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See detailComplete genome sequence of a novel bovine norovirus: Evidence for slow genetic evolution in genogroup III genotype 2 noroviruses
Mauroy, Axel ULg; Scipioni, A.; Mathijs, E. et al

in Journal of Virology (2012), 86(22), 12449-12450

A new genogroup III genotype 2 bovine norovirus, B309/2003/BE, was entirely sequenced and genetically compared to the original Newbury2/1976/UK strain and to Dumfries/1994/UK, detected in 1976 and 1994 ... [more ▼]

A new genogroup III genotype 2 bovine norovirus, B309/2003/BE, was entirely sequenced and genetically compared to the original Newbury2/1976/UK strain and to Dumfries/1994/UK, detected in 1976 and 1994, respectively. Interestingly, except in welldefined coding regions (N-terminal protein, 3A-like protease, hypervariable region of the capsid protein, and C-terminal part of the minor structural protein), very low genetic differences were noted between the entire genomes of these three strains along a 30-year-long period. It allowed some hypotheses of hotspots of genetic evolution through a low genetic evolution background in genotype 2 genogroup III bovine noroviruses. © 2012, American Society for Microbiology. [less ▲]

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See detailCaractérisation des viandes bovines à très longue durée de conservation sous vide
Didimo Imazaki, Pedro Henrique ULg; Nezer, Carine ULg; Taminiau, Bernard ULg et al

Poster (2011, December 09)

Le but de cette étude a été d’évaluer la conservabilité de viandes bovines de différentes origines (Royaume-Uni et Irlande, Australie et Brésil) et l’influence sur celle-ci de la température de ... [more ▼]

Le but de cette étude a été d’évaluer la conservabilité de viandes bovines de différentes origines (Royaume-Uni et Irlande, Australie et Brésil) et l’influence sur celle-ci de la température de conservation (1 °C vs. +4 °C). Des paramètres physico-chimiques (pH, couleur, proportion des différentes formes redox de la myoglobine (FRMb), indice TBARS et acides organiques) et microbiologiques (flore aérobie totale, flore lactique, Enterobacteriaceae, Pseudomonas spp. et Brochothrix thermosphacta) ont été mesurés sur sept lots de contre-filet conditionnés sous vide : aux ⅔ de la DLC et à la fin de la DLC. La diversité bactérienne a été évaluée par galeries API50 CHL et par métagénomique. Le pH a diminué au cours de la conservation dans deux lots. La couleur et la proportion des FRMb sont restées stables. Une augmentation de l’indice TBARS, plus prononcée à +4 °C, a été observée. Les viandes australiennes et brésilienne ont présenté des taux en acides acétique et citrique plus élevés. Tous les lots conservés à 1 °C ont présenté une qualité microbiologique satisfaisante à la fin de leur DLC (viandes britanniques et irlandaises = 35 ~ 45 jours; australiennes = 140 jours et brésilienne = 120 jours). La conservation à +4 °C a favorisé la croissance d’entérobactéries, facteur limitant de la conservation de plusieurs lots. L’identification bactérienne a révélé la présence de bactéries connues pour leur effet bioprotecteur. La phase ultérieure de ce travail consistera à étudier la dynamique de la flore microbienne endogène en fonction des conditions environnementales appliquées (température, atmosphère). [less ▲]

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See detailEvaluation de la diversité bactérienne et de son évolution pendant la conservation de la viande fraîche bovine de différentes origines emballée sous vide
Didimo Imazaki, Pedro Henrique ULg; Maréchal, Aline; Nezer, Carine ULg et al

Poster (2011, November 18)

Un grand nombre de bactéries lactiques associées à la viande sont connues comme d'importants producteurs de bactériocines. Ces bactériocines sont des toxines protéiques présentant une activité bactéricide ... [more ▼]

Un grand nombre de bactéries lactiques associées à la viande sont connues comme d'importants producteurs de bactériocines. Ces bactériocines sont des toxines protéiques présentant une activité bactéricide ou bactériostatique contre des espèces proches de la souche productrice. La présence de certaines bactéries lactiques dans la viande fraîche pourrait donc prolonger la durée de conservation, et améliorer la stabilité microbienne et la sécurité de ce produit. Dans ce contexte, une étude a été réalisée sur des échantillons de contre-filet de différentes origines emballés sous vide, dans le but d’évaluer la diversité bactérienne et son évolution pendant la conservation. L’étude a été réalisée sur trois lots provenant d’Irlande, du Brésil et d’Australie, affichant respectivement une DLC de 35 jours, 120 jours et 140 jours. Après réception dans le laboratoire, les échantillons ont été conservés à 1 °C. Ensuite, pendant le dernier tiers de leur DLC, ils ont été conservés à 1 °C ou à +4 °C. Des dénombrements ont été réalisés : 1) aux ⅔ de la DLC et 2) à la fin de la DLC. Les germes dénombrés ont été : la flore aérobie totale à +22 °C, la flore lactique à +22 °C et les Enterobacteriaceae à +30 °C en utilisant le système automatique de dénombrement TEMPO®. Tous les échantillons conservés à 1 °C ont présenté une qualité microbiologique satisfaisante à la fin de la conservation. Par contre, la conservation à +4 °C a favorisé une croissance plus importante des bactéries lactiques et des Enterobacteriaceae. Dans le cas des Enterobacteriaceae, le seuil défini pour évaluer l’acceptabilité des différents lots a été dépassé. Les dénombrements ont permis de caractériser la dynamique de croissance des populations bactériennes, mais n’ont donné que très peu d’information sur la diversité bactérienne des échantillons. Dans le but de caractériser celle-ci, une étude métagénomique a été réalisée. Ce champ relativement nouveau de la génétique permet d'étudier les communautés de microorganismes dans leur environnement naturel, en contournant la nécessité de culture et isolement en laboratoire. Les résultats préliminaires révèlent qu’aux ⅔ de la DLC, Aquabacterium était le genre dominant dans les lots d’origines irlandaise et brésilienne, Pseudomonas était le genre dominant dans le lot d’origine australienne. A la fin de la DLC (après conservation à +4 °C), Aquabacterium et Escherichia étaient les genres dominants dans le lot d’origine irlandaise. L’ordre Lactobacillales était le plus abondant dans les lots d’origines brésilienne et australienne. Les différences dans la composition de la population bactérienne de la viande, en particulier en ce qui concerne les bactéries lactiques, pourraient expliquer les longues DLC appliquées dans certains pays. Ces recherches doivent être poursuivies pour identifier les populations bactériennes (et leur source) présentes dans ces viandes et pour étudier leur dynamique au cours de la conservation. [less ▲]

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See detailLa métagénomique au service de la microbiologie alimentaire : étude de l’évolution des populations microbiennes lors du vieillissement de deux matrices alimentaires
Taminiau, Bernard ULg; Nezer, Carine; Poullet, Jean-Baptiste et al

Conference (2011, November 17)

La viande et les préparations à base de viande représentent des biotopes de choix pour les bactéries. L’optimisation de la conservation de ces denrées, importante tant du point de vue économique que de ... [more ▼]

La viande et les préparations à base de viande représentent des biotopes de choix pour les bactéries. L’optimisation de la conservation de ces denrées, importante tant du point de vue économique que de santé publique passe par une meilleure connaissance de ces biotopes et des processus de détérioration de leurs qualités. Les microbiologistes ont depuis longtemps abordé ce problème en utilisant différentes approches. Ainsi, les études basées sur les méthodes de culture ont été complétées par des stratégies axées sur des approches indépendantes de la culture microbiologique. Les techniques actuelles de séquençage de nouvelle génération ont permis de donner une nouvelle dimension à l’écologie microbienne à travers l’analyse métagénomique d’un grand nombre d’individus au sein d’une population microbienne mixte. Notre propos est de démontrer que cette méthodologie peut être appliquée avec succès à l’étude des flores microbiennes des denrées alimentaires et pourra être adaptée, à court terme, aux exigences spécifiques de la microbiologie alimentaire. Dans cette étude, un produit de viande crue et une préparation de viande cuite, la viande hachée de porc et le boudin blanc, ont subi un test de vieillissement dans différentes conditions de conservation (Température et packaging). L’ADN ribosomal 16S a été extrait des produits originaux et des échantillons à la date limite de consommation et, après normalisation, les régions hypervariables V5 et V6 de l’ADN 16S ont été séquencées. Un total d’environ 130.000 séquences ont été obtenues et une analyse métagénomique a abouti à la classification taxonomique au genre pour 80% de cette population. L’analyse subséquente des populations microbiennes montre que les populations microbiennes majoritaires à la date limite de conservation sont bien celles qui sont généralement observées lors d’analyse microbiologiques de ces produits de viande. Toutefois, les populations sous-dominantes et surtout plusieurs populations de germes non cultivables ont pu être identifiée. Ces groupes de bactéries, plus difficile à obtenir par d’autres méthodes (culture-dépendante et indépendante confondues), doivent être étudiées car elles participent aux processus de détériorations de ces matrices alimentaires. Enfin, la sensibilité de cette technologie rend possible l’analyse des denrées alimentaires présentant un taux microbien très faible et permet donc l’identification des contaminants microbiens avant leur développement. [less ▲]

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See detailDevelopment of a quantitative risk assessment for cheese made from raw goat milk contaminated by Listeria monocytogenes
Delhalle, Laurent ULg; Ellouze, Mariem; Clinquart, Antoine ULg et al

Poster (2011, September)

A retrospective study was performed to assess the potential risk of human listeriosis following a contamination by L. monocytogenes of cheeses made from goat raw milk reported by the Belgian Federal ... [more ▼]

A retrospective study was performed to assess the potential risk of human listeriosis following a contamination by L. monocytogenes of cheeses made from goat raw milk reported by the Belgian Federal Agency for the Safety of the Food Chain in 2005. The source of the contamination was related to a shedder goat, excreting 2.6 log cfu (colonies forming units) L. monocytogenes / ml without any clinical symptom. On the basis of the collected data, a quantitative microbial risk assessment model was developed covering the production chain from the milking of goats until the consumed products. Predictive microbiology models were used to simulate the growth of L. monocytogenes during the process of cheeses made from goat raw milk. The modular exposure assessment model showed a significant growth of L. monocytogenes during chilling and storage of the milk collected the day before the cheese production (increase of 1.7 log cfu/ml for the median) and during the step of starter and rennet adjunction to milk (increase of 0.8 log cfu/ml for the median). The median estimated final result (in the fresh cheese) was equal to 3.5 log cfu/g. The model estimates (expressed as median final result issued from the exposure assessment) were realistic compared to the number of L. monocytogenes measured in the fresh cheese (3.6 log cfu/g) reported during the cheese contamination period. The average number of expected cases of human listeriosis was between 0 and 1 for a high-risk sub-population and 0 for a low-risk healthy sub-population. Scenario analysis was finally performed to identify the most significant factors and aid in developing priorities for risk mitigation. Thus, by using quantitative risk assessment and predictive microbiology models, this study provided valuable information to identify and to control critical steps in a local production chain of goat cheese made from raw milk. [less ▲]

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See detailInfluence of temperature on conservability of chilled vacuum packed beef from different origins
Didimo Imazaki, Pedro Henrique ULg; Maréchal, Aline; Nezer, Carine ULg et al

Poster (2011, August 07)

The objective of this experiment was to study the conservability of chilled vacuum-packed meat depending on storage temperature (–1 °C vs. +4 °C) during the last third of their shelf life. Physicochemical ... [more ▼]

The objective of this experiment was to study the conservability of chilled vacuum-packed meat depending on storage temperature (–1 °C vs. +4 °C) during the last third of their shelf life. Physicochemical parameters (pH and colour) and microbiological growth (total aerobic bacteria, lactic acid bacteria, Enterobacteriaceae, Pseudomonas spp. and Brochothrix thermosphacta) of Longissimus dorsi samples from different origins (United Kingdom and Ireland, Australia and Brazil) were measured at: i) 2/3 of their shelf life and ii) the end of their shelf life. Sample bacteria population growing on MRS was identified by API 50 CHL strips. Unlike Irish and British samples, pH of some Australian and Brazilian samples decreased during conservation. The colour of the samples remained stable and it did not seem to be influenced by temperature. All samples conserved at –1 °C presented a satisfactory microbiological quality at the end of their shelf life (British and Irish meat = 35~45 days; Australian meat = 140 days and Brazilian meat = 120 days). On Australian and Brazilian samples, temperature did not influence total aerobic bacteria growth, but conservation at +4 °C favoured lactic acid bacteria and Enterobacteriaceae growth. API 50 CHL strip identifications revealed the presence of bacteria like Lactobacillus brevis, Carnobacterium maltaromaticum and Lactobacillus fermentum, which occur naturally in fresh meat and are known for their bioprotective effect against other microorganisms. Further analyses are being carried out using molecular methods in order to study the initial bacteria population diversity and it evolution during storage. [less ▲]

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See detailCase study of contamination by Listeria monocytogenes in raw goat milk cheese: development of a quantitative risk assessment model of the production chain
Korsak Koulagenko, Nicolas ULg; Delhalle, Laurent ULg; Daube, Georges ULg

Conference (2011, August 03)

Introduction: Quantitative Risk assessment could be applied in industries as a tool to control and manage the safety of food products. Purpose: A contamination by Listeria monocytogenes of cheeses made ... [more ▼]

Introduction: Quantitative Risk assessment could be applied in industries as a tool to control and manage the safety of food products. Purpose: A contamination by Listeria monocytogenes of cheeses made from raw milk was reported by the Belgian food agency. This contamination was caused by the presence of an asymptomatic “shedder” goat in the herd. With field and laboratory collected data, a quantitative risk assessment model of the production chain was developed. Methods: A modular risk model was built to simulate the food production pathway covering the milking of goats until the final product for the customers. A dynamic square root model was used to predict the growth rate in relation with the temperature, the pH and the water activity along the production chain with predictive microbiology modules. Results: The shedder goat was identified from the herd and milk samples were taken from the two different parts of the mammary gland with 2.6 log cfu (colonies forming units) Listeria monocytogenes/ml for the right part and absence in 25 ml for the left part of the mammary gland. Numbering of Listeria monocytogenes was carried out on the final products with 3.6 log cfu/g in the fresh not ripened cheeses. The modular risk assessment shows a significant growth of Listeria monocytogenes during chilling and storage of the milk collected the day before the cheese production (increase of 0.6 log cfu/ml) and during adjunction of ferment and rennet to milk (increase of 0.8 log cfu/ml). The model confirms the results obtained in the final products. Significance: The modular risk model gives valuable informations to identify and to control critical steps in the food production chain of goat cheese made from raw milk. Only one shedder goat can cause a high risk to become ill for the consumers of contaminated products. [less ▲]

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See detailTRANSMISSION ROUTES OF NOROVIRUSES, EMERGING HUMAN PATHOGENS IN FOOD “NORISK”
Mathijs, E; Stals, A; Denayer, S et al

Book published by Belgian Science Policy (2011)

A. Context Noroviruses are pathogens causing gastroenteritis and infections result in typical symptoms such as abdominal cramps, fever, watery diarrhea and other symptoms such as headaches, chills and ... [more ▼]

A. Context Noroviruses are pathogens causing gastroenteritis and infections result in typical symptoms such as abdominal cramps, fever, watery diarrhea and other symptoms such as headaches, chills and general myalgias, which usually last for 2 to 3 days. The illness is self-limiting in most cases. The NV genus contains 5 genogroups whereby genogroup I and II (GI and GII) comprise most of the human infective NV genotypes. Bovine and murine NV are classified respectively in genogroup III (GIII) and V (GV), while porcine NV are also classified in GII. Human infective (mainly GI and GII) noroviruses (NV) have increasingly been recognized as a global major cause of acute non-bacterial gastroenteritis, but sensitive detection is only possible by molecular methods, due to the unavailability of a cultivation system. Development of these molecular methods showed that NV could be responsible for 60 % and 77 % of all gastroenteritis cases with known etiology in the USA and in Europe, respectively. The fraction of NV outbreaks caused by consumption of contaminated foods is estimated to be 10 to 20 %. Food products can be contaminated through 2 main transmission routes: either pre-harvest contamination, whereby mostly fresh produce and bivalve shellfish are involved. Shellfish are contaminated by cultivation in contaminated water, while fresh produce can by contaminated by use of contaminated irrigation water or (post-) harvest contamination often involving an infected food handler or food picker. A broad range of food products are related to the latter transmission route. Detection of NV in foods is more difficult because detection of NV present at very low levels on the foods should be possible due to the low infectious dose. Therefore, (genomic material of) NV has to be extracted from the foods and has to be detected subsequently by a molecular detection method. Furthermore, NVs are present in several animal species, raising important questions about zoonotic transmission and potential animal reservoir. B. Objectives 1. The NV RNA detection methodology: elaboration, optimization and evaluation of a real-time PCR format and determination of specificity, sensitivity and robustness. Two protocols will be developed. A real-time RT-PCR protocol directed to detection of the acknowledged GGI and GGII strains involved in outbreaks to be used in the frame of control and surveillance by food authorities and food business operators to verify their products and production process. Another real-time RT PCR protocol directed towards a wide diversity of NV genogroups (including newly reported animal associated NV) to be used for research purposes to establish transmission routes and document circulating strains in the environment. 2. The sample preparation method: to evaluate the effectiveness of several virus concentration / viral RNA extraction and purification protocols from a variety of food matrices in particular seafoods and with emphasis on elaboration of an appropriate extraction producedure in fresh produce/ready-to-eat foods. 3. The routine detection of NVs in food stuffs (seafoods and fresh products): to develop and implement a standard protocol with establishment of appropriate controls for rapid screening of foods for the presence of NVs in accordance with the guidelines for officially approved analysis and harmonization and to generate information on the prevalence of NV strains in foods at retail, products and production processes under the control of food business operators and the primary production. 4. Elucidation of transmission routes (zoonosis hypothesis) through molecular tracing, with a global view on NV strains circulating among human, animal and also in food. 5. The tracing of outbreaks: scenario for coupling clinical data from NV outbreaks to their foodborne cause and risk evaluation. 6. The development of a risk profile on NV present in the food chain and animal species (strain types circulating, potential animal reservoir, zoonose, definition and incidence in at risk foods, link to epidemiological information). 7. Tracing of the genetic evolution of NVs: genetic profiles and emerging of recombinants C. Conclusions Objective 1: A multiplex real-time RT-PCR assay for simultaneous detection of human GI and GII NV in clinical samples was designed, with the successful inclusion of MNV-1 as real-time PCR IAC. Evaluation of this multiplex assay showed a high concordance between the multiplex assay and the corresponding singleplex PCR assays. Specificity analysis of the multiplex assay by testing a NV RNA reference panel and clinical GI and GII NV samples showed that specific amplification of NV GI and GII was possible. In addition, no cross-amplification was observed when subjecting a collection of bovine NV and other (non-NV) enteric viruses to the multiplex assay. Finally, MNV-1 was successfully integrated as IAC, although a sufficiently low concentration was needed to avoid interference with the possibility of the developed multiplex assay to quantitatively and simultaneously detect the presence of GI and GII NV within one sample. Persistent contamination problems leading to false-positive results were encountered, but an investigation was performed towards the source of the contamination. The problem could be controlled and only occasional contamination has been observed. Objective 2: Two protocols for extraction of NV from soft red fruits (selected as fresh produce product) and ready-to-eat (RTE) foods were evaluated towards robustness and sensitivity. For the RTE foods, the protocol for RTE foods made use of a guanidine isothiocyanate containing reagent to extract viral RNA from the food sample (basic protocol called TriShort) with an eventual concentration/purification step (extended protocol called TriConc). The protocol for extraction of NV from soft red fruits consisted of alkaline elution of NV particles from the food, followed by polyethylene glycol precipitation and organic solvent purification. After purification, the RNA was detected by the multiplex real-time RT-PCR assay optimized in objective 1. The influence of (1) the NV inoculum level and (2) different food types on the recovery of NV from these foods was investigated for both protocols. Overall, the elution –precipitation protocol was able to recover NV from soft red fruits with efficiencies of 10 % to 20 % in most cases while the protocol for RTE foods yielded recovery efficiencies of >1% (TriShort protocol) and 0.1 to 10 % (TriConc protocol). For both NV extraction methods, taking into account all dilution factors resulted in a detection limit of approximately 104 genomic copies/10g. Simultaneous recovery of GI and GII NV in similar or 100-fold different concentrations was possible in both food categories. A significant influence of the NV inoculum level on its recovery was noticeable in both protocols as high inoculum levels were recovered more successfully and with a higher efficiency compared to low level inocula in both protocols. This phenomenon, together with the influence of the food type on the recovery was more explicit on the protocol for RTE foods compared to the protocol for soft red fruits. Objective 3: The multiplex real-time RT-PCR assay described in objective 1 and the virus extraction protocols described in objective 2 were combined to two NV detection methods. The murine norovirus 1 (MNV-1), a cultivable genogroups V NV, was in these detection methods used and evaluated as control reagent. MNV-1 was used to control the entire virus detection protocol (process control; PC), the reverse transcription reaction (reverse transcription control; RTC) and the real-time PCR reaction (internal amplification control; IAC) when detecting NV in foods. Evaluation showed that MNV-1 PC and RTC could be used for detection of inefficient extraction and inhibition of the RT-PCR, respectively. On the other hand, the MNV-1 IAC provided only little added value and it was suggested to leave this control out. Objective 4: Screening of 75 fruit samples for NV presence was performed using the protocol for soft red fruits (objective 2) and the multiplex real-time RT-PCR assay (objective 1). MNV-1 was used as PC, RTC and IAC. A total of 18 samples tested positive for GI and/or GII NV despite good bacteriological quality. Results obtained showed the difficulty of expressing positive (real-time) PCR results towards terms of public health threat if no associated diseases or outbreaks are reported. Although these low NV levels might indicate virus contamination at some point during the fresh produce chain, care should be taken to translate these results as a significant risk to the public health. Nevertheless, a possible risk for food borne transmission of NV from these food products cannot be excluded either. Genotyping results from 115 clinical samples originating from gastro-enteritis epidemics reported to the Scientific Institute of Public Health allowed us to characterise the NV strains implicated in these outbreaks between 2007 and 2010. Similarly, the creation of a stool bank with domestic animal clinical samples and NV screening in these samples in the first part of the NORISK project have allowed the characterisation of animal NVs especially in the bovine and porcine species. These results confirm that bovine and porcine NVs may be endemic in our counties but besides these animal NVs, no other animal NV was detected in the other animal species selected for the stool bank. Objective 5: After the introduction of Norovirus specific analysis method in the surveillance of foodborne outbreaks, it became clear, that Norovirus is an important agent causing foodborne outbreaks in Belgium. During the last three years it was even the leading reported agent. It became also clear that it is not so easy to define the transmission routes of Norovirus. By the introduction of a scenario for gastro-enteritis a classification based on the possible transmission route was possible. In all the reported outbreaks no primary contaminated food like bivalve shellfish or red fruits was involved. Secondary contaminated food plays an imported role in the transmission of Norovirus with an infected food handler as a crucial vector. Besides the food related outbreaks it became clear that person-to person transmission and a high environmental contamination are risk factors in the further transmission of Norovirus in the population The fact that many people are living close together in for example youth camps or elderly homes, the common use of sanitary facilities and the common preparation of meals, combined with the high infectivity of Norovirus and the existence of asymptomatic carriers, results in highly vulnerable populations in these conditions. Although Norovirus infections often end up in a positive way, they may have a high impact on the health (eg elderly homes) and may cause a lot of costs (less personnel at work) and sorrow (eg closure of a youth camp). Although both the prevention and decrease of the risk of a Norovirus infection are not evident, some measures have to be taken. A good hand-, toilet- and kitchen hygiene, a good infrastructure and the rapid signaling of gastro-enteritis outbreaks can decrease the risk of Norovirus infection and might restrict further spread of the virus. The knowledge rising from the Norovirus outbreaks reported at the NRL FBO allowed use to formulate and publish specific measures and recommendations for Norovirus outbreaks, which help the inspectors and physicians in the rapid diagnosis and prevention of the further transmission of Norovirus outbreaks. Objective 6: Throughout the NORISK project, NVs were detected in different food matrices available for human consumption, in humans and in animals like cattle and pigs. For a better comprehension of NV transmission routes, sequences of the detected NVs were determined and submitted for further analysis. Genotyping of NVs in food matrices came out to be a real challenge and consisted into a bottleneck as the amount of genetic material on food was insufficient for PCR amplification and sequencing. This obstacle was not overcome during our project and NV sequences were only obtained from clinical samples in humans and animals. Interesting was that no animal NVs were detected in samples originating from humans and no human sequences were amplified from animal clinical samples. Thus, there is no evidence of a potential interspecies NV transmission and zoonotic transmissions seem unlikely to occur. However NV, being an RNA virus, exhibit great genomic plasticity and changes in its genome could lead to the emergence of new NV variant with different biological proprieties that should not be left out (objective 7). Objective 7: Sequences obtained in the human and bovine clinical samples show different NV strains that exhibit incoherent clustering for the partial sequences of the polymerase and the capsid region indicating that they might be recombinant. For the human NV strains, although the majority of the gastroenteritis outbreaks were involved with GII.4 NVs in 2007 and 2008, other GII NVs were detected from the end of 2008 to 2010 along GII.4 NVs. Among these NVs, a variety of new recombinants were detected in different samples from different outbreaks between 2008 and 2010. New « super » polymerase sequences (GII.e and GII.g) related to the previously described GII.b polymerase were detected in the same period. The exact significance of the emergence of these polymerases or their origin has yet to be elucidated but their involvement with different outbreaks might indicate that they have a selective advantage upon the capsid parental strains. Based on sequencing data, norovirus (NV) recombinants have been described, but no experimental evidence of recombination in NVs has been documented. Using the murine norovirus (MNV) model, we investigated the occurrence of genetic recombination between two co-infecting wild-type MNV isolates in RAW cells. The design of a PCR-based genotyping tool allowed accurate discrimination between the parental genomes and the detection of a viable recombinant MNV (Rec MNV) in the progeny viruses. Genetic analysis of Rec MNV identified a homologous-recombination event located at the ORF1–ORF2 overlap. Rec MNV exhibited distinct growth curves and produced smaller plaques than the wild-type MNV in RAW cells. Here, we demonstrate experimentally that MNV undergoes homologous recombination at the previously described recombination hot spot for NVs, suggesting that the MNV model might be suitable for in vitro studies of NV recombination. Moreover, the results show that exchange of genetic material between NVs can generate viruses with distinct biological properties from the parental viruses. [less ▲]

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See detailA new tool to control meat products safety: a web based application of predictive microbiology models
Delhalle, Laurent ULg; Adolphe, Ysabelle ULg; Crevecoeur, Sébastien ULg et al

Conference (2011)

Predictive microbiology is considered by the European legislation as a tool to control food safety. Meat and meat products are particularly sensitive to contamination with pathogens. However, development ... [more ▼]

Predictive microbiology is considered by the European legislation as a tool to control food safety. Meat and meat products are particularly sensitive to contamination with pathogens. However, development of predictive microbiology models and interpretation of results require specific knowledge. A free web based model has been developed for an easy use by people who are not experts in this field as industries and public authorities. The model can simulate the growth of Salmonella spp, Listeria monocytogenes and Escherichia coli O157 in minced pork meat and on pork meat product (white pudding) under different environmental conditions. The model provides simulations under static or dynamic conditions over time. The user also has the opportunity to import the specific growth rate and cardinal parameters of a bacterium. Unlike polynomial models currently available, this free web access model is distinguished by the use of secondary square roots and primary logistic model with delay. This model permits to have a real time process management, to prospect new formulation for safer products or to design safer processes, to estimate the shelf life of a food product, etc [less ▲]

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See detailDiversity, phylogenetic relationship and antibacterial potential of Bifidobacterium species isolated from raw milk production chain in Abidjan (Côte d'Ivoire)
Kouamé-Sina, Sylvie Mireille; Dadié, Adjéhi; Makita, Kohei et al

in African Journal of Microbiology Research [=AJMR] (2011), 5(21), 3394-3403

The local dairy commodity, from farm to retail point, is informal and often escapes safety surveillance and results in high contamination of local milk by pathogens. The objective of this study was to ... [more ▼]

The local dairy commodity, from farm to retail point, is informal and often escapes safety surveillance and results in high contamination of local milk by pathogens. The objective of this study was to determine the biodiversity of Bifidobacterium species in the informal dairy production chain in Abidjan and evaluate their potential antibacterial activity against pathogens. Bifidobacterium species were identified after sequencing of hsp60 genes. Results showed that Bifidobacterium were present in 9% of samples. Milkers' hands (14%) and cows’s udders (14%) were the most contaminated with Bifidobacterium. These isolates belong to five different species. Most Bifidobacterium isolated are Bifidobacterium minimum (53%) and Bifidobacterium pseudolongum subsp. Globosum (24.4%). The other strains are composed of one strain of Bifidobacterium thermophilum, Bifidobacterium thermacidophilum subsp. suis and Bifidobacterium magnum. The isolated Bifidobacterium species have antibacterial activities that are not related to bacteriocins production, but to organic acids production (65%), which exert in vitro inhibitory action against Listeria monocytogenes, Salmonella hadar and Salmonella typhimurium, Staphylococcus aureus, Escherichia coli O27 and Escherichia coli O157H7. However, ensuring milk safety along the local milk production chain requires implementation of good hygiene practices together with adapted technology, such as fermentation. [less ▲]

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See detailBifidobacterium pseudolongum are efficient indicators of animal fecal contamination in raw milk cheese industry
Delcenserie, Véronique ULg; Gavini, Françoise; China, Bernard et al

in BMC Microbiology (2011), 11(178),

Background: The contamination of raw milk cheeses (St-Marcellin and Brie) from two plants in France was studied at several steps of production (raw milk, after addition of rennet - St-Marcellin - or after ... [more ▼]

Background: The contamination of raw milk cheeses (St-Marcellin and Brie) from two plants in France was studied at several steps of production (raw milk, after addition of rennet - St-Marcellin - or after second maturation - Brie -, after removal from the mold and during ripening) using bifidobacteria as indicators of fecal contamination. Results: Bifidobacterium semi-quantitative counts were compared using PCR-RFLP and real-time PCR. B. pseudolongum were detected in 77% (PCR-RFLP; 1.75 to 2.29 log cfu ml-1) at the different production steps) and 68% (real-time PCR; 2.19 to 2.73 log cfu ml-1) of St-Marcellin samples and in 87% (PCR-RFLP; 1.17 to 2.40 log cfu ml-1) of Brie cheeses samples. Mean counts of B. pseudolongum remained stable along both processes. Two other populations of bifidobacteria were detected during the ripening stage of St-Marcellin, respectively in 61% and 18% of the samples (PCR-RFLP). The presence of these populations explains the increase in total bifidobacteria observed during ripening. Further characterization of these populations is currently under process. Forty-eight percents (St-Marcellin) and 70 % (Brie) of the samples were B. pseudolongum positive / E. coli negative while only 10 % (St-Marcellin) and 3 % (Brie) were B. pseudolongum negative / E. coli positive. Conclusions: The increase of total bifidobacteria during ripening in Marcellin’s process does not allow their use as fecal indicator. The presence of B. pseudolongum along the processes defined a contamination from animal origin since this species is predominant in cow dung and has never been isolated in human feces. B. pseudolongum was more sensitive as an indicator than E. coli along the two different cheese processes. B. pseudolongum should be used as fecal indicator rather than E. coli to assess the quality of raw milk and raw milk cheeses. Results: Bifidobacterium semi-quantitative counts were compared using PCR-RFLP and real-time PCR. Bif. pseudolongum were detected in 77% (PCR-RFLP; 1.75 to 2.29 log cfu ml-1) at the different production steps) and 68% (real-time PCR; 2.19 to 2.73 log cfu ml-1) of St-Marcellin samples and in 87% (PCR-RFLP; 1.17 to 2. 40 log cfu ml-1) of Brie cheeses samples. Mean counts of Bif. pseudolongum remained stable along both processes. Two other populations of bifidobacteria were detected during the ripening stage of St-Marcellin, respectively in 61% and 18% of the samples (PCR-RFLP). The presence of these populations explains the increase in total bifidobacteria observed during ripening. Further identification of these species is currently under process. Forty-eight percents (St-Marcellin) and 70 % (Brie) of the samples were Bif. pseudolongum positive / E. coli negative while only 10 % (St-Marcellin) and 3 % (Brie) were Bif. pseudolongum negative / E. coli positive. Conclusions: The increase of total bifidobacteria during ripening in Marcellin’s process does not allow their use as fecal indicator. The presence of Bif. pseudolongum along the processes defined a contamination from animal origin since this species is predominant in cow dung and has never been isolated in human feces. Bif. pseudolongum was more sensitive as an indicator than E. coli along the two different cheese processes. Bif. pseudolongum should be used as fecal indicator rather than E. coli to assess the quality of raw milk and raw milk cheeses. [less ▲]

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See detailChilling of heavy carcasses from double muscled cattle: time-temperature evolution and predictive modelling of growth of Listeria monocytogenes and Clostridium perfringens
Delhalle, Laurent ULg; Collignon, Bertrand; Dehard, Sandrine ULg et al

Poster (2010, August)

The time/temperature combination during carcass chilling is of concern in order to avoid bacterial growth. The chilling speed is lower in carcasses with high muscular development such as large cattle from ... [more ▼]

The time/temperature combination during carcass chilling is of concern in order to avoid bacterial growth. The chilling speed is lower in carcasses with high muscular development such as large cattle from the Belgian Blue breed. Three slaughterhouses were selected for temperature and pH measurements during the chilling process at 6 different days on 4 half carcasses in order to obtain representative data from heavy carcasses with high muscular development. Predictive microbiology was used to evaluate the potential growth of Listeria monocytogenes and Clostridium perfringens on the surface and in the depth of the carcasses. The gamma concept was chosen as secondary model taking into account the effect of temperature, pH and water activity on the selected bacteria during the chilling process. The predicted growth potential of Listeria monocytogenes is influenced by the different environmental conditions of the selected slaughterhouses and could reach 1.4 log CFU/cm² after the chilling process. The potential growth of Clostridium perfringens is limited due to unfavourable conditions during the first hours and to low temperature later. It can be concluded that when the initial level of contaminating bacteria is not excessive the speed at which the carcass is currently chilled is sufficient to limit the growth of these two pathogens and to ensure the product quality [less ▲]

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See detailLa microbiologie prévisionnelle : un nouvel outil de maîtrise de la qualité microbiologique des denrées alimentaires
Delhalle, Laurent ULg; Clinquart, Antoine ULg; Daube, Georges ULg

in Food Science and Law (2010), 2

Les nouveaux outils de microbiologie prévisionnelle permettent de mieux comprendre et de maîtriser le comportement des micro-organismes dans la chaîne alimentaire. La législation européenne récente, dont ... [more ▼]

Les nouveaux outils de microbiologie prévisionnelle permettent de mieux comprendre et de maîtriser le comportement des micro-organismes dans la chaîne alimentaire. La législation européenne récente, dont le but est garantir un niveau élevé de sécurité sanitaire des aliments proposés au consommateur, mentionne cette méthodologie comme outil de référence. Les autorités en charge de la gestion de la santé publique et les industriels ont maintenant à leur disposition des outils leur permettant de comprendre les flux de contamination dans la chaîne alimentaire, de fixer une date limite de consommation, d’optimaliser les processus de transformation et de conservation, et de proposer des aliments sûrs aux consommateurs. Cette discipline offre la possibilité, à travers ses modèles et ses concepts, d’exploiter des données issues d’expérimentations réalisées au laboratoire ou en production et de les convertir en applications qui permettent d’évaluer la stabilité et la sécurité d’une denrée alimentaire. [less ▲]

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See detailModeling Microbial Cross-contamination in Quick Service Restaurants by Means of Experimental Simulations With Bacillus Spores
Baptista Rodrigues, Ana Lúcia ULg; Crevecoeur, Sébastien ULg; Dure, Remi et al

Poster (2010)

Cross contamination has been frequently mentioned as being in the origin of a wide range of food borne outbreaks. Handling of food is one of the ways through which cross contamination may occur. For many ... [more ▼]

Cross contamination has been frequently mentioned as being in the origin of a wide range of food borne outbreaks. Handling of food is one of the ways through which cross contamination may occur. For many different reasons, quick service restaurants are particularly at risk. Due to its importance, cross contamination via the hands should be taken into consideration when carrying out a quantitative risk assessment. The main goal of this study was to determine transfer rates of bacteria to and via the hands, reduction rates of two hand sanitizing procedures and to apply the results to a quantitative microbial risk assessment model. According to our results, handling of a portion of raw minced meat contaminated at 4.104 cfu leads to the presence of 24 cfu on both hands, 3 cfu on ready-to-eat product (RTE) manipulated with unwashed hands, 1 cfu on RTE manipulated with wiped hands and absence on RTE manipulated with washed hands. This study provides adequate quantitative data for quantitative microbial risk assessment. [less ▲]

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See detailNUSAP: a method to evaluate the quality of assumptions in quantitative microbial risk assessment
Boone, Ides; Van der Stede, Yves; Dewulf, Jeroen et al

in Journal of Risk Research (2010), 13

implemented to evaluate assumptions in a quantitative microbial risk assessment (QMRA) model for Salmonella spp. in minced pork meat. This QMRA model allows the testing of mitigation strategies for the ... [more ▼]

implemented to evaluate assumptions in a quantitative microbial risk assessment (QMRA) model for Salmonella spp. in minced pork meat. This QMRA model allows the testing of mitigation strategies for the reduction of human salmonellosis and aims to serve as a basis for science-based policy making. The NUSAP method was used to assess the subjective component of assumptions in the QMRA model by a set of four pedigree criteria: ‘the influence of situational limitations’, ‘plausibility’, ‘choice space’ and ‘the agreement among peers’. After identifying 13 key assumptions relevant for the QMRA model, a workshop was organized to assess the importance of these assumptions on the output of the QMRA. The quality of the assumptions was visualized using diagnostic and kite diagrams. The diagnostic diagram pinpointed assumptions with a high degree of subjectivity and a high ‘expected influence on the model results’ score. Examples of those assumptions that should be dealt with care are the assumptions regarding the concentration of Salmonella on the pig carcass at the beginning of the slaughter process and the assumptions related to the Salmonella prevalence in the slaughter process. The kite diagrams allowed a clear overview of the pedigree scores for each assumption as well as a representation of expert (dis)agreement. The evaluation of the assumptions using the NUSAP system enhanced the debate on the uncertainty and its communication in the results of a QMRA model. It highlighted the model’s strong and weak points and was helpful for redesigning critical modules. Since the evaluation of assumptions allows a more critical approach of the QMRA process, it is useful for policy makers as it aims to increase the transparency and acceptance of management decisions based on a QMRA model. [less ▲]

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See detailChilling of carcasses from double muscled cattle: time-temperature evolution and predictive modelling of growth of Listeria monocytogenes and Clostridium perfringens
Delhalle, Laurent ULg; Collignon, Bertrand; Dehard, Sandrine ULg et al

Poster (2010)

The time/temperature combination during carcass chilling is of concern in order to avoid bacterial growth. The chilling speed is lower in carcasses with high muscular development such as large cattle from ... [more ▼]

The time/temperature combination during carcass chilling is of concern in order to avoid bacterial growth. The chilling speed is lower in carcasses with high muscular development such as large cattle from the Belgian Blue breed. Three slaughterhouses were selected for temperature and pH measurements during the chilling process at 6 different days on 4 half carcasses in order to obtain representative data from heavy carcasses with high muscular development. Predictive microbiology was used to evaluate the potential growth of Listeria monocytogenes and Clostridium perfringens on the surface and in the depth of the carcasses. The gamma concept was chosen as secondary model taking into account the effect of temperature, pH and water activity on the selected bacteria during the chilling process. The predicted growth potential of Listeria monocytogenes is influenced by the different environmental conditions of the selected slaughterhouses and could reach 1.4 log CFU/cm² after the chilling process. The potential growth of Clostridium perfringens is limited due to unfavourable conditions during the first hours and to low temperature later. It can be concluded that when the initial level of contaminating bacteria is not excessive the speed at which the carcass is currently chilled is sufficient to limit the growth of these two pathogens and to ensure the product quality. [less ▲]

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