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Análise de sulfonamidas no mel: validação e optimização de um método de HPLC-fluorescência

Correia, Daniela M.
Fonte: Instituto Politécnico de Bragança, Escola Superior Agrária Publicador: Instituto Politécnico de Bragança, Escola Superior Agrária
Tipo: Dissertação de Mestrado
POR
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Os antibióticos são usados no tratamento de abelhas com o intuito de combater infecções associadas às doenças da Loque Americana e Loque Europeia que atacam as larvas das abelhas levando à destruição da colmeia. As sulfonamidas são um dos grupos de antibióticos mais usados pelos apicultores. Como a Legislação Europeia, proíbe a comercialização do mel na presença de quaisquer resíduos de antibióticos, a implementação de métodos analíticos que permitam a detecção e quantificação destes resíduos afigura-se uma tarefa fundamental. O presente trabalho teve como objectivo a optimização, validação e aplicação de um método de HPLC, com detector de fluorescência e derivatização pré-coluna, para a análise de 11 sulfonamidas em amostras de mel: sulfatiazol, sulfamerazina, sulfametazina, sulfametizol, sulfametoxipiridazina, sulfisoxazol, sulfadoxina, sulfametoxazol, sulfadimetoxina, sulfaquinoxalina e sulfameter (padrão interno). O método de calibração usado foi o de sobreposição de matriz de mel com padrão interno. A separação foi efectuada numa coluna de fase reversa à temperatura de 32 ºC em modo de gradiente usando 2 eluentes preparados com diferentes proporções de tampão acetato (pH 4...

The Portuguese experience with bee pathology laboratories for the diagnosis of bee diseases

Pires, Sância; Valério, Maria José; Almeida, Armandina
Fonte: World Organisation for Animal Health, OIE-Reference Laboratory for Bee Diseases Publicador: World Organisation for Animal Health, OIE-Reference Laboratory for Bee Diseases
Tipo: Conferência ou Objeto de Conferência
ENG
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In Portugal there are two referenced laboratories responsible for the diagnosis of bee diseases, namely the National Laboratory of Veterinary Investigation (LNIV) which is the National Reference Laboratory and the Bee Pathology Laboratory which belongs to the Agrarian Superior School of Bragança (ESAB). This laboratory was created in collaboration with the Apicultural Association of Montesinho Natural Park (AAPNM) and was certified by the Veterinary Authority in 2006. The Bee Pathology Laboratory is located in the city of Bragança, integrated in the Trás – Os – Montes e Alto Douro region. Is a research and also laboratory that provides a service to individual beekeepers when requested either directly from the laboratory or through the Apicultural Association including those of Controlled Zones. These Zones are defined as areas within the national territory where the systematic control of the bee diseases is undertaken. They are regulated at the national level by the Decree – Law nº 203 of November 25th -2005, which also established which diseases are considered to be of mandatory notification in our country. The diseases referred in this regulation are: American Foulbrood (AFB), European Foulbrood (EFB), Acarapidosis, Varroosis...

The first Paenibacillus larvae bacteriophage endolysin (PlyPl23) with high potential to control American foulbrood

Oliveira, A.; Leite, Marta; Kluskens, Leon; Santos, Sílvio; Melo, Luís D. R.; Azeredo, Joana
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em /07/2015 ENG
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Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-pos- itive pathogens. This work describes the characterization of a novel endolysin (PlyPl23) encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restric- tion to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new endolysin described herein has an N-acetylmuramoyl-L-alanine ami- dase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0), showing its feasible applica- tion in the field. At pH 7.0, a concentration of 0.2 M of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly...

A PCR Detection Method for Rapid Identification of Melissococcus pluton in Honeybee Larvae

Govan, V. A.; Brözel, V.; Allsopp, M. H.; Davison, S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/1998 EN
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Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.

Complete Genome Sequence of Melissococcus plutonius ATCC 35311 ▿

Okumura, Kayo; Arai, Rie; Okura, Masatoshi; Kirikae, Teruo; Takamatsu, Daisuke; Osaki, Makoto; Miyoshi-Akiyama, Tohru
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2011 EN
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We report the first completely annotated genome sequence of Melissococcus plutonius ATCC 35311. M. plutonius is a one-genus, one-species bacterium and the etiological agent of European foulbrood of the honeybee. The genome sequence will provide new insights into the molecular mechanisms underlying its pathogenicity.

Diversity of Melissococcus plutonius from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates

Arai, Rie; Tominaga, Kiyoshi; Wu, Meihua; Okura, Masatoshi; Ito, Kazutomo; Okamura, Naomi; Onishi, Hidetaka; Osaki, Makoto; Sugimura, Yuya; Yoshiyama, Mikio; Takamatsu, Daisuke
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 19/03/2012 EN
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European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose...

Complete Genome Sequence of Melissococcus plutonius DAT561, a Strain That Shows an Unusual Growth Profile and Is Representative of an Endemic Cluster in Japan

Okumura, Kayo; Arai, Rie; Okura, Masatoshi; Kirikae, Teruo; Takamatsu, Daisuke; Osaki, Makoto; Miyoshi-Akiyama, Tohru
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2012 EN
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We report the complete genome sequence of Melissococcus plutonius DAT561, which is a causative agent of European foulbrood. M. plutonius DAT561 is a representative of nonfastidious strains isolated in Japan. The addition of potassium phosphate was not required for normal growth, unlike for typical M. plutonius strain/isolates.

Genome Sequence of Paenibacillus alvei DSM 29, a Secondary Invader during European Foulbrood Outbreaks

Djukic, Marvin; Becker, Dominik; Poehlein, Anja; Voget, Sonja; Daniel, Rolf
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2012 EN
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Paenibacillus alvei is known as a secondary invader during European foulbrood of honeybees. Here, we announce the 6.83-Mb draft genome sequence of P. alvei type strain DSM 29. Putative genes encoding an antimicrobial peptide, a binary toxin, a mosquitocidal toxin, alveolysin, and different polyketides and nonribosomal peptides were identified.

Inhibitory Effect of Gut Bacteria from the Japanese Honey Bee, Apis cerana japonica, Against Melissococcus plutonius, the Causal Agent of European Foulbrood Disease

Wu, Meihua; Sugimura, Yuya; Iwata, Kyoko; Takaya, Noriko; Takamatsu, Daisuke; Kobayashi, Masaru; Taylor, DeMar; Kimura, Kiyoshi; Yoshiyama, Mikio
Fonte: University of Wisconsin Library Publicador: University of Wisconsin Library
Tipo: Artigo de Revista Científica
Publicado em 15/09/2014 EN
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46.99%
European foulbrood is a contagious bacterial disease of honey bee larvae. Studies have shown that the intestinal bacteria of insects, including honey bees, act as probiotic organisms. Microbial flora from the gut of the Japanese honey bee, Apis cerana japonica F. (Hymenoptera: Apidae), were characterized and evaluated for their potential to inhibit the growth of Melissococcus plutonius corrig. (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae), the causative agent of European foulbrood. Analysis of 16S rRNA gene sequences from 17 bacterial strains isolated by using a culture-dependent method revealed that most isolates belonged to Bacillus, Staphylococcus, and Pantoea. The isolates were screened against the pathogenic bacterium M. plutonius by using an in vitro growth inhibition assay, and one isolate (Acja3) belonging to the genus Bacillus exhibited inhibitory activity against M. plutonius. In addition, in vivo feeding assays revealed that isolate Acja3 decreased the mortality of honey bee larvae infected with M. plutonius, suggesting that this bacterial strain could potentially be used as a probiotic agent against European foulbrood.

Diversity of honey stores and their impact on pathogenic bacteria of the honeybee, Apis mellifera

Erler, Silvio; Denner, Andreas; Bobiş, Otilia; Forsgren, Eva; Moritz, Robin F A
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
EN
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Honeybee colonies offer an excellent environment for microbial pathogen development. The highest virulent, colony killing, bacterial agents are Paenibacillus larvae causing American foulbrood (AFB), and European foulbrood (EFB) associated bacteria. Besides the innate immune defense, honeybees evolved behavioral defenses to combat infections. Foraging of antimicrobial plant compounds plays a key role for this “social immunity” behavior. Secondary plant metabolites in floral nectar are known for their antimicrobial effects. Yet, these compounds are highly plant specific, and the effects on bee health will depend on the floral origin of the honey produced. As worker bees not only feed themselves, but also the larvae and other colony members, honey is a prime candidate acting as self-medication agent in honeybee colonies to prevent or decrease infections. Here, we test eight AFB and EFB bacterial strains and the growth inhibitory activity of three honey types. Using a high-throughput cell growth assay, we show that all honeys have high growth inhibitory activity and the two monofloral honeys appeared to be strain specific. The specificity of the monofloral honeys and the strong antimicrobial potential of the polyfloral honey suggest that the diversity of honeys in the honey stores of a colony may be highly adaptive for its “social immunity” against the highly diverse suite of pathogens encountered in nature. This ecological diversity may therefore operate similar to the well-known effects of host genetic variance in the arms race between host and parasite.

High quality draft genome of Lactobacillus kunkeei EFB6, isolated from a German European foulbrood outbreak of honeybees

Djukic, Marvin; Poehlein, Anja; Strauß, Juliane; Tann, Fabian Jannik; Leimbach, Andreas; Hoppert, Michael; Daniel, Rolf
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
Publicado em 19/02/2015 EN
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The lactic acid bacterium Lactobacillus kunkeei has been described as an inhabitant of fructose-rich niches. Here we report on the genome sequence of L. kunkeei EFB6, which has been isolated from a honeybee larva infected with European foulbrood. The draft genome comprises 1,566,851 bp and 1,417 predicted protein-encoding genes.

First Insights into the Genome of Fructobacillus sp. EFB-N1, Isolated from Honey Bee Larva Infected with European Foulbrood

Djukic, Marvin; Daniel, Rolf; Poehlein, Anja
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em 30/07/2015 EN
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European foulbrood is a worldwide disease affecting the honey bee brood. Here, we report the draft genome sequence of Fructobacillus sp. EFB-N1, which was isolated from an infected honey bee larva derived from a Swiss European foulbrood outbreak. The genome consists of 68 contigs and harbors 1,629 predicted protein-encoding genes.

The pathogenicity for honey-bee larvae of microorganisms associated with european foulbrood

Bailey, L.
Fonte: INRA - Instituto Nacional de Investigação Agronômica da França Publicador: INRA - Instituto Nacional de Investigação Agronômica da França
Tipo: Journal Article-postprint
EN; ENGLISH
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Tests made on larvae in normal bee colonies with pure cultures of Streptococcus pluton (White), Streptococcus faecalis Andrews and Horder, and Bacillus alvei Cheshire and Cheyne, three bacterial species commonly associated with European foulbrood (EFB), showed that S. pluton was the natural prirnary etiological agent. These results, together with other recent work, indicate that S. pluton is the cause of EFB throughout the world. Of the other associated bacterial species, Bacterium eurydice White, which is the most commnon, and S. faecalis probably have supplementary pathogenic effects. Bacillus alvei and other less common bacilli are saprophytes of the dead larvae.

The brood diseases of bees

Phillips, Everett Franklin, 1878-1951
Fonte: United States Dept. of Agriculture, Bureau of Entomology; United States Dept. of Agriculture, Bureau of Entomology ( Washington, D.C ) Publicador: United States Dept. of Agriculture, Bureau of Entomology; United States Dept. of Agriculture, Bureau of Entomology ( Washington, D.C )
Tipo: Artigo de Revista Científica Formato: 5 p. : ; 24 cm.
Publicado em //1906 ENGLISH
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Caption title.; (Statement of Responsibility) by E.F. Phillips.

The cause of european foul brood

Bailey, L.
Fonte: INRA - Instituto Nacional de Investigação Agronômica da França Publicador: INRA - Instituto Nacional de Investigação Agronômica da França
EN
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36.31%

The epizootiology of european foulbrood of the larval honey bee, Apis mellifera Linnaeus

Bailey, L.
Fonte: INRA - Instituto Nacional de Investigação Agronômica da França Publicador: INRA - Instituto Nacional de Investigação Agronômica da França
Tipo: Journal Article-postprint
EN; ENGLISH
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Larvae, artificially infected when 0 to 1 day old with Streptococcus pluton (White) and placed in colonies, were usually ejected by adult bees. Ejection was delayed from colonies deprived either of unsealed brood or their queen, or which were reinforced with adult bees. The feces of surviving larvae whose weight was subnormal, contained many viable cells of S. pluton. Colonies reinforced with unsealed brood removed more infected larvae than usual. It is concluded that infected larvae are ejected when larval food is merely adequate, as it may be when a colony is growing rapidly ; and they are kept when larval food is more abundant, as it may be when brood rearing is retarded. Natural outbreaks of disease occurred when brood-rearing, in colonies heavily infected with S. pluton, was increasing during nectar flows, which was also when secondary infection with Bacterium eurydice White increased. At such times, larvae heavily infected with both organisms may die quicker than they are being removed. At the same time infection of new larvae with S. pluton seemed to decrease: transmission of S. pluton was presumably checked by the death and ejection of unsealed larvae. Thus outbreaks usually seemed to be self-limiting. Colonies eject introduced larvae even more readily after the main nectar flows which may account for the difficulty in causing disease artificially at this time.

European foul brood : a disease of the larval honeybee (Apis mellifera L.) caused by a combination of streptococcus pluton (Bacillus pluton White) and Bacterium eurydice White

Bailey, L.
Fonte: INRA - Instituto Nacional de Investigação Agronômica da França Publicador: INRA - Instituto Nacional de Investigação Agronômica da França
Tipo: Journal Article-postprint
EN; ENGLISH
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Result of preliminary experiments have shown that European foul brood disease of the larval honeybee can be caused in bee colonies by spraying their brood with suspensions of Streptococcus pluton (Bacillus pluton White) and Bacterium eurydice White if the two organisms are grown together in mixed anaerobic culture ; fifth subcultures of a mixed culture were usually virulent, although virulence diminished rapidly after further sub-cultivation. It was not possible to cause the disease by simultaneous inoculation with separate cultures of the two organisms. As there is a tendency for colonies of S. pluton and B. eurydice to grow within, or upon, each other on agar in anaerobic cultures, the separate cultures of each organism were subcultured five times to try to ensure the exclusion of the other. The results of these experiments showed that virulence was maintained by one or both organisms in mixed culture, but the possibility remained that only one of the organisms was pathogenic; its virulence may have been maintained by the growth of the other which may, in vivo, be a secondary invader of the diseased larva ...

Ætiology of european foul brood ; a disease of the larval honey-bee

Bailey, L.
Fonte: INRA - Instituto Nacional de Investigação Agronômica da França Publicador: INRA - Instituto Nacional de Investigação Agronômica da França
Tipo: Journal Article-postprint
EN; ENGLISH
Relevância na Pesquisa
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THE cause of European foul brood was originally thought to be Bacillus alvei or a mixture of B. alvei and Streptococcus apis. The causative organism was later named and described by White as Bacillus pluton, a lanceolate Gram-positive bacterium. This organism is the first of several which have been found to appear in diseased larvæ. However, White and others failed to culture B. pluton in vitro and it was afterwards considered to be a dissociant form of Bacillus alvei, of Bacterium eurydice, or of both. Others have maintained that B. pluton is a separate organism. It has been pointed out that Streptococcus pluton would be a more suitable designation than Bacillus pluton both on morphological grounds and in the absence of evidence that it forms spores. For these reasons and those given below the organism will be referred to as Streptococcus pluton.

The isolation and cultural characteristics of streptococcus pluton and further observations on bacterium eurydice

Bailey, L.
Fonte: INRA - Instituto Nacional de Investigação Agronômica da França Publicador: INRA - Instituto Nacional de Investigação Agronômica da França
Tipo: Journal Article-postprint
EN; ENGLISH
Relevância na Pesquisa
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An account is given of the development of a reliable method for the isolation of Streptococcus pluton (Bacillus pluton White), an organism associated with European foul-brood disease of the larval honeybee. S. pluton, isolated as an anaerobe, may be trained to grow as an aerobe in rod form. Its principal anaerobic growth requirements are a low molar ratio of Na: K, high inorganic phosphate concentration, glucose or fructose, and undetermined factors provided by yeast extract. Peptones are harmful to growth. Aerobic growth has no very critical requirements other than glucose, fructose or sucrose. Bacterium eurydice White which, together with S. pluton, causes European foul-brood disease grows well anaerobically on a yeast extract + glucose + fructose medium; either sugar alone supports only feeble anaerobic growth. Anaerobic growth of B.eurydice is also accelerated by a low molar ratio of Na: K and is inhibited by peptones. S. pluton and B.eurydice appear to be separate organisms; no evidence has been obtained to support claims by previous workers that S. pluton is a variant of B. eurydice.

European foulbrood

Bailey, L.
Fonte: INRA - Instituto Nacional de Investigação Agronômica da França Publicador: INRA - Instituto Nacional de Investigação Agronômica da França
Tipo: Journal Article-postprint
EN
Relevância na Pesquisa
46.62%