Genetic features differentiating bovine, food, and human isolates of shiga toxin-producing Escherichia coli O157 in The Netherlands.

The frequency of Escherichia coli O157 genotypes among bovine, food, and human clinical isolates from The Netherlands was studied. Genotyping included the lineage-specific polymorphism assay (LSPA6), the Shiga-toxin-encoding bacteriophage insertion site assay (SBI), and PCR detection and/or subtyping of virulence factors and markers [stx1, stx(2a)/stx(2c), q21/Q933, tir(A255T), and rhsA(C3468G)]. LSPA6 lineage II dominated among bovine isolates (63%), followed by lineage I/II (35.6%) and lineage I (1.4%). In contrast, the majority of the human isolates were typed as lineage I/II (77.6%), followed by lineage I (14.1%) and lineage II (8.2%). Multivariate analysis revealed that the tir(A255T) SNP and the stx(2a)/stx(2c) gene variants were the genetic features most differentiating human from bovine isolates. Bovine and food isolates were dominated by stx(2c) (86.4% and 65.5%, respectively). Among human isolates, the frequency of stx(2c) was 36.5%, while the frequencies of stx(2a) and stx(2a) plus stx(2c) were 41.2% and 22.4%, respectively. Bovine isolates showed equal distribution of tir(255A) (54.8%) and tir(255T) (45.2%), while human isolates were dominated by the tir(255T) genotype (92.9%). LSPA6 lineage I isolates were all genotype stx(2c) and tir(255T), while LSPA6 lineage II was dominated by tir(255A) (86.4%) and stx(2c) (90.9%). LSPA6 lineage I/II isolates were all genotype tir(255T) but showed more variation in stx(2) types. The results support the hypothesis that in The Netherlands, the genotypes primarily associated with human disease form a minor subpopulation in the bovine reservoir. Comparison with published data revealed that the distribution of LSPA6 lineages among bovine and human clinical isolates differs considerably between The Netherlands and North America.

Quantitative levels of norovirus and hepatitis A virus in bivalve molluscs collected along the food chain in the Netherlands, 2013-2017.

Contamination of bivalve molluscs with viruses is well recognized as a food safety risk. A microbiological criterion for norovirus (NoV) and hepatitis A virus (HAV) in shellfish, however, does not exist in the European Union currently. The aim of this study was to evaluate the contamination levels of these viruses for fluctuation over a long period (2013-2017) in oyster (n = 266) and mussel samples (n = 490) using a method based on ISO/TS 15216-1: 2013. Samples were taken at different points in the food chain, either directly post-harvest, at Dutch dispatch centers or in retail stores, from September until March of each year. Altogether, 53.1% of the mussel and 31.6% of the oyster samples tested positive for NoV RNA. Simultaneous presence of NoV GI and GII RNA was observed in 31.6% of mussel and 10.2% of oyster samples. Contamination levels in NoV positive mussel samples collected post-harvest from B-areas were significantly higher than in those collected post-harvest from A-areas, or at dispatch centers or retail stores. Levels in oysters from dispatch were significantly lower than those collected in retail stores. Ready for sale mussels and oysters contained 2.04 and 1.76 mean log10 transformed NoV genome copies/gram (gc/g), respectively. GII levels were at a constant level in ready for sale mussels throughout all sampling periods in the study. This seemed to be true for oysters as well. HAV RNA was detected in only one of the tested mussel samples (n = 392) (typed HAV 1A) and in none of the tested oyster samples (n = 228). Critical evaluation of NoV and HAV levels in shellfish can be of help for risk assessment and risk management actions.

Detection and quantification of hepatitis E virus RNA in ready to eat raw pork sausages in the Netherlands.

The aim of the present study was to assess raw pork sausages collected on the Dutch market for the presence of hepatitis E virus (HEV) RNA. 46 of 316 (14.6%) products sampled from Dutch retail stores in 2017-2019 were positive for HEV RNA. HEV RNA was detected in 10.8% of "cervelaat" (n = 74), 18.5% of salami (n = 92), 26.1% of "metworst" (n = 46), 16.3% of "snijworst" (n = 43) samples. This was significantly more often than in other raw pork sausages like dried sausages, fuet or chorizo (3.3%, n = 61). The percentage of HEV RNA positive products was not significantly different for products sold as either sliced or unsliced deli meat. The average viral load in positive tested products was 2.76 log10 genome copies per 5 g, incidentally reaching up to 4.5 log10 genome copies per 5 g. The average HEV RNA level was significantly higher in samples collected in 2017 than those in samples collected in 2018, and most of the samples in 2019. Typing by sequence analysis was successful for 33 samples, all revealing genotype 3c. The results support recent epidemiological studies that identified specific raw pork sausages as risk factor for hepatitis E virus infection in the Netherlands. Persons at risk, including Dutch transplant recipients, have been advised to avoid the consumption of raw pork sausages. The study warrants a continuation of monitoring to follow the HEV RNA levels in pork products for use in risk assessments and risk management.

Monitoring of pork liver and meat products on the Dutch market for the presence of HEV RNA.

The aim of the present study was to assess pork liver and meat products present on the Dutch market for the presence of hepatitis E virus (HEV) RNA. HEV RNA was detected in 27.3% of 521 products sampled from Dutch retail stores in 2016. 12.7% of livers were positive for HEV RNA (n = 79), 70.7% of liverwurst (n = 99), 68.9% of liver pate (n = 90), but in none of the pork chops (n = 98), fresh sausages (n = 103) or wild boar meat (n = 52). The highest level of HEV RNA contamination was observed in a liver (reaching up to 1 × 106 copies/g), followed by ready to eat liverwurst and liver pate (up to 3 × 104 copies/g and 7 × 104 copies/g respectively). Sequence analyses revealed mainly genotype 3c, but also some 3a, 3e and 3f strains. One strain derived from a liver sample was 100% (493 nt) identical with one isolated from a HEV case with onset of disease close in time and geography, although no direct epidemiological link could be established. Despite liverwurst and liver pate undergo heat treatment (information dd. Mid 2017) that may be sufficient to inactivate HEV, persons at risk, including Dutch transplant recipients, have been advised to avoid the consumption of raw liver as well as liverwurst and liver pate.

Porcine blood used as ingredient in meat productions may serve as a vehicle for hepatitis E virus transmission.

The aim of the present study was to investigate whether the use of porcine blood(products) in food could be a risk for a hepatitis E virus (HEV) infection. HEV RNA was detected in 33/36 batches of (non-heated) liquid products and in 7/24 spray dried powder products. Contamination levels varied among the products, but were highest in liquid whole blood, plasma and fibrinogen reaching levels of 2.2×102 to 2.8×102 HEV genome copies per 0.2g. Sequence analyses revealed genotype 3 strains, of which two were 100% (493nt) identical to recently diagnosed HEV cases, although no direct epidemiological link was established. The industry provided information on processing of blood products in (ready-to-eat)-meat. From this, it was concluded that blood products as an ingredient of processed meat may not be sufficiently heated prior to consumption, and therefore could be a vehicle for transmission.

Prevalence of Clostridium difficile in retailed meat in the Netherlands.

Recent reports indicate that a large proportion of community-acquired Clostridium difficile infections (CA-CDI) are not linked to recent antibiotic therapy, older age, significant comorbidity or previous hospitalization. Possible community sources for CA-CDI include animals and food, and therefore a surveillance study on the prevalence of C. difficile in meat was performed. Samples of different meat species were collected from the retail trade and analyzed for the presence of C. difficile using a method that included selective enrichment in C. difficile broth, subsequent alcohol shock-treatment and plating onto C. difficile selective medium. C. difficile isolates were tested for the presence of toxin genes and were typed using PCR ribotyping. Of 500 samples tested, 8 (1.6%) were positive for the presence of C. difficile: 1 from lamb (6.3%) and 7 from chicken meat (2.7%). The isolated strains belonged to PCR ribotypes different from those that are currently most frequently found in patients with CDI in the Netherlands, except for C. difficile PCR ribotype 001 which was found in one chicken meat sample. This observation suggests that other matrices than meat may serve as a source for CA-CDI.

Two outbreaks of campylobacteriosis associated with the consumption of raw cows' milk.

The present paper summarises the investigation of two different outbreaks of milk-associated Campylobacter enteritis in the Netherlands. In 2005, after a school trip to a dairy farm, 22 out of a group of 34 children developed diarrhoeal illness and Campylobacterjejuni was cultured from the stool samples of 11 of the cases. The illness was found to be epidemiologically associated with drinking raw milk during the farm visit; 86% of the cases could be explained by drinking raw milk. C.jejuni was also isolated from three of 10 faecal samples from dairy cattle collected at the farm. The human isolates and C.jejuni isolates from one of these three samples of cattle faeces revealed identical restriction patterns by both pulsed-field gel electrophoresis (PFGE) and flagellin (fla) typing by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Both epidemiological and bacteriological evidence implicated contaminated raw milk as the vehicle of transmission, though C.jejuni was not isolated from the bulk tank milk or the milk filter collected during the farm investigation. In 2007, an outbreak of enteritis was notified among people who had attended a lunch at a dairy farm where bulk tank milk was served. Of the 19 persons who had consumed raw milk, 16 (84%) had become ill. Of the persons who did not drink the raw milk, none became ill. A significant association was found between tasting the raw milk and being ill (risk difference=0.84, p=0.0011). C.jejuni was cultured from four of seven cases who had submitted a stool specimen. C. jejuni was also isolated from a sample of bulk tank milk and the isolate had an identical flaA PCR-RFLP genotype to isolates obtained from patients. Also in this outbreak both the epidemiological and bacteriological findings support raw milk as the vehicle for the enteritis. These two outbreaks highlight the health risks associated with the consumption of raw milk. As long as legislation allows the sale and distribution of untreated milk these risks will continue. Therefore, consumers need to be continuously informed about the dangers inherent in consuming unpasteurised milk or products made from raw milk. Farmers need to be strongly discouraged from serving raw milk to their visitors.