RESUMEN
BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) whole cell vaccines have been widely used tools in the control of Johne's disease in animals despite being unable to provide complete protection. Current vaccine strains derive from stocks created many decades ago; however their genotypes, underlying mechanisms and relative degree of their attenuation are largely unknown. RESULTS: Using mouse virulence studies we confirm that MAP vaccine strains 316 F, II and 2e have diverse but clearly attenuated survival and persistence characteristics compared with wild type strains. Using a pan genomic microarray we characterise the genomic variations in a panel of vaccine strains sourced from stocks spanning over 40 years of maintenance. We describe multiple genomic variations specific for individual vaccine stocks in both deletion (26-32 Kbp) and tandem duplicated (11-40 Kbp) large variable genomic islands and insertion sequence copy numbers. We show individual differences suitable for diagnostic differentiation between vaccine and wild type genotypes and provide evidence for functionality of some of the deleted MAP-specific genes and their possible relation to attenuation. CONCLUSIONS: This study shows how culture environments have influenced MAP genome diversity resulting in large tandem genomic duplications, deletions and transposable element activity. In combination with classical selective systematic subculture this has led to fixation of specific MAP genomic alterations in some vaccine strain lineages which link the resulting attenuated phenotypes with deficiencies in high reactive oxygen species handling.
Asunto(s)
Vacunas Bacterianas/efectos adversos , Vacunas Bacterianas/genética , Variación Genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/patogenicidad , Animales , ADN Bacteriano/genética , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Paratuberculosis/microbiología , Paratuberculosis/patología , Análisis de Supervivencia , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/genéticaRESUMEN
Bovine spongiform encephalopathy (BSE) is acquired orally and the mechanisms involved in the absorption and transportation of infectivity across the gut wall are therefore critical. Isolated gut loops were created in lambs, massaged to remove intestinal contents (flushed) or left non-flushed, inoculated with cattle BSE homogenate and excised at different time-points. Gut loops were examined by immunohistochemistry (IHC) for disease-associated prion protein (PrP(d)), and the contents were analysed by Western blotting (WB) to determine the degradation rate of protease-resistant PrP (PrP(res)). The contents of scrapie-inoculated gut loops from a previous experiment were analysed by WB, and these in vivo digestion results were compared with those of an in vitro experiment on the same transmissible spongiform encephalopathy homogenates. BSE-inoculum-derived PrP(d) was detected by IHC in the gut lumen between 15 min and 3.5 h. It was found in the intestinal lymphatic system from 30 min onwards and was present at the highest frequency at 120 min post-inoculation. In vivo degradation of PrP(res) in the BSE inoculum had a significantly (P=0.006) different pattern compared with scrapie-derived PrP(res), with the BSE PrP(res) degrading more rapidly. However, the overall amount of degradation became similar by 120 min post-challenge. The results of the in vitro digestion experiments showed a similar pattern, although the magnitude of PrP(res) degradation was less than in the in vivo environment where absorption could also take place. BSE-derived PrP(res) is less protease resistant than scrapie PrP over a short time-course and the disappearance of detectable PrP(res) from the gut lumen results from both absorption and digestion by intestinal contents.
Asunto(s)
Encefalopatía Espongiforme Bovina/transmisión , Intestinos/patología , Priones/metabolismo , Enfermedades de las Ovejas/transmisión , Animales , Western Blotting , Bovinos , Inmunohistoquímica , Transporte de Proteínas , OvinosRESUMEN
The purpose of this investigation was to characterise the virulence of two Mycobacterium avium subspecies paratuberculosis (M.a. paratuberculosis) vaccine strains and compare them with a recent virulent isolate in new born calves over a time course of 8 months post-inoculation. Paratuberculosis-free new born calves were inoculated orally with either a vaccine strain (2e or 316F) or a wild type strain (F13) of M.a. paratuberculosis. Blood and faecal samples were collected throughout the experiment to analyse immune responses to infection and assess faecal shedding of M.a. paratuberculosis. Tissue samples were taken at post-mortem for histological examination and bacteriological culture. Cell-mediated immune responses were measured using a Bovigam (CSL) interferon-gamma assay. At 20 weeks post-inoculation there was a significant increase in the cell-mediated immune responses in calves infected with the wild type strain relative to the two vaccine strains. Acid fast bacteria were detected in the faeces of calves in all three groups between 4 and 8 weeks post-inoculation. Histopathology was unrewarding in all three groups. M.a. paratuberculosis was recovered only from tissues of calves inoculated with the wild type strain. Therefore, it appeared that the vaccine strains used in this study had reduced virulence. Identifying the genes responsible for pathogenesis observed in the wild type isolate and reduced or inactive in these vaccine isolates may offer a valuable resource for improving our knowledge of pathogenesis and permit the development of improved diagnostic reagents and vaccines for the control of M.a. paratuberculosis in livestock.
Asunto(s)
Vacunas Bacterianas/inmunología , Enfermedades de los Bovinos/prevención & control , Mycobacterium avium subsp. paratuberculosis/inmunología , Paratuberculosis/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/microbiología , Heces/microbiología , Inmunidad Celular/inmunología , Interferón gamma/sangre , Mycobacterium avium subsp. paratuberculosis/patogenicidad , Paratuberculosis/inmunología , Paratuberculosis/microbiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Virulencia/inmunologíaRESUMEN
Following peripheral exposure, many transmissible spongiform encephalopathy (TSE) agents accumulate first in lymphoid tissues before spreading to the CNS (termed neuroinvasion) where they cause neurodegeneration. Early TSE agent accumulation upon follicular dendritic cells (FDCs) in lymphoid follicles appears critical for efficient neuroinvasion. Most clinical cases of variant Creutzfeldt-Jakob disease have occurred in young adults, although the reasons behind this apparent age-related susceptibility are uncertain. Host age has a significant influence on immune function. As FDC status and immune complex trapping is reduced in aged mice (600 days old), we hypothesized that this aging-related decline in FDC function might impair TSE pathogenesis. We show that coincident with the effects of host age on FDC status, the early TSE agent accumulation in the spleens of aged mice was significantly impaired. Furthermore, following peripheral exposure, none of the aged mice developed clinical TSE disease during their lifespans, although most mice displayed histopathological signs of TSE disease in their brains. Our data imply that the reduced status of FDCs in aged mice significantly impairs the early TSE agent accumulation in lymphoid tissues and subsequent neuroinvasion. Furthermore, the inefficient neuroinvasion in aged individuals may lead to significant levels of subclinical TSE disease in the population.
Asunto(s)
Envejecimiento/fisiología , Encéfalo/inmunología , Células Dendríticas Foliculares/inmunología , Proteínas PrPSc/patogenicidad , Enfermedades por Prión/inmunología , Sistema Nervioso Simpático/inmunología , Administración Oral , Factores de Edad , Animales , Encéfalo/patología , Complemento C4/inmunología , Complemento C4/metabolismo , Células Dendríticas Foliculares/metabolismo , Células Dendríticas Foliculares/patología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Ratones , Ratones Endogámicos C57BL , Proteínas PrPSc/administración & dosificación , Enfermedades por Prión/patología , Bazo/inmunología , Bazo/inervación , Bazo/patología , Sistema Nervioso Simpático/patologíaRESUMEN
In order to gain a better understanding of the pathogenesis of scrapie in sheep an experimental model was developed to characterise immune system cells in the minutes following inoculation with scrapie-brain homogenate. Four 1-year-old susceptible (ARQ/ARQ) sheep were inoculated via the subcutaneous route at four different peripheral lymph node (LNs) drainage sites, at specific time points, prior to euthanasia of the sheep. The LNs were removed post-mortem at 30, 90, 180 and 300min after inoculation. Flow cytometric triple-labelling was carried out on the LN cells and indicated that inoculation of scrapie-brain homogenate adjacent to a lymph node may delay or even inhibit the number of host CD21(+) B cells expressed within the first 5h. Immunohistochemistry was used to attempt detection of the abnormal form of prion protein (PrP(sc)) in draining LNs adjacent to inoculation sites, with negative results at those time points.
Asunto(s)
Linfocitos B/inmunología , Receptores de Complemento 3d/análisis , Scrapie/inmunología , Animales , Citometría de Flujo , Inmunohistoquímica , Proteínas PrPSc/análisis , Priones/análisis , OvinosRESUMEN
Jaagsiekte sheep retrovirus (JSRV) causes a fatal lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). OPA is a significant disease in many sheep-rearing countries and there is no effective method of control. A unique feature of OPA is the overproduction of fluid in the lung of affected animals. This lung fluid contains JSRV and provides a means of transmission through the inhalation of virus. In this study we demonstrated that lung fluid from different OPA cases contained between 10(7) and 10(10) copies of JSRV RNA per ml. Examination of JSRV RNA survival under conditions that mimic natural conditions suggested that intact JSRV virions may persist for several weeks in the environment. These are the first quantitative data on JSRV in lung fluid and provide valuable information for implementing appropriate biosecurity measures to control the spread of JSRV in the field.
Asunto(s)
Líquidos Corporales/virología , Retrovirus Ovino Jaagsiekte/aislamiento & purificación , Retrovirus Ovino Jaagsiekte/fisiología , Pulmón/virología , Adenomatosis Pulmonar Ovina/virología , Animales , Ovinos , Temperatura , Factores de TiempoRESUMEN
Mycobacterium avium subsp. paratuberculosis causes paratuberculosis, a chronic granulomatous enteritis. Detecting animals with paratuberculosis infections is difficult because the currently available tools have low sensitivity and lack specificity; these tools are prone to generating spurious positive test results caused by exposure to environmental M. avium complex organisms. To generate candidate antigens for incorporation into a specific test for paratuberculosis, subspecies-specific proteins were determined by proteomic comparison of M. avium subsp. paratuberculosis and M. avium subsp. avium. Analysis was aimed at revealing proteins only expressed (or predominant) in the protein profile of M. avium subspecies paratuberculosis. Two-dimensional gel electrophoresis resolved approximately 1,000 protein spots from each subspecies. Proteome analysis identified protein spots whose expression profile appeared markedly increased in M. avium subsp. paratuberculosis, and 32 were identified by analysis of their tryptic peptide profile by matrix-assisted laser desorption ionization-time of flight analysis. Thirty of these proteins were cloned, and their recombinant proteins were expressed. Ovine paratuberculosis sera were used to assess their immunoreactivity by enzyme-linked immunosorbent assay (ELISA), Western blotting, and dot blot analysis. Seventeen proteins were detected in at least one of the immunoassays, and eleven proteins were detected by ELISA with an optical density in excess of the cutoff of 0.1 in four of six sera tested. The immunoreactivity of these proteins indicates their potential as unique diagnostic antigens for the development of a specific serological detection of paratuberculosis.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Mycobacterium avium subsp. paratuberculosis/inmunología , Paratuberculosis/diagnóstico , Proteínas Recombinantes/inmunología , Enfermedades de las Ovejas/diagnóstico , Animales , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Proteoma/inmunología , Oveja Doméstica/inmunología , Oveja Doméstica/microbiologíaRESUMEN
BACKGROUND: Particulate air pollution (PM(10)) consists of a mixture of components, including nanoparticles and metals. Studies from our laboratory have demonstrated that transition metals can potentiate the ability of nanoparticles to induce lung inflammation and that the zinc content of PM(10) was largely responsible for their potential to induce inflammation. These results are also relevant to zinc-containing engineered nanoparticles. OBJECTIVES: To investigate the potential of ZnCl(2) and FeCl(3) to interact with nanoparticle carbon black in cell-free and biological systems to generate ROS, express pro-inflammatory mediators and cytotoxic ability. METHODS: ROS production was examined using DCFH-DA. J774 cells were treated for 4 h with 14 nm CB and/or ZnCl(2) before measuring TNF-alpha by ELISA. Cytoskeletal changes were investigated using confocal microscopy. Flow cytometry was used to examine apoptotic/necrotic cells and phagocytic ability. RESULTS: In a cell-free system the particles generated significant ROS, whereas ZnCl(2) did not. Treatment of cells with 100 microM ZnCl(2), but not FeCl(3), increased TNF-alpha. Treatment with 14 nm CB alone induced TNF-alpha, which was synergistically enhanced by ZnCl(2). No interactions were observed in cells treated with 14 nm CB and FeCl(3). Cytoskeletal changes were observed with increasing concentrations of ZnCl(2). These results were confirmed by flow cytometry indicating that ZnCl(2) induced markers of apoptosis and necrosis. The phagocytic ability of cells was also significantly decreased. Nanoparticle carbon black alone did not induce changes in apoptosis/necrosis or the phagocytosis activity of the cells. CONCLUSION: Despite an inability to induce ROS production, ZnCl(2) stimulated TNF-alpha production which was synergistically enhanced by 14 nm carbon black. The ability of zinc to induce morphological changes and cell death was not altered by nanoparticle treatment.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Cloruros/toxicidad , Compuestos Férricos/toxicidad , Nanopartículas/toxicidad , Hollín/toxicidad , Compuestos de Zinc/toxicidad , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inflamación/inducido químicamente , Mediadores de Inflamación/metabolismo , Macrófagos , Ratones , Microscopía Confocal , Necrosis/metabolismo , Fagocitosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Three groups of five calves, namely, V1, V2, and V3, were immunized intramuscularly at 4 and 8 weeks of age with ca. 10(9), 10(8), and 10(7) CFU, respectively, of a derivative of Pasteurella multocida B:2 wild-type strain 85020 containing a deletion in the aroA gene (strain JRMT12). The first and second vaccinations resulted in significantly (P < 0.01) higher rectal temperature responses in groups V1 and V2 than in group V3. Serum immunoglobulin M (IgM) and IgG titers did not increase in any group until after the second vaccination and were then significantly higher in groups V1 and V2 than in group V3 (P = 0.001 for both IgM and IgG). All vaccinated groups and three unvaccinated challenge control calves (group CC) were injected subcutaneously at 10 weeks of age with ca. 10(7) CFU of strain 85020. Vaccinated calves survived the challenge, but two CC animals developed clinical disease and were killed for humane reasons. After challenge, mean serum amyloid A concentrations were significantly higher (P < 0.001) in the CC group than in the vaccinated groups. Postmortem examination revealed that calves in the CC group showed the most extensive range of bacteriologically positive tissues and gross and histopathological lesions. Overall, a clear dose-dependent response was present, with those receiving a higher vaccine dose being less affected clinically, bacteriologically, and pathologically by the wild-type challenge. The V2 treatment appeared to give the best combination of high immune response, protection, and safety.
Asunto(s)
Vacunas Bacterianas/inmunología , Enfermedades de los Bovinos/inmunología , Septicemia Hemorrágica/veterinaria , Infecciones por Pasteurella/veterinaria , Pasteurella multocida/inmunología , Proteína Amiloide A Sérica/inmunología , 3-Fosfoshikimato 1-Carboxiviniltransferasa/metabolismo , Animales , Vacunas Bacterianas/farmacología , Bovinos , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/prevención & control , Septicemia Hemorrágica/inmunología , Septicemia Hemorrágica/microbiología , Septicemia Hemorrágica/prevención & control , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Inyecciones Intramusculares , Infecciones por Pasteurella/inmunología , Infecciones por Pasteurella/microbiología , Infecciones por Pasteurella/prevención & control , Pasteurella multocida/aislamiento & purificación , Proteína Amiloide A Sérica/metabolismo , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/farmacologíaRESUMEN
Expanded CAG x CTG trinucleotide repeat tracts are associated with several human inherited diseases, including Huntington's disease, myotonic dystrophy, and spinocerebellar ataxias. Here we describe a new model system to investigate repeat instability in the Escherichia coli chromosome. Using this system, we reveal patterns of deletion instability consistent with secondary structure formation in vivo and address the molecular basis of orientation-dependent instability. We demonstrate that the orientation dependence of CAG x CTG trinucleotide repeat deletion is determined by the proofreading subunit of DNA polymerase III (DnaQ) in the presence of the hairpin nuclease SbcCD (Rad50/Mre11). Our results suggest that, although initiation of slippage can occur independently of CAG x CTG orientation, the folding of the intermediate affects its processing and this results in orientation dependence. We propose that proofreading is inefficient on the CTG-containing strand because of its ability to misfold and that SbcCD contributes to processing in a manner that is dependent on proofreading and repeat tract orientation. Furthermore, we demonstrate that transcription and recombination do not influence instability in this system.
Asunto(s)
Inestabilidad Cromosómica/genética , ADN Bacteriano/genética , Escherichia coli/genética , Conformación de Ácido Nucleico , Expansión de Repetición de Trinucleótido/genética , Secuencia de Bases , Cromosomas Bacterianos/genética , ADN Polimerasa III/genética , Replicación del ADN , ADN Bacteriano/biosíntesis , ADN Bacteriano/química , Proteínas de Escherichia coli/genética , Exonucleasas , Modelos Genéticos , Datos de Secuencia Molecular , Recombinación Genética , Eliminación de Secuencia/genética , Transcripción GenéticaRESUMEN
Enterohemorrhagic Escherichia coli O157:H7 is an important intestinal pathogen of humans with a main reservoir of domesticated ruminants, particularly cattle. It is anticipated that the risk of human infection can be reduced by controlling the organism within its reservoir hosts. Several options for the control of E. coli O157:H7 in cattle have been proposed, but none have been demonstrated to be successful in the field. Here we describe a novel experimental method, based on the terminal-rectum-restricted colonization described previously, to eliminate fecal carriage of E. coli O157:H7. In experimentally challenged calves, direct application to the rectal mucosa of either of two therapeutic agents, polymyxin B or chlorhexidine, greatly reduced bacterial shedding levels in the immediate posttreatment period. The most efficacious therapeutic agent, chlorhexidine, was compared in orally and rectally challenged calves. The treatment eliminated high-level shedding and reduced low-level shedding by killing bacteria at the terminal rectum. A rapid-detection system based on the ability to identify E. coli O157:H7 from swabs of the rectal mucosa was also assessed. This test was sufficiently sensitive to identify high-level bacterial carriage. Thus, a combination of the detection method and treatment regimens could be used in the field to eliminate high-level fecal excretion of E. coli O157:H7, so greatly reducing its prevalence within this host and the risk of human infection.
Asunto(s)
Antibacterianos/administración & dosificación , Clorhexidina/administración & dosificación , Escherichia coli O157/efectos de los fármacos , Escherichia coli O157/aislamiento & purificación , Heces/microbiología , Polimixina B/administración & dosificación , Recto/microbiología , Animales , Antibacterianos/farmacología , Portador Sano/tratamiento farmacológico , Portador Sano/microbiología , Bovinos , Enfermedades de los Bovinos/tratamiento farmacológico , Enfermedades de los Bovinos/microbiología , Clorhexidina/farmacología , Recuento de Colonia Microbiana , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Polimixina B/farmacología , Recto/efectos de los fármacos , Sensibilidad y Especificidad , Manejo de Especímenes/métodosRESUMEN
Paratuberculosis (Johne's disease) poses a significant economic problem to beef, dairy and sheep industries worldwide, and is caused by Mycobacterium avium subspecies paratuberculosis. In this study, 2D PAGE was used as a tool to investigate the virulent state of M. avium subsp. paratuberculosis, incorporating the technique of beating the organism with zirconium/silica beads to provide a comprehensive representation of its proteome. A direct comparison of the proteomes of M. avium subsp. paratuberculosis scraped from the terminal ileum of ovine paratuberculosis cases, and the identical strain grown in vitro, is presented. These analyses identified a set of 10 proteins whose expression is upregulated during natural infection: 1-pyrroline-5-carboxylate dehydrogenase (RocA), a putative acyl-CoA dehydrogenase (FadE14), 2-methylcitrate dehydratase (2-mcd), arginosuccinate synthase (ArgG), universal stress protein (usp), 30S ribosomal protein S2 (RpsB), peptidyl-prolyl cis-trans isomerase (PpiA), luciferase-like monooxygenase (lmo), thiosulfate sulfurtransferase (SseA) and ATP-dependent Clp protease (ClpB). Most of the proteins identified do not have obviously related functions; however, ArgG and RocA function in the same pathway, and may have a concerted action for energy production in vivo.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium avium subsp. paratuberculosis/metabolismo , Paratuberculosis/microbiología , Proteómica , Enfermedades de las Ovejas/microbiología , 1-Pirrolina-5-Carboxilato Deshidrogenasa/metabolismo , Animales , Argininosuccinato Sintasa/metabolismo , Proteínas Bacterianas/análisis , Proteínas de Escherichia coli , Íleon/microbiología , Mycobacterium avium subsp. paratuberculosis/efectos de los fármacos , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Ovinos , Regulación hacia Arriba , Circonio/farmacologíaRESUMEN
Using a virulent United Kingdom Corynebacterium pseudotuberculosis isolate, an ovine experimental model of caseous lymphadenitis was developed, in which the manifestation of disease was equivalent to the naturally observed infection in this country. Subsequently, the capacity of several experimental vaccines to protect against experimental challenge was determined. Sheep were immunised with a recombinant derivative of phospholipase D, deriving from the virulent UK isolate, a formalin-killed bacterin of the same strain, or a bacterin supplemented with recombinant phospholipase D. Following homologous experimental challenge, the phospholipase D and bacterin vaccines were observed to confer statistically significant protection against infection, and appeared to restrict dissemination of challenge bacteria beyond the inoculation site in the majority of animals. More importantly, the combined vaccine succeeded in providing absolute protection against infection, whereby challenge bacteria were eradicated from all vaccinates. In addition to the experimental vaccines, a commercially available CLA vaccine, unlicensed for use in the European Union, was assessed for its capacity to protect against heterologous challenge. The vaccine conferred significant protection, although the dissemination of infection beyond the inoculation site was not restricted as it had been with the previous vaccines. However, no animals immunised with this vaccine manifested infection within the lungs; thus, a potentially important route of disease transmission was eliminated. The results of this study provide information pertinent to the development of an effective caseous lymphadenitis vaccination strategy in the UK.
Asunto(s)
Vacunas Bacterianas , Infecciones por Corynebacterium/veterinaria , Corynebacterium pseudotuberculosis/inmunología , Enfermedades de las Ovejas/prevención & control , Vacunación/veterinaria , Animales , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Infecciones por Corynebacterium/inmunología , Infecciones por Corynebacterium/prevención & control , Linfadenitis/inmunología , Linfadenitis/prevención & control , Linfadenitis/veterinaria , Masculino , Fosfolipasa D/administración & dosificación , Fosfolipasa D/inmunología , Ovinos , Enfermedades de las Ovejas/inmunología , Reino Unido , Vacunación/métodos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.
Asunto(s)
Escherichia coli O157/genética , Fimbrias Bacterianas/química , Fimbrias Bacterianas/genética , Genes Bacterianos , Operón/genética , Animales , Adhesión Bacteriana/fisiología , Bovinos , Células Cultivadas , Clonación Molecular , Escherichia coli K12/genética , Escherichia coli O157/química , Escherichia coli O157/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Matriz Extracelular/metabolismo , Fimbrias Bacterianas/ultraestructura , Eliminación de Gen , Recto/microbiologíaRESUMEN
Initial laboratory trials of an experimental subunit vaccine against Theileria parva based on the 67-kDa major sporozoite surface antigen revealed a range of responses to challenge. We have analyzed convergence in seven sets of monozygotic twins which suggests that genetic factors may have an influence in determining the degree of protection provided by p67 immunization. In addition, we have examined whether allelic diversity at major histocompatibility complex class II loci influences protection. Analysis of bovine leukocyte antigen DRB3 diversity in 201 animals identified significant associations with vaccine success (DRB3*2703; P = 0.027) and vaccine failure (DRB3*1501; P = 0.013). Furthermore, DRB3*2703 was associated with the likelihood of immunized animals showing little to no clinical signs of disease following challenge. We discuss the acquired and innate immune mechanisms that may be behind the associations described here.
Asunto(s)
Antígenos de Protozoos/administración & dosificación , Bovinos/genética , Bovinos/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/inmunología , Theileria parva/inmunología , Theileriosis/inmunología , Theileriosis/prevención & control , Alelos , Animales , Frecuencia de los Genes , Genes MHC Clase II , Genotipo , Theileriosis/genéticaRESUMEN
The state of anthelmintic resistance in Scottish sheep flocks was evaluated during April-August 2000 using in vitro bioassays, egg hatch assays for detecting thiabendazole (TBZ) resistance and larval development assays for levamisole, ivermectin and TBZ resistance. Anaerobic sampling kits and detailed questionnaires outlining farm demographics and current management practices were mailed to 227 Moredun Foundation member farms, replies were received from 98 farms. Kits received from 90 farms contained sufficient material to conduct one or more of the assays. A majority of the farms examined (64%) exhibited TBZ resistance but there were both farm locality and regional variations in the percentage of resistant farms. Teladorsagia was the predominant genera detected from farms. No resistance to levamisole or ivermectin was detected in any of the samples. There was no strong evidence from this survey that any of the management practices examined greatly affected TBZ resistance.
Asunto(s)
Antihelmínticos/farmacología , Nematodos/efectos de los fármacos , Infecciones por Nematodos/veterinaria , Enfermedades de las Ovejas/tratamiento farmacológico , Crianza de Animales Domésticos , Animales , Antihelmínticos/uso terapéutico , Antinematodos/farmacología , Bioensayo/veterinaria , Resistencia a Medicamentos , Femenino , Ivermectina/farmacología , Larva/efectos de los fármacos , Levamisol/farmacología , Masculino , Infecciones por Nematodos/tratamiento farmacológico , Infecciones por Nematodos/parasitología , Óvulo/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria/veterinaria , Escocia , Ovinos , Enfermedades de las Ovejas/parasitología , Encuestas y Cuestionarios , Tiabendazol/farmacologíaRESUMEN
Both the ultrafine particle and transition metal components of particulate air pollution (PM(10)) have been hypothesized to be important factors in determining toxicity and potential adverse health effects. In this study we aimed to investigate interactions between transition metal salts and a surrogate environmental particle-ultrafine carbon black (ufCB). In all experimental systems employed, the ufCB was found to be more reactive than its fine counterpart (CB). Incubation of ufCB with the reactive oxygen species (ROS)-sensitive probe dichlorofluorescin in the absence of cells generated significantly more ROS than CB. With addition of either cupric sulfate (CuSO(4)), ferrous sulfate (FeSO(4)), or ferric chloride (FeCl(3)), the ROS generation in the presence of ufCB was enhanced in a potentiative manner. In Mono Mac 6 macrophages, ufCB again produced more ROS than CB. However, addition of iron salts had no additive effect over and above that induced in the macrophages by ufCB. In the mouse macrophage cell line J774, ufCB decreased the cellular content of GSH and ATP. Addition of iron further decreased both GSH and ATP and a potentiative interaction between ufCB and FeSO(4) was observed, but only at the highest iron concentrations tested. A concentration-dependent increase in tumor necrosis factor-alpha production by J774 cells was also observed following exposure to ufCB, which was not further enhanced by the addition of iron. J774 cells were also found to sequester or chelate iron without inducing toxicity. In the rat lung ufCB induced a significant neutrophil influx and this inflammatory effect was potentiativelly enhanced by the addition of FeCl(3) (100 microM). These findings suggest that (1) ultrafine particles and metals interact by chemical potentiation in a cell-free environment to generate ROS, (2) potentiation between ultrafine particles and metal salts is not observed in the presence of macrophages as iron is sequestered or chelated by the cells, (3) in the lung, ultrafine particles and iron salts interact in a potentiative manner to generate inflammation.