Salmonella Não Tifóide (SNT) é um dos patógenos que mais causam gastroenterite, representando um problema de saúde pública em todo o mundo. Portanto, a presença de SNT em matrizes ambientais como águas superficiais e esgotos tem sido alvo de pesquisas em todo o mundo. Os objetivos deste estudo foram quantificar e caracterizar SNT em amostras de águas superficiais e esgotos brutos da Região Metropolitana de São Paulo (RMSP). Foram coletadas amostras, quinzenalmente, no ponto de captação de água para o abastecimento público da represa Guarapiranga e do manancial São Lourenço, assim como de esgotos brutos das cidades de Taboão da Serra e São Lourenço da Serra. A quantificação de SNT foi realizada pela técnica do Número Mais Provável (NMP). A caracterização dos isolados (identificação do sorotipo e potencial patogênico) foi realizada pela técnica de Reação em Cadeia da Polimerase (Polimerase Chain Reaction - PCR), com quatro conjuntos de iniciadores, contemplando marcadores genéticos consolidados (fliC, invA e spvC) e regiões gênicas ainda não caracterizadas. As águas superficiais apresentaram concentrações que variaram de Limite de Detecção (LD <0,06473 NMP/100 mL) a 0,67 NMP/100 mL, com frequência de 4 por cento , enquanto nos esgotos brutos as concentrações foram de LD a 54,22 NMP/100 mL, com frequência de 54 por cento . Foram isoladas sete cepas de amostras de águas superficiais, identificadas como Salmonella sp., e 499 de esgotos brutos, dentre as quais se identificaram nove sorotipos potencialmente patogênicos. Os sorotipos mais prevalentes foram Salmonella enterica subsp. enterica sor. Enteritidis e Salmonella enterica subsp. enterica sor. Typhimurium, que se apresentaram quase que exclusivamente nos meses de verão (dezembro a janeiro). Os dois últimos sorotipos apresentaram padrões atípicos de PCR, com regiões gênicas que contêm genes que expressam constituintes fimbriais e outras ainda não caracterizadas. Esta investigação evidencia a presença de Salmonella Não Tifóide potencialmente patogênica nas amostras de esgoto bruto analisadas sugerindo sua circulação na população da região estudada e corrobora com o regime sazonal de salmonelose. DeCS: Salmonella, água superficial, esgoto, sorotipo / Non-Typhoidal Salmonella (NST) is a concern pathogen which is responsible for gastroenteritis outbreaks worldwide. So its presence in environmental sources such as surface waters and sewage has been investigated around the world. The aims of this research were quantify and characterize NST in samples of surface water and raw sewage from Metropolitan Region of São Paulo (MRSP). Samples were collected fortnightly in the catchment point of supply from Guarapiranga reservoir and São Lourenço River, as well as raw sewage from the cities of Taboão da Serra and São Lourenço da Serra. Quantification of NST was performed using Most Probable Number technique (MPN). Characterization of the isolates (serotype identification and pathogenic potential) was carried out by multiplex Polimerase Chain Reaction technique (m-PCR) with four sets of primers, including genetic markers (fliC, invA and spvC) and gene regions not yet characterized. Surface water showed concentrations ranging from detection limit (DL <0.06473 MPN / 100 mL) to 0.67 MPN/100 mL with a frequency of 4 per cent . Raw wastewater concentrations were from DL to 54.22 MPN/100 mL, with a frequency of 54 per cent . Seven strains of surface water samples were isolated and identified as Salmonella sp. and 499 from raw sewage, of which nine were identified as potentially pathogenic serotypes. The most prevalent serotypes were Salmonella enterica subsp. enterica ser. Enteritidis and Salmonella enterica subsp. enterica ser. Typhimurium, which have been found almost exclusively in summer months (December-January). The two serotypes presented atypical patterns of PCR with gene regions containing genes that express fimbrial constituents and other not yet characterized. This research shows the presence of potentially pathogenic Non-Typhoidal Salmonella in raw sewage samples suggesting its circulation in the population of the study area and corroborates with the seasonal cases of salmonellosis. DeCS: Salmonella, surface water, sewage, serotype.

Salmonella Não Tifóide (SNT) é um dos patógenos que mais causam gastroenterite, representando um problema de saúde pública em todo o mundo. Portanto, a presença de SNT em matrizes ambientais como águas superficiais e esgotos tem sido alvo de pesquisas em todo o mundo. Os objetivos deste estudo foram quantificar e caracterizar SNT em amostras de águas superficiais e esgotos brutos da Região Metropolitana de São Paulo (RMSP). Foram coletadas amostras, quinzenalmente, no ponto de captação de água para o abastecimento público da represa Guarapiranga e do manancial São Lourenço, assim como de esgotos brutos das cidades de Taboão da Serra e São Lourenço da Serra. A quantificação de SNT foi realizada pela técnica do Número Mais Provável (NMP). A caracterização dos isolados (identificação do sorotipo e potencial patogênico) foi realizada pela técnica de Reação em Cadeia da Polimerase (Polimerase Chain Reaction - PCR), com quatro conjuntos de iniciadores, contemplando marcadores genéticos consolidados (fliC, invA e spvC) e regiões gênicas ainda não caracterizadas. As águas superficiais apresentaram concentrações que variaram de Limite de Detecção (LD <0,06473 NMP/100 mL) a 0,67 NMP/100 mL, com frequência de 4 por cento , enquanto nos esgotos brutos as concentrações foram de LD a 54,22 NMP/100 mL, com frequência de 54 por cento . Foram isoladas sete cepas de amostras de águas superficiais, identificadas como Salmonella sp., e 499 de esgotos brutos, dentre as quais se identificaram nove sorotipos potencialmente patogênicos. Os sorotipos mais prevalentes foram Salmonella enterica subsp. enterica sor. Enteritidis e Salmonella enterica subsp. enterica sor. Typhimurium, que se apresentaram quase que exclusivamente nos meses de verão (dezembro a janeiro). Os dois últimos sorotipos apresentaram padrões atípicos de PCR, com regiões gênicas que contêm genes que expressam constituintes fimbriais e outras ainda não caracterizadas. Esta investigação evidencia a presença de Salmonella Não Tifóide potencialmente patogênica nas amostras de esgoto bruto analisadas sugerindo sua circulação na população da região estudada e corrobora com o regime sazonal de salmonelose. DeCS: Salmonella, água superficial, esgoto, sorotipo / Non-Typhoidal Salmonella (NST) is a concern pathogen which is responsible for gastroenteritis outbreaks worldwide. So its presence in environmental sources such as surface waters and sewage has been investigated around the world. The aims of this research were quantify and characterize NST in samples of surface water and raw sewage from Metropolitan Region of São Paulo (MRSP). Samples were collected fortnightly in the catchment point of supply from Guarapiranga reservoir and São Lourenço River, as well as raw sewage from the cities of Taboão da Serra and São Lourenço da Serra. Quantification of NST was performed using Most Probable Number technique (MPN). Characterization of the isolates (serotype identification and pathogenic potential) was carried out by multiplex Polimerase Chain Reaction technique (m-PCR) with four sets of primers, including genetic markers (fliC, invA and spvC) and gene regions not yet characterized. Surface water showed concentrations ranging from detection limit (DL <0.06473 MPN / 100 mL) to 0.67 MPN/100 mL with a frequency of 4 per cent . Raw wastewater concentrations were from DL to 54.22 MPN/100 mL, with a frequency of 54 per cent . Seven strains of surface water samples were isolated and identified as Salmonella sp. and 499 from raw sewage, of which nine were identified as potentially pathogenic serotypes. The most prevalent serotypes were Salmonella enterica subsp. enterica ser. Enteritidis and Salmonella enterica subsp. enterica ser. Typhimurium, which have been found almost exclusively in summer months (December-January). The two serotypes presented atypical patterns of PCR with gene regions containing genes that express fimbrial constituents and other not yet characterized. This research shows the presence of potentially pathogenic Non-Typhoidal Salmonella in raw sewage samples suggesting its circulation in the population of the study area and corroborates with the seasonal cases of salmonellosis. DeCS: Salmonella, surface water, sewage, serotype.

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No. of bitstreams: 2 Tese - Aline Pedrosa de Oliveira - 2016.pdf: 3128576 bytes, checksum: bbad449f3830d7359e905a2812b2a74e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-12-22 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Salmonella enterica is a foodborne pathogen with multifactorial and complex pathogenic mechanisms. Identification of the presence of virulence genes and antimicrobial resistance profiles in isolates of poultry origin provides relevant information on the risk attributed to the consumption of products contaminated by the agent. The objective of this study was to verify the susceptibility profile of Salmonella enterica for nalidixic acid (30μg), amicacin (30μg), ampicillin (10mg), ceftiofur (30μg), chloramphenicol (30μg), ciprofloxacin (5μg), enrofloxacin (5μg), streptomycin (10mg), gentamicin (10mg), tetracycline (30μg), tobramycin (10mg) and trimethoprim (5μg) used in both human and animal medicine, to investigate the presence of multiresistant isolates, to detect the presence of the variable region of the class 1 Integron, to analyze the association between the presence of Class 1 Integron and antimicrobial resistance and to evaluate the presence of virulence genes located in the islands of virulence 1 (invA) and 2 (sseD), gene encoding long polar fimbriae (lpfA) and plasmidial spvR, to identify the virulence profiles and pathogenicity potential of Salmonella enterica serovars isolated from carcasses, hearts, livers, gizzards and environment of slaughterhouses located in the State of Goiás and on chicken carcasses marketed in commercial establishments in Goiânia -GO. The highest resistance frequency was observed for ceftiofur, 19.12% (13/68), followed by streptomycin, gentamicin, tobramycin, tetracycline and trimetropic, 16.18% (11/68) both, nalidixic acid 14.71% (10/68), ampicillin 13.24% (9/68), and enrofloxacin 2,94% (2/68). No resistance was observed for ciprofloxacin, only intermediate, 45.59% (31/68), 100% (68/68) of the isolates were sensitive to amikacin and chloramphenicol. Of the 68 isolates 22 (32.35%) were resistant to one or more antimicrobial principles. Twelve profiles of antimicrobial resistance were identified, 54.54% (12/22) of the isolates presented multiresistance. The variable region of Class 1 Integron was detected in 63.23% (43/68) of the isolates. The presence of this region was not associated with antimicrobial resistance. All slaughterhouses and in most commercial establishments it was possible to identify Salmonella enterica carrying the Integron of class 1 demonstrating the ubiquity of the same. The invA gene was identified in 100% (59/59), sseD in 92.53% (54/59), lpfA in 86.51% (52/54) and spvR in 86.18% (49/59) of the serovars of Salmonella enterica. Six virulence profiles were identified, 77.97% of the isolates were grouped in profile A characterized by the presence of the four virulence genes simultaneously. The knowledge of the virulence profiles of the isolates allows to affirm that the serovars identified in the state of Goiás are potentially virulent and capable of triggering disease in poultry production systems and in humans. / Salmonella enterica é um patógeno de veiculação alimentar com mecanismos de patogenicidade multifatoriais e complexos. A identificação da presença de genes de virulência e de perfis de resistência a antimicrobianos em isolados de origem avícola fornece informações relevantes quanto ao risco atribuído ao consumo de produtos contaminados pelo agente. Pelo exposto, objetivou-se com este trabalho verificar o perfil de suscetibilidade de Salmonella enterica para ácido nalidíxico (30μg), amicacina (30μg), ampicilina (10mg), ceftiofur (30μg), cloranfenicol (30μg), ciprofloxacina (5μg), enrofloxacina (5μg), estreptomicina (10mg), gentamicina (10mg), tetraciclina (30μg), tobramicina (10mg) e trimetoprima (5μg), utilizados tanto na medicina humana quanto animal. Investigar a presença de isolados multirresistentes. Detectar a presença do Integron de classe e analisar a associação entre a presença deste e a resistência antimicrobiana. Ainda avaliar a presença de genes de virulência localizados nas ilhas de virulência 1 (invA) e 2 (sseD), gene codificador de fímbria polar longa (lpfA) e o plasmidial spvR em sorovares de Salmonella enterica isolados a partir de carcaças, corações, fígados, moelas e ambiente de abate de abatedouros localizados no estado de Goiás e em carcaças de frango comercializadas em estabelecimentos comerciais de Goiânia -GO. A maior frequência de resistência foi obervada para ceftiofur, 19,12% (13/68), seguido pelos antimicrobianos, estreptomicina, gentamicina, tobramicina, tetraciclina e trimetropima, 16,18% (11/68), ácido nalidíxico 14,71% (10/68), amplicilina 13,24% (9/68), e enrofloxacina 2,94% (2/68). Não foi observado resistência dos isolados para ciprofloxacina, sendo, 45,59% (31/68), considerados apenas intermediários. Entretanto, 100% (68/68) dos isolados foram sensíveis à amicacina e ao cloranfenicol. Dos 68 isolados, 22 (32,35%) foram resistentes a um ou mais princípios antimicrobianos. Foram identificados 12 perfis de resistência à antimicrobianos e 54,54% (12/22) dos isolados apresentaram multirresistência. A região variável do Integron de Classe 1 foi detectado em 63,23% (43/68) dos isolados. A presença desta região não apresentou associação com a resistência aos antimicrobianos. Em todos os abatedouros e na maioria dos estabelecimentos comerciais foi possível identificar Salmonella enterica transportando o Integron de classe 1, demonstrando a ubiquidade do mesmo. O gene invA foi identificado em 100% (59/59), sseD em 92,53% (54/59), lpfA em 86,51% (52/54) e spvR em 86,18% (49/59) dos sorovares de Salmonella enterica. Foram identificados seis perfis de virulência (A, B, C, D, E e F). Ao todo, 77,97 % dos isolados se agruparam no perfil A, caracterizado pela presença dos quatro genes de virulência simultaneamente. O conhecimento dos perfis de virulência dos isolados permite afirmar que os sorovares identificados no estado de Goiás são potencialmente virulentos e capazes de desencadear doença em sistemas de produção avícola e em humanos.

Although traditional microbiological methods are accepted standard for Salmonella detection, they are labor intensive and time consuming. Therefore, for food industry and public health, finding sensitive and rapid methods is required. As a rapid and reliable tool, Real-Time PCR is one of the most common methods in molecular detection and research area. The aim of the current study is to develop rapid, sensitive and quantitative Salmonella detection method using Real-Time PCR technique based on inexpensive, easy to produce, convenient and standardized plasmid based positive control for the first time. To achieve this, two plasmids were constructed as reference molecules by cloning two most commonly used Salmonella specific target regions &lsquo / invA and ttrRSBC&rsquo / into them. Standard curves were constructed for the plasmids and reproducibility, PCR efficiency, amplification efficiency values were calculated. To illustrate the applicability of the developed method, enriched (as used commonly for Salmonella detection with Real-Time PCR) 105 to 100 CFU/ml level (estimated by standard plate counts before enrichment) S. Typhimurium ATCC 14028 cultures were tried to detect and quantify, also compared with traditional culture method. In addition, detection limits of the developed technique were determined by serial dilution of DNA extracted from 105 CFU/ml level. The results revealed much faster detection ability of the developed plasmid based Salmonella detection method (in comparison to traditional culture method, ISO 6579:2004) allowing quantitative evaluation with perfect reproducibility, sensitivity (except for lower concentrations for invA target), detection limit, PCR efficiency, amplification efficiency for both invA and ttrRSBC targets. The detection and quantification ability of the method developed by using S. Typhimurium ATCC 14028 cultures were tested also with 15 Salmonella species using milk as a representative food. The results also revealed much faster (in comparison to traditional culture method, ISO 6579:2004) quantitative detection ability of the developed method. Thus, the developed method has great potential to be used in food industry for rapid and quantitative Salmonella detection.

Die Infektion mit Salmonella und Yersinia (Y.) enterocolitica über Produkte tierischen Ursprungs stellt nach wie vor ein ungelöstes Problem des gesundheitlichen Verbraucherschutzes dar. Will man diese Zoonoseerreger aus der Lebensmittelkette fernhalten, sind moderne und gut validierte Nachweissysteme erforderlich. Eine Infektion von Schweinen erfolgt überwiegend im Mastbetrieb mit Infektionsdosen, die nur zu einer milden klinischen Symptomatik führen. In den meisten Fällen überstehen die Tiere die Infektion mit Salmonella und Y. enterocolitica und werden zu klinisch inapparenten Keimträgern. Solche Schweine stellen ein Reservoir für die Infektion anderer Tiere und für den Eintrag in die Lebensmittelkette dar. Im Rahmen dieser Arbeit wurden zwei PCR-Methoden zum spezifischen Nachweis von Salmonella und Y. enterocolitica im Schlachtschwein entwickelt und anhand von Probenmaterial aus eigens dafür durchgeführten Infektionsversuchen mit S. Typhimuirum und Y. enterocolitica evaluiert. Beide Methoden mussten sich am diagnostischen Goldstandard für den jeweiligen Erreger messen lassen. Für Salmonella Typhimurium wurde die ISO-Norm 6579 und für Y. enterocolitica die ISO-Norm 10273 zum kulturellen Nachweis ausgewählt. Es konnte eine neue PCR-Methodik zum Salmonella-Nachweis in 14 verschiedenen Gewebeproben etabliert werden, die im Vergleich zum kulturellen Nachweis nach ISO 6579 eine Sensitivität von 100 % und eine Spezifität von 96 % aufweist und die Zeitspanne bis zum spezifischen Nachweis des Erregers um mindestens 24 Stunden reduziert. Die Untersuchungen erfolgten anhand von 420 Gewebeproben aus persistent infizierten Schweinen aus Infektionsversuchen mit S. Typhimurium DT104. Dieses neu entwickelte und validierte PCR-Verfahren wurde mit einem bereits etablierten PCR-Nachweissystem nach RAHN et al. (1992) - wie in der DIN 10135 angegeben - verglichen. Beide PCR-Methoden basieren auf dem invA-Virulenzgen von S. Typhimurium. Im Infektionsversuch konnten zwei Gewebeproben (Caecum und Lnn. Ileocolici) bestimmt werden, durch deren Kombination man mit beiden Nachweismethoden 96 % (23 von 24 Tieren) aller im Versuch infizierten Schweine als Salmonella positiv identifizieren konnte. Erstmals gelang in dieser Arbeit der Nachweis des yopT-Gens bei plasmidtragenden Y. pseudotuberculosis-Stämmen sowie die Bestimmung der Sequenz (European Bioinformatics Institute, Accession-Number: AJ304833). Das yopT-Gen kodiert für ein 35,5 kDa großes Effektor-Protein, das einen zytotoxischen Effekt auf HELA-Zellen und Makrophagen besitzt. Durch den Nachweis des yopT-Gens bei Y. pseudotuberculosis-Stämmen war es erstmals möglich, eine für Y. enterocolitica spezifische, auf dem yopT-Gen des Virulenzplasmids basierende PCR-Methode zu etablieren, die auch die Diskriminierung von Y. pseudotuberculosis-Isolaten gestattet. In einem weiteren Infektionsversuch konnte gezeigt werden, dass es die auf dem yopT-Gen von Y. enterocolitica basierende PCR-Methode erlaubt, Carrier-Tiere mit hoher Sensitivität (100 %) und Spezifität (87 %) innerhalb von 56 Stunden in lymphatischen Geweben zu identifizieren. Besonders geeignet für den Nachweis mit der ISO 10273 und dem neu etablierten yopT PCR-Verfahren waren das Ileum und die Lnn. ileocolici. In dieser Arbeit ist der Versuch gelungen, die Diagnostik für zwei der drei wichtigsten beim Schwein vorkommenden humanen Enteritiserreger zu standardisieren, indem Kombinationen aus Gewebeproben bestimmt wurden, die sowohl für den Nachweis mit der jeweiligen Goldstandard-Methode als auch mit den schnelleren und sensitiveren PCR-Methoden geeignet sind. Die Ergebnisse dieser Arbeit tragen zu einer deutlichen Verbesserung der Diagnostik von Salmonella und Y. enterocolitica beim Schlachtschwein bei. Es bleibt zu hoffen, dass somit der Eintrag dieser Zoonoseerreger in die Lebensmittelkette reduziert und der Verbraucherschutz auf diesem Gebiet beträchtlich verbessert werden kann. / The infection with Salmonella and Yersinia (Y.) enterocolitica through foodstuff from slaughter pigs is one of the major problems of hygienic consumer protection. To avoid the contamination of products from pig industry modern and well validated bacteriological identification systems are necessary. An infection predominantly occurs in the fattening pens, showing mild clinical symtoms only. The majority of infected pigs overcome the infection with Salmonella and Y. enterocolitica and become clinically inapparent carrier pigs. Those pigs are a reservoir for the contamination of other animals and pork products. In the context of this work two PCR-assays for the specific detection of Salmonella and Y. enterocolitica have been developed and validated on the basis of tissue samples from experimentally infected pigs. Both methods have been compared with the classical bacterial culture. Two international standards were used for bacterial detection: ISO 6579 for S. Typhimurium and ISO 10273 for pathogenic Y. enterocolitica. It was possible to establish a new PCR-assay for the specific detection of Salmonella in 14 different tissues of experimentally infected pigs. In comparison to the standard ISO 6579 a sensitivity of 100 % and a specificity of 96 % were calculated for the PCR-assay. The investigations were carried out with 420 tissue samples of persistently infected pigs that have been experimentally infected with S. Typhimurium. By using the PCR-method for the detection of Salmonella in positive tissue samples, the detection-time could be reduced around 24 hours. The new PCR-assay developed and validated in this work, was compared with the PCR-method described in DIN 10135, which is based on the studies of RAHN et al. (1992). Both methods were based on the invA-virulence gene of S. Typhimurium. A combination of samples from ileocolic lymph node and caecum was particularly suitable for the detection of 96 % of the experimentally infected pigs (23 off 24 animals) with the PCR-assay and the culture method. In this study, the yopT-gene was proved for the first time to be present in plasmid bearing Y. pseudotuberculosis-Isolates, and the nucleotid sequence was determined (European Bioinformatics Institute, Accession-Number: AJ304833). YopT encodes a 35.5 kDa effector protein (YopT), which induces a cytotoxic effect in HeLa cells and macrophages. This finding was used to develop a specific PCR-assay for the detection of pathogenic Y. enterocolicica strains and the discrimination from pathogenic Y. pseudotuberculosis strains. Embedded in an experimental Y. enterocolitica-infection-model in swine, it was shown that the yopT PCR-assay is suitable for the detection of pathogenic Y. enterocolitica in lymphatic tissue of persistently infected pigs. The yopT PCR-method shows a sensitivity of 100 % and a specificity of 87 % in lymphatic tissue. By the use of the PCR-assay, the detection of Y. enterocolitica was possible within 56 hours. A combination of specimens from the ileum and ileocolic lymph nodes was most suitable for the detection of pathogenic Y. enterocolitica in slaughter pigs with the ISO-Standard 10273 and the yopT PCR. This investigation succeeded in standardizing the identification of two of the three most important zoonotic agents for human enteric disease. The standardization was achived by the use of a combination of samples suitable for the identification with both, the “Goldstandard” and the specific and rapid PCR-method. The results of this work offer a better identification of Salmonella and Y. enterocolitica in slaughter pigs in the future. Based on these facts it is possible to avoid contamination of food products from slaughter pigs and to improve the hygienic consumer protection considerably.

Die Infektion mit Salmonella und Yersinia (Y.) enterocolitica über Produkte tierischen Ursprungs stellt nach wie vor ein ungelöstes Problem des gesundheitlichen Verbraucherschutzes dar. Will man diese Zoonoseerreger aus der Lebensmittelkette fernhalten, sind moderne und gut validierte Nachweissysteme erforderlich. Eine Infektion von Schweinen erfolgt überwiegend im Mastbetrieb mit Infektionsdosen, die nur zu einer milden klinischen Symptomatik führen. In den meisten Fällen überstehen die Tiere die Infektion mit Salmonella und Y. enterocolitica und werden zu klinisch inapparenten Keimträgern. Solche Schweine stellen ein Reservoir für die Infektion anderer Tiere und für den Eintrag in die Lebensmittelkette dar. Im Rahmen dieser Arbeit wurden zwei PCR-Methoden zum spezifischen Nachweis von Salmonella und Y. enterocolitica im Schlachtschwein entwickelt und anhand von Probenmaterial aus eigens dafür durchgeführten Infektionsversuchen mit S. Typhimuirum und Y. enterocolitica evaluiert. Beide Methoden mussten sich am diagnostischen Goldstandard für den jeweiligen Erreger messen lassen. Für Salmonella Typhimurium wurde die ISO-Norm 6579 und für Y. enterocolitica die ISO-Norm 10273 zum kulturellen Nachweis ausgewählt. Es konnte eine neue PCR-Methodik zum Salmonella-Nachweis in 14 verschiedenen Gewebeproben etabliert werden, die im Vergleich zum kulturellen Nachweis nach ISO 6579 eine Sensitivität von 100 % und eine Spezifität von 96 % aufweist und die Zeitspanne bis zum spezifischen Nachweis des Erregers um mindestens 24 Stunden reduziert. Die Untersuchungen erfolgten anhand von 420 Gewebeproben aus persistent infizierten Schweinen aus Infektionsversuchen mit S. Typhimurium DT104. Dieses neu entwickelte und validierte PCR-Verfahren wurde mit einem bereits etablierten PCR-Nachweissystem nach RAHN et al. (1992) - wie in der DIN 10135 angegeben - verglichen. Beide PCR-Methoden basieren auf dem invA-Virulenzgen von S. Typhimurium. Im Infektionsversuch konnten zwei Gewebeproben (Caecum und Lnn. Ileocolici) bestimmt werden, durch deren Kombination man mit beiden Nachweismethoden 96 % (23 von 24 Tieren) aller im Versuch infizierten Schweine als Salmonella positiv identifizieren konnte. Erstmals gelang in dieser Arbeit der Nachweis des yopT-Gens bei plasmidtragenden Y. pseudotuberculosis-Stämmen sowie die Bestimmung der Sequenz (European Bioinformatics Institute, Accession-Number: AJ304833). Das yopT-Gen kodiert für ein 35,5 kDa großes Effektor-Protein, das einen zytotoxischen Effekt auf HELA-Zellen und Makrophagen besitzt. Durch den Nachweis des yopT-Gens bei Y. pseudotuberculosis-Stämmen war es erstmals möglich, eine für Y. enterocolitica spezifische, auf dem yopT-Gen des Virulenzplasmids basierende PCR-Methode zu etablieren, die auch die Diskriminierung von Y. pseudotuberculosis-Isolaten gestattet. In einem weiteren Infektionsversuch konnte gezeigt werden, dass es die auf dem yopT-Gen von Y. enterocolitica basierende PCR-Methode erlaubt, Carrier-Tiere mit hoher Sensitivität (100 %) und Spezifität (87 %) innerhalb von 56 Stunden in lymphatischen Geweben zu identifizieren. Besonders geeignet für den Nachweis mit der ISO 10273 und dem neu etablierten yopT PCR-Verfahren waren das Ileum und die Lnn. ileocolici. In dieser Arbeit ist der Versuch gelungen, die Diagnostik für zwei der drei wichtigsten beim Schwein vorkommenden humanen Enteritiserreger zu standardisieren, indem Kombinationen aus Gewebeproben bestimmt wurden, die sowohl für den Nachweis mit der jeweiligen Goldstandard-Methode als auch mit den schnelleren und sensitiveren PCR-Methoden geeignet sind. Die Ergebnisse dieser Arbeit tragen zu einer deutlichen Verbesserung der Diagnostik von Salmonella und Y. enterocolitica beim Schlachtschwein bei. Es bleibt zu hoffen, dass somit der Eintrag dieser Zoonoseerreger in die Lebensmittelkette reduziert und der Verbraucherschutz auf diesem Gebiet beträchtlich verbessert werden kann. / The infection with Salmonella and Yersinia (Y.) enterocolitica through foodstuff from slaughter pigs is one of the major problems of hygienic consumer protection. To avoid the contamination of products from pig industry modern and well validated bacteriological identification systems are necessary. An infection predominantly occurs in the fattening pens, showing mild clinical symtoms only. The majority of infected pigs overcome the infection with Salmonella and Y. enterocolitica and become clinically inapparent carrier pigs. Those pigs are a reservoir for the contamination of other animals and pork products. In the context of this work two PCR-assays for the specific detection of Salmonella and Y. enterocolitica have been developed and validated on the basis of tissue samples from experimentally infected pigs. Both methods have been compared with the classical bacterial culture. Two international standards were used for bacterial detection: ISO 6579 for S. Typhimurium and ISO 10273 for pathogenic Y. enterocolitica. It was possible to establish a new PCR-assay for the specific detection of Salmonella in 14 different tissues of experimentally infected pigs. In comparison to the standard ISO 6579 a sensitivity of 100 % and a specificity of 96 % were calculated for the PCR-assay. The investigations were carried out with 420 tissue samples of persistently infected pigs that have been experimentally infected with S. Typhimurium. By using the PCR-method for the detection of Salmonella in positive tissue samples, the detection-time could be reduced around 24 hours. The new PCR-assay developed and validated in this work, was compared with the PCR-method described in DIN 10135, which is based on the studies of RAHN et al. (1992). Both methods were based on the invA-virulence gene of S. Typhimurium. A combination of samples from ileocolic lymph node and caecum was particularly suitable for the detection of 96 % of the experimentally infected pigs (23 off 24 animals) with the PCR-assay and the culture method. In this study, the yopT-gene was proved for the first time to be present in plasmid bearing Y. pseudotuberculosis-Isolates, and the nucleotid sequence was determined (European Bioinformatics Institute, Accession-Number: AJ304833). YopT encodes a 35.5 kDa effector protein (YopT), which induces a cytotoxic effect in HeLa cells and macrophages. This finding was used to develop a specific PCR-assay for the detection of pathogenic Y. enterocolicica strains and the discrimination from pathogenic Y. pseudotuberculosis strains. Embedded in an experimental Y. enterocolitica-infection-model in swine, it was shown that the yopT PCR-assay is suitable for the detection of pathogenic Y. enterocolitica in lymphatic tissue of persistently infected pigs. The yopT PCR-method shows a sensitivity of 100 % and a specificity of 87 % in lymphatic tissue. By the use of the PCR-assay, the detection of Y. enterocolitica was possible within 56 hours. A combination of specimens from the ileum and ileocolic lymph nodes was most suitable for the detection of pathogenic Y. enterocolitica in slaughter pigs with the ISO-Standard 10273 and the yopT PCR. This investigation succeeded in standardizing the identification of two of the three most important zoonotic agents for human enteric disease. The standardization was achived by the use of a combination of samples suitable for the identification with both, the “Goldstandard” and the specific and rapid PCR-method. The results of this work offer a better identification of Salmonella and Y. enterocolitica in slaughter pigs in the future. Based on these facts it is possible to avoid contamination of food products from slaughter pigs and to improve the hygienic consumer protection considerably.