Este trabalho teve por objetivo identificar e estudar a expressão de genes envolvidos na resposta imune a endoparasitas gastrintestinais em bovinos. Contagem periódica de ovos por grama de fezes (OPG) e análises de coproculturas foram realizadas para identificar animais resistentes e susceptíveis a endoparasitas gastrintestinais. A contagem de OPG permitiu identificar estes animais, sendo que o grupo susceptível apresentou uma contagem 20 vezes superior ao grupo resistente (P<0,001). Análises de coproculturas permitiram concluir que a infestação em bovinos foi mista, com a predominância dos gêneros Cooperia e Haemonchus e menor incidência de Oesophagostomum. Para a identificação dos genes, foi utilizada a metodologia EST (Expressed Sequence Tag) e foram construídas duas bibliotecas de clones de cDNA obtidos a partir da mucosa do abomaso, intestino delgado e nódulos linfáticos abomasais de bovinos resistentes (ER1) e susceptíveis (ES1) a nematódeos gastrintestinais. Foram geradas 2496 ESTs de cada biblioteca. Destas, 1664 e 1898 foram válidas para as bibliotecas ER1 e ES1, respectivamente. Dentre as 2323 sequências únicas obtidas foram identificados vários produtos gênicos relacionados à resposta imune, tais como moléculas de classe I e II do MHC, imunoglobulinas, interleucinas, lisozima e pepsinogênio. Para a quantificação da expressão de RNA mensageiro, foi utilizada a metodologia de transcrição reversa e reação em cadeia da polimerase em tempo real, onde foram analisados 10 genes relacionados à polarização da resposta imune (as interleucinas IL- 2, IL-4, IL-8, IL-12p35 e IL-13, as citocinas TNF-&#945;, IFN-&#947;, MCP-1&#945; e MCP-2 e a glicoproteína mucina 1-MUC1). As análises de expressão gênica realizadas na mucosa do abomaso revelaram aumento nos níveis de RNAm para IL-4 (P<0,018), IL-13 (P<0,002) e diminuição de TNF-? (P<0,0001) nos animais resistentes; nos nódulos linfáticos abomasais houve aumento de IL-4 (P<0,019) nos animais resistentes e de IFN-&#947; (P<0,007) nos animais susceptíveis; no intestino delgado houve aumento de IL-4 (P<0,01) e IL-13 (P<0,045) nos animais resistentes, ao contrário de IL-2 (P<0,047), IL-12p35 (P<0,029), IFN-&#947; (P<0,004) e MCP-1 (P<0,03), cujos níveis de RNAm foram maiores nos animais susceptíveis. No abomaso dos animais resistentes houve menor expressão de pepsinogênio (P<0,025) e maior de lisozima (P<0,042). Em conclusão, a estratégia utilizada permitiu a identificação de vários genes envolvidos na resposta imune e a descoberta inédita de que Bos indicus resistentes a parasitas gastrintestinais apresentam uma resposta TH2 polarizada, em contraste aos animais susceptíveis, que apresentam uma resposta TH1. / The aim of this work was identify genes related to immune response to gastrointestinal nematodes in cattle. The periodic counts of eggs per gram (EPG) of feces and coproculture analysis were done to identify the resistant and susceptible animals. The EPG counts allowed us to identify these animals. It was twenty-fold higher in susceptible group (P<0.001). The coproculture analysis allowed us to conclude that the infestation is predominantly characterized byCooperia spp. and Haemonchus spp. and a low incidency of Oesophagostomum. To identify the genes, it was used the EST (Expressed Sequence Tags) methodology and constructed two cDNA libraries from abomasum, abomasum lymph nodules and small intestine from resistant (ER1) and susceptible (ES1) cattle. It was generated 2496 ESTs from each library. From these, 1664 and 1898 ESTs were valids to ER1 and ES1 libraries, respectively. Among the 2323 unique sequences were identifyed several genes related to immune response, such as MHC class I and II molecules, immunoglobulins, interleukins, lysozyme and pepsinogen. To study the gene expression, it was used the reverse transcription and real-time polymerase chain reaction methodology to quantify the messager RNA expression of 10 genes related to polarization of immune response (the interleukins IL-2, IL-4, IL-8, IL-12p35 e IL-13, the cytokines TNF-&#946;, IFN- &#947;, MCP-1&#946; and MCP-2 and the glycoprotein mucin 1-MUC1). The gene expression analysis in the abomasum reveled that IL-4 (P<0,018) and IL-13 (P<0,002) were up-regulated and TNF-&#946; (P<0,0001) was down-regulated in resistant group; in the abomasum lymph nodules IL-4 (P<0,019) and IFN-&#947; (P<0,007) were both up-regulated in resistant and susceptible group, respectively; in the small intestine IL-4 (P<0,01) and IL-13 (P<0,045) were up-regulated in resistant group and IL-2 (P<0,047), IL-12p35 (P<0,029), IFN-&#947; (P<0,004) and MCP-1 (P<0,03) were down-regulated in susceptible one. In the abomasum from resistant group, pepsinogen was down-regulated (P<0,025) and lysozyme was up-regulated (P<0,042). In conclusion, the strategy used allowed us to identify several genes involved in immune response and the inedit discovery that Bos indicus resistant to gastrointestinal nematodes present a TH2-type response, in contrast to susceptible animals that present a TH1-type response.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T13:37:26Z No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T13:38:53Z (GMT) No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-25T13:38:53Z (GMT). No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-13 / The norovirus (NoVs) are important viral causative agents of acute gastroenteritis (AGE), affecting individuals of all ages in distinct parts of the word; however, the highest morbi-mortality rates occur mainly in children under five years of age and the elderly. The aim of this study was to to screening NoV by Polymerase Chain Reaction Post Reverse Transcription (RT-qPCR) and real-time (RT-qPCR) in fecal and nasal swab of children up to six years of age, with and without AGE symptoms. Samples were obtained at the Materno Infantil Hospital, from May/2014 to May/2015. Secretor status of children was also determined by enzyme immunoassay and genotyping (FUT2 gene) from the sediment of nasal swab epithelial cells. A global positivity index of 17% (37/219) for NoV in feces, and from these positive children, 48.6% (18/37) had AGE symptoms. Mean viral load in fecal samples was 2.59 x 1010 CG/g from symptomatic and 1.37 x 109 CG/g in asymptomatic. A higher positivity rate (70%) was observed GII NoV, compared to GI NoV (30%) and a higher positivity in children up to 24 month old (67.5%), although not statistically significant. As for the secretor status of children positive for NoV in fecal samples, 94.6% positive secretory status. The NoV were detected in practically every month of the study, and no particular pattern of circulation in relation to dry or rainy seasons was observed. Most children positive for NoV (70%) had the record they have received at least the first dose of the vaccine against Rotavirus, being the highest viral load detected among vaccinated children. The NoV RNA was detected in 8.7% of nasal swab samples of the children and of these, 58% had AGE symptoms. The mean viral load in swab samples from symptomatic children was 2.10 x 108 and in the asymptomatic children was 2.41 x 107 CG/mL. A high NoV genotype variability was found in the study (GI.2, GI.3, GI.5, GII.3, GII.4 and GII.6), with a predominance of GII.4 (28.6%), with this being the first report of NoV GI.5 in Brazil. The data obtained in this study reveal a high frequency, viral load, and genetic variability of NoVs among children attended in a hospital of Goiânia, Goiás. The results are important for a better understanding of NoV epidemiology in nosocomial environment, and may constitute useful information on the advent of the development of an effective vaccine. The viral load in nasal swab samples is a novel data that may contribute for the elucidation of a possible alternative rout of NoV transmission. / Os norovírus (NoVs) são importantes agentes virais causadores de gastroenterite aguda (GEA), atingindo indivíduos de todas as idades em diversas partes do mundo, porém os maiores índices de morbimortalidade ocorrem principalmente nas crianças menores de cinco anos e idosos. O objetivo do presente estudo foi realizar a pesquisa de NoV, através da Reação em Cadeia pela Polimerase Pós-Transcrição Reversa (RT-PCR) e por tempo real (RT-qPCR) em amostras fecais e swab nasal de crianças com até seis anos de idade, com e sem sintomas de GEA. As amostras foram coletadas no Hospital Materno Infantil, entre maio/2014 e maio/2015. Procedeu-se ainda à determinação do status secretor das crianças, por Ensaio Imunoenzimático e genotipagem (gene FUT2), em sedimento de células epiteliais do swab nasal. Foi observado um índice de positividade global de 17% (37/219) para NoV nas fezes, sendo que destas crianças positivas, 48,6% (18/37) apresentavam sintomas de GEA. A carga viral média nas amostras de fezes de crianças com sintomas foi 2,59 x 1010 CG/g e 1,37 x 109 CG/g nas assintomáticas. Observou-se maior positividade para NoV GII (70%) quando comparado ao GI (30%) e maior positividade nas crianças de até 24 meses de idade (67,5%), entretanto este dado não foi estatisticamente significativo. Quanto ao status secretor das crianças positivas para NoV nas fezes, 94,6% status secretor positivo. Os NoV foram detectados em praticamente todos meses do estudo, não sendo observado padrão de circulação definido em relação às estações seca e chuvosa. A maioria das crianças positivas para NoV (70%) tinham o registro de terem recebido pelo menos a primeira dose da vacina contra Rotavírus, sendo a carga viral mais elevada detectada entre crianças vacinadas. O RNA de NoV foi ainda detectado em 8,7% (19/219) das amostras de swab nasal das crianças e destas, 58% apresentavam sintomas de GEA. A carga viral média nas amostras de swab das crianças sintomáticas foi 2,10 x 108 CG/mL e nas assintomáticas 2,41 x 107 CG/mL. Foi observada elevada variabilidade de genótipos de NoV no estudo (GI.2, GI.3, GI.5, GII.3, GII.4 e GII.6), com maior predominância de GII.4 (28,6%), sendo este o primeiro relato de NoV GI.5 no Brasil. Os dados obtidos neste estudo revelam elevada frequência, carga viral e variabilidade genética de NoVs entre crianças atendidas em um hospital de Goiânia, Goiás. Os resultados são importantes para o melhor entendimento da epidemiologia dos NoVs em ambiente nosocomial, e poderão ser uteis como informação no advento do desenvolvimento de uma vacina eficaz. A determinação da carga viral de NoV em xii amostras de swab nasal é um dado novo, podendo este contribuir para a elucidação de uma possível rota alternativa de transmissão dos NoV.

Tropheryma whipplei a longtemps été uniquement considérée comme l’agent responsable de la maladie de Whipple, une affection rare caractérisée notamment par une perte de poids, des diarrhées chroniques et des douleurs abdominales. Toutefois, au cours de ces dernières années, il est apparu que les infections à T. whipplei peuvent présenter des manifestations cliniques communes telles que des pneumonies, des bactériémies fébriles ou des gastroentérites, ce qui montre que la maladie de Whipple ne constitue pas la seule manifestation de l’infection à T. whipplei. Ma thèse a eu deux objectifs. Le premier a été de caractériser l’interaction entre T. whipplei et la cellule dans laquelle elle se réplique, le macrophage. J’ai montré en utilisant diverses techniques (biologie moléculaire à haut débit, biologie cellulaire et biochimie) que T. whipplei induit une réponse macrophagique inédite caractérisée par une polarisation M2 associée à une réponse interféron de type I. J’ai également montré que ces événements sont associés à l’apoptose des macrophages dont l’induction se fait par voie extrinsèque et que l’IL-16, pour laquelle un rôle au cours de l’infection à T. whipplei était avéré, intervient d’une part dans le blocage de la maturation phagosomale et d’autre part interfère avec l’activation des macrophages. Mon second objectif a été de montrer à travers un modèle animal que la primo-infection par T. whipplei se manifeste par une gastroentérite auto-résolutive. Cet objectif découlait directement de travaux récents qui associent T. whipplei à diverses manifestations cliniques et notamment à des épisodes diarrhéiques chez l’enfant. Mes résultats confortent clairement cette hypothèse et montrent également que des dommages préexistants de la muqueuse intestinale permet l’établissement de l’infection à T. whipplei. / Tropheryma whipplei has only been considered as the agent of Whipple‘s disease, a rare disease characterized by weight loss, chronic diarrheas and abdominal pains. It is now believed that infections with T. whipplei result in common clinical manifestations, such as pneumonia, fever, bacteriema or gastroenteritis and, as a consequence, it is likely that the Whipple’s disease is not the only manifestation of T. whipplei infection. During my PhD, I had 2 objectives. The first was to characterize the interaction between T. whipplei and the cell type in which T. whipplei replicates, namely macrophages. I showed using diverse techniques (high throughput molecular biology, cell biology and biochemistry) that T. whipplei induced an unusual macrophage response, characterized by M2 polarization with type I interferon response. I also showed that these events were associated with apoptosis of macrophages induced by the extrinsic pathway and that IL-16, which was already described during T. whipplei infection, was involved in the blockade of the phagosomal maturation and interfered with macrophage activation. The second objective was to show using a murine model that primary infection with T. whipplei results in self-limiting gastroenteritis. This objective directly arose from recent work that associated T. whipplei with various clinical manifestations and, in particular, with diarrheal episodes in children. My results clearly verified this hypothesis and also revealed that pre-existing mucosal damage allowed the establishment of the infection.

MSc (Microbiology) / Department of Microbiology

No description available.

PhD (Microbiology) / Department of Microbiology / Globally, one in ten child deaths before the age of 5 years is due to diarrheal disease, causing almost 800,000 mortalities worldwide, which mostly occur in Sub-Saharan Africa and South Asia. Eighty-eight percent (88%) of diarrheal deaths worldwide are attributable to unsafe water, inadequate sanitation and poor hygiene. Unsanitary environments and poor hygiene practices allow diarrhea causing pathogens including viruses, bacteria and parasites to spread more easily. Norovirus (NoV) are now considered the most common cause of outbreaks of nonbacterial gastroenteritis. However, the factors which control the genetic diversity, the sources of sporadic NoV infections, the transmission and persistence of infection are poorly understood. Limited data are available for NoVs strains in South Africa, especially in rural and peri-urban areas. Despite the excessive burden of diarrhea disease in developing countries, NoVs outbreaks have been to date mostly reported in developed countries. Given that the contribution of the various pathogens to diarrhea may differ substantially between regions depending on local meteorological, geographic, and socio-economic conditions, there is a need to investigate intensively the role of viral agents associated with diarrhea in different settings in Africa continent. How would poor living conditions in rural setting impact the prevalence and genetic characteristics of Norovirus strains circulating Limpopo province is the research question of this study. ix To determine the prevalence and genetic diversity of NoVs strains circulating in the rural communities in the Limpopo Province, South Africa and investigate the genetic relationship between NoVs strains, a cross-sectional study was performed on human stools collected from rural communities. We used qualitative variables of poor living environmental conditions including type of water used at the household of child’s parent or guardian, use of toilet seat, presence of livestock at the household and parent employment status to assess possible environmental risk factors of NoV infection within the study area. Prior to this prospective study, we conducted a systematic review of the PubMed and EMBASE databases for published articles of Human NoVs in Africa between 1990 and 2013 in order to assess the contribution of Human NoVs to diarrhoeal diseases in Africa. This review provides a picture of Human NoVs studies in Africa and reveals that unreported sporadic gastroenteritis cases of Human NoVs are common in Africa. Most are community-associated infections reported from urban settings. Possible environmental transmission routes have been documented. Combined environmental and clinical studies are required for targeted actions to control transmission of Human NoVs in Africa. Between July 2014 and April 2015, outpatient children under 5 years of age from rural communities of Vhembe district, South Africa, were enrolled for the study. A total of 303 stool specimens were collected from those with diarrhea (n=253) and without (n=50) diarrhea. NoVs were identified using real-time one-step RT-PCR. Nucleotide sequencing methods were performed to genotype the strains. Phylogenetic analyses x were performed to compare identified NoVs genotypes to the worldwide circulating strains. One hundred and four (41.1%) NoVs were detected. NoV detection rates in symptomatic and asymptomatic children (OR = 1.24; 95% CI 0.66 – 2.33) were not significantly different. Comparison of the median CT values for NoV in symptomatic and asymptomatic children revealed significant statistical difference of estimated GII viral load from both groups, with a much higher viral burden in symptomatic children to our knowledge this is the first study reporting on the differences in estimated viral load of GII and GI NoV positive cases and controls. The study findings may have implications for the diagnosis of NoV disease and future vaccine development, which may only need to consider GII as the genogroup associated with diarrhea in the South African population. Sequence analyses demonstrated multiple NoV genotypes identified in rural communities of Vhembe district. The most prevalent NoV genotypes were GII.4 Sydney 2012 variants (n=7) among the capsid genotypes, GII.Pe (n=9) among the polymerase genotypes and GII.Pe/GII.4 Sydney 2012 (n=8) putative recombinants among the RdRp/Capsid genotypes. Two unassigned GII.4 variants and an unusual RdRp genotype GII.P15 were found. With note, the rare GII.P15 identified in this study, has a common ancestor with GII.P15 strain from Japan previously reported as GII / untypeable recombinant strain implicated in a gastroenteritis outbreak. To our knowledge this is the first report of this unusual genotype in the African continent. Though not proven predictive of diarrhea disease in this study, the high detection rate of NoV reflects the substantial exposure of children from rural communities to enteric xi pathogens possibly. However in this study no risk factor has been found between NoV positive and qualitative environmental variables of poor living conditions in rural setting. The results also suggest that the difference between asymptomatic and symptomatic children with NoV may be at the level of the viral load of NoV genogroups involved. The findings highlighted NoV genetic diversity and revealed continuous pandemic spread and predominance of GII.Pe/GII.4 Sydney 2012, indicative of increased NoV activity. An unusual RdRp genotype GII.P15 and two unassigned GII.4 variants were also identified from rural settings of the Vhembe district/South Africa. NoV surveillance / NRF

Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Rotavirus diarrhea is still an important cause of mortality in children worldwide, responsible each year for about 500,000 deaths. The introduction of the vaccine against the virus could lead to a reduction in morbidity and mortality associated with rotavirus. In Brazil, the Rotarix ® rotavirus vaccine, which contains the genotype G1P [8], was included in the national programme of immunization since March 2006. This project aims to evaluate clinical and epidemiological data related to infection by RVA, rotavirus genotypes prevalent. The research project is a cross-sectional study in which children with acute diarrhoea were recorded prospectively from 2010 to 2012. The location of collection of fecal samples was the Hospital of urgency of Sergipe in Pediatric Emergency sector, in Aracaju/SE. The clinical and epidemiological information was obtained through a questionnaire. The clinical severity was determined by a scoring system 20, calculated from the survey point. It was verified the episodes of acute diarrhea and stool samples collected for research and genotyping of rotavirus by ELISA and RT-PCR method.Descriptive statistical calculations were carried out to define the epidemiology of rotavirus. EIA positive results were found in 78 of the 790 samples. The most frequent genotype was G2 P [4], followed by the G8 genotype P [4], P [8] G1, G3 P [8], G1 P [6]. Mixed infections were detected G2 P [4] P [8], P [4] and G1G2 G2G8 P [4] and in general there was a cocirculação of different genotypes in the years studied, moreover, shows an alternation between the genotypes every year. The results obtained in this study observed a variability of positive cases distributed, confirming that the seasonality in the region is not remarkable. Therefore, new strains of rotavirus are emerging and associated with severe diarrhea. For this reason, monitoring is required to follow changes in the epidemiology of rotavirus. / A diarreia por rotavírus ainda é uma importante causa de mortalidade infantil em todo o mundo, sendo responsável a cada ano por cerca de 500.000 mortes. A introdução da vacina contra o vírus pode levar a uma redução da morbidade e mortalidade associadas ao rotavírus. No Brasil, a Rotarix®, a vacina contra o rotavírus, que contém o genótipo G1P[8], foi incluído no programa nacional de imunizações em março de 2006. Este projeto teve como objetivo avaliar dados clínicos, epidemiológicos e genótipos predominantes relacionados à infecção por RV-A. O projeto de pesquisa é um estudo transversal em que as crianças com diarreia aguda foram registrados prospectivamente a partir de 2010 a 2012. O local de coleta das amostras fecais foi o Hospital de Urgência de Sergipe no setor de Urgência Pediátrica, em Aracaju/SE. A gravidade clínica foi determinada por um sistema de pontuação 20, calculado a partir do ponto de questionário. Foram verificados os episódios de diarreia aguda e coletadas amostras de fezes para pesquisa e genotipagem de rotavírus pelo método ELISA e RT-PCR. Cálculos estatísticos descritivos foram realizados para definir a epidemiologia do rotavírus. Resultados positivos foram encontrados em 78 das 790 amostras. O genótipo mais frequente foi G2P[4], seguido do genótipo G8P[4], G1P[8], G3P[8], G1P[6]. Foram detectadas infecções mistas G2 P[4] P[8], G1G2 P[4] e G2G8 P[4] e de um modo geral observou-se uma cocirculação de distintos genótipos nos anos estudados, além disso, nota-se uma alternância entre os genótipos a cada ano. O resultado obtido neste estudo observou uma variabilidade dos casos positivos distribuídos, confirmando que a sazonalidade na região não é marcante. Portanto, novas cepas de rotavírus estão surgindo e associados à diarreia grave. Por esta razão, a vigilância contínua é necessária para acompanhar as mudanças na epidemiologia do rotavírus.

D. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Salmonella enterica serovar Typhimurium and Campylobacter jejuni are the leading causes of Salmonellosis and Campylobacteriosis that is characterised by gastroenteritis. These waterborne diseases can be easily prevented by home water treatment methods such as solar disinfection (SODIS). The SODIS process involves placing microbiologically unsafe water in clear plastic or glass bottles and exposing them to direct sunlight for approximately six to eight hours. SODIS kills microbes through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating. The result is microbiologically safe water. Continuous drinking of SODIS treated water may confer some immunological effects on the consumer. These immunological effects have not been thoroughly explored. Therefore, the objectives of this study were to firstly, characterise the effects of solar irradiation on the viability of S. Typhimurium and C. jejuni; secondly, to determine the cytotoxicity and modulation of cell death of solar irradiated S. Typhimurium and C. jejuni on macrophages. Thirdly, to analyse the chemokine and cytokine profiles of macrophages infected with solar irradiated S. Typhimurium and C. jejuni. Lastly, to analyse the host-cell interactions of macrophages infected with solar-irradiated and non-solar irradiated S. Typhimurium and C. jejuni using a proteomic approach. In all the experiments, S. Typhimurium and C. jejuni were (i) heat/chemically treated, (ii) solar and non-solar irradiated for 4 and 8 hours. A murine macrophage cell line RAW264.7 was co-cultured with the differentially treated bacteria species for 3 and 24 hours. Appropriate controls were included. The impact of solar irradiated S. Typhimurium and C. jejuni on intracellular growth, proliferation, cytotoxicity, and apoptosis on macrophages was assessed. Intracellular growth of the both bacterial species was assessed with the gentamicin protection assay, and cytotoxicity was determined by Lactate Dehydrogenase Assay (LDH). The macrophages treated with solar irradiated S. Typhimurium and C. jejuni showed no intracellular growth after 48 hours post-infection. However, the non-irradiated S. Typhimurium survived within the macrophages and were highly toxic to the macrophages (average cytotoxicity of 91%±32). The non-solar irradiated C. jejuni were metabolically active but non-culturable, whereas the solar-irradiated C. jejuni was metabolically inactive. Thus, solar irradiated C. jejuni showed a lower percentage cytotoxicity (2.57% ± 0.32%) in comparison to non-solar irradiated C. jejuni at 24 hours post-infection (p.i.) (30.28% ± 0.05%). Flow cytometric analysis showed that the non-irradiated S. Typhimurium brought about a statistically significant increase in the percentage of necrotic cells (48% ± 2.99%), whereas bacteria irradiated for 8 hours produced a lower percentage of necrotic cells (25% ± 5.87%). The heat/chemical attenuated samples had the lowest percentage of necrotic cells (21.15% ± 5.36%) at 24 h p.i. Macrophages treated with solar irradiated and non-solar irradiated C. jejuni did not induce necrosis, but apoptotic cell death. At 24 h p.i., the highest proportion of apoptotic cell death was observed in macrophages treated with non-solar irradiated C. jejuni whereas the solar irradiated C. jejuni showed a lower percentage of apoptotic cell death. Therefore, there is great possibility that S. Typhimurium and C. jejuni could become avirulent after SODIS treatment and this could prevent gastroenteritis in consumers of SODIS-treated water. The activation of macrophages infected with solar irradiated S. Typhimurium and C. jejuni was also assessed in this study. The production of nitric oxide (NO) was determined using the Greiss Reagent Assay, whereas the production of chemokines, cytokines, and growth stimulating factors by the RAW264.7 cells in vitro was measured using the Luminex 200. The results showed that both solar and non-solar irradiated S. Typhimurium inhibited the production of nitric oxide in the RAW264.7 cells. The heat/chemically attenuated S. Typhimurium induced a significant increase (p<0.0.5) in the production of NO2− in the macrophages when compared to the unstimulated RAW264.7. The chemokine and cytokine levels produced by the macrophages were similar in the solar inactivated S. Typhimurium and the live untreated S. Typhimurium. However, macrophages treated with heat/chemically attenuated S. Typhimurium showed an anti-inflammatory response by inhibiting the production of pro-inflammatory cytokines such as IL-1, IL-1, IL-2, IL-6, and IL-17 in macrophages. The macrophages treated with solar and non-solar irradiated C. jejuni possibly produced an anti-inflammatory effect since the amount of pro-inflammatory cytokines in the samples was significantly reduced during the late infection period (24 h p.i.). This study also analysed the proteomic profiles of macrophages treated with LPS, non-solar irradiated, solar irradiated, heat/ chemical inactivated S. Typhimurium, and C. jejuni. This was carried out using SWATH-mass spectrophotometry-based proteomics. Proteins were extracted from infected macrophages after 24 hours p.i. HILIC-based sample clean-up and digestion, DDA LCMS-MS (spectral library), SWATH LCMS-MS, and data processing were carried out. A total of 15,077 peptides matching to 2,778 proteins were identified at 1% FDR with numerous differentially expressed proteins (DEPs) detected in macrophages treated with lipopolysaccharide (LPS), non-solar irradiated C. jejuni (NS), heat-attenuated C. jejuni (HA) and 4h-solar irradiated (SI4) and 8h-solar irradiated (SI8) C. jejuni, respectively. Pathway analysis revealed that most of the upregulated proteins in macrophages treated with solar irradiated C. jejuni were involved in oxidation-reduction processes, endoplasmic reticulum stress, transport, antigen processing and presentation of exogenous peptide antigens via MHC class I (TAP-dependant) and ATP-biosynthetic processes. The KEGG-pathways also revealed the roles of some upregulated proteins in lysosomal and phagosome pathways. In conclusion, our results revealed that there is coordinated up-regulation of MHC-I processing pathways occurred at 24 h p.i. It is likely that proteins from solar irradiated C. jejuni may undergo proteasomal degradation, and the peptides are transported to the endoplasmic reticulum (ER) and loaded onto MHC-I molecules. Peptide loading results in class I complexes consolidation and transit to the cell surface where antigens can be presented to circulating CD8 + T cells. Additionally, solar irradiated C. jejuni also undergoes degradation in the phagosome. The phagosome has the potential to create antigens that can be expressed on the cell surface of macrophages to stimulate different lymphocytes and induce appropriate immune responses, thus, connecting the innate to adaptive immunity, and this could also have health benefits via the consumption of SODIS treated water. However, proteomic analysis of S. Typhimurium showed no significant differentially expressed proteins in macrophages treated with LPS, non-solar irradiated, and solar irradiated S. Typhimurium. This may be due to an overestimation of the extracted protein. However, DEPs in macrophages treated with heat-attenuated S. Typhimurium showed that macrophages may have adapted an anti-inflammatory M2 phenotype because the IFN-γ signalling pathway was downregulated. This may have contributed to non-expression of the chemokine IFN-γ in RAW264.7 cells. Moreover, proteins such as Hmox1 and Sqstm1 were upregulated, and this is also characteristic of M2 macrophages. This study provided new insights on the effect of solar irradiated Salmonella Typhimurium and Campylobacter jejuni on the proliferation and activation of macrophages in vitro.