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.

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.

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.

En modell för biologisk kontamination av dricksvattensystem är utvecklad som ett komplement till kommersiella mjukvaror. Modellen benämns Biofilm contamination model (BioCon), vilken utgörs av delmodeller för transport och biofilmer. De dominerande processerna beskrivs genom ett system av partiella differentialekvationer. BioCon inkluderar även en numerisk lösare för de biofysikaliska modellerna och visualisering av den tidsupplösta kontaminationen i systemet. BioCon är utformad för att kunna hantera många olika biologiska agens och miljöparametrar genom parametrisering av de styrande ekvationerna. De två patogenerna Escherichia coli och Cryptosporidium spp. utgör pilotagens i denna rapport och relevanta egenskaper hos dem diskuteras. Förslag på parametrisering ges för E. coli baserad främst på publicerad litteratur men också till del från preliminär data från laboratorieexperiment genomförda under detta projekt. Modellstrukturen av de viktigaste komponenterna såsom transport, inbindning/dissociation, utveckling av biofilm, och påverkan av temperatur, desinfektionsmedel och näringsämnen, är utformade för att ge upphov till beteenden som överensstämmer med rapporterade experimentell data och teoretiska modeller. Huvuddragen i BioCon har på detta sätt blivit verifierade med avseende på nuvarande kunskapsläge, vilket presenteras i denna rapport. Däremot har en omfattande och kvantitativ validering av BioCon, genom jämförelse med experimentell data för samma system av agens, miljöfaktorer och flöden, ej kunnat genomföras inom ramen för detta projekt. Detta rekommenderas som en framtida studie.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Background:Salmonellosis is one of the most frequent food-borne infections. It iscaused by contaminated food mainly of animal origin, although humanto human transmission and numerous environmental contaminationsmay also be inflicted. Between 30 and 60% of the reported food-borneoutbreaks in the EU are caused by Salmonella and this is the secondmost commonly reported food-borne infection causing gastroenteritis.Salmonella enterica serovar Enteritidis (S. Enteritidis) is the mostwidespread serovar in Europe and other parts of the world.

Methods:In this study we analysed forty-nine S. Enteritidis isolates from veterinaryand food sources in Bulgaria obtained during the period of eight years.We used multiple-locus variable-number of tandem repeats analysis(MLVA) genotyping and the classical antimicrobial resistance (AMR) discdiffusion method to test the diversity of the isolates.

Results:Results showed that isolates were divided into twenty-four MLVA andnine AMR profiles. The calculated Simpsons diversity index was 0.956for MLVA and 0.754 for AMR, respectively. The AMR testing revealed that47% of the isolates were resistant to one and 4% to ≥4 antimicrobials.The most frequent resistotypes were resistance to sulphonamides (n=21)and sensitive to all compounds (n=9). The most frequent MLVA profileswere 3-5-3-3-11 (n=6); 5-13-2-3-11 (n=5); 5-9-2-3-8 (n=5); 6-12-2-3-11(n=4); 5-10-2-3-11 (n=3); 4-5-3-3-9 (n=3). MLVA profiles are presentedaccording to the order of the loci sequenced: SE1, SE2, SE9, SE3 and SE5.We searched for similar S. Enteritidis MLVA profiles in published data. Apartial match was found for some profiles only.

Conclusions:It could be concluded that the MLVA profiles of S. Enteritidis obtained inthis study (the first ones for Bulgaria) have not been frequently isolatedin other counties.

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.

Salmonellosis is one of the most frequent food-borne infections. It is caused by infected food mainly of animal origin, although human to human transmission and numerous environmental contaminations may also be inflicted. Salmonella enterica serovar Enteritidis (S. Enteritidis) is a common pathogen worldwide. In this study 49 S. Enteritidis isolates from veterinary and food sources in Bulgaria obtained during the years 2004 to 2012 were analysed. The multiple-locus variable-number of tandem repeats analysis (MLVA) genotyping and the antimicrobial resistance (AMR) to compare the isolates were used. Results showed that isolates were divided into twenty-four MLVA and nine AMR profiles. The calculated Simpson's diversity index was 0.956 for MLVA and 0.693 to be changed for AMR, respectively. The most frequent MLVA profiles presented according to the order of the loci sequenced SENTR7 - SENTR5 - SENTR6 - SENTR4 - SE-3 were: 3-11-5-3-3 (n = 6); 2-8-9-5-3 (n = 5); 2-11-13-5-3 (n = 5); 2-11-12-6-3 (n = 4); 2-11-10-5-3 (n = 3); 3- 9-5-4-3 (n = 3). The AMR revealed that 53.1% of the isolates were resistant to one and 4.1% to ≥ 4 antimicrobials. The MLVA profiles obtained in this study were compared to the published data and they have not been isolated on a frequent basis. A partial match was found for isolates in Belgium, Thailand, China, and USA only.

The hospital environment represents an important mediator for the transmission of healthcare-associated infections through direct and indirect hand contact with hard surfaces and textiles. In this study, bacteria on high-touch sites, including textiles and hard surfaces in two care wards in Sweden, were identified using microbiological culture methods and 16S rDNA sequencing. During a cross-sectional study, 176 high-touch hard surfaces and textiles were identified and further analysed using microbiological culture for quantification of total aerobic bacteria, Staphylococcus aureus, Clostridium difficile and Enterobacteriacae. The bacterial population structures were further analysed in 26 samples using 16S rDNA sequencing. The study showed a higher frequency of unique direct hand-textile contacts (36 per hour), compared to hard surfaces (2.2 per hour). Hard surfaces met the recommended standard of ≤ 5 CFU/cm2 for aerobic bacteria and ≤ 1 CFU/cm2 for S. aureus (53% and 35%, respectively) to a higher extent compared to textiles (19% and 30%, respectively) (P = 0.0488). The number of bacterial genera was higher on textiles than on the hard surfaces. Staphylococcus (30.4%) and Corynebacterium (10.9%) were the most representative genera for textiles and Streptococcus (13.3%) for hard surfaces. The fact that a big percentage of the textiles did not fulfil the criteria for cleanliness, combined with the higher bacterial diversity, compared to hard surfaces, are indicators that textiles were bacterial reservoirs and potential risk vectors for bacterial transmission. However, since most of the bacteria found in the study belonged to the normal flora, it was not possible to draw conclusions of textiles and hard surfaces as sources of healthcare associated infections. 2023 Nygren et al.

Detta projekt har utforskat möjligheter att använda sidoströmmar; broccoliblad och stjälk, från broccoliproduktion till nya torkade livsmedelsingredienser. Skördehantering, skördeekonomi, lämpliga processtekniker, livscykelanalys, mikrobiologisk analys och riskbedömning, analys av näringsämnen och bioaktiva ämnen, sensorik och marknadsaspekter har undersökts.

Hälsosamma livsmedel är idag något som många konsumenter prioriterar när de väljer mat till måltider, och intresset är också stort för färdiga rätter. Detta projekt initierades för att öka intresset för konsumtion av broccoli, en produkt med bra hälsovärde beaktat innehåll av näringsämnen och andra nyttiga ämnen, och att möta konsumenters efterfrågan på hälsosamma färdiga rätter. Broccolisorten ’Beneforte’ valdes eftersom denna sort har en dokumenterad högre halt av glukorafanin*. Det pågår kliniska studier i Europa om hälsoeffekter av broccolisorter med hög halt av detta ämne och dessa skulle kunna leda till att ett hälsopåstående blir godkänt av EFSA, den europeiska livssäkerhetsmyndigheten. Produkter baserade på denna broccolisort skulle då kunna marknadsföras med hälsopåståendet.

Vegetable products such as berries, leafy greens and sprouts pose a particular risk of transmittinginfectious diseases since they are eaten raw. Irrigation using faecally contaminated water is the mostlikely source of infectious agents but wild animal faeces and manure can also contaminate the productsfrom soil. The purpose of this project was to provide an overview of irrigation water quality inSweden, link it to microbiological risks and, based on this information, develop data for nationalguidelines with the aim of safe production of edible berries and vegetables that better prevent diseaseoutbreaks.The quality of both water and crops was generally good at the farms included in the project. Pathogensand ESBL-producing E. coli were occasionally found in water. However, these were only isolatedfrom surface water samples that were more polluted with respect to faecal indicators. Only a few cropsamples showed higher faecal contamination. The majority of these samples were from one farmerusing pond water for irrigation. No pathogens were detected on the crops. However, one cabbagesample tested positive for ESBL-producing E. coli.A quantitative risk assessment of Shiga toxin-producing E. coli (STEC) from iceberg lettuce showedthat even low faecal contamination and the absence of detected pathogens in irrigation water can stillpose a risk of gastrointestinal infection. This risk assessment can provide the basis for microbiologicalcriteria, for example, with regard to water quality. However, the uncertainties associated with the riskassessment need to be taken into account. The main uncertainties were 1: the excretion andpathogenicity of STEC from infected cattle, 2: the proportion of these that adhere to the irrigated crop,3: the likelihood of being infected at low doses (single STEC bacterium) and 4: the importance of soilcontamination.Various measures that can reduce the likelihood of foodborne outbreaks and disease cases are 1:criteria for water quality, 2: purification of water not achieving these criteria, 3: residence timebetween irrigation and harvest and 4: consumers rinsing the produce. It is also important for farmers tokeep equipment such as pipes and nozzles clean to avoid contamination and bacterial growth.

If surface water is used for irrigation (for example, in the absence of a safe groundwater supply),during the two weeks prior to harvest, a sampling programme should be implemented to ensure lowfaecal contamination and the absence of Salmonella. However, only water of drinking water qualityshould be used two days prior to harvest. It would be preferable for surface water to undergopurification before irrigation. This can be achieved, for example, by photocatalysis, UV light orfiltration.In a future with decreasing groundwater levels, drier summers and rainier winters, there is a need tocollect water for irrigation during the autumn rains, spring floods and wintertime to be stored inreservoirs for later use. Water from reservoirs may need purification before being used for irrigation,depending on how protected the reservoir is from contamination.

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.

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.

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.

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.

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.

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.

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.