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  • 1. Lofstrom, C.
    et al.
    Eriksson, J.
    Aspan, A.
    Haggblom, P.
    Gunnarsson, A.
    Borch, Elisabeth
    SIK – Institutet för livsmedel och bioteknik.
    Improvement and validation of RAPD in combination with PFGE analysis of Salmonella enterica ssp. enterica serovar Senftenberg strains isolated from feed mills2006In: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 114, no 42067, p. 345-351Article in journal (Refereed)
    Abstract [en]

    In 1995 and 1996 a Swedish feed mill had problems due to a persistent contamination of Salmonella enterica spp. enterica serovar Senftenberg that was difficult to eliminate. Forty-eight strains isolated from the feed mill, together with unrelated strains included to evaluate the discriminatory power and reproducibility, were analysed by pulsed-field gel electrophoresis (PFGE). The source of contamination in the feed mill was identified and preventative measures were taken, that led to a resolution of the problem. A previously developed randomly amplified polymorphic DNA (RAPD) protocol was used, to evaluate a rapid and low-cost alternative to PFGE typing. The use of the alternative thermostable DNA polymerase Tth was shown to increase the reproducibility of the RAPD analysis. The reproducibility, in terms of Pearson's and Dice's similarity coefficients for duplicate runs, increased from 72.0 ± 16.9% and 72.3 ± 12.9% for Taq to 91.6 ± 7.5% and 90.9 ± 5.3% for the fingerprints obtained for the RAPD method employing Tth DNA polymerase. Simpson's index of diversity was calculated and found to be 0.580 for RAPD and 0.896 for PFGE. All of the seven RAPD types could be subdivided into one or more PFGE types, whereas none of the 22 PFGE types was divided into more than one RAPD type. RAPD provides a simple, rapid and powerful screening method that can be used to initially select isolates for further analysis by PFGE. © 2005 Elsevier B.V. All rights reserved.

  • 2.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Hansen, F.
    Hoorfar, J.
    Validation of a 20-h real-time PCR method for screening of Salmonella in poultry faecal samples2010In: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 144, no 3-4, p. 511-514Article in journal (Refereed)
    Abstract [en]

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

  • 3.
    Löfström, Charlotta
    et al.
    DTU Technical University of Denmark, Denmark.
    Hintzmann, Ann-Sofie
    DTU Technical University of Denmark, Denmark.
    Sørensen, Gitte
    DTU Technical University of Denmark, Denmark.
    Baggesen, Dorte Lau
    DTU Technical University of Denmark, Denmark.
    Outbreak of Salmonella enterica serovar Typhimurium phage type DT41 in Danish poultry production2015In: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 178, no 1-2, p. 167-172Article in journal (Refereed)
    Abstract [en]

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

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

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

  • 5.
    Pedersen, Karl
    et al.
    DTU Technical University of Denmark, Denmark.
    Sørensen, Gitte
    DTU Technical University of Denmark, Denmark.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Leekitcharoenphon, Pimlapas
    DTU Technical University of Denmark, Denmark.
    Nielsen, Bent
    Danish Agriculture and Food Council, Denmark.
    Wingstrand, Anne
    DTU Technical University of Denmark, Denmark.
    Aarestrup, Frank M.
    DTU Technical University of Denmark, Denmark.
    Hendriksen, René S.
    DTU Technical University of Denmark, Denmark.
    Baggesen, Dorte Lau
    DTU Technical University of Denmark, Denmark.
    Reappearance of Salmonella serovar Choleraesuis var. Kunzendorf in Danish pig herds2015In: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 176, no 3-4, p. 282-291Article in journal (Refereed)
    Abstract [en]

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

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