A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 305 It is required for tetrathionate respiration in Salmonella The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay The detection probabilities were 70% when a Salmonella cell suspension containing 103 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 104 CFU/ml was used A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella The diagnostic accuracy was shown to be 100% compared to the traditional culture method The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods Studies are planned to assess the interlaboratory performance of this diagnostic PCR method

Diagnostic Real-Time PCR for Detection of Salmonella in Food

A European Union-wide baseline survey on Campylobacter in broiler batches and on Campylobacter and Salmonella on broiler carcasses was carried out in 2008. A total of 10,132 broiler batches were sampled from 561 slaughterhouses in 26 European Union Member States and two countries not belonging to the European Union. From each randomly selected batch the caecal contents of 10 slaughtered broilers were collected, pooled and examined for Campylobacter. From the same batch one carcass was collected after chilling and the neck skin together with the breast skin was examined for the presence of Campylobacter and Salmonella, in addition to the determination of the Campylobacter counts. Campylobacter was detected in pooled caecal contents of broilers and on broiler carcasses in all participating countries. At Community level the prevalence of Campylobacter-colonised broiler batches was 71.2% and that of Campylobacter-contaminated broiler carcasses was 75.8%. The Member State prevalence varied from 2.0% to 100.0% and from 4.9% to 100.0%, for caecal contents and carcasses, respectively. The results of the counts of Campylobacter on broiler carcasses showed substantial variation among the countries in contamination levels. About two-thirds of the Campylobacter isolates from the pooled caecal contents as well as from the broiler carcasses were identified as Campylobacter jejuni, while one-third was Campylobacter coli. Twenty-two Member States and one non-Member State isolated Salmonella on the broiler carcasses, with a Community prevalence of 15.7%. This prevalence varied widely among the Member States, from 0.0% to 26.6%. However, one Member State had an exceptionally high prevalence of 85.6% with the majority of isolates being S. Infantis. The Community prevalence of Salmonella Enteritidis or Salmonella Typhimurium-contaminated broiler carcasses was 3.6%. Salmonella Infantis and Salmonella Enteritidis were the two most frequently isolated serovars on broiler carcasses in the EU and accounted for about onethird and one-sixth of the Salmonella isolates, respectively.

/pdf/analysis-of-the-baseline-survey-on-the-prevalence-of-2xebujo8bi.pdf

Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2008, Part A: Campylobacter and Salmonella prevalence estimates

Salmonellosis is the second leading cause of bacterial foodborne illness in the United States, and the great majority of these infections are associated with the consumption of products such as meat, poultry, eggs, milk, seafood, and fresh produce contaminated with Salmonella. The per capita consumption of meat and poultry in United States has increased significantly over the past century. This increase is especially evident with poultry products, where there has been a nearly 6-fold increase in chicken consumption and 17-fold increase in turkey consumption since 1909. The per capita consumption of pork has also increased over this time from 18.7 to 21.7 kg/yr. With this increase in meat and poultry consumption, the dynamics of animal production and consumer exposure have changed leading to new challenges in limiting salmonellosis. To meet the demands of consumers, more intensive agricultural practices have been adopted, which has likely changed the population characteristics of Salmonella present among poultry flocks and swine populations. In Salmonella isolated from swine in the United States, S. Typhimurium has replaced S. Choleraesuis as the predominant serovar in recent years. Among isolates from turkeys collected in 2004, serovars S. Senftenberg and S. Hadar were most common overall; however, S. Heidelberg was most common from clinical diagnostic sources, potentially indicating increased virulence. Salmonella Heidelberg was also the most commonly detected serovar among chicken isolates from clinically ill birds and Salmonella surveillance samples. Overall among the 10 serovars most commonly associated with human infections, 6 are also found in the top serovars of swine and poultry. These include S. Typhimurium, S. Enteritidis, S. Heidelberg, S. Montevideo, S. Saintpaul, and S. I 4,[5],12:i:-.

Salmonella challenges: prevalence in swine and poultry and potential pathogenicity of such isolates.

ABSTRACT A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 103 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 104 CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.

Diagnostic real-time PCR for detection of Salmonella in food andcharacterization of epidemiologically important Salmonellaenterica subsp. enterica serovars isolated from livestock,food and humans

Salmonella spp. represent one of the most common causes of bacterial foodborne illnesses around the world. The species Salmonella enterica contains more than 2500 serotypes, and emergence of new human pathogenic Salmonella strains and serotypes represents a major public health issue. Salmonella enterica subsp. enterica serotype 4,5,12:i:– represents a monophasic variant of Salmonella Typhimurium, which has rarely been identified before the mid-1990s. The prevalence of this serotype among human salmonellosis cases has increased considerably since the mid-1990s and Salmonella 4,5,12:i:– currently (i.e., the first decade of the 2000s) represents one of the most common serotypes among human cases in many countries around the world. This paper discusses our current knowledge of the global ecology, epidemiology, transmission, and evolution of this emerging Salmonella serotype.

/pdf/emergence-distribution-and-molecular-and-phenotypic-5ezi0gc6op.pdf

Emergence, distribution, and molecular and phenotypic characteristics of Salmonella enterica serotype 4,5,12:i:-.

Pulsed-field gel electrophoresis, plasmid profiling, and phage typing were used to characterize and determine possible genetic relationships between 48 Salmonella enterica subsp. enterica isolates of pig origin collected in Catalonia, Spain, from 1998 to 2000. The strains were grouped into 23 multidrug-resistant fljB-lacking S. enterica serovar 4,5,12:i:- isolates, 24 S. enterica serovar Typhimurium isolates, and 1 S. enterica serovar 4,5,12:-:- isolate. After combining the XbaI and BlnI macrorestriction profiles (XB profile), we observed 29 distinct subtypes which were grouped into seven main patterns. All 23 of the 4,5,12:i:- serovar strains and 10 serovar Typhimurium isolates were found to have pattern AR, and similarities of >78% were detected among the subtypes. Three of the serovar Typhimurium DT U302 strains (strains T3, T4, and T8) were included in the same 4,5,12:i:- serovar cluster and shared a plasmid profile (profile I) and a pattern of multidrug resistance (resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline, gentamicin, and trimethoprim-sulfamethoxazole) commonly found in monophasic isolates. This led us to the conclusion that strains of the S. enterica 4,5,12:i:- serovar might have originated from an S. enterica serovar Typhimurium DT U302 strain.

Several Salmonella enterica subsp. enterica Serotype 4,5,12:i:− Phage Types Isolated from Swine Samples Originate from Serotype Typhimurium DT U302

Classical biotyping characterizes strains by creating biotype profiles that consider only positive and negative results for a predefined set of biochemical tests. This method allows Salmonella subspecies to be distinguished but does not allow serotypes and phage types to be distinguished. The objective of this study was to determine the relatedness of isolates belonging to distinct Salmonella enterica subsp. enterica serotypes by using a refined biotyping process that considers the kinetics at which biochemical reactions take place. Using a Vitek GNI+ card for the identification of gram-negative organisms, we determined the biochemical kinetic reactions (28 biochemical tests) of 135 Salmonella enterica subsp. enterica strains of pig origin collected in Spain from 1997 to 2002 (59 Salmonella serotype Typhimurium strains, 25 Salmonella serotype Typhimurium monophasic variant strains, 25 Salmonella serotype Anatum strains, 12 Salmonella serotype Tilburg strains, 7 Salmonella serotype Virchow strains, 6 Salmonella serotype Choleraesuis strains, and 1 Salmonella enterica serotype 4,5,12:−:− strain). The results were expressed as the colorimetric and turbidimetric changes (in percent) and were used to enhance the classical biotype profile by adding kinetic categories. A hierarchical cluster analysis was performed by using the enhanced profiles and resulted in 14 clusters. Six major clusters grouped 94% of all isolates with a similarity of ≥95% within any given cluster, and eight clusters contained a single isolate. The six major clusters grouped not only serotypes of the same type but also phenotypic serotype variations into individual clusters. This suggests that metabolic kinetic reaction data from the biochemical tests commonly used for classic Salmonella enterica subsp. enterica biotyping can possibly be used to determine the relatedness between isolates in an easy and timely manner.

Use of Biochemical Kinetic Data To Determine Strain Relatedness among Salmonella enterica subsp. enterica Isolates

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Several Salmonella enterica subsp. enterica Serotype 4,5,12:i:− Phage Types Isolated from Swine Samples Originate from Serotype Typhimurium DT U302

Use of Biochemical Kinetic Data To Determine Strain Relatedness among Salmonella enterica subsp. enterica Isolates