeitet. I) Zur Verbesserung der Lyse der mykobakteriellen Zellwand als Voraussetzung für eine Zunahme der Freisetzung von DNA wurden im Vergleich zu einer standardisierten DNA-Isolierungsmethode vier verschiedene Lyseprotokolle (thermische, enzymatische, thermo-enzymatische und mechanische Lyse) entwickelt und mit M. bovis BCG durchgeführt. Die Verbesserung wurde anhand der cycle threshold (Ct)-Werte einer MTC-spezifischen Real-Time (rt) Polymerase-Kettenreaktion (PCR) geprüft. Zwei Lyseprotokolle (thermische und mechanische Lyse) wurden bei zehn Gewebeproben (Lymphknoten, Leber und Lunge) von zehn Tieren (acht Rinder, ein Lama und ein Luchs) mit nachgewiesener Tuberkulose ange-wendet. II) Ausserdem, wurde eine rt-PCR mit dem 16S rRNA Gen als Zielgen (16S-rt-PCR) für den direkten Nachweis von Erregern der Gattung Mycobacterium im Gewebe entwickelt. III) Ein neu entwickelter Spoligotyping-Microarray wurde mit der konventionellen Spoligoty-ping-Methode verglichen, um die neue Methode in Bezug die Sensitivität des Nachweises und des diskriminatorischen Potenzials direkt bei infizierten Gewebeproben zu analysieren. Bei der konventionellen Methode erfolgt die Hybridisierung des PCR-Produktes auf einer Nylon Membran, auf der spezifische Oligonukleotide fixiert sind. Bei der Microarray-Methode sind diese auf einem Microarray-Chip fixiert. Die Ergebnisse der Untersuchungen (I) zur Lyse der Zellwand bei M. bovis BCG zeigten, dass bei der mechanischen Lyse eine Zunahme um 14 % und bei der thermischen Lyse eine Zunahme an PCR-Produkt von 6 % im Vergleich zur Standardlyse erbrachte. Bei beiden Lyseprotokollen wurde eine statistische Signifikanz von α = 1 % (Mann-Whitney-Test) im Vergleich zur Standardlyse errechnet. Bei den tuberkulösen Gewebeproben wurde bei der mechanischen Lyse eine durchschnitliche Zunahme an PCR-Produkt um circa 9 % im Vergleich zur Standardlyse erzielt. Dieser Unterschied war jedoch auf Grund der geringen Probeanzahl nicht statistisch signifikant. II) Bei der Untersuchung von 43 Mykobakterien-Spezies, sechs Mitgliedern des MTC (unter anderen M. bovis BCG) und 37 Non Tuberculous Mycobacteria (NTM) Spezies, konnten alle mit der entwickelten Real-Time PCR (16S-rt-PCR) nachgewiesen werden. DNA-Extrakte von acht nicht zur Gattung Mycobacterium gehörenden Spezies wurden mit der 16S-rt-PCR nicht erfasst. Ein Erreger der Gattung Gordonia und ei-ner der Gattung Rhodococcus wurden auf Grund ihres engen Verwandtschaftsgrades jedoch ebenfalls mit der 16S-rt-PCR detektiert. Die oben erwähnten mittels MTC-spezifischer rt-PCR (Zielgen IS 1081) als infiziert identifizierten zehn Gewebeproben, wurden mittels 16S-rt-PCR untersucht. Die Ergebnisse zeigten, dass beiden rt-PCR Systeme eine vergleichbare Sensitivität aufwiesen. III) Bei dem Vergleich zwischen den Spoligotyping-Methoden zeigte sich die neue Methode um einen Faktor von 100 bei der M. bovis BCG-Reinkultur und um einen Fak-tor von 10 bei DNA-Extrakten aus tuberkulösen Gewebeproben sensitiver als die konventionelle Methode. Im Rahmen dieser Arbeit hat sich der Einsatz der mechanischen Lyse für die Verbesserung der Freisetzung von mykobakterieller DNA als routinefähig erwiesen. Die entwickelte 16S-rt-PCR erwies sich als brauchbare Methode für den Nachweis von Erregern der Gattung Mycobacterium. Die Microarray-Methode stellte sich wesentlich einfacher, sensitiver und schneller dar als die konventionelle Methode. Zusammenfassend kann gesagt werden, dass alle drei Ansätze dieser Arbeit einen Beitrag zur Verbesserung der molekularen Labordiagnostik der Tuberkulose leisten. / Bovine tuberculosis is a chronic disease, that results from infection of Mycobacterium (M.) bovis and M. caprae, members of Mycobacterium tuberculosis complex (MTC), respectively. The laboratory diagnosis of bovine tuberculosis is possible with culture as well as considerate fast molecular methods. The aim of this study was to improve the sensitivity of DNA detec-tion of tuberculosis-causing pathogens. Therefore, three different issues were addressed in the complex molecular procedure targeted. I) four different lytic protocols (thermal, enzymatic, thermo-enzymatic and mechanical lysis) were developed and compared to a standardized DNA isolation protocol that was performed on pure culture of Mycobacterium (M.) bovis BCG in order to improve the mycobacterial cell wall lysis leading to an increase of DNA release. The efficiencies of the lysis protocols were assessed by the resulting cycle threshold (Ct) values of a MTC-specific real time (rt) Polymerase Chain Reaction (PCR). Two lysis protocols (thermal and mechanical) were selected to fur-ther testing of ten tuberculosis-infected tissue samples (lymph nodes, liver and lungs) from ten animals (eight cattle, one lama and one lynx). II) In addition, a real time PCR using the 16S rRNA gene as target sequence was developed, which is also suitable to detect pathogens of Genus Mycobacterium on tissue samples. III) A comparison of a newly developed microarray and the conventional spoligotyping method was realised, to analyse the applicability of the microarray in relation of the sensitivity of the method and to analyse discriminatory potential directly from infected tissue samples. During the conventional spoligotyping method, the PCR product is hybridized with specific oligonucleotides, fixed on a nylon membrane. Using the newly developed method these oligonucleotides are fixed on a microarray-chip. The results I) of the mycobacterial cell wall lysis experiment with pure culture of M. bovis BCG showed an increase of 14 % by using mechanical lysis and an increase by 6 % of the PCR product by using thermal lysis compared to the standard protocol. Using the mechanical lysis as well as the thermal lysis a statistically significant (α = 1 % (Mann-Whitney-Test)) im-provement compared to the standard lysis was achieved. Mechanical lysis was performed on tuberculous tissue samples and the results of the lysis were improved by 9 % compared to the standard lysis. However, the difference between mechanical and standard lysis was not statistically significant due the small sample number. II) Forty-three different mycobacterial species, six members of MTC (among them M. bovis BCG) and 37 Non Tuberculous Mycobacteria (NTM), were detected using the newly developed real time PCR (16S-rt-PCR). Eight non-mycobacterial species were not detected using this rt-PCR, whereas one of genus Rhodococcus and one of genus Gordonia were detected by the 16S-rt-PCR due to their close genetic similarity to the genus Mycobacterium. The ten tuberculosis-infected tissue samples (see above) testing positive using a MTC-specific real time rt-PCR (target gene IS 1081) were sub-jected to the 16S-rt-PCR. Both rt-PCR systems showed a comparable sensitivity. III) By comparing the two spoligotyping-methods, the ArrayStrip™–format method was more sensitive than the conventional method by a factor of 100 applied to pure culture and by a factor of 10 when applied to DNA extracts from infected tissue samples. In conclusion, the mechanical lysis proved to be a practical method to liberate mycobacterial DNA. The newly developed rt-PCR was suitable to detect members of the genus Mycobacterium. The spoligotyping ArrayStrip™–format method appeared to be substantially easier to perform, more sensitive, and less time-consuming than the conventional method. The three methods described were suitable to improve the molecular laboratory diagnosis of tuberculosis.

Thesis (MScMedSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background: The conventional sequence analysis is the most common method used for the detection of drug-resistant mutants. Due to its sensitivity limitations, it is unable to detect these mutants when comprising less than 20% (minor populations) of the total virus population in a sample. However, real-time PCR-based assays offer a rapid, sensitive, specific and easy detection and quantification of such mutants. The HIV-1 variants harbouring the K103N mutation are associated with resistance to nevirapine (NVP) and efavirenz (EFV). The persisting drug-resistant mutants decay slowly to low levels, and therefore they are called minor drug-resistant mutants. Consequently, they affect subsequent treatment with the drugs of the relevant class. Objectives: The objective of this study was to design two TaqMan real-time PCR-based assays called selective-polymerase chain reaction (SPCR), namely the total viral copy SPCR assay and the K103N-SPCR assay. The former detects HIV-1 of subtype C reverse transcriptase sequences, whereas the latter detects K103N drug-resistant variants in these sequences. Design and Methods: In developing the SPCR assays, sets of appropriate primers and probes for the HIV-1 subtype C reverse transcriptase (RT) were developed to use in the K103N-specific reaction and the total copy reaction. Twelve DNA plasmid standards with sequence diversity were constructed for the assay from two HIV-1subtype C samples known to harbour the K103N mutation (AAC or AAT) in our Department‟s Resistance Databank. Their RT regions were amplified, cloned and verified with sequencing. Site-directed mutagenesis was used to induce mutations at 103 amino acid position in some of these clones to generate more standards with either one of the three codons (AAA, AAC and AAT). The two assays were optimized and validated, and a standard curve was generated for each assay using 10-fold serial dilution (5x107-5x100 DNA copy/μL) of a K103N-mutant plasmid standard. The optimized and validated SPCR assays were used to screen 40 nested PCR products of previously genotyped patient samples for minor K103N variants. Results: Two sensitive and reproducible selective real-time PCR (SPCR) assays, with cut-offs of 8.23 and 10.33 and a detection limit of 0.01% for the K103N resistance variants, were successfully developed. The assays detected a prevalence of 25.64-46.15% for the K103N resistance mutation in 39 patient samples. The genotyping (population sequencing) missed 40-53.85% of these variants. Conclusion: In conclusion, sensitive and reliable selective real-time PCR assays to detect and quantify minor K103N variants of HIV-1 in nested PCR products were successfully developed. The assay had a lower detection limit of 0.01%. / AFRIKAANSE OPSOMMING: Agtergrond: Konvensionele volgorde bepaling analise is die mees algemeenste metode wat gebruik word vir die opsporing van middel-weerstandige mutasies, maar weens beperkte sensitiwiteit is dit nie moontlik om hierdie mutante op te spoor wanneer dit minder as 20% (minderheids populasie) van die totale viruspopulasie in `n monster uitmaak nie. Nietemin, kwalitatiewe PKR-gebaseerd toetse bied vinnige, sensitiewe, spesifieke en makliker opsporings en kwantifisering van sulke mutante aan. MIV-1 variante wat die K103N mutasie bevat word geassosieer met weerstand teen nevirapine (NVP) and efavirenz (EFV). Volhoudende middel-weerstandige mutasies vergaan stadig na laer vlakke en word daarom na minderheids middel weerstandige mutasies verwys. Gevolglik affekteer dit opvolgende behandeling met die middel van die relevante klas. Doelwitte: Die doel van die studie was om twee TaqMan kwantifiserende PKR gebaseerde selektiewe polymerase ketting reaksies (SPKR), naamlik totale virale kopie SPKR en K103N-SPKR te ontwikkel. Die voormalige toets het die MIV-1 subtipe C omgekeerde transkriptase volgorde bepaal, waar K103N die middel-weerstand variante in hierdie volgorde opspoor. Ontwerp en Metodes: `n Geskikte stel inleiers en peiler was ontwikkel vir die MIV-1 subtipe C omgekeerde transkriptase (OT) vir gebruik in die K103N-spesifieke en die totaal kopie reaksie. Twaalf DNS plasmied standaarde met volgorde diversiteit was saamgestel vir die toets vanaf twee MIV-1 subtipe C monsters wat volgens ons Departement se weerstand databasis geklassifeer is vir die besit van die K103N mutasie (AAC of AAT). Die OT streke was geamplifiseer, gekloneer en geverifieer deur volgorde bepaling. Punt-gerigte mutagenese is gebruik om `n mutasie by die amino suur posisie 103 van sekere klone te induseer om meer standaarde te genereer wat een van die drie kodons (AAA, AAC en AAT) bevat. Die twee toetse is geoptimiseer en gevalideer en `n standard kurwe is genereer vir elk van die toetse deur die gebruik van tienvoud serie verdunnings (107-1 DNS kopie/μL) van `n algemene K103N-mutante plasmied standard. Die geoptimiseerde en gevalideerde SPKR toets was gebruik om vir die minderheids K103N variante in 40 “nested” PKR produkte van voorheen gegenotipeerde pasiënt te soek. Resultate: Twee sensitiewe en herproduseerbare selektiewe kwantitiewe PKR toetse met `n ΔCt afsnypunt van 8.23 en `n deteksie limiet van 0.006% was ontwikkel vir die K103N weerstand variant. Die toets het `n voorkomsyfer van 25.6 % vir die K103N weerstand mutasie in 40 pasiënt monsters bepaal, waar genotipering (populasie volgorde ) 40% van hierdie variante nie opgespoor het nie. Gevolgtrekking: `n Sensitiewe en betroubare selektiewe kwantitatiewe PKR toets vir die opspoor en kwantifisering van die minderheids K103N variante van MIV-1 in PKR produkte was ontwikkel. Hierdie toets het `n laer opsporings limiet van 0.01%. / Poliomyelitis Research Foundation (PRF) / National Research Fund (NRF) / National Health Laboratory Service Research Trust (NHLS RT)

Thesis (PhD (Microbiology))--Stellenbosch University, 2008. / One of the key selection criteria for potential probiotics is the ability to adhere and colonise the host gastrointestinal tract (GIT). Probiotics compete for receptor sites at the host intestinal surface, preventing the colonisation of pathogens, thereby protecting the host from infection. In addition, several important intestinal functions are mediated by the binding of probiotics to host tissue. However, the molecular mechanisms and genotypic characterization of adhesive elements have not received as much attention as other aspects of probiotic research. The present study aims to contribute to this area of research. The first part of the study focused on monitoring the expression of mucus adhesion genes mub, mapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of Lactobacillus plantarum 423, as well as mub, surface layer protein (slp) and EF-Tu of Lactobacillus acidophilus ATCC 4356 when grown in the presence of mucin, bile, pancreatin and at low pH. Real time PCR was used. mub, mapA and EF-Tu of strain 423 were up-regulated in the presence of mucus and expression increased under increasing concentrations of mucus. Expression of mapA was up-regulated under normal gut conditions (0.3%, w/v, bile; 0.3%, w/v, pancreatin; pH 6.5) and at higher levels of bile (1.0%, w/v) and pancreatin (1.0%, w/v). Expression of mub was downregulated in the presence of bile and pancreatin at pH 6.5, whilst the expression of EFTu and plaA remained unchanged. At pH 4.0, the expression of mub and mapA remained unchanged, whilst EF-Tu and plaA were up-regulated. Expression of mapA was down-regulated in the presence of 0.1% (w/v) cysteine, suggesting that the gene is regulated by a mechanism of transcription attenuation that involves cysteine. In the case of L. acidophilus ATCC 4356, none of the genes were up-regulated under increasing concentrations of mucin, whilst only slp and EF-Tu were up-regulated under normal and stressful gut conditions in vitro. In the second part of the study, male Wistar rats were used to evaluate which section of the gastrointestinal tract are colonised by L. plantarum 423 and Enterococcus mundtii ST4SA and determine the effect of adhesion. Fluorescent in situ hybridization (FISH) incorporating strain specific oilgonucleotide probes indicated strong fluorescent signals for L. plantarum 423 along the intestinal lining of the ileum and the cecum. L. plantarum 423 did not colonise the colon as indicated by real timePCR. Fluorescent signals were recorded for E. mundtii ST4SA across the epithelial barrier of cecum and colonic tissue, suggesting that translocation took place. Real time PCR revealed highest cell numbers of strain ST4SA in the cecum and the colon. Haemotoxylin eosin staining of rat tissue revealed no change in morphology or any toxic effects induced upon adhesion of the strains. 16S rDNA PCR and denaturing gradient gel electrophoresis (DGGE) revealed a decrease in enterobacterial species whilst the lactic acid bacterial content remained unchanged. Strains 423 and ST4SA agglutinated yeast cells in vitro, indicating the possible presence of mannose receptors. It is well known that these receptors play a crucial role in the elimination of type 1 fimbriated strains of E. coli. It is thus safe to speculate that mannose receptors may have played a role in diminishing the enterobacterial content in the gut. The third part of the study encompassed characterization of cell surface proteins of L. plantarum 423 and their role in adhesion to Caco-2 cell lines. The strain lacks the typical surface layer protein whilst a multifunctional “intracellular” protein, elongation factor Tu (EF-Tu) and glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) were detected. Removal of surface proteins reduced adherence of strain 423 to Caco-2 cell lines by 40%, suggesting that these proteins play a role in adhesion. The ability of strain 423 to competitively adhere, exclude and displace Clostridium sporogenes LMG 13570 and Enterococcus faecalis LMG 13566 from Caco-2 cell lines, was studied. Adhesion of C. sporogenes LMG 13570 and E. faecalis LMG 13566 was inhibited by 70% and 90%, respectively. Strain 423 excluded C. sporogenes LMG 13570 from Caco-2 cells by 73% and displaced the pathogen by 80%. E. faecalis LMG 13566 was excluded by 60% and displaced from Caco-2 cells by 90%. Despite removal of the surface proteins, L. plantarum 423 was still capable of competitively adhering to Caco-2 cells and reduced adherence of C. sporogenes LMG 13570 by 50% and E. faecalis LMG 13566 by 70%.

UA Open Access Publishing Fund / Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.

Real-time PCR method is a type of PCR which allows continual monitoring of DNA amplification during every cycle of its process. It is mostly used for gene expression analysis. Based on the results of previous experiments, we decided to test out the effect of anticoagulants EDTA, heparin, sodium citrate and CPDA on the expression of selected genes of the immunological spectrum and further, to test how the time period between drawing the blood and processing of blood sample influences mRNA levels of selected genes that are determined by changes in gene expression and/or mRNA degradation. To quantify mRNA of the studied genes, we isolated total RNA from the peripheral blood leucocytes and transcribed it into cDNA by using the reverse transcription PCR. This cDNA served as a template for the real-time PCR. To examine the changes of the expression caused by the effect of each particular anticoagulants, peripheral blood derived from 10 volunteers was used (each donor's blood was taken into 3 vacuum tubes with EDTA, heparin and sodium citrate anticoagulant agents). Next to that, we obtained 10 buffy coat samples in transfusion blood bags with CPDA anticoagulant agent. Compared to blood cells influenced by one of the three anticoagulant agents present in vacuum tubes, cells from transfusion bags affected...

Plus de 130 espèces de Vibrio, bactéries ubiquitaires des milieux marins et estuariens sont décrites, dont certaines sont des pathogènes d’organismes marins (poissons, coraux, coquillages, …). Douze espèces sont pathogènes pour l’homme, parmi lesquelles V. cholerae, V. parahaemolyticus et V. vulnificus. Les cas d’infections humaines sont essentiellement liés à la consommation de produits de la mer crus ou à l’exposition de plaies cutanées à l’eau de mer. En France, ce risque est faible mais pourrait s’accentuer en raison de la consommation croissante de produits de la mer, de l'augmentation de la part des individus immunodéprimés dans la population, de l'impact des activités anthropiques et du réchauffement climatique sur le milieu marin. Une meilleure compréhension de l'écologie de ces bactéries (distribution, abondance et diversité) est un pré-requis à la mise en place d’une surveillance environnementale de ce risque. L’étude menée au cours de ma thèse en zone Manche-Mer du Nord, dans le cadre du programme Vibrio Manche (2009-2012, partenariat EDFR&D/Ifremer/Institut Pasteur de Lille), avait pour objectif d'évaluer la diversité et la distribution spatio-temporelle côtière des vibrions, dont des espèces potentiellement pathogènes pour l’homme et les animaux, et leur relation avec les paramètres environnementaux biologiques (phytoplancton et zooplancton) et physico-chimiques. La stratégie de suivi a reposé sur le dénombrement et l’isolement de souches de Vibrio, à 22°C et 37°C sur milieu sélectif, à partir d’eau de mer et de sédiments superficiels collectés au cours de neuf campagnes d’échantillonnage et associés à la mesure de paramètres environnementaux. Le développement et la validation d’outils d’identification des souches environnementales par PCR en temps réel et le séquençage de marqueurs génétiques discriminants ont conduit à mettre en évidence une grande diversité d’espèces parmi les isolats à 22°C, celle-ci étant composée principalement de pathogènes d’organismes marins dont l’espèce V. splendidus. Les isolats à 37°C, marqués par la prédominance de deux espèces, V. alginolyticus et V. harveyi, et la rareté des pathogènes humains détectés dans les conditions d’analyses retenues pour cette étude. Au terme de l’étude, nous avons mis en évidence la présence de 20 et 17 espèces de vibrions respectivement à 22°C (deux campagnes) et 37°C (neuf campagnes) sur la zone étudiée, et l’influence probable de conditions environnementales contrastées sur la structure de ces populations. En termes d’abondance, les vibrions cultivables à 37°C ont montré une forte saisonnalité, et une relation significative à la température de l’eau et à l’abondance totale du zooplancton. La saisonnalité des vibrions cultivables à 22°C a été moindre, et leur abondance a pu être reliée de façon significative à la concentration en chlorophylle a totale et également à l’abondance totale en zooplancton. Ces éléments de connaissance et la stratégie d’identification développée pourront, à terme, contribuer au développement d’outils de surveillance microbiologique environnementale et ouvrent des perspectives pour mieux comprendre l’écologie des vibrions et les facteurs structurant non seulement leur abondance, mais aussi leur diversité. / One hundred and thirty species of vibrios, ubiquitous bacteria of marine and estuarine environments have been described, some of them being pathogenic for marine organisms. Twelve species are classified as human pathogens, Vibrio cholerae, V. parahaemolyticus and V. vulnificus being the most frequently reported in cases of infection. Human infections are linked mostly to raw seafood consumption or to the exposure of skin wounds to contaminated seawater. In France, the risk is low but it is expected to increase in the future due to the increase of raw seafood consumption, the part of immuno-compromised people, but also the impact of anthropic activities and global warming on the marine environment. A better understanding of the ecology of these bacteria (distribution pattern, abundance and diversity) is a prerequisite for the monitoring of the Vibrio-risk in the environment. The 2-year study carried out in the Eastern English Channel during my PhD was part of the Vibrio Manche program (2009-2012, funding EDF R&D/Ifremer/Institut Pasteur de Lille), which main objective was to characterize the spatio-temporal distribution of the vibrios, among which the potentially pathogenic species for human and animal, and their relations with the biotic(phytoplankton and zooplankton) and abiotic environmental parameters. The strategy consisted in the enumeration and isolation of Vibrio strains, at 22°C and37°C on a selective medium, from seawater and superficial sediments collected during nine sampling campaigns and associated with the recording of environmental parameters. The protocol developed in this study provides an appropriate and rapid screening tool to identify a large number of bacterial strains routinely isolated from the environment. This was based on real-time PCR assays and the sequencing of discriminant genetic markers. We highlighted the important diversity of species among the 22°C isolates, represented mainly by species pathogenic for marine organisms such as V. splendidus. Contrastingly, the 37°C isolates were mainly represented by two species, V. alginolyticus and V. harveyi, and the potentially pathogenic species for humans were rarely detected using this experimental approach. We detected 20 and 17 species of vibrios among the 22°C (two sampling campaigns) and 37°C(nine sampling campaigns) isolates, respectively, and highlighted the probable influence of contrasted environmental conditions on these populations structure. These two populations presented different seasonal dynamics, as the seawater temperature and the zooplankton abundance showed to be the main drivers for the 37°C population. The 22°C population seemed to be linked to the chlorophyll a concentration and zooplankton abundance. These data and the approach developed in this study could contribute to the development of tools for the monitoring of coastal environment and they give perspectives to better understand the ecology of the vibrios and the environmental factors driving their abundance and diversity.

O objetivo do estudo foi avaliar, o efeito do laser de Diodo 808nm como coadjuvante ao tratamento periodontal na redução de periodontopatógenos Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia pela técnica da Reação em Cadeia da Polimerase em Tempo Real. Foram selecionados vinte e quatro pacientes portadores de periodontite crônica neste estudo de boca dividida, duplo cego e randomizado. Dois sítios uniradiculares de cada paciente foram utilizados e divididos em dois grupos experimentais: TESTE - raspagem alisamento polimento corono radicular (RAPCR) associado à duas aplicações de laser de Diodo de alta potência (comprimento de onda de 808nm, 1,5 Watts, 597,1 W/cm2, durante 20 segundos no modo contínuo). A primeira aplicação foi realizada 24 horas após RAPCR e a segunda após sete dias; CONTROLE foi realizado o mesmo procedimento porém sem a aplicação do laser. O biofilme subgengival foi coletado antes do tratamento e seis semanas após a segunda aplicação do laser. A avaliação microbiológica foi feita através da Reação em Cadeia da Polimerase em tempo real para a quantificação de Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia. A comparação entre os grupos não demonstram diferenças estatisticamente significantes (p<0,05). Concluiu-se que, dentro dos limites deste estudo, a aplicação do laser de Diodo de 808nm como coadjuvante ao tratamento periodontal não reduziu de forma significativa os periodontopatógenos: Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia quando comparado à RAPCR. / The aim of this study was to evaluate, by real-time polymerase chain reaction, the effect of Diode laser 808nm as an adjuvant in the reduction of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia in to periodontal treatment. Twenty-four patients with chronic periodontitis the study designs was split-mouth, double blind and ramdomized controlled trial. Two sites from uniradicular teeth of each patient were used and divided in two experimental groups: TEST scalling and root planing (SRP) associated with two high power Diode laser application (wavelength of 808nm, 1,5W at the display (597,1 W/cm2) for 20 seconds in the continuous-wave mode) the first laser application was 24 hours after SRP and the second seven days later; CONTROL a similar procedure without laser application. The subgingival biofilm was colleted before treatment and six weeks after the second laser application. The microbiologic evaluation was done by real-time polymerase chain reaction for the quantification of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. The comparison between the groups did not show significant differences (p<0,05). Within the limits of this study, it can be concluded that Diode laser 808nm application as an adjuvant in the periodontal treatment did not reduce the periodontal pathogens Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia when compared by scaling root planning.

Nos últimos anos, o Brasil tem liderado a produção e exportação mundial de cana-de-açúcar e seus derivados. Com isso, a produtividade agrícola aumenta necessitando-se ampliar as pesquisas de caráter sócio-ambiental, a fim de modelar um desenvolvimento tecnológico mais próximo do sustentável. Desta forma, torna-se necessário conhecer a contribuição do plantio da cana-de-açúcar com o aumento da concentração de gases de efeito estufa na atmosfera, especialmente no que se refere ao óxido nitroso (N2O), derivado do uso de fertilizantes nitrogenados. Sabe-se que na ausência de oxigênio, bactérias específicas passam a reduzir compostos nitrogenados, formando este poderoso gás de aquecimento global durante as etapas decorrentes de um processo conhecido como desnitrificação biológica. Através dos avanços nas áreas da biologia molecular, tornou-se possível conhecer os microorganismos não cultiváveis e suas respectivas funções em um determinado ambiente. Este trabalho teve como objetivo estimar as emissões de N-N2O nos solos de cana-de-açúcar derivadas da aplicação de fertilizantes nitrogenados durante um período de trinta dias, em duas situações de manejo da cultura: I- Sem queima: colheita mecanizada com a manutenção da palha da cana; II-Com queima: colheita manual precedida pela queima da palha da cana. Simultaneamente, os genes envolvidos na desnitrificação e produção de N2O no solo (norB, nirK, nirS e nosZ) foram quantificados por PCR quantitativo em tempo real em condições de laboratório e de campo. Sob condições de laboratório, pode-se observar que as emissões de NN2O atingiram 0,8 mg.m-2 h-1 em solos com a manutenção da palha, tanto com a aplicação de uréia quanto de nitrato de amônio. Os mesmos produtos, quando aplicados em solos sem a presença da palha emitiram 0,45 mg.m-2 h-1. No campo, os maiores fluxos de N-N2O foram encontrados no período de elevada precipitação pluviométrica, que ocorreu na primeira semana após a aplicação do fertilizante nitrato de amônio, chegando a 0,7 mg.m-2 h-1 nos solos com palha e 0,37 mg.m-2 h-1 nos solos sem a palha. Tanto no campo quanto no laboratório foram encontradas as maiores quantidades dos genes envolvidos na desnitrificação em solos com a permanência da palha, com valores próximos de 107 genes por grama de solo. A atual tendência a substituir a colheita manual da cana pela colheita mecanizada favorece a agregação do solo, diminui o grau de erosão e aumenta os estoques de carbono, mas também pode resultar em aumento das emissões de N-N2O e do fator de emissão dos fertilizantes nitrogenados, conforme encontrado para o nitrato de amônio aplicado no campo, onde o valor obtido foi de 0,3% para os solos sem a palha e 0,7% para solos com palha. Sendo assim, é fundamental estudar as melhores condições do uso da terra e o papel exercido pela microbiota que nela existe, proporcionando um tratamento mais adequado aos resíduos culturais, diminuindo assim as emissões de gases estufas. / In recent years, Brazil has led the production and worldwide export of sugarcane and its products. Thus, agricultural productivity increases need to expand research of socio-environmental, in order to model a technology development closer sustainable. Therefore, it is necessary to know the contribution of planting sugarcane with increasing concentration of greenhouse gases in the atmosphere, especially with regard to nitrous oxide (N2O), derived from nitrogenous fertilizer use. It is known that in the absence of oxygen, bacteria are specific to reduce nitrogen, forming this powerful global warming gas arising during the stages of a biological process known as denitrification. Through advances in molecular biology, it has become possible to know the non-cultivable micro-organisms and their functions in a given environment. This study aimed to measure the emissions of N-N2O in soils of sugarcane derived from the application of nitrogen fertilizers for a period of thirty days, in two situations crop management: I-No burning: mechanized harvesting with maintaining cane straw; II-With burning: manual harvest preceded by burning the straw. Simultaneously, the genes involved in denitrification and N2O production in soil (norB, nirS, nirK, and nosZ) were quantified by real-time quantitative PCR in the laboratory and the field. Under laboratory conditions, it can be seen that the N-N2O reached 0,8 mg.m-2 h-1 in soils with maintaining the straw, either with the application of urea and ammonium nitrate. The same products, when applied to soils without the presence of straw delivered 0,45 mg.m-2 h-1. In the field, the highest N-N2O fluxes were found in the period of high rainfall, which occurred in the first week after application of ammonium nitrate, reaching 0,7 mg.m-2 h-1 in soils with straw and 0,37 mg.m-2 h-1 in the soil without straw. Both in the field and in the laboratory were found greater amounts of genes involved in denitrification in soils fertilized with the permanence and straw, with values close to 107 per gram of soil. The current trend is to replace manual harvesting of sugarcane by mechanized harvesting promotes soil aggregation, decreases the degree of erosion and increases carbon stocks, but can also result in increased emission factor of nitrogen fertilizers, as found for nitrate applied in the field, where the value was 0,3% for the soil without straw and 0.7% for soils with straw. Therefore, it is essential to study the best conditions of land use and the role played by the microbiota that is therein, providing appropriate treatment to crop residues, thus reducing greenhouse gas emissions.

A leishmaniose visceral canina (LVC) é uma grave zoonose, causada pela Leishmania infantum (syn. chagasi). O ciclo deste parasito é heteroxeno e a transmissão acontece principalmente pela picada da fêmea do vetor, dípteros da espécie Lutzomyia longipapis. O cão doméstico é o principal alvo das campanhas de controle da doença por ser a principal fonte de infecção para o vetor no ambiente urbano. O Ministério da Saúde adota testes sorológicos para a detecção de animais positivos; no entanto a sensibilidade e especificidade desses testes são questionáveis. Além das falhas inerentes a qualquer teste diagnóstico, no caso da LVC existem alguns entraves, especialmente pela distribuição geográfica, comum a outras doenças causadas por tripanossomas, e pela similaridade genética com os outros parasitas da mesma família. Nessas áreas de sobreposição pode haver tanto reação cruzada quanto co-infecção, dificultando a interpretação dos testes. No presente estudo, foram coletadas amostras de suabe conjuntival e sangue de cães em inquérito soroepidemiológico realizado no município de Ilha Solteira - SP. A presença de Leishmania spp. e Leishmania infantum foram testadas por PCR convencional e PCR em tempo real com primers direcionados ao kDNA de Leishmania spp. A avaliação sorológica foi realizada através da RIFI, e a identificação e caracterização dos tripanossomatídeos foi realizada através da PCR com primers ITS1. A SC-qPCR foi o teste que detectou o maior número de animais. De 204 cães utilizados no estudo, 19,12% (30/204) foram positivos na SC-qPCR. Na SG-qPCR foram 12,74% (26/204) de animais positivos. O teste que detectou o menor número de animais foi a SC-cPCR, com 10,78% (22/204). Enquanto na SG-cPCR obtivemos 13,23% (27/204) animais positivos. De 28 amostras selecionadas para o sequenciamento do gene ITS1, 19 (67,85%) foram 100 ou 99% similares à L. infantum, sugerindo que a maioria dos cães positivos para LVC estavam realmente infectados com esta espécie. Entretanto, 2 cães (7,14%), que tiveram suas amostras sequenciadas para tal gene, revelaram 99% de similaridade com Crithidia fasciculata. Dos testes avaliados, esses cães foram positivos apenas na SG-cPCR para Leishmania spp. Os resultados indicam que a SC-qPCR foi o teste mais eficaz em detectar amostras realmente positivas para L. infantum, e que se deve atentar ao fato de existirem outros tripanossomatídeos infectando os cães em área endêmica de LVC, podendo dificultar o diagnóstico adequado dos animais infectados por L. infantum. / Canine visceral leishmaniasis (CVL) is a serious zoonosis caused by Leishmania infantum (syn. chagasi). The life cycle of this parasite is heteroxenous and the transmission occurs through the bite of the female sandfly, diptera species Lutzomyia longipalpis. The domestic dog is the main focus disease control campaigns, since it is the most important source of infection for the vector in urban environment. For positive dogs detection the health ministry uses serological tests, however the sensitivity and specificity of these tests are questionable. In addition to flaws inherent in any diagnostic test, in case of CVL there are some obstacles, especially by geographic distribution, common to other diseases caused by trypanosomes, and also by genetic similarity with other parasites of the same family. In areas of disease overlap, cross-reaction or co-infection may occur, making it difficult to interpret the results. In this study, conjunctival swab samples and whole blood of dogs were collected in seroepidemiological survey conducted in Ilha Solteira - SP. The presence of Leishmania spp. and Leishmania infantum were tested by conventional PCR and real-time PCR with Leishmania spp. kDNA-targeted primers. The serological evaluation was carried out by RIFI, and the identification and characterization of trypanosomatids was performed by PCR with ITS1 primers. SC-qPCR was the test that detected the largest number of animals. Of 204 dogs used in this study, 19.12% (30/204) were positive in SC-qPCR. In SG-qPCR 12.74% (26/204) animals were positive. The test that detected the lowest number of animals was SC-cPCR, with 10.78% (22/204). While in the SG-cPCR we obtained 13.23% (27/204) positive animals. From 28 samples selected for ITS1 gene sequencing, 19 (67.85%) were 100 or 99% similar to L. infantum, suggesting that most CVL positive dogs were infected with this species. However, two dogs (7.14%), which had their samples sequenced for the same gene, showed 99% similarity with Crithidia fasciculata. From the evaluated tests, these dogs were only positive in SG-cPCR for Leishmania spp. The results indicate that SC-qPCR was the most effective test to detect L. infantum positive samples , and it should be noted that there are other trypanosomatids infecting dogs in an endemic CVL area, which can difficult to diagnose animals properly infected by L. infantum.