Conselho Nacional de Desenvolvimento Científico e Tecnológico / Horse breeding industry is an activity in ascension worldwide, and is responsible for generating jobs and income. In Brazil, especially in Rio Grande do Sul state, there are several horse breeding farms with high-standard equines. Although these herds are under strict sanitary control, the occurrence of respiratory diseases is an important cause of mortality in foals and reduced athletic performance. Among the respiratory diseases, equine rhodococcosis, caused by the bacterium Rhodococcus equi, is the major cause of pneumonia in foals. Rhodococcus equi is worldwide distributed, and have emerged as an important cause of economic losses due to pneumonia in young animals. However, preventive measures and effective control of the disease are still challenges to be reached. In R. equi infection, iron (Fe) is classified as an essential element not only for the bacterium multiplication, but also as a key for the expression of virulence factors. Studies have shown the presence of specific Fe uptake mechanisms in R. equi, which have been determining its survival in both saprophytic and pathogenic life styles. However, as a type of nutritional immunity, mammals, including horses, reduce the plasmatic concentration of Fe through its binding in proteins, including the transferrin (Tf). In this context, the present thesis was developed to study aspects related to metabolism and acquisition of Fe by R. equi and Fe importance in the pathogenesis of equine rhodococcosis (manuscript 1), control and treatment of infections caused by R. equi through drugs with capability to reduce the availability of intracellular Fe (manuscript 2), and genetic susceptibility to R. equi pneumonia (manuscript 3), including the assessment of polymorphisms in the equine Tf gene as risk factors related to susceptibility and/or resistance to equine rhodococcosis (manuscript 4 ). We conclude that R. equi is evolving to specialize it in the acquisition and utilization of Fe from the host, skills that should be considered as key points for the development of chemotherapeutic agents. Once R. equi encodes redundant mechanisms of acquisition and utilization of Fe, it is likely that chemotherapeutic agents will need act on multiple cellular mechanisms or be used in combination. Furthermore, the term "nutritional immunity" may be considered an important strategy to minimize antimicrobial resistance observed in R. equi. As an example of chemotherapy associated with iron metabolism, we observed that chloroquine inhibits the intracellular multiplication of R. equi, most likely due to intracellular iron deprivation. However, further studies are necessary to evaluate the chloroquine therapeutic potential against R. equi infections. We also observed important chromosomal regions positively associated with R. equi pneumonia, which seem to possess genes associated with immune response against intracellular pathogens. This observation allows us to classify the equine rhodococcosis as a disease of polygenic basis, as postulated by previous studies. Finally, we found that polymorphisms in the Tf gene, including some not described yet in the literature, occur in Brazilian Sport Horses and Brazilian Thoroughbred Horses. There is the occurrence of two alleles between the breeds studied, including heterozygosis for these alleles. We believe that there is a relationship between equine Tf variants, and genetic susceptibility to R. equi pneumonia in the breeds evaluated. Summarizing, we have demonstrated that the modulation of Fe availability may be a useful approach to control the disease. / A equideocultura é uma atividade em ascensão mundial, responsável pela geração de empregos e renda. No Brasil, em especial no Rio Grande do Sul, encontram-se diversos locais de criação de equinos de alto padrão zootécnico. Embora estes rebanhos estejam sob rigoroso controle sanitário, a ocorrência de doenças respiratórias é causa importante de mortalidade em potros e redução de seu desempenho atlético. Dentre as doenças respiratórias, a rodococose equina, causada pela bactéria Rhodococcus equi, é a principal causa de pneumonia nesta categoria animal. R. equi está distribuído mundialmente, e cresce como causa de perdas econômicas devido à pneumonia observada em animais jovens. No entanto, medidas preventivas e efetivo controle da enfermidade são ainda desafios a serem alcançados. Na infecção por R. equi, o ferro (Fe) apresenta-se como um elemento fundamental não somente para multiplicação da bactéria, mas também, como um determinante para a expressão de fatores de virulência. Estudos têm demonstrado a presença de mecanismos específicos de captação de Fe em R. equi, os quais determinam sua sobrevivência tanto durante seu estilo de vida saprófito quanto patogênico. Em contrapartida, como uma forma de imunidade nutricional, mamíferos, entre eles os equinos, diminuem a concentração plasmática de Fe através de sua ligação em proteínas, entre elas, a transferrina (Tf). Neste contexto, esta tese foi elaborada visando contemplar aspectos relacionados ao metabolismo e aquisição de Fe por R. equi e sua importância para patogenia da rodococose equina (manuscrito 1), controle e tratamento de infecções por R. equi através de drogas com capacidade de modular a disponibilidade de Fe intracelular (manuscrito 2), e susceptibilidade genética à pneumonia por R. equi (manuscrito 3), incluindo a avaliação de polimorfismos no gene da Tf equina como fatores de risco relacionados à susceptibilidade e/ou resistência genética à rodococose equina (manuscrito 4). Concluímos que R. equi está evoluindo de forma a especializar-se na aquisição e utilização de Fe a partir do hospedeiro, habilidades que devem ser consideradas como pontos chave no desenvolvimento de agentes quimioterápicos. Uma vez que R. equi codifica redundantes mecanismos de aquisição e utilização de Fe, é provável que agentes quimioterápicos deverão inibir múltiplos mecanismos ou ser utilizados em combinação. Além disso, o conceito de imunidade nutricional pode considerado uma importante estratégia para minimizar a resistência antimicrobiana observada em R. equi. Como um exemplo de quimioterápicos associados ao metabolismo de Fe, observados que chloroquine inibir a multiplicação intracelular de R. equi, muito provavelmente devido à deprivação de Fe intracelular. No entanto, ainda são necessários estudos avaliando o potencial terapêutico de chloroquine como tratamento alternativo de infecções por R. equi. Observou-se, também, importantes regiões cromossômicas positivamente associadas à pneumonia por R. equi, as quais parecem possuir genes associados à resposta imune contra patógenos intracelulares. Esta observação nos permite classificar a rodococose equina como uma enfermidade de base poligênica, como postulado por estudos anteriores. Por fim, verificamos que polimorfismos no gene da Tf, inclusive polimorfismos ainda não descritos na literatura, ocorrem em equinos das raças Brasileiro de Hipismo e Puro Sangue de Corrida, criados no Brasil. Existe a ocorrência de dois alelos entre as raças estudas, incluindo animais heterozigotos para estes alelos. Acredita-se que exista uma relação entre variantes de Tf equina e susceptibilidade genética à pneumonia por R. equi nas raças analisadas. Em suma, demonstrou-se através de diferentes estudos que a modulação da disponibilidade de Fe pode ser uma forma de controle da rodococose equina.

Made available in DSpace on 2014-08-20T13:32:54Z (GMT). No. of bitstreams: 1 tese_leandro_nizoli.pdf: 538110 bytes, checksum: 770c97bed302c0714288a9278ce94694 (MD5) Previous issue date: 2009-03-18 / Equine theileriosis is considered to be one of the most important parasitic diseases that affect horses, and has great economic impact on the equine industry. The disease is caused by the etiologic agent Theileria equi, which is classified as a hematozoan. The losses associated with equine theileriosis are related to clinical manifestation as well as restriction to international travel to positive horses. Chronic infected equines suffer the risk of the disease relapse which leads to losses in reproduction performance and are potentially disseminators of the disease. In the last years, studies on the immunologic diagnosis and vaccination against T. equi have focused to obtain distinct antigenic proteins. On the outer membrane of this protozoan, major surface proteins has been characterized and named as EMAs (equi merozoite antigen). Of these, EMA-1 has been used as antigen for diagnosis due to its conservation in diverse isolates. Its role as a potential immunogen has been well documented due its ability to stimulate a humoral response with production of specific antibodies in infected animals. Through this antibodies one can used as tool for immune diagnostic of this disease. EMA-1 is also a strong candidate to be use as a vaccine in the control of equine theileriosis. In this study we used the Pichia pastoris yeast as expression system for the production of the EMA-1 protein of T. equi and evaluated its antigenicity and immunogenicity. When tested for antigenicity, the recombinant protein was recognized by antibodies form chronic T. equi infected horses, suggesting that epitopes of the native were conserved in the recombinant protein. Also we were able to observe that this protein was immunogenic in mice. The data obtained in this study demonstrated that the yeast P. pastoris is an expression system of heterologous protein suitable for the production of EMA-1 from T. equi. / A Theileriose eqüina é considerada uma das principais doenças parasitárias que acometem os eqüinos, acarretando grande impacto econômico na equinocultura. A doença é causada pelo hematozoário Theileria equi. As perdas econômicas associadas à theileriose eqüina estão relacionadas tanto aos fatores clínicos, quanto à restrição ao trânsito internacional de animais soropositivos, já que animais portadores crônicos são passíveis de reagudizações, gerando perda de performance física e reprodutiva, e são potencialmente disseminadores da enfermidade. Nos últimos anos, os estudos sobre o diagnóstico imunológico e vacinação contra T. equi concentram-se na obtenção de frações antigênicas. Na membrana externa deste protozoário foram caracterizadas proteínas principais de superfície denominadas de EMAs (equi merozoite antigen). Dentre estas, a EMA-1 destaca-se como antígeno para diagnóstico em função de sua conservação entre diversos isolados. Seu papel também tem sido caracterizado como imunógeno por estimular forte resposta humoral com produção de anticorpos em animais infectados, podendo ser usado como ferramenta para imunodiagnóstico dessa doença. EMA-1 é também um potencial candidato como antígeno vacinal no controle da theileriose equina. Neste estudo utilizou-se o sistema eucariótico de expressão baseado na levedura metilotrófica Pichia pastoris, para a produção da proteína EMA-1 de T. equi e a avaliação quanto a sua antigenicidade e imunogenicidade. Quanto a sua antigenicidade, a proteína recombinante foi reconhecida por anticorpos de animais portadores crônicos de T. equi, sugerindo que epítopos nativos foram conservados na proteína recombinante. Também foi observado que a proteína recombinante foi capaz de gerar resposta imune em camundongos vacinados com esta proteína. Os dados obtidos neste estudo demonstram que a levedura P. pastoris é um sistema de expressão heterólogo adequado para a produção da proteína EMA-1 de T. equi, podendo ser utilizada como imunobiológico no desenvolvimento de testes diagnósticos e vacina recombinante.

Rhodococcus equi (R. equi) is one of the most serious causes of pneumonia in young foals. The clinical disease is of great concern to breeding farms worldwide due to the impact of mortality on economic losses. While adult horses are resistant to R. equi, foals exhibit a distinct age-associated susceptibility. The mechanism underlying this susceptibility in foals is not well understood. Interferon-gamma (IFNg) plays an important role in the clearance of R. equi, but its expression is impaired in neonatal foals. Moreover, the regulation of this age-related IFNg expression in foals remains unknown. In humans, IFNg expression has been shown to be regulated by DNA methylation, lymphoproliferation, and influenced by environmental exposure. Therefore, we hypothesized that environmental exposure promotes IFNg expression through regulation of DNA methylation and lymphoproliferation. The objectives were: (1) to estimate the relevance of IFN-g production and R. equi infection in foals; (2) investigate the role of lymphoproliferation and DNA methylation in the regulation of IFN-g expression in foals; (3) to evaluate the effect of environmental exposure on IFN-g expression by housing foals in a barn environment verses pasture.; (4) to investigate the effect of environment exposure on antigen-presenting cells (APC), which sensor the environmental antigens and modulate IFN-g production by T cells. The results demonstrated that the IFN-g expression was inversely correlated with the age-related susceptibility to R. equi infection. lymphoproliferation promoted IFN-g expression in foals, whereas, DNA methylation repressed IFN-g expression. The IFN-g expression was augmented in foals exposed to the barn air which contained higher numbers of aerosol miroorganisms. DNA on the IFN-g promoter was demethylated and the lymphoproliferative activity was elevated in foals with barn-air exposure. The barn-air exposure also promoted the maturation and activation of APC to prime IFN-g expression by T cells in foals. Overall, this body of work demenstrated a relationship between IFN-g expression and R. equi infection, provided novel information on mechanisms that regulate IFN-g expression, and identified the effect of environment on mechanisms responsible for IFN-g expression.

Orientador: Marcio Garcia Ribeiro / Banca: José Paes de Almeida Nogueira Pinto / Banca: Rogerio Giuffrida / Resumo: A rodococose suína compreende doença infecciosa caracterizada por linfadenites piogranulomatosas. Diferentes fatores de virulência são reconhecidos na patogenicidade de Rhodococcus equi. A estrutura da parede celular bacteriana, a viabilidade no interior de fagócitos e na ausência de ferro, a produção de citotoxinas, a resistência aos antimicrobianos convencionais e, recentemente, a presença de proteínas associadas à virulência (Vap) reguladas por plasmídios, são considerados os principais mecanismos de virulência do microrganismo. Diferentes fatores de virulência foram avaliados em 23 (6,1%) linhagens de R. equi isoladas de 378 linfonodos submandibulares e mesentéricos de suínos e javalis (Sus scrofa). Foram realizados exames microbiológicos em 129 linfonodos apresentando lesões (linfadenite) e 129 sem lesões (controle) de suínos, e 60 linfonodos com lesões e 60 sem lesões de javalis. Dentre as 23 linhagens de R. equi, 19 (7,4%) foram isoladas de suínos, das quais 17 obtidas de linfonodos com lesões e duas sem lesões. Das 19 linhagens de suínos, 18 (94,7%) foram obtidos de linfonodos submandibulares e um (5,3%) de mesentérico. As quatro (3.3%) linhagens de R. equi isoladas de javalis foram obtidas exclusivamente de linfonodos com lesões. Destes, três foram obtidos de linfonodos submandibulares e um de mesentérico. Dentre nove antimicrobianos testados, azitromicina (100,0%), gentamicina (100,0%), levofloxacina (100,0%), vancomicina (100,0%), amoxicillina/ácido clavulânico (94,7%), eritromicina (94,7%) e rifampicina (94,7%) foram os fármacos mais efetivos. Baixa ocorrência de resistência aos antimicrobianos nos isolados de suínos foi observada contra os fármacos testados. A concentração inibitória mínima (MIC90) da azitromicina, eritromicina e rifampicina foi observada, respectivamente, em ≤2 µg/mL, ≤0,5 µg/mL and ≤1 µg/mL... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: The rhodococcosis in swine comprise an infectious disease characterized by pyogranulomatous lymphadenitis. Different virulence factors are recognized in pathogenicity of the Rhodococcus equi. The structure of bacterial cell wall, the viability inside of phagocytes and in absence of iron, the production of cytotoxins, the resistance to conventional antimicrobials and recently, the presence of proteins associated to virulence (Vap) regulated by plasmids, are considered the most important virulence mechanisms of microorganism. Different virulence factors were evaluated in 23 (6.1%) R. equi strains isolated from 378 submandibular and mesenteric lymph nodes of swine and wild boars (Sus scrofa). Microbiological exams were performed in 129 lymph nodes presenting lesions (lymphadenitis) and 129 without lesions (controls) from swine, and 60 lymph nodes with lesions and 60 without lesions from wild boars. Among 23 R. equi strains, 19 (7.4%) were isolated from swine and, from these, 17 were obtained from lymph nodes with lesions and two without lesions. From 19 strains isolated from swine, 18 (94.7%) were obtained of submandibular lymph nodes and one (5.3%) from mesenteric. The four (3.3%) R. equi strains isolated from wild boars were obtained exclusively of lymph nodes presenting lesions. From these, three were obtained from submandibular lymph nodes and one of mesenteric. Among nine antimicrobials tested, azithromycin (100.0%), gentamicin (100.0%), levofloxacin (100.0%), vancomycin (100.0%), amoxicillin/clavulanic acid (94.7%), erythromycin (94.7%) and rifampin (94.7%) were the most-effective drugs. Low rates of resistance to antimicrobials in swine isolates were observed against drugs tested. The inhibitory minimal concentration of 90% of isolates (MIC90) with use of azithromycin, erythromycin and rifampin were observed respectively in 2 μg/mL, 0.5 μg/mL and 1 μg /mL... (Complete abstract click electronic access below) / Mestre

<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.

<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 5 uppsatser.

Zugl.: Berlin, Freie Universiẗat, Diss., 2008.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 186 pages. Includes Vita. Includes bibliographical references.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 152 pages. Includes Vita. Includes bibliographical references.