P. Fach

To determine whether plant sequences, including transgenic sequences, are present in animal blood... more To determine whether plant sequences, including transgenic sequences, are present in animal blood, we tested blood samples from Holstein cows fed with either Bt176 genetically modified corn or conventional corn. We used previously described sensitive real-time PCR assays targeting transgenic sequences (35S promoter and Bt176 specific junction sequence), a monocopy maize-specific sequence (ADH promoter), and two multicopy sequences from plant nucleus (26S rRNA gene) and chloroplast (psaB gene). The presence of Cry1A(b) protein in bovine blood samples was also tested using a sandwich ELISA kit. Our study shows the ability of plant nuclear and/or chloroplast DNA fragments to enter bovine blood circulation. However, maize nuclear DNA, both mono- and multicopy sequences, was less detected than chloroplast DNA, probably because the higher number of chloroplast copies and also possibly because nuclear DNA might be less protected by the nuclear membrane. Despite our data confirm the ability of small (ca.150 bp) plant DNA fragments to cross the intestinal barrier, we were unable to demonstrate clearly the presence of transgenic DNA or proteins in bovine blood. No sample tested positive with the two real-time PCR assays targeting transgenic sequences (35S promoter and Bt176 specific junction sequence). Only faint punctual positive results occurred randomly and were probably due to postsample collection or laboratory contamination or can be considered as artifact as they have never been confirmed. Our data highlight the difficulties to detect transgenic sequences in blood of dairy cows fed genetically modified corn (Bt176) silage. Those results show that in order to meet the consumers&#39; demand of animals fed with GM products there is currently no cost-effective analytical procedure to replace documentary traceability.

Flux bactériens et qualité sanitaire des coquillages en baie de la Fresnaye

Actes de colloques- …, 2001

... Auteur(s) / Author(s). DUPRAY E. (1) ; CAPRAIS M.-P. (1) ; DERRIEN A. (1) ; MONFORT P. (2) ; ... more

Shiga Toxin–producingEscherichia coliStrains Negative for Locus of Enterocyte Effacement

Emerging Infectious Diseases, 2009

Out of 174 bovine Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrheic c... more Out of 174 bovine Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrheic calves in Germany and Belgium, 122 strains (70.1%) were selected because of their reactivity with the eae (E. coli attaching and effacing gene) probe ECW1-ECW2. One hundred seven of these eae-positive strains (87.7%) harbored stx1 genes, 13 strains (10.7%) had stx2 genes, and 2 strains (1.6%) had both stx genes. The strains displayed 17 different O types, the majority (97 strains [79.5%]) belonging to O5 (5 strains), O26 (21 strains), O111 (13 strains) O118 (36 strains), O145 (9 strains), and O157 (13 strains). In the HEp-2 cell adhesion assay, 99 strains (81.1%) showed a localized adhesion, and 80 strains (65.6%) stimulated actin accumulation, as determined in the fluorescence actin staining test. None of the strains harbored genes coding for bundle-forming pili (bfpA), clearly differentiating them from enteropathogenic E. coli. espB gene sequences were only detectable in 23 (18.9%) of the eae-positive bovine STEC strains. Three different PCRs were established, differentiating between eae sequences of enteropathogenic E. coli strain E2348/69 (O127:H6) and STEC strain EDL933 (O157: H7). Primers matching in the more heterologous downstream eae sequences gave amplicons in only 8 of the 17 O types (O84:

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Animal Botulism Outcomes in the AniBioThreat Project

Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science, 2013

Botulism disease in both humans and animals is a worldwide concern. Botulinum neurotoxins produce... more Botulism disease in both humans and animals is a worldwide concern. Botulinum neurotoxins produced by Clostridium botulinum and other Clostridium species are the most potent biological substances known and are responsible for flaccid paralysis leading to a high mortality rate. Clostridium botulinum and botulinum neurotoxins are considered potential weapons for bioterrorism and have been included in the Australia Group List of Biological Agents. In 2010 the European Commission (DG Justice, Freedom and Security) funded a 3-year project named AniBioThreat to improve the EU's capacity to counter animal bioterrorism threats. A detection portfolio with screening methods for botulism agents and incidents was needed to improve tracking and tracing of accidental and deliberate contamination of the feed and food chain with botulinum neurotoxins and other Clostridia. The complexity of this threat required acquiring new genetic information to better understand the diversity of these Clostridia and develop detection methods targeting both highly specific genetic markers of these Clostridia and the neurotoxins they are able to produce. Several European institutes participating in the AniBioThreat project collaborated on this program to achieve these objectives. Their scientific developments are discussed here. B otulism is a severe flaccid paralysis disease caused by neurotoxins produced by the Gram-positive anaerobic spore-forming bacteria Clostridium botulinum and some strains of Clostridium baratii and Clostridium butyricum, which are distributed ubiquitously in soil and aquatic environments. 1 They are divided into 7 toxin types (A to G) depending on their antigenic properties. 2 Toxin types A, B, E, and F, produced by C. botulinum group I (proteolytic) and group II (nonproteolytic), are mainly responsible for human botulism. Toxin types C and D, produced by strains belonging to group III, are more associated with animal botulism. 3 Group IV, also referred to as C. argentinensis, has not generally been associated with illness. 4 C. baratii (Type F) and C. butyricum (Type E) can

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The Mobilome; A Major Contributor to Escherichia coli stx2-Positive O26:H11 Strains Intra-Serotype Diversity

Frontiers in Microbiology, 2017

Shiga toxin-producing Escherichia coli of serotype O26:H11/H-constitute a diverse group of strain... more Shiga toxin-producing Escherichia coli of serotype O26:H11/H-constitute a diverse group of strains and several clones with distinct genetic characteristics have been identified and characterized. Whole genome sequencing was performed using Illumina and PacBio technologies on eight stx2-positive O26:H11 strains circulating in France. Comparative analyses of the whole genome of the stx2-positive O26:H11 strains indicate that several clones of EHEC O26:H11 are co-circulating in France. Phylogenetic analysis of the French strains together with stx2-positive and stx-negative E. coli O26:H11 genomes obtained from Genbank indicates the existence of four clonal complexes (SNP-CCs) separated in two distinct lineages, one of which comprises the "new French clone" (SNP-CC1) that appears genetically closely related to stx-negative attaching and effacing E. coli (AEEC) strains. Interestingly, the whole genome SNP (wgSNP) phylogeny is summarized in the cas gene phylogeny, and a simple qPCR assay targeting the CRISPR array specific to SNP-CC1 (SP_O26-E) can distinguish between the two main lineages. The PacBio sequencing allowed a detailed analysis of the mobile genetic elements (MGEs) of the strains. Numerous MGEs were identified in each strain, including a large number of prophages and up to four large plasmids, representing overall 8.7-19.8% of the total genome size. Analysis of the prophage pool of the strains shows a considerable diversity with a complex history of recombination. Each clonal complex (SNP-CC) is characterized by a unique set of plasmids and phages, including stx-prophages, suggesting evolution through separate acquisition events. Overall, the MGEs appear to play a major role in O26:H11 intra-serotype clonal diversification.

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Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

Food Microbiology, 2009

This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright

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Passive immunisation of chicks using an autogenous vaccine when administered in breeders

Animal - science proceedings

Diagnostic PCR: making internal amplification control mandatory

Journal of applied microbiology, 2004

PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples

Applied and Environmental Microbiology, 1995

A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostrid... more A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostridium botulinum types A, B, E, F, and G, and five individual probes allowed identification of each toxinotype by hybridization of the PCR products. The 72 strains of different Clostridium species tested and 11 other bacterial species commonly found in food samples gave an amplification product. This assay was able to detect 1 C. botulinum type A or B and 10 C. botulinum type E strains per reaction. With 184 artificially contaminated food samples, after an 18-h enrichment step, the sensitivity was 10 bacteria per g of sample and the correlation with the mouse bioassay reached 95.6%.

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Procédé de détection et d'identification d'escherichia coli entérohémorragique

Detection of Genetically Modiﬁed Corn (Bt176) in Spiked Cow Blond Samples by Polymerase Chain Reaction and Immunoassay

Duplex real-time PCR detection of type III effector tccP and tccP2 genes in pathogenic Escherichia coli and prevalence in raw milk cheeses

Letters in Applied Microbiology, 2011

Aims: To develop a duplex real-time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC... more Aims: To develop a duplex real-time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP ⁄ TccP2-encoding genes which are pivotal to EHEC-mediated actin cytoskeleton reorganization in human intestinal epithelial cells. Methods and Results: The specificity of the assay was demonstrated with DNA from EHEC reference strains and nonE. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H) , O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H) , O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real-time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and ⁄ or tccP2 genes in 19AE75% of the cheese enrichment suspensions. Conclusions: A highly specific and sensitive duplex real-time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and ⁄ or tccP2. Significance and Impact of the Study: The developed real-time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.

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Micro-array for the identification of Shiga toxin-producing Escherichia coli (STEC) seropathotypes associated with Hemorrhagic Colitis and Hemolytic Uremic Syndrome in humans

International Journal of Food Microbiology, 2010

A micro-array has been developed, based on the GeneDisc ® array, for the genetic identification o... more A micro-array has been developed, based on the GeneDisc ® array, for the genetic identification of 12 O-types and 7 H-types of Shiga toxin-producing Escherichia coli (STEC) including the most clinically relevant enterohemorrhagic E. coli (EHEC) serotypes. The genes selected for determination of the O antigens (rfbE O157 , wzx O26 , wzx O103 , wbd1 O111 , ihp1 O145 , wzx O121 , wzy O113 , wzy O91 , wzx O104 , wzy O118 , wzx O45 , and wbgN O55) and H-types (fliC H2 , fliC H7 , fliC H8 , fliC H11 , fliC H19 , fliC H21 , and fliC H28) showed a high specificity and concordance with serology. The micro-array also had a high specificity for EHEC-associated virulence factors, including Shiga toxins 1 and 2 (stx1 and stx2), intimin (eae), enterohemolysin (ehxA), serine protease (espP), catalase peroxidase (katP), the type II secretion system (etpD), subtilase cytotoxin (subA), autoagglutinating adhesin (Saa) and type III secreted effectors encoded in the genomic islands OI-122 (ent/espL2, nleB, and nleE) and OI-71 (nleF, nleH1-2, and nleA). The eae gene was detected in all typical EHEC strains, and the pattern of nle genes encoded in OI-71 and OI-122 was found to be closely associated with certain serotypes of typical EHEC and emerging EHEC strains. Virulence plasmid associated genes such as katP, espP, and etpD were more common in EHEC than in STEC strains; this supports their association with virulence. This array constitutes a valuable approach for the identification of STEC strains with a high potential for human virulence.

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Revisiting the STEC Testing Approach: Using espK and espV to Make Enterohemorrhagic Escherichia coli (EHEC) Detection More Reliable in Beef

Frontiers in Microbiology, 2016

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef... more Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of "false positive" results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPR O26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPR O26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPR O26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013.

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Diagnostic PCR: making internal amplification control mandatory

Letters in Applied Microbiology, 2004

The explosive increase since the beginning of the 1990s in the number of publications reporting P... more The explosive increase since the beginning of the 1990s in the number of publications reporting PCR-based methods for detection or molecular typing of foodborne pathogens has attracted the attention of end-user laboratories. However, the well-recognized difficulties in reproducing published tests because of variation in performance of PCR thermal cyclers (Schoder et al. 2003), in efficiency of different DNA polymerases, and in the presence of PCR inhibitors in the sample matrix, have hampered implementation in end-user laboratories. This particularly applies to laboratories with quality-assurance programmes. It is necessary to have PCR-based methods available as internationally recognized standards (Hoorfar and Cook 2002). Currently, lack of international standards often forces end-user laboratories to spend substantial resources on adaptation of the published tests. Although many commercial PCR kits are available, it is important that endusers and reference laboratories have access to openformula, noncommercial and nonproprietary PCRs in which the information on target gene and reagents are fully available. The prerequisite for a PCR, published in the scientific literature, to be adopted as a standard is that it has to be nonproprietary, and to have been validated through multicentre collaborative trial according to the international criteria (Anon. 2001, 2002b; Hoorfar and Cook 2002). Multicentre trial validation of noncommercial PCRs for detection of zoonotic pathogens has been performed by a European validation and standardization project (FOOD-PCR: http://www.pcr.dk) involving 35 laboratories from 21 countries (Hoorfar 1999; Malorny et al. 2003). A major drawback of most published PCRs, surprisingly even to date, is that they do not contain an internal amplification control (IAC). An IAC is a nontarget DNA sequence present in the same sample reaction tube, which is co-amplified simultaneously with the target sequence. In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But, it could also mean that the reaction was inhibited, as a result of malfunction of thermal cycler, incorrect PCR mixture, poor polymerase activity and, not least, the presence of inhibitory substances in the sample matrix. Conversely, in a PCR with an IAC, a control signal will always be produced when there is no target sequence present. When neither IAC signal nor target signal is produced, the PCR reaction fails. Thus, when using a PCR-based method in routine analysis, an IAC, if the concentration adjusted correctly, will indicate false-negative results. It is the false-negative results that turn a risk into a threat for the population, whereas a false-positive result merely leads to a clarification of the presumptive results by re-testing the sample. The European Standardization Committee (CEN), in collaboration with International Standard Organization (ISO) has proposed a general guideline for PCR testing that requires the presence of IAC in the reaction mixture (Anon. 2002a). Therefore, only IAC-containing PCRs may undergo multicentre collaborative trials, which is a prerequisite for standardization. Scientific journals must provide the source of new PCRbased methods suitable for standardization. Therefore, we propose that henceforward the editorial boards of applied microbiology journals require inclusion of an IAC in diagnostic PCR reported in submitted manuscripts. This could be performed by providing a specific section devoted to PCR in their Instruction to Authors.

Multicenter Validation of PCR-Based Method for Detection of Salmonella in Chicken and Pig Samples

Journal of AOAC INTERNATIONAL, 2004

As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 ... more As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse and pig swab samples. The 3 levels were 1–10, 10–100, and 100–1000 colony-forming units (CFU)/100 mL. Sample preparations, including inoculation and pre-enrichment in buffered peptone water (BPW), were performed centrally in a German laboratory; the pre-PCR sample preparation (by a resin-based method) and PCR assay (gel electrophoresis detection) were performed by the receiving laboratories. Aliquots of BPW enrichment cultures were sent to the participants, who analyzed them using a thermal lysis procedure followed by a validated Salmonella-specific PCR assay. The results were reported as negative or positive. Outlier results caused, for example, by gross departures from the experimental pro...

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Shiga Toxin-Producing Serogroup O91 Escherichia coli Strains Isolated from Food and Environmental Samples

Applied and Environmental Microbiology, 2017

Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe ... more Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray and other molecular assays to examine 49 serogroup O91 strains (environmental, food, and clinical strains) for their virulence potential and phylogenetic relationships. Most of the isolates were identified to be strains of the O91:H21 and O91:H14 serotypes, with a few O91:H10 strains and one O91:H9 strain being identified. None of the strains had the eae gene, which codes for the intimin adherence protein, and many did not have some of the genetic markers that are common in other STEC strains. The genetic profiles of the strains within each serotype were similar but differed greatly between strains of different serotypes. The genetic profiles of the O91:H21 strains that we tested were identi...

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Genetic Analysis and Detection of fliCH1 and fliCH12 Genes Coding for Serologically Closely Related Flagellar Antigens in Human and Animal Pathogenic Escherichia coli

Frontiers in Microbiology, 2016

The E. coli flagellar types H1 and H12 show a high serological cross-reactivity and molecular ser... more The E. coli flagellar types H1 and H12 show a high serological cross-reactivity and molecular serotyping appears an advantageous method to establish a clear discrimination between these flagellar types. Analysis of fliC H1 and fliC H12 gene sequences showed that they were 97.5% identical at the nucleotide level. Because of this high degree of homology we developed a two-step real-time PCR detection procedure for reliable discrimination of H1 and H12 flagellar types in E. coli. In the first step, a real-time PCR assay for common detection of both fliC H1 and fliC H12 genes is used, followed in a second step by real-time PCR assays for specific detection of fliC H1 and fliC H12 , respectively. The real-time PCR for common detection of fliC H1 and fliC H12 demonstrated 100% sensitivity and specificity as it reacted with all tested E. coli H1 and H12 strains and not with any of the reference strains encoding all the other 51 flagellar antigens. The fliC H1 and fliC H12 gene specific assays detected all E. coli H1 and all E. coli H12 strains, respectively (100% sensitivity). However, both assays showed cross-reactions with some flagellar type reference strains different from H1 and H12. The real-time PCR assays developed in this study can be used in combination for the detection and identification of E. coli H1 and H12 strains isolated from different sources.

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Emerging Multidrug-Resistant Hybrid Pathotype Shiga Toxin–Producing Escherichia coli O80 and Related Strains of Clonal Complex 165, Europe

Emerging Infectious Diseases, 2018

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Emerging Multidrug-Resistant Hybrid Pathotype Shiga Toxin–Producing <i>Escherichia coli</i> O80 and Related Strains of Clonal Complex 165, Europe

Emerging Infectious Diseases, Dec 1, 2018

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Persistence of Plant DNA Sequences in the Blood of Dairy Cows Fed with Genetically Modified (Bt176) and Conventional Corn Silage

by Koenraad Duhem and P. Fach

Journal of Agricultural and Food Chemistry, 2009