Search

Vertical farming is a relatively new fresh fruit and vegetable production system, where lamps (mostly light emitting diodes [LED]) are the sole light source. A high light use efficiency (LUEinc), defined as shoot dry weight per incident photosynthetic photon flux density (PPFD; g mol−1) integral, is crucial for the economic viability of vertical farming. Very different values for LUEinc have been reported in the literature and it is not clear whether LUEinc is higher in vertical farming than in greenhouse or open field cultivation. Values of LUEinc of lettuce grown in a vertical farm (53 studies), greenhouse (13 studies) and open field (8 studies) were collected from literature, as well as relevant cultivation aspects such as lettuce weight at harvest, cultivation period (plant age at harvest), daily light integral, cumulative daily light integral for the whole cultivation period, planting density and CO2 concentration. The average LUEinc for lettuce grown in a vertical farm was 0.55 g mol−1 which was higher than 0.39 g mol−1 for greenhouse-grown lettuce. Both were substantially higher than for field-grown lettuce (0.23 g mol−1). The maximum measured LUEinc for lettuce grown in a vertical farm (1.63 g mol−1) is close to the published maximum theoretical value, which ranges from 1.26 to 1.81 g mol−1. Since all environmental factors can be fully controlled, vertical farming has the capability to achieve the theoretical maximum LUEinc. Using the highest reported LUEinc based on shoot fresh weight (44 g mol−1 at 200 μmol m−2 s−1 PPFD and 16 h photoperiod), it is estimated that each layer of a vertical farm can potentially produce annually up to 700 kg of lettuce per m2 at 500 μmol m−2 s−1 of continuous light.

Vertical farming can produce food in a climate-resilient manner, potentially emitting zero pesticides and fertilizers, and with lower land and water use than conventional agriculture. Vertical farming systems (VFS) can meet daily consumer demands for nutritious fresh products, forming a part of resilient food systems—particularly in and around densely populated areas. VFS currently produce a limited range of crops including fruits, vegetables and herbs, but successful implementation of vertical farming as part of mainstream agriculture will require improvements in profitability, energy efficiency, public policy and consumer acceptance. Here we discuss VFS as multi-layer indoor crop cultivation systems, exploring state-of-the-art vertical farming and future challenges in the fields of plant growth, product quality, automation, robotics, system control and environmental sustainability and how research and development, socio-economic and policy-related institutions must work together to ensure successful upscaling of VFS to future food systems.

Graphical abstract Sheep and goats were domesticated around 9000 BC in the Middle East. Milk from sheep is very popular in many populations and areas. Taste and high fat content of cheeses from sheep milk make them popular. In many countries, dairy products from sheep and goats are consumed by children and people with allergies to cow milk. Other regions of the world, are not suitable for cows and goats, and sheep milk is an essential part of local daily diet. Consumption of raw milk and raw milk products have a zoonotic risk. The main pathogens associated with sheep milk are: Brucella melitensis, Campylobacter spp., Coxiella burnetii, Leptospira spp. Listeria spp., Salmonella spp., Shiga-toxin producing Escherichia coli, tick borne encephalitis, Toxoplasma gondii, and Rift Valley fever. This risk of these pathogens can be reduced by proper flock health management, prevention of contamination during milking and adequate milk processing, transport and storage. In small scale production systems there is a higher risk, compared to industrialized production systems because of a less protocolled and controlled production process. Especially s, young children, elderly people, pregnant women and immunocompromised (YOPI) persons, and those suffering from disease should be aware of the risk of consuming raw milk and raw milk products. Therefore, strict hygiene practices throughout the production process and supply chain in combination with stringent periodic control of these products should be applied. Although pasteurization influences the taste of milk, it sufficiently reduces zoonotic risks while not negatively influencing the nutritional value. This manuscript describes major and minor pathogens that might be transmitted in milk from sheep. Also, unintended contamination and multiplication and prevention thereof are described. We summarize some major human outbreaks caused by consumption of milk from sheep and finally discuss the implications., Sheep were domesticated around 9000 BC in the Middle East, and since then milk from sheep gradually became very popular, not only for drinking but also for making cheeses and other dairy products. Nowadays, these dairy products are also important for people with an allergy to cow milk, and these products are an essential part of the local daily diet in regions of the world that are not suitable for cows and goats. Consumption of raw milk and raw milk products has a zoonotic risk, and with regard to sheep, the main pathogens associated with such dairy products are: Brucella melitensis, Campylobacter spp., Listeria spp., Salmonella spp., Shiga-toxin producing Escherichia coli, Staphylococcus aureus, tick borne encephalitis virus, and Toxoplasma gondii. Especially, young children, elderly people, pregnant women and immunocompromised (YOPI) persons, and those suffering from disease should be aware of the risk of consuming raw milk and raw milk products. This latter risk can be reduced by proper flock health management, prevention of contamination during milking, adequate milk processing, transport, and refrigerated storage. Only processes equaling pasteurization sufficiently reduce zoonotic risks from milk and milk products, but proper cooling is essential and recontamination must be prevented. Therefore, strict hygiene practices throughout the production process and supply chain especially for raw milk and raw dairy products, should be applied. Small scale production systems pose a greater risk compared to industrialized production systems because of a less protocolized and controlled production process. This manuscript describes zoonotic risks of pathogens from sheep and their milk borne transmission. Additionally, routes of contamination, possibilities for multiplication, and prevention measures thereof are described. We summarize some major human outbreaks caused by consumption of sheep milk and products made thereof, and finally discuss their implications.

The aim of this study was to develop a Typhimurium (ST) challenge model in weaned pigs suitable to evaluate effects of water and feed interventions on fecal shedding and growth performance. Two studies were performed. In Exp. 1 weaned pigs were fed either a standard diet (CON) or a diet with a high buffer capacity (HB) and challenged for either 3 or 7 consecutive days in a Latin square design with 4 × 8 individually housed pigs. In Exp. 2, the CON 7-d challenge method was chosen for further model development and validation. Thirty-two individually housed weaned pigs were divided over 4 treatments: a nonchallenged control group (NCON), a challenged positive control group (PCON), a challenged intervention group with acidified water (WATER), and a challenged intervention group with acidified feed (FEED). Pigs were orally challenged once daily on d 7 to 9 or d 7 to 13 after weaning (d 0) with 1 ×10 cfu ST. From d 0 to 28, rectal temperature and occurrence of diarrhea were recorded daily, and BW and feed intake were measured weekly. Fecal samples were collected on d 0, 2, 7, 9, 13, 16, 20, 23, and 27 in Exp. 1 and d 0, 2, 7, 8, 9, 13, 15, and 27 in Exp. 2 for quantification. The results of both experiments showed quantifiable fecal shedding (average peak shedding of approximately 3.5 log and 5.5 log cfu/g, respectively), accompanied by a transient 0.5°C increase in rectal temperature and an increase in occurrence of diarrhea. In Exp. 2 during the week of challenge (i.e., d 7 to 14), a reduction in growth performance (ADG: -157 to 200 g/d and G:F: -0.22 to 0.25 g/d; < 0.01) in PCON and FEED was observed compared to NCON, with WATER showing an intermediate response. The WATER treatment also showed a numerically lower peak shedding (difference of -1.3 to 1.4 log cfu/g) compared to PCON and FEED. To conclude, we repeatedly infected weaned pigs successfully with 1 × 10 cfu of ST for 7 consecutive days, resulting in detectable and quantifiable fecal shedding. This ST challenge model may be suitable for evaluation of effects of water and feed interventions on peak fecal shedding and growth performance.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12 ng/assay) and ∼60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4 × 104 cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.

This chapter focuses on the classification and taxonomy and uses and available cultivars and types of tomatoes. Information on the production and marketing aspects of the global tomato industry is discussed and future perspectives for the tomato sector are highlighted.

This chapter focuses on the various processes that determine the growth and yield of tomatoes, with emphasis on the influence of environmental factors and crop management, such as light, carbon dioxide, temperature, humidity, salinity, plant density and leaf and fruit pruning. Guidelines on how to grow tomato in experiments with a controlled environment are also provided.

Streptococcus suis is an emerging zoonotic pathogen, which causes meningitis and septicaemia in humans and pigs [1, 2]. Ingestion of S. suis serotype 2 (SS2) contaminated food was identified as a risk factor for meningitis caused by SS2 in adult patients in Vietnam and Thailand [2], indicating that the gastrointestinal tract (GIT) might serve as an entry site of SS2 infection in humans [3, 4]. The upper respiratory tract (URT) is considered the main entry site of S. suis infection in pigs [5]. However, recent studies showed that the porcine GIT is rapidly colonized by S. suis after weaning [6] and that bacterial entry through the GIT occurs in piglets after mild stress induction [7], indicating that also the porcine GIT is an entry site of S. suis infection. The capsular polysaccharide (CPS), which determines the serotype, appears to play an essential role during the interaction with the URT epithelium [8]. Suilysin (Sly), a hemolytic pore-forming toxin, may also play a role facilitating translocation across the mucosal epithelium [9]. SS2 is responsible for most reported infections in humans and pigs worldwide [1, 10]. However, S. suis serotype 9 (SS9) is the most common serotype isolated from diseased pigs in Northern Europe [11] and is also prevalent in China [12] but has never been isolated from human patients [1, 10]. It is not known why isolates with serotypes that are highly prevalent among diseased pigs, such as SS9, have never been reported from human cases of SS infections. In addition to serotype, the genotype contributes to virulence of S. suis. Invasive S. suis strains are limited to certain sequence types (ST) and multi locus sequence typing (MLST) clonal complexes (CC) [13]. The majority of the SS2 strains isolated from human patients belong to CC1. However, we recently identified SS2 isolates from human patients in The Netherlands belonging to CC20, which is unrelated to CC1, indicating that multiple zoonotic clones are circulating worldwide [11]. In contrast, Dutch SS9 strains isolated from diseased pigs mainly belong to CC16, which is unrelated to both CC1 and CC20 [11, 14]. We aimed to provide experimental proof of concept that S. suis is capable of translocation in the GIT, in the absence of any invasive procedures. We then studied strain- and host-specific factors that may modulate S. suis infectivity at the intestinal mucosal surface, potentially providing clues to the zoonotic potential of SS2.

The aim of this study was to develop a Typhimurium (ST) challenge model in weaned pigs suitable to evaluate effects of water and feed interventions on fecal shedding and growth performance. Two studies were performed. In Exp. 1 weaned pigs were fed either a standard diet (CON) or a diet with a high buffer capacity (HB) and challenged for either 3 or 7 consecutive days in a Latin square design with 4 × 8 individually housed pigs. In Exp. 2, the CON 7-d challenge method was chosen for further model development and validation. Thirty-two individually housed weaned pigs were divided over 4 treatments: a nonchallenged control group (NCON), a challenged positive control group (PCON), a challenged intervention group with acidified water (WATER), and a challenged intervention group with acidified feed (FEED). Pigs were orally challenged once daily on d 7 to 9 or d 7 to 13 after weaning (d 0) with 1 ×10 cfu ST. From d 0 to 28, rectal temperature and occurrence of diarrhea were recorded daily, and BW and feed intake were measured weekly. Fecal samples were collected on d 0, 2, 7, 9, 13, 16, 20, 23, and 27 in Exp. 1 and d 0, 2, 7, 8, 9, 13, 15, and 27 in Exp. 2 for quantification. The results of both experiments showed quantifiable fecal shedding (average peak shedding of approximately 3.5 log and 5.5 log cfu/g, respectively), accompanied by a transient 0.5°C increase in rectal temperature and an increase in occurrence of diarrhea. In Exp. 2 during the week of challenge (i.e., d 7 to 14), a reduction in growth performance (ADG: -157 to 200 g/d and G:F: -0.22 to 0.25 g/d;0.01) in PCON and FEED was observed compared to NCON, with WATER showing an intermediate response. The WATER treatment also showed a numerically lower peak shedding (difference of -1.3 to 1.4 log cfu/g) compared to PCON and FEED. To conclude, we repeatedly infected weaned pigs successfully with 1 × 10 cfu of ST for 7 consecutive days, resulting in detectable and quantifiable fecal shedding. This ST challenge model may be suitable for evaluation of effects of water and feed interventions on peak fecal shedding and growth performance.

Streptococcus suis is a zoonotic swine pathogen and a major public health concern in Asia, where it emerged as an important cause of bacterial meningitis in adults. While associated with food-borne transmission in Asia, zoonotic S. suis infections are mainly occupational hazards elsewhere. To identify genomic differences that can explain zoonotic potential, we compared whole genomes of 98 S. suis isolates from human patients and pigs with invasive disease in the Netherlands, and validated our observations with 18 complete and publicly available sequences. Zoonotic isolates have smaller genomes than non-zoonotic isolates, but contain more virulence factors. We identified a zoonotic S. suis clone that diverged from a non-zoonotic clone by means of gene loss, a capsule switch, and acquisition of a two-component signalling system in the late 19th century, when foreign pig breeds were introduced. Our results indicate that zoonotic potential of S. suis results from gene loss, recombination and horizontal gene transfer events.

SUMMARYShiga toxin-producing Escherichia coli (STEC) bacteria can cause outbreaks and sporadic cases of gastroenteritis in humans. Ruminants are seen as the main reservoir. The aim of this study was to evaluate the spatial association between reported human STEC O157 infections in The Netherlands and different livestock densities. Data were collected at the municipality level and a spatial regression analysis was performed. Between April 1999 and December 2008, 409 symptomatic sporadic cases were registered. Adding an interaction term between season, age, and livestock density showed an increased risk of STEC cases in summer for living in areas with cattle, in particular for young children. In conclusion, cattle, but not pigs or poultry, are indicated as an important source for human STEC O157 infections in rural areas. The association is probably due to direct or indirect contact with cattle, resulting in symptomatic infections, especially in young children.

In spring 2008, 15 Salmonella Panama laboratory-confirmed cases were reported within 2 weeks, twice the average annual number of reported cases of this infrequent serotype in The Netherlands. To identify the source responsible for this national outbreak, we carried out an epidemiological, microbiological, and trace-back investigation. In total, 33 cases were reported, and a matched case-control study (23 cases/24 controls) identified consumption of fresh (unpasteurized) fruit juice purchased from a large retailer (X) as the only significant risk factor for illness (matched odds ratio: 7.4, 95% confidence interval: 1.5-37.2). Though the bacterium could not be isolated from fruit juice, the minimal pH value for growth of the causative strain of the outbreak (3.4) was compatible with survival in fruit juice from X. The outbreak strain showed acid resistance and adaptive properties that may explain how it could have caused infection through fresh orange juice. To our knowledge, this is the first documented outbreak related to fresh fruit juice consumption in western Europe since 1922. A growing number of consumers who are seeking healthy food practices are exposed to the infectious risks related to unpasteurized fresh fruit juice. Labeling regulations should be adapted to properly indicate to the consumers that unpasteurized fresh fruit juices remain vulnerable to microbial contamination. Frequent microbiological screening and strict compliance with food safety procedures should reduce the infectious hazards of fresh fruit juices.

The major objective of this study was to investigate the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in different types of food samples and to compare their genetic relatedness with STEC strains previously isolated from animal sources in Bangladesh. We investigated a total of 213 food samples, including 90 raw meat samples collected from retail butcher shops, 20 raw milk samples from domestic cattle, and 103 fresh juice samples from street vendors in Dhaka city. We found that more than 68% (n = 62) of the raw meat samples were positive for the stx gene(s); 34% (n = 21) of buffalo meats and 66% (n = 41) of beef. Approximately 10% (n = 2) of the raw milk and 8% (n = 8) of the fresh juice samples were positive for stx. We isolated STEC O157 from seven meat samples (7.8%), of which two were from buffalo meats and five from beef; and no other STEC serotypes could be isolated. We could not isolate STEC from any of the stx-positive raw milk and juice samples. The STEC O157 isolates from raw meats were positive for the stx(2), eae, katP, etpD, and enterohemorrhagic E. coli hly virulence genes, and they belonged to three different phage types: 8 (14.3%), 31 (42.8%), and 32 (42.8%). Pulsed-field gel electrophoresis (PFGE) typing revealed six distinct patterns among seven isolates of STEC O157, suggesting a heterogeneous clonal diversity. Of the six PFGE patterns, one was identical and the other two were ≥90% related to PFGE patterns of STEC O157 strains previously isolated from animal feces, indicating that raw meats are readily contaminated with fecal materials. This study represents the first survey of STEC in the food chain in Bangladesh.

Control of Mycoplasma bovis infections depends on good husbandry practices and antibiotic treatment. To allow more prudent use of antimicrobial drugs, there is a need for information on the susceptibility profile of this pathogen. The objective of the present study was to analyse the in vitro antimicrobial susceptibility of clinical M. bovis isolates in the Netherlands. The collection comprised 95 bovine isolates, originating from lungs (n=56), mastitis milk (n=27), and synovial fluid (n=12), collected between 2008 and 2014. Minimal inhibitory concentrations (MICs) were assessed by broth microdilution, both by using in-house prepared MIC plates and by using commercially available MIC plates. For each antimicrobial agent, the range of MIC results, the MIC50, and MIC90 values were calculated. M. bovis strains recently isolated in the Netherlands appeared to be characterized by relatively high MIC values for antimicrobial agents that, until now, have been recommended by the Dutch Association of Veterinarians for treating pneumonia caused by Mycoplasma species. Fluoroquinolones appeared to be the most efficacious in inhibiting M. bovis growth, followed by tulathromycin and oxytetracycline. The highest MIC values were obtained for erythromycin, tilmicosin, and tylosin. Future studies should be done on determining M. bovis specific clinical breakpoints, standardization of methods to determine MIC values as well as molecular studies on detection of antimicrobial resistance mechanisms of M. bovis isolates to develop PCR assays for determining resistance.

The objective of the present study was to analyse the in vitro antimicrobial susceptibility of Streptococcus suis isolates from post-mortem samples from pigs in the Netherlands. S. suis isolates originated from diagnostic submissions of pigs sent to the Pathology Department of GD Animal Health, from April 2013 till June 2015. Minimal inhibitory concentrations (MICs) of in total 15 antimicrobials were assessed by broth microdilution following CLSI recommendations. MIC50 and MIC90 values were determined and MICs were interpreted as susceptible, intermediate and resistant using CLSI veterinary breakpoints (when available). Emergence of resistance among S. suis (n=1163) derived from clinical submissions of pigs appeared to be limited. Resistance to ampicillin, ceftiofur, clindamycin, enrofloxacin, florfenicol, penicillin, trimethoprim/sulfamethoxazole and tetracycline was 0.3%, 0.5%, 48.1%, 0.6%, 0.1%, 0.5%, 3.0%, and 78.4%, respectively. Cross-resistance between penicillin and ampicillin appeared to be incomplete. MIC values of erythromycin, clindamycin, neomycin, penicillin and tilmicosin for isolates originating from grower/finisher pigs were significantly more often lower than the MIC values of isolates from suckling/weaned piglets. It has to be kept in mind that these results represent only part of the Dutch pig population and it can be discussed whether this is a representative sample. Interpretation of the MIC results of (clinically relevant) antimicrobials tested for treatment of S. suis infection is strongly hampered by the lack of CLSI-defined veterinary clinical breakpoints that are animal species- and body site-specific. Therefore, and to conduct a clinically reliable monitoring of antimicrobial susceptibility of veterinary pathogens, more species- and organ-specific veterinary breakpoints are urgently needed.

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne’s disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold’s egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Lowenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne’s) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.

A PCR assay with an internal amplification control was developed for Listeria monocytogenes. The assay has a 99% detection probability of seven cells per reaction. When tested against 38 L. monocytogenes strains and 52 nontarget strains, the PCR assay was 100% inclusive (positive signal from target) and 100% exclusive (no positive signal from nontarget). The assay was then evaluated in a collaborative trial involving 12 European laboratories, where it was tested against an additional 14 target and 14 nontarget strains. In that trial, the inclusivity was 100% and the exclusivity was 99.4%, and both the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 h of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR. The performance characteristics of the PCR-based method were evaluated in a collaborative trial involving 13 European laboratories. In that trial, a specificity value (percentage of correct identification of blank samples) of 81.8% was obtained; the accordance was 87.9%, and the concordance was 68.1%. The sensitivity (correct identification of milk samples inoculated with 20 to 200 L. monocytogenes cells per 25 ml) was 89.4%, the accordance was 81.2%, and the concordance was 80.7%. This method provides a basis for the application of routine PCR-based analysis to dairy products and other foodstuffs and should be appropriate for international standardization.

Item does not contain fulltext Surveys carried out between 1990 and 2000 indicated that the incidence of STEC O157-associated gastroenteritis in The Netherlands was 1250 cases/year (median), of which 180 visited a general practitioner, 40 are reported and 0.6 are fatal, mainly in the elderly. There are approximately 20 cases of STEC O157-associated haemolytic-uraemic syndrome (HUS) per year, mainly in children. There are 2.5 HUS patients per year who develop end-stage renal disease (ESRD). There are an estimated 2 HUS-related and 0.5 ESRD-related fatalities per year. The mean disease burden associated with STEC O157 in the Dutch population is 116 (90% confidence interval 85-160) Disability Adjusted Life Years (DALYs) per year. Mortality due to HUS (58 DALYs), and ESRD (21 DALYs) and dialysis due to ESRD (21 DALYs) constitute the main determinants of disease burden. Sensitivity analysis indicates that uncertainty associated with model assumptions did not have a major effect on these estimates.

A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project ( http://www.pcr.dk ). Each laboratory analysed by the PCR a set of independent obtained presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole broiler carcass-rinse resulted in a detection limit of less than 5 cells per 25 g meat or 100 ml broiler rinse. A total of 435 samples from four countries, including pig carcass swabs (n=285), whole broiler carcass-rinse (n=25), various raw meat (n=33), and environmental samples (n=92) were investigated. The interlaboratory diagnostic accuracy, i.e. diagnostic specificity and sensitivity, was shown to be 97.5%. The co-amplification of an internal amplification control indicated possible inhibitory substances derived from the sample. This work can contribute to the quality assurance of PCR-based diagnostic methods and is currently proposed as international standard document.

A young child was admitted to hospital with haemolytic-uraemic syndrome caused by infection with a Shiga toxin 2-producing strain of Escherichia coli (STEC) O157. Five days before he became ill, the child had visited a small petting zoo. STEC O157 strains were isolated from faecal samples from goats and sheep housed on the farm. The human and the animal isolates were indistinguishable by molecular subtyping. The petting zoo voluntarily closed temporarily to prevent further cases of infection. Two out of 11 other, randomly selected petting zoos (including one deer park) visited subsequently, tested positive. Furthermore, during the study period there was one more notification of STEC O157 infection possibly linked with a farm visit. Although STEC O157 was indeed found in the petting zoo associated with this patient, transmission through animal contact could not be confirmed because the human isolate was not available for subtyping. The case study and the results of the other on-farm investigations highlight the risk of acquiring severe zoonotic infections during visits to petting zoos.

Contains fulltext : 185329.pdf (Publisher’s version ) (Closed access) The aim of this study was to analyse the results of a programme in the Netherlands for enhanced surveillance of Shiga-toxin-producing Escherichia coli (STEC) O157. In this programme, implemented in January 1999, all laboratories report positive cases to the public health services and submit isolates for typing to the reference laboratory. Public health services collect clinical and risk factor information of patients, using a standardised questionnaire. Results were analysed for the first two and a half years of the programme. In February 2000, a questionnaire was sent to all laboratories to assess (i) the criteria for testing faecal samples for STEC O157, (ii) the diagnostic tools used, and (iii) the level of participation in the surveillance programme. Between January 1999 and June 2001, 93 cases of symptomatic STEC O157 infection were reported, 25% of which occurred in children aged 0-4 years. Serotyping for O, H and stx types showed that two types dominated, O157:H7, s tx2 positive (48%) and O157:H-, stx1 and stx2 positive (24%). Analysis of the 93 isolates by pulsed-field gel electrophoresis showed 17 clusters of isolates with at least 95% fragments in common, including isolates with unknown epidemiological links. Of the patients for whom questionnaire information was reported, 38% were hospitalised, 15% developed haemolytic uraemic syndrome, and 52% reported a known risk factor, such as contact with farm animals or manure, consumption of raw or undercooked beef, consumption of raw milk or cheese made from raw milk, or contact with a symptomatic individual. Response to the laboratory survey was high (97%). Only 6% of the laboratories carried out testing for non-O157 STEC, although 95% performed testing for STEC O157. The majority (88%) used culture on sorbitol MacConkey agar or sorbitol MacConkey agar with cefixime and tellurite as the method of detection of STEC O157. The identity of the strains was confirmed primarily with commercially available latex agglutination assays (95% of laboratories) and biochemical characterisation with the API 20E test (bioMerieux, France) (42% of laboratories). Most laboratories (92%) used selection criteria for testing, especially bloody diarrhoea and other clinical information (81% of laboratories) and young age (10%). It is concluded that STEC O157 is a limited public health problem in the Netherlands, although the selective testing policy and the low sensitivity of the culture techniques used probably caused the incidence of STEC O157 infection to be underestimated.

The Dutch government, the meat producers organisation and the meat industry have recognised O157 VTEC as an important public health hazard, and agreed on the necessity to improve the hygiene in Dutch cattle- and calf-slaughtering establishments. This paper reports activities within a national action programme to achieve this objective, “Zero-tolerance for faecal contamination during slaughter of cattle and calves”. The study included inspection of hygienic performances in slaughterhouses, and visual and microbiological (aerobic plate counts, Enterobacteriaceae counts and O157 VTEC presence/absence on visually clean cattle and calf carcasses) assessment of carcass cleanliness. Initial studies concluded that the hygienic performances in the Dutch cattle and calf slaughterhouses should be immediately improved. In 52% of the slaughterhouses inspected, carcasses were observed to be contaminated with hide, hair or faeces. Around 45% of the slaughterhouses had constructural deficiencies likely to lead to structural cross-contamination of carcasses, by direct carcass–carcass contact, or by indirect contacts with floors, walls or steps. In 39% of the slaughterhouses, cleaning and disinfection procedures were inadequate. Visual inspection of chilled carcasses found that in 11 of the 27 slaughterhouses visited, more than 10% of the carcasses were visibly contaminated. In 6 of the 27 slaughterhouses visited, more than 50% of the carcasses inspected were visibly contaminated. Microbiological analysis of visually clean carcasses noted contamination levels similar to those reported from other countries. O157 VTEC were not isolated during this study. Circulation of these findings lead to increased efforts by all parties to fulfil the requirements of the statutory “Zero-tolerance” programme. A follow-up study noted a significant decrease in the proportions of faecally contaminated carcasses, i.e., 7% of chilled carcasses were visibly contaminated with faeces, as opposed to 22% contamination during the initial study. The follow-up study also noted a greater awareness of the importance of good hygienic practices among slaughterhouse personnel and government meat inspectors.

Shiga toxin-producing Escherichia coli (STEC) are an important cause of haemorrhagic colitis and the diarrhoea-associated form of the haemolytic uraemic syndrome. Of the numerous serotypes of E. coli that have been shown to produce Shiga toxin (Stx), E. coli O157:H7 and E. coli O157:NM (non-motile) are most frequently implicated in human disease. Early recognition of STEC infections is critical for effective treatment of patients. Furthermore, rapid microbiological diagnosis of individual patients enables the prompt notification of outbreaks and implementation of control measures to prevent more cases. Most human infections caused by STEC have been acquired by the consumption of contaminated foods, especially those of bovine origin such as undercooked ground beef and unpasteurized cows' milk, and by person-to-person contacts. To identify the reservoirs of STEC and the routes of transmission to man, sensitive methods are needed as these pathogens may only be present in food, environmental and faecal samples in small numbers. In addition, sensitive and rapid detection methods are necessary for the food industry to ensure a safe supply of foods. Sensitive methods are also needed for surveillance programmes in risk assessment studies, and for studies on survival and growth of STEC strains. Cultural methods for the enrichment, isolation and confirmation of O157 STEC are still evolving. Several selective enrichment media have been described, of which modified tryptone soy broth with novobiocin and modified E. coli broth with novobiocin, seem to be the most appropriate. These media are minimally-selective broths that give a somewhat limited differential specificity favouring isolation of O157 STEC, as opposed to other Gram-negative bacteria, in the sample. An incubation temperature of 41-42 degrees C further enhances selectivity. The occurrence of heat-, freeze-, acid- or salt-stressed STEC in foods means that it is important to be able to detect cells that are in a stressed state, as STEC generally have a very low infectious dose, and injured cells mostly retain their pathogenic properties. For the isolation of stressed O157 STEC, pre-enrichment in a non-selective broth is necessary. The most widely used plating medium for the isolation of typical sorbitol-non-fermenting strains of STEC of serogroup O157 is sorbitol MacConkey agar with cefixime and tellurite (CT-SMAC). As some STEC strains are sensitive for tellurite and/or are sorbitol-fermenting, the use of a second isolation medium, such as one of the newer chromogenic media, is recommended. Immunomagnetic separation (IMS) following selective enrichment, and subsequent spread-plating of the concentrated target cells onto CT-SMAC agar, appears to be the most sensitive and cost-effective method for the isolation of E. coli O157 from raw foods. IMS increases sensitivity by concentrating E. coli O157 relative to background microflora, which may overgrow or mimic O157 STEC cells on selective agars. While cultural isolation of O157 STEC from foods and faeces is time-consuming, labour-intensive and hence, costly, rapid immunological detection systems have been developed which significantly reduce the analysis time. These methods include enzyme-linked immunosorbent assays (ELISAs), colony immunoblot assays, direct immunofluorescent filter techniques, and several immunocapture techniques. Both polyclonal and monoclonal antibodies specific for the O and H antigens are used for these methods. Many of these test systems are able to detect less than one O157 STEC cell g(-1) of raw meat after overnight enrichment. Presumptive results are available after just one day, but need to be completed with the isolation of the organisms. The primary use of these procedures is therefore to identify food and faecal samples that possibly contain O157 STEC.

Rectal contents and tonsils from Dutch slaughter pigs collected immediately after slaughter were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157 (O157 VTEC). In addition, fresh fecal material from poultry layer flocks and turkey flocks collected on poultry farms was examined for the presence of O157 VTEC. E. coli O157 strains were isolated from two (1.4%) of 145 pigs. The strains were isolated from samples of rectal contents, all samples of tonsils being negative. While all 501 fecal samples from chicken flocks were found negative, E. coli O157 strains were isolated from six (1.3%) of 459 pooled fecal samples from turkey flocks. One of the porcine isolates and one of the turkey isolates contained the VT2 gene, the E. coli attaching-and-effacing gene, as well as the enterohemorrhagic E. coli hemolysin gene. Production of VT was confirmed by cytotoxicity tests on Vero cells. Based on these characteristics, the two stains were regarded as potentially pathogenic for humans. The porcine and the turkey isolate were further characterized as being of phage types 4 and 14, respectively. While biochemically typical of E. coli O157, the remaining six isolates were nonverocytotoxigenic and negative for both the E. coli attaching-and-effacing gene and the enterohemorrhagic E. coli hemolysin gene. All eight E. coli O157 isolates did not carry genes that encode E. coli heat-labile and heat-stable enterotoxins. It was concluded that pigs and poultry can be a source of O157 VTEC strains characteristic of those causing illness in man. The extent to which pigs and poultry play a role in the epidemiology of human O157 VTEC infection needs further research.

From May through November 1997, 1,011 samples of raw milk from bulk storage tanks were examined for the presence of verocytotoxin-producing Escherichia coli of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. The samples originated from 1,011 different dairy herds located throughout the Netherlands. O157 VTEC was not isolated from any of the milk samples examined. Additionally, survival of O157 VTEC in raw and UHT-sterilized cow's milk at 7 and 15 degrees C was studied, both in the absence and presence of an activated lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS). Results indicated that the O157 VTEC strain tested was able to grow in raw milk at 7 degrees C as well as at 15 degrees C. Naturally occurring amounts of thiocyanate and hydrogen peroxide in the raw milk tested were not sufficient to activate the LPS. Although the LPS exhibited an antimicrobial activity against O157 VTEC in LPS-activated sterilized milk, O157 VTEC populations were not (or not as obviously) reduced in LPS-activated raw milk. Possibly background microflora were more sensitive to the LPS than the O157 VTEC test strain. It was concluded that raw milk contaminated with O157 VTEC will remain a hazard if kept at 7 or 15 degrees C. Effective pasteurization and avoiding postpasteurization contamination are necessary to ensure the safety of milk.

During the period from September 1996 through November 1996, 10 Dutch dairy farms were visited to collect fecal samples from all cattle present. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. Cattle on 7 of the 10 dairy farms tested positive for O157 VTEC, with the proportion of cattle infected varying from 0.8 to 22.4%. On the seven farms positive for O157 VTEC, the excretion rate was highest in calves ages 4 to 12 months (21.2%). In a follow-up study, two O157 VTEC-positive farms and two O157 VTEC-negative farms identified in the prevalence study were revisited five times at intervals of approximately 3 months. Cattle on each farm tested positive at least once. The proportion of cattle infected varied from 0 to 61.0%. Excretion rates peaked in summer and were lowest in winter. Again, the highest prevalence was observed in calves ages 4 to 12 months (11.8%). O157 VTEC strains were also isolated from fecal samples from horses, ponies, and sheep and from milk filters and stable flies. O157 VTEC isolates were characterized by VT production and type, the presence of the E. coli attaching-and-effacing gene, phage type, and pulsed-field gel electrophoretic genotype. No overlapping strain types were identified among isolates from different farms except one. The predominance of a single type at each sampling suggests that horizontal transmission is an important factor in dissemination of O157 VTEC within a farm. The presence of more than one strain type, both simultaneously and over time, suggests that there was more than one source of O157 VTEC on the farms. Furthermore, this study demonstrated that the O157 VTEC status of a farm cannot be ascertained from a single visit testing a small number of cattle.

In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157. E. coli O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and E. coli attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of E. coli O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of E. coli O157 strains that are potentially pathogenic for humans.

International audience

This study has evaluated the efficacy of selective enrichment and plating media used for the isolation of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157, by examining pure bacterial cultures. In addition, the performance of a variety of commercial test kits for the detection of E. coli O157 strains inoculated into minced beef was compared, using the Ampcor E. coli O157:H7 Kit, 3M Petrifilm™ Test Kit-HEC, Dynabeads anti- E coli O157, EHEC-TEK™, and the Tecra E. coli O157 visual immunoassay. The commercial Verotox F test for the determination of the VT type of VTEC isolates was compared with a polymerase chain reaction (PCR) assay for VT-coding genes. Modified E. coli broth containing novobiocin (mEC + n) and sorbitol MacConkey agar supplemented with cefixime and tellurite (CT-SMAC) were the most efficacious media for selective enrichment and isolation, respectively. After enrichment of the inoculated samples, all kits tested could detect less than one O157 VTEC cell per g of minced beef. While the results of the immunoassays need to be confirmed by isolating the organisms, the use of the immunomagnetic separation technique directly yields isolates. The results of the Verotox F test were consistent with PCR results. A sensitive and cost-effective method for the isolation of O157 VTEC from minced beef in food industry and epidemiological studies involving large numbers of samples is the following: enrichment in mEC + n at 37°C for 6 to 8 h with shaking at 100 rpm, followed by immunomagnetic separation using Dynabeads anti- E. coli O157 and spread plating of the concentrated target cells onto CT-SMAC. The Verotox F test can be used to determine whether the isolates produce VTl and/or VT2.

The importance of transport resistance (distance between source and sink) on assimilate partitioning in tomato is questioned. Slack and Calvert (Journal of Horticultural Science52: 309–315, 1977) concluded that, in tomato, excising of fruit trusses showed a direct influence of distance from source on assimilate partitioning. A dry matter distribution model for tomato, based on the hypothesis that distribution is regulated by the sink strengths of the plant organs and that no influence of transport resistance on partitioning exists, has been described and validated by Heuvelink (Annals of Botany77: 71–80, 1996). Using this model, it is shown that the results of Slack and Calvert (1977) can be explained more simply on the basis of the succession of trusses with growth shifted with respect to time. Therefore, their results do not prove that transport resistance plays a role in assimilate partitioning.

Raw meats obtained from retail outlets in the Netherlands were examined for the presence of Escherichia coli of serogroup O157 and other verocytotoxin (VT)-producing E. coli (VTEC), in three different surveys. In the first survey O157 VTEC were detected and isolated by selective plating onto sorbitol MacConkey agar following selective enrichment in modified tryptone soy broth with acriflavin. The organisms were isolated from 2 (0.3%) of 770 samples of minced mixed beef and pork, but not detected in samples of raw minced beef (n = 1,000), minced pork (n = 260), or poultry products (n = 300). In the second survey an additional 360 raw meats were examined with the 3M Petrifilm™ Test Kit-HEC, after selective enrichment in modified E. coli broth containing novobiocin. VT-negative E. coli O157 strains were isolated from 22 (6.1%) samples. In the third survey 180 enrichment cultures of the first survey were screened for the presence of VT1 and VT2 genes with a polymerase chain reaction (PCR). Twenty-nine (16.1%) of the 180 enrichment cultures showed a positive PCR: one for the VT1 gene only, 17 for the VT2 gene only, and 11 for both the VT1 and VT2 gene. A total of 46 VTEC strains were isolated from 10 randomly selected PCR-positive samples. Serotyping revealed that 41 of the 46 VTEC isolates belonged to nine different O serogroups; the remaining five were unidentifiable. A number of the serogroups recovered have been associated with human disease.

Escherichia coli O157 detection limits in artificially contaminated beef and cattle faeces samples were determined using Dynabeads anti E. coli O157 immunomagnetic beads, VIDAS-UP, VIDAS-ICE, and real-time PCR (GeneDisc and LightCycler) systems. Dynabeads anti-E. coli O157 immunomagnetic separation (IMS) and the GeneDisc cycler were the most sensitive methods, and could detect an initial 1 CFU in 25g beef samples after 6h of incubation in modified tryptone soya broth with novobiocin (mTSB+n) or buffered peptone water (BPW). The VIDAS-UP method could detect an initial 10 CFU, while VIDAS-ICE and the LightCycler methods could only detect an initial 100 CFU. Higher detection rates were achieved with 18 hour incubations, where an initial 1 CFU in a 25g sample could be detected with all five methods. For cattle faeces enrichments, Dynabeads anti-E. coli O157 IMS could detect an initial 1 CFU after a 6 h incubation in mTSB+n, while the VIDAS-UP and VIDAS-ICE methods could detect an initial 10 CFU and both PCR methods could only detect an initial 100 CFU. Detection rates were lower in BPW, compared to mTSB+n, with thresholds of 100 CFU for VIDAS-ICE, VIDAS-UP and GeneDisc methods, and >100 CFU for the LightCycler method.

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.

The species Escherichia coli contains both diarrhoeagenic and non-diarrhoeagenic strains and it is very important to have methods available which can differentiate between them. Adequate culture methods have been developed for the isolation of verocytotoxin-producing E. coli (VTEC) of serogroup 0 157 from foods. However, at present no single isolation procedure is available for the recovery of all VTEC causing severe human disease. Additionally, there are still no simple sensitive procedures available for the direct cultivation of strains of the other groups of diarrhoeagenic E. coli. The isolation of these organisms will best be accomplished by a combination of culture and molecular biological methods. In this review, some comparative studies of the media described for VTEC, especially VTEC O157, are noted and the difficulties associated with the isolation and enumeration of these organisms considered. Modified tryptone soya broth supplemented with novobiocin or modified E. coli broth supplemented with novobiocin and incubated at 41–42°C are the most appropriate selective enrichments. Injured VTEC O157 cells require pre-enrichment in a non-selective broth. Methods for the isolation of VTEC O157 should include sorbitol MacConkey agar supplemented with cefixime and potassium tellurite as the most effective isolation medium for typical sorbitol non-fermenting VTEC O157, and a second isolation medium not based on the fermentation of sorbitol but, for instance, on β-D-glucuronidase activity. Where the numbers of background flora are low, washed sheep blood agar supplemented with calcium (“EHEC agar”) may be used.

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. (C) 2010 Elsevier B.V. All rights reserved.

Between October and December 2009, 23 cases of Salmonella Typhimurium (Dutch) phage type 132, each with an identical multiple-locus variable-number tandem-repeat analysis (MLVA) profile (02-20-08-11-212), were reported from across the Netherlands. A case–control study was conducted using the food-consumption component of responses to a routine population-based survey as a control group. The mean age of cases was 17 years (median: 10 years, range: 1–68). Sixteen cases were aged 16 years or under. Raw or undercooked beef products were identified as the probable source of infection. Consumers, in particular parents of young children, should be reminded of the potential danger of eating raw or undercooked meat.

Cronobacter spp. is an opportunistic pathogen possibly occurring in many different foods and environments. This study reports results from a broad survey of foods manufactured or marketed in The Netherlands, including relevant non-food environments, conducted over a 5-years period (2001–2005). Using a specifically designed real-time polymerase chain reaction method for confirmation, Cronobacter spp. was isolated from milk powders (7/175), powdered formulae for consumers 1 year (1/5), other powdered instant products (1/182), dry cereals (6/123), raw minced meats (7/222), vegetables (2/47), spices (1/28), human faeces (1/98), and human skin (1/116) samples.

The Netherlands experienced a nationwide outbreak of Shiga toxin-producing Escherichia coli (STEC) O157 with onset of symptoms from the end of December 2008 until the end of January 2009. A total of 20 laboratory-confirmed cases were linked to the outbreak strain, serotype O157: H-, stx1, stx2, eae and e-hly positive. The investigation into the source of this outbreak is still ongoing, but evidence so far suggests that infection occurred as a result of consuming contaminated raw meat (steak tartare).