Os trofozoítos de Entamoeba histolytica após se evadirem de uma complexa resposta imune ao nível da mucosa intestinal, invadem o epitélio, podendo disseminar-se pela circulação portal, atingindo o fígado e outros órgãos. A amebíase hepática é a forma mais comum de amebíase extraintestinal e corresponde a menos de 1% dos casos clínicos de amebíase. A modulação das respostas imunológicas frente à administração de substâncias farmacologicamente ativas em modelos experimentais infectados por diferentes patógenos tem contribuído de maneira importante nas investigações de terapias alternativas para colaborar no tratamento das diferentes doenças parasitárias. A melatonina (N-acetil-5- metoxitriptamina) é uma indolamina cuja síntese ocorre na glândula pineal e em vários locais extrapineais, como, as células do sistema imune, retina, medula óssea, pele, leucócitos, e trato gastrointestinal. É uma molécula pleiotrópica que possui atividades imunoestimulatórias e efeitos pró e anti-inflamatórios. O presente estudo teve como objetivo avaliar um possível efeito imunomodulatório subseqüente à administração oral de melatonina em ratos da linhagem Wistar machos infectados no fígado pela cepa HM1-IMSS de Entamoeba histolytica na fase aguda da amebíase hepática experimental. Foram utilizados vários parâmetros como: dosagens das citocinas IFN-?, IL-4, análise por citometria de fluxo das populações celulares TCD3+, TCD4+, TCD8+, CD45+, CD161+ e produção de óxido nítrico. Através dos resultados obtidos, podemos concluir que a melatonina exerceu seus efeitos imunomodulatórios, ora induzindo uma reposta imune mais eficaz de forma a minimizar os efeitos patogênicos da infecção ao mesmo tempo em que exerceu um papel anti-inflamatório na tentativa de manter a homeostase imunológica do hospedeiro, evitando uma exacerbação dessas respostas que certamente iriam influenciar o grau de patogenicidade desta parasitose. / The trophozoites of Entamoeba histolytica after evade a complex immune response at the intestinal mucosal level, invade the epithelial layer and may spread into the portal circulation reaching the liver and other organs. The amoebic liver abscess (ALA) is the most common form of the extraintestinal amebiasis and is less than 1% of clinical cases of human amoebiasis. The modulation of immune responses by the administration of pharmacological active substances in experimental models infected by different pathogens has contributed in investigations of alternative therapies in order to aid the treatment of various parasitic diseases. Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine whose synthesis occurs in the pineal gland and in several extra-pineal locations such as the immune system cells, retina, bone marrow, skin, leukocytes, and gastrointestinal tract. This molecule has pleiotropic effects with immunoenhancing actions besides pro and anti inflammatory effects. The present study aimed to evaluate a possible immunomodulatory effect following the oral administration of melatonin in male Wistar rats infected with the strain HM1-IMSS of E. histolytica during the acute phase of experimental amebic liver abscess. Several immune parameters were evaluated such as of cytokines IFN-?, IL-4, flow cytometry analysis of cell populations TCD3+, TCD4+, TCD8+, CD45+, CD161+, and nitric oxide production. According to our results, we can conclude that melatonin exerted its immunomodulatory actions, once triggering a more accurate host´s immune response reducing the pathogenic effects from the infection and at the same time, exerting an anti-inflammatory role in the attempt to control host´s immune homeostasis, avoiding an exacerbation of these responses that would certainly influence the degree of pathogenicity of this parasitosis.

Made available in DSpace on 2012-09-06T01:11:29Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 913.pdf: 7593708 bytes, checksum: 2f34195bc20a030f18ae9b5e7f19b8e1 (MD5) Previous issue date: 2006 / Sedentarização, poluição do peridomicílio e precariedade de saneamento, são fatores que contribuem para a contaminação ambiental e intensificação da transmissão das enteroparasitoses entre populações indígenas no Brasil. Este inquérito parasitológico foi realizado na etnia Suruí, situada no Estado de Rondônia, Região Amazônica brasileira, em fevereiro e março de 2005. Ênfase foi dado ao diagnóstico da infecção pelo protozoário Entamoeba histolytica, agente etiológico da amebíase, importante causa de morbi-mortalidade por diarréia no mundo. Procurou-se distinguir infecções por Entamoeba histolytica e Entamoeba dispar através de métodos moleculares. (...) Dentre os possíveis determinantes desta transição estão o uso de tratamento anti-helmíntico em massa e transformações nas habitações dos Suruí. Contudo, outros fatores devem ser analisados, uma vez que o emprego irregular do tratamento em massa nesta comunidade não haveria de ser suficiente para manter tamanho controle das helmintíases. A ausência de infecções por Ascaris lumbricoides e Trichuris trichiura indica ainda uma nova situação, onde outros parasitos tendem a ocupar o lugar destes dois agentes comuns, surgindo como infecções emergentes.

Made available in DSpace on 2014-06-12T18:31:29Z (GMT). No. of bitstreams: 2 arquivo8113_1.pdf: 1534236 bytes, checksum: c8ba77e64e07b5daa9db296504c03727 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / A amebíase é uma infecção causada pela Entamoeba histolytica e considerada importante causa de morbi-mortalidade no mundo. O estudo epidemiológico da amebíase tem sido reavaliado desde que a E. histolytica, a causadora da forma patogênica, foi distinta da E. dispar, causadora da forma não patogênica. A recomendação da Organização Mundial de Saúde é fazer o tratamento apenas nos pacientes com diagnóstico específico da infecção pela E. histolytica. O objetivo desta pesquisa foi avaliar a prevalência da E. histolytica em alunos de 4 15 anos das escolas públicas da cidade de Maceió, mediante aplicação em série dos testes de microscopia e ensaios imunoenzimáticos: ENZYMEBA® (Instituto de Medicina Tropical Pedro Kouri ,Havana, Cuba) e E. histolytica II® (TechLab Inc., Blacksburg, Estados Unidos). O ENZYMEBA® confirmou todos os resultados positivos para E. histolytica/E. dispar detectado pela microscopia seguido pela aplicação do teste E. histolytica II® que detectou a presença de E. histolytica. Um total de 1.798 amostras fecais foi analisado de alunos de 18 escolas públicas. A prevalência da infecção para E. histolytica/E. dispar pela microscopia e confirmado pelo teste ENZYMEBA® foi de 3,8% e de E. histolytica foi de 1,0% pelo E. histolytica II®. A aplicação dos dois testes de ELISA permitiu definir a prevalência de 2,8% para E. dispar. Considerando o teste E. histolytica II® como padrão de referência, o baixo valor preditivo positivo da microscopia de 26,4% refletiu o grande número de resultados falso-positivos para amebíase por este método. Todas as amostras fecais foram submetidas ao exame microscópico, indicando que 38,6% dos alunos estavam parasitados por um ou mais parasitos intestinais. Os parasitos mais freqüentes foram Ascaris lumbricoides (16,2%) e Trichuris trichiura (11,7%). Os resultados deste trabalho demonstraram que o exame da microscopia óptica não foi o método adequado para o diagnóstico da amebíase e sugere a aplicação de métodos mais sensíveis para o diagnóstico desta parasitose, utilizando técnicas específicas que distingam a E. histolytica da E. dispar

Intestinal infections with pathogenic protozoa may cause severe disease and remain a neglected problem in regions with inadequate sanitation and hygiene standards, especially in sub-Saharan Africa. Unfortunately, very little data about the prevalence of these infections in risk groups exist from the region. The present study was therefore conducted to assess the prevalence of intestinal protozoan infections in a representative population sample on Ijinga Island, north-western Tanzania. Methods: This was a cross-sectional study which was carried out in 2016 as part of the on-going SchistoControl pilot project on Ijinga Island, north-western Tanzania. A single stool sample was collected from 357 participants and examined microscopically for presence of trophozoites or cysts of intestinal protozoan parasites. In addition, real-time polymerase chain reaction (qPCR) was used to determine the species of intestinal protozoa. Results: Based on microscopy and qPCR, the prevalence of Giardia intestinalis infection was 12% and 15.1%, respectively. Based on microscopy, the prevalence of Entamoeba histolytica/Entamoeba dispar was 26.1%. However, through species identification using qPCR, 21.8% of the study participants were carrying non-pathogenic E. dispar and none of them was infected with E. histolytica. Conclusion: Intestinal protozoan infections are common among the population in the study area. The detection of these infections in different age groups indicates a poor hygienic standard in the community. Improvement in water, sanitation, hygiene and public health education on hand washing will help in controlling these infections. / Intestinale Infektionen mit pathogenen Protozoen können schwere Erkrankungsbilder verursachen und stellen eine oftmals vernachlässigte Problematik, vor allem in Regionen mit geringen Sanitär- und Hygienestandards, dar. Dies betrifft insbesondere südlich der Sahara gelegene Länder. Hier existieren leider keine oder nur unzureichende Prävalenzdaten, was zum Teil auch auf die inadäquaten diagnostischen Möglichkeiten zurückzuführen ist. Die Übertragung erfolgt fäkal-oral über verunreinigte Nahrungsmittel sowie kontaminiertes Trinkwasser und kann symptomatische und asymptomatische, akute und chronische Infektionen hervorrufen. Methoden: Es handelt sich um eine Querschnittsstudie, die 2016 im Rahmen des laufenden SchistoControl-Pilotprojekts auf der Insel Ijinga im Nordwesten Tansanias durchgeführt wurde. Es wurden Stuhlproben von 357 Teilnehmern gesammelt und mikroskopisch auf Trophozoiten oder Zysten von intestinalen Protozoen untersucht. Zusätzlich wurde die Realtime-Polymerasekettenreaktion (qPCR) zur Speziesdifferenzierung verwendet. Ergebnisse: Basierend auf Mikroskopie und qPCR betrug die Prävalenz von Giardia intestinalis 12% bzw. 15,1%. Basierend auf der Mikroskopie betrug die Prävalenz von Entamoeba histolytica / Entamoeba dispar 26,1%. Durch Speziesdifferenzierung mittels qPCR waren jedoch 21,8% der Studienteilnehmer positiv für E. dispar und keiner positiv für E. histolytica. Schlussfolgerung: Intestinale Protozoeninfektionen sind in der Bevölkerung des Studienortes häufig. Der Nachweis dieser Infektionen in verschiedenen Altersgruppen weist auf einen geringen Hygienestandard in der Gemeinde hin. Eine Verbesserung der Wasser-, Sanitär-, Hygienestruktur und des Gesundheitsbewusstseins wird zur Kontrolle dieser Infektionen beitragen.

PhD (Microbiology) / Department of Microbiology / Background: Diarrheal diseases have a major effect on human health, Globally; it is second only to pneumonia as a leading cause of death among children under five. They are due to a variety of infectious and non-infectious agents; including Entamoeba spp. Entamoeba histolytica is an invasive enteric protozoan parasite that causes amebiasis. Amebiasis is frequent in communities without clean water and poor sanitation, which include low-income South African populations in Giyani and Pretoria. In these populations, the amount of diarrhea caused by Entamoeba histolytica inclusive of all ages, sexes and HIV status is uncertain. Diagnosis of the parasite is usually by microscopy. However, microscopy lacks sensitivity and specificity, therefore it is not reliable. Fortunately, molecular diagnostic tests have been developed to detect different Entamoeba species in humans. It is known that the parasite E. histolytica causes asymptomatic and symptomatic diseases. However, the transition from colonization to disease is still unclear. While parasite and host factors, as well as environmental conditions influence the infection outcome, there is currently no clear explanation of wide variation in the presentation of the disease. This could suggest that there are other factors affecting the disease outcome. A better understanding of these factors as well as their role in disease remains target objectives of modern scientists and it will definitely help in the fight against the disease. In spite of the emerging evidence that the host microbiome, parasite burden and the inflammatory response contribute to the virulence of E. histolytica, their roles have never been defined in developing regions such as Giyani and Pretoria. In addition, the present study hypothesized that co-infections with E. histolytica and secretion of extracellular vesicles/exosomes have a significant impact on the virulence of E. histolytica. Little has been explored or elucidated about responses triggered by other enteropathogens/ameba interplay that could be important in the induction of tissue invasion and disease and also how E. histolytica/enteropathogens interplay in these infections has not been determined. Therefore, the knowledge of this interplay could help in understanding how this modifies disease manifestations by modulating pathogen virulence and the host response. The use of secretion systems is an essential biological process exploited by pathogenic microorganisms to promote survival and spread of the pathogen, which in turn exacerbate the infection. The study of extracellular vesicles (EVs) released by pathogens is a new and exciting field that may realistically contribute to a better understanding of the pathogenic process of E. histolytica and provide alternate control strategies. Aim and objective of the study: The overall aim of the study was to determine the impact of enteric pathogens and secreted extracellular vesicles on amebic virulence and the outcome of infection. This aim was addressed in through a series of six primary objectives, which were: a. To investigate the distribution and prevalence of protozoan parasites in South Africa. b. To investigate novel species of Entamoeba circulating in the South African population. ix c. To elucidate the impact of gut microbiota and immune response during amebic infection. d. To determine the role of Entamoeba histolytica macrophage inhibitory factor (EhMIF) during amebic infection. e. To investigate the impact of co-infections on the outcome of amebiasis. f. To determine the presence of secreted extracellular vesicles/exosomes in Entamoeba histolytica. Brief methodology and results: A modified and validated Taqman qPCR assay (with taqman probes and genus specific primers) was used for amplification and target detection. This assay was used to investigate the distribution and prevalence of protozoan parasites (Cryptosporidium spp and Giardia lamblia) in South Africa, the assay was considered superior for this project because it is more sensitive than conventional PCR and it can be used to detect multiple infection targets. This assay allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes. A total of 484 stool samples collected from diarrheal and non-diarrheal patients from rural and urban communities of South Africa were studied. The overall prevalence of parasites (Giardia lamblia and Cryptosporidium spp) in rural and urban patients were found to be 49% (112/227) and 21% (54/257) respectively (p= < 0.0001). The distribution of specific pathogens in rural areas was Cryptosporidium spp (20%) and Giardia lamblia (14%). Our findings showed no significant difference in parasitic infections between gender and the age of the participants (Chapter 3). The discovery of novel species is of great importance to human health. We have recently discovered stools positive for Entamoeba organisms by microscopy but PCR negative for known Entamoeba species. This led to the hypothesis that novel species of Entamoeba are present in the South African population. A comprehensive assay was used which included probes to identify Entamoeba bangladeshi from diarrheal and non-diarrheal participants. A sensitive qPCR assays and amplicon sequencing was used to detect Entamoeba spp, Prevotella copri and Enterobacteriaceae. Interestingly, E. bangladeshi was identified in the South African population. Entamoeba was present in 27% (E. histolytica 8.5% (41/484), E. dispar 8% (38/484), and E. bangladeshi 4.75% (23/484) E. moshkovskii was not detected in the present study. We were also able to observe changes in the host microbiome and the parasite burden associated with E. histolytica infections in S. African diarrhea cases versus asymptomatic controls but not with E. bangladeshi or E. dispar. In E. histolytica positive samples the level of both parasite and P. copri were lower in non-diarrheal samples (p=0.0034) (Chapter 4). There is accumulating evidence that the inflammatory response contributes to injury. Little is known about the key parasite mediators of host mucosal immunopathology. This study hypothesized that migration inhibitory factor (MIF) mediates the destructive host inflammatory response seen in amebic colitis. To determine the role of EhMIF during amebic infection, we used a genetic approach to test the effect of EhMIF on mucosal inflammation. We found that EhMIF induces IL-8 secretion from intestinal epithelial cells. Mice treated with antibodies that specifically block EhMIF had reduced chemokine expression and neutrophil infiltration in the mucosa. In addition to antibody-mediated neutralization, mice infected with parasites overexpressing EhMIF had increased chemokine expression, neutrophil influx and mucosal damage. We also found that the concentration of EhMIF correlated with the level of intestinal inflammation in persons with intestinal amebiasis. Together, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets (Chapter 5). To investigate the impact of co-infections on the outcome of amebiasis, we analyzed the co-occurence of E. histolytica with other enteropathogens known to cause diarrheal infections, such as Shigella/EIEC (IpaH), Campylobacter (cadf), Enterotoxigenic E. coli (STh), Norovirus GII and Adenovirus (Hexon). The results were compared with those obtained with E. histolytica that were not interacted with enteropathogens and with E. histolytica interacted with enteropathogens. The impact of multiple infections on the outcome of the infection was compared between nondiarrheal and diarrheal stool samples. It was found that co-infections with two pathogens were associated with diarrhea compared to single infections. Moreover, Norovirus GII, Campylobacter (Cadf) and co-infections were associated with diarrhea in the study population. This study did not show any significant impact of pathogens co-infecting with E. histolytica on the outcome of amebic infection (Chapter 6). The presence of secreted extracellular vesicles/Exosomes in Entamoeba histolytica was determined by using the Pathogenic ameba strains (HM-1:IMSS or HM-1:IMSS (Sub-strain-US) from petri’s lab to purify exosomes using the commercially available kit to isolate exosomes (total exosomes isolation kit). Our study for the first time revealed that E. histolytica does secrete Evs. This finding increases the appreciation that all organisms are likely to secrete these EVs (Chapter 7). However, the impact of these EVs on the pathogenesis of E. histolytica needs further investigations. Conclusion: This study has contributed significantly to our knowledge on infectious diarrhea and the diversity of Entamoeba species by providing new data on the rate and prevalence of Entamoeba diarrheal infections and their distribution in the South African population. Our study describes for the first time the presence of E. bangladeshi in the South African population. Furthermore, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets. This study also, for the first time revealed that E. histolytica does secrete EVs. The results from this work will undoubtedly open an exciting research to establish a deeper understanding of the function and role of these vesicles in amebic infection. We encourage public health interventions like health education programs and improvement of sanitation and hygiene in these populations. Molecular diagnostics should be used for specific diagnostic in clinical settings. / NRF

PhD (Microbiology) / Department of Microbiology

MSc (Microbiology) / Department of Microbiology

No description available.

Coyotes (Canis latrans) have inhabited Florida (USA) since the 1960s and are currently found throughout the state. The purpose of the present study was to obtain information on enteric parasites and diet of Florida coyotes from two different habitat types. Seasonal variation in diet was also examined. Fresh coyote fecal samples were collected from protected and urban habitats in Pinellas County, Florida (USA; 27°54' n, 82°41'w) from may 2005 to march 2007. A standard fecal flotation examination and formalin-ethyl acetate sedimentation were utilized on fecal samples collected from the protected (n=40) and urban (n=50) habitats. Five novel (newly documented) parasites of coyotes were discovered; one cestode (Hymenolepis spp.), one nematode (Ascaris spp.), and three protozoa (Balantidium coli, Blastocystis spp., and Entamoeba histolytica). Novel parasites of Florida coyotes were also discovered two cestodes (diphyllobothrium latum and dipylidium caninum), two nematodes (toxocara canis and uncinaria stenocephala), one trematode (paragonimus spp.), and four protozoa (cryptosporidium spp., giardia canis, isospora spp., and sarcocystis cruzi). One cestode (Taenia spp.), three nematodes (Ancylostoma caninum, Physaloptera spp., and Trichurus vulpis), and one trematode (Alaria spp.) were also recovered, all of which have previously been documented in Florida coyotes. Diet items were identified to the lowest possible taxonomic level by gross morphological characteristics and medullary configurations of dorsal guard hairs. A poisson regression was utilized to determine the relation between diet items and habitat, season, and interaction. In the protected habitat (n=49), vegetative matter (96%), Insecta (53%), and Rodentia (45%) were recovered most often, as opposed to berries (56%) and Lagomorpha (32%) in the urban habitats (n=71). Overall, vegetative matter, berries, and Lagomorpha were recovered most often from Florida coyote fecal samples. Odocoileus virginianus, Lagomorpha, and berries varied the most between wet and dry seasons. It is suggested that Florida coyotes are more susceptible to reinfection by novel parasites because of their rapid range expansion and lack of acquired immunity. Rapid habitat loss in Florida (i.e., urbanization) lowers survival of adult coyotes, increases the probability of transmission of disease between wild and domestic canids, and alters the diet of coyotes by lowering biological diversity of available prey items.

Interleukin-8 is a chemokine which is chemotactic for neutrophils and T-lymphocytes and plays a crucial role in the pathogenesis of inflammatory bowel disease. Intestinal mucosal epithelial cells produce IL-8 in response to pathogens which mediates bidirectional communication between pathogen and host. The objective of this study was to investigate the molecular mechanisms involved in IL-8 gene regulation in T84 human colonic epithelial cells. To determine if IL-8 plays a role in the pathogenesis of intestinal amebiasis, the effect of Entamoeba histolytica on IL-8 gene expression was investigated. E. histolytica secreted components enhanced IL-8 mRNA expression and protein production in the absence of amebae-enterocyte contact. The proinflammatory cytokines IL-1beta and TNF-alpha were not involved in IL-8 protein production. As PGE2 is central in mucosal inflammation, the effect of PGE2 on IL-8 gene expression was determined. Using purified PGE2 and PGE2 receptor agonists, it was shown that PGE2 coupled to the EP4 receptor and triggered cAMP-dependent PKA signaling which upregulated IL-8 mRNA expression at the posttranscriptional level. Elevation of [Ca 2+]i from intracellular Ca2+ stores by A23187 or thapsigargin stimulated IL-8 mRNA transcription and IL-8 protein production through the activation of calcineurin. Moreover, IL-8 3'-UTR had a strong suppressive effect on CAT reporter gene expression in COS7 cells by reducing its mRNA level. A unique fragment (nt 2387-2743) containing AU rich elements was shown to attenuate CAT mRNA expression by destabilizing the transcripts. Secondary structure but not AU rich elements played a major role in CAT mRNA turnover.