Search

Poster intitulé "Identification of a new pharmacological target for the treatment of vibriosis", présenté durant le 17ème Congrès de la Société Française de Microbiologie "Microbes" (3-5 octobre 2022, Montpellier). https://www.alphavisa.com/sfm/2022/documents/SFM2022_Livre-Resumes.pdf

Submitted by Simone Maisonave (simonemaisonave@hotmail.com) on 2022-09-01T12:36:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Amanda_Barbosa_Atrib.pdf: 727091 bytes, checksum: b916a05153e217c4fed999889710fe03 (MD5) Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2022-09-06T23:43:51Z (GMT) No. of bitstreams: 2 Dissertacao_Amanda_Barbosa_Atrib.pdf: 727091 bytes, checksum: b916a05153e217c4fed999889710fe03 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Made available in DSpace on 2022-09-06T23:43:52Z (GMT). No. of bitstreams: 2 Dissertacao_Amanda_Barbosa_Atrib.pdf: 727091 bytes, checksum: b916a05153e217c4fed999889710fe03 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2021-03-31 Sem bolsa O pescado pode ser contaminado no ambiente em que vive ou durante a manipulação após a captura. A espécie bacteriana Vibrio parahaemolyticus é uma das principais causadoras de surtos associados ao consumo de pescados contaminados e já foi isolada de peixes da espécie Micropogonias furnieri, no entanto, pouco se sabe sobre o comportamento deste patógeno na matriz alimentar do pescado, tampouco sobre métodos de controle. Os óleos essenciais vêm sendo utilizados no preparo de alimentos em virtude do sabor e aroma diferenciados. Dentre outras propriedades, o óleo essencial de Origanum vulgare se destaca pela capacidade de inibir o crescimento de determinados microrganismos indesejáveis. As coberturas contendo óleo essencial de plantas ganharam reconhecimento ultimamente, sendo empregados para uma melhora na qualidade e segurança alimentar, no entanto, ainda são necessários mais estudos acerca do tema. O objetivo deste estudo foi avaliar a atividade antimicrobiana de óleo essencial de O. vulgare associado a cobertura comestível de amido em filé de peixe. O efeito do óleo de O. Vulgare foi testado in vitro pelo teste de disco-difusão. Posteriormente, cobertura de amido com 1% e 1,5% de óleo essencial de O. vulgare foi aplicada em filés de M. furnieri experimentalmente contaminados com V. parahaemolyticus. Como controle, filés sem cobertura e com cobertura sem óleo também foram testados. Foram realizadas contagens de V. parahaemolyticus após 0, 1, 2, 3, 4, 5 e 10 dias de estocagem. No teste de disco-difusão o óleo essencial de O. vulgare apresentou efeito inibitório sobre o crescimento bacteriano. A cobertura de amido associada ao óleo essencial de O. vulgare foi eficiente para reduzir a multiplicação bacteriana nos filés de peixe experimentalmente contaminados nas duas concentrações utilizadas no estudo, mantendo as contagens abaixo do limite máximo admitido pela legislação brasileira. A utilização de cobertura de amido com óleo essencial de O. vulgare no controle de V. parahaemolyticus em pescados é uma alternativa promissora para o consumo seguro desses alimentos. Fish can be contaminated in the environment in which it lives or during handling after being caught. The bacterial species Vibrio parahaemolyticus is one of the main causes of outbreaks associated with the consumption of contaminated fish and has already been isolated from fish of the species Micropogonias furnieri, however, little is known about the behavior of this pathogen in the fish's food matrix, nor under control. The essential oils used in food preparation due to their distinctive flavor and aroma. Among other properties, the essential oil of Origanum vulgare stands out for its ability to inhibit the growth of undesirable microorganisms. The coverings containing essential oil of plants have gained recognition lately, being used for a better quality and food security, however, more studies on the subject are still addressed. The aim of the study was to evaluate an antimicrobial activity of O. vulgare essential oil associated with an edible starch cover in fish fillet. The effect of O. Vulgare oil was tested in vitro disc diffusion test. Subsequently, starch coverage with 1% and 1.5% essential oil of O. vulgare was applied to fillets of M. furnieri experimentally contaminated with V. parahaemolyticus. As a control, fillets without cover and with coverage without oil were also tested. V. parahaemolyticus counts were performed after 0, 1, 2, 3, 4, 5 and 10 days of storage. In the disk-diffusion test, the essential oil of O. vulgare had an inhibitory effect on bacterial growth. The coverage associated with the essential oil of O. vulgare was efficient to reduce bacterial multiplication in fish fillets experimentally contaminated in the two rules used in the study, keeping as counts below the maximum limit allowed by Brazilian legislation. The use of starch cover with O. vulgare essential oil to control V. parahaemolyticus in fish is a promising alternative for safe food consumption.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES A pesquisa objetivou-se em relacionar as análises bacteriológicas de vibrio com a análise presuntiva indicativa de vibriose no camarão marinho Litopenaeus vannamei cultivado em sistema semi-intensivo de baixa salinidade submetidos a dois ciclos de produção a fim de melhorar os resultados nas fazendas de camarão.O estudo foi desenvolvido em campo, utilizando três viveiros denominados V11, V12 e V13 em dois ciclos de produção em sistema semi-intensivo com baixa salinidade. Com coletas semanais, o experimento teve início em abril de 2019 estendendo-se até outubro de 2019. Foram avaliados a correlação entre a contagem de vibrios sacarose positiva e sacarose negativa com as variáveis da análise presuntiva (Preenchimento dos Túbulos do Hepatopâncreas-P1, Necrose no hepatopâncreas -P2, Deposição de cálcio -P3, Cor do urópodes-P4 e Black spot -P5) e com salinidade, temperatura, pH e Oxigênio Dissolvido (O2) da água. No ciclo I, as bactérias sacarose negativa apresentaram correlação positiva com a temperatura da água, com P2 e com P5. E também uma correlação negativa entre sacarose negativa e P3. Já no segundo ciclo apresentou correlação positiva entre as sacarose negativa com o pH da água, P2 e P5 e um correlação negativa entre sacarose negativa com O2 e P4. Foram identificadas em todos os viveiros e ciclos as bactérias sacarose positiva V. Cholera e V. alginolyticus, e as bactérias Sacarose negativaV. parahaemolyticus e V. Vulnificus. Percebeu-se que a contaminação do camarão próxima a de 104 UFC/g associado à presença de necrose no hepatopâncreas e black spot pode confirmar indicativo de vibriose. Concluise que a análise presuntiva com índices elevados das características de necrose no hepatopâncreas e black spot pode suspeitar de vibriose, sendo confirmada pela técnica microbiológica tradicional. Isso irá permitir que os criadores tomem os cuidados de manejo mais rapidamente a partir do exame presuntivo em campo The research aimed to relate the bacteriological analyzes of vibrio with the presumptive indicative analysis of vibriosis in the marine shrimp Litopenaeus vannamei grown in a low-salinity semi-intensive system submitted to two production cycles in order to improve the results in the shrimp farms.The study was developed in the field, using three nurseries called V11, V12 and V13 in two production cycles in a semi-intensive system with low salinity. With weekly collections, the experiment started in April 2019, extending until October 2019. The correlation between the positive sucrose and negative sucrose vibrios count with the variables of the presumptive analysis (Hepatopancreas Tubules-P1, Necrosis in hepatopancreas -P2, calcium deposition -P3, color of uropodsP4 and black spot -P5) and with salinity, temperature, pH and dissolved oxygen (O2) in the water. In cycle I, the negative sucrose bacteria showed a positive correlation with water temperature, with P2 and with P5. And also a negative correlation between negative sucrose and P3.In the second cycle, there was a positive correlation between negative sucrose with water pH, P2 and P5 and a negative correlation between negative sucrose with O2 and P4. In all the nurseries and cycles, the positive sucrose bacteria V. Cholera and V. alginolyticus, and the negative sucrose bacteria were identified V. parahaemolyticus and V. Vulnificus. It was realized that contamination of shrimp close to 104 UFC/g associated with the presence of necrosis in the hepatopancreas and black spot can confirm an indication of vibriosis. It is concluded that the presumptive analysis with high indices of necrosis characteristics in the hepatopancreas and black spot may suspect vibriosis, being confirmed by the traditional microbiological technique. This will allow breeders to take care of handling more quickly from the presumptive examination in the field

Global change, including anthropogenic activities, have resulted in an increase in the incidence of Vibrio-associated illnesses. These diseases not only affect humans but also marine animals. Despite strong ecological and economic consequences, little is known about population structure and virulence mechanisms of vibrios in the environment. To better understand and manage those diseases, we explored the ecology, the evolution and the virulence of these infectious agents. First, we found that some environmental strains were virulent towards oyster and we identified original virulence mechanisms related to their ecology and evolution. In France, oyster-farms are facing massive mortality events associated with the presence of a virus and bacteria of the genus Vibrio. We showed that the virus appears neither essential nor sufficient to cause high mortality rates contrary to the vibrios that play a major role. Juvenile diseased oysters were always co-infected by several populations, but only one, Vibrio crassostreae, was detected systematically and in abundance. Its virulence is dependent on a type VI secretion system that is carried by a conjugative plasmid. Our results suggest that V. crassostreae first differentiated into a low-virulent oyster colonizer and turned into a pathogen after acquisition of the virulence plasmid. The narrow distribution of the plasmid suggests that it has been selected by high density farming areas. Finally, we showed that the plasmid transfer or selection was enhanced in oysters, which suggests that oysters represent a niche for horizontal gene transfer. Overall, this work can lead to the development of diagnostic tools and epidemiological monitoring of pathogenic vibrios.; Les changements globaux provoquent une augmentation des infections associées aux Vibrio. Les vibrioses affectent les humains, mais aussi des animaux marins. Un problème majeur est le manque de connaissances sur les vibrios présents dans l’environnement. Pour mieux comprendre et gérer ces maladies, nous nous sommes intéressés à l’écologie, l’évolution et la virulence de ces agents infectieux. Tout d’abord, nous avons montré que dans la nature, certains génotypes de Vibrio sont virulents pour les huîtres et que leurs mécanismes de virulence sont impliqués dans la différentiation écologique de ces souches. En France, les parcs ostréicoles font face à des évènements de mortalités massives associés à la présence d’un virus et de bactéries du genre Vibrio. Nous avons montré que le virus n’est ni essentiel, ni suffisant pour induire de fortes mortalités, contrairement aux vibrios qui jouent un rôle majeur. Les huîtres juvéniles malades sont toujours infectées par plusieurs populations, mais une seule, Vibrio crassostreae, est détectée en abondance. La virulence de cette population dépend d’un système de sécrétion de type IV codé par un plasmide conjugatif. Nos résultats suggèrent que V. crassostreae était un colonisateur peu virulent et que l’invasion du plasmide l’a rendu hautement pathogène. La distribution restreinte du plasmide de virulence suggère qu’il a été sélectionné par les hautes densités d’élevages. Enfin, nous avons montré que le transfert ou la sélection du plasmide était favorisé dans l’huître, suggérant que l’huître représente une niche propice aux transfert latéraux de gènes. Ce travail pourra permettre le développement d’outils de diagnostic et le suivi épidémiologique des vibrios pathogènes.

Global changes linked to increases in temperature and ocean acidification, but also to more direct anthropogenic influences such as aquaculture, have caused a worldwide increase in the reports of Vibrio-associated illnesses affecting humans and also animals such as shrimp and molluscs. Investigation of the emergence of Vibrio pathogenesis events requires the analysis of microbial evolution at the gene, genome and population levels, in order to identify genomic modifications linked to increased virulence, resistance and/or prevalence, or to recent host shift. From a more applied point of view, the elucidation of virulence mechanisms is a prerequisite to devising prophylactic methods to fight infectious agents. In comparison with human pathogens, fairly little is known about the requirements for virulence in vibrios pathogenic to animals. However, the advent of genome sequencing, especially next-generation technologies,the possibility of genetically manipulating most of the Vibrio strains, and the recent availability of standardised animals for experimental infections have now compensated for the considerable delay in advancement of the knowledge of non-model pathogens such as Vibrio and have led to new scientific questions., Nous assistons actuellement à un accroissement mondial du nombre de maladies imputables à Vibrio rapportées aussi bien chez l’homme que chez des espèces animales comme les crevettes et les mollusques, dû aux changements planétaires liés à l’augmentation des températures et à l’acidification des océans, parmi d’autres influences plus directement associées à l’activité humaine, notamment l’aquaculture. Les études sur la pathogenèse de l’émergence de Vibrio recourent à des analyses de l’évolution microbienne tant au niveau génétique et génomique qu’à l’échelle des populations, de manière à identifier les modifications génomiques associées à l’accroissement de la virulence, de la résistance et/ ou de la prévalence, ainsi que l’extension récente des spécificités d’hôte. D’un point de vue plus pratique, l’élucidation des mécanismes de virulence est une condition préalable à la conception de méthodes prophylactiques permettant de lutter contre les agents infectieux. Les critères de virulence des Vibrio pathogènes pour les animaux sont beaucoup moins connus que ceux des agents pathogènes affectant l’être humain. Néanmoins, l’avènement des techniques de séquençage du génome, en particulier celles de nouvelle génération, la possibilité de soumettre la plupart des souches de Vibrio à des manipulations génétiques et la standardisation récente de modèles animaux pour des essais d’infection expérimentale ont permis de rattraper une grande partie du retard des connaissances sur les agents pathogènes non utilisés comme modèles tels que Vibrio, en suscitant de nouvelles questions scientifiques.

A carcinicultura mundial sofre grandes perdas econômicas por enfermidades virais e bacterianas, sendo importante que sejam incentivados estudos e ações que minimizem os danos provocados por estas doenças em todas as áreas, desde os econômicos, saúde animal e humana até ambientais. Diante destes fatos, foram avaliados: a) o efeito da presença de areia como substrato sobre o comportamento de camarões juvenis Litopenaeus vannamei em laboratório (manuscrito I); b) as atividades comportamentais de camarões juvenis L. vannamei infectados por Vibrio parahaemolyticus (ATCC 17802) e submetidos a tratamentos veterinários em laboratório (manuscrito II); c) identificação dos agentes envolvidos em enfermidades com altas mortalidades em fazendas de carcinicultura (manuscrito III). O estudo foi realizado em três etapas. Na primeira etapa (manuscrito I), os animais foram submetidos a tratamentos com ausência ou presença de areia como substrato, divididos em dois grupos, cujos animais foram colocados em 30 aquários de material plástico com capacidade para quatro litros, 15 deles com areia como substrato e 15 sem areia. Foram avaliados os comportamentos de enterramento somente para o grupo com areia e inatividade, natação, ingestão de alimento, limpeza e rastejamento; sobrevivência e ganho de peso para ambos os grupos. Na segunda etapa (manuscrito II), os animais foram divididos em cinco grupos: 1) Controle 1, 2) Controle 2 -animais infectados e sem tratamento veterinário; 3) Animais infectados, tratados com medicamento isopático ISO; 4) Animais infectados, tratados com medicamento antibiótico (florfenicol) ANTIB; 5) Animais infectados, tratados com medicamentos isopático e antibiótico (florfenicol) simultaneamente ISO+ANTIB. Foram então analisados os comportamentos desses animais, a sobrevivência e o ganho de peso. Na terceira etapa (manuscrito III), foi visitada uma fazenda de carcinicultura com altos índices de mortalidade na cidade de Obregón, em Sonora, no México. Foram coletados 30 animais dessa fazenda, os quais foram levados para análises presuntivas (análise a fresco e bacteriologia) e confirmatórias (bioquímica e qPCR) para identificação de agentes patogênicos virais e/ou bacterianos. Os resultados obtidos no manuscrito 1 mostram que não houve diferença significativa entre os comportamentos observados, também não houve diferença entre crescimento e ganho de peso, indicando que a ausência de areia não interferiu na expressão de comportamento dos animais. Isto possibilita que os experimentos seguintes não usem areia como substrato. No manuscrito 2, os camarões infectados e tratados com isopatia e antibiótico simultaneamente apresentaram desempenho comportamental semelhante ao grupo controle 1, indicando que apresentam comportamento compatível com animais saudáveis. No Manuscrito 3, ficou evidente que a alta mortalidade identificada em viveiros com camarões juvenis, além da cepa patógena causadora da AHPND, Vibrio parahaemolyticus, outras espécies de bactérias dos gêneros Vibrio e Aeromonas estavam associadas. World shrimp culture suffers large economic losses for viral and bacterial diseases, so it is important that studies and actions be encouraged to minimise the damage caused by these diseases in all areas - economy, animal and human health and environment. The present study evaluated the effect of a) presence of the substrate on the behaviour of juvenile shrimp Litopenaeus vannamei in the laboratory (manuscript I); b) behavioral activities of juvenile shrimp L. vannamei infected by Vibrio parahaemolyticus (ATCC 17802) and subjected to veterinary treatments in laboratory (manuscript II); c) identified the pathogenic agents associated to diseases with high mortality in shrimp culture farms (manuscript III). The study was conducted in three stages. In the first stage (manuscript I), the animals were subjected to two treatments, the absence or presence of sand as substrate, and were divided into two groups, placed in 30 plastic aquariums with capacity for four liters, half of them with sand on the bottom as substrate and the other half without sand. Burying behavior was evaluated only for the group whose aquariums had sand as substrate; inactivity, swimming, food intake, cleaning and crawling, survival and weight gain were measured in both groups. In the second stage (manuscript II), the animals were divided into five groups: 1) control, 2) infected animals with no veterinary treatment; 3) infected animals treated with isopathic medicine; 4) infected animals treated with antibiotics (florfenicol); 5) infected animals treated with isopathic medicine plus antibiotics (florfenicol). Then, the behaviors and survival and weight gain of the animals were analysed. In the third stage, we visited a shrimp farm with high rates of mortality in Obregón city, Sonora, Mexico. Thirty animals collected in the ponds were taken to presumptive (fresh smear analysis and bacteriology) and confirmatory (Biochemistry and qPCR) analyses for identification of viral and/or bacterial pathogens. The results in Manuscript 1 showed that for juvenile prawns maintained in aquariums with or without sand as substrate for up to three weeks, behavior, survival and weight gain were not significantly different, which demonstrates that lack of sand on the substrate did not modify behavioral responses of the animals. So, in the next stage, sand was not used as substrate anymore. In Manuscript 2, the infected prawns that were treated with isopathy plus antibiotics showed behavioral performance similar to the control group, indicating that their behavior is compatible with healthy individuals. And in Manuscript 3, concerning the high mortality of juvenile prawns in the ponds, besides the pathogenous that causes AHPND, Vibrio parahaemolyticus, other species of bacteria of the genus Vibrio and Aeromonas were associated to the pathology of the prawns.

For fifteen years, summer mortalities have been observed in wild and farmed populations of European abalone, Haliotis tuberculata, along the north French coast. These mortalities are attributed to the bacterial species Vibrio harveyi and occur in sexually mature animals, when the seawater temperature exceeds 17°C.A multifactorial approach to the study of this host-parasite interaction was done during this thesis, in order to specify the intrinsic abalone conditions in vibriosis mortalities, the infectious cycle of V. harveyi in European abalone and the role of temperature in the fulfillment of this infectious cycle, and finally the physiological response of abalone, at cellular and molecular level, when exposed to V. harveyi.The main results showed a differential gene expression between resistant and susceptible abalone during exposure to V. harveyi, indicating the importance of the physiological status of the host, in survival to vibriosis. This hypothesis is supplemented by the susceptibility of sexually immature abalone at 19°C to this disease, usually resistant, and exposed to manipulation stressor. Moreover, the study of the portal of entry and the dynamics of infection by V. harveyi in European abalone revealed a particular tropism of this vibrio pathogen for gill tissues, in the earlier hours of contact, and its invasion into the circulatory system from the first 24 hours of contact. The study of the response of abalone hemocyte and gill metabolism, at the cellular and molecular level, in the earlier hours of contact, shows 1/ a genesis of oxidative stress in gills of susceptible abalone, and 2/ an alteration of hemocyte functions, which may constitute one of the major strategies of virulence in V. harveyi., Depuis une quinzaine d’années, des mortalités estivales d’ormeaux européens, Haliotis tuberculata, surviennent sur le littoral breton et normand, et en structures aquacoles. Ces mortalités sont attribuées à l’espèce bactérienne Vibrio harveyi, et se produisent chez des ormeaux sexuellement matures lorsque la température de l’eau dépasse 17°C.Ce travail de thèse visait en une approche multifactorielle de l’étude de cette interaction hôte-parasite, afin de spécifier les conditions intrinsèques aux ormeaux dans le déclenchement de cette vibriose, le cycle infectieux de V. harveyi chez l’ormeau européen et le rôle de la température dans l’accomplissement de ce cycle infectieux, et enfin la réponse physiologique de l’ormeau lors d’une exposition à V. harveyi.Les principaux résultats montrent un différentiel d’expression génomique entre des ormeaux résistants et des ormeaux sensibles au cours d’une exposition à V. harveyi, attestant ainsi l’importance du statut physiologique de l’hôte dans la survie à la vibriose chez l’ormeau européen. Ce constat est supplémenté de la mise en évidence de sensibilité à cette maladie chez des ormeaux sexuellement immatures, habituellement résistants, acclimatés à 19°C et exposés à des conditions contraignantes de type manipulation. Par ailleurs, l’étude de la voie d’entrée et de la dynamique d’infection de V. harveyi chez l’ormeau européen a révélé un tropisme particulier de ce vibrion pathogène vers les tissus branchiaux dès les premières heures de contact, et son invasion dans le système circulatoire dès 24h de contact. L’étude de la réponse hémocytaire des ormeaux et du métabolisme branchial, à l’échelle moléculaire et cellulaire, lors des premières heures de contact, démontre 1/ la genèse d’un stress oxydatif au niveau des branchies d’ormeaux sensibles à la vibriose et 2/ une altération du fonctionnement des hémocytes, ce qui présume de l’une des stratégies majeures de virulence de V. harveyi.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. Programa de Pós-Graduação em Aquicultura O objetivo deste trabalho foi verificar se saturações de 30, 60 e 100% de oxigênio dissolvido exercem influência sobre a sobrevivência e sobre os parâmetros imunológicos, contagem total de hemócitos e espécies reativas de oxigênio em camarões da espécie Litopenaeus vannamei, sob desafio do vírus da mancha branca e da bactéria Vibrio alginolyticus. A infecção ocorreu por inoculação direta na musculatura. Os desafios experimentais tiveram duração de 96 horas com vírus e 72 horas com a bactéria e os parâmetros imunológicos foram avaliados a cada 24 horas. Houve uma menor mortalidade no grupo de animais com 100% de saturação de oxigênio no desafio com vírus e uma maior mortalidade na saturação de 30% de oxigênio para o V. alginolyticus. Não foram encontradas diferenças entre os três níveis de oxigênio dissolvido durante o período experimental, tanto para a bactéria como para o vírus, nos parâmetros imunológicos. O grupo de animais não infectados não apresentou mortalidade e os parâmetros imunológicos permaneceram estáveis.

The use of wrasse (Labridae) as cleanerfish in salmonid aquaculture is the only method for removal of salmon lice that does not have any negative impact on the environment. The use of wild-caught wrasse has increased during the past few years due to increasing problems with salmon lice developing resistance to chemotherapeutica. The use of wrasse is not ethically acceptable, due to a continuous loss of wrasse in the salmon farms, resulting in an unacceptably high use of wrasse. One of the species used is the corkwing wrasse (Symphodus melops). Previous studies indicate that this species is susceptible to disease caused by Vibrio bacteria. Detection of Vibrio tapetis (LP2) in Norwegian corkwing wrasse was the first detection of this bacterium in fish and the first detection in Norway. In this Master thesis, V. tapetis LP2 was used in a series of challenge experiments with wild-caught corkwing wrasse. In addition to LP2, two other V. tapetis strains from clams were used; the reference strain CECT 4600 from Manila clams (Ruditapes philippinarum) in France and NRP45, from Manila clams introduced in Norway. Three experiments were performed: Corkwing wrasse were challenged with three doses of LP2 by bath challenge and intraperitonal injections in order to choose the better challenge method. The results were not consistent, but bath challenge with >106 CFU x ml-1was considered a reliable method, and applied in the following experiments. The next experiment was performed in order to study the effect of increasing temperature on LP2 challenge. Fish were challenged with 107 CFU x ml-1 at 6, 9, 12 and 15 °C respectively. The results revealed a trend of increased mortality at increasing temperatures, and a significantly higher mortality was measured at 15 °C than at 6 °C. In the third experiment, fish was challenged with LP2, CECT 4600 and NRP 45. LP2 was re-isolated from dead fish, whereas the other two strains were not. V. tapetis was also isolated from fish that were challenged with CECT 4600 and NRP 45. The results show that V. tapetis may be pathogenic to corkwing wrasse, but mortality was however not higher in the LP2 challenged groups than in the other groups. Other fish that died during the experiment died due to infections with other Vibrio spp, and revealed the same pathology as LP2 infected fish. The results indicate that corkwing wrasse carry Vibrios, and that in particular V. tapetis and V. splendidus strains may cause disease when corkwing wrasse are kept in captivity. Experiments were carried out from June until September. Comparing the results from the different experiments, we found a high mortality of females caught during the spawning season, and the catch of corkwing wrasse should thus be avoided in this period. The results presented in this thesis may be used as a background for further studies of bacterial diseases of wild caught wrasse, improve wrasse welfare and move us a step further on the way to obtain a sustainable use of wrasse as cleaner fish in the growing salmonid aquaculture. I dag er bruk av leppefisk (Labridae) som rensefisk i oppdrett av laks den eneste anvendte metoden for fjerning av lakselus som ikke er forbundet med negative miljømessige konsekvenser. Bruk av villfanget leppefisk har variert, men har økt de siste årene på grunn av problemer med lakselus som utvikler resistens mot lusemidler. Dagens bruk av leppefisk er imidlertid ikke etisk akseptabel. Det er et kontinuerlig tap av leppefisk i merdene som ikke er fullt ut forstått. Tapene fører til et uakseptabelt, kontinuerlig, høyt forbruk av leppefisk. En av leppefiskartene som benyttes er grønngylt (Symphodus melops). Flere tidligere arbeider tyder på at grønngylt er særlig utsatt for sykdom forårsaket av Vibrio-bakterier. Funn av Vibrio tapetis (LP2) hos grønngylt i Norge var det første funnet av denne bakterien hos fisk, og det første funnet av denne bakterien i Norge. I forsøkene som er beskrevet i denne oppgaven ble V. tapetis LP2 brukt som smittebakterie i en serie smitteforsøk med villfanget grønngylt. I tillegg ble det brukt to V. tapetis-isolater fra skjell; referansestammen CECT 4600 fra teppeskjell (Ruditapes philippinarum) i Frankrike og NRP 45, isolert fra norske teppeskjell. Det ble utført tre smitteforsøk: Grønngylt ble smittet med tre forskjellige doser LP2 ved bad og intraperitoneal injeksjon, for å bestemme den beste smittemetoden. Resultatene fra dette forsøket ga ikke helt konsistente resultater, men badsmitte med >106 CFU x ml-1 ble vurdert som pålitelig metode og benyttet i videre forsøk. Neste forsøk ble utført for å studere temperaturens innvirkning ved smitte med LP2. Grønngylt ble badsmittet med 107 CFU x ml-1 ved 6, 9, 12 og 15 °C. Resultatene viste en trend til økt dødelighet ved stigende temperatur, og det ble påvist signifikant høyere dødelighet ved 15 °C enn ved 6 °C. I det tredje forsøket ble fisken i tillegg til V. tapetis LP2 smittet med CECT 4600 og NRP45. I dette forsøket ble kun LP2 reisolert fra døde fisk, mens CECT 4600 og NRP45 ikke ble reisolert. Resultatene tyder på at LP2 kan være patogen for grønngylt. Dødeligheten var imidlertid ikke høyere i LP2-smittegruppene enn i de andre gruppene, og det ble både fra smitte- og kontrollgruppene isolert andre Vibrio spp og V. tapetis som ikke var identisk med smittebakteriene. Resultatene tyder derfor på at grønngylt kan være bærere av Vibrio-bakterier, særlig V. tapetis- og V. splendidus-stammer, og lett utvikler sykdom forårsaket av disse bakteriene når de holdes i fangenskap. Forsøkene ble utført på forskjellig tid på året. Det var særlig høy dødelighet av hunner som var fanget inn i tiden rundt gyting. Dette tilsier at fisket etter grønngylt bør unngås i gyteperioden. Resultatene i denne oppgaven kan brukes som en bakgrunn for videre studier av bakteriesykdommer hos villfanget leppefisk, bedre velferdssituasjonen hos leppefisk og på denne måten bidra til å oppnå en bærekraftig bruk av leppefisk i laksenæringen. Master i Havbruksbiologi - Generell havbruksbiologi MAMN-HAV HAV399

The aim of this study was to evaluate the pathogenicity of Vibrio alginolyticus isolated from an outbreak of sea horse Hippocampus reidi reared in the State of Santa Catarina, Brazil, by experimental infection. Sea horses with necrosis on the mouth epithelium were collected from aquaria at the Aquaculture Department, UFSC and the bacterium isolated from the mouth, liver, heart and blood in tiosulphate citrate bilesalt sucrose agar broth. The strains were identified by API 20E kit with 99.1% probability as Vibrio alginolyticus. Twelve adult sea horses (9.63 ± 2.42 g and 15.12 ± 0.87 cm) were distributed in six aquaria of 10 L capacity with aerated sea water. Fish from three aquaria were submitted to an immersion bath in a solution containing 1.0 × 10(7) CFU of V. alginolyticus/mL for 15 minutes. Fish from the other three aquaria received the same procedure without bacteria. Twenty four hours after this challenge, 100% mortality was observed in the animals infected with V. alginolyticus. No mortality was observed in non-infected fish. Hyperplasia, displacement and fusion of secondary lamellae of the gills; leukocyte infiltration and necrotic foci in the kidney; hyperplasia, sinusoidal deformation and necrotic foci in the liver were observed in histopathological analysis. The V. alginolyticus isolated in this study was pathogenic to H. reidi and constitutes an important sanitary problem to its culture. Foi avaliado por meio de infecção experimental a patogenicidade de Vibrio alginolyticus isolado de um surto de enfermidade em cavalo-marinho Hippocampus reidi cultivado no Estado de Santa Catarina, Brasil. Os peixes com necroses no epitélio bucal foram coletados em aquários do Departamento de Aquicultura, UFSC e as bactérias isoladas da boca, fígado, coração e sangue em meio Agar tiossulfato citrato bile sacarose. Os isolados foram identificados pelo kit API 20E como Vibrio alginolyticus com 99,1% de probabilidade. Doze peixes adultos (9,63 ± 2,42 g e 15,12 ± 0,87 cm) foram distribuídos em seis aquários de 10 L com água marinha e aeração. Peixes de três aquários foram submetidos a um banho de imersão por 15 minutos em uma solução contendo 1,0 × 10(7) UFC de V. alginolyticus/mL. Nos outros três aquários realizou-se o mesmo procedimento sem a bactéria. Vinte e quatro horas após o desafio, 100% de mortalidade foi observada nos animais infectados com V. alginolyticus. Não houve mortalidade nos peixes não infectados. Nas análises histopatológicas, foi observado hiperplasia, deslocamento do epitélio e fusão das lamelas secundárias das brânquias; infiltração de leucócitos e necrose no rim; hiperplasia, deformação sinusoidal e necrose no fígado nos animais desafiados com V. alginolyticus. O V. alginolyticus isolado neste estudo foi patogênico para H. reidi, constituindo-se de um importante problema para seu cultivo.

Atlantisk torsk Gadus morhua er en av de viktigste artene i norsk oppdrettsnæring, og produksjonen er stigende. Sykdomsproblemene er betydelige, og blant annet klassisk vibriose (infeksjoner med Vibrio anguillarum) forårsaker store tap. Vibriosevaksinene som brukes i dag beskytter ikke tilstrekkelig mot alle stammer av V. anguillarum, noe som gjenspeiler behovet for økt kunnskap om sykdommen og bakterien som forårsaker den. To atypiske isolater av Vibrio anguillarum ble virulenstestet med intraperitoneal (i.p.) injeksjonssmitte og badsmitte. I pilotforsøket ble det benyttet fem individer i hvert av de syv forsøkskarene. Testisolatene HI 21413 og HI 21429 ble injisert i tre ulike smittedoser; lavdose, middeldose og høydose på henholdsvis 103, 105 og 107 bakterier ml-1. Det ble registrert dødelighet i to av de syv karene. Kar 3 (infisert med HI 21413, 107 bakterier ml-1) hadde tre av fem døde fisk, hvor det ble reisolert V. anguillarum fra to av de døde fiskene. Kar 5 (infisert med HI 21429, 103 bakterier ml-1) hadde en død fisk (av fem totalt), ingen V. anguillarum ble reisolert fra dette karet. I hovedforsøket ble fisken badsmittet i 1 time med dose 106 bakterier ml-1. Hver testgruppe bestod av 84-92 individer fordelt på tre replikate kar. Fisk smittet med V. anguillarum-stamme HI 610, ble benyttet som positiv kontroll, og usmittet torskeyngel ble benyttet som negativ kontroll. Testisolatene hadde lavere virulens på torskeyngel sammenlignet med tidligere resultater fra badsmitteforsøk på plommesekklarver (Sandlund & Bergh 2008). Testisolatene var også lavvirulente sammenlignet med det positive kontrollisolatet HI 610. Torskeyngel i gruppe 1 (badsmittet med HI21413) og gruppe 2 (badsmittet med HI21429) hadde endelig kumulativ dødelighet på henholdsvis 11 % og 27 % ved forsøksavslutning, dag 26 etter smitte. Kumulativ dødelighet for gruppe 3 (positiv kontroll) og gruppe 4 (negativ kontroll) var henholdsvis 73 % og 11 % ved forsøksavslutning. Ved sekvensanalyse av 16S rRNA- og GyraseB-genet til isolater dyrket fra død fisk i hovedforsøket, ble det reisolert V. anguillarum fra en fisk badsmittet med HI 610, to fisker badsmittet med HI 21413 og fem fisker badsmittet med HI 21429. Fire atypiske V. anguillarum-isolater; HI 21412, HI 21413, HI 21414 og HI 21429, ble MIC-testet. MIC-verdier ble fastsatt og sammenlignet med tidligere resultater fra lignende forsøk. Master i Fiskehelse MAMN-FISK FISKL

P. acidilactici MA 18/5M is a dietary probiotic (BACTOCELL®) which has shown its efficiency on several terrestrial and aquatic species. The present thesis aimed to investigate more fully the effects of this probiotic on cultured penaeid shrimps. For this study, we chose a global approach by replacing the problem studied in a model of interactions between three compartments: the shrimp, its intestinal microflora and the probiotic. Having first defined how the probiotic should be administrated, we studied its effects under controlled laboratory conditions at several levels of Litopenaeus stylirostris biology: (i) Nutritional status and growth, (ii) associated bacterial microflora, and (iii) health status with respect to oxidative stress. Then the probiotic improves the growth of the shrimps and the feed conversion ratio. Our study shows that these effects are partially due to a better use of the feed, particularly of dietary carbohydrates, but also suggest an action on the metabolism, and\or on the growth via a contribution of essential nutrients. On the other hand, although the intestinal bacterial communities seems little affected, we show an antagonism effect of the probiotic toward total and vibrionacea intestinal flora, as well as a decrease in the level of infection of shrimps during challenge with pathogenic V. nigripulchritudo. Beside, our works highlight that infection by this pathogen, led to a reduction in antioxidant defences, to a rise in oxidative damages, and to a release of mortalities within 48 hours. This oxidative stress following bacterial infection was better controlled by animals receiving the probiotic diet, and resistance of the shrimps to infection was significant improved. A nutritional hypothesis is proposed to explain such result, hypothesis based on a link between carbohydrate metabolism and antioxidant status. As a conclusion of these works, we can propose a first explanatory scenario for the effects of P. acidilactici on a penaeid shrimp. Finally probiotic's effects were assessed under a pilot scale in a commercial farm from New Caledonia currently affected by the "summer syndrome ". We confirm the improvements brought in term of zootechnical results, with in particular, a better resistance to this syndrome. This work was made possible thanks to a breeding method in floating cages, developed during this thesis, and therefore transferred, as an experimental tool, to the shrimp industry of New Caledonia., Pediococcus acidilacticii MA18/5M est un probiotique bactérien (BACTOCELL®) qui a démontré son efficacité chez plusieurs espèces d'animaux terrestres et aquatiques. Cette thèse avait pour objectif d'investiguer plus en détails les effets de ce probiotique chez les crevettes pénéides d'élevage. Pour cette étude, nous avons choisi une approche globale replaçant la problématique dans un modèle d'interactions entre trois compartiments : la crevette, sa microflore intestinale et le probiotique. Après avoir défini les modalités d'administration, l'effet de P. acicilactici a été étudié, en conditions de laboratoire, chez la crevette Litopenaeus stylirostris, à différents niveaux: (i) statut nutritionnel et croissance, (ii) microflore bactérienne associée, et (iii) santé de l'animal à travers son statut antioxydant et son niveau de stress oxydant. Ainsi, le probiotique améliore la croissance des crevettes et l'indice de conversion de l'aliment. Notre étude montre que ces effets sont en partie liés à une meilleure utilisation de l'aliment, notamment des carbohydrates alimentaires, mais suggèrent aussi une action sur le métabolisme, et/ou sur la croissance via un apport de nutriments essentiels. D'autre part, bien que les communautés bactériennes intestinales semblent peu affectées, un effet d'antagonisme du probiotique vis-à-vis de la flore intestinale totale et vibrionacée a été montré, ainsi qu'une réduction du niveau d'infection des crevettes au cours de challenge avec le pathogène V. nigripulchritudo. Par ailleurs, nous mettons en évidence que l'infection par ce pathogène, conduit à un abaissement des défenses antioxydantes, à une élévation des dégâts oxydatifs, et au déclenchement de mortalités dans les 48 heures. Le probiotique atténue le niveau de ce stress oxydant, et améliore significativement la résistance des crevettes à l'infection. Une hypothèse nutritionnelle est proposée afin d'expliquer ce résultat, hypothèse reposant sur un lien entre le métabolisme des carbohydrates et le statut antioxydant. A l'issue de ces travaux, nous pouvons alors proposer un premier scénario explicatif des effets de P. acidilactici chez une crevette pénéide. Finalement le probiotique a été éprouvé à l'échelle pilote sur une ferme commerciale de Nouvelle-Calédonie affectée par le « syndrome d'été ». Nous confirmons les améliorations apportées en termes de résultats zootechniques, avec notamment une meilleure résistance au syndrome. Ce travail a été réalisé grâce une méthode d'élevage en cages flottantes, mis au point au cours de cette thèse, et qui a dés lors été transférée, comme outil expérimental, à la filière crevetticole Calédonienne.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-13T12:20:42Z No. of bitstreams: 1 Joanna Dourado.pdf: 721470 bytes, checksum: 1ade18b6e4af854fcb0abb1162f7c2bb (MD5) Made available in DSpace on 2016-10-13T12:20:42Z (GMT). No. of bitstreams: 1 Joanna Dourado.pdf: 721470 bytes, checksum: 1ade18b6e4af854fcb0abb1162f7c2bb (MD5) Previous issue date: 2009-02-13 Shrimp farming all over the world has been affected in the last years by many diseases, that have contributed to the development indexes decline in the production of farmed marine shrimp. On top of the losses due to diseases, it's important to mention that some of the vibrio species are zoonotic agents, meaning they can also cause diseases in humans. Given the importance of bacteria of the genus Vibrio to shrimp farming and public health, the aim of this study was to evaluate marine shrimp, correlate the clinical findings with the presence of the vibrio bacteria and identify the isolated species in the water of the ponds of cultivated marine shrimp in Pernambuco. The samples were collected from four coastal farms and the laboratory analysis consisted of the research and identification of the vibrio bacteria in the hepatopancreas and in the water of the tanks, and identification of any alteration by examination of fresh samples. Thiosulphate citrate bile salt agar (TCBS) was used as the culture media and the selected colonies were later biochemically tested. Four hundred and eighty shrimps were analyzed, of hose 424 (88.33%) showed to have vibrio and one or more alterations. There were identified a total of 903 alterations, the most frequent of muscle tissue (329; 36.43%) and the least frequent of the hepatopancreas tubules (121; 13.40%). When analizing the growth cycle, it was observed the largest number of alterations (503; 55.7%) in the dry season. Taking into account the weight of the shrimps, it was observed 540 (59.8%) alterations when the average weight was 8g. Out of all the analyzed animals, 314 (65.42%) showed to have vibrio, being 38 samples from the ponds water and 605 shrimp isolated. The most abundant species was V. mediterranei, (21.31%), considered an environmental species non pathogenic to shrimp, and the least abundant species were V. campbellii, V. rotiferanus, V. shiloni, V. splendidus, V. tapetis and V. wodanis (0.16 %). A dependency between the alterations found on the exam and the presence of the vibrio was noticed, showing that the aforementioned exam, combined to microbiological analyzes, is an important tool that should be used when monitoring the farms. It was also noticed a great variety of pathogenic and non pathogenic species of vibrio in the studied samples. Such microorganisms could, eventually, be related to shrimp and human infections, which could represent a risk for shrimp farming and public health respectively. A carcinicultura mundial tem enfrentado nos últimos anos vários impactos causados por enfermidades, que contribuíram para a queda dos índices de desenvolvimento na produção de camarão marinho cultivado. Além das perdas causadas devido às enfermidades, ressalta-se que algumas espécies de víbrio são agentes zoonóticos, ou seja, podem causar doenças também em humanos. Dada a importância das bactérias do gênero Vibrio para a carcinicultura e saúde pública, objetivou-se avaliar camarões marinhos, associar os achados clínicos com a presença de bactérias do gênero Vibrio e identificar as espécies isoladas da água dos viveiros e de camarões marinhos (Litopenaeus vannamei) cultivados no litoral do estado de Pernambuco. As amostras foram coletadas em quatro carciniculturas do litoral e as análises laboratoriais compreenderam pesquisa e identificação de bactérias do gênero Víbrio no hepatopâncreas e na água dos viveiros, e identificação de alterações através do exame a fresco. Para o isolamento das espécies foi utilizado o meio de cultivo Ágar Tiossulfato Citrato Sais de Bile Sacarose (TCBS) e as colônias selecionadas foram posteriormente submetidas a provas bioquímicas. Foram analisados no total 480 camarões, destes 424 (88,33%) apresentaram um ou mais tipos de alteração. Foi identificado um total de 903 alterações, sendo a mais freqüente a da musculatura (329; 36,43%) e a menos freqüente a alteração dos túbulos do hepatopâncreas (121; 13,40%). Com relação ao ciclo de cultivo, foi observado o maior número de alterações (503; 55,7%) no período de estio. Levando-se em consideração a média de peso dos camarões verificou-se 540 (59,8%) alterações quando os mesmos estavam com peso médio de 8 g. Do total de animais analisados 314 (65,42%) apresentavam vibrio e uma ou mais alteração no exame a fresco. Foi obtido um total de 643 isolados de Vibrio, sendo 38 de amostras de água dos viveiros e 605 isolados de camarão. A espécie mais abundante foi V. mediterranei (21,31%), considerada espécie ambiental e não patogênica para o camarão, e as espécies de menor predominância foram V. campbellii, V. rotiferianus, V. shiloni, V. splendidus, V. tapetis e V. wodanis (0,16%). Foi verificada uma dependência entre as alterações encontradas no exame a fresco e a presença de vibrio, concluindo-se que o referido exame, associado à análise microbiológica é uma importante ferramenta que deve ser utilizada no monitoramento da saúde dos animais. Verificou-se também uma ampla variedade de espécies patogênicas e não patogênicas de víbrio nas amostras estudadas, que podem eventualmente estar relacionados a infecções nos camarões e nos humanos, representando um risco para a carcinicultura e para a saúde pública respectivamente.

P. acidilactici MA 18/5M is a dietary probiotic (BACTOCELL®) which has shown its efficiency on several terrestrial and aquatic species. The present thesis aimed to investigate more fully the effects of this probiotic on cultured penaeid shrimps. For this study, we chose a global approach by replacing the problem studied in a model of interactions between three compartments: the shrimp, its intestinal microflora and the probiotic. Having first defined how the probiotic should be administrated, we studied its effects under controlled laboratory conditions at several levels of Litopenaeus stylirostris biology: (i) Nutritional status and growth, (ii) associated bacterial microflora, and (iii) health status with respect to oxidative stress. Then the probiotic improves the growth of the shrimps and the feed conversion ratio. Our study shows that these effects are partially due to a better use of the feed, particularly of dietary carbohydrates, but also suggest an action on the metabolism, and\or on the growth via a contribution of essential nutrients. On the other hand, although the intestinal bacterial communities seems little affected, we show an antagonism effect of the probiotic toward total and vibrionacea intestinal flora, as well as a decrease in the level of infection of shrimps during challenge with pathogenic V. nigripulchritudo. Beside, our works highlight that infection by this pathogen, led to a reduction in antioxidant defences, to a rise in oxidative damages, and to a release of mortalities within 48 hours. This oxidative stress following bacterial infection was better controlled by animals receiving the probiotic diet, and resistance of the shrimps to infection was significant improved. A nutritional hypothesis is proposed to explain such result, hypothesis based on a link between carbohydrate metabolism and antioxidant status. As a conclusion of these works, we can propose a first explanatory scenario for the effects of P. acidilactici on a penaeid shrimp. Finally probiotic's effects were assessed under a pilot scale in a commercial farm from New Caledonia currently affected by the "summer syndrome". We confirm the improvements brought in term of zootechnical results, with in particular, a better resistance to this syndrome. This work was made possible thanks to a breeding method in floating cages, developed during this thesis, and therefore transferred, as an experimental tool, to the shrimp industry of New Caledonia., Pediococcus acidilacticii MA18/5M est un probiotique bactérien (BACTOCELL®) qui a démontré son efficacité chez plusieurs espèces d'animaux terrestres et aquatiques. Cette thèse avait pour objectif d'investiguer plus en détails les effets de ce probiotique chez les crevettes pénéides d'élevage. Pour cette étude, nous avons choisi une approche globale replaçant la problématique dans un modèle d'interactions entre trois compartiments : la crevette, sa microflore intestinale et le probiotique. Après avoir défini les modalités d'administration, l'effet de P. acicilactici a été étudié, en conditions de laboratoire, chez la crevette Litopenaeus stylirostris, à différents niveaux: (i) statut nutritionnel et croissance, (ii) microflore bactérienne associée, et (iii) santé de l'animal à travers son statut antioxydant et son niveau de stress oxydant. Ainsi, le probiotique améliore la croissance des crevettes et l'indice de conversion de l'aliment. Notre étude montre que ces effets sont en partie liés à une meilleure utilisation de l'aliment, notamment des carbohydrates alimentaires, mais suggèrent aussi une action surle métabolisme, et/ou sur la croissance via un apport de nutriments essentiels. D'autre part, bien que les communautés bactériennes intestinales semblent peu affectées, un effet d'antagonisme du probiotique vis-à-vis de la flore intestinale totale et vibrionacée a été montré, ainsi qu'une réduction du niveau d'infection des crevettes au cours de challenge avec le pathogène V. nigripulchritudo. Par ailleurs, nous mettons en évidence que l'infection par ce pathogène, conduit à un abaissement des défenses antioxydantes, à une élévation des dégâts oxydatifs, et au déclenchement de mortalités dans les 48 heures. Le probiotique atténue le niveau de ce stress oxydant, et améliore significativement la résistance des crevettes à l'infection. Une hypothèse nutritionnelle est proposée afin d'expliquer ce résultat, hypothèse reposant sur un lien entre le métabolisme des carbohydrates et le statut antioxydant. A l'issue de ces travaux, nous pouvons alors proposer un premier scénario explicatif des effets de P. acidilactici chez une crevette pénéide. Finalement le probiotique a été éprouvé à l'échelle pilote sur une ferme commerciale de Nouvelle-Calédonie affectée par le « syndrome d'été ». Nous confirmons les améliorations apportées en terme de résultats zootechniques, avec notamment une meilleure résistance au syndrome. Ce travail a été réalisé grâce une méthode d'élevage en cages flottantes, mis au point au cours de cette thèse, et qui a dés lors été transférée, comme outil expérimental, à la filière crevetticole Calédonienne

Objetivou-se averiguar o efeito da bactéria Lactobacillus plantarum e células inativas das bactérias Vibrio alginolyticus, Aeromonas salmonicida e Pasteurella multocida na sobrevivência larval de Litoenaeus vannamei, no teste de estresse e infecção experimental com Vibrio harveyi. Foram utilizados tanques cônicos de 30 L, povoados com 400 larvas cada, no estádio de pós-larva cinco. Tratamentos em triplicatas foram consistidos de: 1: ração comercial (controle), 2: ração comercial + bacterina via oral na artemia, 3: ração comercial + bacterina via imersão e 4: ração comercial com inóculo de Lactobacillus plantarum. A aplicação da bacterina ocorreu seis horas antes da infecção e do teste de estresse; enquanto o Lactobacillus plantarum foi administrado por 15 dias antes dos desafios. As pós-larvas do tratamento 4 (ração suplementada com L. plantarum) obtiveram maior índice de sobrevivência no teste de estresse (87,86 ± 2,35%), seguido dos tratamentos 2 e 3 (bacterina via imersão e oral) com 81,54±1,50% e 80,16 ± 2,15% respectivamente, superiores ao índice do controle (72,63 ± 3,34%). Já no desafio com V. harveyi, os animais do grupo tratado com a adição de bacterina via imersão apresentaram maior sobrevivência (79,60 ± 7,12%). As pós-larvas dos tratamentos com bacterina via oral na artêmia e alimentadas com o probiótico L. plantarum, apresentaram sobrevivências de 65,60 ± 5,18% e 69,60 ± 10,43 %, respectivamente, sendo superiores ao controle (56,4 ± 5,58%), quando desafiados com V. harveyi. Os resultados demonstram que o uso de ração com L. plantarum e bacterina aumentam a sobrevivência das pós-larvas de L. vannamei frente aos testes de estresse e infecções experimentais com V. harveyi. This study aimed to verify the effect of probiotics and inactivated cells of bacterias such as Vibrio alginolyticus, Aeromonas salmonicida and Pasteurella multocida in larvae survival of Litopenaeus vannamei, in stress test and experimental infection with Vibrio harveyi. Conic tanks of 30 L, were stocked with 400 post-larvae stage five. Four experimental treatments with triplicates consisted of: 1: commercial feed (control), 2: commercial feed plus bacterin by oral administration in artemia, 3: commercial feed plus bacterin by immersion administration, 4: commercial feed with Lactobacillus plantarum inoculation. Bacterin application was conducted 6h before the infection and stress test, while probiotic administration was for 15 days before challenges. In stress test, post-larvae of treatment 4 (commercial feed supplemented with Lactobacillus plantarum) with reached the highest survival rate (87,86 ± 2,35%) followed by the ones of treatment 3 and 2 (bacterim by immersion and bacterim by oral administration in artemia) with 81,54±1,50% and 80,16 ± 2,15%, respectively, which were superior to the control treatment (72,63 ± 3,34%). Next to V. harveyi challenge, animals from treatment 3 presented the highest survival rate (79,60 ± 7,12%) followed by treatments 4 (69,60 ± 10,43%), 2 (65,60 ± 5,18%) and control (56,4 ± 5,58%). All treatments were different from control. The present results demonstrate the possible use of L. plantarum and bacterin as promoters in survival rates of L. vannamei post-larvae in the stress tests and challenges with Vibrio harveyi.

Objetivou-se averiguar o efeito da bactéria Lactobacillus plantarum e células inativas das bactérias Vibrio alginolyticus, Aeromonas salmonicida e Pasteurella multocida na sobrevivência larval de Litoenaeus vannamei, no teste de estresse e infecção experimental com Vibrio harveyi. Foram utilizados tanques cônicos de 30 L, povoados com 400 larvas cada, no estádio de pós-larva cinco. Tratamentos em triplicatas foram consistidos de: 1: ração comercial (controle), 2: ração comercial + bacterina via oral na artemia, 3: ração comercial + bacterina via imersão e 4: ração comercial com inóculo de Lactobacillus plantarum. A aplicação da bacterina ocorreu seis horas antes da infecção e do teste de estresse; enquanto o Lactobacillus plantarum foi administrado por 15 dias antes dos desafios. As pós-larvas do tratamento 4 (ração suplementada com L. plantarum) obtiveram maior índice de sobrevivência no teste de estresse (87,86 ± 2,35%), seguido dos tratamentos 2 e 3 (bacterina via imersão e oral) com 81,54±1,50% e 80,16 ± 2,15% respectivamente, superiores ao índice do controle (72,63 ± 3,34%). Já no desafio com V. harveyi, os animais do grupo tratado com a adição de bacterina via imersão apresentaram maior sobrevivência (79,60 ± 7,12%). As pós-larvas dos tratamentos com bacterina via oral na artêmia e alimentadas com o probiótico L. plantarum, apresentaram sobrevivências de 65,60 ± 5,18% e 69,60 ± 10,43 %, respectivamente, sendo superiores ao controle (56,4 ± 5,58%), quando desafiados com V. harveyi. Os resultados demonstram que o uso de ração com L. plantarum e bacterina aumentam a sobrevivência das pós-larvas de L. vannamei frente aos testes de estresse e infecções experimentais com V. harveyi.

Since 1997, a new pathology seasonally occurs in new caledonian shrimp farms during the warm season and was named Summer Syndrome. Diseased Litopenaeus stylirostris shrimp suffer from a septicemic vibriosis which was attributed to V. nigripulchritudo. Preliminary studies based on a collection of V. nigripulchritudo strains have brought to light different virulence levels according to experimental infections results; three virulence statuses were defined: highly (HP), moderately (MP) and non pathogenic (NP). The aim of this work was to genetically characterize virulent V. nigripulchritudo strains. In a first step the genetic diversity of 58 V. nigripulchritudo strains was analyzed by MLST and AP-PCR, revealing a cluster of HP and MP strains, characterized by a low genetic variability and that includes all Summer Syndrome-associated isolates. This confirms the emergence of one cluster of pathogenic V. nigripulchritudo simultaneously with the emergence of the Summer Syndrome ; in a second step, 368 genetic markers of virulence were identified by a Suppressive Subtractive Hybridization performed between the genomes of a HP strain and a genetically close, NP isolate; the distribution of the screened SSH fragments was studied in 58 V. nigripulchritudo isolates by macro-array: 78 DNA fragments were selected, allowing to characterize clusters identified and pathogenic statuses; 13 are specific of the HP strains involved in Summer Syndrome. Interestingly, 10 of these markers are carried by a plasmid pSFn1 that contains sequences highly similar to those of a plasmid pAK1, detected in Vibrio shilonii, a coral pathogen. The origin and consequences of this plasmid acquisition are discussed., Depuis 1997, les élevages de crevettes en Nouvelle-Calédonie sont confrontés à une nouvelle maladie, le Syndrome d'été, une vibriose septicémique dont l'agent étiologique est Vibrio nigripulchritudo. Les résultats d'infection expérimentale sur une collection de souches, ont montré l'existence de trois pathotypes distincts : hautement (HP), moyennement (MP) et non pathogène (NP). L'étude du polymorphisme génétique de 58 souches par typage moléculaire en MLST et AP-PCR, a mis en évidence un groupe phylogénétique particulier caractérisé par un très faible degré de variabilité génétique (confirmant l'émergence de ce groupe en parallèle à l'émergence du Syndrome d'été) et constitué uniquement de souches HP (dont toutes celles associées au Syndromed'été) et de souches MP. Afin d'identifier des marqueurs génétiques de la virulence des souches responsables du Syndrome d'été, et parmi ces marqueurs des gènes codant potentiellement pour des effecteurs de la virulence, une approche soustractive par SSH a été développée entre une souche HP de type Syndrome d'été et une souche NP : 368 marqueurs génétiques ont ainsi été mis en évidence ; la distribution de ces marqueurs a été étudiée chez les 58 souches de la collection par une approche en macroarray : 78 marqueurs ont été sélectionnés, qui permettent de caractériser les différents groupes phylogénétiques et les différents pathotypes, dont 13 fragments spécifiques des souches HP type Syndrome d'été. Parmi ces 13 fragments, 10 ont été localisés sur le plasmide pSFn1 qui a été entièrement séquencé. Ce même plasmide a été purifié uniquement des souches HP de type Syndrome d'été. Par ailleurs, une très forte homologie a été mise en évidence entre pSFn1 et pAK1, un autre plasmide également séquencé et retrouvé chez la souche V. shilonii AK1, responsable du blanchiment du corail Oculina patagonica en Méditerranée. Ces résultats ont ouvert la discussion sur le rôle de pSFn1 dans la virulence de V. nigripulchritudo.

Submitted by (edna.saturno@ufrpe.br) on 2016-08-11T16:51:11Z No. of bitstreams: 1 Carlos Andre Bezerra Alves.pdf: 107646 bytes, checksum: c97f6b2e6468643c3953a5613da56559 (MD5) Made available in DSpace on 2016-08-11T16:51:11Z (GMT). No. of bitstreams: 1 Carlos Andre Bezerra Alves.pdf: 107646 bytes, checksum: c97f6b2e6468643c3953a5613da56559 (MD5) Previous issue date: 2007-03-22 Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq Diseases means any adverse alteration in the health or acting in the productivity of the animals. In shrimps, the diseases can be unchained when it occur an unbalance potentially among the environmental conditions of nurseries, the health condition of the cultivated shrimps and the pathogenic agents, associated with the employed handling. This way, it was aimed at to evaluate the sanitary conditions and to determine which the relevant factors in the attack of the vibrioses in shrimp cultivated Litopenaeus vannamei. In the period of December of 2005 to November of 2006, were collected and analyzed shrimp samples, water and nursery soil, as well as technical information on the handling adopted in a commercial shrimp culture located in the north of Pernambuco/ Brazil. The handling parameters were correlated through mathematical models (P

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. Programa de Pós-Graduação em Aqüicultura. Avaliou-se o tempo de atuação da bactéria Lactobacillus B6 na flora bacteriana intestinal de Litopenaeus vannamei. Utilizaram-se dois grupos experimentais, um alimentado com dieta comercial e outro alimentado dieta comercial suplementada com o probiótico Lactobacillus B6. As avaliações iniciaram-se 8 dias após o início do experimento e repetiram-se 2, 4, 6 e 8 dias após a substituição do fornecimento da dieta suplementada com Lactobacillus B6 por dieta comercial. A contagem de bactérias lácticas no trato digestivo dos camarões foi superior no tratamento que foi alimentado com dieta suplementada com Lactobacillus B6 somente até o 4o dia de avaliação. A contagem de vibrios e enterobactérias no trato digestivo dos camarões foi superior no controle em relação ao tratamento probiótico apenas nas avaliações nos dias 0 e 2. Nos dias 0 e 2 e 4, a contagem total de hemócitos (THC) dos tratamentos alimentado com ração suplementada com probiótico e controle 3 horas após a inoculação com solução salina e do tratamento probiótico 3 horas após a inoculação com V. harveyi não diferiram e foram superiores ao controle 3 horas após a inoculação com V. harveyi. Nas avaliações dos dias 6 e 8, os tratamentos probiótico e controle 3 horas após a inoculação com solução salina não apresentaram diferenças quanto a THC que foram superiores a dos tratamentos probiótico e controle 3 horas após a inoculação com Vibrio harveyi. Não foi observada diferença significativa entre os tratamentos quanto à atividade da enzima fenoloxidase. No sexto dia após a substituição da alimentação suplementada com probióticos por dieta comercial, todos os parâmetros avaliados foram semelhantes nos camarões alimentados ou não com dieta suplementada com probióticos, sugerindo que o período de ação da bactéria Lactobacillus B6 no trato digestivo dos camarões é curto.

Sous la pression accrue des instances visant à protéger l'environnement, les organophosphorés, carbonates et pyrethrinoïdes, remplacent depuis une vingtaine d'années les pesticides plus persistants dans l'environnement tels que les organochlorés. S'ils sont rapidement dégradables dans l'environnement, ces composés sont néanmoins susceptibles d'affecter des espèces non cibles comme les crevettes. Ils peuvent induire une réduction de la croissance et une sensibilité accrue à des infections bactériennes et virales ou même une mortalité directe. Après différentes études préliminaires visant à préciser les colorations tissulaires à effectuer et les concentrations sub-létales en toxique, des crevettes blanches (Litopenaeus vannamei) juvéniles ont été exposées oralement avec 6,48 ug de méthylparathion (MPAR) par gramme de nourriture pendant 4 jours. Ces animaux ont ensuite subi une injection intramusculaire de Vibrio parahaemolyticus le jour 5. Les groupes contrôle ont été constitués de : crevettes nourries avec le MPAR puis injectées avec une solution saline stérile, de crevette nourries avec de l'acétonitrile (solvant du MPAR) et injectées avec la bactérie (V), et de crevettes recevant de l'acétonitrile puis injectées avec une solution saline stérile. La mortalité a été enregistrée et tous les animaux ont été collectés pour un examen histologique à la fin des 8 jours d'expérience. L'analyse des mortalités cumulées au jour 8 a montré que le traitement combiné (MPAR/V) était significativement différent des autres traitements (X2 = 35,6, DL = 3, p

The control of diseases remains a question of major importance in order to reduce pathological risks in bivalve larval rearings. For such a purpose, the knowledge of diseases, pathogens and routes of infection is required. A new species of vibrio pathogenic to P. maximus larvae, Vibrio pectenicida, was described by phenotypic and genotypic characters. It belongs to the V. splendidus group, where some other new pathogenic species were already found. The pathogenesis of V. pectenicida was studied by histological methods and experimental exposures in conventional and axenic larval rearings. These studies assessed the pathogenic character of V. pectenicida strains and the lack of particular symptoms during the disease. The location of bacteria in the tissues can be explained by a translocation phenomenon. The toxic activity of the pathogenic vibrio seems to be due to bacterial internal factors. In fact, vibrio bacterial lysats, mainly cytoplasmic extracts, exhibited a lethal activity in axenic larvae. To confirm the occurence of these toxic internal factors, a chemiluminescent (CL) test with adult hemocytes was perfected and used. The pathogenic vibrios inhibited the CL activity of hemocytes from bivalve adult. The inhibitory effect, observed with intact bacteria, was found again in the cytoplasmic fraction. This fact confirmed the existence of internal toxic factor(s). According to the partial purification, one of them may be a small molecule (below 3 kDa), hydrophilic, protease and acid resistant and thermo-stable. This toxin was different of previously described toxins. The relationships between the immunity of adult, very resistant to bacterial infection, and the larvae were investigated by two experiments: reproduction of disease in P. maximus adults by a strain related to V. splendidus and the injection of V. pectenicida to adults inducing a decrease of the hemocytes activity. Some applications were proposed by the end of this work to avoid the use of antibiotics, detrimental to the environment, during the first steps of the mollusc production., Le contrôle des pathologies est un élément primordial pour réduire les risques en élevage larvaire de mollusques bivalves. Il requiert une connaissance précise des maladies, des pathogènes et de leur mode d'action. Une nouvelle espèce de vibrio pathogène pour les larves de P. maximus, Vibrio pectenicida a été décrite par des caractères phénotypiques et génotypiques. Elle appartient au groupe de V. splendidus dans lequel se retrouvent d'autres nouvelles espèces pathogènes. La pathogénie de V. pectenicida a été étudiée en histologie et par des infections expérimentales sur des élevages larvaires conventionnels et axéniques. Ces études ont confirmé le caractère pathogène des souches de V. pectenicida et l'absence de symptômes spécifiques de la maladie. En ultrastructure, la localisation des bactéries à l'intérieur des tissus et de cellules peut être expliquée par un phénomène de translocation. L'activité toxique des vibrios pathogènes se manifeste vraisemblablement par des facteurs internes à la bactérie. En effet, une action létale de lysats bactériens de Vibrionaceae, et principalement de la fraction cytoplasmique, a été mise en évidence sur des larves axéniques. L'existence de facteurs toxiques internes a été confirmée par l'utilisation d'un test de chimioluminescence (CL) sur des hémocytes d'adultes. En effet, l'activité inhibitrice observée avec les bactéries entières, a été retrouvée dans la fraction cytoplasmique des vibrios pathogènes. D'après des essais de purification partielle, un des facteurs toxiques doit être une petite molécule (moins de 3kDa), hydrophile, résistante aux protéases, aux acides et à la chaleur. Cette toxine est éloignée des toxines bactériennes déjà décrites. Les relations entre l'immunité des adultes, très résistants aux attaques bactériennes, et celles des larves ont été abordées à partir de deux expériences: une infection expérimentale provoquée par une souche proche de V. splendidus sur des P. maximus adultes et l'injection de V. pectenicida à des animaux adultes entraînant la baisse de réactivité des hémocytes. Enfin quelques applications pratiques ont été proposées pour éviter l'utilisation d'antibiotiques durant la phase larvaire.