El objetivo del presente estudio fue detectar la presencia de Salmonella spp. en el total (n igual a 30) de la población de tortugas motelo (Geochelone denticulata) de un criadero de 25 m2 de área, construido con materiales de la misma zona y piso de tierra; la alimentación de los animales a base de frutas y verduras, agua Ad libitum y se observó presencia de roedores, la crianza se realizo en condiciones de cautiverio en la ciudad de Iquitos, Región Loreto. Las muestras de heces se obtuvieron por hisopado rectal, luego llevadas al laboratorio de la Estación Experimental IVITA-Iquitos y se colocaron en un medio de enriquecimiento Agar Soya Tripticasa para luego ser remitidas en condiciones de refrigeración a la Sección de Bacteriología y Micología del Laboratorio de Microbiología y Parasitología, de la Facultad de Medicina Veterinaria de la Universidad Nacional Mayor de San Marcos; para su aislamiento e identificación mediante pruebas de cultivo bacteriológico y bioquímicas. El 6.7% (2/30) de las muestras fueron positivas a Salmonella spp. Adicionalmente se encontraron otras bacterias como Escherichia coli y Pseudomona aeruginosa en un 80% (24/30), Proteus vulgaris en un 26.6% (8/30), Proteus mirabilis en 6.7% (2/30) y Citrobacter spp. 16.7% (5/30). La Salmonella spp se detectó en tortugas sanas y enfermas, además se realizó la tipificación por el método Who Global Salm Surv, obteniendo como resultado Salmonella enterica subespecie enterica serotipo typhimurium en las dos muestras positivas a dicha bacteria. Se concluye que existe presencia de Salmonella spp. (Salmonella typhimurium) en muestras de hisopados cloacales de tortugas motelo criadas en cautiverio, y que los demás microorganismos hallados forman parte de la familia enterobacteriaceae propio del tracto intestinal de éstos animales. / The objective of the present study was to detect the presence of Salmonella spp. in the total (n same 30) of the population of turtles motelo (Geochelone denticulata) of a hatchery of 25 m2 of area, built with materials of the same area and earth floor; the feeding of the animals with the help of fruits and vegetables, dilutes Ad libitum and presence of rodents, the upbringing one was observed I carry out under captivity conditions in the city of Iquitos, Región Loreto. The samples of grounds were obtained by rectal hisopado and then taken to the laboratory of the Experimental Station IVITA-Iquitos and they were placed in a means of enrichment Agar Soya Tripticasa for then to be remitted under refrigeration conditions to the Section of Bacteriology and Micología of the Laboratory of Microbiology and Parasitología, of the Faculty of Veterinary Medicine of the University National Major of than San Marcos; for their isolation and identification by means of bacteriological and biochemical cultivation tests. The 6. 7% (2/30) of the samples they went positive to Salmonella spp. Additionally they were other bacterias like Escherichia coli and Pseudomona aeruginosa in 80% (24/30), Proteus vulgaris in a 26. 6% (8/30), Proteus mirabilis in 6. 7% (2/30) and Citrobacter spp. 16. 7% (5/30). The Salmonella spp was detected in healthy and sick turtles, she was also carried out the tipificación for the method Who Global Salm Surv, obtaining Salmonella enterica subspecies enterica serotipo typhimurium in the two positive samples to this bacteria. You concludes that presence of Salmonella spp exists. (Salmonella typhimurium) in samples of hisopados cloacales of turtles motelo raised in captivity, and that the other found microorganisms are part of the family enterobacteriaceae characteristic of the intestinal tract of these animals.

Thèse d'exercice : Pharmacie : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 105-118).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 88-102).

Salmonella infections remain one of the most common food borne diseases worldwide. Gastroenteritis, which can be caused by many non-typhoidal Salmonella (NTS) serovars, is relatively common in North America. One of the main risk factors of NTS gastroenteritis is travel to endemic areas in the developing world. The current treatment of NTS infections with antibiotics is reserved for severe cases. A growing concern with antibiotic use is that clinical isolates are becoming drug resistant. Although most NTS infections are self-limiting in nature, the burden on the body and recovery can take several months. Thus, it is vital to prevent NTS infections rather than solely rely on treatment. We have previously discovered two novel surface associated polysaccharides in Salmonella: O-Antigen capsule and X-factor. Not only O-Antigen Capsule is considered a common surface antigen, but its’ genes were found to be expressed during in vivo infections in mice. Such an antigen would be a suitable candidate in developing a vaccine against Salmonella induced gastroenteritis. The goal of this research was to evaluate the use of O-Antigen capsule to develop a traveler’s vaccine for NTS associated gastroenteritis. Results and Conclusions: We have developed a purification protocol and purified the capsule and X-factor from Salmonella Typhimurium, Enteritidis, and Heidelberg. Lipopolysaccharide (LPS) was co-isolated with O-Antigen capsule, but removed using Triton extraction. Salmonella LPS is strain-specific and an adaptive immune response against LPS will not provide cross-protection. We generated specific immune sera in rabbits to recognize O-Antigen capsule and X-factor produced by Salmonella Typhimurium and Enteritidis. We used a mouse model to determine the immunization dose of O-Antigen capsule and showed that conjugation is necessary to enhance the immune response in mice. To boost capsule production, we analyzed PyihUTSRQPO activity using a luciferase-based reporter system. Deletion of a putative transcriptional repressor (YihW) resulted in over 100-fold increase in PyihUTSRQPO confirming YihW as a repressor. We have also looked at the effect of growth media, temperature, and sugar precursors on PyihUTSRQPO activity, and were able to show that PyihUTSRQPO has highest activity in Tryptone broth at 30oC in the absence of any additional sugars.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

In recent decades salmonellosis has been on the rise as a food related illness worldwide. Causing over 24% of all non-typhoidal Salmonellosis cases, SE is the most frequently isolated serovar of Salmonella. Increased isolation of SE from eggs has paralleled an increase in the number of transovarian infections associated with laying hens in the poultry industry. This route of infection is a fairly new line of study when compared to the more traditional path where SE originates from fecal contamination through the shell. Salmonella Typhimurium (ST) is another concern for the egg industry. ST has caused 23.5% of all non-typhoidal salmonellosis cases. Understanding these two egg pathogens requires an in depth look at the mechanisms by which an egg may support infection and bacterial growth. Eggs were inoculated with both SE and ST onto the vitelline membrane and incubated for 24 hours. It was hoped that by gathering samples from the interior of the egg membrane, the albumen of the egg, and the membrane itself, some clarification as to when Salmonella is allowed to grow within the egg could be gathered. Albumen and membrane were found to be more hospitable environments to bacterial growth with increased storage times. In order to better understand the movement of bacteria into pre-ovulatory tissues, samples were gathered from mature laying hens. Follicular tissues were separated into divisions based on maturity, and bacteria were added to an in vitro cell culture broth containing the follicles. The point of this experiment was to determine if either species of Salmonella preferentially moved into follicles of different maturity when inoculated in vitro. A third experiment looked into the role of developmental stages of the vitelline membrane in exclusion of bacteria from the nutrient rich yolk. Tissues were gathered in the method described above. The follicular sack was removed from half of these samples and left intact for the other half. Another treatment group included was the yolks of eggs which had been laid by the same flock of birds. Results showed that follicles with intact follicular sacks were more susceptible to bacterial colonization than other treatment groups.

La salmonelosis es una de las enfermedades transmitidas por alimentos más frecuente en el mundo. El Código Alimentario Argentino establece para carne molida la ausencia de Salmonella spp. y para su análisis recomienda la norma BAM. Esta metodología requiere tiempo hasta obtener el resultado final. La PCR se utiliza como método rápido para la detección de patógenos. El objetivo de este trabajo fue prevenir la presencia de Salmonella spp. en la carne molida destinada a consumo minorista. Se desarrolló una técnica de PCR en tiempo real (RT- PCR) con agentes intercalantes para la detección de Salmonella spp. y se realizó una validación intra-laboratorio. El límite de detección fue 104 UFC/20 μl de mezcla de reacción. En esta etapa la técnica presentó 100% de inclusividad y exclusividad. Se comparó el desempeño de esta técnica contra una PCR de punto final y una RT-PCR comercial mediante el análisis de 92 muestras de carne molida obtenidas de carnicerías de la ciudad de Berisso. Durante el muestreo se realizó una encuesta al responsable del comercio. Se obtuvieron 13 (14%) muestras positivas por aislamiento y por RT-PCR comercial y ocho (8,7%) muestras positivas al utilizar PCR de punto final y la técnica desarrollada. Se aislaron 5 serotipos de Salmonella. El desempeño de la RT-PCR desarrollada se puede mejorar mediante el rediseño de los oligonucleótidos cebadores en base a los clones circulantes aislados en la carne molida. En las encuestas se identificaron problemas como: falta de trazabilidad de la carne, falta de procedimientos de sanitización estandarizado y carencias edilicias. El Programa “Carnicerías Saludables” permitió detectar los puntos críticos en las etapas de triturado y envasado de la carne molida y de esta forma implementar estrategias de prevención y control de salmonelosis por medio de la capacitación de 167 manipuladores.

6 Zusammenfassung In-vitro- und In-vivo-Studien zur Wirksamkeit von Eigelbantikörpern gegen Salmonella Enteritidis Michael Gürtler Institut für Lebensmittelhygiene der Veterinärmedizinischen Fakultät der Universität Leipzig Infektionen mit Salmonellen gehören weltweit zu den wichtigsten von Tieren auf den Menschen übertragbaren Erkrankungen. Anteilmäßig besitzen dabei die durch kontaminierte Lebensmittel, insbesondere Eier und Eiprodukte, hervorgerufenen Infektionen die größte Bedeutung. Dabei ist nach wie vor Salmonella Enteritidis (S. Enteritidis) in den Geflügelbeständen vorherrschend. Als Möglichkeit zur Bekämpfung von Salmonella-Infektionen bietet sich die passive Immunisierung der Legehennen durch orale Applikation von Eigelbantikörpern an. Bereits mehrfach wurde das Verfahren der Verabreichung von antikörperhaltigem Volleipulver zur Bekämpfung von bakteriellen Infektionskrankheiten, vor allem bei Kälbern und Ferkeln, getestet. Untersuchungen über die Einsatz solcher Eigelbantikörper mit dem Ziel der Zurückdrängung von Salmonellen in Legehennenbeständen sind bisher nicht bekannt geworden. Ziel dieser Arbeit war es, im Rahmen von In-vitro- und In-vivo-Studien den Einfluss von Eigelbantikörpern auf Vermehrung und Vorkommen von S. Enteritidis bei Legehennen zu untersuchen. Im Versuchskomplex A wurde in vitro ermittelt, ob Anti-S. Enteritidis-Antikörper im Eidotter in der Lage sind, die Vermehrung von Salmonellen zu hemmen. Im Versuchskomplex B wurde untersucht, ob es gelingt, durch die orale Verabreichung von Anti-S. Enteritidis-Eigelbantikörpern die Kontaminationsrate der gelegten Eier mit Salmonellen bei experimentell infizierten Legehennen zu beeinflussen. Im Versuchskomplex C sollte geprüft werden, ob Eigelbantikörper die Fähigkeit besitzen, das Adhäsions- und Invasionsvermögen von Salmonellen an bzw. in Epithelzellen einer permanenten Zelllinie zu beeinflussen. Im Rahmen von Versuchskomplex A wurden Legehennen mit einem inaktivierten S. Enteritidis-Stamm parenteral hyperimmunisiert, um hohe Konzentrationen an Antikörpern im Dotter zu induzieren. Die Eidotter wurden mit zwei S. Enteritidis-Stämmen in drei verschiedenen Kontaminationsdosen beimpft und das Vermehrungsverhalten durch Keimzählung als Generationszeit ermittelt. Im Versuchskomplex B wurden in zwei Infektionsversuchen Anti-S. Enteritidis-Eigelbantikörper in Form von Volleipulver in einer Menge von 3 g pro Tier und Tag über das Futter an experimentell mit S. Enteritidis infizierte Legehennen verabreicht und die Ergebnisse mit denen einer Kontrollgruppe ohne Volleipulver verglichen. Die gelegten Eier wurden getrennt nach Eischale, Eiklar und Eigelb auf den Infektionsstamm untersucht. Im Versuchskomplex C wurden die Adhäsivität und Invasivität verschiedener Salmonella-Stämme an bzw. in Epithelzellen bei An- bzw. Abwesenheit von Anti-S. Enteritids-Eigelb- bzw. -Serumantikörpern unter Verwendung einer permanenten Dünndarmepithelzelllinie von der Ratte (IEC-6) nach Inkubation durch Keimzählung überprüft. Die Ergebnisse in Versuchskomplex A zeigten, dass Eigelbantikörper nicht in der Lage sind, im Eigelb befindliche Salmonellen in ihrer Vermehrung zu hemmen oder gar abzutöten. Die Infektionsversuche im Versuchskomplex B ergaben, dass bei einer Infektionsdosis von 2x109 koloniebildenden Einheiten (kbE)/Tier die Salmonella-Kontaminationsrate der Eier durch das antikörperhaltige Eipulver nicht gesichert verändert wird. Dagegen konnte beim Einsatz einer solchen von 2x108 kbE/Tier eine Wirkung des antikörperhaltigen Eipulvers auf die Kontamination der einzelnen Eikompartimente nachgewiesen werden. Dabei wurden in der Versuchsgruppe mit 13,3 % gesichert weniger Eier als positiv detektiert als in der Kontrollgruppe mit 29,4 % (p ≤ 0,001), wobei mindestens in einem der drei Kompartimente Eischale, Eiklar oder Eigelb Salmonellen nachweisbar waren. In der Versuchsgruppe wies jedes Salmonella-positive Ei auch Keime auf der Eischale auf. In der Kontrollgruppe waren mit 29,9 % signifikant mehr Eischalen positiv als in der Versuchsgruppe mit 13,3 % (p ≤ 0,001). Beim Eiklar ließ die geringe Nachweisrate sowohl in der Versuchs- als auch in der Kontrollgruppe eine statistische Beurteilung des Eipulvereinflusses nicht zu. Aus dem Eidotter wurden in der Versuchsgruppe bei 0,6 % der Eier Salmonellen isoliert, während es in der Kontrollgruppe mit 2,4 % signifikant mehr waren (p ≤ 0,05). Die Ergebnisse des Versuchskomplexes C zeigten, dass Volleiantikörper und Serumantikörper die Invasion von S. Enteritidis in intestinale Epithelzellen serovarspezifisch vermindern können. Insgesamt lassen die Ergebnisse die Schlussfolgerung zu, dass bei einer oralen Verabreichung von Volleipulver mit S. Enteritidis-spezifischen Antikörpern an Legehennen bei einer Infektionsbelastung von 2x108 kbE/Tier mit einer gesicherten Reduzierung des Vorkommens von Salmonellen im Nahrungsmittel Ei im Vergleich zu Tieren ohne Zugabe von Eipulver zu rechnen ist. / In vitro and in vivo studies on the efficiency of egg yolk antibodies against Salmonella Enteritidis Michael Gürtler Institute of Food Hygiene, Faculty of Veterinary Medicine, University of Leipzig Infection of humans by Salmonella are among the most important zoonotic diseases. Contaminated food, especially eggs and egg products, represents the highest risk. Still Salmonella Enteritidis (S. Enteritidis) dominates, a serovar that occurs especially in poultry flocks. A possibility to combat Salmonella infection is the passive immunisation of laying hens by oral administration of egg yolk antibodies. The application of antibody-containing full-egg powder against bacterial diseases was tested previously especially on calves and piglets. The suitability of administration of egg-yolk antibodies to protect laying-hens flocks against Salmonella was not tested previously. It was the aim of the study to investigate the influence of egg-yolk antibodies of S. Enteritidis in egg laying hens by in-vivo and in-vitro studies. In part A, we investigated the influence of anti-S. antibodies in egg yolk on the multiplication of Salmonella in vitro. In part B, the impact of oral application of anti-Salmonella full-egg antibodies on the in-vivo Salmonella-contamination rate of eggs layed by experimentally infected hens was investigated. In part C the ability of egg-yolk antibodies and serum antibodies were tested with regard to the rate of adhesion and invasion of Salmonella on or in epithelial cells of a permanent cell culture line (two major pathogenic mechanisms). In part A, laying hens were hyper-immunised parenterally by an inactivated S. Enteritidis strain to induce a high concentration of antibodies in the egg yolk. Egg yolk was than inoculated with two S. Enteritidis strains in three different contamination doses. Multiplication of Salmonella cells was detected by cell counts and calculated as generation time. In part B, laying hens were immunised passively with anti-Salmonella egg yolk antibodies (by feeding 3 g of full egg powder per day and per animal) and the effects were compared with a control group without full-egg powder. The inoculation strain of S. Enteritidis was detected on egg shell, in egg white and in egg yolk separately. In part C, a suspension of S. Enteritidis was added to a permanent duodenal epithelial cell line of the rat (IEC-6). After incubation the adhesivity of Salmonella strains and their ability to invade these cells in the presence or in the absence of anti-S. Enteritidis egg yolk and serum antibodies, respectively, was detected by cell count. The results of part A demonstrated that egg-yolk antibodies are not able to inhibit multiplication of Salmonella in egg yolk. Experiments of part B showed that the Salmonella contamination rate of eggs could not be influenced by antibody-containing egg powder at a Salmonella infection dose of 2x109 colony forming units (cfu)/animal, whereas the antibody-containing egg powder at an Salmonella infection dose of 2x108 cfu/animal proved effective (i.e. contamination rate of egg components was reduced). In the experimental group 13.3 % of the eggs were Salmonella positive, which was significantly different from 29.4 % in the control group (p ≤ 0,001). At least in one of the three compartiments egg shell, egg white or egg yolk Salmonella was detectable. In the experimental group Salmonella could be isolated from the shell of every positive egg. The rate of Salmonella isolation from egg shells of the control group was significantly higher those in the experimental group (p ≤ 0,001). With regard to egg white, the low number of Salmonella detections in both groups did not allow statistical evaluation of the effect of full-egg powder. From egg yolk of the experimental group in 0.6 % of the eggs Salmonella could be isolated which was significantly lower than the detection rate in the control group with 2.4 % (p ≤ 0,05). The results of part C demonstrated that full-egg antibodies and serum antibodies can inhibit the serovar-specific invasion of S. Enteritidis into intestinal epithelial cells. As a whole, the results support the conclusion that oral administration of full-egg powder containing S. Enteritidis specific antibodies to egg-laying hens exposed to a administration of 2x108 cfu/animal reduce the occurrence of Salmonella in the egg compared to eggs of animals without egg powder administration.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.