A diarréia aguda é a segunda causa de morte em crianças < 5 anos em países emergentes. Anaeróbios estritos intestinais como B. fragilis enterotoxigênico e Clostridium difficile estão associados a este quadro infeccioso, assim como Escherichia coli diarreiogênica, Salmonella spp., Shigella spp. e Yersinia enterocolitica. Neste estudo, foi avaliada quantitativamente a presença de enteropatógenos diretamente das fezes. Amostras fecais de crianças com (110) e sem (150) diarréia foram analisadas. Todos os enteropatógenos avaliados foram detectados nas crianças com e sem diarréia, à exceção de Salmonella spp. e Y. enterocolitica que foram observados somente em fezes diarréicas. Os resultados sugerem uma relação bacteriana estreita e presentes em casos diarréicos ou não diarréicos. A presença de espécies EPEC/EHEC e de Shigella spp./EIEC mostraram valores estatisticamente significativos. / Diarrhea is a major cause of mortality and morbidity worldwide achieving children < 5 years old, and is second cause of death in emerging countries. Intestinal anaerobes like enterotoxigenic B. fragilis and Clostridium difficile are associated to acute diarrhea, as well as diarreiogenic Escherichia coli, Salmonella spp., Shigella spp. and Yersinia enterocolitica. In this study, it was evaluated quantitatively the presence of enteropathogens directly from fezes. Stools specimens from children with (110) and without (150) diarrhea were collected. All enteropathogens were detected in children with and without diarrhea except Salmonella spp. and Y. enterocolitica that were only observed in diarrheal fezes. This results suggest a tight bacterial relationship in both groups evaluated. The presence of EPEH/EHEC and Shigella spp./EIEC show statistically significant values.

The porcine proliferative enteropathies (PPE) are a group of diseases ranging from intestinal adenomatosis (PIA), a chronic condition causing reduced growth rates in post weaning pigs, to the often fatal proliferative haemorrhagic enteropathy (PHE), resulting in intestinal haemorrhage. PHE predominantly occurs in older and heavier pigs than the chronic disease PIA. This thesis examined whether the age when susceptible pigs are infected affects the clinical response to L.intracellularis infection. The characteristic pathologic lesion of PPE is the abnormal proliferation of crypt epithelial cells in the ileum and colon. Closely associated with this proliferation is the presence of an obligately intracellular bacterium, Lawsonia intracellularis. Characterisation of L.intracellularis was performed in in-vitro co-cultures of L.intracellularis extracted from PHE-affected mucosa. The efficacy of antimicrobials to inhibit the growth of L.intracellularis in-vitro was evaluated and compared with isolates cultured in the United Kingdom. The results were analysed with respect to medication strategies currently used to control PPE in piggeries. PPE occurs in virtually all piggery management systems, including newly developed systems that are aimed at improving the herd health, such as segregated early weaning and multiple site production. PPE is currently controlled in Australia with the routine addition of antimicrobials in pig feed, in particular olaquindox. Recommendations to reduce the use of feed-based antibiotics in Australia require the development of alternate strategies to control diseases such as PPE. Sequential outbreaks of PHE reported in minimal disease herds suggested that pigs could develop immunity to disease. An experimental model of L.intracellularis infection was developed in this thesis to demonstrate that immunity to re-infection with L.intracellularis could be developed. Infection was monitored by detection of faecal shedding of L.intracellularis and serum IgG antibodies against L.intracellularis. Two in-feed antimicrobial strategies were analysed in this thesis for their ability to induce the development of immunity to L.intracellularis, while avoiding clinical signs of disease. The first strategy evaluated the use of low levels of in-feed antimicrobials to allow subclinical infection and the development of immunity. The second strategy evaluated the use of high levels of in-feed antimicrobials to terminate infection two weeks after exposure to L.intracellularis. Gaining a greater understanding of how L.intracellularis infection is spread both within and between piggeries will enable the development of management strategies to control the spread of infection. This thesis examined the possibility that other species in contact with pigs and piggeries such as rats, mice and birds may transmit infection to pigs. The transmission of infection between pigs via the faecal/oral route was also examined, as was the survival and infectivity of L.intracellularis over time. Ultimately this thesis aimed to understand the pattern of L.intracellularis infection and the survival and transmission of L.intracellularis in order to develop effective control measures for PPE, especially in minimal disease herds.

The porcine proliferative enteropathies (PPE) are a group of diseases ranging from intestinal adenomatosis (PIA), a chronic condition causing reduced growth rates in post weaning pigs, to the often fatal proliferative haemorrhagic enteropathy (PHE), resulting in intestinal haemorrhage. PHE predominantly occurs in older and heavier pigs than the chronic disease PIA. This thesis examined whether the age when susceptible pigs are infected affects the clinical response to L.intracellularis infection. The characteristic pathologic lesion of PPE is the abnormal proliferation of crypt epithelial cells in the ileum and colon. Closely associated with this proliferation is the presence of an obligately intracellular bacterium, Lawsonia intracellularis. Characterisation of L.intracellularis was performed in in-vitro co-cultures of L.intracellularis extracted from PHE-affected mucosa. The efficacy of antimicrobials to inhibit the growth of L.intracellularis in-vitro was evaluated and compared with isolates cultured in the United Kingdom. The results were analysed with respect to medication strategies currently used to control PPE in piggeries. PPE occurs in virtually all piggery management systems, including newly developed systems that are aimed at improving the herd health, such as segregated early weaning and multiple site production. PPE is currently controlled in Australia with the routine addition of antimicrobials in pig feed, in particular olaquindox. Recommendations to reduce the use of feed-based antibiotics in Australia require the development of alternate strategies to control diseases such as PPE. Sequential outbreaks of PHE reported in minimal disease herds suggested that pigs could develop immunity to disease. An experimental model of L.intracellularis infection was developed in this thesis to demonstrate that immunity to re-infection with L.intracellularis could be developed. Infection was monitored by detection of faecal shedding of L.intracellularis and serum IgG antibodies against L.intracellularis. Two in-feed antimicrobial strategies were analysed in this thesis for their ability to induce the development of immunity to L.intracellularis, while avoiding clinical signs of disease. The first strategy evaluated the use of low levels of in-feed antimicrobials to allow subclinical infection and the development of immunity. The second strategy evaluated the use of high levels of in-feed antimicrobials to terminate infection two weeks after exposure to L.intracellularis. Gaining a greater understanding of how L.intracellularis infection is spread both within and between piggeries will enable the development of management strategies to control the spread of infection. This thesis examined the possibility that other species in contact with pigs and piggeries such as rats, mice and birds may transmit infection to pigs. The transmission of infection between pigs via the faecal/oral route was also examined, as was the survival and infectivity of L.intracellularis over time. Ultimately this thesis aimed to understand the pattern of L.intracellularis infection and the survival and transmission of L.intracellularis in order to develop effective control measures for PPE, especially in minimal disease herds.

A diarréia aguda é a segunda causa de morte em crianças < 5 anos em países emergentes. Anaeróbios estritos intestinais como B. fragilis enterotoxigênico e Clostridium difficile estão associados a este quadro infeccioso, assim como Escherichia coli diarreiogênica, Salmonella spp., Shigella spp. e Yersinia enterocolitica. Neste estudo, foi avaliada quantitativamente a presença de enteropatógenos diretamente das fezes. Amostras fecais de crianças com (110) e sem (150) diarréia foram analisadas. Todos os enteropatógenos avaliados foram detectados nas crianças com e sem diarréia, à exceção de Salmonella spp. e Y. enterocolitica que foram observados somente em fezes diarréicas. Os resultados sugerem uma relação bacteriana estreita e presentes em casos diarréicos ou não diarréicos. A presença de espécies EPEC/EHEC e de Shigella spp./EIEC mostraram valores estatisticamente significativos. / Diarrhea is a major cause of mortality and morbidity worldwide achieving children < 5 years old, and is second cause of death in emerging countries. Intestinal anaerobes like enterotoxigenic B. fragilis and Clostridium difficile are associated to acute diarrhea, as well as diarreiogenic Escherichia coli, Salmonella spp., Shigella spp. and Yersinia enterocolitica. In this study, it was evaluated quantitatively the presence of enteropathogens directly from fezes. Stools specimens from children with (110) and without (150) diarrhea were collected. All enteropathogens were detected in children with and without diarrhea except Salmonella spp. and Y. enterocolitica that were only observed in diarrheal fezes. This results suggest a tight bacterial relationship in both groups evaluated. The presence of EPEH/EHEC and Shigella spp./EIEC show statistically significant values.

Background: Idiopathic inflammatory bowel disease (IBD) and food-responsive diarrhea (FRD) are common categories of chronic inflammatory enteropathy (CIE) in dogs. The intestinal microbiota is considered a major contributing factor to the pathogenesis of IBD. Intestinal dysbiosis has been identified in dogs with IBD, but only little information is available on differences in the mucosal microbiota of dogs diagnosed with IBD and FRD. In humans, dyslipidemia has also been described in patients with IBD. However, studies on the lipid profile in dogs with IBD or FRD are currently lacking. Objectives: This is the first study to (1) compare the intestinal mucosal microbiota of dogs with IBD and FRD in the duodenum and the colon, (2) evaluate differences in the mucosal microbiota of each dog before and after the induction phase of treatment, and (3) evaluate the systemic phospholipid profile of dogs with IBD or FRD also both prior to and after the induction phase of treatment. Materials and Methods: Duodenal and colonic mucosal biopsies from 24 dogs with CIE (15 FRD, 9 IBD) and EDTA-plasma and whole blood from 32 dogs (16 FRD, 16 IBD) were retrieved from a former study on canine CIE. All client-owned dogs were prospectively enrolled in the study. All dogs received a standardized diagnostic work-up and treatment including a dietary trial. Dogs that responded to the elimination diet within 14 days were classified as FRD; the remaining dogs requiring additional immunosuppressant treatment were classified as IBD. Biopsy specimens of duodenum and colon were obtained endoscopically both before and after standard therapy. DNA was extracted from these biopsies and the intestinal mucosal microbiota of the duodenum and colon were evaluated by Illumina sequencing of the bacterial 16S rRNA gene. The phospholipids in whole blood and EDTA plasma, collected both before and after treatment, were analyzed by hydrophilic interaction liquid chromatography (HILIC). Differences in the composition were statistically assessed by alpha diversity indices, principal coordinate analysis, analysis of similarities (ANOSIM) and linear discriminant (LDA) analysis effect size (LEfSe) for the microbiota and by principal component analysis (PCA), analysis of variance (ANOVA) and random forest analysis for the phospholipid profile. Results: All differences in the microbial composition and phospholipid profile described below are statistically confirmed with significance set at p-value < 0.05 or LDA score > 2.0. No difference in the global bacterial composition was identified neither between the two disease groups of dogs nor with the treatment status. However, abundances of several bacterial taxa varied between disease groups and also with the treatment status. When comparing disease groups, an unclassified genus of Neisseriaceae was more abundant in the duodenum in the IBD group, whereas Bilophila spp. occurred more frequently in the duodenum of the FRD group. Comparison before and after treatment revealed Enterococcus spp., Corynebacterium spp. and Proteobacteria to be enriched in the duodenum of FRD dogs before treatment. Bacteroides spp. was more abundant in the colon in the FRD group post-treatment. In the IBD dogs, Unclassified_Neisseriaceae was more abundant in the duodenum and mainly Proteobacteria (Burkholderia spp., Citrobacter spp.) in the colon prior to treatment. Bacteroides spp. was significantly more abundant in the colon after treatment. The phospholipidome differed dependent on the type of specimen (whole blood vs. plasma). In addition, treatment and disease severity presented the most significant factors determining the variance of the phospholipid profile. An increase in lysolipids and a significant shift of the phosphatidylcholine (PC) species from PC 38:4 (18:0 / 20:4) to mainly lysophosphatidylcholine 18:0 was observed after treatment. Conclusions: Some differences in individual bacterial taxa were identified both between disease groups of dogs and with regard to treatment status. The role of these bacterial groups in the pathogenesis of IBD and FRD is still unknown. However, Bacteroides spp. might be of importance, and this species could potentially serve as marker of treatment response. Furthermore, significant variances were identified in the phospholipid profiles of dogs with IBD and FRD, which were particularly associated with the type of specimen used, disease severity, and treatment status. These alterations in the phospholipid profile could potentially aid in monitoring the response to treatment. Subsequently, specific modulation of the intestinal microbiota and the phospholipid profile might also present novel therapeutic strategies in dogs with CIE.

Escherichia coli enteroinvasora (EIEC) e Shigella sp causam disenteria bacilar que é caracterizada pela invasão e destruição da mucosa do cólon humano. Amostras de EIEC possuem características bioquímicas, genéticas patogênicas semelhantes às espécies de Shigella, porém a doença causada por EIEC se apresenta numa forma mais branda e autolimitante. As células do epitélio intestinal participam ativamente da imunidade da mucosa, expressando e secretando uma série de mediad ores inflamatórios como citocinas, quimiocinas, moléculas de adesão e óxido nítrico. Para melhor entendimento da patogênese de EIEC, estudamos a resposta inflamatória modulada por este microrganismo em células epiteliais intestinais da linhagem Caco-2, comparando-a com Shigella flexneri. Células Caco-2 foram infectadas com EIEC ou S. flexneri por diferentes intervalos de tempo, para posterior analise da capacidade de invasão e disseminação bacterianas (UFC, PLAQUE ASSA Y), indução de morte celular (FACS), analise relativa de genes envolvidos no reconhecimento bacteriano e na resposta inflamatória (RT-PCR, RPA), dosagem de citocinas e quimiocinas pró-inflamatórias (ELISA) e óxido nítrico (GRIESS). Neste trabalho foi possível observar que: (i) a capacidade de disseminação e (ii) a indução da morte celular em células Caco-2 foi significativamente maior na infecção por S. flexneri do que EIEC; (iii) há diferenças em relação à expressão relativa de genes das células Caco-2 envolvidos no reconhecimento das duas cepas bacterianas. Foi evidenciado o papel essencial dos receptores intracelulares no reconhecimento bacteriano das células Caco-2, sendo a expressão relativa do mRNA do receptor intracelular Nod1 foi maior para EIEC quando comparado com S. flexneri; (iv) há diferenças significativas na cinética de produção de NO pelas células Caco¬2 infectadas, em que EIEC induziu mais precocemente a produção de NO quando comparado com S. flexneri. Estes dados sinalizam que as células epiteliais intestinais reconhecem e respondem de forma diferente frente a essas duas espécies bacterianas, apresentando uma resposta inflamatória mais eficiente no controle da infecção induzida por EIEC. / Escherichia coli enteroinvasive (EIEC) and Shigella sp cause bacillary dysentery which is characterized by the invasion and destruction of the human colon mucosa. Samples of EIEC have characteristics biochemical, genetic and pathogenic similar to those of Shigella species, however the disease caused by EIEC is more lenient. The cells of the intestinal epithelium actively participate in the mucosal immunity by expression and production of several inflammatory mediators such as cytokines, chemokines, adhesion molecules and nitric oxide. For better understanding of the EIEC pathogenesis, we studied the inflammatory response modulated by this microorganism in intestinal epithelial cells Caco-2, comparing it with Shigella flexneri. Caco-2 cells were infected with EIEC or S. flexneri during different intervals of time and analyzed the invasiveness and spread bacteria capacity (CFU, PLAQUE ASSAY), induction of cell death (FACS), analysis of genes involved in the recognition of bacterial and inflammatory response (RT-PCR, RPA), production of pro-inflammatory cytokines and chemokines (ELISA) and nitric oxide (NO) (GRIESS). In this work was possible to observe that: (i) the ability to spread and (ii) the induction of cell death in Caco-2 cells was significantly higher in S. flexneri infection than EIEC, (iii) there are differences regarding the relative expression of genes of Caco-2 cells involved in the recognition of two bacterial strains. It was highlighted the essential role of intracellular receptors in recognition of bacterial by Caco-2 cells, and the expression of mRNA of the intracellular receptor Nod 1 was higher for EIEC when compared with S. flexneri, (iv) there are significant differences in the kinetics of NO production by Caco-2 infected cells, EIEC induced a early NO production when compared with S. flexneri. These data indicate that the intestinal epithelial cells recognize and respond in a different way to these bacterial species and induce an inflammatory response more efficient in control of the infection induced by EIEC.

Escherichia coli enteroinvasora (EIEC) e Shigella sp causam disenteria bacilar que é caracterizada pela invasão e destruição da mucosa do cólon humano. Amostras de EIEC possuem características bioquímicas, genéticas patogênicas semelhantes às espécies de Shigella, porém a doença causada por EIEC se apresenta numa forma mais branda e autolimitante. As células do epitélio intestinal participam ativamente da imunidade da mucosa, expressando e secretando uma série de mediad ores inflamatórios como citocinas, quimiocinas, moléculas de adesão e óxido nítrico. Para melhor entendimento da patogênese de EIEC, estudamos a resposta inflamatória modulada por este microrganismo em células epiteliais intestinais da linhagem Caco-2, comparando-a com Shigella flexneri. Células Caco-2 foram infectadas com EIEC ou S. flexneri por diferentes intervalos de tempo, para posterior analise da capacidade de invasão e disseminação bacterianas (UFC, PLAQUE ASSA Y), indução de morte celular (FACS), analise relativa de genes envolvidos no reconhecimento bacteriano e na resposta inflamatória (RT-PCR, RPA), dosagem de citocinas e quimiocinas pró-inflamatórias (ELISA) e óxido nítrico (GRIESS). Neste trabalho foi possível observar que: (i) a capacidade de disseminação e (ii) a indução da morte celular em células Caco-2 foi significativamente maior na infecção por S. flexneri do que EIEC; (iii) há diferenças em relação à expressão relativa de genes das células Caco-2 envolvidos no reconhecimento das duas cepas bacterianas. Foi evidenciado o papel essencial dos receptores intracelulares no reconhecimento bacteriano das células Caco-2, sendo a expressão relativa do mRNA do receptor intracelular Nod1 foi maior para EIEC quando comparado com S. flexneri; (iv) há diferenças significativas na cinética de produção de NO pelas células Caco¬2 infectadas, em que EIEC induziu mais precocemente a produção de NO quando comparado com S. flexneri. Estes dados sinalizam que as células epiteliais intestinais reconhecem e respondem de forma diferente frente a essas duas espécies bacterianas, apresentando uma resposta inflamatória mais eficiente no controle da infecção induzida por EIEC. / Escherichia coli enteroinvasive (EIEC) and Shigella sp cause bacillary dysentery which is characterized by the invasion and destruction of the human colon mucosa. Samples of EIEC have characteristics biochemical, genetic and pathogenic similar to those of Shigella species, however the disease caused by EIEC is more lenient. The cells of the intestinal epithelium actively participate in the mucosal immunity by expression and production of several inflammatory mediators such as cytokines, chemokines, adhesion molecules and nitric oxide. For better understanding of the EIEC pathogenesis, we studied the inflammatory response modulated by this microorganism in intestinal epithelial cells Caco-2, comparing it with Shigella flexneri. Caco-2 cells were infected with EIEC or S. flexneri during different intervals of time and analyzed the invasiveness and spread bacteria capacity (CFU, PLAQUE ASSAY), induction of cell death (FACS), analysis of genes involved in the recognition of bacterial and inflammatory response (RT-PCR, RPA), production of pro-inflammatory cytokines and chemokines (ELISA) and nitric oxide (NO) (GRIESS). In this work was possible to observe that: (i) the ability to spread and (ii) the induction of cell death in Caco-2 cells was significantly higher in S. flexneri infection than EIEC, (iii) there are differences regarding the relative expression of genes of Caco-2 cells involved in the recognition of two bacterial strains. It was highlighted the essential role of intracellular receptors in recognition of bacterial by Caco-2 cells, and the expression of mRNA of the intracellular receptor Nod 1 was higher for EIEC when compared with S. flexneri, (iv) there are significant differences in the kinetics of NO production by Caco-2 infected cells, EIEC induced a early NO production when compared with S. flexneri. These data indicate that the intestinal epithelial cells recognize and respond in a different way to these bacterial species and induce an inflammatory response more efficient in control of the infection induced by EIEC.