Thesis (MPH.) -- University of Limpopo, 2015 / Cholera is an acute enteric infection caused by the ingestion of bacterium Vibrio cholerae present in faecally contaminated water or food. Primarily linked to insufficient access to safe water and proper sanitation, its impact can be even more dramatic in areas where basic environmental infrastructures are disrupted or have been destroyed. The aim of the study was to investigate the factors contributing to the prevalence of cholera and the environmental risk factors associated with cholera in the Vhembe district of Limpopo province between 2008 and 2012. The objectives of the study were to identify environmental risk factors for cholera and to determine the number of cholera cases in the Vhembe district. The study used a quantitative, retrospective and cross-sectional research method. The records of 317 patients who met the study criteria were reviewed using an audit tool. The Statistical Package for Social Sciences (SPSS) version 22 was used to analyze the data. The results revealed that lack of adequate hygiene practices, limited access to safe drinking water, lack of safe food preparation and handling, and inadequate sanitation system are risk factors associated with cholera. The study recommends prevention, control of cholera outbreak and case management. Keywords: Cholera, outbreak, Vibrio cholerae 01 and 0139, Watery diarrhea (ricewater), Prevalence, Risk factors.

Marine aquaculture is already vital to global food security and will continue to become more important in the coming years. Crassostrea gigas (Pacific oysters) is the primary oyster species cultivated worldwide. The FAO and IPCC predict that climate change will create uncertainty and challenges for marine aquaculture. Baynes Sound, British Columbia, is a productive region for aquaculture, producing >50% of British Columbia’s total annual bivalve production by live weight and value. Major summer mortality events have been documented in farmed Crassostrea gigas globally since the 1950’s. These events are believed to be caused by a multiplicity of factors including changes induced by anthropogenic climate change. One of the major contributors to summer mortality is the proliferation of Vibrio bacteria, specifically Vibrio aestuarianus, which has been shown to increase in abundance and virulence when seawater temperatures rise. Despite this connection and the economic importance of oyster farming in the region, little is known about the presence of V. aestuarianus in Baynes Sound. Our 17-month study sampled 7 sites in Baynes Sound on 33 occasions from May 2019 to September 2020. We found a positive correlation between seawater temperature and total Vibrio detected in water samples in Baynes Sound, an association that was stronger when the overall temperature regime was warmer. We found no significant correlation between any of the bacterial assays tested and salinity, pH, or Ωarag saturation. We also did not identify a geographic pattern to bacterial abundance or virulence amongst test C. gigas in the field. Understanding that flagellates are the predominant type of microalgae present in Baynes Sound when summer mortality events occur, in lab trials, we found that incorporating V. aestuarianus into marine aggregates with flagellate microalgae caused higher mortality than aggregates with diatoms or planktonic V. aestuarianus. These results were not statistically significant but led us to look at how exposure to husbandry stress pre and post inoculation with V. aestuarianus incorporated into marine aggregates affects mortality. We found that stress was a significant driver of mortality, particularly when administered 24h post inoculation, suggesting that farmers should avoid sorting or tumbling their oysters in the summer, and particularly immediately after a marine heatwave. Oysters lack adaptive immune systems and are grown in an open ocean environment where it is not possible to eliminate their exposure to pathogens. These factors make it impossible to use vaccines or antibacterial disinfectants to combat diseases. Therefore, breeding genetic resistance to V. aestuarianus may be the most effective way to fight summer mortality. Creating a repeatable and accurate protocol for inoculating oysters with marine bacteria is key to accurate heritability measurements and the estimation of breeding values of different families. Key factors include controlling for dose per animal, laboratory efficiency, and inoculation via a mechanism that mimics real-world infection and does not bypass the animal’s immune defenses. We designed a protocol which controls for these factors, separating each animal into individual containers and adding a controlled dose of planktonic bacteria to each. Previously used methods of injection or using an infected “donor” animal in a group tank do not control for dose or bypass parts of the oysters’ natural immune system, potentially creating inaccuracies in survival data generated with these methods. After designing this protocol, we tested 32 full-sib families and estimated the heritability of survival to V. aestuarianus on the observed and underlying liability scales to be 0.095 (SE = 0.043), and 0.15 (SE = 0.068) respectively. We also found a strong negative correlation between oyster size and survival, with a gram of additional weight creating a 73% increase in the risk of death. Fast growth and large size are two traits which have been explicitly bred into C. gigas stock. Our work suggests that breeding a slower-growing, smaller animal may reduce summer mortality. / Graduate / 2022-09-15

Mittels Quorum sensing können Bakterienzellen die Expression von Genen Zelldichte-abhängig steuern. Dies spielt eine besondere Rolle bei der Expression von Virulenzfaktoren oder Antibiotikaproduktion, der Biofilmentwicklung oder Phänomenen wie genetischer Kompetenz, Sporulation oder Biolumineszenz. Vibrio harveyi, ein Gram-negativer, mariner, frei lebender Organismus, reguliert die Expression von Biolumineszenz-Genen Zelldichte-abhängig nach dem Prinzip des Quorum sensing und sollte im Rahmen dieser Arbeit als Modell für die Mechanismen der Signaltransduktion untersucht werden. Dazu wurden die Proteine der Lux-Signaltransduktionskaskade heterolog in E. coli überexprimiert und teilweise gereinigt. Mittels in vitro Phosphorylierung konnten die enzymatischen Aktivitäten der Proteine erstmals biochemisch charakterisiert werden. Für die Hybridsensorkinase LuxN konnte neben einer Kinase-Aktivität ein Phosphotransfer auf das Histidin-Phosphotransferprotein LuxU gezeigt werden. Die Autophosphorylierungsaktivität ist dabei eindeutig von der Konzentration des Signalmoleküls, eines Acyl-Homoserinlaktons, abhängig. Eine ebenfalls eindeutig nachgewiesene Phosphatase-Aktivität von LuxN, die zur Dephosphorylierung von LuxU führt, war dagegen konstitutiv. Damit konnte ein auf biochemischen Daten basierendes Modell der Signaltransduktion von V. harveyi postuliert werden. Basierend auf den Ergebnissen von Topologieuntersuchungen mittels luxN-Reportergenfusionen und Protease-Zugänglichkeitsstudien konnten neue Hinweise auf die Membrantopologie der Hybridsensorkinase ermittelt werden. Diese lassen auf ein Modell mit neun Transmembranhelices schließen, bei der der N-terminus des Proteins im Periplasma der Zelle lokalisiert zu sein scheint.

The primary contributor to Vibrio fischeri aminopeptidase activity is aminopeptidase N, PepN. Colonization assays revealed the pepN mutant strain to be deficient at forming dense aggregates and populating the host’s light organ compared to wildtype within the first 12 hours of colonization; however the mutant competed normally at 24 hours. To address the role of PepN in colonization initiation and establish additional phenotypes for the pepN mutant strain, stress response and other physiological assays were employed. Marked differences were found between pepN mutant and wildtype strain in response to salinity, acidity, and antibiotic tolerance. This study has provided a foundation for future work on identifying a putative role for V. fischeri PepN in regulating stress response.

Prebiotics and probiotics, proposed alternatives to antibiotics in shrimp aquaculture, are reported to improve growth parameters, promote disease resistance, and influence the gut microbial community. This study aimed to investigate the influence of prebiotic- or probiotic-coated feed and/or exposure to the Early Mortality Syndrome-causing strain of Vibrio parahaemolyticus ( VP-EMS) on the mid and hindgut microbiome of Pacific white shrimp (Litopenaeus vannamei). A monoculture probiotic strain of Bacillus subtilis spores: O14VRQ, and a prebiotic product of cultured Saccharomyces cerevisiae cell walls: MOS, were administered to shrimp as feed additives for 14 days, before a pathogen challenge to VP-EMS. Based on previous efforts, animals in this study were fed experimental diets for 14 days to allow ample amount of time for the prebiotic to be metabolized by health-promoting bacteria and for the probiotic spores to germinate. The pathogen challenge consisted of negative disease control (no VP-EMS exposure, commercial feed), positive disease control (VP-EMS exposure, commercial feed) and two treatment groups, probiotic (VP-EMS exposure) and prebiotic (VP-EMS exposure). DNA extraction, 16S rRNA gene amplicon sequencing, polymerase chain reaction (PCR), and sequencing were utilized to create an overview of the mid and hindgut microbial composition. No significant differences in survival were shown between experimental diets following exposure to sublethal levels of VP-EMS. Bioinformatic analyses revealed no distinct shifts in the mid and hindgut microbiome of shrimp across experimental diets and time points. Results of this data revealed that dominant members of the intestinal microbiome, Proteobacteria, Actinobacteriota, Bacteroidota, Verrucomicrobiota, Flavobacteriaceae, Demequinaceae, Vibrionaceae, Shewanellaceae, Rhodobacteriaceae, and Rubritaleaceae were relatively stable across treatments and time points. Sequencing methods such as metagenomics or metatranscriptomics should be utilized for a higher microbiome resolution. Furthermore, the use of quantitative polymerase chain reaction to quantify ingested probiotic spores, prebiotic-associated bacteria, and VP-EMS is recommended. / Master of Science in Life Sciences / Shrimp aquaculture is one of the fastest-growing aquaculture sectors that provides another supply of feed that save wild fish populations. The use of prebiotics and probiotics are reported to improve growth, provide disease protection, influence the gut community, improve the immune system, and serve as substitutes to antibiotics. In this study, the effect of probiotic-, or prebiotic-coated diets and/or exposure to the Early Mortality Syndrome-causing Vibrio parahaemolyticus (VP-EMS) strain on the gut communty of shrimp (Litopenaeus vannamei) was investigated. DNA extraction, 16S rRNA gene amplification, and sequencing were utilized to identify the microbes in the intestines of shrimp. Based on previous studies, animals in this study were fed experimental diets for 14 days to allow enough time for prebiotic-associated bacteria and probiotic spores to multiply within the intestines. Exposure to a sublethal level of VP-EMS did not significantly affect shrimp survival between treatments. Analyses showed no noticeable differences in the intestinal microbial communities between treatments and time points. This research provided initial proof of what microbes occupy the mid and hindgut microbiome. A higher resolution sequencing method is recommended to gain a better understanding of the microbes and their roles in the intestines. The use of quantitative polymerase chain reaction is warranted to evaluate the amount of ingested probiotic spores, prebiotic-associated bacteria, and VP-EMS.

Graduation date: 2006

Depuis 1987, la palourde japonaise est sujette à une infection récurrente : la Maladie de l'Anneau Brun (MAB), provoquée par la bactérie Vibrio tapetis. Cette infection est caractérisée par la formation d'un anneau brun sur la face interne des valves et provoque souvent la mort de la palourde, suite à l'invasion de son système circulatoire par la bactérie. Cependant, quelques spécimens peuvent guérir de cette infection en sécrétant une nouvelle couche minéralisée qui recouvre l'anneau brun. Le but du travail de cette thèse est de comprendre et caractériser comment les palourdes japonaises sont capables de résister à la MAB, principalement en étudiant le mécanisme de réparation coquillière. Pour ce faire, des techniques comme la Microscopie Electronique à Balayage (MEB), la micro-spectrométrie confocale Raman, la microsonde WDS (Wave-length Dispersive Spectrometry), ainsi que des colorations cellulaires, la biochimie et la sérologie ont été utilisées. La couche de réparation est composée d'aragonite, de polyènes, de molécules luminescentes au Raman et de molécules sulfatées. La microstructure de la couche de réparation est souvent différente de celle de la couche interne saine et montre une grande variabilité. De plus, la matrice organique coquillière de la couche de réparation est quantitativement et qualitativement différente de celle de la couche interne saine et montre également une grande variabilité. Ces différences saines-réparées et ces variabilités pourraient être expliquées par une modification du régime sécrétoire des cellules calcifiantes du manteau, ainsi que par une mobilisation d'hémocytes préalablement chargés en sels insolubles de carbonate de calcium au niveau des sites de réparation coquillière.

Plusieurs études ont examiné la sensibilité aux antimicrobiens chez les bactéries d’organismes provenant de produits issus de l’aquaculture ou de leur environnement. Aucune information n’est cependant disponible concernant la résistance aux antimicrobiens dans les bactéries de la flore de poissons ou de fruits de mer vendus au détail au Canada. C’est particulièrement vrai en ce qui a trait aux bactéries des genres Aeromonas et Vibrio, dont certaines espèces sont des agents pathogènes zoonotiques connus. Au cours de cette étude, la sensibilité aux antimicrobiens d’isolats d’Aeromonas spp. et de Vibrio spp. provenant de poissons et de crevettes domestiques et importés a été mesurée à l’aide de techniques de micro dilution en bouillon et/ou de diffusion sur disque. Les classes d’antimicrobiens examinés comprenaient les tétracyclines (TET), les inhibiteurs de la voie des folates (sulfadiméthoxine-triméthoprime, SXT), le florfenicol (FLO), et les quinolones (acide nalidixique / enrofloxacine, NA/ENO). Des valeurs seuils épidémiologiques pour Aeromonas et Vibrio ont été établies en utilisant la méthode d’interprétation normalisée des données de résistance provenant de diffusion sur disque. La recherche de gènes de résistance associés au profil de résistance des isolats a été effectuée en utilisant des PCRs et des puces ADN. Le nombre d’isolats résistants aux divers antimicrobiens parmi les 201 isolats d’Aeromonas et les 185 isolats de Vibrio étaient respectivement les suivants: TET (n=24 et 10), FLO (n=1 et 0), SXT (n=2 et 8), NA (n=7 et 5) et ENO (n= 5 et 0). Diverses associations de gènes tet(A), tet(B), tet(E), floR, sul1, sul2, et intI1 ont été détectées, les gènes tet(E), intI1, sul2 et tet(B) étant les plus communs. Les espèces d’Aeromonas et de Vibrio isolées de poissons au détail et de fruits de mer peuvent héberger une variété de gènes de résistance, bien que peu fréquemment. Le risque que représente ces gènes de résistance reste à évaluer en considérant le potentiel infectieux des bactéries, l’utilisation des ces agents antimicrobiens pour le traitement des maladies en aquaculture et en médecine humaine et leur rôle en tant que réservoir de la résistance antimicrobienne. / Multiple studies have examined antimicrobial susceptibility in bacteria from aquacultured products microorganisms and their environment. However, no information is available concerning antimicrobial resistance in bacterial flora of fish and seafood available at the retail level in Canada. This is particularly true for the common aquatic commensals, Aeromonas and Vibrio, for which some species are known zoonotic pathogens. In the course of this study, the antimicrobial susceptibility among Aeromonas spp. and Vibrio spp. from domestic and imported fish and seafood was characterized. Aeromonas and Vibrio spp. isolates cultured from finfish and shrimp samples were evaluated for antimicrobial susceptibility by broth microdilution and/or disk diffusion techniques. Antimicrobial classes examined in detail included: tetracyclines (TET), folate pathway inhibitors (sulfadimethoxine-trimethoprim, SXT), florfenicol (FLO), and the quinolones (nalidixic acid / enrofloxacin, NA/ENO). Epidemiological cut-off values (ECV’s) for Aeromonas/Vibrio were established using normalized resistance interpretation (NRI) of disk diffusion data. Isolates were further examined by PCR and microarray for genes associated with their antimicrobial resistance. Of 201 Aeromonas and 185 Vibrio isolates, those classified as resistant were as follows, respectively: TET (n=24 and 10), FLO (n=1 and 0), SXT (n=2 and 8), NA (n=7 and 5) and ENO (n=5 and 0). Various combinations of tet(A), tet(B), tet(E), floR, sul1, sul2 and intI1 genes were detected with tet(E), intI1, sul2 and tet(B) being the most common. Vibrio and Aeromonas species isolated from retail fish and seafood sources can harbor a variety of resistance determinants, although their occurrence is not high. The risk represented by these resistances remains to be evaluated in view of the potential for bacterial infection and their role as a reservoir for antimicrobial resistance.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-07T11:44:28Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_DiversidadeGeneticaIsolados.pdf: 1765217 bytes, checksum: e159b664332d4cd1ed8d170a0a2c6559 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-12T15:56:27Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_DiversidadeGeneticaIsolados.pdf: 1765217 bytes, checksum: e159b664332d4cd1ed8d170a0a2c6559 (MD5) / Made available in DSpace on 2014-02-12T15:56:27Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_DiversidadeGeneticaIsolados.pdf: 1765217 bytes, checksum: e159b664332d4cd1ed8d170a0a2c6559 (MD5) Previous issue date: 2009 / Vibrio cholerae, agente etiológico da cólera, é uma bactéria nativa de ambientes aquáticos de regiões temperadas e tropicais em todo o mundo. A cólera é endemica e epidemica em países da África, Ásia e Americas Central e do Sul. Neste trabalho o objetivo foi estudar a diversidade genética de isolados desta espécie, de ambientes aquáticos da Amazônia brasileira. Um total de 148 isolados de V.cholerae não-O1 e não-O139 (NAGs) e O1 ambientais da Amazônia, obtidos entre 1977 e 2007, foram caracterizados e comparados a linhagens clínicas de V.cholerae O1 da sexta e sétima pandemias. Utilizou-se os perfis de macrorestrição definidos em eletroforese em gel de agarose em campo pulsado (PFGE), para determinar a relação clonal entre V.cholerae non-O1 e O1 ambientais e clínicos. A presença de genes de virulência (hlyA/hem, hlyB, hlyC, rtxA, rtxC, tcp, ctx, zot, ace, stn/sto) e integrons de classe 1, 2 e 3 (intI 1, 2 e 3), foi analisada utilizando-se a reação em cadeia da polimerase. A análise dos perfis de macrorestrição revelou que os NAGs apresentaram uma grande diversidade genética comparada aos V.cholerae O1. Isolados de NAGs e O1 segregaram em distintos grupos e a maioria dos O1 ambientais apresentou relação clonal com isolados clínicos da sétima pandemia de cólera. A distribuição dos genes de virulência entre os NAGs é diferente a dos O1, os quais, em geral, foram positivos para todos os genes de virulência estudados exceto stn/sto e integrons de classe 1, 2 e 3. Alguns V.cholerae O1 ambientais pertencentes a linhagem da sétima pandemia, apresentaram uma extensiva perda de genes. Diferentes NAGs foram stn/sto+ e intI 1+. Dois alelos do gene aadA foram encontrados: aadA2 e aadA7. De modo interessante os V.cholerae O1 ambientais pertencentes à linhagem pandêmica, só foram isolados durante o período da última epidemia de cólera na região Amazônica brasileira (1991-1996). / Vibrio cholerae, the etiologic agent of cholera, is an autochtonus bacterium of aquatic environment in temperate and tropical regions of the world. Cholera is endemic and epidemic in many countries in Africa, Asia and Central and South America. In this study our goal was to detemine the genetic diversity of V.cholerae environmental isolates from aquatic ecosystems in the Amazon region of Brazil. A total of 148 environmental strains of V.cholerae non-O1 and non-O139 (NAG) and O1 serogroups, isolated from the Amazon region since 1977 to 2007, were characterized and compared with clinical strains of V.cholerae O1 from sixty and seventh cholera pandemic. PFGE (pulsed-field gel electrophoresis) was performed to determine the clonal relationships between V.cholerae non-O1, O1 environmental and clinical strains. The presence of virulence genes (hlyA/hem, hlyB, hlyC, rtxA, rtxC, tcp, ctx, zot, ace, stn/sto) and class 1, 2 and 3 integrons (intI 1, 2 e 3) was analyzed by polymerase chain reaction. Whole genome macrorestriction analysis revealed that the environmental V.cholerae NAGs were more diverse than the environmental O1 strains, both groups segregate in distinct clusters and most of environmental O1 strains show a clonal relationship with seventh cholera pandemic strains. The distribution of virulence genes in NAGs strains is largely different from that of O1 strains which, in general, were positive for all virulence genes analyzed excepting for stn/sto and class 1, 2 and 3 integrons. Some O1 environmental strains, belonging to the seventh pandemic lineage, went through an extensive gene loss. Distinct NAGs strains were stn/sto+ and intI 1+. Two alleles of aadA were found: aadA2 and aadA7. Interestingly, V.cholerae O1 environmental strains belonging to the pandemic lineage were only isolated during the period of cholera epidemic in the Amazon region of Brazil (1991-1996).

Submitted by (edna.saturno@ufrpe.br) on 2017-02-09T14:06:18Z No. of bitstreams: 1 Danielli Matias de Macedo Dantas.pdf: 243863 bytes, checksum: 12196d11f6515cb7163e69e8e9c45af9 (MD5) / Made available in DSpace on 2017-02-09T14:06:18Z (GMT). No. of bitstreams: 1 Danielli Matias de Macedo Dantas.pdf: 243863 bytes, checksum: 12196d11f6515cb7163e69e8e9c45af9 (MD5) Previous issue date: 2008-02-08 / The fast expansion of farmed shrimp industry during the last decades has been offering high profitable deal to many countries. However, diseases appearing have caused lots of serious economic damage on productive sector. The effect of probiotic in Litopenaeus vannamei juveniles when submitted to the infection by Vibrio harveyi was evaluated in the present study The exposure to V. harveyi was carried out during 15 days using individuals reared for one month in a closed recirculation system with a commercial probiotic (Bacillus sp.) added to water and food (PAR), only in the water (PA), only in food (PR) and without probiotic (SP). Were examined the responses of food consumption, total concentration of hemocytes, growth and survival. The food consumption did not differ significantly among the treatments, but in the SP treatment a significant reduction occurred in the second week. The PAR and PA treatments showed inferior and more constants values in the total hemocits concentrations during the experimental period. The highest growth rates were observed in the PAR (0.34 ± 0.19 g/week) and SP (0.45 ± 0.04 g/week) treatments, but did not differ statistically within these treatments. Shrimp survival values were higher than 98% in all treatments. The results suggest that the addition of Bacillus sp. to the water or food separately, could increase the food consumption during a longer period of time in shrimp infected by V. harveyi and interfere positively in the immune response of L. vannamei. / A rápida expansão da indústria do camarão na aqüicultura durante poucas décadas tem proporcionado a muitos países altos rendimentos. Entretanto, o surgimento de doenças tem causado sérios prejuízos econômicos ao setor produtivo. Este trabalho verificou o efeito do uso de probiótico no cultivo de juvenis de Litopenaeus vannamei quando submetidos à infecção ao Vibrio harveyi. A exposição ao V. harveyi foi realizada durante 15 dias nos camarões provenientes de um cultivo de 30 dias em sistema fechado sem renovação de água com utilização de um probiótico comercial (Bacillus sp.) adicionado na água e na ração (PAR), somente na água (PA), somente na ração (PR) e sem adição de probiótico (SP). Foram analisadas as respostas de consumo alimentar, concentração total de hemócitos, crescimento e sobrevivência. A análise do consumo alimentar dos camarões não mostrou diferença significativa entre os tratamentos, sendo a única exceção o tratamento SP onde ocorreu uma redução no consumo na segunda semana. Os tratamentos PAR e PA apresentaram valores menores e mais constantes de concentração total de hemócitos durante o período do experimento. As maiores taxas de crescimento foram verificadas nos tratamentos PAR (0,34 ± 0,19 g/semana) e SP (0,45 ± 0,04 g/semana), os quais não diferiram estatisticamente entre si. Os camarões apresentaram valores de sobrevivência acima de 98% em todos os tratamentos. Os resultados sugerem que a utilização do Bacillus sp. adicionados na água ou na ração separadamente, aumentam o consumo alimentar por um maior período de tempo em camarões infectados pelo V. harveyi e interferem positivamente na resposta imune de L.vannamei.