Thesis (Ph.D.)--Murdoch University, 2010. / Thesis submitted to the Faculty of Health Sciences. Includes bibliographical references (leaves 106-130)

Memoria para optar al Título Profesional de Médico Veterinario / Corynebacterium pseudotuberculosis es el microorganismo causante de la linfoadenitis caseosa en pequeños rumiantes, así como de la linfangitis ulcerativa y abscesos en equinos y bovinos, causando pérdidas económicas importantes ya que disminuye el rendimiento productivo del ganado. Uno de los principales factores de virulencia de esta bacteria es la presencia de una esfingomielinasa específica, la fosfolipasa D, que favorece la diseminación de la bacteria dentro del hospedero. En este estudio se determina la presencia del gen que codifica esta exotoxina y se cuantifica la capacidad hemolítica de la misma, en aislados de C. pseudotuberculosis obtenidos desde ovinos, caprinos y equinos. Como resultado se obtuvo que existen diferencias dependiendo del origen, lo que puede estar generando las características de expresión de la enfermedad, ya que las cepas de origen ovino tienen mayor capacidad hemolítica que las de otras especies. Se sugiere que la capacidad hemolítica de las diferentes cepas puede estar relacionada con presentaciones clínicas diferentes en las especies animales en estudio y se propone un método de medición cualitativa de hemólisis en placas de agar sangre

Memoria para optar al Título Profesional de Médico Veterinario / Se ha observado que cepas de Corynebacterium pseudotuberculosis provenientes de diferentes especies animales tienen características fenotípicas y genotípicas particulares. C. pseudotuberculosis en medios líquidos, se desarrolla formando grumos, esto se debe a que el microorganismo se organiza en agregados bacterianos embebidos en una matriz de exopolisacáridos. El objetivo de este trabajo fue determinar diferencias en la capacidad de producción de exopolisacáridos entre cepas de C. pseudotuberculosis provenientes de equinos, ovinos y caprinos. Se estudiaron 4 cepas de cada especie, las que fueron cultivadas para cuantificar su producción de exopolisacáridos mediante el método fenol-ácido sulfúrico. Se determinó que no existen diferencias estadísticamente significativas (p>0,05) entre cepas provenientes de equinos, ovinos y caprinos. Sin embargo, la variabilidad en producción de exopolisacárido de las cepas de C. pseudotuberculosis estudiadas, independientemente de la especie animal de origen, sugiere que se realicen investigaciones posteriores para identificar las causas de esa variabilidad

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A escassez de dados sobre tuberculose e micobacterioses em ovinos (Ovis aries) motivou o presente trabalho de isolamento e tipificação de microorganismos presentes em linfonodos e lesões macroscópicas sugestivas de tuberculose. Foram avaliados pelo teste tuberculinico, 353 ovinos das raças Santa Inês e Texel de duas propriedades da região de Pindamonhangaba - São Paulo. Dos 57 animais selecionados para abate, 31 apresentavam reação ao PPD bovino maior que ao PPD aviário e 26 com reação ao PPD bovino menor que ao PPD aviário. Onze animais (19,3%) apresentaram na necrópsia lesões sugestivas de tuberculose. Os órgãos afetados foram o fígado, linfonodo submandibular, intestino, pulmão, linfonodo mediastino e glândula mamária. Foram isoladas micobactérias de sete (12,3%) animais e a tipificação genética pelo método de PRA demonstrou cinco (71,42%) infectados pelo Mycobacterium flavescens 1, um (14,28%) pelo M. kansasi, e um (14,28%) por micobactéria pertencente ao Complexo M. tuberculosis. Exames bacteriológicos para outras bactérias e/ou fungos isolaram Corynebacterium pseudotuberculosis em quatro (7,01%) dos 57 animais abatidos. Houve isolamento simultâneo de micobactérias e de Corynebacterium pseudotuberculosis em dois (3,5%) dos 57 animais abatidos. Os exames histopatológicos apontaram em nove (15,78%) animais a presença de granuloma e coloração de Ziehl-Neelsen positivo. A análise dos resultados obtidos permitiram concluir que, neste trabalho, os testes imunoalérgicos (Teste Cervical Simples e Teste Cervical Comparativo) não foram capazes de diferenciar infecção provocada pelo M. flavescens 1, M. kansasi, complexo M. tuberculosis e C. pseudotuberculosis. Nos exames macroscópico e histopatológico lesões provocadas por M. flavescens 1, M. kansasi, e C. pseudotuberculosis não foram diferenciáveis das provocadas pelo complexo M. tuberculosis. / The occurrence of few data on ovine (Ovis aries) tuberculosis and mycobacteriosis has motivated this work of isolation and typing microorganism found in lymph nodes and tuberculosis-like gross lesions. Tuberculin skin test was performed in 353 Santa Ines and Texel ovine breeds of two properties located at Pindamonhangaba Municipality - Sao Paulo State. Fifty seven animals were selected to be slaughtered and 31 of them had the bovine PPD skin test higher than avian PPD and other 26 presented bovine PPD reaction lower than avian PPD. Eleven animals (19.3%) showed tuberculosis-like gross lesions at necropsy. Most affected organs were liver, submandibular lymph nodes, intestines, lungs, mediastinic lymph nodes and mammary gland. It was possible to isolate mycobacteria from seven (12.3%) animals and genetic typing by the PRA method showed that five animals (71.42%) were infected with Mycobacterium flavescens 1, one (14.28%) with M. kansasi, and one (14.28%) with M. tuberculosis complex mycobacteria. Bacteriological culture isolation for other bacteria and/or fungi were positive for Corynebacterium pseudotuberculosis in four (7.01%) of 57 slaughtered animals. There was a concomitant isolation of mycobacteria and Corynebacterium pseudotuberculosis in two (3.5%) of 57 slaughtered animals. Histopathologic examination demonstrated the presence of granuloma and positive Ziehl-Neelsen staining in nine (15.78%) animals. Results analysis allowed concluding that in this work, immuno-allergic tests (Simple Cervical Test and Comparative Cervical Test) were not capable to differentiate infection caused by M. flavescens 1, M. kansasi, M. tuberculosis complex and C. pseudotuberculosis. During gross examination and histopathology, lesions caused by M. flavescens 1, M. kansasi, and C. pseudotuberculosis were not distinguishable from those caused by M. tuberculosis complex.

A escassez de dados sobre tuberculose e micobacterioses em ovinos (Ovis aries) motivou o presente trabalho de isolamento e tipificação de microorganismos presentes em linfonodos e lesões macroscópicas sugestivas de tuberculose. Foram avaliados pelo teste tuberculinico, 353 ovinos das raças Santa Inês e Texel de duas propriedades da região de Pindamonhangaba - São Paulo. Dos 57 animais selecionados para abate, 31 apresentavam reação ao PPD bovino maior que ao PPD aviário e 26 com reação ao PPD bovino menor que ao PPD aviário. Onze animais (19,3%) apresentaram na necrópsia lesões sugestivas de tuberculose. Os órgãos afetados foram o fígado, linfonodo submandibular, intestino, pulmão, linfonodo mediastino e glândula mamária. Foram isoladas micobactérias de sete (12,3%) animais e a tipificação genética pelo método de PRA demonstrou cinco (71,42%) infectados pelo Mycobacterium flavescens 1, um (14,28%) pelo M. kansasi, e um (14,28%) por micobactéria pertencente ao Complexo M. tuberculosis. Exames bacteriológicos para outras bactérias e/ou fungos isolaram Corynebacterium pseudotuberculosis em quatro (7,01%) dos 57 animais abatidos. Houve isolamento simultâneo de micobactérias e de Corynebacterium pseudotuberculosis em dois (3,5%) dos 57 animais abatidos. Os exames histopatológicos apontaram em nove (15,78%) animais a presença de granuloma e coloração de Ziehl-Neelsen positivo. A análise dos resultados obtidos permitiram concluir que, neste trabalho, os testes imunoalérgicos (Teste Cervical Simples e Teste Cervical Comparativo) não foram capazes de diferenciar infecção provocada pelo M. flavescens 1, M. kansasi, complexo M. tuberculosis e C. pseudotuberculosis. Nos exames macroscópico e histopatológico lesões provocadas por M. flavescens 1, M. kansasi, e C. pseudotuberculosis não foram diferenciáveis das provocadas pelo complexo M. tuberculosis. / The occurrence of few data on ovine (Ovis aries) tuberculosis and mycobacteriosis has motivated this work of isolation and typing microorganism found in lymph nodes and tuberculosis-like gross lesions. Tuberculin skin test was performed in 353 Santa Ines and Texel ovine breeds of two properties located at Pindamonhangaba Municipality - Sao Paulo State. Fifty seven animals were selected to be slaughtered and 31 of them had the bovine PPD skin test higher than avian PPD and other 26 presented bovine PPD reaction lower than avian PPD. Eleven animals (19.3%) showed tuberculosis-like gross lesions at necropsy. Most affected organs were liver, submandibular lymph nodes, intestines, lungs, mediastinic lymph nodes and mammary gland. It was possible to isolate mycobacteria from seven (12.3%) animals and genetic typing by the PRA method showed that five animals (71.42%) were infected with Mycobacterium flavescens 1, one (14.28%) with M. kansasi, and one (14.28%) with M. tuberculosis complex mycobacteria. Bacteriological culture isolation for other bacteria and/or fungi were positive for Corynebacterium pseudotuberculosis in four (7.01%) of 57 slaughtered animals. There was a concomitant isolation of mycobacteria and Corynebacterium pseudotuberculosis in two (3.5%) of 57 slaughtered animals. Histopathologic examination demonstrated the presence of granuloma and positive Ziehl-Neelsen staining in nine (15.78%) animals. Results analysis allowed concluding that in this work, immuno-allergic tests (Simple Cervical Test and Comparative Cervical Test) were not capable to differentiate infection caused by M. flavescens 1, M. kansasi, M. tuberculosis complex and C. pseudotuberculosis. During gross examination and histopathology, lesions caused by M. flavescens 1, M. kansasi, and C. pseudotuberculosis were not distinguishable from those caused by M. tuberculosis complex.

Made available in DSpace on 2014-06-11T19:32:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-11-22Bitstream added on 2014-06-13T20:04:04Z : No. of bitstreams: 1 monnazzi_lgs_dr_arafcf.pdf: 1628105 bytes, checksum: 439e61c2131f392d13c659297a95020d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / As três espécies patogênicas do gênero Yersinia, Y. pestis, Y. enterocolitica e Y. pseudotuberculosis, compartilham um tropismo pelos tecidos linfóides e um plasmídeo de 70-kb que é essencial para a virulência. O plasmídeo codifica um sistema de secreção do tipo III e proteínas efetoras chamadas Yops (Yersinia outer proteins). Este sistema de secreção é responsável por translocar as Yops para dentro das células do hospedeiro, onde elas interagem com alvos específicos e alteram as funções destas células. As Yops são capazes de modular a resposta imune do hospedeiro, permitindo à bactéria se replicar extracelularmente nos tecidos e órgãos linfóides. Embora haja muita informação sobre os mecanismos usados pela Yersinia para evadir do sistema imune inato de defesa, pouco se sabe sobre como ela afeta a resposta imune adaptativa in vivo. O objetivo deste trabalho foi analisar a influência das Yops E, H e M, translocadas pela Y. pseudotuberculosis, na colonização e persistência da bactéria no baço e fígado dos animais infectados, nas quantidades de LT-CD4 e LT-CD8 durante a infecção e na produção das principais citocinas Th1 e Th2 por estas subpopulações de linfócitos. Além disso, foi verificado o papel destas Yops sobre a ativação do fator nuclear κB (NF-κB) e sobre a atividade citotóxica dos LT-CD8. Para isso, camundongos BALB/c fêmeas foram infectados intravenosamente com a amostra selvagem de Y. pseudotuberculosis (WT), ou com amostras mutantes incapazes de secretar as Yops E, H e M (YopE-, YopH- e YopM-), ou ainda com a amostra curada do plasmídeo de virulência (YpIII). No 5°, 7°, 14° e 21° dia pós-infecção (pi), os animais foram sacrificados e as células esplênicas foram obtidas de camundongos infectados e de camundongos não infectados (grupo controle). Os níveis de colonização no baço e no fígado foram determinados por... / The three pathogenic species of the genus Yersinia, Y. pestis, Y. enterocolitica and Y. pseudotuberculosis, share a tropism for lymphoid tissues and a 70-kb plasmid essential for virulence. The plasmid encodes a type III secretion system and effector proteins called Yops (Yersinia outer proteins). This secretion system is responsible for translocating the Yops into the host cells, where they interact with specific host targets and alter the functions of these cells. Yops are able to modulate the host immune defenses allowing the bacteria to replicate extracellularly in lymphoid tissues and organs. Although there is ample information on the mechanisms used by Yersinia to evade the innate immune system, very little is known about how it affects the adaptive immune response in vivo. The aim of this research was to analyze the influence of translocated Yops E, H and M of Y. pseudotuberculosis on the colonization and persistence of the bacterium in the spleen and liver of infected animals, on the quantities of CD4 and CD8 T cells during the infection and on the production of the main Th1 and Th2 cytokines by these lymphocyte subpopulations. In addition, it was verified the role of these same Yops on the activation of nuclear factor κB (NF- κB) and on the CD8 T cells cytotoxic activity. To this end, female BALB/c mice were infected intravenously with the wild type Y. pseudotuberculosis (WT), or with the mutant strains unable to secrete the Yops E, H and M (YopE-, YopH- and YopM-) or with the plasmid-cured strain (YpIII). On the 5th, 7th, 14th and 21st days post-infection (pi), the animals were sacrificed and the spleen cells were isolated from infected and uninfected mice (control group). The levels of colonization in the spleen and liver were determined by counting the number of colony-forming units. Both the phenotypic analysis of lymphocytes and the intracellular... (Complete abstract click electronic access below)

Corynebacterium pseudotuberculosis is a pathogenic bacterium which cause suppurative infection mainly in sheep, goats, horses, and other species. It plays a major role in the economy of sheep production worldwide. It produces a toxic phospholipase D which may be involved in pathogenesis. The relationship of physiologic differences between the two biovars of the organism to rates of growth and toxin production are not known. In this work, isolates of C.pseudotuberculosis were examined for relative ability to grow in brain heart infusion broth and a semidefined medium. The effects of carbohydrate source and the presence or absence of various ions were also examined. Results revealed no statistically significant differences between isolates or biovars in any characteristic studied, including growth rate, pH change during growth and PLD production. The semidefined medium provides a reasonable alternative to more complex media for further work in purification of PLD and study of its structure and function.

The objective of this thesis was to better understand the epidemiology of caseous lymphadenitis (CLA) and its economic effects on the Australian sheep industries. The work also sought to apply this knowledge to better understand how to control the disease in Australian sheep flocks. The occurrence of CLA in some other species, as a comparison to CLA in sheep, is described in the literature review. However a comprehensive review of research on CLA in all livestock species is not needed to meet the aims of the thesis. The research used serological tests developed in the 1970s and ‘80s to examine the epidemiology and costs of CLA in Western Australian sheep flocks. Epidemiological techniques were used to determine the main risk factors associated with high incidences of CLA. Controlled experimentation was used to better understand the spread of CLA within flocks. Prevalence data collected at abattoirs and survey techniques were employed to better understand how CLA was being controlled in Australian sheep flocks. It was shown that toxin and cell wall ELISA tests for CLA were useful tools to study the spread of CLA in sheep flocks. Using the serological tests it was estimated that 80 per cent of CLA spread occurs after the second and third shearings. This knowledge was used to design a risk factor study which identified two significant factors associated with high CLA spread, that is in dips and close contact after shearing. These tests also facilitated the study of new CLA infection on wool production. It was found that sheep produce approximately five per cent less clean wool in the year in which they are infected. The main routes for the spread of CLA were from sheep with lung abscesses to those with skin cuts and through dips, rather than from an environment contaminated with Corynebacterium pseudotuberculosis bacteria. A survey of sheep producers found that the prevalence of CLA had decreased from over 50% in the 1970s to approximately 20% in the late 1990s. It also found that current vaccines were being used inefficiently. Large increases in the efficiency of control can be achieved by approximately 70 per cent of producers by using commercially available vaccines and the recommended vaccination programs of two