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Papers by Rickard Knutsson

Zenodo (CERN European Organization for Nuclear Research), Dec 21, 2021

Zenodo (CERN European Organization for Nuclear Research), Dec 22, 2021

COHESIVE general meeting Room Background Various organizations have addressed that foresight stud... more COHESIVE general meeting Room Background Various organizations have addressed that foresight studies regarding one health is of interest. However, looking into the future and foresight studies are complex. There are many options for futures, but it generally involves identifying areas of uncertainty, challenges, underlying drivers and opportunities. In other words, futures are about a structured manner to explore possible and preferable futures. Foresight is a process to conduct futures work and horizon scanning is a specific technique and various horizon scanning tools are available (UK GOV, 2018). There are also various terms and definitions and one that will be used for this workshop is following: "Horizon scanning is a specific foresight methodology that utilizes various steps to identify issues at the edge of current thinking that may have significant impact in the medium to long term future" (FAO, 2014).

European journal of wildlife research, Mar 14, 2024

The food security value of wild meat is calculated by combining proxy methods for quantifying gam... more The food security value of wild meat is calculated by combining proxy methods for quantifying game animal abundance with shadow pricing techniques for assessing the unit values of food security. This study calculated the food security values of moose, roe deer, wild boar, and fallow deer for Sweden overall and for individual counties. The results showed that meat from these animal populations accounts for approximately 9% of meat consumption in Sweden and for 1.2% of the minimum energy food consumption during periods of crisis for the whole of Sweden, while in some counties it can be as much as 8%. The calculated unit value, or shadow price, of the minimum energy requirement ranged between € 0.1 and € 4.2/mcal, depending on the magnitude of the crisis scenario. At most, the total food security value of actual animal population sizes amounted to 0.50 billion euros, but this was unevenly distributed, with high values in counties that have an abundance of moose and wild boar.

Health Security, 2017

The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration a... more The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration among the 4 agencies with responsibility for the laboratory diagnostics of high-consequence pathogens, covering animal health and feed safety, food safety, public health and biodefense, and security. The aim of the network is to strengthen capabilities and capacities for diagnostics at the national biosafety level-3 (BSL-3) laboratories to improve Sweden's biopreparedness, in line with recommendations from the EU and WHO. Since forming in 2007, the FBD network has contributed to the harmonization of diagnostic methods, equipment, quality assurance protocols, and biosafety practices among the national BSL-3 laboratories. Lessons learned from the network include: (1) conducting joint projects with activities such as method development and validation, ring trials, exercises, and audits has helped to build trust and improve communication among participating agencies; (2) rotating the presidency of the network steering committee has fostered trust and commitment from all agencies involved; and (3) planning for the implementation of project outcomes is important to maintain gained competencies in the agencies over time. Contacts have now been established with national agencies of the other Nordic countries, with an aim to expanding the collaboration, broadening the network, finding synergies in new areas, strengthening the ability to share resources, and consolidating long-term financing in the context of harmonized European biopreparedness.

CRC Press eBooks, May 13, 2013

A review describes the sample prepn. for biochem. methods, enrichment methods, immunol. methods, ... more A review describes the sample prepn. for biochem. methods, enrichment methods, immunol. methods, and phys. methods. The pretreatment of a complex biol. sample is crucial, and for successful PCR the following requirements have to be fulfilled: complete lack or low concn. of PCR-inhibitory components in the sample and sufficient concn. of target DNA. The aim of the pre-PCR treatment is to convert a complex biol. sample contg. the target microorganisms into PCR-amplifiable samples.

CRC Press eBooks, Apr 11, 1994

Humana Press eBooks, Nov 15, 2003

Case Studies in Food Safety and Authenticity, 2012

Abstract: This chapter describes an unexpected anthrax outbreak in a group of Swedish beef cattle... more Abstract: This chapter describes an unexpected anthrax outbreak in a group of Swedish beef cattle kept indoors during the winter season of 2008. Anthrax is a serious zoonotic disease, caused by the pathogenic agent Bacillus anthracis. The way in which the outbreak was managed, the costs involved and the lessons learned are discussed. Research needs, crisis management and preparedness gaps are also identified.

Humana Press eBooks, Nov 15, 2003

Biosecurity and Bioterrorism-biodefense Strategy Practice and Science, Sep 1, 2013

Laboratory response networks (LRNs) have been established for security reasons in several countri... more Laboratory response networks (LRNs) have been established for security reasons in several countries including the Netherlands, France, and Sweden. LRNs function in these countries as a preparedness measure for a coordinated diagnostic response capability in case of a bioterrorism incident or other biocrimes. Generally, these LRNs are organized on a national level. The EU project AniBioThreat has identified the need for an integrated European LRN to strengthen preparedness against animal bioterrorism. One task of the AniBioThreat project is to suggest a plan to implement laboratory biorisk management CWA 15793:2011 (CWA 15793), a management system built on the principle of continual improvement through the Plan-Do-Check-Act (PDCA) cycle. The implementation of CWA 15793 can facilitate trust and credibility in a future European LRN and is an assurance that the work done at the laboratories is performed in a structured way with continuous improvements. As a first step, a gap analysis was performed to establish the current compliance status of biosafety and laboratory biosecurity management with CWA 15793 in 5 AniBioThreat partner institutes in France (ANSES), the Netherlands (CVI and RIVM), and Sweden (SMI and SVA). All 5 partners are national and/or international laboratory reference institutes in the field of public or animal health and possess highcontainment laboratories and animal facilities. The gap analysis showed that the participating institutes already have robust biorisk management programs in place, but several gaps were identified that need to be addressed. Despite differences between the participating institutes in their compliance status, these variations are not significant. Biorisk management exercises also have been identified as a useful tool to control compliance status and thereby implementation

ASM Press eBooks, Apr 9, 2014

Biosecurity and Bioterrorism-biodefense Strategy Practice and Science, Sep 1, 2013

Botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishe... more Botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishes. The disease in animals is mainly caused by toxins produced by Clostridium botulinum strains belonging to group III, although outbreaks due to toxins produced by group I and II organisms have been recognized. Group III strains are capable of producing botulinum toxins of type C, D, and C/D and D/C mosaic variants. Definitive diagnosis of animal botulism is made by combining clinical findings with laboratory investigations. Detection of toxins in clinical specimens and feed is the gold standard for laboratory diagnosis. Since toxins may be degraded by organisms contained in the gastrointestinal tract or may be present at levels below the detection limit, the recovery of C. botulinum from sick animal specimens is consistent for laboratory confirmation. In this article we report the development and in-house validation of a new multiplex real-time PCR for detecting and typing the neurotoxin genes found in C. botulinum group III organisms. Validation procedures have been carried out according to ISO 16140, using strains and samples recovered from cases of animal botulism in Italy and France. B otulism is a severe and potentially lethal illness caused by exposure to botulinum neurotoxins (BoNTs) produced by Clostridium botulinum and other BoNT-producing clostridia strains. 1,2 All warm-blooded animals (including humans), birds, and some fishes, can be affected by the disease but show different levels of susceptibility to the different types of BoNTs. 3 Human botulism is mainly associated with types A, B, E, and F toxins, whereas animal botulism is primarily associated with types C, D, C/D, and D/C toxins, although some outbreaks due to types A, B, and E have also been recognized. 4-8 Mammals, such as cattle, horses, and sheep, as well as waterfowl and poultry are the animals most often involved in outbreaks. 4,9 Cattle botulism is most frequently caused by types D or D/C toxin, followed by types C, A, B, and C/D toxins. 4,8,10 Equine botulism is caused by types B, C, and A toxins. 11,12 Fur farm animals, such as foxes and minks, seem to be susceptible to types C and C/D toxin. 13,14 Birds Fabrizio Anniballi, CLT, is a Laboratory Technician; Bruna Auricchio, CLT, is a Laboratory Technician; Alfonsina

Biosecurity and Bioterrorism-biodefense Strategy Practice and Science, Sep 1, 2013

Microbial forensics is an important part of a strengthened capability to respond to biocrime and ... more Microbial forensics is an important part of a strengthened capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. The goal of a microbial forensic investigation is to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon-that is, the pathogen. The recent development of next-generation sequencing (NGS) technologies has greatly increased the resolution that can be achieved in microbial forensic analyses. It is now possible to identify, quickly and in an unbiased manner, previously undetectable genome differences between closely related isolates. This development is particularly relevant for the most deadly bacterial diseases that are caused by bacterial lineages with extremely low levels of genetic diversity. Whole-genome analysis of pathogens is envisaged to be increasingly essential for this purpose. In a microbial forensic context, whole-genome sequence analysis is the ultimate method for strain comparisons as it is informative during identification, characterization, and attribution-all 3 major stages of the investigation-and at all levels of microbial strain identity resolution (ie, it resolves the full spectrum from family to isolate). Given these capabilities, one bottleneck in microbial forensics investigations is the availability of high-quality reference databases of bacterial whole-genome sequences. To be of high quality, databases need to be curated and accurate in terms of sequences, metadata, and genetic diversity coverage. The development of whole-genome sequence databases will be instrumental in successfully tracing pathogens in the future. I ntentional introduction of diseases (human, animal, or plant) caused by biological agents in acts of bioterrorism is a global threat that could have profound implications. 1-4 Methods for the early detection and analysis of organisms that could potentially be used in this way are rapidly evolving. In particular, the past decade has seen major advances in a new scientific discipline, microbial forensics, whose core objective is attribution: the investigative process aimed at identifying the perpetrators of a bioterrorism or biocrime event and bringing them to

International Journal of Food Microbiology, Mar 1, 2011

Available strain collections of Bacillus anthracis and Bacillus cereus were screened for B. cereu... more Available strain collections of Bacillus anthracis and Bacillus cereus were screened for B. cereus strains sharing major genotypic characteristics with B. anthracis. Based on the comparison of partial spoIIIAB sequences, whole genome sequences and MLST, a strain set representing different lineages including candidate model strains for B. anthracis was compiled. Spores from the selected strain set and two B. anthracis strains were prepared according to a newly optimized protocol transferable to biosafety level-3 (BSL3) conditions and phenotypic characteristics including scanning electron microscopy (SEM), heat inactivation, and germination were evaluated. Two B. cereus isolates were identified that were genetically related to B. anthracis and showed high similarity to B. anthracis spores in their heat inactivation profile and their response to the germinants L-alanine and inosine. In addition, these isolates were also mimicking B. anthracis on modified PLET, a selective plating medium for B. anthracis, and shared various other biochemical characteristics with B. anthracis. Therefore these two strains are not only appropriate models for B. anthracis in experiments based on spore characteristics but also in trials working with plating media. These two strains are now used within the BIOTRACER consortium as validated models for B. anthracis and will facilitate the development and optimization of tracing and detection systems for B. anthracis in the food and feed chain.

Biosecurity and Bioterrorism-biodefense Strategy Practice and Science, Sep 1, 2014

The objectives of the Global Health Security Agenda (GHSA) will require not only a ''One Health''... more The objectives of the Global Health Security Agenda (GHSA) will require not only a ''One Health'' approach to counter natural disease threats against humans, animals, and the environment, but also a security focus to counter deliberate threats to human, animal, and agricultural health and to nations' economies. We have termed this merged approach ''One Health Security.'' It will require the integration of professionals with expertise in security, law enforcement, and intelligence to join the veterinary, agricultural, environmental, and human health experts essential to One Health and the GHSA. Working across such different professions, which occasionally have conflicting aims and different professional cultures, poses multiple challenges, but a multidisciplinary and multisectoral approach is necessary to prevent disease threats; detect them as early as possible (when responses are likely to be most effective); and, in the case of deliberate threats, find who may be responsible. This article describes 2 project areas that exemplify One Health Security that were presented at a workshop in January 2014: the US government and private industry efforts to reduce vulnerabilities to foreign animal diseases, especially foot-and-mouth disease; and AniBioThreat, an EU project to counter deliberate threats to agriculture by raising awareness and implementing prevention and response policies and practices.

International Journal of Food Microbiology, Feb 1, 2002

A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for... more A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10 5-10 8 (CFU/ml), the coefficient of variation < 5%. When a background flora was present at concentrations ! 10 6 (CFU/ml), the DNA amplification was influenced and a change in the log-linear slope leading to a lower amplification efficiency was observed. To study the dynamic detection range and relative amplification precision during enrichment, Y. enterocolitica and background flora were inoculated at various concentrations. It was possible to detect inoculation concentrations of 10 1 (CFU/ml) Y. enterocolitica in the presence of at least an inoculation concentration of 10 3 (CFU/ml) of an undefined background flora and the optimal conditions for sample withdrawal was in the range of 9 to 18 h enrichment. The YPCE medium can, especially for swab samples, form part of a simple analysis procedure allowing high throughput PCR.

This thesis deals with the methodological advances of diagnostic PCR including reliability of PCR... more This thesis deals with the methodological advances of diagnostic PCR including reliability of PCR and pre-PCR processing of food and feed samples. Diagnostic PCR has been greatly improved by the introduction of the second generation of PCR, so-called real-time PCR. Automated closed-tube quantitative real-time PCR favours the analysis of food-borne pathogenic bacteria. However, in common with conventional PCR, the real-time PCR technology requires pre-PCR processing of the food/feed sample (i) to remove PCR-inhibitory substances, (ii) facilitate detection of low concentrations of target bacteria, (iii) to convert a heterogeneous bulk sample to a more homogeneous PCR sample, and (iv) to restrict competitive background flora. The aim of the research presented in this thesis was to adapt microbiological food sampling to PCR by designing pre-PCR processing strategies for future routine analysis of Yersinia enterocolitica and Salmonella in complex samples from the food-chain. To enable the detection of low concentrations of Y. enterocolitica and Salmonella in PCR-inhibitory samples, such as pork and animal feed, enrichment PCR procedures have been employed. The reliability of PCR detection of pathogenic Y. enterocolitica using a developed multiplex PCR assay was studied using a logistic regression model for determination of the detection probability. The probability of detecting 1´104 CFU/ml Y. enterocolitica was estimated to be 85.4%. A Yersinia-PCR-compatible enrichment (YPCE) medium was developed to remove the necessity for sample preparation prior to PCR detection of Y. enterocolitica. The pre-PCR processing strategy allows detection of low concentrations (101 CFU/ml) in the presence of background flora in concentrations up to three orders of magnitude higher than Y. enterocolitica. The YPCE medium can, form part of integrated and automated pre-PCR processing protocol, especially for swab samples. To complement the Yersinia assay a RAPD protocol was developed for inter-laboratory use. The stringent RAPD protocol was evaluated on 70 Yersinia strains and allowed discrimination at serotype level, and the sub-clusters of Y. enterocolitica correlated with the geographic origin of isolates, where especially O:3 strains from Scandinavia formed a homogeneous sub-cluster. Enrichment PCR of Salmonella enterica was studied using real-time PCR. A model was developed to describe the 5' nuclease real-time PCR performance in the presence buffered peptone water (BPW) and brain heart infusion. Using the model it was found that the rTth DNA polymerase mixture was more resistant to the presence of BPW than AmpliTaq Gold. Accurate detection of 1 CFU/ml S. Enteritidis inoculated in BPW required 8.4 hours’ enrichment using the rTth DNA polymerase mixture, while AmpliTaq Gold required 11.6 h. Using an alternative DNA polymerase, Tth instead of Taq, facilitated the PCR detection of Salmonella in animal feed. The PCR protocol was more sensitive than the traditional culture-based standard method (NMKL-71), since out of 155 feed samples, 8% were positive for PCR detection of Salmonella in comparison with 3% with the NMKL-71 method. (Less)

Journal of defense management, 2015

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagn... more 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 PCRinhibitory 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 PCRamplifiable 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.

Journal of Clinical Microbiology, 2002

The performance of a 5 nuclease real-time PCR assay was studied to optimize an automated method o... more 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 (C T) and the fluorescence intensity by a normalized reporter value (⌬R n). The C T 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 H 2 O (ddH 2 O) 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 ddH 2 O. 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 ؊13 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 (C T < 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.

Zenodo (CERN European Organization for Nuclear Research), Dec 21, 2021

Zenodo (CERN European Organization for Nuclear Research), Dec 22, 2021

COHESIVE general meeting Room Background Various organizations have addressed that foresight stud... more COHESIVE general meeting Room Background Various organizations have addressed that foresight studies regarding one health is of interest. However, looking into the future and foresight studies are complex. There are many options for futures, but it generally involves identifying areas of uncertainty, challenges, underlying drivers and opportunities. In other words, futures are about a structured manner to explore possible and preferable futures. Foresight is a process to conduct futures work and horizon scanning is a specific technique and various horizon scanning tools are available (UK GOV, 2018). There are also various terms and definitions and one that will be used for this workshop is following: "Horizon scanning is a specific foresight methodology that utilizes various steps to identify issues at the edge of current thinking that may have significant impact in the medium to long term future" (FAO, 2014).

European journal of wildlife research, Mar 14, 2024

The food security value of wild meat is calculated by combining proxy methods for quantifying gam... more The food security value of wild meat is calculated by combining proxy methods for quantifying game animal abundance with shadow pricing techniques for assessing the unit values of food security. This study calculated the food security values of moose, roe deer, wild boar, and fallow deer for Sweden overall and for individual counties. The results showed that meat from these animal populations accounts for approximately 9% of meat consumption in Sweden and for 1.2% of the minimum energy food consumption during periods of crisis for the whole of Sweden, while in some counties it can be as much as 8%. The calculated unit value, or shadow price, of the minimum energy requirement ranged between € 0.1 and € 4.2/mcal, depending on the magnitude of the crisis scenario. At most, the total food security value of actual animal population sizes amounted to 0.50 billion euros, but this was unevenly distributed, with high values in counties that have an abundance of moose and wild boar.

Health Security, 2017

The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration a... more The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration among the 4 agencies with responsibility for the laboratory diagnostics of high-consequence pathogens, covering animal health and feed safety, food safety, public health and biodefense, and security. The aim of the network is to strengthen capabilities and capacities for diagnostics at the national biosafety level-3 (BSL-3) laboratories to improve Sweden's biopreparedness, in line with recommendations from the EU and WHO. Since forming in 2007, the FBD network has contributed to the harmonization of diagnostic methods, equipment, quality assurance protocols, and biosafety practices among the national BSL-3 laboratories. Lessons learned from the network include: (1) conducting joint projects with activities such as method development and validation, ring trials, exercises, and audits has helped to build trust and improve communication among participating agencies; (2) rotating the presidency of the network steering committee has fostered trust and commitment from all agencies involved; and (3) planning for the implementation of project outcomes is important to maintain gained competencies in the agencies over time. Contacts have now been established with national agencies of the other Nordic countries, with an aim to expanding the collaboration, broadening the network, finding synergies in new areas, strengthening the ability to share resources, and consolidating long-term financing in the context of harmonized European biopreparedness.

CRC Press eBooks, May 13, 2013

A review describes the sample prepn. for biochem. methods, enrichment methods, immunol. methods, ... more A review describes the sample prepn. for biochem. methods, enrichment methods, immunol. methods, and phys. methods. The pretreatment of a complex biol. sample is crucial, and for successful PCR the following requirements have to be fulfilled: complete lack or low concn. of PCR-inhibitory components in the sample and sufficient concn. of target DNA. The aim of the pre-PCR treatment is to convert a complex biol. sample contg. the target microorganisms into PCR-amplifiable samples.

CRC Press eBooks, Apr 11, 1994

Humana Press eBooks, Nov 15, 2003

Case Studies in Food Safety and Authenticity, 2012

Abstract: This chapter describes an unexpected anthrax outbreak in a group of Swedish beef cattle... more Abstract: This chapter describes an unexpected anthrax outbreak in a group of Swedish beef cattle kept indoors during the winter season of 2008. Anthrax is a serious zoonotic disease, caused by the pathogenic agent Bacillus anthracis. The way in which the outbreak was managed, the costs involved and the lessons learned are discussed. Research needs, crisis management and preparedness gaps are also identified.

Humana Press eBooks, Nov 15, 2003

Biosecurity and Bioterrorism-biodefense Strategy Practice and Science, Sep 1, 2013

Laboratory response networks (LRNs) have been established for security reasons in several countri... more Laboratory response networks (LRNs) have been established for security reasons in several countries including the Netherlands, France, and Sweden. LRNs function in these countries as a preparedness measure for a coordinated diagnostic response capability in case of a bioterrorism incident or other biocrimes. Generally, these LRNs are organized on a national level. The EU project AniBioThreat has identified the need for an integrated European LRN to strengthen preparedness against animal bioterrorism. One task of the AniBioThreat project is to suggest a plan to implement laboratory biorisk management CWA 15793:2011 (CWA 15793), a management system built on the principle of continual improvement through the Plan-Do-Check-Act (PDCA) cycle. The implementation of CWA 15793 can facilitate trust and credibility in a future European LRN and is an assurance that the work done at the laboratories is performed in a structured way with continuous improvements. As a first step, a gap analysis was performed to establish the current compliance status of biosafety and laboratory biosecurity management with CWA 15793 in 5 AniBioThreat partner institutes in France (ANSES), the Netherlands (CVI and RIVM), and Sweden (SMI and SVA). All 5 partners are national and/or international laboratory reference institutes in the field of public or animal health and possess highcontainment laboratories and animal facilities. The gap analysis showed that the participating institutes already have robust biorisk management programs in place, but several gaps were identified that need to be addressed. Despite differences between the participating institutes in their compliance status, these variations are not significant. Biorisk management exercises also have been identified as a useful tool to control compliance status and thereby implementation

ASM Press eBooks, Apr 9, 2014

Biosecurity and Bioterrorism-biodefense Strategy Practice and Science, Sep 1, 2013

Botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishe... more Botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishes. The disease in animals is mainly caused by toxins produced by Clostridium botulinum strains belonging to group III, although outbreaks due to toxins produced by group I and II organisms have been recognized. Group III strains are capable of producing botulinum toxins of type C, D, and C/D and D/C mosaic variants. Definitive diagnosis of animal botulism is made by combining clinical findings with laboratory investigations. Detection of toxins in clinical specimens and feed is the gold standard for laboratory diagnosis. Since toxins may be degraded by organisms contained in the gastrointestinal tract or may be present at levels below the detection limit, the recovery of C. botulinum from sick animal specimens is consistent for laboratory confirmation. In this article we report the development and in-house validation of a new multiplex real-time PCR for detecting and typing the neurotoxin genes found in C. botulinum group III organisms. Validation procedures have been carried out according to ISO 16140, using strains and samples recovered from cases of animal botulism in Italy and France. B otulism is a severe and potentially lethal illness caused by exposure to botulinum neurotoxins (BoNTs) produced by Clostridium botulinum and other BoNT-producing clostridia strains. 1,2 All warm-blooded animals (including humans), birds, and some fishes, can be affected by the disease but show different levels of susceptibility to the different types of BoNTs. 3 Human botulism is mainly associated with types A, B, E, and F toxins, whereas animal botulism is primarily associated with types C, D, C/D, and D/C toxins, although some outbreaks due to types A, B, and E have also been recognized. 4-8 Mammals, such as cattle, horses, and sheep, as well as waterfowl and poultry are the animals most often involved in outbreaks. 4,9 Cattle botulism is most frequently caused by types D or D/C toxin, followed by types C, A, B, and C/D toxins. 4,8,10 Equine botulism is caused by types B, C, and A toxins. 11,12 Fur farm animals, such as foxes and minks, seem to be susceptible to types C and C/D toxin. 13,14 Birds Fabrizio Anniballi, CLT, is a Laboratory Technician; Bruna Auricchio, CLT, is a Laboratory Technician; Alfonsina

Biosecurity and Bioterrorism-biodefense Strategy Practice and Science, Sep 1, 2013

Microbial forensics is an important part of a strengthened capability to respond to biocrime and ... more Microbial forensics is an important part of a strengthened capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. The goal of a microbial forensic investigation is to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon-that is, the pathogen. The recent development of next-generation sequencing (NGS) technologies has greatly increased the resolution that can be achieved in microbial forensic analyses. It is now possible to identify, quickly and in an unbiased manner, previously undetectable genome differences between closely related isolates. This development is particularly relevant for the most deadly bacterial diseases that are caused by bacterial lineages with extremely low levels of genetic diversity. Whole-genome analysis of pathogens is envisaged to be increasingly essential for this purpose. In a microbial forensic context, whole-genome sequence analysis is the ultimate method for strain comparisons as it is informative during identification, characterization, and attribution-all 3 major stages of the investigation-and at all levels of microbial strain identity resolution (ie, it resolves the full spectrum from family to isolate). Given these capabilities, one bottleneck in microbial forensics investigations is the availability of high-quality reference databases of bacterial whole-genome sequences. To be of high quality, databases need to be curated and accurate in terms of sequences, metadata, and genetic diversity coverage. The development of whole-genome sequence databases will be instrumental in successfully tracing pathogens in the future. I ntentional introduction of diseases (human, animal, or plant) caused by biological agents in acts of bioterrorism is a global threat that could have profound implications. 1-4 Methods for the early detection and analysis of organisms that could potentially be used in this way are rapidly evolving. In particular, the past decade has seen major advances in a new scientific discipline, microbial forensics, whose core objective is attribution: the investigative process aimed at identifying the perpetrators of a bioterrorism or biocrime event and bringing them to

International Journal of Food Microbiology, Mar 1, 2011

Available strain collections of Bacillus anthracis and Bacillus cereus were screened for B. cereu... more Available strain collections of Bacillus anthracis and Bacillus cereus were screened for B. cereus strains sharing major genotypic characteristics with B. anthracis. Based on the comparison of partial spoIIIAB sequences, whole genome sequences and MLST, a strain set representing different lineages including candidate model strains for B. anthracis was compiled. Spores from the selected strain set and two B. anthracis strains were prepared according to a newly optimized protocol transferable to biosafety level-3 (BSL3) conditions and phenotypic characteristics including scanning electron microscopy (SEM), heat inactivation, and germination were evaluated. Two B. cereus isolates were identified that were genetically related to B. anthracis and showed high similarity to B. anthracis spores in their heat inactivation profile and their response to the germinants L-alanine and inosine. In addition, these isolates were also mimicking B. anthracis on modified PLET, a selective plating medium for B. anthracis, and shared various other biochemical characteristics with B. anthracis. Therefore these two strains are not only appropriate models for B. anthracis in experiments based on spore characteristics but also in trials working with plating media. These two strains are now used within the BIOTRACER consortium as validated models for B. anthracis and will facilitate the development and optimization of tracing and detection systems for B. anthracis in the food and feed chain.

Biosecurity and Bioterrorism-biodefense Strategy Practice and Science, Sep 1, 2014

The objectives of the Global Health Security Agenda (GHSA) will require not only a ''One Health''... more The objectives of the Global Health Security Agenda (GHSA) will require not only a ''One Health'' approach to counter natural disease threats against humans, animals, and the environment, but also a security focus to counter deliberate threats to human, animal, and agricultural health and to nations' economies. We have termed this merged approach ''One Health Security.'' It will require the integration of professionals with expertise in security, law enforcement, and intelligence to join the veterinary, agricultural, environmental, and human health experts essential to One Health and the GHSA. Working across such different professions, which occasionally have conflicting aims and different professional cultures, poses multiple challenges, but a multidisciplinary and multisectoral approach is necessary to prevent disease threats; detect them as early as possible (when responses are likely to be most effective); and, in the case of deliberate threats, find who may be responsible. This article describes 2 project areas that exemplify One Health Security that were presented at a workshop in January 2014: the US government and private industry efforts to reduce vulnerabilities to foreign animal diseases, especially foot-and-mouth disease; and AniBioThreat, an EU project to counter deliberate threats to agriculture by raising awareness and implementing prevention and response policies and practices.

International Journal of Food Microbiology, Feb 1, 2002

A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for... more A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10 5-10 8 (CFU/ml), the coefficient of variation < 5%. When a background flora was present at concentrations ! 10 6 (CFU/ml), the DNA amplification was influenced and a change in the log-linear slope leading to a lower amplification efficiency was observed. To study the dynamic detection range and relative amplification precision during enrichment, Y. enterocolitica and background flora were inoculated at various concentrations. It was possible to detect inoculation concentrations of 10 1 (CFU/ml) Y. enterocolitica in the presence of at least an inoculation concentration of 10 3 (CFU/ml) of an undefined background flora and the optimal conditions for sample withdrawal was in the range of 9 to 18 h enrichment. The YPCE medium can, especially for swab samples, form part of a simple analysis procedure allowing high throughput PCR.

This thesis deals with the methodological advances of diagnostic PCR including reliability of PCR... more This thesis deals with the methodological advances of diagnostic PCR including reliability of PCR and pre-PCR processing of food and feed samples. Diagnostic PCR has been greatly improved by the introduction of the second generation of PCR, so-called real-time PCR. Automated closed-tube quantitative real-time PCR favours the analysis of food-borne pathogenic bacteria. However, in common with conventional PCR, the real-time PCR technology requires pre-PCR processing of the food/feed sample (i) to remove PCR-inhibitory substances, (ii) facilitate detection of low concentrations of target bacteria, (iii) to convert a heterogeneous bulk sample to a more homogeneous PCR sample, and (iv) to restrict competitive background flora. The aim of the research presented in this thesis was to adapt microbiological food sampling to PCR by designing pre-PCR processing strategies for future routine analysis of Yersinia enterocolitica and Salmonella in complex samples from the food-chain. To enable the detection of low concentrations of Y. enterocolitica and Salmonella in PCR-inhibitory samples, such as pork and animal feed, enrichment PCR procedures have been employed. The reliability of PCR detection of pathogenic Y. enterocolitica using a developed multiplex PCR assay was studied using a logistic regression model for determination of the detection probability. The probability of detecting 1´104 CFU/ml Y. enterocolitica was estimated to be 85.4%. A Yersinia-PCR-compatible enrichment (YPCE) medium was developed to remove the necessity for sample preparation prior to PCR detection of Y. enterocolitica. The pre-PCR processing strategy allows detection of low concentrations (101 CFU/ml) in the presence of background flora in concentrations up to three orders of magnitude higher than Y. enterocolitica. The YPCE medium can, form part of integrated and automated pre-PCR processing protocol, especially for swab samples. To complement the Yersinia assay a RAPD protocol was developed for inter-laboratory use. The stringent RAPD protocol was evaluated on 70 Yersinia strains and allowed discrimination at serotype level, and the sub-clusters of Y. enterocolitica correlated with the geographic origin of isolates, where especially O:3 strains from Scandinavia formed a homogeneous sub-cluster. Enrichment PCR of Salmonella enterica was studied using real-time PCR. A model was developed to describe the 5' nuclease real-time PCR performance in the presence buffered peptone water (BPW) and brain heart infusion. Using the model it was found that the rTth DNA polymerase mixture was more resistant to the presence of BPW than AmpliTaq Gold. Accurate detection of 1 CFU/ml S. Enteritidis inoculated in BPW required 8.4 hours’ enrichment using the rTth DNA polymerase mixture, while AmpliTaq Gold required 11.6 h. Using an alternative DNA polymerase, Tth instead of Taq, facilitated the PCR detection of Salmonella in animal feed. The PCR protocol was more sensitive than the traditional culture-based standard method (NMKL-71), since out of 155 feed samples, 8% were positive for PCR detection of Salmonella in comparison with 3% with the NMKL-71 method. (Less)

Journal of defense management, 2015

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagn... more 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 PCRinhibitory 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 PCRamplifiable 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.

Journal of Clinical Microbiology, 2002

The performance of a 5 nuclease real-time PCR assay was studied to optimize an automated method o... more 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 (C T) and the fluorescence intensity by a normalized reporter value (⌬R n). The C T 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 H 2 O (ddH 2 O) 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 ddH 2 O. 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 ؊13 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 (C T < 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.