Inulin-type fructans modulate intestinal Bifidobacterium species populations and decrease fecal short-chain fatty acids in obese women
Salazar, N. ; Dewulf, E.M. ; Neyrinck, A.M. ; Bindels, L.B. ; Cani, P.D. ; Mahillon, J. ; Vos, W.M. de; Thissen, J.P. ; Gueimonde, M. ; Reyes-Gavilán, C.G. de los; Delzenne, N.M. - \ 2015
Clinical Nutrition 34 (2015)3. - ISSN 0261-5614 - p. 501 - 507.
gradient gel-electrophoresis - protein-coupled receptor - gut microbiota - mice - prebiotics - increases - glucose - diet - pcr - fermentation
Background & aims : Inulin-type fructans (ITF) prebiotics promote changes in the composition and activity of the gut microbiota. The aim of this study was to determine variations on fecal short chain fatty acids (SCFA) concentration in obese women treated with ITF and to explore associations between Bifidobacterium species, SCFA and host biological markers of metabolism. Methods Samples were obtained in a randomized, double blind, parallel, placebo-controlled trial, with 30 obese women randomly assigned to groups that received either 16 g/day ITF (n = 15) or maltodextrin (n = 15) for 3 months. The qualitative and quantitative analysis of Bifidobacterium spp. was performed in feces by PCR-DGGE and q-PCR, and SCFA profile was analyzed by gas chromatography. Spearman correlation analysis was performed between the different variables analyzed. Results The species Bifidobacterium longum, Bifidobacterium pseudocatenulatum and Bifidobacterium adolescentis were significantly increased at the end of the treatment in the prebiotic group (p <0.01) with being B. longum negatively correlated with serum lipopolysaccharide (LPS) endotoxin (p <0.01). Total SCFA, acetate and propionate, that positively correlated with BMI, fasting insulinemia and homeostasis model assessment (HOMA) (p <0.05), were significantly lower in prebiotic than in placebo group after the treatment period. Conclusions ITF consumption selectively modulates Bifidobacterium spp. and decreases fecal SCFA concentration in obese women. ITF could lessen metabolic risk factors associated with higher fecal SCFA concentration in obese individuals.
Extended-spectrum and AmpC B-lactamase-producing Escherichia coli in broilers and peoplelivingand/or working on broiler farms: prevalence, risk factors and molecular characteristics
Huijbers, P.M.C. ; Graat, E.A.M. ; Haenen, A.P.J. ; Santen, M.G. van; Essen-Zandbergen, A. van; Mevius, D.J. ; Duijkeren, E. van; Hoek, A.H.A.M. van - \ 2014
Journal of Antimicrobial Chemotherapy 69 (2014)10. - ISSN 0305-7453 - p. 2669 - 2675.
livestock-associated mrsa - enterobacteriaceae - netherlands - humans - identification - plasmids - poultry - genes - meat - pcr
OBJECTIVES: The objectives of this study were to: estimate the prevalence of extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase-producing Escherichia coli carriage among broiler farmers, their family members and employees; identify and quantify risk factors for carriage, with an emphasis on contact with live broilers; and compare isolates from humans and broilers within farms with respect to molecular characteristics to gain insight into transmission routes. METHODS: A cross-sectional prevalence study was conducted on 50 randomly selected Dutch broiler farms. Cloacal swabs were taken from 20 randomly chosen broilers. Faecal swabs were returned by 141 individuals living and/or working on 47 farms. ESBL/AmpC-producing E. coli were isolated and, for selected isolates, phylogenetic groups, plasmids and sequence types were determined. Questionnaires were used for risk factor analysis. RESULTS: All sampled farms were positive, with 96.4% positive pooled broiler samples. The human prevalence was 19.1%, with 14.3% and 27.1% among individuals having a low and a high degree of contact with live broilers, respectively. Five pairs of human-broiler isolates had identical genes, plasmid families and E. coli sequence types, showing clonal transmission. Furthermore, similar ESBL/AmpC genes on the same plasmid families in different E. coli sequence types in humans and broilers hinted at horizontal gene transfer. CONCLUSIONS: The prevalence among people on broiler farms was higher than in previous studies involving patients and the general population. Furthermore, an increased risk of carriage was shown among individuals having a high degree of contact with live broilers. The (relative) contribution of transmission routes that might play a role in the dissemination of ESBL/AmpC-encoding resistance genes to humans on broiler farms should be pursued in future studies.
Prevalence of Toxoplasma gondii in common moles (Talpa europaea)
Krijger, I.M. ; Cornelissen, J.B.W.J. ; Wisselink, H.J. ; Meerburg, B.G. - \ 2014
Acta Veterinaria Scandinavica 56 (2014). - ISSN 0044-605X
brain-tissue - costa-rica - oocysts - cysts - farms - hosts - pcr
Background: The prevalence of Toxoplasma gondii in common moles, Talpa europaea, was investigated in order to determine whether moles can serve as an indicator species for T. gondii infections in livestock. Findings: In total, 86 moles were caught from 25 different sites in the Netherlands. Five different trapping habitats were distinguished: pasture, garden, forest, roadside, and recreation area. No positive samples (brain cysts) were found during microscopic detection (n¿=¿70). Using the Latex Agglutination Test (LAT), sera of 70 moles were examined, whereby no sample reacted with T. gondii antigen. Real Time-PCR tests on brain tissue showed 2 positive samples (2.3%). Conclusions: Because of the low number of positives in our study, the use of the common mole as an indicator species for livestock infections is currently not recommended.
Development and evaluation of Taqman assays for the differentiation of Dickeya (sub)species
Wolf, J.M. van der; Haas, B.H. de; Hoof, R.A. van; Haan, E.G. de; Bovenkamp, G.W. van den - \ 2014
European Journal of Plant Pathology 138 (2014)4. - ISSN 0929-1873 - p. 695 - 709.
complete genome sequence - syn. erwinia-chrysanthemi - potato - strains - pcr - relatedness - polymerase - clade - crops - hosts
TaqMan assays were developed for the detection of seven Dickeya species, namely D. dianthicola, D. dadantii, D. paradisiaca, D. chrysanthemi, D. zeae, D. dieffenbachiae and D. solani. Sequences of the gene coding for dnaX were used for the design of primers and probes. In studies with axenic cultures of bacteria, the assays were highly specific and only reacted with strains of the target species, and not with non-target bacteria, including those belonging to other Dickeya species and other genera. The detection thresholds for DNA extracted from pure cultures of target strains ranged from 10 to 100 fg. The TaqMan assays for D. dianthicola and D. solani were more extensively evaluated as part of a method validation procedure. The threshold level for target bacteria added to a potato peel extract diluted ten-times in a semi-selective broth, was strain dependent and ranged from 1,000 to 100,000 cfu/ml. The coefficients of variation for repeatability and reproducibility were low and results were largely independent of the type of substrate, i.e. potato tuber or carnation leaf extracts. However, during routine testing of seed potatoes, false-positive reactions were found with the assay for D. solani. The use of the TaqMan assays for inspection of plant propagation material, ecological studies and studies on the effect of control strategies in disease management strategies is discussed
Denitrification in restored and unrestored Danish streams
Veraart, A.J. ; Audet, J. ; Rocha Dimitrov, M. ; Hoffmann, C.C. ; Gillissen, F. ; Klein, J.J.M. de - \ 2014
Ecological Engineering 66 (2014). - ISSN 0925-8574 - p. 129 - 140.
16s ribosomal-rna - denitrifying bacteria - headwater streams - nosz genes - nutrient-uptake - restoration - water - sediments - nirk - pcr
Stream restoration often aims at mitigating nutrient pollution in aquatic ecosystems. However, despite recent research efforts, effects of restoration practices on in-stream nitrogen removal remain unclear. In this study, denitrification rates as well as factors controlling denitrification in unrestored and restored sections of two Danish streams (S1 and S2) were compared. The 15N isotope pairing technique was used to measure denitrification in situ. Denitrifier presence was analyzed by denaturing gradient gel electrophoresis (DGGE) and quantitative PCR of nitrite reductase (nirK and nirS) and nitrous oxide reductase (nosZ) genes. Denitrification rates were highly variable, with denitrification rates of 3106 µmol N m-2 h-1 in the unrestored section of S1, but no detectable denitrification in the restored section of S1, whereas in S2 restored and unrestored sections had similar denitrification rates of around 250 µmol N m-2 h-1. These large differences in denitrification rates were mainly due to differences in hydrologic conditions and sediment characteristics. High nitrate fluxes from upwelling groundwater created denitrification hotspots in the unrestored section of S1. Moreover, a lack of organic matter in the restored section of S1 likely caused a low abundance of denitrifiers and consequently no detectable denitrification. Our results indicate the importance of hydrology and sediment organic matter for stream nitrogen dynamics, which should be considered in restoration design
A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis
Thierry, S. ; Hamidjaja, R.A. ; Girault, G. ; Lofstrom, C. ; Ruuls-van Stalle, E.M.F. ; Sylviane, D. - \ 2013
Journal of Microbiological Methods 95 (2013)3. - ISSN 0167-7012 - p. 357 - 365.
tandem-repeat analysis - large-scale - listeria-monocytogenes - pathogen detection - genotyping assays - dna - probes - discrimination - amplification - pcr
Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex (R) xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2 ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis. (C) 2013 Elsevier B.V. All rights reserved.
Evaluation of a Commercial ELISA for Detection of Ruminant Processed Animal Proteins in Non-Ruminant Processed Animal Proteins
Bremer, M.G.E.G. ; Margry, R.J.C.F. ; Vaessen, J.C.H. ; Doremalen, A.M.H. van; Palen, J.G.P. van der; Kaathoven, R.G.C. van; Kemmers-Voncken, A.E.M. ; Raamsdonk, L.W.D. van - \ 2013
Journal of AOAC International 96 (2013)3. - ISSN 1060-3271 - p. 552 - 559.
plant sterilization conditions - shrimp litopenaeus-vannamei - linked-immunosorbent-assay - bone meals - classical microscopy - rendered meat - by-products - feed - pcr - identification
Due to a growing aquaculture industry, demand for high-quality proteins for aquatic feeds is increasing. Non-ruminant processed animal proteins (PAPs) have shown great potential for this purpose. Safe reintroduction of non-ruminant PAPs in aqua feed requires methods that can discriminate ruminant and non-ruminant PAPs at contamination levels at or below 2%. Because the official European Union method lacks species specificity, the performance of MELISA-TEK™ Ruminant, a commercial immunoassay, combined with the MELISA-TEK High Sensitivity Sample Extraction kit was evaluated. Various non-ruminant PAPs spiked with ruminant PAPs (processed at 133, 137, 141, and 145°C) were analyzed. Results showed an overall specificity of 99%, indicating no cross-reaction with non-ruminant PAPs. The sensitivity of the assay strongly depended on both processing temperature and proportion of muscle fibers of the ruminant PAPs. Overall sensitivity of samples with 1 and 2% ruminant PAPs was 92 and 100%, respectively. For ruminant PAPs processed at 133 and 137°C, the sensitivity was 100% for both 1 and 2% ruminant spikes. Overall accuracies were 96 and 99% for 1 and 2% ruminant spikes, respectively. In conclusion, the MELISA-TEK Ruminant assay showed satisfactory results, which makes it a suitable candidate method to enable safe reintroduction of non-ruminant PAPs in aqua feed.
Large scale variation in DNA copy number in chicken breeds
Crooijmans, R.P.M.A. ; Fife, M. ; FitzGerald, R.J. ; strickland, S. ; Groenen, M. - \ 2013
BMC Genomics 14 (2013). - ISSN 1471-2164
human genome - structural variation - mareks-disease - evolution - map - genes - resistance - feather - loci - pcr
Background Detecting genetic variation is a critical step in elucidating the molecular mechanisms underlying phenotypic diversity. Until recently, such detection has mostly focused on single nucleotide polymorphisms (SNPs) because of the ease in screening complete genomes. Another type of variant, copy number variation (CNV), is emerging as a significant contributor to phenotypic variation in many species. Here we describe a genome-wide CNV study using array comparative genomic hybridization (aCGH) in a wide variety of chicken breeds. Results We identified 3,154 CNVs, grouped into 1,556 CNV regions (CNVRs). Thirty percent of the CNVs were detected in at least 2 individuals. The average size of the CNVs detected was 46.3 kb with the largest CNV, located on GGAZ, being 4.3 Mb. Approximately 75% of the CNVs are copy number losses relatively to the Red Jungle Fowl reference genome. The genome coverage of CNVRs in this study is 60 Mb, which represents almost 5.4% of the chicken genome. In particular large gene families such as the keratin gene family and the MHC show extensive CNV. Conclusions A relative large group of the CNVs are line-specific, several of which were previously shown to be related to the causative mutation for a number of phenotypic variants. The chance that inter-specific CNVs fall into CNVRs detected in chicken is related to the evolutionary distance between the species. Our results provide a valuable resource for the study of genetic and phenotypic variation in this phenotypically diverse species.
Prediction of Bacillus weihenstephanensis acid resistance: The use of gene expression patterns to select potential biomarkers
Desriac, N. ; Postollec, F. ; Coroller, L. ; Sohier, D. ; Abee, T. ; Besten, H.M.W. den - \ 2013
International Journal of Food Microbiology 167 (2013)1. - ISSN 0168-1605 - p. 80 - 86.
gram-positive bacteria - oxidative stress - staphylococcus-aureus - virulence factors - risk-assessment - cereus group - subtilis - quantification - pcr - temperature
Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-resistance towards otherwise lethal stresses. In this study, an approach was followed to select molecular biomarkers (quantitative gene expressions) to predict induced acid resistance after exposure to various mild stresses, i.e. exposure to sublethal concentrations of salt, acid and hydrogen peroxide during 5 min to 60 min. Gene expression patterns of unstressed and mildly stressed cells of Bacillus weihenstephanensis were correlated to their acid resistance (3D value) which was estimated after exposure to lethal acid conditions. Among the twenty-nine candidate biomarkers, 12 genes showed expression patterns that were correlated either linearly or non-linearly to acid resistance, while for the 17 other genes the correlation remains to be determined. The selected genes represented two types of biomarkers, (i) four direct biomarker genes (lexA, spxA, narL, bkdR) for which expression patterns upon mild stress treatment were linearly correlated to induced acid resistance; and (ii) nine long-acting biomarker genes (spxA, BcerKBAB4_0325, katA, trxB, codY, lacI, BcerKBAB4_1716, BcerKBAB4_2108, relA) which were transiently up-regulated during mild stress exposure and correlated to increased acid resistance over time. Our results highlight that mild stress induced transcripts can be linearly or non-linearly correlated to induced acid resistance and both approaches can be used to find relevant biomarkers. This quantitative and systematic approach opens avenues to select cellular biomarkers that could be incremented in mathematical models to predict microbial behaviour. Keywords: Bacillus; Acid resistance; Gene expression; Modelling; Biomarker; Food safety
Bacterial Diversity in Meconium of Preterm Neonates and Evolution of Their Fecal Microbiota during the First Month of Life
Moles, L. ; Gómez, M. ; Heilig, G.H.J. ; Bustos, G. ; Fuentes Enriquez de Salamanca, S. ; Vos, W.M. de; Fernandez, L. ; Rodriguez, J.M. ; Jimenez, E. - \ 2013
PLoS ONE 8 (2013)6. - ISSN 1932-6203
16s ribosomal-rna - birth-weight infants - intestinal microbiota - necrotizing enterocolitis - amniotic-fluid - colonization - gut - pcr - communities - complex
The establishment and succession of bacterial communities in infants may have a profound impact in their health, but information about the composition of meconium microbiota and its evolution in hospitalized preterm infants is scarce. In this context, the objective of this work was to characterize the microbiota of meconium and fecal samples obtained during the first 3 weeks of life from 14 donors using culture and molecular techniques, including DGGE and the Human Intestinal Tract Chip (HITChip) analysis of 16S rRNA amplicons. Culture techniques offer a quantification of cultivable bacteria and allow further study of the isolate, while molecular techniques provide deeper information on bacterial diversity. Culture and HITChip results were very similar but the former showed lower sensitivity. Inter-individual differences were detected in the microbiota profiles although the meconium microbiota was peculiar and distinct from that of fecal samples. Bacilli and other Firmicutes were the main bacteria groups detected in meconium while Proteobacteria dominated in the fecal samples. Culture technique showed that Staphylococcus predominated in meconium and that Enterococcus, together with Gram-negative bacteria such as Escherichia coli, Escherichia fergusonii, Klebsiella pneumoniae and Serratia marcescens, was more abundant in fecal samples. In addition, HITChip results showed the prevalence of bacteria related to Lactobacillus plantarum and Streptococcus mitis in meconium samples whereas those related to Enterococcus, Escherichia coli, Klebsiella pneumoniae and Yersinia predominated in the 3(rd) week feces. This study highlights that spontaneously-released meconium of preterm neonates contains a specific microbiota that differs from that of feces obtained after the first week of life. Our findings indicate that the presence of Serratia was strongly associated with a higher degree of immaturity and other hospital-related parameters, including antibiotherapy and mechanical ventilation
Replication of hepatitis E virus in three-dimensional cell culture
Berto, A. ; Poel, W.H.M. van der; Honing-Hakze, R.W. van der; Martelli, F. ; Ragione, R.M. La; Grierson, S.S. ; Johne, R. ; Inglese, N. ; Reetz, J. ; Collins, J. ; Dastjerdi, A. ; Banks, M. - \ 2013
Journal of Virological Methods 187 (2013)2. - ISSN 0166-0934 - p. 327 - 332.
simulated microgravity - thermal-stability - hev - assay - transmission - infectivity - system - pcr
Hepatitis E is an acute, viral hepatitis epidemic in developing regions, but which is detected with increasing frequency in sporadic form in developed regions. Pigs and possibly some other mammals are considered reservoirs of zoonotic infection with hepatitis E virus (HEV). However, whilst the relative significance of potential transmission routes from pigs to people is still unclear, the consumption of raw or undercooked pig meat has been implicated as a source of HEV infection. The lack of information about HEV zoonotic transmission is due in part to the difficulties of in vitro propagation of HEV. The Rotating Wall Vessel (RVW) has been described as a useful tool for the culture of cell lines in a 3-dimensional (3D) configuration. The aim of this work was to develop a 3D cell culture system for HEV to facilitate studies into the viability of virions contaminating pig tissues. This study, demonstrated that HEV can replicate efficiently in the RWV in human hepatoblastoma PLC/PRF/5 cells for up to 5 months not only by real time RT-PCR but also by detection of complete virions via electron microscopy. Furthermore, the replication of HEV progeny was observed by detecting HEV RNA by RT-PCR. The progeny were able to infect fresh 3D cultures, showing that this method is able to produce infectious hepatitis E virions.
Characterization of low-molecular-weight-glutenin subunit genes from the D-genome of Triticum aestivum, Aegilops crassa, Ae. cylindrica and Ae. tauschii
Naghavi, M.R. ; Ahmadi, S. ; Shanejat-Boushehri, A.A. ; Komaei, G. ; Struik, P.C. - \ 2013
Biochemical Systematics and Ecology 50 (2013). - ISSN 0305-1978 - p. 23 - 29.
endosperm storage proteins - group-1 chromosomes - wheat endosperm - hexaploid wheat - common wheat - cloning - pcr - electrophoresis - identification - polymorphism
Twenty low-molecular-weight-glutenin subunit (LMW-GS) gene sequences from the D-genome from Aegilops crassa (2n ¼ 4x ¼ 28), Ae. cylindrica (2n ¼ 4x ¼ 28), Ae. tauschii (2n ¼ 2x ¼ 14) and Triticum aestivum (2n ¼ 6x ¼ 42) were obtained using five sets of specific allele primer pairs. Only the sequences of the first primer pair were complete coding sequences (cds) of LMW-GS, and had 305, 304, 306 and 305 LMW-m amino acid residues in Ae. crassa, Ae. cylindrica, Ae. tauschii and T. aestivum, respectively. The repetitive domain and repeat motif numbers of all LMW glutenin subunits showed eight conserved cysteine residues that lead to the same functional activity in different genome. Based on DNA and predicted protein sequences, phylogenetic trees for all sets of sequences were drawn. At the DNA level, the species closest to T. aestivum for the second, third, fourth and fifth set of sequences were Ae. cylindrica, Ae. tauschii and Ae. crassa, respectively. At the protein level, the species closest to T. aestivum based on the first, second and fifth set of sequences were Ae. cylindrica, Ae. crassa and Ae. crassa, respectively. For other sets of sequences, bread wheat proved to be a distinct species. The LMW-GS gene sequences have been recorded in the GenBank with accession numbers JQ726549–JQ726568.
Detection and Quantification of Leveillula taurica Growth in Pepper Leaves
Zheng, Z. ; Nonomura, T. ; Bóka, K. ; Matsuda, Y. ; Visser, R.G.F. ; Toyoda, H. ; Kiss, L. ; Bai, Y. - \ 2013
Phytopathology 103 (2013)6. - ISSN 0031-949X - p. 623 - 632.
internal transcribed spacer - powdery-mildew - genus leveillula - capsicum-annuum - resistance - pcr - infections - sequences - fungi - dna
Leveillula taurica is an obligate fungal pathogen that causes powdery mildew disease on a broad range of plants, including important crops such as pepper, tomato, eggplant, onion, cotton, and so on. The early stage of this disease is difficult to diagnose and the disease can easily spread unobserved; for example, in pepper and tomato production fields and greenhouses. The objective of this study was to develop a detection and quantification method of L. taurica biomass in pepper leaves with special regard to the early stages of infection. We monitored the development of the disease to time the infection process on the leaf surface as well as inside the pepper leaves. The initial and final steps of the infection taking place on the leaf surface were consecutively observed using a dissecting microscope and a scanning electron microscope. The development of the intercellular mycelium in the mesophyll was followed by light and transmission electron microscopy. A pair of L. taurica-specific primers was designed based on the internal transcribed spacer sequence of L. taurica and used in real-time polymerase chain reaction (PCR) assay to quantify the fungal DNA during infection. The specificity of this assay was confirmed by testing the primer pair with DNA from host plants and also from another powdery mildew species, Oidium neolycopersici, infecting tomato. A standard curve was obtained for absolute quantification of L. taurica biomass. In addition, we tested a relative quantification method by using a plant gene as reference and the obtained results were compared with the visual disease index scoring. The real-time PCR assay for L. taurica provides a valuable tool for detection and quantification of this pathogen in breeding activities as well in plant-microbe interaction studies.
Efficient multiplex simple sequence repeat genotyping of the oomycete plant pathogen Phytophthora infestans
Li, Y. ; Cooke, D.E.L. ; Jacobsen, E. ; Lee, T.A.J. van der - \ 2013
Journal of Microbiological Methods 92 (2013)3. - ISSN 0167-7012 - p. 316 - 322.
genetic-linkage maps - microsatellite markers - population-genetics - netherlands - migrations - origin - potato - blight - pcr
Genotyping is fundamental to population analysis. To accommodate fast, accurate and cost-effective genotyping, a one-step multiplex PCR method employing twelve simple sequence repeat (SSR) markers was developed for high-throughput screening of Phytophthora infestans populations worldwide. The SSR markers reported for this species were evaluated and the twelve most informative and easily scored were selected. To accomplish a single step genotyping procedure, we optimized primers, fluorescent labels and PCR conditions to genotype using a capillary electrophoresis system with four fluorescent labels (FAM, NED, PET and VIC) and a labeled LIZ standard for sizing of the SSR fragments. The results obtained using commercially available multiplex PCR kits on a set of reference isolates were in agreement with that obtained using primer pairs in simplex reactions. In testing on many thousands of isolates, we have found the markers appropriate for resolving distinct multilocus genotypes (MLGs) of isolates of European and wider populations. Here we demonstrate the utility of the assay on a set of 19 reference isolates plus 77 others sampled from The Netherlands and Great Britain. In most isolates one to two alleles were observed at each locus but the presence of three alleles at a single locus in some isolates was consistent with increased ploidy. Methods are presented that are appropriate for the analysis of datasets comprising isolates of mixed ploidy levels. We also report on the direct P. infestans genotyping from infected field material to collect, store and extract pathogen DNA. A critical step in this multiplex method was the standardization of the protocol between two laboratories in The Netherlands and Great Britain. Reference isolates were exchanged and an allele nomenclature and scoring system agreed. Such co-operation is facilitating the genotyping of international collections of P. infestans isolates in wider networks of laboratories and providing the data required to expand an existing international database of pathogen diversity
Screening for new sources of resistance to Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato
Sen, Y. ; Zhu, F. ; Vandenbroucke, H. ; Wolf, J.M. van der; Visser, R.G.F. ; Heusden, A.W. van - \ 2013
Euphytica 190 (2013)2. - ISSN 0014-2336 - p. 309 - 317.
bacterial canker - corynebacterium-michiganense - lycopersicon-esculentum - ssp michiganensis - seeds - pcr - quantification - crosses
Bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), is considered the most serious bacterial threat, resulting in high damages in production areas. Worldwide, Cmm is subjected to quarantine regulations.There is no cultivar in market containing Cmm resistance genes. This project aimed to screen tomatoes or wild relatives of tomato for resistance to Cmm, to be used for starting breeding programs. We have screened 24 different wild accessions of tomato and found several new tolerant sources: Solanum pimpinellifolium GI.1554, S. parviflorum LA735 and S. parviflorum LA2072. We also confirmed the tolerance which was reported previously in S. peruvianum LA2157, S. peruvianum PI127829, S. peruvianum LA385, S. habrochaites LA407 and S. lycopersicum cv. IRAT L3. No immunity was found. Also accessions showing a low disease score still contained high titers of bacteria as determined by a dilution plating method, using tow selective media. These results were confirmed with a TaqMan real time PCR assay, which was developed to determine and quantify Cmm in planta
Characterization of Dickeya strains isolated from potato grown under hot-climate conditions
Tsror, L. ; Ben-Daniel, B. ; Chalupowicz, L. ; Wolf, J.M. van der; Lebiush, S. ; Erlich, O. ; Dror, O. ; Barel, V. ; Nijhuis, E.H. ; Manulis-Sasson, S. - \ 2013
Plant Pathology 62 (2013)5. - ISSN 0032-0862 - p. 1097 - 1105.
soft-rot erwinias - chrysanthemi - population - israel - crops - pcr
Dickeya strains isolated in Israel in 2006–2010 were characterized by dnaX sequence analysis, pulsed-field gel electrophoresis (PFGE), biochemical assays and pectolytic activity, and found to be homogeneous: most of them could be classified as ‘Dickeya solani’. Of the 34 strains isolated from imported seed tubers or potato plants grown from imported seed, 32 were typed as ‘D. solani’ and only two were characterized as Dickeya dianthicola. Biovar typing indicated that all ‘D. solani’ strains were biovar 3. ‘Dickeya solani’ strains were most closely related to Dickeya dadantii subsp. dieffenbachiae according to PFGE and dnaX analyses and both species exhibited high pectolytic activity. Expression levels of two putative virulence genes, pelL (encoding a pectic enzyme) and dspE (encoding a type III effector) were significantly induced in ‘D. solani’ strains isolated from potato plants or tubers grown in hot climates such as the Negev region in Israel, compared to those isolated from seed tubers imported from the Netherlands, France or Germany. Results of this study support the hypothesis that ‘D. solani’ strains isolated in Israel are also clonal; however, they appear to be more virulent than strains isolated in Europe
Halal assurance in food supply chains: Verification of halal certificates using audits and laboratory analysis
Spiegel, M. van der; Fels-Klerx, H.J. van der; Sterrenburg, P. ; Ruth, S.M. van; Scholtens-Toma, I.M.J. ; Kok, E.J. - \ 2012
Trends in Food Science and Technology 27 (2012)2. - ISSN 0924-2244 - p. 109 - 119.
hazard analysis - lard adulteration - meat - haccp - pcr - pork - authentication - identification - products - system
The global halal market is increasing. Worldwide a large number of standardization and certification organizations has been established. This article discusses halal requirements, summarizes applied standards and certification, and explores current verification of halal certificates using audits and laboratory analysis. Successive research can use the insights to achieve appropriate assurance of halal food products and proper labelling for consumers and buyers.
A multi-locus phylogenetic evaluation of Diaporthe (Phomopsis)
Udayanga, D. ; Liu, X. ; Crous, P.W. ; McKenzie, E.H.C. ; Chukeatirote, E. ; Hyde, K.D. - \ 2012
Fungal Diversity 56 (2012)1. - ISSN 1560-2745 - p. 157 - 171.
multiple sequence alignment - plant-pathogenic fungi - species concepts - south-africa - primer sets - genes - phaseolorum - longicolla - pcr - phylogeography
The genus Diaporthe (Phomopsis) includes important plant pathogenic fungi with wide host ranges and geographic distributions. In the present study, phylogenetic species recognition in Diaporthe is re-evaluated using a multi-locus phylogeny based on a combined data matrix of rDNA ITS, and partial sequences from the translation elongation factor 1-a (EF 1-a), ß tubulin (TUB) and calmodulin (CAL) molecular markers. DNA sequences of available ex-type cultures have been included, providing a multi-locus backbone tree for future studies on Diaporthe. Four utilizable loci were analyzed individually and in combination, and ITS, EF 1-a and multi-locus phylogenetic trees are presented. The phylogenetic tree inferred by combined analysis of four loci provided the best resolution for species as compared to single gene analysis. Notes are provided for nine species previously known in Phomopsis that are transferred to Diaporthe in the present study. The unraveling of cryptic species complexes of Diaporthe based on Genealogical Concordance Phylogenetic Species Recognition (GCPSR) is emphasized.
A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching
Kretzschmar, T. ; Kohlen, W. ; Sasse, J. ; Borghi, L. ; Schlegel, M. ; Bachelier, J.B. ; Reinhardt, D. ; Bours, R.M.E.H. ; Bouwmeester, H.J. ; Martinoia, E. - \ 2012
Nature 483 (2012)7389. - ISSN 0028-0836 - p. 341 - 344.
arbuscular-mycorrhizal fungi - medicago-truncatula - auxin transport - abscisic-acid - gene family - arabidopsis - pcr - germination - inhibition - pathway
Strigolactones were originally identified as stimulators of the germination of root-parasitic weeds1 that pose a serious threat to resource-limited agriculture2. They are mostly exuded from roots and function as signalling compounds in the initiation of arbuscular mycorrhizae3, which are plant–fungus symbionts with a global effect on carbon and phosphate cycling4. Recently, strigolactones were established to be phytohormones that regulate plant shoot architecture by inhibiting the outgrowth of axillary buds5, 6. Despite their importance, it is not known how strigolactones are transported. ATP-binding cassette (ABC) transporters, however, are known to have functions in phytohormone translocation7, 8, 9. Here we show that the Petunia hybrida ABC transporter PDR1 has a key role in regulating the development of arbuscular mycorrhizae and axillary branches, by functioning as a cellular strigolactone exporter. P. hybrida pdr1 mutants are defective in strigolactone exudation from their roots, resulting in reduced symbiotic interactions. Above ground, pdr1 mutants have an enhanced branching phenotype, which is indicative of impaired strigolactone allocation. Overexpression of Petunia axillaris PDR1 in Arabidopsis thaliana results in increased tolerance to high concentrations of a synthetic strigolactone, consistent with increased export of strigolactones from the roots. PDR1 is the first known component in strigolactone transport, providing new opportunities for investigating and manipulating strigolactone-dependent processes.
Genotypic Diversity of Coxiella burnetii in the 2007-2010 Q Fever Outbreak Episodes in The Netherlands
Tilburg, J.J.H.C. ; Rossen, J.W.A. ; Hannen, E.J. van; Melchers, W.J.G. ; Hermans, M.H.A. ; Bovenkamp, J. van de; Roest, H.I.J. ; Bruin, A. de; Nabuurs-Fransen, M.H. ; Horrevorts, A.M. ; Klaassen, C.H.W. - \ 2012
Journal of Clinical Microbiology 50 (2012)3. - ISSN 0095-1137 - p. 1076 - 1078.
The genotypic diversity of Coxiella burnetii in clinical samples obtained from the Dutch Q fever outbreak episodes of 2007-2010 was determined by using a 6-locus variable-number tandem repeat analysis panel. The results are consistent with the introduction of one founder genotype that is gradually diversifying over time while spreading throughout The Netherlands.