Dissertationen zum Thema „Disease bacteriosis“

The colicin and lectin-like bacteriocins are a broad class of antimicrobial proteins produced by Gram-negative bacteria. They are generally narrow spectrum, killing or inhibiting the growth of closely related bacteria. Numerous Gram-negative bacteria that are important pathogens of both animals and plants produce and are susceptible to these bacteriocins. As such, these proteins represent an attractive alternative to traditional small molecule antibiotics for controlling bacterial infection. Very little is known about bacteriocins produced by Gram-negative plant pathogens and so the aim of this work was to discover novel bacteriocins active against globally important plant pathogens from the genera Pectobacterium and Pseudomonas. The bacteriocins discovered in this study were then structurally and functionally characterised and assessed for their ability to impart disease resistance when expressed in a model transgenic system. This study presents the discovery and characterisation of the bacteriocins syringacin M, syringacin L1 and pyocin L1 from the genus Pseudomonas, As well as the discovery and characterisation of the unusual ferredoxin containing pectocins from the genus Pectobacterium. Also presented is the discovery of a novel virulence related ferredoxin/iron-uptake system in Pectobacterium, which is parasitised by the pectocins for cell entry. Additionally, the transgenic expression of the bacteriocin putidacin L1 in both Arabidopsis thaliana and Nicotiana benthamiana was shown to provide these plants with resistance to infection by strains of the plant pathogen P. syringae.

CAPES
A bacteriose do pessegueiro causada por Xanthomonas arboricola pv. pruni é uma das principais doenças da cultura no Brasil e na maioria das regiões produtoras do mundo. A utilização de cultivares resistentes é um instrumento para reduzir o progresso da doença. Este trabalho teve como objetivo avaliar e classificar genótipos de pessegueiro quanto à sensibilidade a Xanthomonas arboricola pv. pruni. Assim, foram realizados três experimentos visando avaliar a reação de genótipos de pessegueiro em diferentes situações. No experimento em condições naturais se avaliou a incidência, a severidade e a desfolha ocasionada pela doença. Os genótipos avaliados apresentaram níveis diferentes de sensibilidade, sendo que os genótipos Conserva 985, Conserva 1129, Conserva 871 e ‘Tropic Snow’ apresentaram-se como os mais resistentes, enquanto que ‘Bonão’, Conserva 1125, ‘Atenas’ e Conserva 1153 os mais suscetíveis. Em laboratório se avaliou a severidade da bacteriose em folhas destacadas e inoculadas com Xanthomonas arboricola pv. pruni em oito genótipos de pessegueiro, selecionados no experimento em condições naturais Mensurou-se, também, a atividade de alguns compostos bioquímicos relacionados à patogênese em dois genótipos mais resistentes e dois mais suscetíveis. Com base nos resultados obtidos conclui-se que a técnica de inoculação por infiltração em folhas destacadas é viável para identificar genótipos resistentes à bacteriose; os genótipos Conserva 985 e Conserva 1129 apresentaram-se como os mais resistentes, enquanto que Conserva 1125, ‘Atenas’ e Conserva 1153 os mais suscetíveis; não foi possível relacionar a resistência à bacteriose com os compostos bioquímicos estudados. Em ambiente controlado (estufa) se avaliou a progressão da doença em plantas juvenis, através dos níveis de severidade nas folhas, utilizando os mesmos genótipos do experimento em laboratório. Os resultados obtidos foram similares aos experimentos anteriores e ratificaram a resistência dos genótipos Conserva 985 e Conserva 1129 e de suscetibilidade dos genótipos Conserva 1153, ‘Bonão’ e ‘Antenas’ a bacteriose causada por Xanthomonas arboricola pv. pruni.
The bacterioses of peach caused by Xanthomonas arboricola pv. pruni is an of the majors diseases of the peach orchard in Brazil and in the most of producing regions of the world. The use of resistant cultivars is a tool to reduce disease progresses in commercial orchards. This work aimed to evaluate and classify peaches genotypes which the sensitivity to Xanthomonas arboricola pv. pruni. Thus, three experiments were performed to evaluate the reaction of genotypes of peach in different situations. In the experiment under natural conditions, it was evaluated the incidence, severity and defoliation caused by bacterial spot. The genotypes showed different levels of sensitivity, and the genotypes Conserva 985, Conserva 1129, Conserva 871 and 'Tropic Snow' were the most resistant, while ‘Bonão’, Conserva 1125, 'Atenas' and Conserva 1153 was the most susceptible ones. In laboratory, it was evaluated the severity of bacterial spot in detached peach leaf, inoculated with Xanthomonas arboricola pv. pruni in eight genotypes of peach, selected in the experiment under natural conditions. It was measured the activated of some biochemical compounds related to the pathogenesis in two resistant genotypes and two susceptible ones. Based on the results, it was concluded that the detached-leaf bioassay, inoculated by infiltration, is possible to use for identify resistant bacterial spot genotypes. The genotypes that showed the lowest rates of disease were Conserva 985 and Conserva 1129, while Conserva 1153, Atenas and Conserva 1125 showed the highest rates; it was not possible to identify a biochemical compound related to bacterial spot resistance. In a controlled environment (greenhouse), it was evaluated the progression of the disease in young plants through the levels of bacterial spot severity on the leaves, using the same genotypes of the experiment in the laboratory. The results were similar with the previous results confirming the resistance of Conserva 985 and Conserva 1129, and the susceptibility of Conserva 1153 and ‘Atenas’ to bacterial spot caused by Xanthomonas arboricola pv. pruni.

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: The effect of the bacteriocin-like peptide AS-48, produced by Enterococcus faecalis FAIRE 92, was tested against a mastitis isolate of Staphylococcus aureus in an in vivo and in vitro study. During initial tests peptide AS-48 showed no significant activity towards S. aureus, even with a ten-fold concentrated cell-free supernatant. Activity was obtained only after purification with Triton X-114 phase partitioning, followed by cation exchange chromatography. Titers for the purified peptide varied between 3200 and 12800 AU/ml. The purified peptide also exhibited activity towards Streptococcus agalactiae and Streptococcus dysgalactiae, but not against Escherichia coli. The size of peptide AS-48 was determined at 7150 Da, based on electronspray mass spectrometry and SDS-PAGE. Complete inhibition of cell growth was obtained by adding 1ml of the purified peptide (3200 AU/ml) to 100 ml of cells of S. aureus in the lag growth phase. When the same concentration of peptide AS-48 was added to a culture of S. aureus in mid-exponential growth, a slight decrease in viable cell numbers was recorded, which lasted for only 30 min. Cell growth commenced thereafter. In situ experiments in cows were done with purified peptide AS-48, encapsulated in liposomes. These in vivo studies were conducted by administering peptide AS-48 (6400 AU/ml) to different udder quarters. In a prevention trial, i.e. where quarters were pretreated with peptide AS-48, a reduction close to 90% in the viable cell numbers of S. aureus was recorded relative to the control quarters, which were not treated with the peptide. A 50% reduction in somatic cell count (SCC) was recorded. In the treatment trial, i.e. infected quarters treated with peptide AS-48, a reduction of up to 94% in viable cell numbers of S. aureus was recorded. In the same quarters, a reduction in SCC amounted to almost 80%. A recombinant strain was constructed by conjugating plasmid 92 (p92), encoding peptide AS-48, from Enterococcus faecalis FAIRE 92 to E. faecalis FA2/Ent, which produces enterocins 1071A and 1071B. Southern blot hybridization experiments revealed thepresence of plasmid p92 in the recipient strain without the loss of plasmid pEF1071, which encodes enterocins 1071A and 1071B. All three antimicrobial peptides, i.e. enterocin 1071A, enterocin 1071B and peptide AS-48, were produced in transconjugant FA2/Ent/AS-48. The spectrum of antimicrobial activity of the transconjugant was greater than that recorded for strains FA2/Ent and FAIRE 92, respectively and included E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus and S. aureus. These organisms are not inhibited by strain FA2/Ent. However, low levels of peptide AS-48 was produced by strain FA2/Ent/AS-48. Further research in fermentation and gene expression will be needed before the transconjugant E. faecalis FA2/Ent/AS-48 may be used in the treatment of mastitis.
AFRIKAANSE OPSOMMING: Die effek van die bakteriosien-agtige, peptied AS-48, geproduseer deur Enterococcus faecalis FAIRE 92, is gedurende ‘n in vivo en in vitro studie teen ‘n mastitiese Staphylococcus aureus-isolaat getoets. Aanvanklike toetse met peptied AS-48, selfs tienvoudig gekonsentreerde selvrye supernatant, het geen beduidende aktiwiteit teen S. aureus getoon nie. Aktiwiteit is eers verkry na suiwering met Triton X-114 fase-skeiding gevolg deur katioon uitruilingschromatografie. Titers vir die gesuiwerde peptied het tussen 3200 en 12800 AE/ml gewissel. Die gesuiwerde peptied het ook aktiwiteit teen Streptococcus agalactiae en Streptococcus dysgalctiae getoon, maar nie teen Escherichia coli nie. Peptied AS-48 het ‘n molekulêre massa van 7150 Da, soos bepaal met elektronsproeimassa spektrometrie en SDS-PAGE. Totale inhibisie van selgroei is verkry deur 1 ml gesuiwerde peptied AS-48 (3200 AE/ml) by ‘n 100 ml kultuur van S. aureus in die sloerfase te voeg. Dieselfe konsentrasie peptied AS-48, toegevoeg tydens die mideksponensiële groeifase, het egter slegs ‘n klein vermindering in die aantal lewende selle teweeg gebring en het ook vir slegs ‘n 30 min geduur. Selgroei het hierna weer normaal voort gegaan. In situ eksperimente op koeie is uitgevoer met gesuiwerde peptied AS-48, geenkapsuleerd in liposome. Hierdie In vivo studies is onderneem deur peptied AS-48 (6400 AE/ml) in verskillende kwarte van die uier, kunsmatig of reeds geïnfekteerd met S. aureus, toe te dien. In ‘n voorkomings-eksperiment waar kwarte vooraf met peptied AS- 48 behandel is, is ‘n verlaging van byna 90% in die lewende seltelling van S. aureus relatief tot die kontrole kwarte, sonder behandeling met peptied AS-48, verkry. ‘n 50% verlaging in die somatiese seltelling (SST) is verkry. In die behandelings-eksperiment, waar geïnfekteerde kwarte met peptied AS-48 behandel is, is ‘n verlaging van byna 90% in lewende S. aureus selle gevind. In dieselfde kwarte is ‘n verlaging van byna 80% in die SST genoteer.‘n Rekombinante ras is gekonstrueer deur plasmied 92 (p92), wat kodeer vir peptied AS- 48, vanaf Enterococcus faecalis FAIRE 92 na E. faecalis FA2/Ent, wat enterosien 1071A en 1071B produseer, te konjugeer. Southern-klad hibridisasie het die teenwoordigheid van plasmied p92 in die ontvanger ras, sonder die verlies van plasmied pEF1071 wat enterosien 1071A en 1071B kodeer, getoon. Al drie antimikrobiese peptiede, nl. enterosien 1071A, enterosien 1071B en peptied AS-48, is deur die transkonjugant FA2/Ent/AS-48 geproduseer. Die spektum van antimikrobiese aktiwiteit van die transkonjugant vand die transkonjugant is breër as dié van rasse FA2/Ent en FAIRE 92, onderskeidelik en het ook E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus en S. aureus ingesluit. Hierdie organismes word nie deur ras FA2/Ent geïnhibeer nie. Lae vlakke van peptied AS-48 is egter deur ras FA2/Ent/AS-48 geproduseer. Verdere navorsing in fermentasie en geenuitdrukking is nodig voordat E. faecalis FA2/Ent/AS-48 in die behandeling van mastitis gebruik kan word.

Streptococcus uberis, an environmental organism also associated with dairy animals, is a common and persistent cause of bovine mastitis. New approaches to control these infections need to be identified. One such strategy may be the application of bacteriocins; proteinaceous antimicrobials elaborated by bacteria that typically inhibit the growth of strains closely related to the producer organism. The well-characterized lactococcal bacteriocin nisin is the active ingredient in two commercial products currently in use for the prevention of mastitis. However, reports of resistance development have prompted the investigation of alternative bacteriocins to be used in conjunction with nisin in 'bacteriocin cocktails' designed to have more comprehensive inhibitory activity against mastitis pathogens. The bacteriocins of gram-positive bacteria have been divided into four distinct classes: (I) lantibiotics, (II) non-lantibiotic peptides, (III) large proteins, and (IV) circular peptides. Although it has been known for more than twenty years that S. uberis commonly produce bacteriocin-like inhibitory substances (BLIS), none had been characterised prior to the present study. The first step in the current investigation was a survey of the BLIS activities of a set of fifteen S. uberis and S. bovis strains against a set of standard indicators as well as common gram-positive mastitis pathogens. Additional tests using a deferred antagonism agar plate-based assay showed that some of the BLIS activities were heat-sensitive and their production was influenced by the presence of either blood or a fermentable carbohydrate source in the test medium. On the basis of the results obtained from these tests it became apparent that S. uberis and S. bovis may commonly produce more than a single inhibitory agent. S. uberis 42 became the focus of this study because (a) it had broad inhibitory activity against mastitis-associated bacteria, (b) it did not display cross-resistance to nisin, and (c) from the preliminary screening results it appeared to produce both heat-stable and heat-labile inhibitory agents. Acid extracts of S. uberis 42 cells yielded inhibitory activity that, when fractionated by reversed-phase HPLC, yielded a peptide of 3029 Da. Although this peptide was blocked to Edman degradation at position 2, following propanethiol-modification a 20-amino acid sequence was obtained. Degenerate primers to lantibiotic biosynthesis gene homologs were used to initiate inverse PCR and primer walking, ultimately yielding a 15-kb contiguous sequence encompassing 11 genes typical of those involved in lantibiotic synthesis, regulation and immunity. Due to the close similarities to nisin of the S. uberis 42 lantibiotic precursor (78%), and the organisation and composition of the locus, this inhibitor was named nisin U. Nucleotide sequences homologous to insertion sequences were detected in the vicinity of the nisin U locus, and indicate a possible mechanism of acquisition of this locus by S. uberis. The locus was detected in ten other S. uberis, and also in two S. agalactiae and two S. thoraltensis strains, and in one S. porcinus and one S. pluranimalium strain. The amino acid sequences of some of these differed in one or two amino acids, and these variants were named nisin U2 and nisin U3 accordingly. Nisin U, the two nisin U variants, and nisin A exhibited cross-immunity (i.e. all of the producer strains were insensitive to each form of nisin) and cross-inducibility (i.e. all of the producer strains displayed enhanced production when exposed to each form of nisin). Nisin U did not contribute to the entire spectrum of inhibitory activity of S. uberis 42. Freeze thaw extracts of S. uberis 42 agar cultures yielded heat-labile inhibitory activity that was inhibitory to L. lactis A5, a producer of nisin Z. Subsequent purification by cation-exchange chromatography, gel filtration, and reversed-phase HPLC yielded a peptide of mass 7048 Da, which was resistant to Edman degradation. Digestion with chymotrypsin released an 819 Da peptide fragment of sequence NH₂-KAQAVIW-COOH. Tn916 mutagenesis of S. uberis 42 enabled the identification of the genetic locus of the inhibitor, comprising six genes potentially involved in its biosynthesis and immunity. The detection of a pair of flanking 159-bp direct repeats indicates possible acquisition of the locus by 'long target duplication'. The inhibitor was inferred to be a circular peptide, on the basis of its behaviour to Edman degradation, and by comparison of its locus with that of other circular bacteriocins. On the basis that the purified peptide appears to induce lysis in sensitive bacteria, although by an as-yet unidentified mechanism, the inhibitor was named uberolysin. The uberolysin structural gene was detected in eight other strains of S. uberis, however not all of these appeared to be producing active inhibitor. No bacteriocins closely resembling the two reported in this thesis have been demonstrated previously to be produced by members of the genus Streptococcus. The remarkable diversity in the structures, activity spectra and basic modes of action of these two bacteriocins produced by a single strain of S. uberis, combined with the observation of apparent greater heterogeneity in properties of a preliminary sampling of BLIS-producing strains, indicates that these bacteria may be an important source of novel antimicrobials of potential value for the treatment of mixed bacterial infections and for minimising potential resistance development.

La sequence ontogenetique de la rhizogenese transformee et les effets cellulaires induits par trois souches d'a. Rhizogenes (1855, 2659 et 8196) chez l'epicotyle de pois sont recherches. Apres inoculation par la souche 1855, on enregistre une large gamme de symptomes comparables a ceux produits par des auxines exogenes. Il s'agit de symptomes comparables a ceux produits par des auxines exogenes. Il s'agit de symptomes cytophysiologiques (hyperhydrie, synthese d'adn, fragmentations nucleaires), histologiques (cambiogenese, tracheogenese) et morphogenetiques (rhizogenese transformee aux points d'inoculation et non transformee dans les regions sous-jacentes aux inoculations). La sequence morphogenetique de la mise en place des racines transformees a ete etablie

O presente trabalho teve por objetivo avaliar a reação de oito variedades comerciais e dois acessos selvagens de maracujá amarelo (P.edulis Sims f. flavicarpa), quanto à resistência a Xanthomonas campestris pv. passiflorae e elaborar uma escala diagramática de sintomas para auxiliar na avaliação da severidade da mancha bacteriana. A escala foi desenvolvida a partir de 100 folhas com sintomas da doença. Desta amostra foram estabelecidos cinco níveis de severidade utilizados na escala (2%, 5%; 11%, 26% e 59%). A escala foi validada por sete avaliadores, que utilizaram 48 folhas com diferentes níveis de severidade. A validação da escala mostrou que os avaliadores apresentaram alta precisão nas suas avaliações, com coeficientes de determinação (R2) variando de 0,86 a 0,95. A maioria dos avaliadores apresentou uma leve tendência em superestimar a severidade da doença. A escala mostrou-se útil ao trabalho, permitindo avaliações com alta precisão e boa acurácia. Para avaliar a reação das dez populações (oito variedades comerciais e dois acessos selvagem) de maracujá amarelo em relação a X. O presente trabalho teve por objetivo avaliar a reação de oito variedades comerciais e dois acessos selvagens de maracujá amarelo (P.edulis Sims f. flavicarpa), quanto à resistência a Xanthomonas campestris pv. passiflorae e elaborar uma escala diagramática de sintomas para auxiliar na avaliação da severidade da mancha bacteriana. A escala foi desenvolvida a partir de 100 folhas com sintomas da doença. Desta amostra foram estabelecidos cinco níveis de severidade utilizados na escala (2%, 5%; 11%, 26% e 59%). A escala foi validada por sete avaliadores, que utilizaram 48 folhas com diferentes níveis de severidade. A validação da escala mostrou que os avaliadores apresentaram alta precisão nas suas avaliações, com coeficientes de determinação (R2) variando de 0,86 a 0,95. A maioria dos avaliadores apresentou uma leve tendência em superestimar a severidade da doença. A escala mostrou-se útil ao trabalho, permitindo avaliações com alta precisão e boa acurácia. Para avaliar a reação das dez populações (oito variedades comerciais e dois acessos selvagem) de maracujá amarelo em relação a X. campestris pv. passiflorae, dois experimentos foram conduzidos em casa de vegetação, o primeiro entre os meses de setembro a dezembro de 2002 e o segundo entre os meses de janeiro a março de 2003. O delineamento experimental utilizado foi o de blocos ao acaso, com nove tratamentos e quatro repetições em ambos os ensaios, sendo que as parcelas experimentais consistiram de 30 e 25 plantas de cada material, respectivamente, no primeiro e segundo experimentos. As avaliações de severidade da doença foram realizadas aos 7, 14 e 21 dias após inoculação, através da escala diagramática de sintomas elaborada neste trabalho. Com os dados das três avaliações, estimou-se a área abaixo da curva do progresso da doença (AACPD) para cada material estudado. Os resultados permitiram detectar diferentes níveis de resistência entre as populações avaliadas. Os materiais mais resistentes a X. campestris pv. passiflorae foram as variedades Sul Brasil e IAC- serie 270, enquanto que as variedades IAC-277, Maguary e Flora foram as mais suscetíveis.
This study aimed to evaluate the reaction of eight populations of commercial yellow passion fruit and two of wild passion fruit (P.edulis Sims f. flavicarpa) to Xanthomonas campestris pv. passiflorae and elaborate a diagrammatic scale of symptoms for the evaluation of the severity of the disease. This scale was developed based on 100 leaves with different levels of disease. Out of this sample, five levels of severity were depicted in the scale (2%, 5%, 11%, 26%, and 59%). The scale was used by seven evaluators to assess the symptoms of 48 leaves with different degrees of severity. The evaluators showed high precision judging by the high correlation coefficients (R2) which ranged from 0.86 to 0.95. Most evaluators showed a tendency to super-estimate the severity of the disease. Notwimstanding, the scale allowed precise and accurate evaluations. Two experiments were performed in the greenhouse to determine the level of resistance of ten populations (eight commercial and two wild) of yellow passion fruit to X. campestris pv passiflorae. The first trial was conducted between September and December 2002, and the second between January and March 2003. The experimental design consisted of random blocks, with nine treatments and four replicates in both experiments. The experimental plot consisted of 30 and 25 plants of each variety, respectively in the first and second trials. Disease severity was evaluated 7, 14, and 21 days after inoculation using the diagrammatic scale. With the data of the three evaluations, the area under the disease progress curve (AUDPC) for each material was estimated and used in the analysis of variance. The results detected different levels of resistance among populations. The most resistant materials were the varieties Sul Brasil and IAC- serie 270, while the varieties IAC-277, Maguary e Flora were the most susceptible.

Dando continuidade aos trabalhos realizados no Departamento de Genética da ESALQ/USP, referentes aos estudos de mapeamento de genes de resistência à bacteriose em maracujá-amarelo, foram realizados dois novos experimentos de inoculação envolvendo uma população obtida a partir do cruzamento entre ‘IAPAR-06’ e ‘IAPAR- 123’, ambos acessos pertencentes ao Instituto Agronômico do Paraná (IAPAR). Essa população F1, composta de 160 indivíduos, foi utilizada para a construção dos mapas de ligação com base em marcadores AFLP, utilizando tanto marcas com segregação 1:1 quanto marcas bi-parentais 3:1, as quais serviram de ponte para a integração dos mapas construídos para cada genitor. Para a avaliação fenotípica, 104 indivíduos, além dos genitores, foram inoculados por dois isolados de Xanthomonas axonopodis pv. passiflorae, em dois experimentos distintos. Foi constatado que a resistência à bacteriose é poligênica e há, pelo menos, três locos quantitativos (QRL) envolvidos. Testes quanto à metodologia de mensuração das lesões foliares também foram realizados. Foi verificado que, independente da metodologia de avaliação fenotípica, os resultados foram equivalentes quanto ao mapeamento de QRL. De acordo com as diferentes datas de avaliação e/ou idades das folhas, QRL com diferentes efeitos foram detectados ou, até mesmo, QRL foram alocados em diferentes grupos de ligação. Os dados oriundos do presente estudo corroboram resultados obtidos em trabalho anterior envolvendo a mesma população de mapeamento. A utilização de um segundo isolado bacteriano possibilitou a detecção de um novo QRL. As limitações do uso de marcadores dominantes para fins de mapeamento de locos quantitativos são discutidas.
Continuing the studies carried out in the Genetics Department at ESALQ/USP concerning the mapping of resistance genes for the bacterial disease in yellow passion fruit, two new assays were conducted involving a population derived from a cross between ‘IAPAR-06’ and ‘IAPAR-123’, both accessions belonging to Instituto Agronômico do Paraná (IAPAR). This F1 population, composed of 160 individuals, was used for constructing genetic maps based on AFLP markers, using both markers with segregation 1:1 and with biparental 3:1, which act as a bridge to integrate the maps constructed for each of the parents. For phenotypic evaluation, 104 individuals plus the parents were inoculated with two isolates of Xanthomonas axonopodis pv. passiflorae in two different assays. It was seen that the resistance to bacterial disease is polygenic, and there are at least three quantitative loci (QRL) involved. Tests regarding methods for measuring leaf lesions were also carried out. It was seen that, irrespective of the methodology used for phenotypic evaluation, the results were identical for QRL mapping. Different evaluation times and/or leaf ages resulted in detection of QRL with different effects, and even QRL allocated on different linkage groups. Data derived from the present study corroborate results obtained previously involving the same mapping population. The use of a second bacterial isolate did allow a new QRL to be detected. The limitations of using of dominant markers for mapping quantitative loci are discussed.

A ingestão de lipídios e seus constituintes, os ácidos graxos, são essenciais na manutenção do crescimento, na eficiência alimentar, na higidez do organismo, no funcionamento adequado dos rins e das brânquias e na reprodução. Este estudo visa identificar a influência exercida pelos lipídios dietéticos sobre as variáveis hematológicas e desempenho imunológico de pacus Piaractus mesopotamicus expostos à ação bacteriana. Juvenis de pacu (14,4 ± 0,4g) foram alimentados com dietas contendo níveis crescentes (20, 40, 60, 80, 100%) de óleo de linhaça (OL), óleo de girassol (OG) e sebo bovino (SB), ricas em ômega-3, ômega-6 e gordura saturada, respectivamente e, posteriormente, comparadas a uma dieta isenta destas fontes e a uma ração comercial. Os resultados obtidos foram submetidos a uma análise das variâncias, rearranjados em grupos e analisados sob o teste de Dunnett. Os níveis dietéticos de 20% de óleo de linhaça, 80% de óleo de girassol ou 80% de sebo bovino condicionaram as melhores taxas de desempenho zootécnico; o uso de 80% de óleo de girassol foi o mais adequado para ganho de peso (67,51±4,950g), taxa de conversão alimentar (1,05±0,088) e taxa de crescimento específico (2,19±0,085%), assim como para o aumento no número de linfócitos (1.964,13 ± 413,550), concomitante ao aumento dos leucócitos (1.986,00 ± 256,700), o que conferiu maior resistência aos animais, quando expostos à bactéria Aeromonas hydrophila.
The intake of lipids and its components, the fatty acids, are essential for proper growth, feed efficiency, health, appropriate kidney and gills function and reproduction performance. This study aims at identifying effects of dietary lipids on hematological variables and immunological performance of pacu Piaractus mesopotamicus exposed to bacterial challenges. Performance of juvenile pacu (14.4 ± 0.4 g) fed a diet with increasing levels (20, 40, 60, 80, 100 %) of linseed oil (LO); sunflower oil (80) and beef tallow (BT), rich in omega-3, omega-6 fatty acids, and saturated fat, respectively, was contrasted to the performance of fish fed diets containing soybean oil as lipid source, and a commercial aquafeed. Recorded results were subjected to ANOVA, grouped and submitted to Dunnett\'s test. Dietary levels of 20 % LO, 80% 80 or 80% beef tallow yielded the best growth performance; fish fed 80% 80 had the best weight gain (67.51 ± 4.95 g), best feed conversion ratio (1.05 :t 0.10), specific growth rate (2.10 ± 0.10 %), and also increased number of Iymphocytes (1,964.13 ± 413.55) concomitantly to increased number of leukocytes (1,986.00 ± 256.70), which also elicited the best immunological performance to fish challenged with Aeromonas hydrophila.

Xylella fastidiosa é o agente causal de uma série de doenças que ocorrem em plantas economicamente importantes como laranjeiras, videiras e cafeeiros, causando no Estado de São Paulo prejuízos relevantes à indústria citrícola. Esta bactéria Gram-negativa é restrita ao xilema das plantas e à porção anterior do trato digestório dos insetos vetores das famílias Cicadellidae e Cercopidae, conhecidos como cigarrinhas. Tentativas de elucidar os mecanismos de virulência e patogenicidade adotados por esta bactéria apontam a formação do biofilme como etapa fundamental para o estabelecimento da infecção e o consequente desenvolvimento da doença na planta, mas fatores adicionais parecem contribuir, tais como a produção de toxinas. As bacteriocinas são proteínas com atividade antibiótica contra cepas próximas à espécie produtora e que já foram associadas à virulência e patogenicidade de outras bactérias. Uma varredura in silico no genoma de X. fastidiosa 9a5c revelou 13 sequências codificadoras de microcinas putativas no cromossomo. Transcritos de todos esses genes foram detectados por RT-qPCR em culturas de X. fastidiosa 9a5c, e análises comparativas com genomas públicos (cepas Temecula1, Dixon, Ann-1, M23, M12, EB92-1 e GB514) e recém sequenciados por nosso grupo de pesquisa (cepas U24d, J1a12, 3124, Hib4, Pr8x e Fb7) revelaram que cada cepa possui seu próprio arsenal de bacteriocinas. Diferenças encontradas in silico entre os loci de bacteriocinas nas cepas foram demonstradas experimentalmente. Nossos resultados comprovam a variabilidade predita nos quatro clusters de bacteriocinas que identificamos, o que é esperado para genes relacionados à adaptação e patogenicidade. Destes loci, três foram detectados por RT-PCR como transcritos policistrônicos. Nossa tentativa de detectar essas proteínas em culturas de X. fastidiosa (através de sequenciamento de polipeptídeos por HPLC-MS/MS) foi capaz de identificar uma das bacteriocinas putativas e, portanto, o conjunto de nossas observações apóia a continuidade dos estudos para elucidar o papel das bacteriocinas na fisiopatologia de X. fastidiosa.
Xylella fastidiosa is the causal agent of diseases that affect several economically important crops such as sweet orange trees, grapevines and coffee trees, causing in the State of São Paulo considerable losses mainly to the citrus industry. This Gram-negative bacterium is restricted to the plant xylem and to the upper gastrointestinal tract of its insect vectors, the sharpshooters from the Cicadellidae and Cercopidae families. Attempts to elucidate the virulence and pathogenicity pathways employed by this bacterium point the biofilm formation as a fundamental step for the establishment of the infection and the consequent development of the plant disease, but additional factors seem to contribute to these processes, such as the production of toxins. Bacteriocins are proteinaceous antibiotics that act against closely-related species and have been previously associated with virulence and pathogenicity in other bacteria. An in silico screening of the X. fastidiosa 9a5c genome revealed 13 coding sequences as putative microcins in the chromosome. Transcripts from all those genes were detected through RT-qPCR in X. fastidiosa 9a5c cultures, and comparative analyses on the public genomes (Temecula1, Dixon, Ann-1, M23, M12, EB92-1 and GB514) plus the ones recently sequenced by our group (U24d, J1a12, 3124, Hib4, Pr8x and Fb7) revealed that each strain possesses its own arsenal of bacteriocins. Differences found in silico among the loci in all strains were experimentally confirmed. Our results demonstrated the predicted variability in the four bacteriocins clusters as expected for adaptation and pathogenicity-related genes. Three out of the four bacteriocins loci were detected by RT-PCR as polycistronic transcripts. Our attempt to detect these proteins in X. fastidiosa cultures (using HPLC-MS/MS polypeptide sequencing) identified one of the putative bacteriocins, and therefore our observations warrant further efforts to elucidate the role of bacteriocins in the X. fastidiosa physiopathology.

A bacteriose, ou mancha oleosa, doença causada por Xanthomonas axonopodis pv. passiflorae, é um sério problema em muitas áreas de produção de maracujá no Brasil, especialmente se associada à antracnose. A transformação genética é uma alternativa para obter plantas resistentes. Proteínas bactericidas, como as atacinas encontradas na hemolinfa de insetos, têm sido usadas para conferir resistência a espécies vegetais. Como as atacinas têm um peptídeo sinal que as direciona para o espaço extracelular em insetos, nós iniciamos este estudo investigando o direcionamento da atacina A em plantas. A seqüência do gene da atacina A (attA) com e sem o peptídeo sinal foi fusionada com os genes repórteres uidA e gfp e epidermes de cebola foram transformadas, via biobalística, com essas construções gênicas. A atacina A, de fato, é acumulada no apoplasto onde, justamente, bactérias fitopatogênicas se multiplicam antes de invadir as células vegetais. Visando obter plantas transgênicas resistentes à bacteriose, foram transformados tecidos foliares e hipocotiledonares com as linhagens LBA 4404 e EHA 105 de Agrobacterium tumefaciens contendo o gene attA. De um total de 313 explantes infectados, foram obtidos 31 brotos PCR+, o que representa uma eficiência de transformação da ordem de 10%. A expressão do transgene foi confirmada por RT-PCR e a resistência ao patógeno foi avaliada pela inoculação de X. axonopodis pv. passiflorae em folhas destacadas de plantas mantidas in vitro. Em dez plantas não houve formação de lesão foliar, indicando uma possível resistência ao patógeno.
Bacterial spot disease caused by Xanthomonas axonopodis pv. passiflorae is a serious problem in many passion fruit production areas in Brazil, especially if associated with anthracnose. Genetic transformation provides an alternative for obtaining resistant plants. Bactericide proteins such as attacins, found in the haemolymph of insects, have been used to confer resistance on plant species. As the attacins have a sign peptide that dispatches them to extracellular space in insects, we initiated our studies investigating the attacin A directing in plants. The attacin A gene (attA) sequence, with and without the sign peptide, was fused to uidA and gfp reporter genes, and onion epidermis were transformed using bioballistics with gene constructions. The protein did accumulate in the apoplast, where bacteria multiply before attacking plant cells. With the aim of obtaining transgenic plants of yellow passion fruit resistant to bacterial disease, leaf and hypocotyl-derived tissues were transformed with LBA 4404 and EHA 105 strains of Agrobacterium tumefaciens containing the attA gene. From a total of 313 infected explants, we obtained 31 PCR+ shoots, a transformation efficiency of 10%. Expression of the attA gene was confirmed by RT-PCR, and pathogen resistance evaluated by X. axonopodis pv. passiflorae inoculation in leaves obtained from in vitro plants. Leaf lesions were not observed in 10 shoots, suggesting a possible resistance to pathogen.

O Brasil é o principal produtor de maracujá amarelo. Entretanto, a produtividade é baixa, cerca de 1 0.000 t por hectare. A produção de frutos varia com o cultivar, condições climáticas, manejo e outros fatores, principalmente doenças causadas por bactérias e vírus. Metodologias de transformação genética são alternativas modernas para obter plantas resistentes. A proteína derivada de inseto, atacina A atua como bactericida, e tem sido utilizada para conferir resistência a espécies vegetais. Os objetivos do presente estudo foram (i) obter a regeneração de brotos in vitro, (ii) testar a eficiência de agentes seletivos durante o processo organogênico, (iii) construir o cassete contendo o gene atacína A, e (iv) determinar as condições físicas e biológicas para a transformação genética de plantas de maracujá amarelo utilizando o método de biobaiística. Em relação a estudos in vitro, três recipientes de cultura foram avaliados como também diferentes concentrações de benzylaminopurina (BA) e água de coco que foram adicionadas ao meio basal. Phytagei e agar também foram testados como agentes solidificantes. As culturas foram avaliadas quanto à resposta morfogênica dos discos foliares. O gene atacina A foi sequenciado e clonado para receber o promotor CAMV 35S com um enhancer duplicado e o terminador 35S. Este vetor foi denominado pFFatacina. O cassete de expressão foi cionado nos vetores pcambia 1300 e pcambia 2300 que contêm os genes higromicina (hpt) e canamicina (nptll), respectivamente. Discos foliares, assim como segmentos entrenodais e hipocotiledonares que induzem calos, foram usados nos experimentos de biobaiística. A expressão do gene uida foi avaliada para testar os parâmetros de bombardeamento, pressão de gás Hélio (psi) e a distância da tela de retenção até o tecido alvo (cm). A resposta organogênica dos discos foliares não diferiu quando placas de petrí, tubos ou frascos foram usados, embora os tubos (2,4 x 8,5 cm, 30 mi) mostraram uma resposta ligeiramente melhor. O meio MS (Murashige & Skoog, Physiologia Plantarum, 15, 1962) solidificado com agar (0,6%) e suplementado com 0,5 mg/L BA e 5% de água de coco (w/v) provou ser eficiente na indução de organgênese. Brotos foram obtidos após 30 dias. Segmentos entrenodais e hipocotiledonares produziram 300 estruturas semelhantes a gemas por explante. Microscopia de varredura e análises histológicas demonstraram ser estruturas foliares, as quais evoluíram em brotos após 50 dias em Y2 MS. Higromicina a 5 mg/L provou ser um agente seletivo apropdado, inibindo organogênese em 60% dos explantes. Canamicina a 50 mg/L foi também efetiva. Calos morfogênicos de até 10 dias e discos foliares de 3 dias de cultivo mostraram elevados níveis de expressão transiente sob 80016,5 ou 100019,5 (psi de gás Héliolcm de distância de võo dos microprojéteis). Foram realizados experimentos de co-transformação com pB[426 (8,7 kb) que contém o gene npdi e pFFatacina (5,25 kb), como também utilizando-se um único vetor (pcatacina 1300), Freqüência de transformação estável de 0,85% foi obtida. A integração do transgene foi confirmada por PCR para o gene atacina A. Este é o primeiro trabalho que relata a transferência de um gene de interesse para plantas de maracujá amarelo por biobalística.
Brazil is the leading producer of the yellow passion fruit. However, the productivity is fairly low, about 10,000 t per hectare. Fruit yields vary with cultivars, climatic conditions, management and other factors, namely bacterial and virus díseases. Genetic transformation methodologies are modern alternatives to obtain plant resistance. The insectderived protein, attacin A acts as bacterícide, and it has been used to confer resistance to plant species. The objectives of the present study were (i) to obtain in vitro shoot regeneration, (ii) to test the efficiency of certain selective agents during the organogenesis process, (iií) to construct the cassette containing the attacin A gene, and (iv) to determine the physical and biological conditions for genetic transformation of passion fruit plants by using the biolistic approach. Regarding the ín vítro studies, three culture recipients were evaluated as well as different concentrations of benzylaminopurine (BA) and coconut water that suppiemented the basal medium. Phytagei and agar were aiso tested as solidifying agents. Cultures were evaluated with respect to leaf dises morphogenic responses. The attacín A gene was sequenced, and cioned to receive the CAMV 35S promoter with a duplicated enhancer sequence, and the 35S terminator. This vector was denoted pFFatacina. The cassette was cioned in pcambia 1300 and pcambia 2300 vectors that contain the hygromícin (hpt) and kanamycin (nptil) genes, respectively. Leaf discs, as well as internodal segments and hypocotyl-derived sections chosen to índuce calii, were used in the biolistic experiments. The uida gene expression was evaluated for testing bombardment parameters, namely the helium pressure (psi) and the distance from the stopping screen to the target tissue (cm). The organogenic response of the leaf discs did not differ when petri dishes, tubes or culture vesseis were used although the tubes (2,4 x 8,5 cm, 30 ml capacity) showed to be slightly better. The agar (0.6%)-solidifíed MS (Murashige & Skoog, Physíología Plantarum, 15, 1962) medium suppiemented with 0.5 mg/L BA and 5% (wlv) coconut water proved to be efficient to induce organogenesis. Shoots were obtained after 30 days. Internada[ and hypocotyl-derived segments produced 300 bud-like structures per explant. Scanning microscopy and histologícal anaiyses provided evidences that they were leaf structures, which last 50 days in 1/2 MS to evolve into shoots. Hygromicin at 5 mg/L proved to be proper as selective agent, inhibiting organogenesis in 60% of the explants. Kanamycin at 50 mg/L was also effective. Morphogenic calli up to 10 days old and 3 d-leaf discs showed high levels of transient uida gene expression under 80016.5 or 1000/9.5 helium pressureldistance from the stopping screen to the target tissue. Cotransformation experiments with pBI426 (8.7 kb) that contain the nptil gene and pFFatacina (5.25 kb) were carried on as well singre vector transformation tríals (pcatacina 1300). Stable transformation frequency of 0.85% was obtained. Transgene integration was confirmei by PCR for the attacin A gene. This is the first report on agronomicaily useful-gene transfer to yeilow passion fruit plants by biolistics.

Thesis (MSc)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Strain ST4SA, isolated from soya beans, was identified as Enterococcus mundtii. BacST4SA, a bacteriocin produced by strain ST4SA inhibited the growth of Acinetobacter baumannii, Bacillus cereus, Clostridium tyrobutyricum, Enterococcus faecalis, Enterococcus faecium, Lactobacillus sakei, Propionibacterium spp., Streptococcus caprinus, Pediococcus sp., Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, and unidentified middle ear isolates A, BW, DW, F, G, and H. BacST4SA was active against Pseudomonas aeruginosa G, BG, I, J, B and E, although variable degrees of resistance were observed for some strains. BacST4SA is positively charged, hydrophobic, contains the YGNGV sequence in the N-terminal, a double-glycine processing site and a disulphide bridge, all of which is typical of a class IIa bacteriocin. The operon, which contains a structural-, ATP-dependent transporter- and immunity gene, is located on a 50-kb plasmid. The 58-amino acid prepeptide is homologous to mundticin KS, mundticin AT06 and bacteriocin QU 2, and differs from enterocin CRL35 by only two amino acids. The 674-amino acid ATP-dependent transporter, consisting of a peptidase C39B domain, an ABC-transporter and an ABC-DLP family domain, displayed 98.9% homology to mundticin KS and 99.25% to enterocin CRL35. The 98-amino acid immunity gene of bacST4SA is completely homologous to enterocin CRL35 and 96.9% to mundticin KS. BacST4SA is 3.950 kDa in size, based on electron spray mass spectrometry. The peptide was isolated from the cell-free supernatant, precipitated with 80% saturated ammonium sulphate, dialysed and freeze-dried to 1 638 400 AU (arbitrary units) per ml. No change in antimicrobial activity was recorded when bacST4SA was incubated in buffer ranging from pH 2 to 12, heated to 100 °C for 90 min and 121 °C for 20 min, and when incubated in the presence of Tween 20, Tween 80, Triton X-100, SDS, urea, EDTA, middle ear fluid and blood. Optimal levels of bacST4SA production (51 200 AU/ml) was recorded after 14 h of growth in MRS broth at 30°C. Maximum production (102 400 AU/ml) was recorded in modified MRS media supplemented with tryptone, yeast extract, a combination of tryptone and yeast extract, K2HPO4 (10.0 or 20.0 g/l), or with the addition of DL-6,8-thoictic acid, L-ascorbic acid, and thiamine, respectively. BacST4SA is bactericidal towards E. faecium HKLHS and bacteriostatic towards S. pneumoniae 40 and middle ear isolates F, BW and H. The peptide adsorbed maximal (94%) to S. pneumoniae 40, P. aeruginosa 25 and E. faecium HKLHS. BacST4SA forms pores in the cytoplasmic membrane of sensitive cells, leading to dissipation of the cell membrane and leakage of cytoplasmic material. BacST4SA was compared with various other antimicrobial treatment agents, and revealed similar to a higher activity towards a number of these agents. BacST4SA revealed a similar level of activity against E. faecium HKLHS and middle ear pathogens P. aeruginosa J and S. pneumoniae 27 when compared with tetracycline (30μg). However, bacST4SA revealed much higher activity when compared to nasal sprays, aminoglycosides, cephalosporins, fluoroquinolones, lincosamides, macrolides, nitroimidazole, penicillin, quinolones, sulfonamides, chloramphenicol, furanzolidone, fusidic acid, rifampicin, trimethoprim, trimethoprim-sulfamethoxazole and vancomycin when tested in vitro.
AFRIKAANSE OPSOMMING: Stam ST4SA, geïsoleer uit sojabone, is as Enterococcus mundtii geidentifiseer. BacST4SA, ‘n bakteriosien geproduseer deur stam ST4SA het die groei van Acinetobacter baumannii, Bacillus cereus, Clostridium tyrobutyricum, Enterococcus faecalis, Enterococcus faecium, Lactobacillus sakei, Propionibacterium spp., Streptococcus caprinus, Pediococcus sp., Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae en ongeïdentifiseerde middeloor isolate A, BW, DW, F, G, en H geinhibeer. BacST4SA is aktief teen Pseudomonas aeruginosa stamme G, BG, I, J, B en E, alhoewel effense weerstand soms waargeneem is. BacST4SA het ‘n netto positiewe lading, is hidrofobies, bevat die YGNGV-volgorde in die N-terminaal, ‘n dubbel-glisien prosesserings setel en ‘n disulfied brug, kenmerkend van klas IIa bakteriosiene. Die operon, wat bestaan uit ‘n strukturele geen, ‘n ATP-afhanklike transport sisteem geen en ‘n immuniteits-geen, is op ‘n 50 kb plasmied gelokaliseer. Die voorloper peptied (58 aminosure lank), is homoloog aan mundticin KS, mundticin AT06 en bakteriosien QU 2 en verskil van enterocin CRL35 met slegs twee aminosure. Die ATP-afhanklike transporter (674 aminosure lank) bestaan uit ‘n peptidase C39B domein, ‘n ABC-transporter en ‘n ABC-DLP tipe domein en is 98.9% homoloog aan mundticin KS and 99.25% aan enterocin CRL35. Die immuniteits-geen (98 aminosure lank) van bacST4SA is ten volle homoloog aan enterocin CRL35 en 96.9% homoloog aan mundticin KS. BacST4SA is 3.950 kDa groot, gebaseer op elektrosproei-massa spektrometrie. Die peptied is uit selvrye supernatant geïsoleer, met 80% versadigde ammonium sulfaat gepresipiteer, gedialiseer en gevriesdroog tot ’n finale konsentrasie van 1 638 400 AE (arbitrêre eenhede) per ml. Geen verandering in antimikrobiese aktiwiteit is waargeneem tydens inkubasie van bacST4SA in buffer van pH 2 tot 12, tydens verhitting (100 °C vir 90 min en 121 °C vir 20 min) en tydens inkubasie in die teenwoordigheid van Tween 20, Tween 80, Triton X-100, SDS, ureum, EDTA, middeloor vloeistof en bloed. Optimale vlakke van bacST4SA produksie (51 200 AE/ml) is na 14 h groei in MRS media by 30°C waargeneem. Maksimale vlakke van die peptied (102 400 AE/ml) is geproduseer in gemodifiseerde MRS medium, aangevul met triptoon, gisekstrak, ‘n kombinasie van triptoon en gisekstrak, K2HPO4 (10.0 of 20.0 g/l), of met byvoeging van DL-6,8-thioktiensuur, L-askorbiensuur, en tiamien onderskeidelik. BacST4SA is bakteriosidies teenoor E. faecium HKLHS en bakteristaties teenoor S. pneumoniae 40 en middeloor isolate F, BW en H. Die peptied adsorbeer optimaal (94%) aan S. pneumoniae 40, P. aeruginosa 25 en E. faecium HKLHS. BacST4SA vorm porieë in die selmembraan van sensitiewe selle en lei tot vernietiging van die selmembraan en lekkasie van die sitoplasma inhoud. In vergelykende studies het bacST4SA ‘n soortgelyke en selfs hoër antimikrobiese aktiwiteit teenoor ‘n aantal bekende antimikrobiese middels getoon. Die aktiwiteit van bacST4SA is soortgelyk aan dié van tetrasiklien (30μg) in toetse teen E. faecium HKLHS en middeloor patogene P. aeruginosa J en S. pneumoniae 27. BacST4SA het egter in ’n in vitro vergelyking met neussproeie, aminoglisiedes, cephalosporiene, fluoroquinolone, lincosamides, makroliede, nitroimidazole, penisilien, quinolone, sulfonamide, chloramphenicol, furanzolidone, fusiensuur, rifampisien, trimethoprim, trimethoprim-sulfamethoxazool en vankomisien ‘n baie hoër aktiwiteit teen patogene getoon.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The indiscriminate use of antimicrobial drugs in aquaculture may cause development of drug-resistant bacteria, which become more difficult to be controlled and eliminated. Probiotics may represent an alternative prophylactic disease control, replacing the use of antibiotics. In this respect, this study isolated, tested and identified potential probiotic bacteria from the gut of cobia, Rachycentron canadum, a potential candidate for marine aquaculture. 40 animals were captured, 10 from a private hatchery and 30 from an offshore culture system (PE, Brazil) between November 2010 and July 2011. Fishes from the hatchery had weight of 139.30 ± 31.52 g and length of 27.13 ± 1.46 cm, while those from offshore culture system weighed 456.77 ± 264.46 g and had length of 37.29 ± 6.05 cm. 45 bacterial were isolated and tested in vitro against five known pathogenic species, Aeromonas hydrophila (IOC/FDA 110-36), Pseudomonas aeruginosa (ATCC 15442), Streptococcus agalactiae (ATCC 13813), Vibrio parahaemolyticus (ATCC 17802) and Vibrio vulnificus (ATCC 27562). Fifteen strains (33.33%) had antibacterial activity to at least one pathogen, while eight (17.77%) were inhibited all pathogens tested. There was significant difference (P<0.05) between percentage of potential probiotic obtained from different seasons, rainy and dry. Strains that presented the best results antagonism test in vitro were identified as Lactobacillus plantarum, Bacillus coagulans, Klebsiella spp., Bacillus circulans, Lactococcus lactis subsp. lactis and Bacillus firmus. The largest inhibition zone observed in the antagonism test was produced by B. circulans against V. vulnificus. Some potential probiotic species identified were characterized by others authors, but isolated from the intestine of other fish species. It is suggested in vivo antagonism tests are performed to prove the effectiveness of these bacteria as probiotic to cobia.
O uso indiscriminado de drogas antimicrobianas na aquicultura pode contribuir para o desenvolvimento de bactérias resistentes aos medicamentos, o que torna esses micro-organismos mais difíceis de serem controlados e eliminados. Probióticos podem representar uma alternativa profilática no controle de doenças, em substituição ao uso de antibióticos. Neste contexto, o estudo foi realizado com o objetivo de isolar, testar e identificar bactérias com potencial probiótico do beijupirá, Rachycentron canadum, potencial candidato para a piscicultura marinha. Foram coletados 40 animais, 10 na fase de berçário e 30 na fase de engorda em sistema offshore (PE, Brasil), no período de novembro de 2010 a julho de 2011. Os peixes coletados do berçário apresentaram peso de 139,30 ± 31,52 g e comprimento de 27,13 ± 1,46 cm, enquanto os animais provenientes do sistema offshore apresentaram peso de 456,77 ± 264,46 g e comprimento de 37,29 ± 6,05 cm. Foram obtidos 45 isolados bacterianos testados in vitro frente a cinco espécies patogênicas conhecidas, Aeromonas hydrophila (IOC/FDA 110-36), Pseudomonas aeruginosa (ATCC 15442); Streptococcus agalactiae (ATCC 13813); Vibrio parahaemolyticus (ATCC 17802) e Vibrio vulnificus (ATCC 27562). Quinze isolados (33,33%) apresentaram atividade antibacteriana a pelo menos um patógeno e oito (17,77%) inibiram todos os patógenos testados. Houve diferença significativa (P<0,05) na proporção de isolados potenciais probióticos obtidos nas distintas épocas do ano, chuvosa e seca. Os isolados que apresentaram os melhores resultados no teste de antagonismo in vitro foram identificados como Lactobacillus plantarum, Bacillus coagulans, Klebsiella spp., Bacillus circulans, Lactococcus lactis subsp. lactis e Bacillus firmus. O maior halo de inibição observado no teste de antagonismo foi produzido por B. circulans frente ao V. vulnificus. Algumas espécies potenciais probióticas identificadas já foram caracterizadas por outros autores, porém isoladas de intestino de outras espécies de peixes. Sugere-se a realização de testes de antagonismo in vivo para que seja comprovada a efetividade das bactérias como probióticas para o beijupirá.

Orientador: Saulo Santesso Garrido
Resumo: Os peptídeos antimicrobianos (PAMs) são uma alternativa interessante como bioconservantes de alimentos devido a sua eficiência e, principalmente, à baixa toxicidade quando comparados aos conservantes químicos tradicionais. As bacteriocinas, uma classe de PAMs, têm atividade antimicrobiana em espécies responsáveis pela degradação de alimentos e, portanto, atualmente são exploradas como bioconservadores. A Leucocina C-TA33a (LeuC), um tipo de bacteriocina produzida pela cepa bacteriana Leuconostoc mesenteroides TA33a, é conhecida por ter um amplo espectro antimicrobiano. Estudos de alinhamento da estrutura primária de diferentes bacteriocinas revelaram que LeuC conserva em sua estrutura regiões homólogas também compartilhadas por outras bacteriocinas, como Sacacina P, Bavaricina A e Enterocina A, possivelmente revelando uma importante relação entre essas sequências de aminoácidos e suas atividades antimicrobianas. Este estudo tem como objetivo sintetizar diferentes peptídeos baseados na estrutura primária da Leucocina CTA33a, a fim de identificar as principais regiões responsáveis pela atividade antimicrobiana. Os peptídeos LeuC-Nt, LeuC-0Cys, LeuC-Ala9, LeuC-Ala14 e LeuC-Cys9,14 foram sintetizados por metodologia de fase sólida, purificados e analisados por HPLC e caracterizados por ESI-MS. Ensaios em meio líquido foram realizados para a determinação do percentual de inibição de crescimento microbianos dos peptídeos sobre espécies patogênicas. Os micro-organismos testados fora... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Antimicrobial peptides (PAMs) are an interesting alternative, because biopreservatives present high efficiency and low toxicity when compared to classic preservatives. Bacteriocins, a class of PAMs, have antimicrobial activity in species responsible for food degradation and are currently being exploited as biopreservatives. The Leucocin C-TA33a (LeuC), a type of bacteriocin, is produced by the Leuconostoc mesenteroides TA33a strain and is known for a broad antimicrobial spectrum. Studies of alignment of the primary structure of different bacteriocins revealed that LeuC retains in its structure homologous regions also shared by other bacteriocins, such as Sacacin P, Bavaricin A and Enterocin A, possibly revealing an important relationship between these amino acid sequences and their antimicrobial activities. This study aims to synthesize different peptides based on the primary structure of Leucocin C-TA33a, with the objective of identifying the main regions responsible for antimicrobial activity. The peptides LeuC-Nt, LeuC-0Cys, LeuC-Ala9, LeuC-Ala14 and LeuC-Cys9,14 were synthesized by solid phase methodology, purified by HPLC and characterized by ESI-MS. Liquid assays were performed to determine the percentage of inhibition of microorganisms of the peptides on pathogenic species. The microorganisms tested were Salmonella serotype Typhimurium, Staphylococcus aureus, Escherichia coli and Listeria monocytogenes. Studies were also conducted through circular dichroism, molecular ... (Complete abstract click electronic access below)
Mestre

La bacteriose a e. Chrysanthemi pv. Dianthicola (echr) facteur limitant de la production du dahlia est transmise par la multiplication vegetative. Afin de proposer une methode de diagnostic plus precise que la detection visuelle, les methodes immunoenzymatiques ont ete etudiees et adaptees pour la detection d'echr dans les tissus du dahlia. La methode das-elisa (double antibody sandwich) est evaluee par rapport aux methodes de diagnostic de reference (isolement et immunofluorescence). Son utilisation, pour l'analyse sanitaire du materiel de propagation vis-a-vis d'echr seul et associe eventuellemnt a la mosaique du dahlia (damy), est etudiee en vue d'une selection sanitaire. Les etudes effectuees pour optimiser les reactifs, pour determiner les parametres pouvant modifier la reaction antigene-anticorps

碩士
國立屏東科技大學
生物科技系所
105
Aquaculture is an important industry in the world. However, bacteria-induced diseases such as septicemia induced by Aermonas hydrophila, often result in severe economic loss in aquaculture. Probiotics in one of strategies for preventing disease in aquaculture. Laboratory previous study , the bacteria was isolated from the lake and named as Chromobacterium aquaticum according to 16s RNA sequencing and biotechemistry analysis. After four days of culture, the supernatant has antibacterial activity. The antimicrobial activity in supernatant were remained in the presens of different temperature (40~100℃) and pH (2~11).Dietary supplementation of the c.aquaticum for one month increase IL、IL、IL、F-、F-kB、Lysozyme、c3b expression in whole body；IL、IL、IL、F-、F-kB、Lysozyme、c3b expression in intestine of zebrafish. The survival rate of A. hydrophila-infection zebrafish was increase after Chromobacterium aquaticum administration. This result to demonstrate that Chromobacterium aquaticum can be used as a potential probiotic in aquaculture for diseae control.