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Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: A 2010 Food and Agriculture Organization (FAO) report indicated that 85% of the world´s fish stocks overexploited. This highlights the importance of commercial aquaculture as a sustainable alternative to capture fisheries. Growth rates for international aquaculture production are slowing, although an average annual increase of 12.6% occurred in Africa between 1970 and 2008. Sub-Saharan Africa accounted for 0.50% of the total world aquaculture production in 2008. The South African aquafeed industry recognized the need for aquaculture research in alternative feed ingredients to ensure a more sustainable future. With routine use of antibiotics in aquaculture, antibiotic resistance have emerged, and novel strategies are needed. Currently, alternative feed ingredients and practical substitution of antibiotics are two of the major challenges faced in aquaculture. The use of plant proteins is preferred over animal proteins such as fish meal. By developing sustainable aquafeeds, that have both nutritional and health benefits, the gastrointestinal tract (GIT) microbiota of the host should be considered. The fish GIT is an open system and acts as a route of infection for pathogenic bacteria. A comparative study was done to determine the structure of the GIT microbiota in Mozambique tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss). Tilapia and trout are important freshwater aquaculture species in South Africa. Tilapia is a warmwater herbivorous fish with a long coiled GIT, whereas trout is a coldwater carnivore and its GIT is short and compartmentalized. Tilapia was reared in recirculation systems with lower water pH and trout in flow-through systems with higher water pH. Tilapia diets consisted of plant-based protein, whereas trout diets contained animal-based proteins. To understand the role of the autochthonous bacteria in the GITs of these fish, we determined community structure along the length of the respective GITs. A highly reproducible DNA fingerprinting technique, automated ribosomal intergenic spacer analysis (ARISA), was used that targeted the intergenic spacer region (ITS) in bacteria. Community analysis based on operational taxonomic units (OTUs) was used to statistically compare the microbiota in different segments. The different GIT compartments in trout also showed distinct bacterial community structures. However, the degree of variation between identical segments between different fish was shown, and consequently, individual microbiotas was not seen. ARISA was further used to determine if formulations containing dietary phytogenics will modify the GIT microbiota. The GIT of fish plays a role in food digestion, while the resident GIT bacteria are important contributors to disease control and nutrient utilization. During two feeding trials tilapia was fed 16 and trout six different diets. Microbial diversity was lower in the GIT of tilapia as compared to trout and no significant shifts in community structure were detected in both species. With a high-throughput DNA sequencing technique, we followed a metagenomic approach to classify the autochthonous microbiota of tilapia and trout on a phylogenetic level. Pyrosequencing was used to define a core microbiota that would enable comparative feeding studies to be carried out. The proposed microbiota of tilapia consisted of genera from the Proteobacteria (5), Firmicutes (1), Actinobacteria (1), Fusobacteria (1) and Cyanobacteria (2). The microbiota of trout comprised genera of the Proteobacteria (3), Firmicutes (3) and Actinobacteria (1). Three genera from the Proteobacteria were common in tilapia and trout, confirming the dominance of this group in the GIT of freshwater fish. The variability and distribution of Lactobacillus in the GITs of both species was determined. Culturing of lactic acid bacteria (LAB) from the GIT of tilapia proves to be difficult and this was confirmed by the low-abundance of particularly Lactobacillus. A number of other LAB genera were identified that confirmed LAB as part of the autochthonous microbiota in freshwater cultured fish. The detection of secondary bacterial fish pathogens as part of the resident microbiota was also established. The use of 454 pyrosequencing in this study, gave a deeper insight into the structure of bacterial communities in the GITs of tilapia and trout. This research is novel and based on our knowledge, the first study to determine the core microbiota in two South African aquaculture species. Data based on community diversity and structure can be used as a basis for further studies. One important study would be to determine the effect environmental stressors would have on the core microbiota. / AFRIKAANSE OPSOMMING: Volgens `n 2010-verslag van die FAO (Food and Agriculture Organization) is 85% van die wêreld se visbronne oorbenut. Dit beklemtoon die belangrikheid van kommersiële akwakultuur as `n alternatief tot visvangste. Internasionaal neem die groeitempo van akwakultuur produksie af, alhoewel groei in Afrika tussen 1970 en 2008 jaarliks met `n gemiddeld van 12.6% toegeneem het. In 2008 was lande suid van die Sahara verantwoordelik vir 0.50% van die wêreld se akwakultuur produksie. Die Suid-Afrikaanse akwavoer industrie het die behoefte raakgesien vir akwakultuur navorsing in alternatiewe voerbestanddele om 'n meer volhoubare toekoms te verseker. Die roetine gebruik van antibiotika in visboerderye het bygedra tot die ontstaan van weerstandbiedende organismes, en daarom is nuwe strategieë nodig. Tans is alternatiewe voeding en die praktiese vervanging van antibiotiese stowwe belangrike uitdagings in akwakultuur. Die gebruik van plantaardige proteïne in viskos word verkies bo dierlike proteïene soos vismeel. Deur die ontwikkeling van volhoubare viskos, wat beide voedingswaarde en voordele vir die gesondheid van visse inhou, moet die mikrobiota in die spysverteringskanaal (SVK) van die gasheer oorweeg word. Die SVK van visse is `n oop sisteem en dien sodoende as `n infeksie kanaal vir patogeniese bakterieë. `n Vergelykende studie is uitgevoer om SVK mikrobiota struktuur te bepaal in Mosambiek tilapia (Oreochromis mossambicus) en reënboogforêl (Oncorhynchus mykiss). Tilapia en forêl is twee belangrike varswater spesies in Suid-Afrikaanse akwakultuur. Tilapia is `n warmwater herbivoor met `n lang, gekronkelde SVK. In teenstelling is forêl `n kouewater vis met `n kort SVK, wat uit verskillende segmente bestaan. Tilapia is in hersirkulasie waterstelsels met `n lae pH gekweek terwyl forêl in deurvloei waterstelsels met `n hoër pH gekweek is. Tilapia diëte het plantaardige proteïne bevat tewyl forêl diëte proteïne vanaf dier-afkoms bevat het. Om die rol van SVK bakterieë beter te verstaan is populasie struktuur bepaal langs die lengte van die SVK. Geoutomatiseerde ribosomale intergeniese spasie analise (ARISA) is `n herhaalbare DNS vingerafdruk tegniek wat die intergeniese spasies gebied (ITS) in bakterieë teiken. Analise gebaseer op operasionele taksonomiese eenhede (OTEs) is gebruik om die mikrobiota van SVK segmente statisties te vergelyk. Die SVK kompartemente in forêl het ook duidelike bakteriële populasie strukture getoon. `n Groot variasie tussen identiese segmente van verskillende vise is waargeneem en individuele mikrobiota kon nie uitgesonder word nie. ARISA is gebruik om die effek van plantaardige dieet formulasies op die SVK mikrobiota te toets. Die SVK van visse speel `n rol in voedselvertering, terwyl die natuurlike SVK bakterieë `n belangrike rol speel in siektebeheer en die benutting van voedingstowwe. Twee voedingsproewe is op tilapia en forêl uitgevoer, met onderskeidelik 16 en 6 verskillende diëte,. Mikrobiese diversiteit was laer in die tilapia SVK in vergeleke met die SVK van forêl en geen beduidende verskuiwings in populasie struktuur is in beide visse waargeneem nie. `n Hoë deurvloei DNS-volgorde bepalings tegniek, gekombineer met `n metagenomiese benadering is gebruik om die mikrobiota van tilapia en forêl op `n taksonomiese vlak te bepaal. “Pyrosequencing” is gebruik om `n kern microbe populasie te identifiseer ten einde vergelykende studies te vergemaklik. Die voorgestelde kern mikrobiota in tilapia het bestaan uit genera van die Proteobakterieë (5), Firmikute (1), Aktinobakterieë (1), Fusobakterieë (1) en Sianobakterieë (2). Die voorgestelde kern mikrobiota van forêl het bestaan uit genera van die Proteobakterieë (3), Firmikutes (3) en Aktinobakterieë (1). Drie genera van die Proteobakterieë het algemeen in tilapia en forêl voorgekom, wat die oorheersing van hierdie groep in die SVK van varswatervisse bevestig. Die veranderlikheid en verspreiding van Lactobacillus in die SVKs van beide visse is bepaal. Dit is moelik om melksuurbakterieë (MSB) uit die SVK van tilapia te isoleer en `n lae voorkoms van Lactobacillus is waargeneem. Ander MSB genera is ook geïdentifiseer wat MSB as deel van die plaaslike mikrobiota in varswater gekweekte visse bevestig. Verder is daar ook vasgestel dat sekondêre patogene deel uitmaak van die plaaslike mikrobiota. In hierdie studie het 454 “pyrosequencing” `n dieper insig gegee oor die struktuur van bakterie-populasies in die SVK van tilapia en forêl. Hierdie navorsing is oorspronklik en volgens die literatuur die eerste studie wat `n kern mikrobe populasie in twee Suid-Afrikaanse akwakultuur spesies waargeneem het. Data wat gebasseer is op populasie diversiteit en struktuur kan gebruik word as basis vir verdere studies. Een so `n studie kan die effek van omgewings stresfaktore op die kern mikrobiota in hierdie vise bepaal.

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Phenobarbital (PB) is one of the most important antiseizure drugs (ASDs) to treat canine idiopathic epilepsy (IE). The effect of PB on the taxonomic changes in gastrointestinal microbiota (GIM) and their functions is less known, which may explain parts of its pharmacokinetic and pharmacodynamic properties, especially its antiseizure effect and drug responsiveness or drug resistance as well as its effect on behavioral comorbidities. Fecal samples of 12 dogs with IE were collected prior to the initiation of PB treatment and 90 days after oral PB treatment. The fecal samples were analyzed using shallow DNA shotgun sequencing, real-time polymerase chain reaction (qPCR)-based dysbiosis index (DI), and quantification of short-chain fatty acids (SCFAs). Behavioral comorbidities were evaluated using standardized online questionnaires, namely, a canine behavioral assessment and research questionnaire (cBARQ), canine cognitive dysfunction rating scale (CCDR), and an attention deficit hyperactivity disorder (ADHD) questionnaire. The results revealed no significant changes in alpha and beta diversity or in the DI, whereas only the abundance of Clostridiales was significantly decreased after PB treatment. Fecal SCFA measurement showed a significant increase in total fecal SCFA concentration and the concentrations of propionate and butyrate, while acetate concentrations revealed an upward trend after 90 days of treatment. In addition, the PB-Responder (PB-R) group had significantly higher butyrate levels compared to the PB-Non-Responder (PB-NR) group. Metagenomics of functional pathway genes demonstrated a significant increase in genes in trehalose biosynthesis, ribosomal synthesis, and gluconeogenesis, but a decrease in V-ATPase-related oxidative phosphorylation. For behavioral assessment, cBARQ analysis showed improvement in stranger-directed fear, non-social fear, and trainability, while there were no differences in ADHD-like behavior and canine cognitive dysfunction (CCD) scores after 90 days of PB treatment. While only very minor shifts in bacterial taxonomy were detected, the higher SCFA concentrations after PB treatment could be one of the key differences between PB-R and PB-NR. These results suggest functional changes in GIM in canine IE treatment.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-26T14:39:58Z No. of bitstreams: 1 joseeugenioriosriccijunior.pdf: 958378 bytes, checksum: f20499edf79931ac9fe901f9233cd200 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:34:06Z (GMT) No. of bitstreams: 1 joseeugenioriosriccijunior.pdf: 958378 bytes, checksum: f20499edf79931ac9fe901f9233cd200 (MD5) / Made available in DSpace on 2016-09-26T20:34:06Z (GMT). No. of bitstreams: 1 joseeugenioriosriccijunior.pdf: 958378 bytes, checksum: f20499edf79931ac9fe901f9233cd200 (MD5) Previous issue date: 2016-08-12 / O supercrescimento bacteriano do intestino delgado (SCBID) é definido como um aumento no número e/ou alteração no tipo de bactérias no intestino delgado. Na sua patogênese incluem dano à válvula ileocecal e anormalidades anatômicas, comuns na doença de Crohn (DC). O presente estudo tem como objetivo estudar a prevalência e preditores de SCBID em pacientes com DC, assim como avaliar a relação entre SCBID e inflamação sistêmica e/ou intestinal. Neste estudo transversal, entre Junho de 2013 e Janeiro de 2015, 92 pacientes com DC e 97 controles com queixas gastrointestinais não crônicas foram avaliados quanto à presença de SCBID usando o teste respiratório (TR) com hidrogênio e metano exalados. Regressão logística multivariada foi realizada para investigar a potencial associação entre SCBID com dados demográficos, características da doença, marcadores sistêmicos de inflamação (proteína C reativa - PCR - e velocidade de hemossedimentação de eritrócitos - VHS), e biomarcador fecal de inflamação intestinal (calprotectina fecal - CF). A taxa de SCBID foi significativamente maior em pacientes com DC do que nos controles (32,6% vs. 12,4%, respectivamente; p = 0,0008). Os pacientes com e sem SCBID foram semelhantes de acordo com os dados demográficos, marcadores inflamatórios sistêmicos e características da doença, exceto o fenótipo estenosante, mais comum nos pacientes com DC positivos para SCBID (43,3% vs. 19,3%; p = 0,015). Notavelmente, a concentração de CF foi significativamente mais elevada em pacientes positivos para SCBID (mediana de 485,8 vs.132,7 ug/g; p = 0,004). Os pacientes que apresentaram aumento da CF e doença estenosante tiveram um odds de 9,43 (IC de 95%, 3,04-11,31; p <0,0001) e 3,83 (95% CI, 1,54-6,75; p = 0,025), respectivamente, para o diagnóstico de SCBID. Em pacientes com DC, o SCBID é uma condição altamente prevalente. O fenótipo estenosante e o aumento da concentração de CF foram fortemente e independentemente associados com a presença de SCBID. O diagnóstico de SCBID seguido de tratamento direcionado é recomendado em pacientes com DC, especialmente no cenário de fenótipo estenosante e/ou na presença concomitante de inflamação intestinal. / Introduction: The small intestine bacterial overgrowth (SIBO) is defined as an increase in the number and/or change in the type of bacteria in the small intestine. In its pathogenesis it is important the damage on the ileocecal valve and anatomical abnormalities, both common in Crohn's disease (CD). The aim of this study was to determine the prevalence and predictors of SIBO in CD patients as well as to assess the relationship between SIBO and systemic and/or intestinal inflammation. In this cross-sectional study, between June 2013 and January 2015, 92 CD patients and 97 controls with non-chronic gastrointestinal complaints were assessed for presence of SIBO using the H2/CH4 glucose breath test. Multivariate logistic regression was performed to investigate the potential association between SIBO and demographic, disease-related data, systemic markers of inflammation (C-reactive protein and erythrocyte sedimentation rate), and biomarker of intestinal inflammation (fecal calprotectin concentration - FCC). The SIBO rate was significantly higher in CD patients than in the controls (32.6% vs. 12.4%, respectively, P=0.0008). Patients with and without SIBO were comparable according to demographics, systemic inflammatory biomarkers, and disease characteristics, except to the stricturing phenotype more common in the SIBO-positive CD patients (43.3% vs. 19.3%, P=0.015). Notably, FCC was significantly higher in SIBO-positive patients (median of 485.8 vs.132.7 μg/g; P = 0.004). Patients presenting increased FCC and stricturing disease had an odds of 9.43 (95% CI, 3.04-11.31; P <0.0001) and 3.83 (95% CI, 1.54-6.75; P=0.025) respectively, for SIBO diagnosis. Conclusions: In CD patients, SIBO is a highly prevalent condition. Stricturing phenotype and increased FCC were strongly and independently associated with the presence of SIBO. SIBO diagnostic work-up followed by directed treatment is recommended in CD patients, especially if stricturing disease or concurrent intestinal inflammation is present.