Con el objetivo de caracterizar la fosfolipasa A de la membrana externa (OMPLA E.C. 3.1.1.32) de Campylobacter jejuni aislado de pollos de carne, se estandarizaron métodos para el aislamiento e identificación de la bacteria a partir de hisopados rectales de aves comerciales. Para ello, primero se analizó la presencia de C. jejuni en hisopados cloacales mediante técnicas bacteriológicas y la reacción en cadena de la polimerasa anidada en base a los genes ribosómicos 16S e hipO, las cuales permitierón detectar Campylobacter jejuni en 29/50 (58%) y 48/50 (96%) de las muestras respectivamente. Después, se determinó la secuencia nucleotídica de los genes ribosómicos 16S y flaA del aislado MP4 con la finalidad de tener una cepa nativa de C. jejuni bien caracterizada que se utilice como modelo de estudio para la fosfolipasa A, las secuencias nucleotídicas de estos dos genes mostraron 98% y 92% de similiitud nucleotídica con las de Campylobacter jejuni subs. jejuni ATCC 33560. Las actividades hemolítica y de fosfolipasa A de C. jejuni MP4 fue dependiente de calcio, cuando este ión se reemplazó con EDTA, las actividades disminuyeron de 4119.4 a 44.78 U/mg de proteína. Utilizando cebadores específicos se amplificó y secuenció la parte central del gen pldA de C. jejuni MP4, se obtuvo 225 nucleótidos y 75 aminoácidos. La comparación de la secuencia aminoácidica por el ClustalW mostró 99% y 98% de identidad con las proteínas OMPLA de las cepas de Campylobacter jejuni RM1221 y C.jejuni subsp. jejuni ATCC 33560 respectivamente; esta alta conservación de secuencia aminoacídica de la fosfolipasa A la convierte en una candidata potencial para el diseño de vacunas y pruebas diagnósticas. / --- In order to characterize the Campylobacter jejuni’s outer membrane phospholipase A (OMPLA E.C. 3.1.1.32) from broiler chickens, it was standarized suitable methods for isolation and identification of this bacterium from cloacal swabs of commercial chickens. To that end, first it was analized the presence of C. jejuni in cloacal swabs by using bacteriological techniques and nested-PCR based on 16S ribosomal and hipO genes. The approaches used detected C. jejuni in the 29/50 (58%) and 48/50 (96%) of the samples respectively. Then, it was determined the nucleotidic sequence of 16S ribosomal gene to have a well-characterized native strain for using it as a model in the study of phospholipase A. The nucleotidic sequence of both genes showed 98% and 92% of similarity with Campylobacter jejuni subs. jejuni ATCC 33560. The haemolytic and phospholipase A enzymatic activities of the C. jejuni MP4 strain was calcium-depended, when calcium was replaced by EDTA both activities significantly decreased of 4119.4 to 44.78 U/mg protein. It was amplified and sequenced the central side from pldA gen of C. jejuni MP4 using specific primers. By means of that, it was obtained 225 nucleotides and 75 aminoacids. The comparison of aminoacidic sequences by CLUSTALW showed 99% and 98% of identity with OMPLA proteins from C. jejuni RM1221 and C. jejuni subsp. jejuni ATCC 33560. This high conservation of the aminoacidic sequence of phospholipase A become it into a potential target to design vaccines and diagnostic tests.

Memoria para optar al Título Profesional de Médico Veterinario / Aunque la presencia de Campylobacter jejuni ha disminuido considerablemente en productos avícolas, esta bacteria sigue siendo un riesgo importante para la salud humana. Es por ello que se analizó la presencia de C. jejuni en carne de pollo congelado obtenido de una planta procesadora en Santiago, Chile. Las muestras provinieron de dos plantas faenadoras (A y B) ubicadas en la Región Metropolitana, pertenecientes a la misma planta procesadora. Un total de 300 muestras fueron analizadas, siendo la unidad de muestra una bolsa sellada de 500 gramos, la cual contenía distintos cortes de ave congelada. Las bolsas fueron transportadas manteniendo la cadena de frío y procesadas inmediatamente después de su descongelación. Submuestras de cada bolsa fueron sembradas en caldo de enriquecimiento (caldo Exeter modificado), y luego se determinó la presencia de C. jejuni utilizando paralelamente dos medios de cultivo (mCCDA y Skirrow). Todos los cultivos se observaron macroscópicamente, siendo aquellas colonias sospechosas, verificadas con tinción de gram (bacilo curvo negativo). La determinación de la especie se realizó mediante el esquema de bio-tipificación descrita por Lior (Nachamkin y Blaser, 2000). Según los análisis realizados, la presencia de C. jejuni en carne de ave congelada alcanzó un 12% (36/300). Los porcentajes de presencia en cada planta faenadora fue de 15,15% (20/132) y 9,52% (16/168), para A y B, respectivamente. De acuerdo con los análisis estadísticos realizados, la diferencia entre estas proporciones no fue significativa (p>0,05). Estos valores son menores a los encontrados por Figueroa et al. (1996), quienes determinaron un 37% para muestras similares, lo que podría deberse a un mejor manejo sanitario en el faenamiento de las aves u otros procedimientos realizados a las muestras evaluadas, como por ejemplo en la crianza o transporte. Es posible entonces concluir, según los resultados obtenidos, que C. jejuni estuvo presente en las muestras de carne de ave analizadas, en similar nivel e independientemente de la fuente muestreada (planta faenadora A y B). Los 2 resultados obtenidos muestran la urgente necesidad de determinar la situación actual nacional y específicamente definir los puntos de contaminación dentro del faenamiento, para establecer las posibles medidas de control.

No description available.

Verschiedene mögliche Pathomechanismen einer Campylobacter jejuni-spezifischen Immunantwort bei der Entstehung akuter Immunneuropathien wurden untersucht. Neben anderen wurden für die Untersuchungen auch C. jejuni-Stämme eingesetzt, welche von Guillain-Barré- (GBS) und Miller-Fisher-syndrome (MFS) Patienten isoliert worden waren. Es wurden Ultraschall-Gesamt-Homogenate der C. jejuni Stämme sowie von Salmonella typhimurium als Kontrollbakterium hergestellt. Anschließend wurden verschiedene Proteinfraktionen isoliert und die Lipopolysaccharide (LPS) der Bakterien isoliert. Durch Immunisierung von Ratten mit diesen C. jejuni-Präparationen konnten keine Krankheitszeichen der experimentellen autoimmunen Neuritis (EAN) ausgelöst werden. Trotz Produktion hoher Titer C. jejuni-spezifischer Antikörper verlief in diesen Tieren eine anschließend durch P2-spezifische T-Lymphozyten induzierte adoptiv transferierte EAN (AT-EAN) nicht schwerer als in mit komplettem Freund´schen Adjuvans (CFA) kontrollimmunisierten Ratten. Nach Immunisierung mit C. jejuni-Protein wurden C. jejuni-spezifische T-Zellen von Lewis-Ratten gewonnen, die mit allen getesteten C. jejuni-Stämmen als Antigen reagieren, jedoch zeigten C. jejuni-spezifische Ratten-T-Zellen in vitro keine Kreuzreaktivität mit PNS-Antigenen und induzierten in vivo keine Neuritis. Im Modell der EAN läßt sich durch Füttern des Antigens eine natürliche orale Toleranz induzieren, welche die Tiere gegen eine aktiv induzierte EAN resistent macht. Die immunologische Auswirkung der enteralen Gabe von C. jejuni-LPS auf die natürliche Immuntoleranz wurde untersucht. Dabei konnte bei diskrepanten Ergebnissen keine pathogene Bedeutung von enteralen C. jejuni-Antigenen in der Ratte festgestellt werden. Zur Generation und Untersuchung C. jejuni-spezifischer monoklonaler Antikörper wurden Balb/c-Mäuse mit C. jejuni-LPS-Präparationen in CFA immunisiert und die Milzzellen dieser Tiere mit Maus-Myelomzellen fusioniert. Es konnte eine Vielzahl von monoklonalen Antikörpern etabliert werden. Selektive Spezifitäten der monoklonalen Antikörper für C. jejuni-LPS oder -protein wurden detektiert, die meisten der monoklonalen Antikörper als IgM, einige als IgG charakterisiert. Die Antikörper reagieren mit allen getesteten C. jejuni-Stämmen sowohl im ELISA als auch im Western Blot kreuz. Eine Reaktivität der Antikörper mit verschiedenen Gangliosiden konnte nicht nachgewiesen werden. Zur Untersuchung eines elektrophysiologisch fassbaren blockierenden Effektes von C. jejuni-spezifischen Antikörpern wurden Makro-patch-clamp-Untersuchungen am Mäusezwerchfell mit dialysierten Seren von C. jejuni-immunisierten Ratten durchgeführt. Einige der C. jejuni-Antiseren blockierten die präsynaptische Quantenfreisetzung partiell. Dieser Effekt war C. jejuni-spezifisch und durch Salmonella-Antiserum oder Kontrollseren CFA-immunisierter Tiere nicht induzierbar. Ein von uns generierter monoklonaler IgG-Antikörper gegen C. jejuni-LPS wurde ebenfalls in Makro-patch-clamp-Untersuchungen getestet und blockierte die Quantenfreisetzung. Weiterhin wurden humane T-Zellen gegen C. jejuni HB 93-13 generiert. Es konnte erstmals gezeigt werden, daß diese Zellen mit anderen C. jejuni-Stämmen, jedoch nicht mit Salmonellen, kreuzreagieren und ausschließlich Proteine jedoch nicht LPS erkennen. Die generierten Zellen sind alle HLA-DR restringiert und der Phänotyp wurde als CD 4+/CD 8-, /-TZR+ identifiziert. Einige der C. jejuni-spezifischen T-Zell-Linien zeigten eine starke oder partielle Kreuzreaktivität mit humanem rekombinantem P2-Protein des PNS und mit einzelnen P2-Peptiden. Dieser Befund belegt erstmals, dass durch Konfrontation mit C. jejuni eine zelluläre Immunantwort angestoßen werden kann, die in autoimmuner Weise mit Myelinprotein des PNS kreuzreagiert. / The present study evaluates the putative pathogenic role of a Campylobacter jejuni directed immune response in the pathogenesis of acute neuropathies. Among other C. jejuni strains, strains isolated from Guillain-Barré- (GBS) and Miller-Fisher syndrome (MFS) patients were used for this investigation. By sonication, total homogenate of different C. jejuni strains and Salmonella typhimurium, which served as a control, were prepared. Additionally, different protein fractions and bacterial lipopolysaccharides (LPS) were isolated. Immunization of rats with C. jejuni preparations did not lead to clinical manifestation of active experimental autoimmune neuritis (EAN). Furthermore, the severity of adoptive transfer-EAN (AT-EAN), induced by adoptively transferred P2-specific T cells was not altered in rats that had been previously immunized with C. jejuni for production of high anti-C. jejuni antibody titers. C. jejuni-specific T cell lines were generated from Lewis rats immunized with C. jejuni proteins. These T cells proliferated in an antigen-specific manner in the presence of extracts from different C. jejuni strains. C. jejuni-specific rat T cells did not show any cross-reactive proliferation to peripheral nervous system (PNS) antigens. Furthermore, it was not possibe to induce neuritis by adoptive transfer of C. jejuni-specific T cells in vivo. Oral application of myelin antigens induces oral tolerance which renders rats resistant to actively induced EAN. This observation lead to analyse the immunological consequences of oral administration of C. jejuni LPS with respect to the induction of tolerance. C. jejuni/myelin-fed rats developed accelerated clinical sings of EAN compared to control animals. Thus, oral administration of C. jejuni HB 93-13 LPS inhibited the induction of myelin-specific oral tolerance. In order to investigate the humoral immune response, monoclonal C. jejuni-specific antibodies were isolated by immunization of Balb/c mice with C. jejuni LPS preparations emulsified in complete Freund´s adjuvant (CFA). Splenocytes from primed animals were fused with myeloma cells. A number of monoclonal antibodies were characterized. These monoclonal antibodies were either specific for C. jejuni LPS or C. jejuni proteins. These immunoglobulins were characterized to be predominantly IgM, but also IgG antibodies could be found. ELISA and western blot analysis verified cross-reactivity of antibodies with different C. jejuni strains. However, the antibodies were not able to recognize gangliosides. Electrophysiological investigations were used to determine a possible blocking effect of C. jejuni-specific antibodies at the neuromusculare endplate. Alteration of neuromuscular transmission at the diaphragm of mice after appling dialysed sera of C. jejuni immunized rat, were investigated using patch-clamp. Several C. jejuni antisera were able to partially block the pre-synaptic quantal release. This effect was C. jejuni-specific and was not inducible by Salmonella typhimurium antisera or control sera, obtained from CFA immunized animals. Additionally, one of the generated monoclonal C. jejuni LPS specific IgG antibodies was able to block the quantal release. Finally, we were able to generate human T cells reacting specifically with C. jejuni HB 93-13. For the first time it could be shown, that these cells respond to homogenates of other C. jejuni strains but not to Salmonella typhimurium homogenate. Specifically C. jejuni proteins but not C. jejuni LPS were recognized by the human T cell lines. The generated T cells were all HLA-DR restricted and identified to be CD4+/CD8-, /-TCR+. A few of the C. jejuni-specific T cell lines demonstrated a strong cross-reactivity to a PNS-component, the recombinant human P2-protein and single P2-peptides. This observation shows that C. jejuni induces a variety of antigen-specific and non-specific immune responses which are able to facilitate or even trigger autoimmunity against the PNS as occuring in GBS or the MFS.

This research used classical and molecular epidemiology tools to assess the potential importance of feedlot cattle as Campylobacter reservoirs. The project was conducted from November 2004 to September 2005 in southern Alberta.<p> Fresh pen-floor fecal samples were collected from commercial feedlot cattle near slaughter weight in seven feedlots. Overall, 87% of 2,776 fecal samples were culture positive for Campylobacter species (86% of 1,400 in winter, 88% of 1,376 in summer), and 69% of 1,486 Campylobacter positive isolates were identified as <i>Campylobacter jejuni</i>. After accounting for clustering within pen and feedlot, the number of days-on-feed and feedlot size were associated (p ¡Ü 0.05) with Campylobacter species isolation rates.<p> Retail ground beef was collected from 60 grocery stores (four chains, three cities). None of the 1,200 packages were culture positive for Campylobacter species. Polymerase chain reaction (PCR) results from a subset of samples (n=142) indicated that 48% of packages were positive for Campylobacter DNA. By species, 14.8% (21/142), 26.8% (38/142) and 1.4% (2/142) of packages were PCR positive for <i>C. jejuni</i>, <i>C. coli</i> and <i>C. hyointestinalis</i> DNA, respectively. The collection period (1, 2, 3 or 4) was associated (p ¡Ü 0.05) with the odds of detecting Campylobacter species DNA using PCR.<p> Oligonucleotide DNA microarrays were used as a platform for comparative genomic hybridization (CGH) analysis of 87 C. jejuni isolates (46 bovine, 41 human) obtained within the same geographical regions and time frame. Of the 13 CGH clusters identified based on overall comparative genomic profile similarity, nine contained human and cattle isolates, three contained only human isolates, and one contained only cattle isolates. In addition, human clinical and feedlot cattle C. jejuni isolates were compared on a gene-by-gene basis and only a small number of the 1,399 genes tested were unequally distributed between the two groups (p ¡Ü 0.05).<p> The high isolation rates of Campylobacter species and <i>C. jejuni</i> reported here may have implications for food safety, public health and environmental contamination. Our findings suggest that feedlot cattle and human <i>C. jejuni</i>strains are very similar and may be endemic within southern Alberta.

This research used classical and molecular epidemiology tools to assess the potential importance of feedlot cattle as Campylobacter reservoirs. The project was conducted from November 2004 to September 2005 in southern Alberta.<p> Fresh pen-floor fecal samples were collected from commercial feedlot cattle near slaughter weight in seven feedlots. Overall, 87% of 2,776 fecal samples were culture positive for Campylobacter species (86% of 1,400 in winter, 88% of 1,376 in summer), and 69% of 1,486 Campylobacter positive isolates were identified as <i>Campylobacter jejuni</i>. After accounting for clustering within pen and feedlot, the number of days-on-feed and feedlot size were associated (p ¡Ü 0.05) with Campylobacter species isolation rates.<p> Retail ground beef was collected from 60 grocery stores (four chains, three cities). None of the 1,200 packages were culture positive for Campylobacter species. Polymerase chain reaction (PCR) results from a subset of samples (n=142) indicated that 48% of packages were positive for Campylobacter DNA. By species, 14.8% (21/142), 26.8% (38/142) and 1.4% (2/142) of packages were PCR positive for <i>C. jejuni</i>, <i>C. coli</i> and <i>C. hyointestinalis</i> DNA, respectively. The collection period (1, 2, 3 or 4) was associated (p ¡Ü 0.05) with the odds of detecting Campylobacter species DNA using PCR.<p> Oligonucleotide DNA microarrays were used as a platform for comparative genomic hybridization (CGH) analysis of 87 C. jejuni isolates (46 bovine, 41 human) obtained within the same geographical regions and time frame. Of the 13 CGH clusters identified based on overall comparative genomic profile similarity, nine contained human and cattle isolates, three contained only human isolates, and one contained only cattle isolates. In addition, human clinical and feedlot cattle C. jejuni isolates were compared on a gene-by-gene basis and only a small number of the 1,399 genes tested were unequally distributed between the two groups (p ¡Ü 0.05).<p> The high isolation rates of Campylobacter species and <i>C. jejuni</i> reported here may have implications for food safety, public health and environmental contamination. Our findings suggest that feedlot cattle and human <i>C. jejuni</i>strains are very similar and may be endemic within southern Alberta.

Con el objetivo de caracterizar la fosfolipasa A de la membrana externa (OMPLA E.C. 3.1.1.32) de Campylobacter jejuni aislado de pollos de carne, se estandarizaron métodos para el aislamiento e identificación de la bacteria a partir de hisopados rectales de aves comerciales. Para ello, primero se analizó la presencia de C. jejuni en hisopados cloacales mediante técnicas bacteriológicas y la reacción en cadena de la polimerasa anidada en base a los genes ribosómicos 16S e hipO, las cuales permitierón detectar Campylobacter jejuni en 29/50 (58%) y 48/50 (96%) de las muestras respectivamente. Después, se determinó la secuencia nucleotídica de los genes ribosómicos 16S y flaA del aislado MP4 con la finalidad de tener una cepa nativa de C. jejuni bien caracterizada que se utilice como modelo de estudio para la fosfolipasa A, las secuencias nucleotídicas de estos dos genes mostraron 98% y 92% de similiitud nucleotídica con las de Campylobacter jejuni subs. jejuni ATCC 33560. Las actividades hemolítica y de fosfolipasa A de C. jejuni MP4 fue dependiente de calcio, cuando este ión se reemplazó con EDTA, las actividades disminuyeron de 4119.4 a 44.78 U/mg de proteína. Utilizando cebadores específicos se amplificó y secuenció la parte central del gen pldA de C. jejuni MP4, se obtuvo 225 nucleótidos y 75 aminoácidos. La comparación de la secuencia aminoácidica por el ClustalW mostró 99% y 98% de identidad con las proteínas OMPLA de las cepas de Campylobacter jejuni RM1221 y C.jejuni subsp. jejuni ATCC 33560 respectivamente; esta alta conservación de secuencia aminoacídica de la fosfolipasa A la convierte en una candidata potencial para el diseño de vacunas y pruebas diagnósticas. / In order to characterize the Campylobacter jejuni’s outer membrane phospholipase A (OMPLA E.C. 3.1.1.32) from broiler chickens, it was standarized suitable methods for isolation and identification of this bacterium from cloacal swabs of commercial chickens. To that end, first it was analized the presence of C. jejuni in cloacal swabs by using bacteriological techniques and nested-PCR based on 16S ribosomal and hipO genes. The approaches used detected C. jejuni in the 29/50 (58%) and 48/50 (96%) of the samples respectively. Then, it was determined the nucleotidic sequence of 16S ribosomal gene to have a well-characterized native strain for using it as a model in the study of phospholipase A. The nucleotidic sequence of both genes showed 98% and 92% of similarity with Campylobacter jejuni subs. jejuni ATCC 33560. The haemolytic and phospholipase A enzymatic activities of the C. jejuni MP4 strain was calcium-depended, when calcium was replaced by EDTA both activities significantly decreased of 4119.4 to 44.78 U/mg protein. It was amplified and sequenced the central side from pldA gen of C. jejuni MP4 using specific primers. By means of that, it was obtained 225 nucleotides and 75 aminoacids. The comparison of aminoacidic sequences by CLUSTALW showed 99% and 98% of identity with OMPLA proteins from C. jejuni RM1221 and C. jejuni subsp. jejuni ATCC 33560. This high conservation of the aminoacidic sequence of phospholipase A become it into a potential target to design vaccines and diagnostic tests.

No description available.

No description available.

Thesis (M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographical references.