Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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The use of Metabolomics to Elucidate Resistance Markers Against Damson-Hop Aphid
Undas, Anna K. ; Weihrauch, Florian ; Lutz, Anton ; Tol, Rob van; Delatte, Thierry ; Verstappen, Francel ; Bouwmeester, Harro - \ 2018
Journal of Chemical Ecology (2018). - ISSN 0098-0331 - 16 p.
Damson-hop aphid - Hop metabolites - Plant defense - Plant resistance - Untargeted metabolite profiling

Phorodon humuli (Damson-hop aphid) is one of the major pests of hops in the northern hemisphere. It causes significant yield losses and reduces hop quality and economic value. Damson-hop aphid is currently controlled with insecticides, but the number of approved pesticides is steadily decreasing. In addition, the use of insecticides almost inevitably results in the development of resistant aphid genotypes. An integrated approach to pest management in hop cultivation is therefore badly needed in order to break this cycle and to prevent the selection of strains resistant to the few remaining registered insecticides. The backbone of such an integrated strategy is the breeding of hop cultivars that are resistant to Damson-hop aphid. However, up to date mechanisms of hops resistance towards Damson-hop aphids have not yet been unraveled. In the experiments presented here, we used metabolite profiling followed by multivariate analysis and show that metabolites responsible for hop aroma and flavor (sesquiterpenes) in the cones can also be found in the leaves, long before the hop cones develop, and may play a role in resistance against aphids. In addition, aphid feeding induced a change in the metabolome of all hop genotypes particularly an increase in a number of oxidized compounds, which suggests this may be part of a resistance mechanism.

Engineering storage capacity for volatile sesquiterpenes in Nicotiana benthamiana leaves
Delatte, Thierry L. ; Scaiola, Giulia ; Molenaar, Jamil ; Sousa Farias, Katyuce de; Alves Gomes Albertti, Leticia ; Busscher, Jacqueline ; Verstappen, Francel ; Carollo, Carlos ; Bouwmeester, Harro ; Beekwilder, Jules - \ 2018
Plant Biotechnology Journal (2018). - ISSN 1467-7644
Nicotiana benthamiana - Lipid bodies - Secondary metabolites - Sesquiterpene - Storage - Triacylglycerol

Plants store volatile compounds in specialized organs. The properties of these storage organs prevent precarious evaporation and protect neighbouring tissues from cytotoxicity. Metabolic engineering of plants is often carried out in tissues such as leaf mesophyll cells, which are abundant and easily accessible by engineering tools. However, these tissues are not suitable for the storage of volatile and hydrophobic compound such as sesquiterpenes and engineered volatiles are often lost into the headspace. In this study, we show that the seeds of Arabidopsis thaliana, which naturally contain lipid bodies, accumulate sesquiterpenes upon engineered expression. Subsequently, storage of volatile sesquiterpenes was achieved in Nicotiana benthamiana leaf tissue, by introducing oleosin-coated lipid bodies through metabolic engineering. Hereto, different combinations of genes encoding diacylglycerol acyltransferases (DGATs), transcription factors (WRINKL1) and oleosins (OLE1), from the oil seed-producing species castor bean (Ricinus communis) and Arabidopsis, were assessed for their suitability to promote lipid body formation. Co-expression of α-bisabolol synthase with Arabidopsis DGAT1 and WRINKL1 and OLE1 from castor bean promoted storage of α-bisabolol in N. benthamiana mesophyll tissue more than 17-fold. A clear correlation was found between neutral lipids and storage of sesquiterpenes, using synthases for α-bisabolol, (E)-β-caryophyllene and α-barbatene. The co-localization of neutral lipids and α-bisabolol was shown using microscopy. This work demonstrates that lipid bodies can be used as intracellular storage compartment for hydrophobic sesquiterpenes, also in the vegetative parts of plants, creating the possibility to improve yields of metabolic engineering strategies in plants.

Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance
Kema, Gerrit H.J. ; Mirzadi Gohari, Amir ; Aouini, Lamia ; Gibriel, Hesham A.Y. ; Ware, Sarah B. ; Den Bosch, Frank van; Manning-Smith, Robbie ; Alonso-Chavez, Vasthi ; Helps, Joe ; M’Barek, Sarrah Ben; Mehrabi, Rahim ; Diaz-Trujillo, Caucasella ; Zamani, Elham ; Schouten, Henk J. ; Lee, Theo A.J. van der; Waalwijk, Cees ; Waard, Maarten A. de; Wit, Pierre J.G.M. de; Verstappen, Els C.P. ; Thomma, Bart P.H.J. ; Meijer, Harold J.G. ; Seidl, Michael F. - \ 2018
Nature Genetics 50 (2018). - ISSN 1061-4036 - p. 375 - 380.
Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici–wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.
Modification of chrysanthemum odour and taste with chrysanthemol synthase induces strong dual resistance against cotton aphids
Li, Jinjin ; Delatte, Thierry ; Vervoort, Jacques ; Gao, Liping ; Verstappen, Francel ; Xiong, Wei ; Gan, Jianping ; Jongsma, Maarten A. ; Wang, Caiyun - \ 2018
Plant Biotechnology Journal (2018). - ISSN 1467-7644
Aphid resistance - Chrysanthemol synthase - Chrysanthemum - Double bioactivity - Glycoside - Terpene volatile
Aphids are pests of chrysanthemum that employ plant volatiles to select host plants and ingest cell contents to probe host quality before engaging in prolonged feeding and reproduction. Changes in volatile and nonvolatile metabolite profiles can disrupt aphid-plant interactions and provide new methods of pest control. Chrysanthemol synthase (CHS) from Tanacetum cinerariifolium represents the first committed step in the biosynthesis of pyrethrin ester insecticides, but no biological role for the chrysanthemol product alone has yet been documented. In this study, the TcCHS gene was over-expressed in Chrysanthemum morifolium and resulted in both the emission of volatile chrysanthemol (ca. 47 pmol/h/gFW) and accumulation of a chrysanthemol glycoside derivative, identified by NMR as chrysanthemyl-6-O-malonyl-β-D-glucopyranoside (ca. 1.1 mM), with no detrimental phenotypic effects. Dual-choice assays separately assaying these compounds in pure form and as part of the headspace and extract demonstrated independent bioactivity of both components against the cotton aphid (Aphis gossypii). Performance assays showed that the TcCHS plants significantly reduced aphid reproduction, consistent with disturbance of aphid probing activities on these plants as revealed by electropenetrogram (EPG) studies. In open-field trials, aphid population development was very strongly impaired demonstrating the robustness and high impact of the trait. The results suggest that expression of the TcCHS gene induces a dual defence system, with both repellence by chrysanthemol odour and deterrence by its nonvolatile glycoside, introducing a promising new option for engineering aphid control into plants.
Methicillin-resistant Staphylococcus pseudintermedius among dogs in the description of novel SCCmec variants
Duim, Birgitta ; Verstappen, Koen M.H.W. ; Kalupahana, Ruwani S. ; Ranathunga, Lakmali ; Fluit, Ad C. ; Wagenaar, Jaap A. - \ 2018
Veterinary Microbiology 213 (2018). - ISSN 0378-1135 - p. 136 - 141.
S. pseudintermedius - SCCmec variants - Sri Lanka - ΨSCCmec
The presence and genetic characteristics of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in Sri Lanka was investigated to add additional insight into global spread, emergence and evolution of MRSP. A total of 234 samples from dogs visiting veterinary clinics were cultured for staphylococci and the genomes of the MRSP isolates were sequenced, to identify resistance genes, the multilocus sequence types (MLST) and spa types. From a questionnaire the history of antimicrobial treatment and patient information was obtained. S. pseudintermedius was isolated from 116/229 samples, eight of these were MRSP. Six MRSP CC45 isolates contained a pseudo-SCC element ΨSCCmec57395. Two isolates belonging to ST429 (CC761) and ST121 (CC121) contained novel variants of the SCCmec Type V(T) element. The elements were designated SCCmecV(T)SL/066, that carried additional transposon-related genes, and SCCmecV(T)SL/154 that carried a type III restriction-modification system, a type 7 ccr gene complex, and a cadA coding sequence. Thirty-seven percent of the dogs received antimicrobial treatment at the time of sampling of which four dogs were MRSP-positive. The proportion of MRSP among S. pseudintermedius is low compared to other countries, despite the fact that in Sri Lanka antimicrobials for treatment of dogs are available over the counter. Important is the finding of novel type V(T) SCCmec elements, which further underlines the high recombination frequency of SCC elements. The ΨSCCmec57395 was found in isolates of CC45, which is the only sequence type of MRSP known to contain this pseudo-cassette.
Staphylococcus aureus nasal colonization differs among pig lineages and is associated with the presence of other staphylococcal species
Verstappen, Koen M. ; Willems, Eveline ; Fluit, Ad C. ; Duim, Birgitta ; Martens, Marc ; Wagenaar, Jaap A. - \ 2017
Frontiers in Veterinary Science 4 (2017)JUN. - ISSN 2297-1769
Colonization - Methicillin-resistant S. aureus - Pigs - Staphylococci - Staphylococcus aureus
Staphylococcus aureus is a common colonizer in pigs, with methicillin-resistant S. aureus (MRSA) in particular being a potential health risk to humans. To reduce the exposure to humans, the colonization in pigs should be reduced. The aim of this study was to quantitatively compare the susceptibility of pig lineages for S. aureus colonization, and if the absence of S. aureus could be associated with the presence or absence of other staphylococcal species. Nasal samples (n = 129) were obtained from seven different pig lineages in the Netherlands, France, and Germany. S. aureus and other staphylococci were enumerated from these samples by real-time (RT)-PCR and culture. Associations were explored between the presence of S. aureus and other staphylococci. S. aureus was detected by RT-PCR on all farms and in samples from pigs of all lineages. Twenty-five percent of the pigs from lineage F (from two farms) were colonized with S. aureus, while in all other lineages it was more than 50% (p < 0.01). Moreover, in S. aureus-positive samples from pigs of lineage F smaller amounts of S. aureus were found than in other lineages. Staphylococcus sciuri, Staphylococcus cohnii, and Staphylococcus saprophyticus were usually not found in combination with S. aureus in these samples. In conclusion: (i) pigs from different genetic lineages have different susceptibilities for colonization with S. aureus. These pigs might contain a genetic factor influencing nasal colonization. (ii) Colonization of S. aureus is also associated with the absence of S. sciuri, S. cohnii, or S. saprophyticus. (iii) The farm environment seems to influence the presence of S. aureus in pigs.
Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences
Verstappen, Koen M. ; Huijbregts, Loes ; Spaninks, Mirlin ; Wagenaar, Jaap A. ; Fluit, Ad C. ; Duim, Birgitta - \ 2017
PLoS One 12 (2017)8. - ISSN 1932-6203 - 10 p.

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74) and non-pseudintermedius genomes (n = 138). Six sequences specific for S. pseudintermedius were identified (sequence length between 300–500 nt). One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54), and eight other staphylococcal species (n = 43). In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. pseudintermedius.

Beyond cattle and communal land : how the Maasai accommodate privatisation in Kenya
Wairimu, W.W. ; Hebinck, P.G.M. - \ 2017
In: Land Law and Governance / Mostert, H., Verstappen, L.C.A., Zevenbergen, J., Cape Town : Juta (Contemporary Studies in Legal and Applied Research series ) - ISBN 9781485120063 - p. 40 - 62.
This chapter discusses the dynamics brought about by privatising land ownership. Changes in land tenure are analysed here from a socio-relational perspective. Changes in land tenure do not just concern ownership but also transform the properties of land space as a result of which new forms of land use emerge. Moreover, this chapter argues that the Maasai are not passive receivers of new land tenure policies enacted by the Kenyan state. They accommodate land tenure changes in many different and unexpected ways. The case discussed here involves the (further) space subdivision of once communal land to group ranches and to individual Maasai families. The further subdivision of land creates space for new practices such as cultivation of crops, notably by women. Men on the other hand build alliances to aggregate smaller pieces of land to allow continuation of a pastoral lifestyle. The chapter also draws attention to land leasing which initiated unsustainable land use practices
Targeting trichothecene biosynthetic genes
Wei, Songhong ; Lee, Theo van der; Verstappen, Els ; Gent, Marga van; Waalwijk, Cees - \ 2017
In: Methods in Molecular Biology Humana Press Inc. (Methods in Molecular Biology ) - p. 173 - 189.
PCR - Quantitative PCR - Trichothecenes
Biosynthesis of trichothecenes requires the involvement of at least 15 genes, most of which have been targeted for PCR. Qualitative PCRs are used to assign chemotypes to individual isolates, e.g., the capacity to produce type A and/or type B trichothecenes. Many regions in the core cluster (consisting of 12 genes) including intergenic regions have been used as targets for PCR, but the most robust assays are targeted to the tri3 and tri12 genes. Quantitative PCRs, that work across trichothecene-producing members of the Fusarium head blight complex, are described along with procedures to quantify the amount of fungal biomass in wheat samples. These assays are directed to the chemotype(s) present in field samples and quantify the total fungal biomass of trichothecene-producing fungi, irrespective of their genetic identity.
Simultaneous Quantification and Differentiation of Streptococcus suis Serotypes 2 and 9 by Quantitative Real-time PCR, Evaluated in Tonsillar and Nasal Samples of Pigs
Dekker, Niels ; Daemen, Ineke ; Verstappen, K.M. ; Greeff, A. de; Smith, H.E. ; Duim, Birgitta - \ 2016
Pathogens 5 (2016)3. - ISSN 2076-0817
Abstract Invasive Streptococcus suis (S. suis) infections in pigs are often associated with serotypes 2 and 9. Mucosal sites of healthy pigs can be colonized with these serotypes, often multiple serotypes per pig. To unravel the contribution of these serotypes in pathogenesis and epidemiology, simultaneous quantification of serotypes is needed. A quantitative real-time PCR (qPCR) targeting cps2J (serotypes 2 and 1/2) and cps9H (serotype 9) was evaluated with nasal and tonsillar samples from S. suis exposed pigs. qPCR specifically detected serotypes in all pig samples. The serotypes loads in pig samples estimated by qPCR showed, except for serotype 9 in tonsillar samples (correlation coefficient = 0.25), moderate to strong correlation with loads detected by culture (correlation coefficient > 0.65), and also in pigs exposed to both serotypes (correlation coefficient > 0.75). This qPCR is suitable for simultaneous differentiation and quantification of important S. suis serotypes
Chicken immune response after in ovo Immunization with Chimeric TLR5 activating flagellin of campylobacter jejuni
Radomska, Katarzyna A. ; Vaezirad, Mahdi M. ; Verstappen, Koen M. ; Wösten, Marc M.S.M. ; Wagenaar, Jaap A. ; Putten, Jos P.M. van - \ 2016
PLoS One 11 (2016)10. - ISSN 1932-6203

Campylobacter jejuni is the main cause of bacterial food-borne diseases in developed countries. Chickens are the most important source of human infection. Vaccination of poultry is an attractive strategy to reduce the number of C. jejuni in the intestinal tract of chickens. We investigated the immunogenicity and protective efficacy of a recombinant C. jejuni flagellin-based subunit vaccine with intrinsic adjuvant activity. Toll-like receptor activation assays demonstrated the purity and TLR5 stimulating (adjuvant) activity of the vaccine. The antigen (20-40 μg) was administered in ovo to 18 day-old chicken embryos. Serum samples and intestinal content were assessed for antigen-specific systemic and mucosal humoral immune responses. In ovo vaccination resulted in the successful generation of IgY and IgM serum antibodies against the flagellin-based subunit vaccine as determined by ELISA and Western blotting. Vaccination did not induce significant amounts of flagellin-specific secretory IgA in the chicken intestine. Challenge of chickens with C. jejuni yielded similar intestinal colonization levels for vaccinated and control animals. Our results indicate that in ovo delivery of recombinant C. jejuni flagellin subunit vaccine is a feasible approach to yield a systemic humoral immune response in chickens but that a mucosal immune response may be needed to reduce C. jejuni colonization.

The effectiveness of bacteriophages against methicillin-resistant Staphylococcus aureus ST398 nasal colonization in pigs
Verstappen, Koen M. ; Tulinski, Pawel ; Duim, Birgitta ; Fluit, Ad C. ; Carney, Jennifer ; Nes, Arie Van; Wagenaar, Jaap A. - \ 2016
PLoS One 11 (2016)8. - ISSN 1932-6203

Methicillin-resistant Staphylococcus aureus (MRSA) is an important colonizer in animals and an opportunistic pathogen in humans. In humans, MRSA can cause infections that might be difficult to treat because of antimicrobial resistance. The use of bacteriophages has been suggested as a potential approach for the control of MRSA colonization to minimize the - often occupational - exposure of humans. The aim of this study was to assess the efficacy of bacteriophage treatment on porcine nasal colonization with MRSA in vitro, in vivo, and ex vivo. The effectiveness of a bacteriophage combination of phage K∗710 and P68 was assessed in vitro by incubating them with MRSA V0608892/1 (ST398) measuring the OD600 hourly. To study the in vivo effect, bacteriophages were administered in a gel developed for human application, which contain 109 plaque-forming units (pfu)/mL (K and P68 in a 19.25:1 ratio) for 5 days to piglets (N = 8) that were experimentally colonized with the MRSA strain. Eight piglets experimentally colonized were used as a negative control. The MRSA strain was also used to colonize porcine nasal mucosa explants and bacteriophages were applied to assess the ex vivo efficacy of treatment. Bacteriophages were effective in vitro. In vivo, sixteen piglets were colonized with MRSA but the number of CFU recovered after the application of the bacteriophages in 8 piglets was not reduced compared to the control animals (approx. 105 CFU/swab). In the ex vivo model, 108 CFU were used to establish colonization with MRSA; a reduction of colonization was not observed after application of bacteriophages. However, application of mupirocin both in vivo and ex vivo resulted in a near eradication of MRSA. In conclusion: i) The MRSA strain was killed in the presence of the bacteriophages phage K∗710 and P68 in vitro. ii) Bacteriophages did not reduce porcine nasal colonization in vivo or ex vivo. Physiological in vivo and ex vivo conditions may explain these observations. Efficacy in the ex vivo model matched that of the in vivo system.

Transmission through air as a possible route of exposure for MRSA
Bos, Marian E.H. ; Verstappen, Koen M. ; Cleef, Brigitte A.G.L. Van; Dohmen, Wietske ; Dorado-García, Alejandro ; Graveland, Haitske ; Duim, Birgitta ; Wagenaar, Jaap A. ; Kluytmans, Jan A.J.W. ; Heederik, Dick J.J. - \ 2016
Journal of Exposure Science and Environmental Epidemiology 26 (2016)3. - ISSN 1559-0631 - p. 263 - 269.
air - exposure - livestock - MRSA - transmission

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is highly prevalent in pigs and veal calves. The environment and air in pig and veal calf barns is often contaminated with LA-MRSA, and can act as a transmission source for humans. This study explores exposure-response relationships between sequence type 398 (ST398) MRSA air exposure level and nasal ST398 MRSA carriage in people working and/or living on farms. Samples and data were used from three longitudinal field studies in pig and veal calf farm populations. Samples consisted of nasal swabs from the human participants and electrostatic dust fall collectors capturing airborne settled dust in barns. In both multivariate and mutually adjusted analyses, a strong association was found between nasal ST398 MRSA carriage in people working in the barns for >20 h per week and MRSA air levels. In people working in the barns <20 h per week there was a strong association between nasal carriage and number of working hours. Exposure to ST398 MRSA in barn air seems to be an important determinant for nasal carriage, especially in the highly exposed group of farmers, next to duration of contact with animals. Intervention measures should therefore probably also target reduction of ST398 MRSA air levels.

Floral volatiles in parasitic plants of the orobanchaceae. Ecological and taxonomic implications
Tóth, Peter ; Undas, Anna K. ; Verstappen, Francel ; Bouwmeester, Harro - \ 2016
Frontiers in Plant Science 7 (2016). - ISSN 1664-462X
Broomrapes - Floral scents - Orobanche - Phelipanche - Phylogenetic patterns - Taxonomy - Volatile organic compounds - Weeds

The holoparasitic broomrapes, Orobanche spp. and Phelipanche spp. (Orobanchaceae), are root parasites that completely depend on a host plant for survival and reproduction. There is considerable controversy on the taxonomy of this biologically and agronomically important family. Flowers of over 25 parasitic Orobanchaceae and a number of close, parasitic and non-parasitic, relatives emitted a complex blend of volatile organic compounds (VOCs), consisting of over 130 VOCs per species. Floral VOC blend-based phylogeny supported the known taxonomy in internal taxonomic grouping of genus and eliminated the uncertainty in some taxonomical groups. Moreover, phylogenetic analysis suggested separation of the broomrapes into two main groups parasitizing annual and perennial hosts, and for the annual hosts, into weedy and non-weedy broomrapes. We conclude that floral VOCs are a significant tool in species identification and possibly even in defining new species and can help to improve controversial taxonomy in the Orobanchaceae.

Health and health-related quality of life in pig farmers carrying livestock-associated methicillin-resistant Staphylococcus aureus
Cleef, B.A.G.L. van; Benthem, B.H.B. van; Verkade, E.J.M. ; Rijen, M.M.L. van; Kluytmans-van den Bergh, M.F.Q. ; Graveland, H. ; Bosch, T. ; Verstappen, K.M.H.W. ; WAGENAAR, J.A. ; Heederik, D. ; Kluytmans, J.A.J.W. - \ 2016
Epidemiology and Infection (2016). - ISSN 0950-2688 - p. 1774 - 1783.
Infectious disease epidemiology - methicillin-resistant S. aureus (MRSA) - public health - zoonoses

There is limited knowledge about the effect of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) carriage on health-related quality of life (QoL). With this study, we explored whether LA-MRSA causes infections or affects health-related QoL in pig farmers. This prospective cohort study surveyed persons working on 49 farrowing pig farms in The Netherlands for 1 year (2010–2011). On six sampling moments, nasal swabs, environmental samples and questionnaires on activities and infections were collected. At the end of the study year, persons were asked about their QoL using the validated SF-36 and EQ-5D questionnaires. Of 120 persons, 44 (37%) were persistent MRSA carriers. MRSA carriage was not associated with infections, use of antimicrobials, healthcare contact and health-related QoL items in univariate or multivariate analysis, most likely due to the ‘healthy worker effect’. Despite high carriage rates, the impact of LA-MRSA carriage in this population of relatively healthy pig farmers on health and health-related QoL appears limited; more research is needed for confirmation.

Multiplex detection and identification of Phytophthora spp. using target-specific primer extension and Luminex xTAG technology
Kostov, K. ; Verstappen, E.C.P. ; Bergervoet, J.H.W. ; Weerdt, M. de; Schoen, C.D. ; Slavov, S. ; Bonants, P.J.M. - \ 2016
Plant Pathology 65 (2016)6. - ISSN 0032-0862 - p. 1008 - 1021.
Detection - Identification - Luminex - Multiplex - Phytophthora - TSPE

There are more than 100 species that belong to the fungus-like genus Phytophthora, many of which can cause severe damage to plants in both natural and agricultural ecosystems. The availability of techniques for detection and identification are crucial for monitoring and control of these pathogens. In recent years, new methods using molecular approaches have been developed. However, the majority of them are designed to detect single Phytophthora species. Techniques that are able to target multiple species in one sample would offer advantages, especially for the assessment of Phytophthora diversity in the environment. This paper describes a multiplex assay for simultaneous detection and identification of 26 members of Phytophthora down to species level and another 22 to clade or subclade level through target-specific primer extension (TSPE) and the Luminex xTAG array detection system. The assay starts with PCR amplification of two genomic regions, ITS and coxI, followed by a multiplex TSPE reaction with clade-, subclade- and species-specific probes. As a result, biotin-dCTP labelled products are generated and subsequently detected through hybridization with a set of anti-TAG coupled, colour-coded paramagnetic beads. The specificity of the method has been tested using DNA extracts from over 400 isolates representing 110 Phytophthora species and subspecies. The sensitivity and robustness have been determined by the use of DNA mixtures, dilution series and environmental samples. Thus the developed technique allows simultaneous identification of multiple Phytophthora species, particularly useful for the detection of these pathogens in environmental samples such as soil, water and plant tissue.

Changes in the population of methicillin-resistant Staphylococcus pseudintermedius and dissemination of antimicrobial-resistant phenotypes in the Netherlands
Duim, Birgitta ; Verstappen, Koen M. ; Broens, E.M. ; Laarhoven, Laura M. ; Duijkeren, Engeline Van; Hordijk, Joost ; Heus, Phebe De; Spaninks, Mirlin ; Timmerman, Arjen J. ; Wagenaar, J.A. - \ 2016
Journal of Clinical Microbiology 54 (2016)2. - ISSN 0095-1137 - p. 283 - 288.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP), which is often multidrug resistant (MDR), has recently emerged as a threat to canine health worldwide. Knowledge of the temporal distribution of specific MRSP lineages, their antimicrobial resistance phenotypes, and their association with clinical conditions may help us to understand the emergence and spread of MRSP in dogs. The aim of this study was to determine the yearly proportions of MRSP lineages and their antimicrobial-resistant phenotypes in the Netherlands and to examine possible associations with clinical conditions. MRSP was first isolated from a canine specimen submitted for diagnostics to the Faculty of Veterinary Medicine of Utrecht University in 2004. The annual cumulative incidence of MRSP among S. pseudintermedius increased from 0.9% in 2004 to 7% in 2013. MRSP was significantly associated with pyoderma and, to a lesser extent, with wound infections and otitis externa. Multilocus sequence typing (MLST) of 478 MRSP isolates yielded 39 sequence types (ST) belonging to 4 clonal complexes (CC) and 15 singletons. CC71 was the dominant lineage that emerged since 2004, and CC258, CC45, and several unlinked isolates became more frequent during the following years. All but two strains conferred an MDR phenotype, but strains belonging to CC258 or singletons were less resistant. In conclusion, our study showed that MDR CC71 emerged as the dominant lineage from 2004 and onward and that less-resistant lineages were partly replacing CC71.

NGS applications in plant pathogen diagnostics
Dullemans, Annette - \ 2015
NGS applications in plant pathogen diagnosticsDullemans Annette, Verstappen Els, Houwers Ilse, Verbeek Martin, Van der Lee Theo, Van der Vlugt René and Bonants Peter. Wageningen UR, BU Biointeractions & Plant Health, PO. Box 16, 6700 AA Wageningen, The Netherlands, annette.dullemans@wur.nlRoutine diagnostics of plant pathogens often involve methods such as ELISA and real-time (TaqMan) PCR. These methods only detect known pathogens. Next Generation Sequencing (NGS) is a valuable method to identify new pathogens. We developed a (semi) automated pipeline to use HiSeq Illumina Sequencing to detect pathogens in different hosts. Total DNA or RNA was isolated from symptomatic plant tissue to generate sequence libraries. Individual samples were labelled with tags to pool multiple samples in one run. The sequence reads generated from a single sample were analyzed in a CLC Genomics Work Bench pipeline. Host reads were removed and the remaining reads were used in a de novo assembly. The resulting contig sequences were compared with sequences in public and in-house databases to identify the origin of the non-host sequences. General knowledge on plant pathogens is essential to identify the pathogen contigs. The read coverage of the pathogen genomes expressed as the Reads per Kb per million reads, and therefore the analytical sensitivity, depends on number of reads, genome size, and the abundance of the pathogen in the host. To investigate whether NGS can be a useful tool in routine screening, RNA samples from an inspection service were sequenced. Samples infected with known pathogens, detected by real-time (Taqman) PCR could be confirmed by NGS. Several examples of NGS will be presented.
Protecting the environment through insect farming as a means to produce protein for use as livestock, poultry, and aquaculture feed
Tomberlin, J.K. ; Huis, A. van; Benbow, M.E. ; Jordan, H. ; Astuti, D.A. ; Azzollini, D. ; Banks, I. ; Bava, V. ; Borgemeister, C. ; Cammack, J.A. ; Chapkin, R.S. ; Cickova, H. ; Crippen, T.L. ; Day, A. ; Dicke, M. ; Drew, D.W.J. ; Emhart, C. ; Epstein, M. ; Finke, M. ; Fischer, C.H. ; Gatlin, D. ; Grabowski, N.Th. ; He, C. ; Heckman, L. ; Hubert, A. ; Jacobs, J. ; Josephs, J. ; Khanal, S.K. ; Kleinfinger, J.F. ; Klein, G. ; Leach, C. ; Liu, Y. ; Newton, G.L. ; Olivier, R. ; Pechal, J.L. ; Picard, C.J. ; Rojo, S. ; Roncarati, A. ; Sheppard, C. ; Tarone, A.M. ; Verstappen, B. ; Vickerson, A. ; Yang, H. ; Yen, A.L. ; Yu, Z. ; Zhang, J. ; Zheng, L. - \ 2015
Journal of Insects as Food and Feed 1 (2015)4. - ISSN 2352-4588 - p. 307 - 309.
Securing protein for the approximate 10 billion humans expected to inhabit our planet by 2050 is a major priority for the global community. Evidence has accrued over the past 30 years that strongly supports and justifies the sustainable use of insects as a means to produce protein products as feed for pets, livestock, poultry, and aquacultured species. Researchers and entrepreneurs affiliated with universities and industries, respectively, from 18 nations distributed across North and South America, Europe, Asia, Africa and Australia contributed to the development of this article, which is an indication of the global interest on this topic. A brief overview of insects as feed for the aquaculture industry along with a review of the black soldier fly, Hermetia illucens (Diptera: Stratiomyidae), as a model for such systems is provided.
Effects of reducing antimicrobial use and applying a cleaning and disinfection program in veal calf farming : Experiences from an intervention study to control livestock-associated MRSA
Dorado-García, Alejandro ; Graveland, Haitske ; Bos, M.E.H. ; Verstappen, K.M. ; Cleef, B.A.G.L. Van; Kluytmans, J.A.J.W. ; Wagenaar, J.A. ; Heederik, D.J.J. - \ 2015
PLoS One 10 (2015)8. - ISSN 1932-6203 - 21 p.

With the ultimate aim of containing the emergence of resistant bacteria, a Dutch policy was set in place in 2010 promoting a reduction of antimicrobial use (AMU) in food-producing animals. In this context, a study evaluated strategies to curb livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA). Fifty-one veal calf farms were assigned to one of 3 study arms: RAB farms reducing antimicrobials by protocol; RAB-CD farms reducing antimicrobials by protocol and applying a cleaning and disinfection program; and Control farms without interventions. MRSA carriage was tested in week 0 and week 12 of 2 consecutive production cycles in farmers, family members and veal calves. Interventions were validated and a cyclic rise in MRSA-prevalence in animals was shown with a more moderate increase in RAB farms. Prevalence in humans declined parallel over time in the study arms but RAB farms were at the lowest MRSA levels from the beginning of the study. In RAB-CD farms, human and animal prevalence did not differ from Control farms and MRSA air loads were significantly higher than in the other study arms. Mimicking the national trend, an overall AMU decrease (daily dosages per animal per cycle (DDDA/C)) was observed over 4 pre-study and the 2 study cycles; this trend did not have a significant effect on a set of evaluated farm technical parameters. AMU was positively associated with MRSA across study arms (ORs per 10 DDDA/C increase = 1.26 for both humans (p = 0.07) and animals (p = 0.12 in first cycle)). These results suggest that AMU reduction might be a good strategy for curbing MRSA in veal calf farming, however the specific cleaning and disinfecting program in RAB-CD farms was not effective. The drop in MRSA prevalence in people during the study could be attributed to the observed long-term AMU decreasing trend.

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