Browsing by Subject "Enzyme-linked immunosorbent assay"

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    Entwicklung und Testung neuer DNA- und Protein-basierter Multikomponentenvakzinen sowie regulatorischer Adjuvanzien gegen eine Infektion mit B. anthracis in Auszucht-Mäusen und Ziegen
    (2015) Köhler, Susanne Melanie; Beyer, Wolfgang
    The discovery of the Sterne spore live vaccine (SSLV) and subsequently its application in a veterinary context contributed to the global reduction of Anthrax related outbreaks since 1930. Nonetheless the causative agent Bacillus anthracis is still prevalent in some mediterranean countries, South and Central America, Africa and Central Asia, as well as the USA and Canada. Reasons for this are the persistence of the pathogen in the soil, as well as still undefined factors for an ongoing cycle of outbreak and spread of the disease and the limited applicability of the SSLV. This includes the necessity to revaccinate annually, the residual virulence in certain sensitive species (e. g. goats and llamas) and the incompatibility to treat and vaccinate simultaneously. To participate in the ongoing search for alternative vaccines this work was dedicated to evaluate protein- and DNA-based components as potential ingredients for a multi-component non-living vaccine formulation (NLV). For the protein-based NLV these included rPA83 as part of the Anthrax toxin, rBclA and Formalin inactivated spores (FIS) as spore specific antigens, a Capsule-Lipopeptide conjugate as part of the vegetative form of the pathogen and a Lipopeptide-adjuvant. The DNA-vaccines consisted of vector-backbones comprising signal sequences able to direct the integrated antigens (rPA83, PAD4LFD1 and BclAD1D3) to the MHCI, MHCII and the secretory pathway. A sperate vector encoding for a positive MHCII-regulator (CIITA) and a vector internal sequence for the Interferon-ß promotor stimulator (mIPS1) served as adjuvants for the DNA-vaccines. The majority of the groups showed detectable antibody titres against their respective antigens, with protein vaccines generally eliciting higher titres against rPA83 than the DNA-vaccines. Regarding rBclA equivalent high titres were measured for protein- and DNA-vaccines alike, which also corresponded to the anti-FIS titres for groups immunized with rBclA, FIS or both. The Capsule-Lipopeptide conjugate did not elicit high titres against the capsule, possibly due to an immune suppressing epitope. Survival rates ranged between 10 and 100 %, with full protection only achieved in a combination of all antigens including FIS. All DNA-vectors induced 30 – 50 % protectiveness when given alone. Notably DNA-vectors including BclAD1D3 elicited 50 % survival and sterile immunity. A combination of the most promising vectors encoding for toxin and spore specific antigens achieved 90 % protectiveness in mice. According to the results from the mice trials, the auspicious protein- and DNA-vaccine combinations were tested in goats in comparison to the SSLV in cooperation with our project partners in South Africa and Turkey. The efficacy of the SSLV was assessed in 3 groups which were challenged shortly after the first immunisation, one year after the first immunisation or after the revaccination. Apart from the comparison of immunogenicity and protectiveness between SSLV and NLV in goats, assessment of data concerning the titre development of SSLV-immunized goats during the course of a year as well as detailed diagnostic data during the infection (behavior, temperature, bacterial loads, correlations and minimal infective dose) were integral part of this study. Compared to one another the SSLV-immunized animals showed equal or higher antibody titres against the measured antigens, with FIS and rPA83 being the most immunogen antigens. Utilizing a higher dose (75 µg) the protein-based NLV protected equivalently to the SSLV (60 – 100 %) yielding 50 % protectiveness without FIS and 80 % if FIS was included. The DNA-vaccines showed little to no immunogenicity in goats, thus no challenge was performed on these animals. The humoral reaction against BclA was generally poor in goats, which has not been noted before and could be a basis for further improvements concerning the SSLV and NLV alike. The different immunizations with the SSLV revealed a broad range for the efficacy of the first vaccination as well as a notable difference in the antibody spectrum between first vaccination and revaccination. Together with the recorded data of the antibody titre development throughout a year a more optimal protocol for immunisation with the SSLV, possibly in combination with an NLV was postulated.
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    Molecular and phenotypic analyses of pathogenicity, aggressiveness, mycotoxin production, and colonization in the wheat-Gibberella zeae pathosystem
    (2004) Cumagun, Christian Joseph R.; Miedaner, Thomas
    Fusarium head blight (FHB), caused by Gibberella zeae (Schwein.) Petch (anamorph: Fusarium graminearum Schwabe), is one of the principal diseases responsible for extensive damage in wheat fields and contamination of grain with the mycotoxins deoxynivalenol (DON) and nivalenol (NIV), rendering the harvest unsafe for human and animal consumption. Control of FHB is difficult because of the complex nature of host-pathogen-environment interaction and the nonavailability of highly effective fungicides. Agronomic practices and resistance breeding, therefore, offer the best strategies for disease management. Mapping by molecular markers provides an accurate approach for genetic analyses of simple and complex traits particularly pathogenicity, aggressiveness, and mycotoxin production. Pathogenicity, as defined here, is the ability to cause disease whereas aggressiveness is the quantity of disease induced by a pathogenic isolate on a susceptible host in which isolates do not interact differentially with host cultivars. The project aims to (1) map pathogenicity and aggressiveness of G. zeae based on a published genetic map (2) estimate genetic diversity of four parent isolates by PCR-based markers (3) examine the inheritance of pathogenicity, aggressiveness, mycotoxin type (DON/NIV), and DON production on a phenotypic basis, (4) analyse genetic covariation among aggressiveness, DON, and fungal colonization, (5) and compare aggressiveness of 42 isolates in greenhouse and field environments. Two crosses of G. zeae using nit (nitrate nonutilizing) marker technique were performed: (1) pathogenic DON-producing Z-3639 (Kansas, USA) x nonpathogenic NIV-producing R-5470 (Japan) belonging to lineage 7 and 6, respectively, and (2) DON-producing FG24 (Hungary) x FG3211 (Germany), both aggressive lineage 7 isolates. For the first cross, 99 progeny segregated in a consistent 61:38 for pathogenicity: nonpathogenicity in a two-year greenhouse experiment. Among the 61 pathogenic progeny, disease severity, measured as percentage infected spikelets, varied significantly (P = 0.01). Heritability for aggressiveness was high. Pathogenicity locus was mapped on linkage group IV near loci PIG1 (red pigment production), TOX1 (trichothecene toxin amount), and PER1 (perithecial production) explaining 60%, 43%, and 51% of the phenotypic variation, respectively. Two large aggressiveness QTLs were mapped on linkage group I linked to the locus TRI5 (trichodiene synthase in the trichothecene gene cluster) and an amplified fragment length polymorphism (AFLP) marker (EAAMTG0655K), explaining 51% and 29% of the observed phenotypic variation, respectively. These unlinked loci suggest that genetic basis between pathogenicity and aggressiveness were different. TRI5 is located in the same gene cluster as a previously identified gene known as TRI13, which determines whether DON or NIV will be produced. DON-producing progeny were, on average, twice as aggressive as were those producing NIV. Loci were only detected in the two linkage groups mentioned from the nine linkage groups present in the map. For the second cross FG24 x FG3211 with 153 progeny, head blight rating and relative plot yield were used as aggressiveness traits. DON production was measured by a commercial kit enzyme immunoassay. These three traits were quantitatively inherited among 153 progeny across three environments. Repeatabilities within each environment were medium to high but heritabilities across environments were medium only due to high progeny-environment interaction. DON was a less environmentally stable trait than aggressiveness. Transgressive segregants were detected frequently. This implies that even a cross within a lineage could lead to an increase in aggressiveness. Mapping of this cross was not initiated because the parents were not polymorphic enough to construct a genetic map. Instead, the parents were analysed for polymorphism in comparison to the parents of the first cross using 31 AFLP primer combinations and 56 random amplified polymorphic DNA (RAPD) primers. Polymorphism between Z-3639 and R-5470 was about three to four times higher than between FG24 and FG3211. Cluster analysis revealed that R-5470 was genetically separated from the other three parents, thus confirming the lineage assignments. Among preselected 50 progeny from the same field experiments that showed normal distribution for aggressiveness - head blight rating, fungal colonization, and DON production were correlated (r = 0.7, P = 0.01). Fungal colonization measured as Fusarium exoantigen (ExAg) content using enzyme-linked immunosorbent assay (ELISA) varied also quantitatively, but heritability was lower due to high progeny-environment interaction and error. Strong correlations among all traits indicate control by similar genes or gene complexes. No significant variation was observed for DON/ExAg ratio. Aggressiveness traits and DON production were more environmentally stable compared to Fusarium ExAg content. Our findings imply that aggressiveness may have other components apart from mycotoxin production. Genotypic variation for aggressiveness among the 42 progeny in one greenhouse and three field environments was significant and their correlation was moderate (r = 0.7, P = 0.01). High heritability in both environments again indicates that aggressiveness was a relatively stable trait, although methods of inoculation differed, i.e., injection for greenhouse and spraying for field experiments. Greenhouse aggressiveness could predict aggressiveness in the field, and thereby should reduce costs for resistance and phytopathological studies. In conclusion, we consider G. zeae as medium-risk pathogen with the potential to evolve to a higher level of aggressiveness due to sexual recombination. Erosion of quantitative resistance in FHB cannot be ignored, especially if host resistances with oligogenic inheritance, e.g. Sumai 3 from China, are used on a large acreage. Consequently, the rather simple inheritance of pathogenicity and aggressiveness in G. zeae could lead to a gradual increase of aggressiveness. These results should enhance efforts of plant breeders to use several, genetic distinct sources of resistance in order to avoid possible FHB outbreaks in the future.