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Botulinum neurotoxins (BoNTs) are highly toxic proteins that require high-affinity immunocapture reagents for use in endopeptidase-based assays. Here, 30 novel and 2 earlier published llama single-domain antibodies (VHHs) against the veterinary-relevant BoNT serotypes C and D were yeast-produced. These VHHs recognized 10 independent antigenic sites, and many cross-reacted with the BoNT/DC and CD mosaic variants. As VHHs are highly suitable for genetically linking to increase antigen-binding affinity, 52 VHH multimers were produced and their affinity for BoNT/C, D, DC, and CD was determined. A selection of 15 multimers with high affinity (KD < 0.1 nM) was further shown to be resilient to a high salt wash that is used for samples from complex matrices and bound native BoNTs from culture supernatants as shown by Endopep-MS. High-affinity multimers suitable for further development of a highly sensitive Endopep-MS assay include four multimers that bind both BoNT/D and CD with KD of 14–99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (<1–19 pM), six of which also bind BoNT/DC with lower affinity (93–508 pM). In addition to application in diagnostic tests, these VHHs could be used for the development of novel therapeutics for animals or humans.

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population.

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.

Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 103-107 CFU/mL were added to healthy bean seed extracts. A flow cytometry sorting procedure was developed to separate immuno-stained Xap cells from crude seed extracts and confirming by PCR. FCM was evaluated for direct viable counting (DVC) of Xap using combinations of propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) or PI and SYTO 9 and also the combination of immuno-FCM and PI. Dilution plating and IF allowed detection of Xap in bean seed extracts in a range of 103-106 CFU/mL and immuno-FCM from 104-106 CFU/mL. Sorted cells could be detected in crude seed extracts by PCR without further extraction. FCM also allowed quantification of viable cells of Xap after DVC procedures; the red fluorescent dye propidium iodide was used to identify dead cells in combination with the green fluorescent dyes cFDA or SYTO 9, these identifying live cells. The combination of immuno-FCM and PI could be more promising and reliable to detect this pathogen in seeds. Key words: seed pathology, flow sorting, PCR-amplification, viability probes, immunofluorescence, bacteria.

An enrichment microsphere immunoassay (MIA) was developed, based on the Luminex xMAP® technology, for the simultaneous (duplex) detection of Pectobacterium atrosepticum (former name Erwinia carotovora subsp. atroseptica) (Pca) and Dickeya dianthicola (former name Erwinia chrysanthemi) (Dcd) in potato plant extracts. Target bacteria in the extracts were enriched for 48 h in a semi-selective broth containing polypectate under low oxygen conditions. Samples were subsequently incubated with antibody-coated colour-coded microspheres (beads) and with secondary antibodies conjugated with Alexa Fluor® 532, a reporter dye. Samples were analyzed with the Luminex analyzer, in which one laser identified each microsphere and another laser the reporter dye conjugated to the secondary antibodies. The assay required minimal sample preparation, could be completed in 1 h, was performed in 96 wells microtitreplates and required no wash steps. The limit of detection for the duplex enrichment MIA was 100¿1000 cfu ml¿1, which was a hundred times lower than of an enrichment-ELISA. Without enrichment, the sensitivity of MIA and ELISA was largely similar and ranged between 106 and 107 cells ml¿1. No difference in sensitivity was found between a MIA in a single or duplex format. In a comparative test with non-infected potato plant extracts and extracts from plants infected with Pca or Dcd, results of the enrichment MIA correlated well with those of the enrichment ELISA and enrichment PCR. These results indicate that MIA can be reliably used for multiplex detection of soft rot Enterbacteriaceae in crude potato plant extracts. The technology is an attractive and cost-effective alternative to other detection methods, including ELISA.

Samenvatting van de voordracht te houden op 30 november 2005 tijdens de Najaarsvergadering van de KNPV (Koninklijke Nederlandse Plantenziektekundige Vereniging).

The continuous growth of the plant embryo is interrupted during the seed maturation processes which results in a dormant seed. The embryo continues development after germination when it grows into a seedling. The embryo growth phase starts after morphogenesis and ends when the embryo fills the seed sac. Very little is known about the processes regulating this phase. We describe mutants that affect embryo growth in two sequential developmental stages. Firstly, embryo growth arrest is regulated by the FUS3/LEC type genes, as mutations in these genes cause a continuation of growth in immature embryos. Secondly, a later stage of embryo dormancy is regulated by ABI3 and abscisic acid; abi3 and aba1 mutants exhibit premature germination only after embryos mature. Mutations affecting both developmental stages result in an additive phenotype and double mutants are highly viviparous. Embryo growth arrest is regulated by cell division activities in both the embryo and the endosperm, which are gradually switched off at the mature embryo stage. In the fus3/lec mutants, however, cell division in both the embryo and endosperm is not arrested, but rather is prolonged throughout seed maturation. Furthermore ectopic cell division occurs in seedlings. Our results indicate that seed dormancy is secured via at least two sequential developmental processes: embryo growth arrest, which is regulated by cell division and embryo dormancy.