Rhodococcus equi é um importante patógeno bacteriano em medicina veterinária, associado, sobretudo, a pneumonias piogranulomatosas em potros no primeiro semestre de vida. São descritos neste trabalho vinte casos de infecção por R. equi em potros recebidos para necropsia no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul entre janeiro de 1997 e janeiro de 2013. Os históricos clínicos obtidos com os veterinários apresentaram grande variabilidade e, mesmo os sinais clássicos de comprometimento respiratório e febre só foram vistos em metade dos casos. Os dados obtidos em uma visita a uma propriedade demonstram que a superpopulação de potros e a introdução de fêmeas no grupo de parição contribuíram para a ocorrência de um surto. Exames de necropsia e de histologia revelaram que pneumonia piogranulomatosa multifocal foi a forma de apresentação mais constante (dezenove casos), seguida por linfadenite piogranulomatosa (dez casos) e tiflocolite piogranulomatosa e ulcerativa (cinco casos). Três animais apresentaram osteomielite piogranuloamatosa, dos quais, dois em vértebras. Uveítes e polissinovites assépticas foram constatadas em três casos. Exame imuno-histoquímco anti-Rhodococcus equi revelou-se positivo em todos os pulmões com lesões, embora os linfonodos tenham sido positivos em apenas três das nove amostras testadas. O exame bacteriológico das amostras de necropsia foi positivo em quinze casos clínicos, assim como em uma amostra de solo da propriedade visitada. O exame de reação da polimerase em cadeia (PCR) revelou o gene de virulência VapA de R. equi em todos os isolados clínicos, mas não na amostra de solo. Adicionalmente os pulmões foram testados por imuno-histoquímica para Pneumocystis sp.e apresentaram marcação em treze dos vinte casos. / Rhodococcus equi is an important bacterial pathogen in veterinary medicine, especially associated with piogranulomatous pneumonia in foals under six months of age. Twenty cases of R. equi infection in foals received for necropsy at the Pathology Veterinary Sector (SPV) of the Federal University of Rio Grande do Sul (UFRGS) between January 1997 and January 2013 are described in this paper. Clinical history obtained with veterinary practitioners presented high variability, and even classical respiratory signs and fever were only observed in half of the cases. Data collected in an investigative visiting to a breeding farm showed that the foal superpopulation and the introduction of females to the parturition group contributed to the occurrence of an outbreak. Necropsy and histologic examinations revealed that multifocal piogranulomatous pneumonia was the most constant presentation (nineteen cases), followed by piogranulomatous lymphadenitis (ten cases) and piogranulomatous and ulcerative typhlocolitis (five cases). Three animals presented piogranulomatous osteomyelitis, two of them in vertebrae. Aseptic uveitis and polisynovitis were verified in three cases. Anti-Rhodococcus equi immunohistochemical examination stained positive in all lungs containing lesions, although lymphnodes have stained positive in only three of nine samples tested. Bacteriologic examination of the necropsy samples was positive in fifteen cases and in a soil sample from the visited breeding farm. Polymerase chain reaction (PCR) test revealed the VapA virulence factor of R.equi in all clinical isolates, but not in the soil sample. Additionally, the lungs were tested to the presence of Pneumocystis sp. by immunohistochemistry, and stained positive in thirteen of twenty cases.

Rhodococcus equi é um importante patógeno bacteriano em medicina veterinária, associado, sobretudo, a pneumonias piogranulomatosas em potros no primeiro semestre de vida. São descritos neste trabalho vinte casos de infecção por R. equi em potros recebidos para necropsia no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul entre janeiro de 1997 e janeiro de 2013. Os históricos clínicos obtidos com os veterinários apresentaram grande variabilidade e, mesmo os sinais clássicos de comprometimento respiratório e febre só foram vistos em metade dos casos. Os dados obtidos em uma visita a uma propriedade demonstram que a superpopulação de potros e a introdução de fêmeas no grupo de parição contribuíram para a ocorrência de um surto. Exames de necropsia e de histologia revelaram que pneumonia piogranulomatosa multifocal foi a forma de apresentação mais constante (dezenove casos), seguida por linfadenite piogranulomatosa (dez casos) e tiflocolite piogranulomatosa e ulcerativa (cinco casos). Três animais apresentaram osteomielite piogranuloamatosa, dos quais, dois em vértebras. Uveítes e polissinovites assépticas foram constatadas em três casos. Exame imuno-histoquímco anti-Rhodococcus equi revelou-se positivo em todos os pulmões com lesões, embora os linfonodos tenham sido positivos em apenas três das nove amostras testadas. O exame bacteriológico das amostras de necropsia foi positivo em quinze casos clínicos, assim como em uma amostra de solo da propriedade visitada. O exame de reação da polimerase em cadeia (PCR) revelou o gene de virulência VapA de R. equi em todos os isolados clínicos, mas não na amostra de solo. Adicionalmente os pulmões foram testados por imuno-histoquímica para Pneumocystis sp.e apresentaram marcação em treze dos vinte casos. / Rhodococcus equi is an important bacterial pathogen in veterinary medicine, especially associated with piogranulomatous pneumonia in foals under six months of age. Twenty cases of R. equi infection in foals received for necropsy at the Pathology Veterinary Sector (SPV) of the Federal University of Rio Grande do Sul (UFRGS) between January 1997 and January 2013 are described in this paper. Clinical history obtained with veterinary practitioners presented high variability, and even classical respiratory signs and fever were only observed in half of the cases. Data collected in an investigative visiting to a breeding farm showed that the foal superpopulation and the introduction of females to the parturition group contributed to the occurrence of an outbreak. Necropsy and histologic examinations revealed that multifocal piogranulomatous pneumonia was the most constant presentation (nineteen cases), followed by piogranulomatous lymphadenitis (ten cases) and piogranulomatous and ulcerative typhlocolitis (five cases). Three animals presented piogranulomatous osteomyelitis, two of them in vertebrae. Aseptic uveitis and polisynovitis were verified in three cases. Anti-Rhodococcus equi immunohistochemical examination stained positive in all lungs containing lesions, although lymphnodes have stained positive in only three of nine samples tested. Bacteriologic examination of the necropsy samples was positive in fifteen cases and in a soil sample from the visited breeding farm. Polymerase chain reaction (PCR) test revealed the VapA virulence factor of R.equi in all clinical isolates, but not in the soil sample. Additionally, the lungs were tested to the presence of Pneumocystis sp. by immunohistochemistry, and stained positive in thirteen of twenty cases.

Em função da proximidade cada vez maior entre o cavalo e o homem, é de extrema importância ter conhecimentos das doenças que acometem os equinos, que por ventura, podem acometer seres humanos. Dentre muitas doenças, pode-se citar duas, que promovem grandes perdas econômicas aos rebanhos eqüinos, tanto no tratamento desses rebanhos, como com a morte dos mesmos, dificultando a importação e exportação de animais: a babesiose e a erliquiose (anaplasmose), que podem estar ou não associadas, acometendo um animal, concomitantemente. A presente pesquisa teve como objetivo investigar e diagnosticar doença concomitante babesiose (por Babesia equi ou Theileria equi) e Erliquiose (por Erliquia equi ou Anaplasma phagocytophilum), no estado de São Paulo, em rebanhos eqüinos, utilizando as técnicas de Nested PCR (Nested polymerase chain reaction reação em cadeia pela polimerase para diagnóstico de T. equi e A. phagocytophilum) e c-ELISA (competitive enzyme-linked immunosorbent assay para diagnóstico de T. equi) ou ELISA indireto (para diagnóstico de A. phagocytophilum) e comparar os resultados obtidos nas diferentes técnicas em 250 amostras de eqüino (sangue total e soro). Como resultado, obteve-se 38,4%, 46% e 36% de positividade, respectivamente, nos testes de pesquisa de hematozoário, c-ELISA e Nested PCR para Theileria equi e 0%, 3% e 0% de positividade, respectivamente, nos testes de pesquisa de hemoparasita, ELISA indireto e Nested PCR para Anaplasma phagocytophilum, não sendo observada a co-infecção de Babesiose e Anaplasmose no rebanho estudo / Due to the increasing proximity between horse and man, it is of extreme importance to understand the diseases that affect horses which by chance may affect humans. Among many diseases, babesiosis and ehrlichiosis (anaplasmosis) promote high economic losses to horses herds in consequence of costs of treatment and also death, making it difficult to import and export animals: They can or not be linked affecting an animal at the same time. This study aimed to investigate and diagnose concomitant babesiosis (Babesia equi and Theileria equi) and ehrlichiosis (for ehrlichia equipment or Anaplasma phagocytophilum) in equine herds of the state of Sao Paulo, using the techniques of Nested PCR (Nested polymerase chain reaction for the diagnosis of T. equi and A. phagocytophilum) and c-ELISA (competitive enzyme-linked immunosorbent assay for diagnosis of T. equi) or ELISA (for diagnosis of A. phagocytophilum). Also to compare results obtained in these different techniques in 250 samples of horse (whole blood and serum). Results showed 38.4%, 46% and 36% positivity, respectively, in tests for the detection of Theileria equi through hematozoan, c-ELISA and Nested PCR and 0%, 3% and 0% positivity, respectively, in tests for the detection of Anaplasma phagocytophilum through blood parasites, indirect ELISA and Nested PCR. It was not observed co-infection Babesiosis and anaplasmosis in the herd study

Infecções pulmonares de potros jovens por Rhodococcus equi resultam em grave pneumonia levando à morte um grande número de animais todos os anos. Até o momento não há nenhuma vacina aceita globalmente para a prevenção da rodococose equina. O único tratamento preconizado é baseado em antibioticoterapia, porém os protocolos clínicos são longos, de alto custo, com efeitos colaterais e tem favorecido a seleção de cepas resistentes aos antibióticos. Diversas estratégias no desenvolvimento de uma vacina segura e eficiente contra a rodococose foram propostas, porém, não induziram um efeito protetor considerável. O principal fator de virulência do R. equi descrito e amplamente estudado é a proteína vapA, no entanto, outras proteínas localizadas na ilha de patogenicidade estão presentes em amostras de R. equi virulento extraídos de animais infectados. Trabalhos recentes têm demonstrado a presença do gene vapG em todas as cepas virulentas e este gene é altamente expresso quando a bactéria reside no interior de macrófagos, tornando-o um possível alvo vacinal. Nosso grupo já possui experiência prévia no uso de linhagem de Salmonella enterica Typhimurium atenuada carreando a proteína vapA. Baseado nos resultados positivos obtidos, foi construída uma linhagem atenuada de S. enterica Typhimurium 3987 expressando a proteína vapG. A administração desta linhagem em camundongos foi capaz de induzir proteção contra R. equi virulento. Os resultados observados foram a colonização e persistência da Salmonella nos órgãos alvo, a redução da carga bacteriana de R. equi, e a indução de um perfil imune protetor semelhante ao observado em animais adultos resistentes. Observou-se o aumento da produção de IL-12p70 e IFN-, alem da presença de níveis aumentados de IL-4 e a redução dos níveis de TNF-. Observou-se também o aumento na subpopulação de células T auxiliares (CD4+) com perfil de memória, além de aumento na população de linfócitos B totais quando comparado aos grupos controles. Este conjunto de resultados indica que a imunização com Salmonella enterica Typhimurium expressando a proteína vapG gera uma resposta imune celular eficiente tornando esta linhagem uma possível candidata a vetor vacinal. Alem disso, sugere que outros antígenos do R. equi podem ser / Pulmonary infections in young foals by Rhodococcus equi result in severe pneumonia, leading to death a large number of animals every year. There is no globally accepted vaccine for the prevention of equine rhodococosis so far. To date, the only acceptable treatment is based on antibiotics, but the clinical protocols are long lasting, expensive, having side effects and favoring the emergence of drug resistant strains. Several strategies for developing a safe and effective vaccine against rhodococosis have been proposed, however, none has induced a significant protective effect. The main virulence factor of R. equi described and extensively studied is the protein vapA, although other proteins encoded by genes in pathogenicity islands are detected in samples of virulent R. equi, isolated from infected animals. Recent works have demonstrated that the vapG gene is present in all virulent strains, and that this gene is highly expressed when bacteria reside within macrophages, making it a potential vaccine target. Our group has already been working with an attenuated Salmonella enterica Typhimurium strain expressing the VapA protein. Even though our results were very promising, we decided to construct a second vaccine strain expressing the VapG protein. Based on the positive results, it was constructed an attenuated strain of S. enterica Typhimurium 3987 expressing the vapG protein. Interestingly, the VapG-expressing strain induced protection against virulent R. equi in mouse model of infection. We could observe colonization and persistence of Salmonella vaccine cells in target organs, with reduction of bacterial loads of R. equi and induction of a protective immune profile similar to that seen in resistant adult animals. We could also observe increased production of IL-12p70 and IFN- in addition to the presence of increased levels of IL-4 and reduced levels of TNF-. Moreover, we detected an increase in the subpopulation of T helper cells (CD4), with a profile of memory, as well as in the population of B lymphocytes, when compared to control groups. This set of results shows that immunization with Salmonella enterica Typhimurium expressing the VapG protein raises an efficient cellular immune response, making this strain a potential candidate for vaccine vector. Furthermore, this work suggests that other R. equi antigens may be taken into account for vaccine construction, besides the VapA protein utilized in the majority of studies.

Orientador: Márcio Garcia Ribeiro / Banca: Antonio Carlos Paes / Banca: José Paes de Almeida Nogueira Pinto / Banca: Adolfo Carlos Barreto Santos / Banca: Rogério Giuffrida / Resumo: A linfadenite infecciosa em suínos representa uma das afecções mais preocupantes na criação de suínos em todo mundo, causada por patógenos de origem bacteriana, geralmente diagnosticada na linha de abate. Acarreta prejuízos econômicos com a condenação total ou parcial das carcaças, bem como reflexos em saúde pública, devido ao potencial zoonótico dos agentes causais. O presente estudo investigou a ocorrência e as principais espécies do gênero Mycobacterium, assim como marcadores de virulência plasmidial em linhagens de Rhodococcus equi (R. equi) isoladas de linfonodos e das fezes de suínos de abatedouros do interior do estado de São Paulo, com e sem linfadenite. Foram examinados 150 linfonodos (50 mesentéricos, 50 mediastínicos e 50 submandibulares) com lesões, 150linfonodos (50 mesentéricos, 50 mediastínicos e 50 submandibulares) sem lesões aparentes e 150 fezes de suínos, provenientes de animais destinados ao abate, em dois frigoríficos do interior do estado de São Paulo. As amostras de linfonodos e fezes foram submetidas ao cultivo microbiológico simultaneamente nos meios de ágar acrescido de sangue bovino (5%) desfibrinado, e meios seletivos de CAZ-NB, TCP e TVP para R. equi, e Stonebrink-Lesslie e Lowenstein-Jensen para micobactérias. As colônias sugestivas de R. equi e positivas no teste de CAMP, foram enviadas ao Japão para detecção de linhagens VapA ou VapB, associadas a virulência. As linhagens sugestivas no cultivo microbiano para o gênero Mycobacterium foram submetidas a caracterização de espécies por PCR pela técnica de PRA. Foram identificados nos linfonodos de suínos com lesões 48 (32,0%) linhagens de Mycobacterium spp.e 6 (4,0%) Rhodococcus equi. Nos linfonodos de suínos sem lesões foram identificados 11 (7,3%) isolados de Mycobacterium spp.e nenhuma linhagem de R. equi. Nas fezes foram identificadas 40 (26,6%) linhagens de Rhodococcus equi e ... / Abstract: The infectious lymphadenitis represents one of the most important diseases in pigs worldwide caused by bacterium, usually diagnosed on the slaughterhouses.The disease leads to economic losses due to total or partial condemnation of carcasses, as well as public health concern due to the zoonotic potential of microorganisms. The present study investigated the occurrence and the main species of the genus Mycobacterium as well as virulence markers of plasmid in Rhodococcus equi (R. equi) strains isolated from lymph nodes and feces from pigs of slaughterhouses in the state of São Paulo, with and without lymphadenitis. Were sampled 150 lymph nodes (50 mesenteric, 50 mediastinal and 50 submandibular) with lesions, 150 lymph nodes (50 mesenteric, 50 mediastinal and 50 submandibular) without visible lesions, and 150 feces from pigs of slaughterhouses of state of São Paulo, Brazil. The lymph nodes samples and feces were subjected to microbiological culture simultaneously indefibrinated bovine blood agar (5%), selective media of CAZ-NB, TCP, TVP for R. equi, and Stonebrink-Lesslie, and Lowenstein-Jensen for mycobacteria . The suggestive colonies of R. equi and positive to CAMP test were sent to Japan for evaluation of plasmid profile (VapA or VapB). The suggestive of Mycobacterium sp. were subjected to polymerase chain reaction (PCR) - based species identification using restriction enzyme pattern analysis (PRA). Among lymph nodes of pigs with lesions, 48 (32.0%) Mycobacterium spp. and 6 (4.0%) R. equi strains were identified. In the lymph nodes of pigs without lesions were identified 11 (7.3%) Mycobacterium spp. and none R. equi strain. From the fecal samples, 40 (26.6%) R. equi and 2 (1.3%) Mycobacterium spp. isolates were identified. From 48 Mycobcterium isolates from pigs with lesions, 37 (77.0%) were identified by PRA as M. avium type 1, and 11 (23.0%) M. avium type 2. Among limph nodes with lesions wer ... / Doutor

The soil-dwelling gram-positive coccobacillus Rhodococcus equi is a well-known veterinary pathogen and emerging human pathogen. Although Rhodococcus infection is primarily associated with pyogranulomatous pneumonia in foals, these bacteria can also infect other animal species including humans. R. equi pathogenicity is mediated by the conjugative virulence plasmid (pVAP) which promotes intracellular proliferation in host macrophages. Currently R. equi is endemic in horse breeding farms worldwide. No R. equi vaccine is available and both treatment and prophylaxis rely on the administration of a prolonged course with a combination of macrolide antibiotics (typically erythromycin) and rifampicin. These antimicrobials were introduced in the therapy against R. equi in the 1980s and multiresistance has now emerged among foal isolates, increasing the risk of zoonotic transmission. In this thesis, the role of conjugal extrachromosomal replicons in the host adaptation of R. equi was explored. A previous epidemiological study indicated that different variants of the pVAP virulence plasmid are associated with different animal hosts. This PhD project provides experimental confirmation of a R. equi plasmid-driven host tropism. In vivo and in vitro competition assays were performed using a set of isogenic strains only different in the virulence plasmid type, in adapted (horse) and non-adapted (mouse) model species. The data obtained in the horse model provides clear evidence of a significant negative selection of the non-equine virulence plasmids both at a cell and at the animal level, while no selection was observed in the non-adapted mouse model. Furthermore, this project characterized the determinant responsible for macrolide resistance in R. equi, a novel erm methylase gene, erm(46). The erm(46) determinant was shown to be transferable between strains by conjugation and herein the underlying mechanism and how erm(46) becomes stabilized in R. equi is described. PacBio SMRT-sequencing based analysis revealed that the erm(46) gene is carried in a self-replicating conjugative plasmid of about 80 kb, that we designated pRErm46. The conjugation machinery of pRErm46 was hypothesized to be responsible for bringing the erm(46) determinant into R. equi. However, some erythromycin resistant isolates lack pRErm46 but erm(46) transfer is still observed. This reflects the observation that erm(46) is present in a mobile element that, upon acquisition with the pRErm46 replicon, transposes at a high frequency and to multiple locations of the host genome. If the erm(46) mobile element transposes to the chromosome, no further transfer of the resistance is observed at a detectable frequency in the absence of pRErm46. On the other hand, if the erm(46) element transposes to the R. equi virulence plasmid, the erm(46) determinant co-opts the pVAPA conjugal transfer machinery and gets transferred at the same high frequency as the virulence plasmid (10-2). This constitutes a unique example of efficient co-transfer, in the same genetic vehicle, of virulence and antimicrobial determinants, two key niche-adaptive traits required for within-host survival of bacterial pathogen.

The focus of this study is scattering matrices in the framework of quantum graphs,more precisely the matrices which describe equi-transmission. They are unitary andHermitian and are independent of the energies of the associated system. In the firstarticle it is shown that in the case where reflection does not occur, such matrices existonly in even dimensions. A complete description of the matrices in dimensions 2, 4,and 6 is given. In dimension 6, 60 five-parameter families are obtained. The 60 matricesyield a combinatorial bipartite graph K62. In the second article it is shown that whenreflection is allowed, the standard matching conditions matrix is equi-transmitting forany dimension n. All equi-transmitting matrices up to order 6 are described. For oddn (3 and 5), the standard matching conditions matrix is the only equi-transmitting matrix.For even n (2, 4 and 6) there exists other equi-transmitting matrices apart fromthose equivalent to the standard matching conditions. All such additional matriceshave zero trace.

While Rhodococcus equi (R. equi) remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals, development of a relevant model to study this bacterium has proven difficult. As a result, the reasons for the underlying foal’s susceptibility to this disease are not well understood. Furthermore, data regarding the immune response of foals to R. equi infection remains controversial. We hypothesized that foals are susceptible to R. equi early in life and that this susceptibility decreases with age. Also, we hypothesized that specific subclasses of IgG antibodies to the virulence-associated protein of R. equi, VapA, predict the outcome of exposure. The objectives of this study were: (1) to develop an R. equi challenge model that resulted in slow progressive disease in some foals as well as spontaneous regression of lesions in others, (2) using the developed model, to investigate the age-related susceptibility of young foals to R. equi, (3) to describe the humoral immune response of foals following experimental challenge and natural infection. The use of a low dose of R. equi to challenge neonatal foals resulted in slow, progressive disease characterized by pulmonary abscessation and spontaneous regression in approximately 50% of the foals. When this low dose was used in 1, 2 or 3-week-old foals, a marked decrease in disease susceptibility was observed as the foals aged. The immunological responses seen after experimental challenge reflect those observed after natural infection. While there was a significant increase of VapA-specific IgG and IgG subclasses over time in both pneumonic and healthy foals, use of VapA-specific IgG(T) showed good sensitivity and specificity when used as a diagnostic tool for R. equi pneumonia. In summary, this study shows that foal susceptibility to R. equi occurs early in life and decreases with age. Whereas all foals developed VapA-specific IgG antibodies post-exposure, IgG(T) appeared to be predictive of infection.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 4 uppsatser.

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