Epidemiological and molecular investigations of Salmonella isolated from duck farms in southwest and around area of Shandong, China.

Salmonella is a zoonotic pathogen posing a serious risk to the farming industry and public health due to food animals serving as reservoirs for future contamination and spread of Salmonella. The present study is designed to monitor the contamination status of Salmonella in duck farms and the main control points during breeding. 160 strains of duck-derived Salmonella were isolated from the 736 samples (cloacal swabs, feces, water, feed, soil, air and dead duck embryos) collected in southwest Shandong Province and the province's surrounding area. The percentage of Salmonella-positive samples collected was 21.74 % (160/736), and the greatest prevalence from duck embryo samples (40.00 %, 36/90). These Salmonella were classified into 23 serotypes depending on their O and H antigens, in which S. Typhimurium (30.15 %), S. Kottbus (13.97 %) and S. Enteritidis (10.29 %) were the prevailing serotypes. Subsequently, the molecular subtyping was done. Clustered regularly interspaced short palindromic repeats (CRISPR) analysis showed that 41 strains of S. Typhimurium and 14 strains of S. Enteritidis were classified into 13 and 3 genotypes, respectively. 19 S. Kottbus isolates from different sources featured ST1546, ST198, ST321, and ST1690 by multilocus sequence typing (MLST) analysis, among which ST1546 belongs to S. Kottbus was a new ST. The minimum spanning tree analysis based on the two CRISPR loci and seven MLST loci from all S. Typhimurium, S. Enteritidis and S. Kottbus isolates revealed that duck embryos, feed and water were key control points to the spread of Salmonella along the breeding chain. Meanwhile, the emergence of S. Kottbus in duck flocks was considered a potential public health hazard.

Challenges Associated with Investigating Salmonella Enteritidis with Low Genomic Diversity in New York State: The Impact of Adjusting Analytical Methods and Correlation with Epidemiological Data.

Defining investigation-worthy genomic clusters among strains of Salmonella Enteritidis is challenging because of their highly clonal nature. We investigated a cluster identified by core genome multilocus sequence typing (cgMLST) consisting of 265 isolates with isolation dates spanning two and a half years. This cluster experienced chaining, growing to a range of 14 alleles. The volume of isolates and broad allele range of this cluster made it difficult to ascertain whether it represented a common-source outbreak. We explored laboratory-based methods to subdivide and refine this cluster. These methods included using cgMLST with a narrower allele range, whole genome multilocus sequence typing (wgMLST) and high-quality single-nucleotide polymorphism (hqSNP) analysis. At each analysis level, epidemiologists retroactively reviewed exposures, geography, and temporality for potential commonalities. Lowering the threshold to 0 alleles using cgMLST proved an effective method to refine this analysis, resulting in this large cluster being subdivided into 34 smaller clusters. Additional analysis by wgMLST and hqSNP provided enhanced cluster resolution, with the majority of clusters being further refined. These analysis methods combined with more stringent allele thresholds and layering of epidemiologic data proved useful in helping to subdivide this large cluster into actionable subclusters.

Comparison of conventional molecular and whole-genome sequencing methods for subtyping Salmonella enterica serovar Enteritidis strains from Tunisia.

We sought to determine the relative value of conventional molecular methods and whole-genome sequencing (WGS) for subtyping Salmonella enterica serovar Enteritidis recovered from 2000 to 2015 in Tunisia and to investigate the genetic diversity of this serotype. A total of 175 Salmonella Enteritidis isolates were recovered from human, animal, and foodborne outbreak samples. Pulsed-field gel electrophoresis (PFGE), multiple locus variable-number tandem repeat analysis (MLVA), and whole-genome sequencing were performed. Eight pulsotypes were detected for all isolates with PFGE (DI = 0.518). Forty-five Salmonella Enteritidis isolates were selected for the MLVA and WGS techniques. Eighteen MLVA profiles were identified and classified into two major clusters (DI = 0.889). Core genome multilocus typing (cgMLST) analysis revealed 16 profiles (DI = 0.785). Whole-genome analysis indicated 660 single-nucleotide polymorphism (SNP) divergences dividing these isolates into 43 haplotypes (DI = 0.997). The phylogenetic tree supported the classification of Salmonella Enteritidis isolates into two distinct lineages subdivided into five clades and seven subclades. Pairwise SNP differences between the isolates ranged between 302 and 350. We observed about 311 SNP differences between the two foodborne outbreaks, while only less or equal to 4 SNP differences within each outbreak. SNP-based WGS typing showed an excellent discriminatory power comparing with the conventional methods such as PFGE and MLVA. Besides, we demonstrate the added value of WGS as a complementary subtyping method to discriminate outbreak from non-outbreak isolates belonging to common subtypes. It is important to continue the survey of Salmonella Enteritidis lineages in Tunisia using WGS.

Genome sequence analysis of 91 Salmonella Enteritidis isolates from mice caught on poultry farms in the mid 1990s.

A total of 91 draft genome sequences were used to analyze isolates of Salmonella enterica serovar Enteritidis obtained from feral mice caught on poultry farms in Pennsylvania. One objective was to find mutations disrupting open reading frames (ORFs) and another was to determine if ORF-disruptive mutations were present in isolates obtained from other sources. A total of 83 mice were obtained between 1995-1998. Isolates separated into two genomic clades and 12 subgroups due to 742 mutations. Nineteen ORF-disruptive mutations were found, and in addition, bigA had exceptional heterogeneity requiring additional evaluation. The TRAMS algorithm detected only 6 ORF disruptions. The sefD mutation was the most frequently encountered mutation and it was prevalent in human, poultry, environmental and mouse isolates. These results confirm previous assessments of the mouse as a rich source of Salmonella enterica serovar Enteritidis that varies in genotype and phenotype.

Analysis of Salmonella enterica serovar Enteritidis isolates from chickens and chicken meat products in Malaysia using PFGE, and MLST.

BACKGROUND: Salmonella is a very important foodborne pathogen causing illness in humans. The emergence of drug-resistant strains also constitutes a serious worry to global health and livestock productivity. This study investigated Salmonella isolates from chicken and chicken meat products using the phenotypic antimicrobial screening as well as the molecular characteristics of Salmonella isolates. Upon serotyping of the isolates, the antimicrobial susceptibility profiling using a panel of 9 commonly used antimicrobials was done. Subsequently, the molecular profiles of all the isolates were further determined using Pulsed Field Gel Electrophoresis (PFGE) and the Whole Genome Multi-Locus Sequence Type (wgMLST) analysis in order to obtain the sequence types. RESULTS: The PFGE data was input into FPQuest software, and the dendrogram generated was studied for possible genetic relatedness among the isolates. All the isolates were found to belong to the Salmonella Enteritidis serotype with notable resistance to tetracycline, gentamycin, streptomycin, and sulfadimidine. The S. Enteritidis isolates tested predominantly subtyped into the ST11 and ST1925, which was found to be a single cell variant of ST11. The STs were found to occur in chicken meats, foods, and live chicken cloacal swabs, which may indicate the persistence of the bacteria in multiple foci. CONCLUSION: The data demonstrate the presence of S. Enteritidis among chickens, indicating its preference and reservoir status for enteric Salmonella pathogens.

Rapid molecular identification and differentiation of common Salmonella serovars isolated from poultry, domestic animals and foodstuff using multiplex PCR assay.

Salmonella is widely distributed throughout the world and can be found in poultry industry, animal breeding centers, food and feedstuffs of all geographical regions. This study was conducted to determine and identify Salmonella serovars isolated from poultry, calves and foodstuffs (poultry and animals products such as egg and meat). A total of one hundred isolates of Salmonella serovars including Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Infantis, Salmonella Gallinarum and Salmonella Pullorum consecutively were subjected to the conventional culture, biochemical and serological assays. The utility of molecular multiplex PCR was investigated to identify and differentiate among five Salmonella serovars which were identified according to the presence of rfbJ, fljB, invA, and fliC genes in S. Typhimurium, sefA, invA and spv genes in Salmonella Enteritidis, fljB, fliC and invA genes in Salmonella Infantis, hut and slgC genes in both Salmonella Gallinarum and Salmonella Pullorum and speC gene specifically in Salmonella Gallinarum. Biochemical assays and serotyping are complicated to directly differentiate between Salmonella Gallinarum and Salmonella Pullorum because of their antigenic similarity. According to the results, Multiplex PCR can be considered as simple, rapid, accurate and useful test to identify and differentiate among Salmonella serovars.

Whole genome sequencing reveals an outbreak of Salmonella Enteritidis associated with reptile feeder mice in the United Kingdom, 2012-2015.

Analysis of whole genome sequencing data uncovered a previously undetected outbreak of Salmonella Enteritidis that had been on-going for four years. Cases were resident in all countries of the United Kingdom and 40% of the cases were aged less than 11 years old. Initial investigations revealed that 30% of cases reported exposure to pet snakes. A case-control study was designed to test the hypothesis that exposure to reptiles or their feed were risk factors. A robust case-definition, based on the single nucleotide polymorphism (SNP) profile, increased the power of the analytical study. Following univariable and multivariable analysis, exposure to snakes was the only variable independently associated with infection (Odds ratio 810 95% CI (85-7715) p < 0.001). Isolates of S. Enteritidis belonging to the outbreak profile were recovered from reptile feeder mice sampled at the retail and wholesale level. Control measures included improved public health messaging at point of sale, press releases and engagement with public health and veterinary counterparts across Europe. Mice destined to be fed to reptiles are not regarded as pet food and are not routinely tested for pathogenic bacteria. Routine microbiological testing to ensure feeder mice are free from Salmonella is recommended.

Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples.

The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA- and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method.IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples.

Genetic diversity and virulence genes of Salmonella enterica subspecies enterica serotype Enteritidis isolated from meats and eggs.

Salmonella enterica subspecies enterica serotype Enteritidis (S. Enteritidis) is one of the leading causes of food-borne gastroenteritis associated with the consumption of contaminated food products of animal origin. Little is known about the genetic diversity and virulence content of S. Enteritidis isolated from poultry meats and eggs in Iran. A total of 34 S. Enteritidis strains were collected from different food sources of animal origin in Tehran from May 2015 to July 2016. All of the S. Enteritidis strains were serotyped, antimicrobial susceptibility tested, and characterized for virulence genes. Pulsed-field gel electrophoresis (PFGE) was also applied for comparison of genetic relatedness. All of the strains harbored invA, hilA, ssrA, sefA, spvC, and sipA genes. A high prevalence of resistance against certain antibiotics such as cefuroxime (79.4%), nalidixic acid (47%), and ciprofloxacin (44.2%) was also observed. Regarding PFGE, S. Enteritidis strains from different sources showed considerable overlap, suggesting the lack of diversity among these isolates. Moreover, no correlation between virulence profiles or antibiotypes and PFGE clusters was observed. In conclusion, our study provided valuable information on virulence gene content, antibiotic resistance, and genetic diversity of S. Enteritidis isolated from food sources.

Outbreak of Salmonella enteritidis phage type 1B associated with frozen pre-cooked chicken cubes, Finland 2012.

In August to October 2012, a nationwide outbreak of Salmonella enteritidis phase type (PT) 1B with 53 cases occurred in Finland. Hypothesis generating interviews pointed toward ready-to-eat chicken salad from a Finnish company and at the same time Estonian authorities informed of a S. enteritidis PT 1B outbreak linked to chicken wrap prepared at an Estonian restaurant. We found that chicken salad was associated with the infection (odds ratio (OR) 16·1, 95% confidence interval (CI) 1·7-148·7 for consumption and OR 17·5. 95% CI 4·0-76·0 for purchase). The frozen pre-cooked chicken cubes used in Finnish salad and in Estonian wraps were traced back to a production plant in China. Great Britain made two Rapid Alert Systems for Food and Feed notifications on chicken cubes imported to the UK from the same Chinese production plant. Microbiological investigation confirmed that the patient isolates in Estonia and in Finland were indistinguishable from the strains isolated from chicken cubes in Estonia and in the UK. We recommend that despite certificates for tested Salmonella, food items should be analyzed when Salmonella contamination in outbreak investigations is suspected. In outbreak investigations, electronically implemented case-case study saves time, effort, and money compared with case-control study.

Prospective use of whole genome sequencing (WGS) detected a multi-country outbreak of Salmonella Enteritidis.

Since April 2015, whole genome sequencing (WGS) has been the routine test for Salmonella identification, surveillance and outbreak investigation at the national reference laboratory in England and Wales. In May 2015, an outbreak of Salmonella Enteritidis cases was detected using WGS data and investigated. UK cases were interviewed to obtain a food history and links between suppliers were mapped to produce a food chain network for chicken eggs. The association between the food chain network and the phylogeny was explored using a network comparison approach. Food and environmental samples were taken from premises linked to cases and tested for Salmonella. Within the outbreak single nucleotide polymorphism defined cluster, 136 cases were identified in the UK and 18 in Spain. One isolate from a food containing chicken eggs was within the outbreak cluster. There was a significant association between the chicken egg food chain of UK cases and phylogeny of outbreak isolates. This is the first published Salmonella outbreak to be prospectively detected using WGS. This outbreak in the UK was linked with contemporaneous cases in Spain by WGS. We conclude that UK and Spanish cases were exposed to a common source of Salmonella-contaminated chicken eggs.

Re-evaluation of a 2014 multi-country European outbreak of Salmonella Enteritidis phage type 14b using recent epidemiological and molecular data.

A European multi-country outbreak of Salmonella Enteritidis phage type (PT) 14b occurred from March to November 2014 associated with the consumption of eggs. The outbreak involved more than 400 human cases from France, Luxembourg, Austria and the United Kingdom. In 2016-2017, it has been re-evaluated combining recent epidemiological results with latest molecular data. The outbreak was traced back to one large Bavarian egg producer with four distinct premises, three located in Bavaria, one in the Czech Republic. The outbreak isolates of S. Enteritidis PT 14b were grouped into three closely related clades by whole genome sequencing. Two of these clades could be referred to two Bavarian premises of the egg producer on the basis of epidemiological and molecular data, while epidemiological data presumably linked the third clade to another premises of the egg producer. Interestingly and in contrast to the situation in other European countries where several outbreaks were documented, all notified 91 laboratory-confirmed cases of S. Enteritidis PT 14b from Bavaria were sporadic, singular cases not belonging to any epidemiological outbreaks. In conclusion, as demonstrated here, the resolution of food-related outbreaks with such a high discriminatory power is rare in outbreak investigation.

Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015.

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data.

Outbreak of Salmonella enteritidis PT14b gastroenteritis at a restaurant in England: the use of molecular typing to achieve a successful prosecution.

OBJECTIVES: To describe an outbreak of Salmonella enteritidis phage type (PT) 14b in people who had eaten at a restaurant, and the investigation and subsequent prosecution of the food business operator (FBO). STUDY DESIGN: The local health protection team and environmental health department formed an outbreak control team to investigate the outbreak. METHODS: Epidemiological, microbiological, and environmental investigations were undertaken. Epidemiological investigations involved case finding and interviews. Microbiological investigation: stool samples from the suspected cases and environmental samples from the implicated food business were investigated. Salmonella isolates obtained were subjected to multiple locus variable-number tandem repeat analysis (MLVA) profiling and whole genome sequencing. In addition, adenosine triphosphate (ATP) hygiene swab tests were used to verify the quality of cleaning procedures and data loggers were used to determine the water temperature of the mechanical dishwasher. RESULTS: Fifteen cases of illness where the causative agent was shown to be S. enteritidis PT14b were identified, all of whom had eaten at the same restaurant. S. enteritidis PT14b was also identified from three of the 11 food and environmental samples taken at the restaurant and found to have the same MLVA profile as the cases. A case for prosecution was built and the FBO was successfully prosecuted in July 2015. CONCLUSIONS: This investigation highlighted that the use of molecular typing as part of thorough epidemiological, microbiological, and environmental investigations can present a robust case for prosecution against restaurants which pose a risk to public health.

Magnetic nanoparticle-enhanced PCR for the detection and identification of Staphylococcus aureus and Salmonella enteritidis.

This paper evaluated magnetic nanoparticle-enhanced PCR for the detection and identification of Staphylococcus aureus and Salmonella enteritidis. Two different types of magnetic nanoparticles designated MPIO (iron concentration 2.5 mg/ml, size 1 µm) and NP (iron concentration 8.7 mg/ml, size 60 nm), both conjugated with S. aureus or S. enteritidis antibodies were evaluated as an enrichment procedure for PCR-detection of the pathogens in Trypticase Soy Broth, milk, blood and meat broth. Bacterial suspensions (1.5x108 cfu/ml) were prepared and serial diluted 10-1. The MPIO and NP nanoparticles were added, followed by incubation for 1 hour at room temperature, magnetic separation of the pellet, DNA extraction and PCR, targeting the femA and invA sequences. The nanoparticle-free and the NP-supplemented dilutions were positive down to the 1.5x102 cfu/ml concentration for both bacteria. The MPIO-supplemented dilutions were positive down to approx. 2x100 cfu/ml concentration, respectively. Bacteria-free TSB was negative by PCR. MPIO nanoparticles (size 1 µm) enhanced the detection of S. aureus and S. enteritidis by PCR, whilst NP nanoparticles (size 60 nm) did not, thus indicating that the size of the magnetic nanoparticles play a significant role in the enrichment procedure.

[FORMATION OF POPULATION MORBIDITY WITH SALMONELLOSIS CAUSED BY SALMONELLA ENTERITIDIS IN REGIONS WITH INCOMPLETE SUPPLY OF LOCAL POULTRY PRODUCTS].

AIM: Study plasmid characteristics of S. enteritidis strains in patients and features of epidemi- ology of the infection in regions with incomplete supply of population with local poultry produc- tion. MATERIALS AND METHODS: Plasmid analysis of microbe strains isolated from 382 patients and 8 samples of products was carried out, and significance of plasmid types in population morbidity was evaluated. Identification of salmonella was carried out by conventional methods, plasmid 41 specter - by Kado C.I. and Liu S.T (1981) method. RESULTS: 98.4% of strains contained virulence plasmid p38, and 80.1% of strains also had small plasmids. Sakhalin strains were divided into 16 plasmid types (D=0.794), and strains from Jewish AO - 10 (D=0.834). Uniformity of strains in patients during infection outbreaks and in transmission factors was detected. CONCLUSION: Features of salmonellosis in. the studied subjects of Russian Federation are determined by higher risk of import of products containing salmonella. Monitoring based on plasmid analysis is an effective base for epidemiologic control.

Comparative study of all Salmonella enterica serovar Enteritidis strains isolated from food and food animals in Greece from 2008 to 2010 with clinical isolates.

The aim of the present work was to study the epidemiology of Salmonella enterica serovar Enteritidis (S. Enteritidis) in Greece, comparing all the food and food animal isolates during a 3-year period with clinical isolates. Submission of the generated data to the PulseNet Europe database was carried out in order to study the population structure of this particular serovar and indicate possible connections with European strains. One hundred and sixty-eight (168) S. Enteritidis strains of human, animal, and food origin, isolated during the period 2008-2010 in Greece, were studied. Strains were characterized by phenotypic (antibiotic resistance) and molecular [pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)] methods. PFGE revealed 39 XbaI, 48 BlnI, and 80 XbaI-BlnI distinct pulsotypes, suggesting several clones circulating through the food chain and multiple sources of transmission. Submission to the PulseNet Europe database indicated that PFGE profile SENTXB.0001, the most common PFGE profile in Europe, was also predominant in Greece (33.3 %). MLST showed that all the strains studied shared the same sequence type (ST11), representing the most common ST in Europe. High rates of resistance to nalidixic acid were observed among human and poultry isolates (~25 %), indicating the potential fluoroquinolone treatment failure. Our data suggest that strains originating from multiple reservoirs circulated in Greece through the food chain during the study period. Predominant profiles in Greece were common to PulseNet Europe profiles, indicating similarities between the S. Enteritidis populations in Greece and Europe.

The evaluation and application of multilocus variable number tandem repeat analysis (MLVA) for the molecular epidemiological study of Salmonella enterica subsp. enterica serovar Enteritidis infection.

BACKGROUND: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the most prevalent Salmonella serotypes that cause gastroenteritis worldwide and the most prevalent serotype causing Salmonella infections in China. A rapid molecular typing method with high throughput and good epidemiological discrimination is urgently needed for detecting the outbreaks and finding the source for effective control of S. Enteritidis infections. METHODS: In this study, 194 strains which included 47 from six outbreaks that were well-characterized epidemiologically were analyzed with pulse field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA). Seven VNTR loci published by the US Center for Disease Control and Prevention (CDC) were used to evaluate and develop MLVA scheme for S. Enteritidis molecular subtyping by comparing with PFGE, and then MLVA was applied to the suspected outbreaks detection. All S. Enteritidis isolates were analyzed with MLVA to establish a MLVA database in Shenzhen, Guangdong province, China to facilitate the detection of S. Enteritidis infection clusters. RESULTS: There were 33 MLVA types and 29 PFGE patterns among 147 sporadic isolates. These two measures had Simpson indices of 0.7701 and 0.8043, respectively, which did not differ significantly. Epidemiological concordance was evaluated by typing 47 isolates from six epidemiologically well-characterized outbreaks and it did not differ for PFGE and MLVA. We applied the well established MLVA method to detect two S. Enteritidis foodborne outbreaks and find their sources successfully in 2014. A MLVA database of 491 S. Enteritidis strains isolated from 2004 to 2014 was established for the surveillance of clusters in the future. CONCLUSIONS: MLVA typing of S. Enteritidis would be an effective tool for early warning and epidemiological surveillance of S. Enteritidis infections.

Outbreak of Salmonella Enteritidis linked to the consumption of frozen beefburgers received from a food bank and originating from Poland: northern France, December 2014 to April 2015.

A prolonged outbreak of Salmonella enterica serotype Enteritidis occurred in northern France between December 2014 and April 2015. Epidemiological investigations following the initial notification on 30 December 2014 of five cases of salmonellosis (two confirmed S. Enteritidis) in young children residing in the Somme department revealed that all cases frequented the same food bank A. Further epidemiological, microbiological and food trace-back investigations indicated frozen beefburgers as the source of the outbreak and the suspected lot originating from Poland was recalled on 22 January 2015. On 2 March 2015 a second notification of S. Enteritidis cases in the Somme reinitiated investigations that confirmed a link with food bank A and with consumption of frozen beefburgers from the same Polish producer. In the face of a possible persistent source of contamination, all frozen beefburgers distributed by food bank A and from the same origin were blocked on 3 March 2015. Microbiological analyses confirmed contamination by S. Enteritidis of frozen beefburgers from a second lot remaining in cases' homes. A second recall was initiated on 6 March 2015 and all frozen beefburgers from the Polish producer remain blocked after analyses identified additional contaminated lots over several months of production.

Loop-Mediated Isothermal Amplification of the sefA Gene for Rapid Detection of Salmonella Enteritidis and Salmonella Gallinarum in Chickens.

Salmonella spp. pose a threat to both human and animal health, with more than 2600 serovars having been reported to date. Salmonella serovars are usually identified by slide agglutination tests, which are labor intensive and time consuming. In an attempt to develop a more rapid screening method for the major poultry Salmonella serovars, we developed a loop-mediated isothermal amplification (LAMP) assay, which directly detected the sefA gene, a fimbrial operon gene existing in several specific serovars of Salmonella enterica including the major poultry serovars, namely Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) and Salmonella enterica serovar Gallinarum (Salmonella Gallinarum). With the 177 bacterial strains we tested, positive reactions were only observed with 85 strains of serovar Salmonella Enteritidis and Salmonella Gallinarum. The detection limit of the LAMP assay was 4 CFU/reaction with genomic DNAs of Salmonella Enteritidis (ATCC 13076) from pure culture and 400 CFU/ reaction with DNA extracted from spiked chicken feces. The LAMP assay was more sensitive than conventional culture, especially without enrichment, in detecting Salmonella Enteritidis (CMCC 50041) in the spiked fecal samples. The results show the sefA LAMP method is a rapid, sensitive, specific, and practical method for directly detection of Salmonella Enteritidis and Salmonella Gallinarum in chickens. The sefA LAMP assay can potentially serve as new on-site diagnostics in the poultry industry.