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.

Orientador: Angela Cleusa de Fatima Banzatto de Carvalho / Banca: Eliana Scarcelli Pinheiro / Banca: Vera Cecilia Annes Ferreira / Banca: Adolorata Aparecida Bianco Carvalho / Banca: Luís Antonio Mathias / Resumo: Os objetivos do presente trabalho foram verificar a ocorrência de Campylobacter jejuní, Campylobacter calí, Salmonella spp., Salmonella Enteritidis e Salmonella Typhimurium em abatedouros de aves e avaliar a resistência das amostras de Salmonella spp. isoladas frente a antimicrobianos de uso comum. Foram colhidas amostras de fezes; penas; água de escaldamento, evisceração e resfriamento; e amostras de carcaça não eviscerada, eviscerada e resfriada em seis abatedouros de aves. Campylobacter jejuni foi detectada em 14 amostras (5%), a maior porcentagem foi em amostras de fezes oito amostras (22%), e C. calí em foi isolada em uma amostra de pena (0,35%). Salmonella spp. foi detectada em 18% (52/288) dos isolados, enquanto os sorotipos S. Enteritidis em 5,6% (16/288) e S. Typhimurium em 2,4% (7/288). Os testes de resistência aos antimicrobianos apontaram 25 amostras resistentes ao aztreonam e à ampicilina (86,2%), 21 à tetraciclina (72,4%) e 16 à amoxicilina/ácido clavulânico e sulfazotrim (55,2%). Nenhum dos isolados testados apresentou 100% de resistência ou sensibilidade aos antimicrobianos utilizados. Os resultados indicam que há uma necessidade de melhorar a qualidade higiênico-sanitária em linhas de abate de aves e o cuidado com o uso indiscriminado de antibióticos na avicultura, oferecendo aos consumidores produtos livres de agentes zoonóticos. / Abstract: The present study was carried out to report the occurrence of Campylobacter jejuní, Campylobacter calí, Salmonella spp., Salmonella Enteritidis e Salmonella Typhimurium in chicken abattoirs and to evaluate the resistance of Salmonella spp. isolated to antibiotics of common use. Samples of feces; feathers; scald, evisceration, and chiller water; and non-eviscerated, eviscerated, and chilted carcasses were coUeded from six chicken abattoirs. Campylobacter jejuni was isolated in 14 samples (5%), isolation was greater in feces, eight samples (22%), one feather sample was positive for the species C. calí (0.35%). Salmonella spp. was detected in 18% (52/288) of the isolates, whereas serotypes S. Enteritidis were identified in 5.6% (16/288) and S. Typhimurium and 2.4% (7/288). Antibiotic tests indicate 25 resistant samples to aztreonam and to ampicilin (86.2%), 21 to tetracycline (72.4%) and 16 to amoxicilin/clavulanic acid and to sulfazotrim (55.2%). None sample tested were 100% resistant or sensitive to ali the antibiotics tested. The results exhibit the need to improve hygiene and sanitary standards in poultry sfaughter fines, and care with the indiscriminate use of antibiotics in avicultura, offering to the consumers products free of zoonotic agents. / Doutor

No description available.

Twenty species and six subspecies of the genera Arcobacter and Campylobacter have been described to date. All are Gram-negative, microaerophilic, curved, spiral or S-shaped cells, and are members of the order Campylobacterales, class Epsilonproteobacteria phylum Proteobacteria. Though most members are pathogenic, C. jejuni, C. coli and A. butzleri are the most frequently isolated species from patients suffering from gastrointestinal illness. The current methods for their detection, identification, and differentiation are cumbersome, time consuming and lack specificity. DNA based molecular techniques including real-time Polymerase Chain Reaction (PCR) and Fingerprinting methods Terminal Restriction Fragments Length Polymorphism (T-RFLP) and Ligase Detection Reaction (LDR) have been used in this project to develop rapid detection and identification methods for Campylobacter and Arcobacter species. Five real-time PCR methods were developed which include: (a) rapid detection and identification of Campylobacter species using real-time PCR adjacent hybridisation probes, (b) rapid identification of C. jejuni using SYBR Green I, (c) rapid detection and differentiation of Arcobacter species using adjacent hybridisation probes, (d) rapid detection and differentiation of Arcobacter species and the Campylobacter group (C. coli, C. jejuni, C. lari, C. hyoilei, C. helviticus, C. hyointestinalis, C. insulaenigrae, C lanienae) using melting temperature (Tm) of adjacent hybridisation probes, and (e) a one tube real-time PCR multiplex for the rapid detection and identification of Campylobacter species, C. coli and C. jejuni using a TaqMan Probe, in an iCycler iQTM (BioRad, USA) and Light CyclerTM (Idaho Technology, USA). [Continued ...]

Les campylobacters sont des bactéries zoonotiques responsables d'entérites chez l'homme. La viande de volaille est considérée comme une source importante de contamination. Il a été suggéré que les désinfectants et les stress subis par les bactéries au cours des procédures de nettoyage et désinfection et des procédés d'abattage des volailles puissent favoriser la sélection de gènes de résistance aux antibiotiques. Afin d'explorer cette hypothèse, des prélèvements dans 4 abattoirs de volailles ont été réalisés au cours de 9 visites. Les niveaux de résistance des campylobacters isolés ont été déterminés par la méthode de dilution en milieu gélosé pour 6 antibiotiques et 2 substances actives entrant dans la composition de désinfectants. Des souches isolées dans l'environnement des abattoirs après nettoyage et désinfection et sur les carcasses de volailles avant l'entrée en salle de ressuage, ont été génotypées avec la technique de PCR-RFLP des gènes pfla/gyrA et flaA. Nos résultats montrent d'une part que les campylobacters sont capables de survivre aux opérations de nettoyage et de désinfection dans les abattoirs de volailles, et que ces souches sont susceptibles de contaminer les carcasses de volailles. D'autre part, nos résultats indiquent que les opérations d'abattage et les procédures de nettoyage et désinfection dans les abattoirs de volailles ne semblent pas favoriser la sélection de souches de campylobacter résistantes aux antibiotiques.

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.

Poultry slaughtering encompasses a series of processing steps with the objective of harvesting the consumable meat. The scalding process consists of the submersion of carcasses in hot water tanks to facilitate the removal of feathers during slaughter. However, the use of a common scalding tank increases the likelihood of carcass cross contamination considering that dirt, fecal material and even digestive and intestinal contents carrying pathogens and other bacteria are widely spread during this operation. Similar cross contamination occurs in the process of chilling carcasses, which also requires submersion of broilers in communal tanks filled with ice and cold water. A plausible approach to reduce contamination in scalders or chillers is the use of Pulsed Electric Fields (PEF) to decontaminate scalder/ chiller water. PEF uses electricity to kill bacteria suspended in liquid media and could be utilized in poultry scalders and chillers to reduce bacterial contamination on carcasses and reduce the potential risk of pathogens reaching the final consumer. A pilot scale system was assembled by the use of a pulse electric field generator (Model SF-700, Simmons. Eng. Co., Dallas, GA) coupled with a commercial scalding tank (Dunkmaster®, Knase Company Inc, MI). C. coli and C. jejuni along with marker strains of Novobiocin and Nalidixic acid resistant S. typhimurium and S. enteritidis strains were used in challenge studies evaluating the effects of the PEF on carcasses, scalder and chiller water contamination. The system was evaluated with 0, 0.5, and 1% sodium chloride in the water with 40 volts of electric current and 0.54 of amperage. Samples were collected at 0, 40, 80, 160, 200 s of treatment with a 10 s on, 5 s off cyclical pulses. The use of PEF in regular scalder/chiller water showed little effect on Salmonella and Campylobacter reductions. However, with the addition of 0.5% NaCl caused a significant (P<0.5) log CFU/ml reduction of Salmonella and Campylobacter within the scalder/chiller water at 40, 80, and 160 seconds respectively.

One of the most overlooked areas in forensic entomology is urban, which applies to insects and their arthropod relatives that have interactions with humans, their associated structures, and companion animals. American cockroaches, Periplaneta americana (L.), are common pests of urban environments. Analyzing spatial distribution of P. americana populations in an artificial, outdoor environment provided insight of gene flow among populations collected in central Texas. This information provides for a better understanding of how and if populations were segregated, or if there was a single unified population. Populations can be genetically differentiated through determining variation of specific gene regions within populations. This study revealed a ubiquitous distribution of cockroach populations, and their ability to indiscriminately inhabit areas within an urban environment. Overall, cockroaches were identified from a large interbreeding population with no discernable relationship between genetic variation of P. americana and spatial distribution. Identifying cockroach populations is relative to understanding the ability of surrogate species indirectly affecting man by their ability to transfer disease-causing organisms including bacteria. This may have potentially deleterious health consequences on animal and/or human populations. There are several pathogens associated with cockroaches which are overlooked during diagnosis of sudden ailments with symptoms being similar to food-borne illnesses, including abdominal cramping, diarrhea, nausea, and fever. Analyzing spatial distributions of Escherichia coli and Campylobacter spp. in relationship to collected cockroaches allowed for prevalence of bacteria species to be identified among populations. The prevalence of bacteria isolated from total populations collected indicated a high prevalence (92.3%) of bacteria carried by the exoskeleton of P. americana. Gram-negative bacteria acquisition and dissemination of organisms such as E. coli was prevalent on campus. Screening for E. coli 1057:H7 and Campylobacter spp. resulted in no positive colony growth. The lack of Campylobacter spp. growth from cuticular surfaces may have resulted from undesirable conditions required to sustain colony growth. Data from this study corroborates the potential ability of cockroaches to mechanically transmit pathogens.