University, Diss., 2005--Würzburg.

Resumo: As bactérias Gram-positivas que contêm ácidos micólicos na parede celular estão classificadas no grupo dos actinomicetos aeróbios ou bactérias corineformes e nocardioformes. Nesse grupo encontra-se o Rhodococcus equi, o qual é relevante à medicina veterinária e humana tal como as micobactérias, causando doença pulmonar que pode mimetizar casos de tuberculose. R. equi é considerado como agente patogênico em potros e tem emergido como oportunista em humanos, especialmente, associado à infecção pelo vírus da imunodeficiência humana. Assim como nos animais, a rodococose humana afeta principalmente, os pulmões, com características clínicas e patológicas, similares à tuberculose pulmonar, em pacientes imunocomprometidos ou não. A identificação de Rhodococcus equi pode ser realizada com base numa variedade de características, fenotípicas, genotípicas e técnicas cromatográficas. Morfologia das colônias, morfologia celular e resistência parcial ao álcool ácido são características chaves para a caracterização inicial. R. equi não oxida ou fermenta carboidratos e nem utiliza acetato, citrato e malonato, como única fonte de carbono, produz catalase, o fator equi (teste de CAMP) e lipase. Não produz amilase, b-galactosidase (ONPG), casease, DNase, esculinase, gelatinase, H2S, indol, lecitinase e oxidase. Demonstra comportamento variável para as provas de nitrato redutase, urease e redução do hipurato, decompõe a adenina, mas não hipoxantina, tirosina e xantina. O método molecular para a identificação de R. equi utiliza a PCR para amplificar um fragmento de 959 pares de base do gene choE, o qual codifica a enzima cholesterol oxidase (COX). Na identificação química, semelhante às espécies do gênero Mycobacterium, membros do gênero Rhodococcus contêm ácidos micólicos plausíveis de serem identificados pela cromatografia... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Gram-positive bacteria, containing mycolic acids in the cellular wall are classified in aerobic actinomycetes or corineform and nocardioform bacteria group. Rhodococcus equi is included in this group, and it is very important to the veterinary and human medicine. Rhodococcus equi is a well-recognized bacterial pathogen in veterinary medicine. First isolated from foals, it causes an important chronic granulomatous pneumonia and lung abscesses. The infection also occurs in humans, often following immunosuppression of various causes. The increased number of human cases reported recently is partly the result of the spread of AIDS but may also reflect the increasing awareness by medical laboratories of this opportunistic pathogen and their improved ability to identify it rather than to dismiss it as a contaminating "micrococcus" or "diphtheroid." R. equi can be identified on the basis of a variety of conventional phenotypic characteristics including microscopic (Gram and acidfast staining) and macroscopic morphologies, growth requirements, metabolism of glucose, and phenotypic molecular characteristics including the presence of mycolic acid composition, which is detected by thin-layer chromatography. The colonial morphology of R. equi is diverse and consists of three major varieties: pale pink and slimy, coral and non-slimy, and pale yellow in color, non-slimy. Colorless colonial variants may also occur. R. equi is a non-motile gram-positive pleomorphic coccobacillus, varying from distinctly coccoid to bacillary depending on growth conditions. All of the rhodococci from clinical specimens are generally weakly acid fast when stained either by the modified Kinyoun method or by the Ziehl-Neelsen method. Regarding biochemical characteristics, the organism is generally biochemically unreactive. It fails to oxidize or ferment carbohydrates, neither uses sodium... (Complete abstract click electronic access below) / Orientador: Clarice Queico Fujimura Leite / Coorientador: Sergio Roberto de Andrade Leite / Banca: Carmo Elias Andrade Melles / Banca: Rosilene Fressatti Cardoso / Banca: Carlos Henrique Gomes Martins / Banca: Antonio Carlos Pizzolitto / Doutor

Orientador: Márcio Garcia Ribeiro / Banca: Carlos Roberto Teixeira / Banca: Jean Carlos Ramos da Silva / Resumo: O presente estudo investigou a ocorrência de Escherichia coli, Rhodococcus equi, Salmonella sp., Coronavírus e Rotavírus nas fezes de Passeriformes e Psitaciformes pertencentes à fauna nacional, de 29 diferentes espécies, sem sinais entéricos. Foram investigados também marcadores de virulência nas linhagens de E. coli (cnf1, hly, papC, papGI, papGII, papGIII, fimH, afa, sfa, iucD, usp, vt1, vt2, eae, k88) e R. equi (genes vapA e vapB). As aves utilizadas no estudo foram provenientes do Centro de Medicina e Pesquisa em Animais Silvestres (CEMPAS) FMVZ - UNESP/ Botucatu, SP, do Parque Zoológico Municipal "Quinzinho de Barros" (PZMQB) de Sorocaba, SP e de criadores particulares com aves registradas no Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis (IBAMA) da região de Botucatu, SP. Do total de 152 amostras avaliadas foram isoladas 46 (30,26%) linhagens de E. coli das quais 37 (80%) foram provenientes de amostras de Psitaciformes e 9 (20%) de Passeriformes. Houve diferença significante (p<0,05) entre os grupos para o maior isolamento de E. coli nos Psitaciformes. Dentre os marcadores de virulência de E. coli foram detectados os genes fim H (58,69%) e eae (4,34%). Foram isoladas 2 (1,32%) linhagens de R. equi, todas de Psitaciformes. Nestes isolados de R. equi não foram identificados os genes vapA e vapB associados à virulência. Foi encontrado material genético de Rotavírus bovino em três (1,97%) amostras de Psitaciformes. Salmonella sp. e Coronavírus não foram identificados nas aves amostradas. A presença de E. coli, R. equi e Rotavírus em amostras de fezes de aves silvestres, sem sinais entéricos, reforça o potencial destas espécies de servirem como reservatórios de patógenos de eliminação entérica para os humanos, devido à presença destes animais no ambiente domiciliar e peridomiciliar / Abstract: The present study investigated the occurrence of Escherichia coli, Rhodococcus equi, Salmonella sp., Coronavirus and Rotavirus in the feces of Passeriformes and psittaciformes belonging to Brazilian wildlife, from 29 different species, without enteric signs. Virulence markers were also investigated in strains of E. coli (cnf1, hlyA, papC, papGI, papGII, papGIII, fimH, afa, sfa, iucD, usp, vt1, vt2, eae, k88) and R. equi (vapA and vapB genes). The birds used in the study came from the Centro de Medicina e Pesquisa em Animais Silvestres (CEMPAS) FMVZ - UNESP / Botucatu, SP, Parque Zoológico Municipal "Quinzinho de Barros" (PZMQB) Sorocaba, SP and private breeders with birds recorded in Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis (IBAMA) from Botucatu region, SP. Of the total 152 fecal samples evaluated were isolated 46 (30.26%) strains of E. coli. From these, 37 (80%) were from psittaciformes samples and 9 (20%) of Passeriformes. There was a statistical difference (p <0.05) between groups with greater isolation of E. coli in psittaciformes. Among the virulence markers of E. coli were detected the genes fimH (58,69%) and eae (4,34%). Were isolated 2 (1.32%) R. equi strains, all from psittaciformes. Among these R. equi isolates any vapA and vapB genes associated with virulence were founded. Genetic material of bovine Rotavirus was found in three (1.97%) psittaciformes samples. Salmonella sp. and Coronavírus weren't identified in any of the sampled birds. The presence of E. coli, R. equi and Rotavirus in fecal samples of wild birds without enteric signs from Brazil wildlife, reinforces the potential of these birds as a reservoirs of pathogens of enteric elimination for humans, due to the presence of these animals in the domestic and peridomestic, environment of human / Mestre

The pathogenic actinomycete Rhodococcus equi is a facultative intracellular parasite that replicates within macrophages. This ability is dependent on the pVAP virulence plasmid, and more specifically, on the laterally acquired vap pathogenicity island (vap PAI) carried by it. R. equi has two contrasting lifestyles as a soil-dwelling microbe and as an inhabitant of the intracellular macrophage compartment. In the first part of this thesis work we analysed the nature of the signals recognised by R. equi to adapt the expression of the virulence genes of the plasmid during the transition from soil saprotroph to intracellular parasite. The expression profile of virulence plasmid genes in response to temperature and pH in vitro and to the macrophage environment was investigated by microarray analysis. A shift to 37ºC was the main stimulus involved in vap PAI gene activation and macrophage-derived signals did not further modulate the expression of the PAI genes contrary to previous suggestions. In a second part of the thesis we investigated the role of a horizontally acquired island encoding exopolysaccharide biosynthesis in the R. equi saprotroph-intracellular parasite dual lifestyle. Mutational analysis of this locus showed that it is responsible for the typical mucoid colony morphology of R. equi and the ability to produce a polysaccharide capsule. Mutations in the capsule locus favoured macrophage uptake but had no effect on intracellular proliferation and in vivo survival in mice. However, the capsule mutants showed significantly increased susceptibility to desiccation, ultraviolet radiation and heat and were outcompeted by capsulated wild-type R. equi in dry soil. Thus, while having a minor role in virulence, the R. equi capsule appears to be primarily required for survival in soil and to act as a transmission factor. The third part of this work followed the identification of a horizontally acquired locus that encodes pili appendages that promote association with macrophages and colonization of the mouse lung. The ability of a component of this structure, the RplB pilin subunit, to act as a vaccine antigen was investigated in mice and horses. Vaccinated mice produced high levels of anti-RplB IgG and showed significant protection against pulmonary challenge with virulent R. equi. The experimental RplB subunit vaccine proved also to be immunogenic in horses, eliciting a strong IgG response in pregnant mares and foals. We also demonstrated passive transfer of high levels of maternal anti-RplB antibodies from the mares to the foals via colostrum. Our results indicate that the RplB pilin subunit is a promising novel candidate R. equi vaccine antigen.

Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2009. / Titre provenant de l'écran titre. Bibliogr. p. 101-104.

Environmental samples (air and soil) were collected from thoroughbred breeding farms with different prevalences of R. equi pneumonia to increase our understanding of the ecology of virulent R. equi on horse farms. The airborne population of virulent R. equi was a major focus of this research, as inhalation of the pathogen from the environment is considered the primary route of pulmonary infection. Air sampling was performed using an air monitoring system with selective media to facilitate the recovery of R. equi, allowing quantitative measurement of airborne virulent R. equi. Polymerase chain reaction and DNA hybridisation techniques were used to evaluate environmental samples to identify and differentiate R. equi.

Tierärztl. Hochsch., Diss., 2004--Hannover.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-02Bitstream added on 2014-06-13T18:44:31Z : No. of bitstreams: 1 silva_p_dr_arafcf.pdf: 2320520 bytes, checksum: 5b67cf713d666bdba286bd22e1e7c114 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As bactérias Gram-positivas que contêm ácidos micólicos na parede celular estão classificadas no grupo dos actinomicetos aeróbios ou bactérias corineformes e nocardioformes. Nesse grupo encontra-se o Rhodococcus equi, o qual é relevante à medicina veterinária e humana tal como as micobactérias, causando doença pulmonar que pode mimetizar casos de tuberculose. R. equi é considerado como agente patogênico em potros e tem emergido como oportunista em humanos, especialmente, associado à infecção pelo vírus da imunodeficiência humana. Assim como nos animais, a rodococose humana afeta principalmente, os pulmões, com características clínicas e patológicas, similares à tuberculose pulmonar, em pacientes imunocomprometidos ou não. A identificação de Rhodococcus equi pode ser realizada com base numa variedade de características, fenotípicas, genotípicas e técnicas cromatográficas. Morfologia das colônias, morfologia celular e resistência parcial ao álcool ácido são características chaves para a caracterização inicial. R. equi não oxida ou fermenta carboidratos e nem utiliza acetato, citrato e malonato, como única fonte de carbono, produz catalase, o fator equi (teste de CAMP) e lipase. Não produz amilase, b-galactosidase (ONPG), casease, DNase, esculinase, gelatinase, H2S, indol, lecitinase e oxidase. Demonstra comportamento variável para as provas de nitrato redutase, urease e redução do hipurato, decompõe a adenina, mas não hipoxantina, tirosina e xantina. O método molecular para a identificação de R. equi utiliza a PCR para amplificar um fragmento de 959 pares de base do gene choE, o qual codifica a enzima cholesterol oxidase (COX). Na identificação química, semelhante às espécies do gênero Mycobacterium, membros do gênero Rhodococcus contêm ácidos micólicos plausíveis de serem identificados pela cromatografia... / Gram-positive bacteria, containing mycolic acids in the cellular wall are classified in aerobic actinomycetes or corineform and nocardioform bacteria group. Rhodococcus equi is included in this group, and it is very important to the veterinary and human medicine. Rhodococcus equi is a well-recognized bacterial pathogen in veterinary medicine. First isolated from foals, it causes an important chronic granulomatous pneumonia and lung abscesses. The infection also occurs in humans, often following immunosuppression of various causes. The increased number of human cases reported recently is partly the result of the spread of AIDS but may also reflect the increasing awareness by medical laboratories of this opportunistic pathogen and their improved ability to identify it rather than to dismiss it as a contaminating micrococcus or diphtheroid. R. equi can be identified on the basis of a variety of conventional phenotypic characteristics including microscopic (Gram and acidfast staining) and macroscopic morphologies, growth requirements, metabolism of glucose, and phenotypic molecular characteristics including the presence of mycolic acid composition, which is detected by thin-layer chromatography. The colonial morphology of R. equi is diverse and consists of three major varieties: pale pink and slimy, coral and non-slimy, and pale yellow in color, non-slimy. Colorless colonial variants may also occur. R. equi is a non-motile gram-positive pleomorphic coccobacillus, varying from distinctly coccoid to bacillary depending on growth conditions. All of the rhodococci from clinical specimens are generally weakly acid fast when stained either by the modified Kinyoun method or by the Ziehl-Neelsen method. Regarding biochemical characteristics, the organism is generally biochemically unreactive. It fails to oxidize or ferment carbohydrates, neither uses sodium... (Complete abstract click electronic access below)

Rhodococcus equi is a Gram-positive intracellular pathogen which can cause severe bronchopneumonia in foals. In recent years, the role of this bacterium as human pathogen has been noted, as R.equi infections in humans have increase in frequency. This increase is associated with the rise in immunosupressed individuals, specially AIDS patients, where infection leads to symptoms and pathology similar to those seen in foals with a high mortality rate. Due to its capability to survive and multiply in murine and equine macrophages, R.equi has been classified as a facultative intracellular bacterium. R.equi is found frequently in macrophages in alveolar infiltrate from infected animals. The pathogenicity of R.equi depends on its ability to exist and multiply inside macrophages and has been associated with the presence of virulence plasmids. It has been observed that, inside foal alveolar macrophages, R.equi-containing vacuoles (RCVs) do not mature into phagolysosomes. However, most of the intracellular events during R.equi infection have not been investigated in detail. The aim of this study was to elucidate the intracellular compartmentation of R.equi and the mechanism by which the bacteria avoid destruction in host macrophages. The importance of the virulence-associated plasmids of R.equi for the establishment of RCVs was also evaluated. Furthermore, the intracellular fate of viable and non-viable R.equi was compared in order to study whether viability of R.equi influeciantes the establishment of RCVs. In this study, the RCV was characterized by using a variety of endocytic markers to follow the path of the bacteria trhough murine macropages. Transmission electron microscopy-base analysis showed that R.equi was found equally frequently in phagosomes with loosely or thightly apposed membranes, and RCV often contains numerous membranous vesicles. Laser scanning microscopy of infected macrophages showed that the majority of phagosomes containing R.equi acquired transiently the early endosomal markers Rab5, Ptlns3P, and EEA-1, suggesting initially undisturbed phagosome maturation. Although the RCV acquired some late endosomal markers, such as Rab7, LAMP-1, and Lamp-2, they did not acquired vATPase, did not interact with pre-labeled lysosomes, and failed to acidify. These data clearly suggest that the RCV is a compartment which has left vacuoles that resemble multivesicular body compartments (MVB), which are transport intermediates between early and late endosomes and display internal vesicles very similar to the ones observed within RCVs. Analyisis of several R.equi strains containing either VapA- or VapB-expressing plasmids or neither demonstrated that the possession of the virulence-associated plasmids does not affect phagosome trafficking over a two hour period of infection. The finding that non-viable R.equi was still able to inhibit phagosome maturation (although not to the same extent as viable R.equi did) suggests that heat-insensitive factors, such as cell periphery lipids, may play a major role in inhibition of phagosome maturation, although heat-sensitive factors may also be involved. / Rhodococcus equi ist ein Gram-positives, fakultativ intrazelulläres Bakterium, das unter anderem die Ursache von Bronchopneumonien bei Fohlen ist. Menschen und andere Säugetiere können ebenfalls von Infektionen mit R. equi betroffen sein. In den letzten Jahren ist die Häufigkeit klinischer Infektionen mit R. equi bei Menschen gestiegen. Die wachsende Anzahl an mmunosupprimierten Patienten (hauptsächlich AIDS-Patienten) liegt dieser Zunahme an Infektionen zugrunde. Die Symptomatologie und Pathologie der Infektion mit R. equi ist bei AIDS-Patienten und Fohlen ähnlich. Die Sterblichkeitsrate ist in beiden Fällen hoch. Die Fähigkeit der Rhodokokken, innerhalb von Makrophagen zu überleben und sich zu vermehren, ist mit dem Vorhandensein von Virulenzplasmiden (virulence-associated plasmids) verbunden. Innerhalb des Makrophagen befinden sich die Rhodokokken in einem Phagosom, das nicht mit Lysosomen fusioniert. Die genaue Kompartimentierung der Rhodococcus equi-enthaltenden Phagosomen in Makrophagen war bisher unbekannt und wurde deshalb in der vorliegenden Promotionsarbeit untersucht. Mit Hilfe mehrerer endozytischer Marker wurde das R. equi-enthaltende Kompartiment charakterisiert. Mögliche Unterschiede zwischen der Kompartimentierung von R. equi(+)- und R. equi(-)-enthaltenden Phagosomen ist ebenfalls Thema dieser Promotionsarbeit. Weiterhin wurde die Etablierung des phagosomalen Kompartiments für jeweils lebende und tote Rhodokokken verglichen. Transmissionselektronenmikroskopische Analysen haben gezeigt, dass die Phagosomenmembran Rhodococcus equi-enthaltender Phagosomen sowohl locker als auch eng anliegend sein kann (50%). Darüber hinaus wurden häufig zahlreiche, membranöse Vesikel in R. equi-enthaltenden Phagosomen gefunden. Diese Phagosomen zeigen somit Ähnlichkeiten zu Multivesicular Bodies. Multivesicular Bodies sind intermediäre Kompartimente zwischen frühen und späten Endosomen und zeigen ebenfalls eine Vielzahl von internen Vesikeln. Untersuchungen am konfokalen Lasermikroskop ergaben, dass die Mehrheit der R. equi-enthaltenden Phagosomen die früh endosomalen Marker Rab5, PtIns3P und EEA-1 transient akquirieren. Dieser Befund deutet auf eine ungestörte phagosomale Reifung im frühen Stadium hin. Trotz der beobachteten Akquisition der spät endosomalen Marker Rab7, LAMP-1 und LAMP-2 konnte keine Akquisition der vATPase, keine Interaktion mit vormarkierten Lysosomen und keine Ansäuerung von R.equi-enthaltenden Phagosomen nachgewiesen werden. Diese Ergebnisse weisen darauf hin, dass R. equi-enthaltende Phagosomen das früh endosomale Stadium abschließen, aber einen typisch spät endosomale Zustand nicht erreichen. Die Analyse unterschiedlicher R. equi-Stämme, die entweder vapA- oder vapB-exprimierende Virulenzplasmide enthalten, hat gezeigt, dass die Anwesenheit von Virulenzplasmiden die phagosomale Reifung über eine Infektionsperiode von zwei Stunden nicht beeinflusst. Getötete Rhodokokken waren in der Lage, die phagosomale Reifung zu inhibieren, aber in geringerem Ausmaß als lebende Rhodokokken. Das weist darauf hin, dass hitze-insensitive Faktoren (wie zum Beispiel Lipide der Zellwand) zur Inhibierung der phagosomalen Reifung entscheidend sind, obwohl dazu auch hitze-sensitive Faktoren (wie Proteine) relevant sein können.

Rhodococcus equi causes severe pneumonia, resulting in disease and sometimes death of foals. Infection is thought to occur by inhalation of dust contaminated with virulent R equi. A recent study of 3 horse breeding farms in Ireland found airborne concentrations of virulent R equi to be significantly higher in stables than in paddocks. More importantly, another study from Australia established an association of airborne concentrations of virulent R equi with the prevalence of R equi pneumonia at 28 farms. The extent to which these associations extend to other farms in different parts of the world is not known. Two farms in central Kentucky with recurrent R equi pneumonia in foals were studied from February through July 2008. Air samples were collected and environmental factors were measured hourly for a 24-hour period each month from stalls and paddocks used to house mares and their foals at each farm. In 2009, samples were collected from 47 foals from stalls at a single horse-breeding farm in central Kentucky on days 1-2, days 7-9, and days 14-16 of life. Concentrations of airborne virulent R equi were determined via a modified colony immunoblot technique. Airborne concentrations of virulent R equi were significantly higher (P = 0.016) from 6:00 A.M. through 11:59 P.M. than for the period from midnight through 5:59 A.M. Presence of the mare and foal at the time of sampling was significantly (P < 0.001) associated with increased airborne concentrations of virulent R equi in stalls. The presence of virulent R equi in stalls was significantly (P = 0.045) more likely at 7 days of age for foals subsequently found to be affected by rhodococcal pneumonia. These findings suggest that recovery of airborne virulent R equi is less likely between 12:00 A.M. and 5:59 A.M., relative to other times, that airborne concentrations of virulent R equi are significantly increased when horses are present at the site for collection of air samples, and that environments containing airborne virulent R equi during the first week of life may influence the risk of subsequent disease for a foal.