RESUMO
AIMS: The purpose of this study was to investigate the occurrence of Escherichia coli O157 and O26 on Belgian dairy cattle farms, the presence of virulence genes in the confirmed isolates and the association of E. coli O26 presence with calf diarrhoea. METHODS AND RESULTS: In total, 233 recto-anal mucosal swabs (RAMS) were obtained from healthy and diarrheic dairy calves on three farms, each alternately visited three consecutive times. RAMS were analysed for presence of E. coli O157 and O26, and stx1, stx2 and eae virulence genes. Overall, 19% of RAMS tested positive for E. coli O157, while 31% tested positive for E. coli O26. The majority of isolates possessed both stx and eae, denoting a high pathogenic potential to humans. While both serogroups persisted at farm level, persistence within the same animal over time appeared to be relatively rare. Interestingly, E. coli O26 was already abundantly present at a younger age compared to E. coli O157. Calf diarrhoea could not be associated with presence of E. coli O26. CONCLUSIONS: Young dairy calves are important on-farm reservoirs of potentially pathogenic E. coli O157 and O26. A role of E. coli O26 in calf diarrhoea could not be confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: O157 and O26 are responsible for the majority of human STEC infections. Gaining more epidemiological information regarding their occurrence and persistence on cattle farms will contribute to a better understanding of STEC ecology and risk of human transmission.
Assuntos
Doenças dos Bovinos , Infecções por Escherichia coli , Escherichia coli O157 , Proteínas de Escherichia coli , Escherichia coli Shiga Toxigênica , Animais , Bélgica , Bovinos , Infecções por Escherichia coli/veterinária , Escherichia coli O157/genética , Fezes , Humanos , Masculino , VirulênciaRESUMO
Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens responsible for global outbreaks. This study was conducted to investigate the occurrence of 'gang of five' STEC serogroups (O26, O103, O111, O145, O157) on Belgian dairy cattle farms by overshoe (OVS) sampling, and to evaluate the presence of virulence genes in the obtained isolates. A total of 88 OVS, collected from the pen beddings of 19 Belgian dairy cattle farms, were selectively enriched in mTSBn, followed by immunomagnetic separation and plating onto CT-SMAC for O157 STEC isolation, as well as in Brila broth, followed by a selective acid treatment and plating onto CHROMagarTM STEC and chromIDTM EHEC for non-O157 STEC isolation. Overall, 11 of 19 farms (58%) tested positive for presence of 'gang of five' STEC. O26 STEC was most frequently isolated from OVS (11/88; 12·5%), followed by O157 (10/88; 11·5%), O145 (3/88; 3·5%) and O103 (3/88; 3·5%). Additionally, 35% of the OVS collected from pens housing young cattle 1-24 months of age tested positive for 'gang of five' STEC, indicating that this age category is more likely to harbour STEC compared to new-born and adult cattle. Importantly, half of the obtained 'gang of five' STEC isolates (48%) possessed the eae and stx2 gene, suggesting a high pathogenic potential to humans.
Assuntos
Adesinas Bacterianas/genética , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Toxina Shiga II/genética , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/genética , Animais , Bélgica , Bovinos , Fazendas , Fezes/microbiologia , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Separação Imunomagnética , Sorogrupo , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/isolamento & purificação , VirulênciaRESUMO
In many plant species nonzygotic embryos can develop from diploid somatic cells grown in tissue culture. Extracellular glycoproteins have been identified that can rescue arrested somatic embryos. One of these glycoproteins may be part of a mechanism that controls the expansion of plant cells.
Assuntos
Proteínas de Plantas/metabolismo , Sementes/crescimento & desenvolvimentoRESUMO
A continuous culture of Saccharomyces cerevisiae strain IFO 0233 growing aerobically at pH 3.4 shows persistent high-amplitude respiratory oscillations with a period of about 45 min. These robust autonomous cycles are accompanied by changes of product accumulation (acetaldehyde and acetic acid), intracellular pH, and intracellular redox state, as indicated by continuously monitored NADH fluorescence and the glutathione content of cell-free extracts. Perturbation of the oscillation of dissolved O2 was produced on addition of 100 microM glutathione, > 10 nM Na nitroprusside, 8 microM NaNO2, or 10 microM S-nitrosoglutathione. NO gas, putative NO.-releasing agents, or an inhibitor of NO synthase were ineffective. We suggest that nitrosation by NO+ of a component of a redox switch can account for these data, and we emphasise the different modes of action of the different redox forms of nitrogen monoxide.
Assuntos
Óxido Nítrico/metabolismo , Saccharomyces cerevisiae/metabolismo , Cátions/metabolismo , Etanol/metabolismo , Concentração de Íons de Hidrogênio , NAD/metabolismo , Óxido Nítrico Sintase/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
A multiple choice questionnaire was submitted to medical technologists in three medical laboratories, at varying times after obtaining an EN 45001 accreditation. A large majority (85-90 per cent) considered that their workload was increased by the accreditation process. In two laboratories, the technologists did not think that the accreditation process had improved the quality of the results. The major advantages were the fact that everything was traceable, that the technologists felt more sure about the procedures to follow, received more responsibilities and had a better knowledge of the tests they performed. The major disadvantages were the increased paperwork, discrepancies between the procedures and the reality, the fact that more attention is paid to the formalities than to the quality of the results and that the accreditation process decreased the adaptability. The number of advantages mentioned seemed to increase with the interval since the accreditation. A small majority of the technologists preferred working in an accredited laboratory than in a non accredited one.
Assuntos
Acreditação/estatística & dados numéricos , Atitude do Pessoal de Saúde , Laboratórios/normas , Pessoal de Laboratório Médico/psicologia , Coleta de Dados , Humanos , Laboratórios/estatística & dados numéricos , Pessoal de Laboratório Médico/estatística & dados numéricos , Controle de Qualidade , Reino Unido , Carga de TrabalhoRESUMO
Respiratory oscillation occurred during aerobic continuous culture of Saccharomyces cerevisiae. During oscillation, phase-related changes in NAD(P)H and GSH levels occur. Perturbation of oscillation and inhibition of respiration occurred when GSH or GSSG was injected; however, there was a phase delay in perturbation in the case of an injection during high respiration. The perturbation phase delay was not apparent when a combination of DL-buthionine-(S,R)-sulphoximine, GSH and 5-nitro-2-furaldehyde was injected. Perturbation by GSH injection caused the intracellular GSH concentration to increase, the GSSG concentration to decrease and the cessation of ethanol uptake. NAD(P)H during perturbation was inversely related to dissolved oxygen. Perturbation by calcium pantothenate and pyridoxal-HCl caused a period of enhanced respiration before oscillation returned. These results suggest that the NAD+/NADH redox is not directly involved in oscillation control and regulation involves glutathione metabolism. Possible regulation points include alcohol dehydrogenase inhibition and/or respiratory-chain inhibition.
Assuntos
Glutationa/metabolismo , Consumo de Oxigênio , Saccharomyces cerevisiae/metabolismo , Etanol/metabolismo , Glutationa/farmacologia , Dissulfeto de Glutationa/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Ácido Pantotênico/farmacologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de TempoRESUMO
Nine doses of oral spiramycin were administered to 59 adult healthy nasopharyngeal carriers of meningococci: a 2-gram loading dose followed by 1 g every 12 h for 4 days. There was a reduction in carriage of 85 and 59%, respectively, on the 2nd and 12th post-treatment day. There was no difference in vitro sensitivity to spiramycin and erythromycin of the meningococci isolated before and after treatment. Spiramycin may be advocated as an effective and safe alternative drug for the chemoprophylaxis of secondary meningococcal disease when sulphonamide resistance is suspected.
Assuntos
Portador Sadio/microbiologia , Leucomicinas/farmacologia , Nasofaringe/microbiologia , Neisseria meningitidis/efeitos dos fármacos , Humanos , Leucomicinas/efeitos adversos , Testes de Sensibilidade MicrobianaRESUMO
A set of cDNA clones have been characterized that represent early nodulin mRNAs from pea root nodules. By RNA transfer blot analyses, the different early nodulin mRNAs were found to vary in time course of appearance during the development of the indeterminate pea root nodule. In situ hybridization studies demonstrated that the transcripts were located in different zones, representing subsequent steps in development of the central tissue of the root nodule. ENOD12 transcripts were present in every cell of the invasion zone, whereas ENOD5, ENOD3, and ENOD14 transcripts were restricted to the infected cells in successive but partially overlapping zones of the central tissue. We conclude that the corresponding nodulin genes are expressed at subsequent developmental stages. The amino acid sequence derived from the nucleotide sequences of the cDNAs, in combination with the localization data, showed that ENOD5 is an arabinogalactan-like protein involved in the infection process, whereas ENOD3 and ENOD14 have a cysteine cluster suggesting that these are metal-binding proteins. Furthermore, we showed that there is a clear difference in the way Rhizobium induced the infection-related early nodulin genes ENOD5 and ENOD12. A factor acting over a long distance induced the ENOD12 gene, whereas a factor acting over a short distance activated the ENOD5 gene.
Assuntos
Fabaceae/genética , Genes de Plantas , Proteínas de Membrana , Proteínas de Plantas/genética , Plantas Medicinais , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA/genética , Expressão Gênica , Regulação da Expressão Gênica , Leghemoglobina/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , Alinhamento de Sequência , SimbioseRESUMO
Somatic embryogenesis of carrot (Daucus carota L.) is inhibited by the glycosylation inhibitor tunicamycin. This inhibition is reversible by the addition of correctly glycosylated glycoproteins which have been secreted into the culture medium. To identify the proteins responsible for complementation, glycoproteins present in the medium of embryo cultures were purified and tested for their activity in the tunicamycin inhibition/ complementation assay. A 38-kDa glycoprotein was purified that could restore embryogenesis to more than 50% of that in untreated controls. This 38-kDa glycoprotein was identified as a heme-containing peroxidase on the basis of its A405/A280 ratio (Reinheit Zahl or RZ) and enzyme activity. The 38-kDa peroxidase consisted of four different cationic isoenzymes of which only one or possibly two appeared active in the complementation assay. The cationic peroxidase isoenzymes from the carrot medium could be effectively replaced by cationic horseradish peroxidases which depended on their catalytic properties for their ability to restore tunicamycin-inhibited somatic embryogenesis.
RESUMO
We describe the construction of a new plant transformation vector, pBINPLUS, based on the popular pBIN19 vector. Improvements over pBIN19 include location of the selectable marker gene at the left T-DNA border, a higher copy number in E. coli, and two rare restriction sites around the multiple cloning site for easier cloning and analysis of T-DNA insertions in plant genomes.
Assuntos
Agrobacterium tumefaciens/genética , DNA Bacteriano/genética , Vetores Genéticos , Plantas/genética , Transformação Genética , Aminoácido Oxirredutases/genética , Sequência de Bases , Clonagem Molecular/métodos , Canamicina Quinase , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plantas Geneticamente Modificadas , Plasmídeos , Origem de ReplicaçãoRESUMO
EP1, an extracellular protein from carrot (Daucus carota) cell suspensions, has been partially characterized by means of an antiserum and a cDNA clone. In both embryo and suspension cultures different molecular mass EP1 proteins were detected, some of which (31, 32, 52, and 54 kilodaltons) were bound to the cell wall and released into the medium, whereas others (49, 60, and 62 kilodaltons) were more firmly bound to the cell wall and could be extracted with a salt solution. Immunoprecipitation of in vitro translation products revealed a single primary translation product of 45 kilodaltons, suggesting that EP1 heterogeneity is due to differential posttranslational modification. In seedlings organ-specific modification of EP1 proteins was observed, a phenomenon which did not persist in suspension cultures initiated from different seedling organs. In culture EP1 proteins were only found to be associated with vacuolated, nonembryogenic cells, and on these cells they were localized in loosely attached, pectin-containing cell wall material. Purified 52/54 kilodaltons EP1 proteins did not alleviate the inhibitory effect of the glycosylation inhibitor tunicamycin on somatic embryogenesis.
RESUMO
Non-embryogenic carrot suspension cells secrete the EP1 glycoprotein. A cDNA clone encoding EP1 was isolated and sequenced. The EP1 sequence revealed a region of homology with Brassica S-locus glycoprotein genes, an Arabidopsis S-like gene and putative S-like receptor protein kinases from maize and Arabidopsis. EP1 gene expression, analysed by in situ mRNA localization, was detected in cells located at the surface of the seedling: in the epidermis of the root, the hypocotyl and the cotyledons, in the root cap, and in a crescent of cells in the apical dome of the shoot. In developing seeds, expression was most pronounced in both the inner and outer integument epidermis.
Assuntos
Genes de Plantas , Glicoproteínas/genética , Proteínas de Plantas/genética , Verduras/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , Glicoproteínas/metabolismo , Hibridização In Situ , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , RNA Mensageiro/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
To explore the feasibility of employing antibodies to obtain disease resistance against plant root pathogens, we have studied the expression of genes encoding antibodies in roots of transgenic plants. A model monoclonal antibody was used that binds to a fungal cutinase. Heavy and light chain cDNAs were amplified by PCR, fused to a signal sequence for secretion and cloned behind CaMV 35S and TR2' promoters in a single T-DNA. The chimeric genes were cloned both in tandem and in a divergent orientation. The roots of tobacco plants transformed with these constructs produced antibodies that were able to bind antigen in an ELISA. Immunoblotting showed assembly to a full-size antibody. In addition, a F(ab')2-like fragment was observed, which is probably formed by proteolytic processing. Both antibody species were properly targeted to the apoplast, but the full-size antibody was partially retained by the wall of suspension cells. The construct with divergent promoters showed a better performance than the construct with promoters in tandem. It directed the accumulation of functional antibodies to a maximum of 1.1% of total soluble protein, with half of the plants having levels higher than 0.35%. The high efficiency of this construct probably results from coordinated and balanced expression of light and heavy chain genes, as evidenced by RNA blot hybridization.
Assuntos
Anticorpos Antifúngicos/biossíntese , Hidrolases de Éster Carboxílico/imunologia , Nicotiana/imunologia , Raízes de Plantas/imunologia , Plantas Tóxicas , Sequência de Aminoácidos , Anticorpos Antifúngicos/genética , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Reações Antígeno-Anticorpo , Sequência de Bases , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Engenharia Genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Dados de Sequência Molecular , Micoses/prevenção & controle , Doenças das Plantas , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional , Nicotiana/genética , Nicotiana/metabolismo , Transformação GenéticaRESUMO
The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.
Assuntos
Anticorpos/metabolismo , Compartimento Celular , Fragmentos de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Oligopeptídeos , Sequência de Aminoácidos , Anticorpos/genética , Sequência de Bases , Transporte Biológico , Hidrolases de Éster Carboxílico/imunologia , Citosol/metabolismo , Fragmentos de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Dados de Sequência Molecular , Plantas Tóxicas , Engenharia de Proteínas/métodos , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Nicotiana/genética , Nicotiana/metabolismoRESUMO
A 47 kDa glycoprotein, termed EP4, was purified from carrot cell suspension culture medium. An antiserum raised against EP4 also recognized a protein of 45 kDa that was ionically bound to the cell wall. EP4 was detected in culture media from both embryogenic and non-embryogenic cell lines and was found to be secreted by a specific subset of non-embryogenic cells. Secretion of the 47 kDa glycoprotein by embryogenic cells was not evident. The 45 kDa cell wall-bound EP4 protein was specific for non-embryogenic cells and was shown by immunolocalization to occur in the walls of clustered cells, with the highest levels in the walls separating adjacent cells. In seedlings, EP4 proteins were mainly found in roots. EP4 cDNA was cloned by screening a cDNA library with an oligonucleotide derived from an EP4 peptide sequence. The EP4 cDNA sequence was found to be 55% homologous to ENOD8, an early nodulin gene from alfalfa.