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  • 1.
    Alsanius, Beatrix
    et al.
    SLU Swedish University of Agricultural Sciences, Sweden.
    Löfström, Charlotta
    RISE - Research Institutes of Sweden, Bioscience and Materials, Agrifood and Bioscience.
    Vattenrening för ökad hygien vid odling av frilandsgrönsaker och bär2017Other (Other (popular science, discussion, etc.))
    Abstract [sv]

    Under senare år har ett flertal utbrottmed magsjuka kopplats till konsumtionav grönsaker, frukt och bär. Sjukdomsframkallandebakterier och virus, såsomnorovirus, Salmonella, toxinproducerandeE. coli, Campylobacter och Listeria. kanspridas från bevattningsvatten via grö-dan till människor och orsaka sjukdom.Smittat bevattningsvatten kan därförförorena frilandsproducerade grönsakeroch bär. Det är alltås viktigt att hakontroll på bevattningsvattnets kvalitet.Dessutom är det viktigt att känna tillvilken typ av kultur som vattnet skaanvändas till, eftersom risken för vidaresmitta till människor varierar mellanolika typer av kulturer. T.ex. är det störrerisk att använda kontaminerat vatten tillkulturer som äts råa utan uppvärmninghos livsmedelsproducenten eller konsument,eftersom det då inte finns nå-gon möjlighet att avdöda de oönskademikroorganismerna i ett efterföljandesteg. Genom rätt hantering och adekvatbehandling av bevattningsvattnetkan dess hygieniska kvalitet förbättras.Ibland finns det möjlighet för odlarenatt byta vattenkälla, men då detta inte ärpraktiskt möjligt kan det kontamineradevattnet renas innan bevattning. I dettafaktablad beskrivs två grundläggandetekniker för rening av bevattningsvattenvid frilandsproduktion, nämligen fotokemi(fotokatalys, UV) och filtrering(mekanisk filtrering, långsamfiltrering).Dessa används för att minska risken försmittspridning med bevattningsvattnet.

  • 2. Andersson, M. G.
    et al.
    Tomuzia, K.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Appel, B.
    Bano, L.
    Keremidis, H.
    Knutsson, R.
    Leijon, M.
    Lövgren, S. E.
    De Medici, D.
    Menrath, A.
    Van Rotterdam, B. J.
    Wisselink, H. J.
    Barker, G. C.
    Separated by a common language: Awareness of term usage differences between languages and disciplines in biopreparedness2013In: Biosecurity and bioterrorism, ISSN 1538-7135, E-ISSN 1557-850X, Vol. 11, no SUPPL. 1, p. S276-S285Article in journal (Refereed)
    Abstract [en]

    Preparedness for bioterrorism is based on communication between people in organizations who are educated and trained in several disciplines, including law enforcement, health, and science. Various backgrounds, cultures, and vocabularies generate difficulties in understanding and interpretating terms and concepts, which may impair communication. This is especially true in emergency situations, in which the need for clarity and consistency is vital. The EU project AniBioThreat initiated methods and made a rough estimate of the terms and concepts that are crucial for an incident, and a pilot database with key terms and definitions has been constructed. Analysis of collected terms and sources has shown that many of the participating organizations use various international standards in their area of expertise. The same term often represents different concepts in the standards from different sectors, or, alternatively, different terms were used to represent the same or similar concepts. The use of conflicting terminology can be problematic for decision makers and communicators in planning and prevention or when handling an incident. Since the CBRN area has roots in multiple disciplines, each with its own evolving terminology, it may not be realistic to achieve unequivocal communication through a standardized vocabulary and joint definitions for words from common language. We suggest that a communication strategy should include awareness of alternative definitions and ontologies and the ability to talk and write without relying on the implicit knowledge underlying specialized jargon. Consequently, cross-disciplinary communication skills should be part of training of personnel in the CBRN field. In addition, a searchable repository of terms and definitions from relevant organizations and authorities would be a valuable addition to existing glossaries for improving awareness concerning bioterrorism prevention planning. © 2013, Mary Ann Liebert, Inc.

  • 3. Anniballi, F.
    et al.
    Fiore, A.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Skarin, H.
    Auricchio, B.
    Woudstra, C.
    Bano, L.
    Segerman, B.
    Koene, M.
    Båverud, V.
    Hansen, T.
    Fach, P.
    Åberg, A.T.
    Hedeland, M.
    Engvall, E. O.
    De Medici, D.
    Management of animal botulism outbreaks: From clinical suspicion to practical countermeasures to prevent or minimize outbreaks2013In: Biosecurity and bioterrorism, ISSN 1538-7135, E-ISSN 1557-850X, Vol. 11, no SUPPL. 1, p. S191-S199Article in journal (Refereed)
    Abstract [en]

    Botulism is a severe neuroparalytic disease that affects humans, all warm-blooded animals, and some fishes. The disease is caused by exposure to toxins produced by Clostridium botulinum and other botulinum toxin-producing clostridia. Botulism in animals represents a severe environmental and economic concern because of its high mortality rate. Moreover, meat or other products from affected animals entering the food chain may result in a public health problem. To this end, early diagnosis is crucial to define and apply appropriate veterinary public health measures. Clinical diagnosis is based on clinical findings eliminating other causes of neuromuscular disorders and on the absence of internal lesions observed during postmortem examination. Since clinical signs alone are often insufficient to make a definitive diagnosis, laboratory confirmation is required. Botulinum antitoxin administration and supportive therapies are used to treat sick animals. Once the diagnosis has been made, euthanasia is frequently advisable. Vaccine administration is subject to health authorities' permission, and it is restricted to a small number of animal species. Several measures can be adopted to prevent or minimize outbreaks. In this article we outline all phases of management of animal botulism outbreaks occurring in wet wild birds, poultry, cattle, horses, and fur farm animals. © 2013, Mary Ann Liebert, Inc.

  • 4.
    de Knegt, Leonardo V.
    et al.
    DTU Technical University of Denmark, Denmark.
    Pires, Sara M.
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    RISE, SP – Sveriges Tekniska Forskningsinstitut, SP Food and Bioscience, Microbiology. DTU Technical University of Denmark, Denmark.
    Sörensen, Gitte
    DTU Technical University of Denmark, Denmark.
    Pedersen, Karl
    DTU Technical University of Denmark, Denmark.
    Torpdahl, Mia
    Statens Serum Institut, Denmark.
    Nielsen, Eva M.
    Statens Serum Institut, Denmark.
    Hald, Tine
    DTU Technical University of Denmark, Denmark.
    Application of Molecular Typing Results in Source Attribution Models: The Case of Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) of Salmonella Isolates Obtained from Integrated Surveillance in Denmark2016In: Risk Analysis, ISSN 0272-4332, E-ISSN 1539-6924, Vol. 36, no 3, p. 571-588Article in journal (Refereed)
    Abstract [en]

    Salmonella is an important cause of bacterial foodborne infections in Denmark. To identify the main animal-food sources of human salmonellosis, risk managers have relied on a routine application of a microbial subtyping-based source attribution model since 1995. In 2013, multiple locus variable number tandem repeat analysis (MLVA) substituted phage typing as the subtyping method for surveillance of S. Enteritidis and S. Typhimurium isolated from animals, food, and humans in Denmark. The purpose of this study was to develop a modeling approach applying a combination of serovars, MLVA types, and antibiotic resistance profiles for the Salmonella source attribution, and assess the utility of the results for the food safety decisionmakers. Full and simplified MLVA schemes from surveillance data were tested, and model fit and consistency of results were assessed using statistical measures. We conclude that loci schemes STTR5/STTR10/STTR3 for S. Typhimurium and SE9/SE5/SE2/SE1/SE3 for S. Enteritidis can be used in microbial subtyping-based source attribution models. Based on the results, we discuss that an adjustment of the discriminatory level of the subtyping method applied often will be required to fit the purpose of the study and the available data. The issues discussed are also considered highly relevant when applying, e.g., extended multi-locus sequence typing or next-generation sequencing techniques.

  • 5.
    Fachmann, M S R
    et al.
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    RISE - Research Institutes of Sweden, Bioscience and Materials, Agrifood and Bioscience. DTU Technical University of Denmark, Denmark.
    Hoorfar, J
    DTU Technical University of Denmark, Denmark.
    Hansen, F
    DMRI Danish Technological Institute, Denmark.
    Christensen, J
    DTU Technical University of Denmark, Denmark.
    Mansdal, S
    DTI Danish Technological Institute, Denmark.
    Josefsen, M H
    DTU Technical University of Denmark, Denmark.
    Detection of Salmonella in meat in less than 5 hours by a low-cost and non-complex sample preparation method.2017In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 83, no 5, article id e03151-16Article in journal (Refereed)
    Abstract [en]

    Salmonella is recognised as one of the most important foodborne bacteria, and has a wide health and socioeconomical impact worldwide. Fresh pork meat is one of the main sources of Salmonella and efficient and fast methods for detection are therefore necessary. Current methods for Salmonella detection in fresh meat usually include >16 h of culture enrichment, in few cases < 12 h, thus requiring at least two working shifts. Here, we report a rapid (< 5 h) and high throughput method, for screening of Salmonella in samples from fresh pork meat, consisting of a 3-h enrichment in standard buffered peptone water, and a real-time PCR compatible sample preparation method, based on filtration, centrifugation, and enzymatic digestion, followed by fast cycling real-time PCR detection. The method was validated in an un-paired, comparative study against the Nordic Committee on Food Analysis (NMKL) reference culture method 187. Pork meat samples (n=140) were either artificially contaminated with Salmonella at levels: 0, 1-10 and 10-100 CFU/25 g, or naturally contaminated. Cohen's Kappa for degree of agreement between the rapid method and the reference was 0.64 and the relative accuracy, sensitivity and specificity for the rapid method were 81.4, 95.1 and 97.9 %, respectively. The limit of detection (LOD50) was 8.8 CFU/25 g for the rapid method and 7.7 CFU/25 g for the reference method. Implementation of this method will enable faster release of Salmonella low risk meat, providing savings for meat producers, and help contribute to improved food safety.

    IMPORTANCE: While the cost of analysis and hands-on time of the presented rapid method were comparable to reference culture methods, the fast product release by this method can provide the meat industry with a competitive advantage. Not only will the abattoirs save costs for work hours and cold storage; consumers as well as retailers will also benefit from fresher meat with a longer shelf life. Furthermore, the presented sample preparation might be adjusted for application in detection of other pathogenic bacteria in different sample types.

  • 6.
    Fachmann, Mette S. R.
    et al.
    DTU Technical University of Denmark, Denmark.
    Josefsen, Mathilde Hasseldam
    DTU Technical University of Denmark, Denmark.
    Hoorfar, Jeffrey
    DTU Technical University of Denmark, Denmark.
    Nielsen, Mette T.
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Cost-effective optimization of real-time PCR-based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases2015In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 119, no 5, p. 1391-1402Article in journal (Refereed)
    Abstract [en]

    Aims: The aim of this study was to cost-effectively improve detection of foodborne pathogens in PCR inhibitory samples through the use of alternative DNA polymerases. Methods and Results: Commercially available polymerases (n = 16) and PCR master mixes (n = 4) were screened on DNA purified from bacterial cells in two validated real-time PCR assays for Campylobacter and Salmonella. The five best performing (based on: limit of detection (LOD), maximum fluorescence, shape of amplification curves and amplification efficiency) were subsequently applied to meat and faecal samples. The VeriQuest qPCR master mix performed best for both meat and faecal samples (LODs of 102 and 104 CFU ml-1 in the purest and crudest DNA extractions respectively) compared with Tth (LOD = 102-103 and 105-106 CFU ml-1). AmpliTaqGold and HotMasterTaq both performed well (LOD = 102-104 CFU ml-1) with meat samples and poorly (LOD = 103-106 CFU ml-1/not detected) with faecal samples. Conclusions: Applying the VeriQuest qPCR master mix in the two tested real-time PCR assays could allow for simpler sample preparation and thus a reduction in cost. Significance and Impact of the Study: This work exemplifies a cost-effective strategy for optimizing real-time PCR-based assays. However, a DNA polymerase suitable for one assay and sample type is not necessarily optimal for other assays or sample types.

  • 7. Grønlund, H. A.
    et al.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Helleskov, J. B.
    Hoorfar, J.
    The use of infrared thermography as a novel approach for real-time validation of PCR thermocyclers2010In: Food Analytical Methods, ISSN 1936-9751, E-ISSN 1936-976X, Vol. 3, no 2, p. 116-119Article in journal (Refereed)
    Abstract [en]

    Validation of PCR thermocycler performance is crucial to obtain reliable results. In this study, infrared (IR) thermography was evaluated as a novel validation tool. After stabilisation, no significant difference in the temperatures recorded using thermography and a reference block-based system was found. By employing IR thermography, information about the length of the time until temperature stabilisation in the sample could be obtained. This study shows the potential of using IR thermography for validation of thermocyclers. © Springer Science + Business Media, LLC 2009.

  • 8. Grønlund, H.
    et al.
    Riber, L.
    Vigre, H.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Folling, L.
    Huehn, S.
    Malorny, B.
    Rådström, P.
    Rudi, K.
    Hoorfar, J.
    Microarray-based genotyping of Salmonella: Inter-laboratory evaluation of reproducibility and standardization potential2011In: International Journal of Food Microbiology, ISSN 0168-1605, E-ISSN 1879-3460, Vol. 145, no SUPPL. 1, p. S79-S85Article in journal (Refereed)
    Abstract [en]

    Bacterial food-borne infections in humans caused by Salmonella spp. are considered a crucial food safety issue. Therefore, it is important for the risk assessments of Salmonella to consider the genomic variation among different isolates in order to control pathogen-induced infections. Microarray technology is a promising diagnostic tool that provides genomic information on many genes simultaneously. However, standardization of DNA microarray analysis is needed before it can be used as a routine method for characterizing Salmonella isolates across borders and laboratories. A comparative study was designed in which the agreement of data from a DNA microarray assay used for typing Salmonella spp. between two different labs was assessed. The study was expected to reveal the possibility of obtaining the same results in different labs using different equipment in order to evaluate the reproducibility of the microarray technique as a first step towards standardization. The low-density array contains 281 57-60-mer oligonucleotide probes for detecting a wide range of specific genomic marker genes associated with antibiotic resistance, cell envelope structures, mobile genetic elements and pathogenicity. Several critical methodology parameters that differed between the two labs were identified. These related to printing facilities, choice of hybridization buffer, wash buffers used following the hybridization and choice of procedure for purifying genomic DNA. Critical parameters were randomized in a four-factorial experiment and statistical measures of inter-lab consistency and agreement were performed based on the kappa coefficient. A high level of agreement (kappa = 0.7-1.0) in microarray results was obtained even when employing different printing and hybridization facilities, different procedures for purifying genomic DNA and different wash buffers. However, less agreement (Kappa = 0.2-0.6) between microarray results were observed when using different hybridization buffers, indicating this parameter as being highly critical when transferring a standard microarray assay between laboratories. In conclusion, this study indicates that DNA microarray assays can be reproduced in at least two different facilities, which is a pre-requisite for the development of standard guidelines. © 2010 Elsevier B.V.

  • 9. Hansen, T.
    et al.
    Skånseng, B.
    Hoorfar, J.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Evaluation of Direct 16S rDNA sequencing as a metagenomics-based approach to screening bacteria in bottled water2013In: Biosecurity and bioterrorism, ISSN 1538-7135, E-ISSN 1557-850X, Vol. 11, no SUPPL. 1, p. S158-S165Article in journal (Refereed)
    Abstract [en]

    Deliberate or accidental contamination of food, feed, and water supplies poses a threat to human health worldwide. A rapid and sensitive detection technique that could replace the current labor-intensive and time-consuming culture-based methods is highly desirable. In addition to species-specific assays, such as PCR, there is a need for generic methods to screen for unknown pathogenic microorganisms in samples. This work presents a metagenomics-based direct-sequencing approach for detecting unknown microorganisms, using Bacillus cereus (as a model organism for B. anthracis) in bottled water as an example. Total DNA extraction and 16S rDNA gene sequencing were used in combination with principle component analysis and multicurve resolution to study detection level and possibility for identification. Results showed a detection level of 10 5 to 106 CFU/L. Using this method, it was possible to separate 2 B. cereus strains by the principal component plot, despite the close sequence resemblance. A linear correlation between the artificial contamination level and the relative amount of the Bacillus artificial contaminant in the metagenome was observed, and a relative amount value above 0.5 confirmed the presence of Bacillus. The analysis also revealed that background flora in the bottled water varied between the different water types that were included in the study. This method has the potential to be adapted to other biological matrices and bacterial pathogens for fast screening of unknown bacterial threats in outbreak situations. © 2013, Mary Ann Liebert, Inc.

  • 10.
    Hellmér, Maria
    et al.
    DTU Technical University of Denmark, Denmark.
    Hjelmsø, Mathis Hjort
    DTU Technical University of Denmark, Denmark.
    Fernandez-Cassi, Xavier
    University of Barcelona, Spain.
    Timoneda, Natalia
    University of Barcelona, Spain.
    Lukjancenko, Oksana
    DTU Technical University of Denmark, Denmark.
    Seidel, Michael
    Technical University of Munich, Germany.
    Aarestrup, Frank Møller
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    RISE, SP – Sveriges Tekniska Forskningsinstitut, SP Food and Bioscience. DTU Technical University of Denmark, Denmark.
    Girones, Rosina
    University of Barcelona, Spain.
    Schultz, Anna Charlotte
    DTU Technical University of Denmark, Denmark.
    Evaluation of methods for the concentration and extraction of viruses from sewage water in the context of metagenomic sequencing2016In: The Danish Microbiological Society Annual Congress 2016: Programme & Abstracts, Copenhagen, 2016, p. 76-76, article id P61Conference paper (Other academic)
  • 11.
    Hintzmann, Ann-Sofie
    et al.
    DTU Technical University of Denmark, Denmark.
    Sørensen, Gitte
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    RISE - Research Institutes of Sweden, Bioscience and Materials, Agrifood and Bioscience. DTU Technical University of Denmark, Denmark.
    Baggesen, Dorte Lau
    DTU Technical University of Denmark, Denmark.
    Whole genome sequencing data confirm transmission of Salmonella enterica serovar Typhimurium phage type DT41 in the Danish poultry production chain2017Conference paper (Other academic)
  • 12.
    Hintzmann, Ann-Sofie
    et al.
    DTU Technical University of Denmark, Denmark.
    Sørensen, Gitte
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    RISE, SP – Sveriges Tekniska Forskningsinstitut, SP Food and Bioscience. DTU Technical University of Denmark, Denmark.
    Baggesen, Dorte Lau
    DTU Technical University of Denmark, Denmark.
    Whole genome sequencing data confirm transmission of Salmonella enterica serovar Typhimurium phage type DT41 in the Danish poultry production chain2016In: Technical meeting on the Impact of Whole Genome Sequencing on food safety management: Poster abstracts, Rome, Italy: Food and Agriculture Organization of the United Nations (FAO), 2016, p. 12-12, article id 11Conference paper (Other academic)
  • 13.
    Hjelmsø, Mathis Hjort
    et al.
    DTU Technical University of Denmark, Denmark.
    Hellmér, Maria
    DTU Technical University of Denmark, Denmark.
    Fernandez-Cassi, Xavier
    University of Barcelona, Spain.
    Timoneda, Natàlia
    University of Barcelona, Spain.
    Lukjancenko, Oksana
    DTU Technical University of Denmark, Denmark.
    Seidel, Michael
    Technical University of Munich, Germany.
    Elsässer, Dennis
    Technical University of Munich, Germany.
    Aarestrup, Frank M
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    RISE - Research Institutes of Sweden, Bioscience and Materials, Agrifood and Bioscience. DTU Technical University of Denmark, Denmark.
    Bofill-Mas, Sílvia
    University of Barcelona, Spain.
    Abril, Josep F
    University of Barcelona,Spain.
    Girones, Rosina
    University of Barcelona, Spain.
    Schultz, Anna Charlotte
    DTU Technical University of Denmark, Denmark.
    Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing.2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 1Article in journal (Refereed)
    Abstract [en]

    Viral sewage metagenomics is a novel field of study used for surveillance, epidemiological studies, and evaluation of waste water treatment efficiency. In raw sewage human waste is mixed with household, industrial and drainage water, and virus particles are, therefore, only found in low concentrations. This necessitates a step of sample concentration to allow for sensitive virus detection. Additionally, viruses harbor a large diversity of both surface and genome structures, which makes universal viral genomic extraction difficult. Current studies have tackled these challenges in many different ways employing a wide range of viral concentration and extraction procedures. However, there is limited knowledge of the efficacy and inherent biases associated with these methods in respect to viral sewage metagenomics, hampering the development of this field. By the use of next generation sequencing this study aimed to evaluate the efficiency of four commonly applied viral concentrations techniques (precipitation with polyethylene glycol, organic flocculation with skim milk, monolithic adsorption filtration and glass wool filtration) and extraction methods (Nucleospin RNA XS, QIAamp Viral RNA Mini Kit, NucliSENS® miniMAG®, or PowerViral® Environmental RNA/DNA Isolation Kit) to determine the viriome in a sewage sample. We found a significant influence of concentration and extraction protocols on the detected viriome. The viral richness was largest in samples extracted with QIAamp Viral RNA Mini Kit or PowerViral® Environmental RNA/DNA Isolation Kit. Highest viral specificity were found in samples concentrated by precipitation with polyethylene glycol or extracted with Nucleospin RNA XS. Detection of viral pathogens depended on the method used. These results contribute to the understanding of method associated biases, within the field of viral sewage metagenomics, making evaluation of the current literature easier and helping with the design of future studies.

  • 14. Jakočiune, D.
    et al.
    Pasquali, F.
    Silva, C. S.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Hoorfar, J.
    Klein, G.
    Manfreda, G.
    Olsen, J. E.
    Enumeration of salmonellae in table eggs, pasteurized egg products, and egg-containing dishes by using quantitative real-time PCR2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 5, p. 1616-1622Article in journal (Refereed)
    Abstract [en]

    Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (Cq) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products. © 2014, American Society for Microbiology.

  • 15. Josefsen, M. H.
    et al.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Hansen, T. B.
    Christensen, L. S.
    Olsen, J. E.
    Hoorfar, J.
    Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time pcr and propidium monoazide treatment, as a tool for quantitative risk assessment2010In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 15, p. 5097-5104Article in journal (Refereed)
    Abstract [en]

    A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (CT.) values (R2 = 0.993), with a quantification range of 1 × 102 to 1 × 107 CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R 2 = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment. Copyright © 2010, American Society tor Microbiology. All Rights Reserved.

  • 16. Josefsen, M. H.
    et al.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Sommer, H. M.
    Hoorfar, J.
    Diagnostic PCR: Comparative sensitivity of four probe chemistries2009In: Molecular and Cellular Probes, ISSN 0890-8508, E-ISSN 1096-1194, Vol. 23, no 3-4, p. 201-203Article in journal (Refereed)
    Abstract [en]

    Three probe chemistries: locked nucleic acid (LNA), minor groove binder (MGB) and Scorpion were compared with a TaqMan probe in a validated real-time PCR assay for detection of food-borne thermotolerant Campylobacter. The LNA probe produced significantly lower Ct-values and a higher proportion of positive PCR responses analyzing less than 150 DNA copies than the TaqMan probe. Choice of probe chemistry clearly has an impact on the sensitivity of PCR assays, and should be considered in an optimization strategy.

  • 17. Karlsson, O. E.
    et al.
    Hansen, T.
    Knutsson, R.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Granberg, F.
    Berg, M.
    Metagenomic detection methods in biopreparedness outbreak scenarios2013In: Biosecurity and bioterrorism, ISSN 1538-7135, E-ISSN 1557-850X, Vol. 11, no SUPPL. 1, p. S146-S157Article in journal (Refereed)
    Abstract [en]

    In the field of diagnostic microbiology, rapid molecular methods are critically important for detecting pathogens. With rapid and accurate detection, preventive measures can be put in place early, thereby preventing loss of life and further spread of a disease. From a preparedness perspective, early detection and response are important in order to minimize the consequences. During the past 2 decades, advances in next-generation sequencing (NGS) technology have changed the playing field of molecular methods. Today, it is within reach to completely sequence the total microbiological content of a clinical sample, creating a metagenome, in a single week of laboratory work. As new technologies emerge, their dissemination and capacity building must be facilitated, and criteria for use, as well as guidelines on how to report results, must be established. This article focuses on the use of metagenomics, from sample collection to data analysis and to some extent NGS, for the detection of pathogens, the integration of the technique in outbreak response systems, and the risk-based evaluation of sample processing in routine diagnostics labs. The article covers recent advances in the field, current debate, gaps in research, and future directions. Examples of metagenomic detection, as well as possible applications of the methods, are described in various biopreparedness outbreak scenarios. © 2013, Mary Ann Liebert, Inc.

  • 18. Knutsson, R.
    et al.
    Löfström, Charlotta
    Lund University, Sweden.
    Grage, H.
    Hoorfar, J.
    Rådström, P.
    Modeling of 5′ nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica2002In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 40, no 1, p. 52-60Article in journal (Refereed)
    Abstract [en]

    The performance of a 5′ nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched Salmonella enterica cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, the threshold cycle (Cτ) and the fluorescence intensity by a normalized reporter value (ΔRn). The Cτ response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from S. enterica serovar Enteritidis was studied in double-distilled H2O (ddH2O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on AmpliTaq Gold or rTth. A descriptive model was proposed and fitted to the available experimental data. Equivalent PCR performances for the two PCR mixtures were obtained when DNA was diluted in ddH2O. However, the level of detection of DNA was affected when BPW was present during amplification. Use of the rTth mixture generated a 1-log-unit wider linear range of amplification, and the DNA detection levels were 2 × 10-12 g/microwell for the rTth mixture and 2 × 10-12 g/microwell for the AmpliTaq Gold mixture. To verify the improved amplification capacity of the rTth mixture, BPW was inoculated with 1 CFU of S. enterica serovar Enteritidis per ml and the mixture was incubated at 30°C. Samples for PCR were withdrawn every 4 h during a 36-h enrichment. Use of the rTth mixture resulted in an earlier PCR detection during enrichment than use of the AmpliTaq Gold mixture. For accurate detection (Cτ ≤ 30) of S. enterica serovar Enteritidis inoculated in BPW, the rTth mixture required 8.4 h of enrichment, while the AmpliTaq Gold mixture needed 11.6 h. In conclusion, the principle applied can improve the methodology of 5′ nuclease real-time PCR for numerical optimization of sample pretreatment strategies to provide automated diagnostic PCR procedures.

  • 19. Knutsson, R.
    et al.
    van Rotterdam, B.
    Fach, P.
    De Medici, D.
    Fricker, M.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Ågren, J.
    Segerman, B.
    Andersson, G.
    Wielinga, P.
    Fenicia, L.
    Skiby, J.
    Schultz, A. C.
    Ehling-Schulz, M.
    Accidental and deliberate microbiological contamination in the feed and food chains - How biotraceability may improve the response to bioterrorism2011In: International Journal of Food Microbiology, ISSN 0168-1605, E-ISSN 1879-3460, Vol. 145, no SUPPL. 1, p. S123-S128Article in journal (Refereed)
    Abstract [en]

    A next frontier of the global food safety agenda has to consider a broad spectrum of bio-risks, such as accidental and intentional contaminations in the food and feed chain. In this article, the background for the research needs related to biotraceability and response to bioterrorism incidents are outlined. Given the current scale of international trade any response need to be considered in an international context. Biotraceability (e.g. the ability to use downstream information to point to processes or within a particular food chain that can be identified as the source of undesirable agents) is crucial in any food-born outbreak and particular in the response to bioterrorism events. In the later case, tested and proven biotraceability improves the following: (i) international collaboration of validated tracing tools and detection methods, (ii) multi-disciplinary expertise and collaboration in the field of food microbiology and conceptual modeling of the food chain, (iii) sampling as a key step in biotracing (iv) optimized sample preparation procedures, including laboratory work in Biosafety level 3 (BSL-3) laboratories, (v) biomarker discovery for relevant tracing and tracking applications, and (vi) high-throughput sequencing using bio-informatic platforms to speed up the characterization of the biological agent. By applying biotraceability, the response phase during a bioterrorism event may be shortened and is facilitated for tracing the origin of biological agent contamination. © 2010 Elsevier B.V.

  • 20. Koyuncu, S.
    et al.
    Andersson, M. G.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Skandamis, P. N.
    Gounadaki, A.
    Zentek, J.
    Häggblom, P.
    Organic acids for control of Salmonella in different feed materials2013In: BMC Veterinary Research, ISSN 1746-6148, E-ISSN 1746-6148, Vol. 9, article id 81Article in journal (Refereed)
    Abstract [en]

    Background: Salmonella control in animal feed is important in order to protect animal and public health. Organic acids is one of the control measures used for treatment of Salmonella contaminated feed or feed ingredients. In the present study, the efficacy of formic acid (FA) and different blends of FA, propionic acid (PA) and sodium formate (SF) was investigated. Four Salmonella strains isolated from feed were assayed for their acid tolerance. Also, the effect of lower temperatures (5°C and 15°C) compared to room temperature was investigated in rape seed and soybean meal.Results: The efficacy of acid treatments varied significantly between different feed materials. The strongest reduction was seen in pelleted and compound mash feed (2.5 log10 reduction) followed by rapeseed meal (1 log10 reduction) after 5 days exposure. However, in soybean meal the acid effects were limited (less than 0.5 log10 reduction) even after several weeks' exposure. In all experiments the survival curves showed a concave shape, with a fast initial death phase followed by reduction at a slower rate during the remaining time of the experiment.No difference in Salmonella reduction was observed between FA and a blend of FA and PA, whereas a commercial blend of FA and SF (Amasil) was slightly more efficacious (0.5-1 log10 reduction) than a blend of FA and PA (Luprocid) in compound mash feed. The Salmonella Infantis strain was found to be the most acid tolerant strain followed by, S. Putten, S. Senftenberg and S. Typhimurium. The tolerance of the S. Infantis strain compared with the S. Typhimurium strain was statistically significant (p&lt;0.05). The lethal effect of FA on the S. Typhimurium strain and the S. Infantis strain was lower at 5°C and 15°C compared to room temperatures.Conclusions: Acid treatment of Salmonella in feed is a matter of reducing the number of viable bacterial cells rather than eliminating the organism. Recommendations on the use of acids for controlling Salmonella in feed should take into account the relative efficacy of acid treatment in different feed materials, the variation in acid tolerance between different Salmonella strains, and the treatment temperature. © 2013 Koyuncu et al.; licensee BioMed Central Ltd.

  • 21. Krämer, N.
    et al.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Vigre, H.
    Hoorfar, J.
    Bunge, C.
    Malorny, B.
    A novel strategy to obtain quantitative data for modelling: Combined enrichment and real-time PCR for enumeration of salmonellae from pig carcasses2011In: International Journal of Food Microbiology, ISSN 0168-1605, E-ISSN 1879-3460, Vol. 145, no SUPPL. 1, p. S86-S95Article in journal (Refereed)
    Abstract [en]

    Salmonella is a major zoonotic pathogen which causes outbreaks and sporadic cases of gastroenteritis in humans worldwide. The primary sources for Salmonella are food-producing animals such as pigs and poultry. For risk assessment and hazard analysis and critical control point (HACCP) concepts, it is essential to produce large amounts of quantitative data, which is currently not achievable with the standard cultural based methods for enumeration of Salmonella. This study presents the development of a novel strategy to enumerate low numbers of Salmonella in cork borer samples taken from pig carcasses as a first concept and proof of principle for a new sensitive and rapid quantification method based on combined enrichment and real-time PCR. The novelty of the approach is in the short pre-enrichment step, where for most bacteria, growth is in the log phase. The method consists of an 8h pre-enrichment of the cork borer sample diluted 1:10 in non-selective buffered peptone water, followed by DNA extraction, and Salmonella detection and quantification by real-time PCR. The limit of quantification was 1.4 colony forming units (CFU)/20cm2 (approximately 10g) of artificially contaminated sample with 95% confidence interval of±0.7 log CFU/sample. The precision was similar to the standard reference most probable number (MPN) method. A screening of 200 potentially naturally contaminated cork borer samples obtained over seven weeks in a slaughterhouse resulted in 25 Salmonella-positive samples. The analysis of salmonellae within these samples showed that the PCR method had a higher sensitivity for samples with a low contamination level (&lt;6.7CFU/sample), where 15 of the samples negative with the MPN method was detected with the PCR method and 5 were found to be negative by both methods. For the samples with a higher contamination level (6.7-310CFU/sample) a good agreement between the results obtained with the PCR and MPN methods was obtained. The quantitative real-time PCR method can easily be applied to other food and environmental matrices by adaptation of the pre-enrichment time and media. © 2010 Elsevier B.V.

  • 22.
    Leekitcharoenphon, Pimlapas
    et al.
    National Food Institute, Denmark.
    Srensen, Gitte
    National Food Institute, Denmark.
    Löfström, Charlotta
    RISE - Research Institutes of Sweden, Bioscience and Materials, Agrifood and Bioscience.
    Battisti, Antonio
    National Reference Laboratory for Antimicrobial Resistance, Italy.
    Szabo, Istvan
    Federal Institute for Risk Assessment, Germany.
    Wasyl, Dariusz
    National Veterinary Research Institute Department of Microbiology, Poland.
    Slowey, Rosemarie
    Food and the Marine Laboratories, Ireland.
    Zhao, Shaohua
    FDA, USA.
    Brisabois, Anne
    FDA, USA.
    Kornschober, Christian
    NRC Salmonella, Austrian Agency for Health and Food Safety, Austria.
    Kärssin, Age
    Veterinaar- ja Toidulaboratoorium, Estonia.
    Szilárd, Janosi
    Státní Veterinární Ústav Praha, Czech Republic.
    Cerny, Tomas
    Státní Veterinární Ústav Praha, Czech Republic.
    Svendsen, Christina Aaby
    National Food Institute, Denmark.
    Pedersen, Karl
    DTU Technical University of Denmark, Denmark.
    Aarestrup, Frank M.
    National Food Institute, Denmark.
    Hendriksen, Rene S.
    National Food Institute, Denmark.
    Cross-Border Transmission of Salmonella Choleraesuis var. Kunzendorf in European Pigs and Wild Boar: Infection, Genetics, and Evolution2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 00179Article in journal (Refereed)
    Abstract [en]

    Salmonella enterica subspecies enterica serotype Choleraesuis is a swine adapted serovar. S. Choleraesuis variant Kunzendorf are responsible for the majority of outbreaks among pigs. S. Choleraesuis is rare in Europe, although there have been serious outbreaks in pigs including two outbreaks in Denmark in 1999-2000 and 2012-2013. Here, we elucidate the epidemiology, possible transmission routes and sources, and clonality of European S. Choleraesuis isolates including the Danish outbreak isolates. A total of 102 S. Choleraesuis isolates from different European countries and the United States of America, covering available isolates from the last two decades were selected for whole genome sequencing. We applied a temporally structured sequence analysis within a Bayesian framework to reconstruct a temporal and spatial phylogenetic tree. MLST type, resistance genes, plasmid replicons and accessory genes were identified using bioinformatics tools. Fifty-eight isolates including 11 out of 12 strains from wild boars were pan-susceptible. The remaining isolates carried multiple resistance genes. Eleven different plasmid replicons in eight plasmids were determined among the isolates. Accessory genes were associated to the identified resistance genes and plasmids. The European S. Choleraesuis was estimated to have emerged in 1837 (95% credible interval, 1733 - 1983) with the mutation rate of 1.02 SNPs/genome/year. The isolates were clustered according to countries and neighbour countries. There were transmission events between strains from the USA and European countries. Wild boar and pig isolates were genetically linked suggesting cross-border transmission and transmission due to a wildlife reservoir. The phylogenetic tree shows that multiple introductions were responsible for the outbreak of 2012-2013 in Denmark, and suggests that poorly disinfected vehicles crossing the border into Denmark were potentially the source of the outbreak. Low levels of single nucleotide polymorphisms (SNP) differences (0-4 SNPs) can be observed between clonal strains isolated from different organs of the same animal. Proper disinfection of livestock vehicles and improved quality control of livestock feed are recommended to prevent future spread of S. Choleraesuis or other more serious infectious diseases such as African swine fever (ASF) into the European pig production system.

  • 23.
    Löfström, Charlotta
    RISE - Research Institutes of Sweden, Bioscience and Materials, Agrifood and Bioscience.
    Pre-PCR processing strategies for rapid detection of Listeria monocytogenes in food and environmental samples2017Conference paper (Other academic)
  • 24.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Hansen, F.
    Hoorfar, J.
    Validation of a 20-h real-time PCR method for screening of Salmonella in poultry faecal samples2010In: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 144, no 3-4, p. 511-514Article in journal (Refereed)
    Abstract [en]

    Efficient and rapid monitoring of Salmonella in the poultry production chain is necessary to assure safe food. The objective was to validate an open-formula real-time PCR method for screening of Salmonella in poultry faeces (sock samples). The method consists of incubation in buffered peptone water for 18 ± 2 h, centrifugation of a 1-ml subsample, DNA extraction on the pellet and PCR. The total analysis time is 20 h. The validation study included comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal). The comparative trial was performed against a reference method from the Nordic Committee on Food Analysis (NMKL187, 2007) using 132 artificially and naturally contaminated samples. The limit of detection (LOD50) was found to be 24 and 33. CFU/sample for the PCR and NMKL187 methods, respectively. The relative accuracy, relative sensitivity and relative specificity were all 100%, when including naturally contaminated samples and samples artificially contaminated with 10-100. CFU/sample. The collaborative trial included six laboratories and valid results were obtained from five of them. Apart from one of the samples that was artificially contaminated with 1-10. CFU/sample being a false negative with PCR for one of the laboratories, no false-positive or false-negative results were reported. This test supplies the growing demand for validated diagnostic PCR methods for screening of samples in the meat production chain to assure safe food. © 2010 Elsevier B.V.

  • 25.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Hansen, F.
    Mansdal, S.
    Hoorfar, J.
    Detection of salmonella in meat: Comparative and interlaboratory validation of a noncomplex and cost-effective pre-PCR protocol2012In: Journal of AOAC International, ISSN 1060-3271, E-ISSN 1944-7922, Vol. 95, no 1, p. 100-104Article in journal (Refereed)
    Abstract [en]

    Cost-effective and rapid monitoring of Salmonella in the meat production chain can contribute to food safety. The objective of this study was to validate an easy-to-use pre-PCR sample preparation method based on a simple boiling protocol for screening of Salmonella in meat and carcass swab samples using a real-time PCR method. The protocol included incubation in buffered peptone water, centrifugation of an aliquot, and a boiling procedure. The validation study included comparative and interlaboratory trials recommended by the Nordic Organization for Validation of Alternative Microbiological Methods (NordVal). The comparative trial was performed against a reference method (NMKL 187, 2007) and a PCR method previously approved by NordVal with a semiautomated magnetic bead-based DNA extraction step using 122 artificially contaminated samples. The LOD was found to be 3.0, 3.2, and 3.4 CFU/sample for the boiling, magnetic bead-based, and NMKL 187 methods, respectively. When comparing the boiling method with the magnetic beads, the relative accuracy (AC), relative sensitivity (SE), and relative specificity (SP) were 98, 102, and 98%, respectively (Cohen's kappa index 0.95). When comparing results obtained by the boiling to the culture-based method, the AC, SE, and SP were found to be 98, 102, and 98%, respectively (kappa index 0.93). In the interlaboratory trial including valid results from 11 laboratories, apart from two falsepositive samples by the boiling method combined with PCR, no deviating results were obtained (SP, SE, and AC were 100, 95, and 97%, respectively). This test is under implementation by the Danish meat industry, and can be useful for screening of large number of samples in the meat production, especially for fast release of minced meat with a short shelf life. © 2012 Publishing Technology.

  • 26.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Hansen, T.
    Maurischat, S.
    Malorny, B.
    Salmonella: Salmonellosis2015In: Encyclopedia of Food and Health, Elsevier Inc. , 2015, p. 701-705Chapter in book (Other academic)
    Abstract [en]

    Salmonella remains one of the most important zoonotic pathogenic bacteria and is the causative agents of salmonellosis. The aim of this article is to give an overview of Salmonella and salmonellosis, starting by describing the characteristics of the microorganism Salmonella, including biochemical properties, physiology, classification, and nomenclature. Thereafter, the epidemiology of the organism is introduced, including the routes of transmission. Finally, the disease salmonellosis, the virulence mechanisms, and the occurrence in different types of food are described. © 2016 Elsevier Ltd. All rights reserved.

  • 27.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Hintzmann, Ann-Sofie
    DTU Technical University of Denmark, Denmark.
    Sørensen, Gitte
    DTU Technical University of Denmark, Denmark.
    Baggesen, Dorte Lau
    DTU Technical University of Denmark, Denmark.
    Outbreak of Salmonella enterica serovar Typhimurium phage type DT41 in Danish poultry production2015In: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 178, no 1-2, p. 167-172Article in journal (Refereed)
    Abstract [en]

    Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) is one of the most prevalent serovars in Europe - where both poultry and poultry related products are common sources of human salmonellosis. Due to efficient control programs, the prevalence of S. Typhimurium in Danish poultry production is very low. Despite this, during the past decades there has been a reoccurring problem with infections with S. Typhimurium phage type DT41 in the Danish poultry production without identifying a clear source. In the end of 2013 and beginning of 2014 an increased isolation of S. Typhimurium DT41 was noted mainly in this production, but also in other samples. To investigate this is in more detail, 47 isolates from egg layers (n=. 5, 1 flock), broilers (n=. 33, 13 flocks), broiler breeding flocks and hatches (n=. 5; 2 flocks and 1 environmental hatchery sample), feed (n=. 1), poultry slaughter house (n=. 3, environmental sample and meat) were typed with multi locus variable number of tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) to investigate the epidemiology of the outbreak. Based on PFGE results isolates were divided into four groups (Simpson's index of diversity (DI). =. 0.24. ±. 0.15). Due to the low DI, PFGE was not sufficient to provide information to unravel the outbreak. Based on MLVA typing the DT41 (42/47 isolates) and the RDNC isolates (5/47) were split into nine groups (DI. =. 0.65. ±. 0.14). When a maximum divergence at one locus was permitted these could be gathered into four groups. Using this criterion, combined with epidemiological information, a spread of one type from broiler breeders to broilers and further to the poultry slaughter house was plausible. In conclusion, although it could be concluded that a spread within the broiler production pyramid had taken place the source of the sudden increase of S. Typhimurium DT41 remains unclear. To investigate this in more detail, further studies using whole genome sequencing to obtain a higher discriminatory strength and including isolates from a longer period of time and from various sources are in progress.

  • 28.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Hoorfar, J.
    Validation of an open-formula, diagnostic real-time PCR method for 20-h detection of Salmonella in animal feeds2012In: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 158, no 3-4, p. 431-435Article in journal (Refereed)
    Abstract [en]

    A comparative study of a 20-h, non-commercial, open-formula PCR method and the standard culture-based method NMKL 187, for detection of Salmonella, was performed according to the validation protocol from the Nordic organisation for validation of alternative microbiological methods (NordVal) on 81 artificially or naturally contaminated animal feed samples. The PCR method is based on culture enrichment in buffered peptone water for 16 ± 2. h followed by a magnetic beads based semi automated DNA extraction and real-time PCR analysis, including an internal amplification control. The limit of detection (LOD50) was found to be 7.19 and 7.24. CFU/sample for the PCR method and NMKL187, respectively. A very good correlation between results obtained by the two methods was found (Cohen's kappa = 0.92). The relative accuracy, relative sensitivity and relative specificity were found to be 97.5%, 102.0% and 96.6%, respectively. This method is the fastest open PCR based analysis protocol for detection of Salmonella in feed samples. Implementing rapid methods such as the one validated in this study can speed up Salmonella testing of feed for food-producing animals. © 2012 Elsevier B.V.

  • 29.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Josefsen, M. H.
    Hansen, T.
    SØndergaard, M. S. R.
    Hoorfar, J.
    Fluorescence-based real-time quantitative polymerase chain reaction (qPCR) technologies for high throughput screening of pathogens2014In: High Throughput Screening for Food Safety Assessment: Biosensor Technologies, Hyperspectral Imaging and Practical Applications, Elsevier Inc. , 2014, p. 219-248Chapter in book (Other academic)
    Abstract [en]

    Real-time quantitative polymerase chain reaction (qPCR) technology is being widely used for high throughput diagnostics of pathogens in the food production chain as an alternative to the more time-consuming and laborious culture-based standard methods. This chapter discusses the basics of qPCR, including data analysis, PCR instruments, and detection chemistries. The importance of choosing appropriate pre-PCR processing strategies - i.e., sampling, sample preparation, and PCR chemistry - to avoid PCR inhibition and achieve correct quantification is furthermore included. Major novel trends such as portable PCR instruments for in-field diagnostics, high-resolution melting curve analysis, and digital PCR are addressed. Finally, some perspectives on future trends are given. © 2015 Elsevier Ltd. All rights reserved.

  • 30.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Josefsen, M. H.
    Hoorfar, J.
    Hansen, F.
    Boosting exports of fresh meat by using faster laboratory methods in Denmark2012In: Case Studies in Food Safety and Authenticity: Lessons from Real-Life Situations, Elsevier Ltd , 2012, p. 267-275Chapter in book (Other academic)
    Abstract [en]

    As Denmark is one of the largest pork producers worldwide, exports of pork meat play an important role in the Danish economy. During the slaughter process fresh meat may become contaminated with enteric pathogens that can pose a risk to public health. Certain countries have strict legislative demands when importing meat, especially fresh minced meat due to its very short shelf-life. This case study describes the successful development and implementation of a rapid Salmonella detection method at the largest slaughterhouses in Denmark. © 2012 Woodhead Publishing Limited All rights reserved.

  • 31.
    Löfström, Charlotta
    et al.
    Lund University, Sweden.
    Knutsson, R.
    Axelsson, C. E.
    Rådström, P.
    Rapid and Specific Detection of Salmonella spp. in Animal Feed Samples by PCR after Culture Enrichment2004In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 1, p. 69-75Article in journal (Refereed)
    Abstract [en]

    A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting 1 CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain.

  • 32.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Krause, M.
    Josefsen, M. H.
    Hansen, F.
    Hoorfar, J.
    Validation of a same-day real-time PCR method for screening of meat and carcass swabs for Salmonella2009In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 9, article id 85Article in journal (Refereed)
    Abstract [en]

    Background. One of the major sources of human Salmonella infections is meat. Therefore, efficient and rapid monitoring of Salmonella in the meat production chain is necessary. Validation of alternative methods is needed to prove that the performance is equal to established methods. Very few of the published PCR methods for Salmonella have been validated in collaborative studies. This study describes a validation including comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal) of a same-day, non-commercial real-time PCR method for detection of Salmonella in meat and carcass swabs. Results. The comparative trial was performed against a reference method (NMKL-71:5, 1999) using artificially and naturally contaminated samples (60 minced veal and pork meat samples, 60 poultry neck-skins, and 120 pig carcass swabs). The relative accuracy was 99%, relative detection level 100%, relative sensitivity 103% and relative specificity 100%. The collaborative trial included six laboratories testing minced meat, poultry neck-skins, and carcass swabs as un-inoculated samples and samples artificially contaminated with 110 CFU/25 g, and 10100 CFU/25 g. Valid results were obtained from five of the laboratories and used for the statistical analysis. Apart from one of the non-inoculated samples being false positive with PCR for one of the laboratories, no false positive or false negative results were reported. Partly based on results obtained in this study, the method has obtained NordVal approval for analysis of Salmonella in meat and carcass swabs. The PCR method was transferred to a production laboratory and the performance was compared with the BAX Salmonella test on 39 pork samples artificially contaminated with Salmonella. There was no significant difference in the results obtained by the two methods. Conclusion. The real-time PCR method for detection of Salmonella in meat and carcass swabs was validated in comparative and collaborative trials according to NordVal recommendations. The PCR method was found to perform well. The test is currently being implemented for screening of several hundred thousand samples per year at a number of major Danish slaughterhouses to shorten the post-slaughter storage time and facilitate the swift export of fresh meat. © 2009 Löfström et al; licensee BioMed Central Ltd.

  • 33.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Schelin, J.
    Norling, B.
    Vigre, H.
    Hoorfar, J.
    Rådström, P.
    Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR2011In: International Journal of Food Microbiology, ISSN 0168-1605, E-ISSN 1879-3460, Vol. 145, no SUPPL. 1, p. S103-S109Article in journal (Refereed)
    Abstract [en]

    To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant density of two Salmonella strains in different growth conditions was determined to be 1.065-1.092g/ml. Based on these data, an optimal discontinuous flotation with three different density layers, ~1.200, 1.102 and 1.055g/ml, was designed for extracting intact Salmonella cells from pig carcass swabs. The method allowed accurate quantification from 4.4×102 to at least 2.2×107CFU Salmonella per swab sample using qPCR (without preceding DNA extraction) or selective plating on xylose lysine deoxycholate agar. Samples with 50CFU could be detected occasionally but fell outside the linear range of the standard curve. The swab samples showed a broad biological diversity; for seven samples not inoculated with Salmonella, the microbial background flora (BGF) was determined to 5.0±2.2 log CFU/ml sample withdrawn after flotation. It was determined that the proceeding PCR step was inhibited by BGF concentrations of ≥6.1×108CFU/swab sample, but not by concentrations ≤6.1×106CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture-independent quantification method considerably. © 2010 Elsevier B.V.

  • 34.
    Mateva, Gergana
    et al.
    National Diagnostic Research Veterinary Institute, Bulgaria.
    Pedersen, Karl
    DTU Technical University of Denmark, Denmark.
    Sørensen, Gitte
    DTU Technical University of Denmark, Denmark.
    Asseva, Galina
    National Center of Infectious and Parasitic Diseases, Bulgaria.
    Daskalov, Hristo
    National Diagnostic Research Veterinary Institute, Bulgaria.
    Petrov, Petar
    National Center of Infectious and Parasitic Diseases, Bulgaria.
    Kantardjiev, Todor
    National Center of Infectious and Parasitic Diseases, Bulgaria.
    Alexandar, Irina
    Bulgarian Academy of Sciences, Bulgaria.
    Löfström, Charlotta
    RISE - Research Institutes of Sweden, Bioscience and Materials, Agrifood and Bioscience. DTU Technical University of Denmark, Denmark.
    Use of multiple-locusvariable-numberof tandem repeatsanalysis (MLVA) to investigate genetic diversity of Salmonellaenterica subsp. enterica serovar Typhimurium isolates fromhuman, food, and veterinary sources2018In: MicrobiologyOpen, ISSN 2045-8827, E-ISSN 2045-8827, Vol. 7, no 1, article id e00528Article in journal (Refereed)
    Abstract [en]

    Salmonella enterica subspecies enterica serovar Typhimurium is the most common zoonotic pathogen in Bulgaria. To allow efficient outbreak investigations and surveillance in the food chain, accurate and discriminatory methods for typing are needed. This study evaluated the use of multiple-locus variable-number of tandem repeats analysis (MLVA) and compared results with antimicrobial resistance (AMR) determinations for 100 S. Typhimurium strains isolated in Bulgaria during 2008–2012 (50 veterinary/ food and 50 human isolates). Results showed that isolates were divided into 80 and 34 groups using MLVA and AMR, respectively. Simpson’s index of diversity was determined to 0.994 ± 0.003 and 0.945 ± 0.012. The most frequently encountered MLVA profiles were 3-11-9-NA-211 (n = 5); 3-12-9-NA-211 (n = 3); 3-12-11-21-311 (n = 3); 3-17-10-NA-311 (n = 3); 2-20-9-7-212 (n = 3); and 2-23-NA-NA-111 (n = 3). No clustering of isolates related to susceptibility/resistance to antimicrobials, source of isolation, or year of isolation was observed. Some MLVA types were found in both human and veterinary/food isolates, indicating a possible route of transmission. A majority (83%) of the isolates were found to be resistant against at least one antimicrobial and 44% against ≥4 antimicrobials. Further studies are needed to verify MLVA usefulness over a longer period of time and with more isolates, including outbreak strains.

  • 35.
    Mogren, Lars
    et al.
    SLU Swedish University of Agricultural Sciences, Sweden.
    Löfström, Charlotta
    RISE - Research Institutes of Sweden, Bioscience and Materials, Agrifood and Bioscience.
    Alsanius, Beatrix
    SLU Swedish University of Agricultural Sciences, Sweden.
    Håll bevattningsrören rena2017Other (Other (popular science, discussion, etc.))
  • 36.
    Pedersen, Karl
    et al.
    DTU Technical University of Denmark, Denmark.
    Sørensen, Gitte
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Leekitcharoenphon, Pimlapas
    DTU Technical University of Denmark, Denmark.
    Nielsen, Bent
    Danish Agriculture and Food Council, Denmark.
    Wingstrand, Anne
    DTU Technical University of Denmark, Denmark.
    Aarestrup, Frank M.
    DTU Technical University of Denmark, Denmark.
    Hendriksen, René S.
    DTU Technical University of Denmark, Denmark.
    Baggesen, Dorte Lau
    DTU Technical University of Denmark, Denmark.
    Reappearance of Salmonella serovar Choleraesuis var. Kunzendorf in Danish pig herds2015In: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 176, no 3-4, p. 282-291Article in journal (Refereed)
    Abstract [en]

    Salmonella enterica serovar Choleraesuis is a porcine adapted serovar which may cause serious outbreaks in pigs. Here we describe outbreaks of salmonellosis due to S. Choleraesuis in four Danish pig farms in 2012-2013 by clinic, serology, and microbiology and compare the isolates to those of a previous outbreak in 1999-2000. The infection was in some herds associated with high mortality and a moderate to high sero-prevalence was found. In 2012-2013 the disease contributed to increased mortality but occurred concomitant with other disease problems in the herds, which likely delayed the diagnosis by up to several months. Nine isolates from the four farms in 2012-2013 and 14 isolates obtained from the outbreak in Denmark in 1999-2000 were subjected to typing using pulsed-field gel electrophoresis (PFGE). Seven isolates were selected for whole genome sequencing (WGS). The PFGE results of 23 isolates displayed five different profiles. The isolates from 2012 to 2013 revealed two distinct profiles, both different from the isolates recovered in 1999-2000. Two of the 2012-2013 farms shared PFGE profiles and had also transported pigs between them. The profile found in the two other 2012-2013 farms was indistinguishable but no epidemiological connection between these farms was found. Analysis of the number of single nucleotide polymorphisms (SNPs) from the WGS data indicated that the isolates from the farms in 2012-2013 were more closely related to each other than to isolates from the outbreak in 1999. It was therefore concluded that the infection was a new introduction and not a persistent infection since the outbreak in 1999. It may further be suggested that there were two or three independent rather than a single introduction. The re-introduction of S. Choleraesuis in Denmark emphasizes the importance of strict hygiene measures in the herds. Further investigations using WGS are now in progress on a larger collection of isolates to study clonality at European level and trace the origin of the infections.

  • 37. Pin, C.
    et al.
    Hansen, T.
    Muñoz-Cuevas, M.
    de Jonge, R.
    Rosenkrantz, J. T.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Aarts, H.
    Olsen, J. E.
    The Transcriptional Heat Shock Response of Salmonella Typhimurium Shows Hysteresis and Heated Cells Show Increased Resistance to Heat and Acid Stress2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 12, article id e51196Article in journal (Refereed)
    Abstract [en]

    We investigated if the transcriptional response of Salmonella Typhimurium to temperature and acid variations was hysteretic, i.e. whether the transcriptional regulation caused by environmental stimuli showed memory and remained after the stimuli ceased. The transcriptional activity of non-replicating stationary phase cells of S. Typhimurium caused by the exposure to 45°C and to pH 5 for 30 min was monitored by microarray hybridizations at the end of the treatment period as well as immediately and 30 minutes after conditions were set back to their initial values, 25°C and pH 7. One hundred and two out of 120 up-regulated genes during the heat shock remained up-regulated 30 minutes after the temperature was set back to 25°C, while only 86 out of 293 down regulated genes remained down regulated 30 minutes after the heat shock ceased. Thus, the majority of the induced genes exhibited hysteresis, i.e., they remained up-regulated after the environmental stress ceased. At 25°C the transcriptional regulation of genes encoding for heat shock proteins was determined by the previous environment. Gene networks constructed with up-regulated genes were significantly more modular than those of down-regulated genes, implying that down-regulation was significantly less synchronized than up-regulation. The hysteretic transcriptional response to heat shock was accompanied by higher resistance to inactivation at 50°C as well as cross-resistance to inactivation at pH 3; however, growth rates and lag times at 43°C and at pH 4.5 were not affected. The exposure to pH 5 only caused up-regulation of 12 genes and this response was neither hysteretic nor accompanied of increased resistance to inactivation conditions. Cellular memory at the transcriptional level may represent a mechanism of adaptation to the environment and a deterministic source of variability in gene regulation. © 2012 Pin et al.

  • 38. Rådström, P.
    et al.
    Knutsson, R.
    Wolffs, P.
    Dahlenborg, M.
    Löfström, Charlotta
    Lund University, Sweden.
    Pre-PCR processing of samples.2003In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 216, p. 31-50Article in journal (Refereed)
  • 39. Rådström, P.
    et al.
    Knutsson, R.
    Wolffs, P.
    Lövenklev, M.
    Löfström, Charlotta
    Lund University, Sweden.
    Pre-PCR processing: Strategies to generate PCR-compatible samples2004In: Molecular Biotechnology, ISSN 1073-6085, E-ISSN 1559-0305, Vol. 26, no 2, p. 133-146Article in journal (Refereed)
    Abstract [en]

    Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

  • 40. Rådström, P.
    et al.
    Löfström, Charlotta
    Lövenklev, M.
    Knutsson, R.
    Wolffs, P.
    Strategies for overcoming PCR inhibition2008In: Cold Spring Harbor Protocols, ISSN 1940-3402, E-ISSN 1559-6095, Vol. 3, no 3Article in journal (Refereed)
    Abstract [en]

    To design a reliable and sensitive conventional or real-time PCR method, it is crucial to select the optimal DNA polymerase(s) and/or amplification facilitator(s) for the chemicals present in the samples during amplification. PCR optimization currentlytends to focus on primer design, buffer, and thermocycling conditions to obtain specific PCR products. The amplification mixture canbe modified by the addition of PCR facilitators or by the choice of the appropriate polymerase to improve proofreading activity, PCR yield, length of amplicon, etc. Modifications can also be made to suit specific applications, such as cloning of PCR products, in vitro mutagenesis, in situ PCP, multiplex PCR, PCR ELISA, or reverse-transcription PCR, However, less effort has been devoted to overcoming the effects of PCR-inhibitory compounds. Ideally, the number of steps required to generate PCR samples should be minimized. The use of appropriate DNA polymerases and amplification facilitators, in combination with optimized sampling techniques reduces the amount of sample handling involved in the analysis. As new PCR technology develops, research inpre-PCR processing is likely to expand in response to the growing demand for rapid, robust, and simple PCR protocols. A future challenge in pre-PCR processing strategies is to design PCR protocols that integrate sampling and DNA amplification in anautomated manner. Furthermore, to monitor PCR performance in the presence of biological compounds, mathematical models are required for the objective interpretation of PCR results. Once the validation parameters of the PCR assay of interest have been studied, the specific sample matrix can be characterized with respect to VCR interference. After obtaining this information, pre-PCR processing strategies can be designed to optimize the protocol for a specific sample. Copyright © 2008 by Cold Spring Harbor Laboratory Press.

  • 41. Rådström, P.
    et al.
    Lövenklev, M.
    Wolffs, P.
    Löfström, Charlotta
    AnalyCen Nordic AB, Sweden.
    Knutsson, R.
    Pre-PCR processing strategies2003In: PCR Technology: Current Innovations, Second Edition, CRC Press , 2003, p. 37-47Chapter in book (Other academic)
    Abstract [en]

    PCR is particularly valuable for monitoring gene expression, quantifying food-borne pathogens, testing viral load, and also for clinical diagnosis. However, although it can be extremely effective with pure solutions of nucleic acids, its usefulness is limited, in part, by the presence of inhibitory substances. Originating from the samples or from DNA extraction protocols, these can reduce or even block amplification. Although many biological samples have been reported to inhibit PCR amplification, the biochemical and physical mechanisms and identities of many inhibitors remain unclear. © 2004 by CRC Press LLC.

  • 42. Schelin, J.
    et al.
    Andersson, G.
    Vigre, H.
    Norling, B.
    Häggblom, P.
    Hoorfar, J.
    Rådström, P.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Evaluation of pre-PCR processing approaches for enumeration of Salmonella enterica in naturally contaminated animal feed2014In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 116, no 1, p. 167-178Article in journal (Refereed)
    Abstract [en]

    Aims: Three pre-PCR processing strategies for the detection and/or quantification of Salmonella in naturally contaminated soya bean meal were evaluated. Methods and Results: Methods included: (i) flotation-qPCR [enumeration of intact Salmonella cells prior to quantitative PCR (qPCR)], (ii) MPN-PCR (modified most probable number method combined with qPCR) and (iii) qualitative culture enrichment PCR. The limit of quantification was 1·8 × 102 CFU g-1 (flotation-qPCR) and 0·02 MPN g-1 (MPN-PCR). Fifteen naturally contaminated Salmonella positive soya bean meal samples from one lot were analysed in parallel with the three methods, using 2·5, 50 and 25 g of feed, respectively, resulting in detection of Salmonella in 6, 15 and 9 bags. Enumeration resulted in 1·8 × 102-7·8 × 103 CFU g-1 (flotation-qPCR) and 0·024 to &gt;5·2 MPN g-1 (MPN-PCR). Conclusions: Except for differences in methodology, results obtained with the three techniques could be due to the presence of nonculturable Salmonella and/or a heterogeneous distribution of Salmonella in the material. Significance and Impact of the Study: The evaluated methods provide different possibilities to assess the prevalence of Salmonella in feed, together with the numbers of culturable, as well as nonculturable cells, and can be applied to generate data to allow more accurate quantitative microbial risk assessment for Salmonella in the feed chain. © 2013 The Society for Applied Microbiology.

  • 43. Skarin, H.
    et al.
    Tevell Åberg, A.
    Woudstra, C.
    Hansen, T.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Koene, M.
    Bano, L.
    Hedeland, M.
    Anniballi, F.
    De Medici, D.
    Olsson Engvall, E.
    The workshop on animal botulism in europe2013In: Biosecurity and bioterrorism, ISSN 1538-7135, E-ISSN 1557-850X, Vol. 11, no SUPPL. 1, p. S183-S190Article in journal (Refereed)
    Abstract [en]

    A workshop on animal botulism was held in Uppsala, Sweden, in June 2012. Its purpose was to explore the current status of the disease in Europe by gathering the European experts in animal botulism and to raise awareness of the disease among veterinarians and others involved in biopreparedness. Animal botulism is underreported and underdiagnosed, but an increasing number of reports, as well as the information gathered from this workshop, show that it is an emerging problem in Europe. The workshop was divided into 4 sessions: animal botulism in Europe, the bacteria behind the disease, detection and diagnostics, and European collaboration and surveillance. An electronic survey was conducted before the workshop to identify the 3 most needed discussion points, which were: prevention, preparedness and outbreak response; detection and diagnostics; and European collaboration and surveillance. The main conclusions drawn from these discussions were that there is an urgent need to replace the mouse bioassay for botulinum toxin detection with an in vitro test and that there is a need for a European network to function as a reference laboratory, which could also organize a European supply of botulinum antitoxin and vaccines. The foundation of such a network was discussed, and the proposals are presented here along with the outcome of discussions and a summary of the workshop itself. © 2013, Mary Ann Liebert, Inc.

  • 44. Søndergaard, M. S. R.
    et al.
    Josefsen, M. H.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Christensen, L. S.
    Wieczorek, K.
    Osek, J.
    Hoorfar, J.
    Low-cost monitoring of Campylobacter in poultry houses by air sampling and quantitative PCR2014In: Journal of Food Protection, ISSN 0362-028X, E-ISSN 1944-9097, Vol. 77, no 2, p. 325-330Article in journal (Refereed)
    Abstract [en]

    The present study describes the evaluation of a method for the quantification of Campylobacter by air sampling in poultry houses. Sampling was carried out in conventional chicken houses in Poland, in addition to a preliminary sampling in Denmark. Each measurement consisted of three air samples, two standard boot swab fecal samples, and one airborne particle count. Sampling was conducted over an 8-week period in three flocks, assessing the presence and levels of Campylobacter in boot swabs and air samples using quantitative real-time PCR. The detection limit for air sampling was approximately 100 Campylobacter cell equivalents (CCE)/m3. Airborne particle counts were used to analyze the size distribution of airborne particles (0.3 to 10 mm) in the chicken houses in relation to the level of airborne Campylobacter. No correlation was found. Using air sampling, Campylobacter was detected in the flocks right away, while boot swab samples were positive after 2 weeks. All samples collected were positive for Campylobacter from week 2 through the rest of the rearing period for both sampling techniques, although levels 1- to 2-log CCE higher were found with air sampling. At week 8, the levels were approximately 104 and 105 CCE per sample for boot swabs and air, respectively. In conclusion, using air samples combined with quantitative realtime PCR, Campylobacter contamination could be detected earlier than by boot swabs and was found to be a more convenient technique for monitoring and/or to obtain enumeration data useful for quantitative risk assessment of Campylobacter. © International Association for Food Protection.

  • 45. Thierry, S.
    et al.
    Hamidjaja, R. A.
    Girault, G.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Ruuls, R.
    Sylviane, D.
    A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis2013In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 95, no 3, p. 357-365Article in journal (Refereed)
    Abstract [en]

    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® 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. © 2013 Elsevier B.V.

  • 46. van Hoek, A. H. A. M.
    et al.
    de Jonge, R.
    van Overbeek, W. M.
    Bouw, E.
    Pielaat, A.
    Smid, J. H.
    Malorny, B.
    DTU Technical University of Denmark, Denmark.
    Junker, E.
    Löfström, Charlotta
    Pedersen, K.
    Aarts, H. J. M.
    Heres, L.
    A quantitative approach towards a better understanding of the dynamics of Salmonella spp. in a pork slaughter-line2012In: International Journal of Food Microbiology, ISSN 0168-1605, E-ISSN 1879-3460, Vol. 153, no 1-2, p. 45-52Article in journal (Refereed)
    Abstract [en]

    Pork contributes significantly to the public health disease burden caused by Salmonella infections. During the slaughter process pig carcasses can become contaminated with Salmonella. Contamination at the slaughter-line is initiated by pigs carrying Salmonella on their skin or in their faeces. Another contamination route could be resident flora present on the slaughter equipment. To unravel the contribution of these two potential sources of Salmonella a quantitative study was conducted. Process equipment (belly openers and carcass splitters), faeces and carcasses (skin and cutting surfaces) along the slaughter-line were sampled at 11 sampling days spanning a period of 4. months.Most samples taken directly after killing were positive for Salmonella. On 96.6% of the skin samples Salmonella was identified, whereas a lower number of animals tested positive in their rectum (62.5%). The prevalence of Salmonella clearly declined on the carcasses at the re-work station, either on the cut section or on the skin of the carcass or both (35.9%). Throughout the sampling period of the slaughter-line the total number of Salmonella per animal was almost 2log lower at the re-work station in comparison to directly after slaughter.Seven different serovars were identified during the study with S. Derby (41%) and S. Typhimurium (29%) as the most prominent types. A recurring S. Rissen contamination of one of the carcass splitters indicated the presence of an endemic 'house flora' in the slaughterhouse studied. On many instances several serotypes per individual sample were found.The enumeration of Salmonella and the genotyping data gave unique insight in the dynamics of transmission of this pathogen in a slaughter-line. The data of the presented study support the hypothesis that resident flora on slaughter equipment was a relevant source for contamination of pork. © 2011 Elsevier B.V.

  • 47. Woudstra, C.
    et al.
    Skarin, H.
    Anniballi, F.
    Auricchio, B.
    De Medici, D.
    Bano, L.
    Drigo, I.
    Hansen, T.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Hamidjaja, R.
    van Rotterdam, B. J.
    Koene, M.
    Bäyon-Auboyer, M. -H
    Buffereau, J. -P
    Fach, P.
    Validation of a real-time PCR based method for detection of clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial2013In: Anaerobe, ISSN 1075-9964, E-ISSN 1095-8274, Vol. 22, p. 31-37Article in journal (Refereed)
    Abstract [en]

    Two real-time PCR arrays based on the GeneDisc® cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc® arrays developed as part of the DG Home funded European project 'AnibioThreat' were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%-100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C.botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C.botulinum C and D that have been evaluated in a European multicenter collaborative trial. © 2013 Elsevier Ltd.

  • 48. Woudstra, C.
    et al.
    Tevell Åberg, A.
    Skarin, H.
    Anniballi, F.
    De Medici, D.
    Bano, L.
    Koene, M.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Hansen, T.
    Hedeland, M.
    Fach, P.
    Animal botulism outcomes in the ani bio threat project2013In: Biosecurity and bioterrorism, ISSN 1538-7135, E-ISSN 1557-850X, Vol. 11, no SUPPL. 1, p. S177-S182Article in journal (Refereed)
    Abstract [en]

    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. © 2013, Mary Ann Liebert, Inc.

  • 49. Ågren, J.
    et al.
    Hamidjaja, R. A.
    Hansen, T.
    Ruuls, R.
    Thierry, S.
    Vigre, H.
    Janse, I.
    Sundström, A.
    Segerman, B.
    Koene, M.
    Löfström, Charlotta
    Wageningen University, Netherlands.
    Van Rotterdam, B.
    Derzelle, S.
    In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences2013In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 4, no 8Article in journal (Refereed)
    Abstract [en]

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays. © 2013 Landes Bioscience.

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