Tierärztliche Hochsch., Diss., 2005--Hannover.

Tierärztliche Hochsch., Diss., 2005--Hannover.

Rhodococcus equi é uma importante causa de broncopneumonia em potros com menos de 6 meses de idade, sendo responsável por 3% das mortes de eqüinos no mundo. É um microrganismo intracelular capaz de sobreviver e multiplicar no interior de macrófagos. Apresenta 3 níveis de virulência de acordo com os diferentes antígenos expressos em sua superfície. Cepas virulentas apresentam um plasmídeo que codifica para a proteína de superfície VapA e são isoladas principalmente de potros com pneumonia e de alguns pacientes humanos. Cepas com virulência intermediária expressam a proteína VapB e predominam em suínos e humanos com AIDS. Cepas avirulentas não expressam antígeno de superfície e são encontradas principalmente no ambiente e em pacientes humanos. Um dos fatores responsáveis pela ampla distribuição da enfermidade em potros, é a imaturidade do sistema imunológico dos animais acometidos pela infecção, que pode tornar-se endêmica em alguns criatórios. Para humanos, as formas de infecção são ainda desconhecidas, mas o contato com eqüinos é relatado em um terço das infecções. Devido à importância clínica da doença, são necessários métodos diagnósticos que promovam sua identificação precoce, facilitando e aumentando as chances de sucesso com o tratamento. Os métodos mais utilizados atualmente são o cultivo microbiológico da bactéria, testes sorológicos para detecção de anticorpos no soro dos animais e técnicas de PCR que detectam a região 16S do rDNA e o fragmento do gene vapA do microrganismo. O objetivo desse trabalho foi utilizar uma técnica de PCR multiplex para detectar simultaneamente os fragmentos dos genes vapA e 16S do rDNA, e gerar um método rápido, específico e sensível para o diagnóstico e caracterização molecular de cepas de R. equi provenientes de eqüinos e seus ambientes. Foram utilizados 118 isolados, sendo 74 amostras de fezes de eqüinos (41 de adultos e 33 de potros), 21 do solo, 10 das instalações utilizando swabs e 3 de outros animais domésticos. Isolados clínicos (10) foram cultivados do pulmão de potros com pneumonia causada por R. equi. Todas as cepas testadas foram confirmadas pela amplificação do gene 16S rDNA, sendo que 16 destes (10 de x potros doentes e 6 de seus ambientes) também foram positivos na amplificação do gene vapA. Quatro dos isolados ambientais que mostraram amplificação do gene vapA foram de haras endêmicos para a doença. A análise desses dados mostra o grande potencial da técnica de PCR multiplex para caracterização molecular de isolados de R. equi. / Rhodococcus equi is an important cause of pyogranulomatous pneumonia in 1-6- month-old foals, being responsible by 3% horse death around the world. It is an intracellular microorganism able to survive and to multiply itself in the macrophages. Three virulence levels have been identified in R. equi, by the presence of virulence associated antigens on the bacteria surface. Virulent strains have a plasmid encoding VapA protein and are isolated from diseased foals and some human patients. Intermediate virulent strains show VapB protein and are commonly founded in swines and humans with AIDS isolates. Avirulent strains don’t show virulence antigens and are founded in environmental samples and human. The immature immune system is the major cause of the susceptibility of foals for the R.equi pneumonia. To humans, the infection routes are unknown yet, but the contact with horses is related with one third of human infections. Due the clinical importance of the disease, diagnostics methods for early identification in animals are necessary, increasing the chances for treatment. The more common diagnostic methods are microbiologic culture, serologic tests and PCR techniques for 16S rDNA and vapA detection. The mainly purpose of this study was apply a multiplex PCR for simultaneous detection and characterization of 16S rDNA and vapA gene fragments in R. equi. Were analyzed 118 R. equi isolates, being 74 eqüine fecal isolates (41 from horses and 33 from foals), 21 from soil, 10 from breed stuffs and 3 from other domestic animals. Ten clinical isolates were cultured from lungs of diseased foals. All 118 isolates characterized as R. equi, were confirmed by 16S rDNA, being 16 isolates positive to vapA gene PCR amplification (10 from diseased foals and six from horse environment). Four environment R. equi isolates positive to both 16S rDNA e vapA gene amplification was from an endemic horse breeding farm. The results show the great potential of multiplex PCR to molecular characterization of R. equi isolates.

Made available in DSpace on 2014-08-13T14:50:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-27Bitstream added on 2014-08-13T17:59:58Z : No. of bitstreams: 1 000768326.pdf: 417220 bytes, checksum: f010ef190b0548a2bbadaa27034a0255 (MD5) / 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 / 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

Rhodococcus equi é uma importante causa de broncopneumonia em potros com menos de 6 meses de idade, sendo responsável por 3% das mortes de eqüinos no mundo. É um microrganismo intracelular capaz de sobreviver e multiplicar no interior de macrófagos. Apresenta 3 níveis de virulência de acordo com os diferentes antígenos expressos em sua superfície. Cepas virulentas apresentam um plasmídeo que codifica para a proteína de superfície VapA e são isoladas principalmente de potros com pneumonia e de alguns pacientes humanos. Cepas com virulência intermediária expressam a proteína VapB e predominam em suínos e humanos com AIDS. Cepas avirulentas não expressam antígeno de superfície e são encontradas principalmente no ambiente e em pacientes humanos. Um dos fatores responsáveis pela ampla distribuição da enfermidade em potros, é a imaturidade do sistema imunológico dos animais acometidos pela infecção, que pode tornar-se endêmica em alguns criatórios. Para humanos, as formas de infecção são ainda desconhecidas, mas o contato com eqüinos é relatado em um terço das infecções. Devido à importância clínica da doença, são necessários métodos diagnósticos que promovam sua identificação precoce, facilitando e aumentando as chances de sucesso com o tratamento. Os métodos mais utilizados atualmente são o cultivo microbiológico da bactéria, testes sorológicos para detecção de anticorpos no soro dos animais e técnicas de PCR que detectam a região 16S do rDNA e o fragmento do gene vapA do microrganismo. O objetivo desse trabalho foi utilizar uma técnica de PCR multiplex para detectar simultaneamente os fragmentos dos genes vapA e 16S do rDNA, e gerar um método rápido, específico e sensível para o diagnóstico e caracterização molecular de cepas de R. equi provenientes de eqüinos e seus ambientes. Foram utilizados 118 isolados, sendo 74 amostras de fezes de eqüinos (41 de adultos e 33 de potros), 21 do solo, 10 das instalações utilizando swabs e 3 de outros animais domésticos. Isolados clínicos (10) foram cultivados do pulmão de potros com pneumonia causada por R. equi. Todas as cepas testadas foram confirmadas pela amplificação do gene 16S rDNA, sendo que 16 destes (10 de x potros doentes e 6 de seus ambientes) também foram positivos na amplificação do gene vapA. Quatro dos isolados ambientais que mostraram amplificação do gene vapA foram de haras endêmicos para a doença. A análise desses dados mostra o grande potencial da técnica de PCR multiplex para caracterização molecular de isolados de R. equi. / Rhodococcus equi is an important cause of pyogranulomatous pneumonia in 1-6- month-old foals, being responsible by 3% horse death around the world. It is an intracellular microorganism able to survive and to multiply itself in the macrophages. Three virulence levels have been identified in R. equi, by the presence of virulence associated antigens on the bacteria surface. Virulent strains have a plasmid encoding VapA protein and are isolated from diseased foals and some human patients. Intermediate virulent strains show VapB protein and are commonly founded in swines and humans with AIDS isolates. Avirulent strains don’t show virulence antigens and are founded in environmental samples and human. The immature immune system is the major cause of the susceptibility of foals for the R.equi pneumonia. To humans, the infection routes are unknown yet, but the contact with horses is related with one third of human infections. Due the clinical importance of the disease, diagnostics methods for early identification in animals are necessary, increasing the chances for treatment. The more common diagnostic methods are microbiologic culture, serologic tests and PCR techniques for 16S rDNA and vapA detection. The mainly purpose of this study was apply a multiplex PCR for simultaneous detection and characterization of 16S rDNA and vapA gene fragments in R. equi. Were analyzed 118 R. equi isolates, being 74 eqüine fecal isolates (41 from horses and 33 from foals), 21 from soil, 10 from breed stuffs and 3 from other domestic animals. Ten clinical isolates were cultured from lungs of diseased foals. All 118 isolates characterized as R. equi, were confirmed by 16S rDNA, being 16 isolates positive to vapA gene PCR amplification (10 from diseased foals and six from horse environment). Four environment R. equi isolates positive to both 16S rDNA e vapA gene amplification was from an endemic horse breeding farm. The results show the great potential of multiplex PCR to molecular characterization of R. equi isolates.

The present study investigates Acanthamoeba-bacteria interaction and how this relation can influence human health aiming at the influence of bacteria on Acanthamoeba in terms of their isolation and diversity, and the effect of Acanthamoeba on bacteria focusing on two emerging human bacterial pathogens Arcobacter butzleri and Rhodococcus equi. To first objective was investigated by the test question “can the presence of a particular type of bacteria play role in the diversity of Acanthamoeba by masking and/or favouring certain genotypes of Acanthamoeba?” To answer this, two different bacteria the Gram+ve Enterococcus the Gram-ve Arcobacter were used as food source for isolation of Acanthamoeba from 102 soil samples while E. coli was used as control. It was found that the presence of different bacteria could affect the isolation of genotypes specially the subgroups and subtypes of Acanthamoeba as manifested by greater diversity of 18S rRNA sequences of Acanthamoeba isolated from environmental samples on Arcobacter (Arc) and Enterococcus (Ent) than those isolated on E. coli (Eco). The Eco isolates consisted of only T4 > T11=T13 compared to Ent isolates with T4 > T16 > T13/16 and the Arc isolates which comprised of T4 > T2 > T2/6 = T13 > T13/16. The T13/16 were the intermediate sequence types with no match to any T types. There were also considerable differences among the T4 subgroups; the Eco isolates consisted of T4-A > T4-B > T4-N > T4- E > T4-D > T4-C while Ent isolates comprised of T4-A > T4-C=T4-D=T4-E=T4-N > T4-B and the Arc isolates had only T4-E > T4-A > T4-B > T4-N. In both Eco and Ent isolates 11 subtypes were recovered with T4-36 being the most abundant, however, in Arc isolates eight subtypes were recovered with T4-12 as the most abundant. The non-Eco isolates were also different in their bacterial endosymiotic profile from Eco isolates with Arc isolates having the greatest proportion of bacterial endosymbionts (15.7%) as compared to 7.8% of Eco and 12.9% of Ent isolates. Together these results indicate a prominent role of prey bacteria on favouring certain genotypes and thus compelling consideration for use of different types of bacteria for isolation of Acanthamoeba to help surface the masked populations as well for more realistic prevalence that will help in better designing of prevention and control strategies. The influence of Acanthamoeba on bacteria was investigated for A. butzleri and R. equi both of which appeared to exploit the former as an environmental reservoir and for modulation of their pathogenic potential. A. butzleri which are closely related to Campylobacter, appeared to have a smooth interaction with Acanthamoeba. They were shown to be easily located through chemotaxis, readily attached and internalized using monosaccharide receptors and a complex phagocytic process, and could survive/proliferate in Acanthamoeba by defying the intra-vacuolar killing processes. Intracellular survival in Acanthamoeba did play a role in promoting the pathogenicity of these bacteria enabling them to survive more than three times longer. Co-culturing of the two organisms also seemed to benefit the bacteria but not Acanthamoeba. A. butzleri were found to be able to sense the environmental changes and thus modulate their virulence, a feature that together with selection pressure for intracellular survival in Acanthamoeba can cause rapid adaptation to intra-amoebal environment and enhance the pathogenic potential of these bacteria for humans and animals. Exploitation of Acanthamoeba for survival was also found to be exhibited by the Mycobacterium-resembling Gram+ve R. equi by utilizing similar strategies for survival/proliferation as used for macrophages, which involved the definite presence of virulence plasmid and its activation at higher temperatures. Moreover, similar genes (vapA, vapC and vapF) were found to play role in intracellular survival in both the macrophages and amoeba cells. The intra-amoebal survival/proliferation capabilities of A. butzleri and R. equi appear to support the notion that free living protists like Acanthamoeba act as environmental reservoirs/virulence trait selectors and are strong candidates for the “missing link” between the ecology and pathology of these emerging pathogenic pathogens. Overall, the observations made in this study explore the vital role of Acanthamoeba-bacteria interaction not only mutually on each other but as a consequence the impact on human health either as a result of masked genotypes in clinical diagnosis of Acanthamoeba or due to environmental reservoir role of Acanthamoeba in selecting virulence traits of bacteria, can pose serious challenges leaving ample opportunities for more emerging bacterial pathogens. These observations call for revising the protocols for Acanthamoeba prevalence, eradication and control strategies.

Rhodococcus equi é uma importante causa de broncopneumonia em potros com menos de 6 meses de idade, sendo responsável por 3% das mortes de eqüinos no mundo. É um microrganismo intracelular capaz de sobreviver e multiplicar no interior de macrófagos. Apresenta 3 níveis de virulência de acordo com os diferentes antígenos expressos em sua superfície. Cepas virulentas apresentam um plasmídeo que codifica para a proteína de superfície VapA e são isoladas principalmente de potros com pneumonia e de alguns pacientes humanos. Cepas com virulência intermediária expressam a proteína VapB e predominam em suínos e humanos com AIDS. Cepas avirulentas não expressam antígeno de superfície e são encontradas principalmente no ambiente e em pacientes humanos. Um dos fatores responsáveis pela ampla distribuição da enfermidade em potros, é a imaturidade do sistema imunológico dos animais acometidos pela infecção, que pode tornar-se endêmica em alguns criatórios. Para humanos, as formas de infecção são ainda desconhecidas, mas o contato com eqüinos é relatado em um terço das infecções. Devido à importância clínica da doença, são necessários métodos diagnósticos que promovam sua identificação precoce, facilitando e aumentando as chances de sucesso com o tratamento. Os métodos mais utilizados atualmente são o cultivo microbiológico da bactéria, testes sorológicos para detecção de anticorpos no soro dos animais e técnicas de PCR que detectam a região 16S do rDNA e o fragmento do gene vapA do microrganismo. O objetivo desse trabalho foi utilizar uma técnica de PCR multiplex para detectar simultaneamente os fragmentos dos genes vapA e 16S do rDNA, e gerar um método rápido, específico e sensível para o diagnóstico e caracterização molecular de cepas de R. equi provenientes de eqüinos e seus ambientes. Foram utilizados 118 isolados, sendo 74 amostras de fezes de eqüinos (41 de adultos e 33 de potros), 21 do solo, 10 das instalações utilizando swabs e 3 de outros animais domésticos. Isolados clínicos (10) foram cultivados do pulmão de potros com pneumonia causada por R. equi. Todas as cepas testadas foram confirmadas pela amplificação do gene 16S rDNA, sendo que 16 destes (10 de x potros doentes e 6 de seus ambientes) também foram positivos na amplificação do gene vapA. Quatro dos isolados ambientais que mostraram amplificação do gene vapA foram de haras endêmicos para a doença. A análise desses dados mostra o grande potencial da técnica de PCR multiplex para caracterização molecular de isolados de R. equi. / Rhodococcus equi is an important cause of pyogranulomatous pneumonia in 1-6- month-old foals, being responsible by 3% horse death around the world. It is an intracellular microorganism able to survive and to multiply itself in the macrophages. Three virulence levels have been identified in R. equi, by the presence of virulence associated antigens on the bacteria surface. Virulent strains have a plasmid encoding VapA protein and are isolated from diseased foals and some human patients. Intermediate virulent strains show VapB protein and are commonly founded in swines and humans with AIDS isolates. Avirulent strains don’t show virulence antigens and are founded in environmental samples and human. The immature immune system is the major cause of the susceptibility of foals for the R.equi pneumonia. To humans, the infection routes are unknown yet, but the contact with horses is related with one third of human infections. Due the clinical importance of the disease, diagnostics methods for early identification in animals are necessary, increasing the chances for treatment. The more common diagnostic methods are microbiologic culture, serologic tests and PCR techniques for 16S rDNA and vapA detection. The mainly purpose of this study was apply a multiplex PCR for simultaneous detection and characterization of 16S rDNA and vapA gene fragments in R. equi. Were analyzed 118 R. equi isolates, being 74 eqüine fecal isolates (41 from horses and 33 from foals), 21 from soil, 10 from breed stuffs and 3 from other domestic animals. Ten clinical isolates were cultured from lungs of diseased foals. All 118 isolates characterized as R. equi, were confirmed by 16S rDNA, being 16 isolates positive to vapA gene PCR amplification (10 from diseased foals and six from horse environment). Four environment R. equi isolates positive to both 16S rDNA e vapA gene amplification was from an endemic horse breeding farm. The results show the great potential of multiplex PCR to molecular characterization of R. equi isolates.

The objective of this study was to determine if R. equi IgG purified from commmercially available hyperimmune R. equi plasma and delivered to the lungs of adult ponies would cause a local inflammatory response and if increases in total and R. equi specific IgG occurred post administration. IgG was purified and concentrated from plasma via protein G affinity chromatography. A cross over study was performed. Eight healthy adult ponies were randomly assigned to two groups of four; each pony acted as its own control. Either the IgG product or 0.9% Saline was delivered via a vibrating mesh nebulizer during the first treatment phase. During the second treatment phase ponies recieved the oppostie treatment. A 4 week washout period was allowed between phases. Bronchoalveolar fluid was recovered using a low volume endoscopic technique prior to aerosolization (time 0), and at 1 hr, 6 hrs, and 24 hours post administration. The BAL fluid total IgG concentration and R. equi specific IgG titers were determined via ELISA and cytologic analysis was performed. No clinically significant local inflammatory response was identified in response to IgG treatment. While total IgG concentrations were increased at T1 compared to T0, no significant effects of time were found (P=0.19). However, overall significantly higher concentrations of total IgG were found after administration of saline when compared to IgG administration (P=0.023).  While the R. equi specific titer increased at T1 after IgG administration, no significant difference was identified between treatment or time (P=0.261). Overall the individual response to IgG was variable. It is possible that the protein rich IgG acted as a relatively hypertonic solution and caused fluid influx from the pulmonary parenchyma after treatment thereby diluting the total IgG present when compared to saline administration. This conclusion cannot be verified as BAL dilution correction was not performed. However, it is unknown what titer or level of increased IgG is nessecary to assist with prevention of disease. Future research should focus on the effect of R. equi specific IgG on pulmonary cells to determine if administration of local R. equi specific IgG would alter intrapulmonary immune responses to R. equi. / Master of Science

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.

A infecção por Rhodococcus equi é a causa mais comum de pneumonia em potros durante os primeiros 6 meses de idade e as medidas utilizadas no controle da infecção são freqüentemente ineficientes. Os protocolos de tratamento são longos, de alto custo e nem sempre eficazes. A prevenção da rodococose através da imunização de potros com vacina eficaz e segura tem sido um desafio. Estudos recentes se baseiam na utilização da proteína A associada a virulência (VapA), que tem sido descrita como importante indutora da imunidade contra R. equi. Este estudo avaliou a resposta clínica e imunitária desenvolvida por potros imunizados com cepa atenuada de S. enterica Typhimurium capaz de expressar o antígeno VapA (grupo teste) ou apenas S. enterica Typhimurium sem o gene vapA (grupo controle), antes e após o desafio experimental. Duas etapas experimentais foram estabelecidas de acordo com a via de imunização: intranasal ou oral em até 12 horas após o nascimento e aos 14 dias de idade. Os animais do grupo teste e do grupo controle foram desafiados experimentalmente com cepa virulenta de R. equi 28 dias após o nascimento. Para acompanhar o desenvolvimento dos sinais clínicos, foram realizados exames clínicos, hemogramas e exames complementares de imagem. O padrão de resposta imune desenvolvida foi avaliada através da dosagem de munoglobulinas, mensuração da expressão de citocinas, ensaio de proliferação de linfócitos, isolamento de R. equi e perfil citológico do LTB. As manifestações clínicas foram observadas de maneira menos intensa nos animais do grupo teste da segunda etapa experimental e óbitos decorrentes da rodococose ocorreram apenas nos animais do grupo controle (2/3). Os animais do grupo teste apresentaram uma resposta de IgGb mais intensa quando comparada aos animais do grupo controle, porém não foi observado diferença estatística. A linfoproliferação e a expressão de citocinas de padrão Th1 foram superiores nos animais do grupo teste. Em contraste, os animais do grupo controle apresentaram uma resposta caracterizada pela maior expressão da citocina IL-4. A produção de IgA local foi mais significativa nos animais imunizados com Salmonella carreando vapA. Não foram observados efeitos tóxicos graves resultantes da imunização em nenhuma das etapas experimentais. A vacinação de potros neonatos com S. enterica Typhimurium expressando VapA foi considerada segura, capaz de modular eficientemente a resposta imune e aparentemente capaz de proteger contra a infecção experimental com R. equi. / Rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. Treatment protocols are long, expensive and do not always produce good results. Rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. Recent studies are based on the use of the virulence associated protein A (vapA) which has been described as an important inducer of immunity against R. equi. The present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of S. enterica Typhimurium expressing VapA antigen (test group) or S. enterica Typhimurium without the vapA gene (control group), previous to and following experimental challenge. Two experimental phases were established according to the immunization route: intranasal or oral vaccination up to 12 hrs following birth and at 14 days of age. The experimental and control groups were challenge on day 28 with a virulent stain of R. equi. Clinical examination, complete hemograms and image complementary exams were used to evaluate the development of clinical signs. Immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation essays, isolation of R. equi and cytological profiles of TBW. Clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group (2/3) and was due to R. equi pneumonia. The test group produced a more intense IgGb response when compared to controls however no statistical difference was observed. Lymphoproliferation and Th1 cytokine expression were higher in the test group. In contrast, controls produced an IL-4 response. Local IgA was significantly higher in animals immunized with Salmonella carrying vapA. Immunization protocols produced no severe toxic effect. The vaccination of neonatal foals with S. enterica Typhimurium expressing VapA was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental R.equi infection.