Pasteurella multocida é um dos principais patógenos envolvidos nas broncopneumonias infecciosas em suínos. Apesar de ser considerada agente secundário à pneumonia enzoótica causada pelo Mycoplasma hyopneumoniae e por agentes virais como o vírus da influenza suína, há evidências do seu envolvimento como agente primário. Neste contexto, o primeiro estudo desenvolvido teve como objetivo desenvolver um método de reprodução experimental de pneumonia por P. multocida A cepa 11246 em suínos infectados com diferentes concentrações de inóculo. Um segundo estudo foi desenvolvido com o objetivo de demonstrar diferenças fenotípicas, moleculares e patogênicas entre cepas de P. multocida A isoladas de casos clínicos de pneumonia em granjas comerciais de suínos de vários estados brasileiros. No primeiro experimento os suínos foram desafiados por gotejamento intranasal lento com inóculo de diferentes concentrações de P. multocida A cepa 11246 [Grupo (G1): 108 Unidades Formadoras de Colônias (UFC)/ml; G2: 107 UFC/ml; G3: 106 UFC/ml e G4: 105 UFC/ml]. Foram utilizados dois suínos por grupo com aproximadamente 100 dias de idade. Neste, todas as concentrações de inóculo demonstraram a capacidade da bactéria em causar doença respiratória grave e septicemia nos animais inoculados. Utilizando-se a mesma metodologia de desafio, com inóculo de 107 UFC/ml, o segundo estudo, ao desafiar 64 suínos igualmente distribuídos em oito grupos (G1 a G8) com oito diferentes cepas de P. multocida A (uma cepa por grupo), os resultados demonstraram a presença de cepas muito patogênicas (G1-11246, G2-11229, G3-16614 e G7-17044); pouco patogênicas (G4-16618 e G5-16972); e apatogênicas (G6-17034 e G8-17078), de acordo com a gravidade das alterações clinico-patológicas desenvolvidas. Na avaliação patológica dos animais desafiados, observaram-se três padrões de lesões distintas, associadas ou não entre si: 1. broncopneumonia fibrinonecrótica cranioventral com pleurite fibrinosa (G1, G3, G7); 2. pleurite difusa uni ou bilateral, associada ou não com pericardite e peritonite (G3, G5, G7) e; 3. pleuropneumonia necrossupurativa focal, geralmente no lobo cardíaco (G1, G2; G3, G4, G7). Na análise genotípica, nos padrões de PFGE obtidos após a macro-restrição com a enzima ApaI, as cepas patogênicas (Nos 11246, 11229, 16614, 16618, 16972 e 17044) foram classificadas no mesmo grupo, com homologia variando de 67,3 a 100%, diferenciando-se das cepas apatogências (Nos 17034 e 17078), que pertenceram a outro grupo, com homologia de apenas 52,7% com as demais amostras. Coletivamente, os resultados demonstraram padrões distintos de patogenicidade de diferentes cepas de P. multocida, os quais podem estar associados à características genéticas das cepas. Adicionalmente o estudo demonstrou a atuação primária de algumas cepas de P. multocida em pneumonias, pleurites e septicemias em suínos. / Pasteurella multocida is one of the main pathogens involved in infectious bronchopneumonia in swine. Although considered a secondary agent to the enzootic pneumonia caused by Mycoplasma hyopneumoniae and viral agents as the swine influenza, there are evidences related to its involvement as a primary agent. In this context, the first study undertaken aimed at developing an experimental reproduction method of pneumonia caused by P. multocida A Strain 11246 in swine infected with different inoculum concentrations. A second study was conducted aiming demonstrating phenotypic, molecular and pathogenic differences between the strains of P. multocida A isolated from clinical cases of pneumonia in commercial swine farms in several Brazilian states. In the first experiment, swine were challenged by slow intranasal drip with different inoculum concentrations of P. multocida A strain 11246 [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml]. Two swine per group with approximately 100 days of age were used. In these animals all inoculum concentrations demonstrated the bacteria capability to cause severe respiratory disease and septicemia in the inoculated animals. Using the same challenge methodology inoculating 107 CFU/ml at the second study when challenging 64 swine equally distributed into eight groups (G1 to G8) with eight different strains of P. multocida A (one strain per group) results showed the presence of highly pathogenic strains (G1-11246, G2- 11229, G3-16614 e G7-17044); less pathogenic (G4-16618 e G5-16972); and apathogenic (G6-17034 e G8-17078), according to the severity of the clinical and pathological alterations developed. In the pathologic evaluation of challenged animals, we observed three distinct patterns of injuries associated or not with each other: 1. Cranioventral fibrinonecrotic bronchopneumonia with fibrinous pleuritis (G1, G3, G7); 2. Difuse uni or bilateral pleuritis pleuritis, associated or not with pericarditis and peritonitis (G3, G5, G7) and; 3. Necrosuppurative focal pleuropneumonia, generally in the cardiac lobe (G1, G2, G3, G4, G7). In genotypic analysis, the PFGE patterns obtained after the macro-restriction with ApaI enzyme, the pathogenic strains (# 11246, 11229, 16614, 16618, 16972 and 17044) were classified in the same group, with homology ranging from 67.3 to 100%, differing from the apathogenic strains (# 17034 and 17078), which belonged to another group, with only 52.7% homology with the other samples. Collectively, the results showed distinct patterns in different pathogenic strains of P. multocida, which may be associated with the genetic features of the strains. Additionally, the reasearch demonstrated the primary role of some strains of P. multocida in pneumonia, pleuritis and septicemia in swine.

Further functional investigation revealed that knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells, concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium Conversely, overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta 1 (IFN-beta1). In this connection, IFN-beta1-mediated 2', 5'-oligoadenylate synthetase (OAS) and ribonuclease L (RNase L) signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover, GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p= 0.019) as compared with their counterpart pre-treatment liver biopsies. / Hepatitis B virus (HBV) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Although considerable progress has been made over the past decade, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. / In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via IFN-beta1-OAS-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection. / In this study, we applied a two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach to globally analyze the host early response to HBV by using an inducible HBV-producing cell line HepAD38. Twenty-three proteins were identified as differentially expressed, with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, as well as in HBV-infected human liver biopsies by immunohistochemistry. / Ma, Yan. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lau, Chi Chiu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 133-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

O leite produzido a ultra alta temperatura denominado oficiamente como UHT é o leite de maior aceitação no mercado consumidor brasileiro. Com a finalidade de ampliar o conhecimento sobre a segurança microbiológica do leite UHT, objetivou-se estudar tanto os Bacillus spp, que vêm sendo relatados como contaminantes do leite UHT no Brasil, como os eventuais esporos sobreviventes ao processamento. A partir de 65 análises de leite UHT para a determinação de microrganismos mesófilos aeróbios estritos e facultativos viáveis, no leite UHT procedente das indústrias sob Inspeção Federal no estado de São Paulo, 7 apresentaram contagens acima de 100 UFC/ml, indicando um percentual de 10,76 % de provas fora dos padrões para essa determinação, no período de jul a nov de 2003. Entretanto por atuação dos controles de qualidade das indústrias partidas foram inutilizadas e o leite que foi ao consumo apresentou 7,69% de microrganismos mesofílicos aeróbios acima dos padrões, sendo que 6.15% destes microrganismos foram considerados como Bacillus sporothermodurans. Dos isolamentos das contagens de microrganismos mesófilos acima dos padrões, foram efetuados estudos de seis cepas de Bacillus spp do leite UHT das indústrias, além de três cepas isoladas do leite UHT alvo de reclamações de consumidores procedente do Instituto Adolfo Lutz da capital de São Paulo. O objetivo do presente projeto foi, após a identificação cultural, microscópica, bioquímica e genética de culturas do Bacillus spp, estudar a patogenicidade das amostras em diversos modelos experimentais como: Teste do camundongo neonatos, alça ligada de coelho, inoculação intraperitoneal em camundongo BALB C e culturas celulares (Vero, HEp2 e Fibroblastos de Embrião de Galinha). As 3 amostras isoladas de leite de reclamações dos consumidores do Instituto Adolfo Lutz de São Paulo foram tipificadas preliminarmente pela técnica de Espectroscopia Infra-Vermelho a Transformada Fourier (FT- IR) como Bacillus flexus, enquanto que as amostras isoladas do leite procedente de três indústrias de São Paulo apresentaram presença de B. flexus e B.oleronius e Bacillus sporothermodurans. Exemplares de B.flexus testados quanto a patogenicidade apresentaram efeito citopático nas culturas celulares Vero, HEP2 e Fibroblastos de Embrião de Galinha e morbidade em camundongo BALB C. O Bacillus sporothermodurans não apresentou patogenicidade a nenhum dos modelos biológicos citados. / The milk produced at ultra high temperature, denominated UHT, is the most popular milk in the Brazilian market. With the intention of increasing the knowledge of microbiologic safety in UHT milk, we concentrated our study on the Bacillus spp, described as the most important contaminants of UHT milk in Brazil, and on possible survivors of the processing procedures. From the 65 analysis of UHT milk collected to determine the presence of strict, aerobe, mesophile microorganisms and viable options, coming from industries under Federal Inspection in São Paulo state, 7 presented a microbial count higher than 100CFU/ml, indicating a percentage of 10.76% samples outside the standard for this determination, in the period between July and November 2003. However, due to the quality control in industries, batches were rendered useless and the milk that went for consumption presented 7.69% mesophile microorganisms above standard, being these organisms 6.15% was considered Bacillus sporothermodurans. From the isolation of mesophile Bacillus counts above average, 5 strains Bacillus spp species from UHT industries were studied, as well as 3 strains isolated from UHT milk denounced by consumers of São Paulo city\'s Adolfo Lutz Institute. The aim of the project, following cultural, microscopic, biochemical and genetic identification of the Bacillus spp, was to study pathogenicity in samples from several experimental models, such as: neonatal mice; rabbit ileum loops, intraperitoneal inoculation of BALB C mice and cell cultures: Vero, Hep2 and Chick Embryo Fibroblasts. The three isolated samples of milk denounced by São Paulo consumers were typified as Bacillus flexus by the Fourier Infrared Transformed Espectroscopy (IF-FR) technique, while isolated samples of milk from 3 industries in São Paulo, accused the presence of the B. flexus, B. oleronius and Bacillus sporothermodurans. Samples of the B. flexus tested in terms of pathogenicity presented a citopatic effect upon cell cultures Vero, HeP2 and Chick Embryo Fibroblasts and a morbidity in mice BalbC. The Bacillus Sporothermodurans did not present pathogenicity in any of the biological models mentioned.

Thesis (M.Sc. (Plant Protection )) --University of Limpopo, 2011 / Crude extracts of wild watermelon (Cucumis africanus) and wild cucumber (C. myriocarpus) fruits are widely used for both medicinal and ritual purposes in South Africa. Fruits are collected fresh from the wild, but have high incidence of post-harvest decay. A study was conducted to isolate and identify the pathogen responsible for post-harvest fruit decay, followed by the pathogenicity tests. Decayed fruits were individually surface-sterilised using 0.5% NaOCl, incubated at 25ºC to allow for decay, small rotten pieces were severed and placed on solidified plates of potato dextrose agar and incubated. At harvest, seven days after incubation, isolated fungus was repeatedly cultured for 21 days for verification of diagnostic characteristics. Based on the morphological characteristics, the pathogen associated with fruit rot of both Cucumis species was identified through the assistance of an expert as Penicillium simplicissimum (Oudem) Thom. Pathogenicity results suggested that P. simplicissimum was responsible for the observed fruit decay in both species, with the higher incidence being in C. africanus, probably due to its low pH. Due to the antibiotics that P. simplicissimum releases and its reduction of medium pH, the culture retained its purity, without any contamination. In conclusion, the pathogen that induces post-harvest fruit decay in C. africanus and C. myriocarpus is P. simplicissimum, which has the ability to reduce the pH of the growing medium and also produce antibiotics. / National Research Foundation

AU COURS DE LA THESE, L'ETUDE DU TRANSCRIPTOME DE SOUCHES GARDEL ET SENEGAL VIRULENTES ET ATTENUEES D'E. RUMINANTIUMA ETE REALISEE. UNE ANALYSE DU TRANSCRIPTOME A DIFFERENTS STADES DE DEVELOPPEMENT, A D'ABORD ETE EFFECTUEE POUR LA SOUCHE GARDEL VIRULENTE. AU STADE CORPS RETICULE (FORME INTRACELLULAIRE NON INFECTIEUSE), UNE SUREXPRESSION DES GENES CODANT POUR DES PROTEINES IMPLIQUEES DANS LE METABOLISME, LE TRANSPORT ET L'ECHANGE DE NUTRIMENTS ET DANS LA RESISTANCE AU STRESS OXYDATIF ETAIT OBSERVEE. IL SEMBLERAIT QUEE. RUMINANTIUMMETTE EN PLACE UN PANEL DE MECANISMES POUR SA SURVIE ET SON DEVELOPPEMENT A L'INTERIEUR DE LA CELLULE HOTE. AU STADE CORPS ELEMENTAIRE (FORME EXTRACELLULAlRE INFECTIEUSE), LE GENE DKSA CODANT POUR UN FACTEUR DE TRANSCRIPTION ETAIT SUREXPRIME. CE GENE A ETE MONTRE COMME ETANT IMPLIQUE DANS LA REGULATION DE FACTEURS DE VIRULENCE. IL SEMBLERAIT . DONC, QU'AU STADE CORPS ELEMENTAIRE, IL Y AIT UNE INDUCTION DE MECANISMES DE VIRULENCE. LA COMPARAISON DE L'EXPRESSION DES GENES AU STADE CORPS ELEMENTAIRE ENTRE SOUCHES VIRULENTES ET ATTENUEES A AUSSI ETE EFFECTUEE. NOS RESULTATS ONT MONTRE UNE MODIFICATION IMPORTANTE DE LA MEMBRANE POUR LES SOUCHES VIRULENTES ET ATTENUEES. POUR LES SOUCHES ATTENUEES, IL A ETE MONTRE UNE SUREXPRESSION DES GENES IMPLIQUES DANS LA BIOGENESE MEMBRANAlRE ET UNE SOUS-EXPRESSION·DES PROTEINES DE LA FAMILLE MULTIGENIQUE MAP. CES RESULTATS SUGGERENT QUE LES PROTEINES MAP JOUENT UN ROLE DE LEURRE VIS-A-VIS DE LA REPONSE IMMUNITAIRE PROTECTRICE. DES PROTEINES MEMBRANAlRES HYPOTHETIQUES SONT SUREXPRIMEES A LA FOIS CHEZ LES SOUCHES VIRULENTES ET ATTENUEES. CERTAINES D'ENTRE ELLES SUREXPRIMEES CHEZ LES SOUCHES ATTENUEES SEMBLENT ETRE DE BONS CANDIDATS VACCINAUX ET DEVRAIENT ETRE ETUDIEES / Transcriptomic study of gardel and senegal both virulent and attenuated e. ruminantium strains was conducted during my phd. an analysis of transcriptome at different stages of development has been first conducted for virulent gardel strain. at reticulate body stage (intracellular form non-infectious), over-expression of genes coding for proteins involved in metabolism, transport and exchange of nutrients and resistance to oxidative stress was observed. at this stage of development, e. ruminantium seems to activate mechanisms for its survival and development within the host cell. at elementary body stage, dksa the gene encoding for a transcription factor was over-expressed. this gene has been shown to be involved in the regulation of virulence factors. it seems, therefore, at the elementary body stage, e. ruminantium induces its virulence factors. secondly, we compare the transcriptome of elementary body between virulent and attenuated strains. our results showed an important membrane modification of attenuated and virulent strains. for attenuated strains, we observed an over-expression of genes involved in membrane biogenesis and a diminution of expression of map multigenic family. it seems that map proteins subvert the protective immune response. hypothetical membrane proteins are over-expressed in both virulent and attenuated strains. some over-expressed proteins in attenuated strains seem to be good vaccine candidates and willstudied.

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. The SPI-2 T3SS has been identified as vital for survival and replication of S. Typhimurium and S. Enteritidis in mouse macrophages, as well as full virulence in mice. In order to test the ability of SE SPI-2 mutants to survive in vivo we used a chicken isolate of SE (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type (WT) strain Sal18: Sal18 attTn7::tet and Sal18 attTn7::cat, while the other two groups received the WT strain (Sal18 attTn7::tet) and one of two mutant strains (Sal18 attTn7::cat ÄspaSÄssaU or Sal18 ÄSPI-1ÄSPI-2::cat). From this study we conclude that S. Enteritidis deficient in the SPI-1 and SPI-2 systems are out-competed by the WT strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains; a WT strain and three other strains missing either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge (PC) we observed a reduced systemic spread of the SPI-2 mutants, but by day 3 the mutants systemic distribution levels matched that of the WT strain. Based on these two studies, we conclude that the SPI-2 T3SS facilitates invasion and systemic spread of S. Enteritidis in chickens, but alternative mechanisms for these processes appear to exist. Several structural components of the T3SSs encoded by SPI-1 and SPI-2 are exposed to the hosts immune system prior to/during the infection/invasion process, making them potential vaccine candidates. Several of these candidates genes were cloned, the proteins overproduced, purified, and formulated as vaccines for use in further studies. SPI-2 T3SS proteins used for vaccine studies included the secretin, SsaC, the needle, SsaG, the filament, SseB, and a part of the translocon, SseD, as well as a number of effectors, SseI, SseL, SifA, and SifB. The first vaccine study involved vaccination of SPF chickens with SseB and SseD, followed by challenge with the WT S. Enteritidis strain Sal18. Additional studies evaluated whether hens vaccinated with SPI-2 T3SS structural or effector components could mount a significant humoral immune response (as measured by serum immunoglobulin Y [IgY] titres), whether these antibodies could be transferred to progeny (as measured by egg yolk IgY titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with the WT S. Enteritidis strain Sal8. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgY that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered SE per bird in most situations. In order to better identify the role of the SPI-2 T3SS in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a WT S. Typhimurium strain, a SPI-2 mutant S. Typhimurium strain, a WT S. Enteritidis strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-infection (PI) up to 24 h PI, while the E. coli strain was no longer recoverable by 3 h PI. We can conclude from these observations that the SPI-2 T3SS is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment as E. coli is effectively eliminated.

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. The SPI-2 T3SS has been identified as vital for survival and replication of S. Typhimurium and S. Enteritidis in mouse macrophages, as well as full virulence in mice. In order to test the ability of SE SPI-2 mutants to survive in vivo we used a chicken isolate of SE (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type (WT) strain Sal18: Sal18 attTn7::tet and Sal18 attTn7::cat, while the other two groups received the WT strain (Sal18 attTn7::tet) and one of two mutant strains (Sal18 attTn7::cat ÄspaSÄssaU or Sal18 ÄSPI-1ÄSPI-2::cat). From this study we conclude that S. Enteritidis deficient in the SPI-1 and SPI-2 systems are out-competed by the WT strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains; a WT strain and three other strains missing either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge (PC) we observed a reduced systemic spread of the SPI-2 mutants, but by day 3 the mutants systemic distribution levels matched that of the WT strain. Based on these two studies, we conclude that the SPI-2 T3SS facilitates invasion and systemic spread of S. Enteritidis in chickens, but alternative mechanisms for these processes appear to exist. Several structural components of the T3SSs encoded by SPI-1 and SPI-2 are exposed to the hosts immune system prior to/during the infection/invasion process, making them potential vaccine candidates. Several of these candidates genes were cloned, the proteins overproduced, purified, and formulated as vaccines for use in further studies. SPI-2 T3SS proteins used for vaccine studies included the secretin, SsaC, the needle, SsaG, the filament, SseB, and a part of the translocon, SseD, as well as a number of effectors, SseI, SseL, SifA, and SifB. The first vaccine study involved vaccination of SPF chickens with SseB and SseD, followed by challenge with the WT S. Enteritidis strain Sal18. Additional studies evaluated whether hens vaccinated with SPI-2 T3SS structural or effector components could mount a significant humoral immune response (as measured by serum immunoglobulin Y [IgY] titres), whether these antibodies could be transferred to progeny (as measured by egg yolk IgY titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with the WT S. Enteritidis strain Sal8. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgY that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered SE per bird in most situations. In order to better identify the role of the SPI-2 T3SS in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a WT S. Typhimurium strain, a SPI-2 mutant S. Typhimurium strain, a WT S. Enteritidis strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-infection (PI) up to 24 h PI, while the E. coli strain was no longer recoverable by 3 h PI. We can conclude from these observations that the SPI-2 T3SS is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment as E. coli is effectively eliminated.

Staphylococcus aureus ist einer der bedeutendsten Erreger boviner Mastitiden. Die Vielgestaltigkeit der Resistenzmuster und Virulenzfaktoren seiner Stämme macht ihn zu einem Problemkeim aus therapeutischer und prophylaktischer Sicht. Seine Fähigkeit zur Bildung hitzestabiler Enterotoxine verleiht ihm lebensmittelhygienische Relevanz. Mehrfachresistente Stämme stellen gefährliche Zoonose-Erreger dar. Ziel der durchgeführten Untersuchung war es daher, Aufschluss über Resistenzdeterminanten und Virulenzfaktoren der in Thüringer Milchviehherden vorkommenden Staphylococcus aureus zu erhalten, wobei eine Microarray-gestützte Genotypisierung zum Einsatz kam. Weiterhin sollte analysiert werden, ob der Genotyp der Isolate mit dem Phänotyp korreliert. In 34 Thüringer Milchviehherden wurde der gesamte Bestand der laktierenden Kühe zweimal auf Basis von Viertelgemelksproben bakteriologisch untersucht. Die Beurteilung der Kulturen erfolgte im Nativausstrich nach 48-stündiger Bebrütung und zusätzlich nach Voranreicherung in einer Glucose-Bouillon mit anschließender 24-stündiger Bebrütung. Staphylococcus aureus-positiv waren 1902 von insgesamt 81 567 Milchproben. Aus diesen wurden 189 für die Herden repräsentative Isolate ausgewählt und mittels Microarray-Technologie umfassend charakterisiert und klassifiziert. Zudem wurde der Phänotyp der Isolate auf Äskulin- und Columbia-Blutagar erfasst und das Resistenzverhalten mittels Agardiffusionstest ermittelt. Die 189 typisierten Staphylococcus aureus konnten elf verschiedenen klonalen Komplexen (CC) zugeordnet werden. Der Großteil der Isolate (80,4 %) zählte zu CC133, CC151 und CC479. Diese Isolate besaßen mit einer Ausnahme das Leukozidin-Gen lukF-P83/lukM. Die übrigen Isolate, die negativ auf lukF-P83/lukM getestet wurden, gehörten acht vergleichsweise sporadisch vorkommenden CC (CC7, CC9, CC20, CC45, CC50, CC97, CC101, CC398) an. In nur 0,7 % der zu den drei dominanten CC zählenden Isolate war das Beta-Laktamase-Gen blaZ vorhanden, während es bei 54,1 % der sporadisch vorkommenden CC detektiert wurde. Das Methicillin-Resistenzgen mecA wurde bei lediglich vier Isolaten (2,1 %) nachgewiesen, die alle CC398 angehörten. Sie verfügten neben Resistenzen gegenüber β-Laktam-Antibiotika über eine Tetrazyklin-Resistenz. Darüber hinaus wurde in einem Isolat das Makrolid/Lincosamid/Streptogramin-Resistenz vermittelnde vgaA und in einem Isolat das Aminoglykosid-Resistenz vermittelnde aacA-aphD detektiert. Humanmedizinisch relevante Enterotoxin-, Exfoliatin- oder PVL-Gene wurden in den vier Methicillin-resistenten Staphylococcus aureus (MRSA) nicht gefunden. Im Agardiffusiontest zeigten diese Isolate eine Penicillin- und eine Tetrazyklin-Resistenz, jedoch keine Resistenz gegenüber Oxacillin, welches als MRSA-Marker gilt. Die Gene der klassischen, humanmedizinisch bedeutsamen Enterotoxine A, B und C waren bei 12,7 % der Isolate vorhanden, wohingegen die Gene von Enterotoxin D und E nicht vorkamen. Insgesamt fanden sich Enterotoxin-Gene bei 78,3 % der typisierten Staphylococcus aureus, wobei die für Enterotoxin G, I, M, N, O und U kodierenden dominierten. Phänotypisch unterschieden sich die CC bezüglich Hämolyse und Pigmentierung, wobei alle CC398-Isolate als eierschalenfarben mit doppelzoniger Hämolyse auftraten. Hämolysin-Gene besaßen alle Isolate, ein Zusammenhang zu den phänotypisch ausgeprägten Hämolysezonen bestand jedoch nicht. Die vorliegende Untersuchung zeigt, dass in Thüringer Milchviehbeständen zwei epidemiologisch unterschiedliche Varianten von Staphylococcus aureus existieren. Die in dieser Studie dominierenden, lukF-P83/lukM-positiven CC133, CC151 und CC479 verursachten einen Großteil der Infektionen und gelten als auf das Euter beschränkte Erreger. Sie können daher als „euterassoziiert“ angesehen werden. Dagegen verfügten die anderen in dieser Untersuchung detektierten, lukF-P83/lukM-negativen CC über Charakteristika „umweltassoziierter“ Keime. Sie besitzen ein breites Wirtsspektrum und treten auch außerhalb des bovinen Euters in der Umgebung der Kühe auf. Die Prüfung auf lukF-P83/lukM erwies sich als zuverlässige Methode, zwischen beiden epidemiologischen Varianten zu unterscheiden. Folglich lässt die An- oder Abwesenheit dieser Genkombination einen Rückschluss auf die in der Herde verbreiteten CC zu. Das ermöglicht die Berücksichtigung der CC-spezifischen Erreger-Eigenschaften bei der Etablierung von Sanierungsprogrammen, die somit effizient gestaltet werden können. MRSA waren in Thüringer Milchviehbeständen wenig verbreitet und nur schwach mit Resistenzdeterminanten und humanmedizinisch bedeutsamen Pathogenitätsfaktoren ausgestattet. Diese MRSA aus Rohmilchproben sind daher nicht mit multiresistenten Isolaten aus der Humanmedizin zu vergleichen. Gene für humanmedizinisch relevante Enterotoxine, für die ein Zusammenhang mit Lebensmittelintoxikationen belegt ist, wurden selten, andere Enterotoxin-Gene jedoch häufig nachgewiesen.

The dimorphic fungus Ustilago maydis is a pathogen of maize and it was used for decades to understand the molecular basis of plant pathogenicity aspects. Recently, much effort went into understanding the cell biology that underlies the virulence of U. maydis. It was shown previously that early endosomes (EEs) move bidirectionally within fungal hyphal cells. Although it was shown that the motility of EEs facilitates growth of the infectious hypha and mutants defective for kinesin-3 (Kin3), the major EE transporter, exhibit impaired polarized growth, the importance of EEs and their motility in plant colonization is not known. The first part of this thesis is focused on the role of EE motility during plant infection. In collaboration with Natalie Steinberg, who performed the plant infection assays, I used a synthetic molecular anchor, K1rPX, to block the motility of EEs at early and late stages during the host plant infection and I found that EE motility is essential during the first two days of pathogenic development, when infectious hyphae exhibit most prominent elongation, whereas blockage of EE motility after 3 days post infection does not inhibit plant colonization. Moreover, I documented that the blockage of EE motility during early stages of the infection causes high plant defence response, which means that the pathogen becomes recognized by the host plant defence system. These results indicate that EE motility is crucial during initial stages of the plant host infection and enables colonization by U. maydis and additionally suggests involvement of EEs in some defence response machinery. The second part of the thesis addresses the relationship between Kin3, the major motor for EE motility, and the microtubule (MT) array. I demonstrate here that Kin3 uses all MT tracks available in the cell, which is in contrast to published results in other systems. In the third part I focused on the interaction between Kin3 and the EEs. I found that the pleckstrin homology (PH) domain localized at the distal part of the Kin3 tail is of minor importance for EE association. This conclusion is supported by in vivo experiments, showing that truncated Kin3PH, which lacks the PH domain, was still able to bind to the organelles. By systematic truncation of parts of the Kin3 tail I found two adjacent regions, a DUF3694 domain and a "linker" region, that are important for binding of Kin3 to EEs. By using a synthetic anchor composed of Kin1 rigor domain and selected Kin3 domains I proved that both domains anchor the EEs to MTs and inhibit EE motility. I also showed that the PH domain is not able to block EE motility. In collaboration with Dr. Nicholas Harmer, who performed structural modelling of selected PH domains, I demonstrated that the PH domain is likely to interact with the motor domain of Kin3. This result was confirmed by using a yeast-two hybrid approach and a protein affinity assay. This indicates a globular organization of the Kin3 motor, which was confirmed by a split-YFP assay in living cells. Deletion of the PH domain and most probably lack of intramolecular interaction between the tail and motor domain reduces Kin3 motility parameters like velocity, frequency and run length indicating that the interaction of the PH domain with the motor domain has a role in the control of Kin3 motility.