Colophon This report is published under the acronym MARAN-2008 by VANTURES, the Veterinary Antibiotic Usage and Resistance Surveillance Working Group. The information presented in MARAN-2008 is based on a collation of data from ongoing surveillance systems on the use of antimicrobial agents in animal husbandry and the development of antimicrobial resistance in bacteria of animal origin and of relevance to public health. The authors thank the members of the Taskforce Masterplan Rational Use of Antibiotics in the Veal Calf Sector for their contribution to the antibiotic usage surveillance in veal calves.

In the period October 2003 to August 2005, 897 faecal samples were collected from wild animals and examined for Salmonella spp., Campylobacter spp., and Shiga toxin producing Escherichia coli (STEC) O157, the prevalence of which was found to be 0.1%, 13.8%, and 0.5 %, respectively. Campylobacter spp. were isolated mainly from faecal samples collected from corvidae (59.8%), and meadow birds and waterfowl (22.4%). A subset of these samples was also examined for Cryptosporidium and Giardia oocysts and cysts. None of the 247 samples examined contained C. parvum oocysts, and only 1 sample (roe faeces) contained G. lamblia assemblage A cysts. In the period September to November 2006, samples of running or still surface water were collected at 10 sites on 5 days, to investigate the presence of Salmonella spp., Campylobacter spp., and STEC O157. Twenty (40.8%) of the surface water samples were positive for one or more bacterial pathogens. Seven (14.3%) samples were positiveforSalmonella spp., 14 (28.6%) samples were positive for Campylobacter spp., and 1 (2.0%) sample was positivefor E. coli O157. Samples collected at only 2 of the 10 sites were negative for the pathogens tested; samples collected at the other 8 sites were positive for the pathogens at least once. To gain a better picture of the potential human health risk, this study should be followed up with a more quantitative study of the occurrence of human pathogens in wildlife, taking into account the different natural habitats and behaviour of the different animal populations and a possible seasonal effect. Furthermore, the contamination of surface water with human pathogens should be investigated more extensively.

In three successive years, we visited petting farms (n=132), care farms (n=91), and farmyard campsites (n=84), respectively, and completed a standard questionnaire with the objective of determining the hygienic status of these farms and describing hygiene measures implemented to reduce the risk of transmission of zoonotic agents from the animals to humans. For at least 85% of the farms, the overall impression of hygiene was recorded as good. However, more attention must be paid to: informing visitors on hygiene and handwashing, provision of handwashing facilities, and a footwear cleaning facility. Examination of samples of freshly voided faeces resulted in the detection of Shiga toxin-producing Escherichia coli O157 and/or Salmonella spp. and/or Campylobacter spp. at almost two-thirds (64.9%) of the petting farms, and around half of the care farms (56.0%) and farmyard campsites (45.2%). These data reinforce the need for control measures for both public and private farms to reduce human exposure to livestock faeces and thus the risk of transmission of zoonotic diseases. Public awareness of the risk associated with handling animals or faecal material should be increased.

In September 2005, the first national food-related outbreak of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157 was investigated in the Netherlands. A total of 21 laboratory-confirmed cases (including one secondary case), and another 11 probable cases (two primary and nine secondary cases) were reported in patients who became ill between 11 September and 10 October 2005. Preliminary investigation suggested consumption of a raw beef product, steak tartare (in the Netherlands also known as "filet americain"), and contact with other symptomatic persons as possible risk factors. A subsequent case-control study supported the hypothesis that steak tartare was the source of the outbreak (matched odds ratio (OR) 272, 95% confidence interval (CI) 3-23,211). Consumption of ready-to-eat vegetables was also associated with STEC O157 infection (matched OR 24, 95% CI 1.1-528), but was considered a less likely source, as only 40% of the cases were exposed. Samples of steak tartare collected from one chain of supermarkets where it is likely that most patients (67%) bought steak tartare, all tested negative for STEC O157. However, sampling was done three days after the date of symptom onset of the last reported case. Since 88% of the cases became ill within a two week period, point source contamination may explain these negative results. It is concluded that steak tartare was the most likely cause of the first national food-related outbreak of STEC O157 in the Netherlands.

In September 2005, the first nationwide outbreak of Shiga toxin-producing Escherichia coli (STEC) O157 infections was observed. A total of 21 confirmed and 11 probable patients were reported, who fell ill between September 11 and October 10. Preliminary investigation by the local public health services revealed two possible risk factors: consumption of steak tartare and contact with other persons with gastroenteritis. The results of the subsequent case-control study suggested steak tartare as the most likely cause of the outbreak. Samples of steak tartare taken at a supermarket chain where most of the patients bought the product, tested negative for STEC O157. However, sampling took place 3 days after the date of symptom onset of the last outbreak case. Because 88% of the cases became ill within a two-week period and samples taken shortly afterwards tested negative, point source contamination of steak tartare was considered most plausible.

En septiembre de 2005, se investigo en los Paises Bajos el primer brote de Escherichia coli O157 productor de shigatoxina (Stx) (STEC) asociado a alimentos. Entre el 11 de septiembre y el 10 de octubre de 2005, se identificaron 21 casos confirmados microbiologicamente (de los cuales uno era caso secundario) y 11 casos probables (dos casos primarios y nueve casos secundarios). Segun las encuestas preliminares, los factores de riesgo posibles eran el consumo de carne de vaca cruda (steak tartaro, tambien llamado « filete americano » en los Paises Bajos), y el contacto con otras personas sintomaticas. El estudio caso-control subsiguiente apoyaba la hipotesis del steak tartaro como fuente del brote (odds-ratio apareado (OR) 272, intervalo de confianza al 95% (IC) 3-23.211). Tambien se asocio el consumo de vegetales listos para consumir con las infecciones por STEC O157 (OR apareado 24, IC 95% 1,1–528), pero fue considerado menos probable, pues solo el 40% de los casos habian sido expuestos. Las muestras de steak tartaro recogidas en una cadena de supermercados donde la mayoria de los pacientes (67%) probablemente lo habian adquirido, dieron todas negativo para STEC O157. Sin embargo, las muestras se tomaron tres dias despues del inicio de sintomas en los ultimos casos declarados. Como la enfermedad ocurrio en un periodo de dos semanas para el 88% de los casos, una contaminacion puntual de la fuente puede ser la explicacion de estos resultados negativos. Se concluyo que el steak tartaro era la causa mas probable de este primer brote por STEC O157 asociado a alimentos en los Paises Bajos.

En septembre 2005, la premiere epidemie d’Escherichia coli O157 producteur de shigatoxines (Stx) (STEC) associee aux aliments a fait l’objet d’une investigation aux Pays-Bas. Un total de 21 cas confirmes microbiologiquement (dont un cas secondaire) et 11 cas probables (deux cas primaires et neuf cas secondaires) ont ete identifies entre le 11 septembre et le 10 octobre 2005. D’apres les enquetes preliminaires, les facteurs de risque possibles etaient la consommation de viande de boeuf crue, (steak tartare, aussi appele « filet americain » aux Pays-Bas), et le contact avec d’autres personnes symptomatiques. L’etude cas-temoin qui a suivi etayait l’hypothese du steak tartare comme source de l’epidemie (odds-ratio apparie (OR) 272, 95% intervalle de confiance (IC) 3-23 211). La consommation de legumes prets a consommer a egalement ete associee a l’infection a STEC O157 (OR apparie 24, 95% IC 1,1–528), mais a ete consideree comme une source moins probable car seuls 40% des cas y avaient ete exposes. Les echantillons de steak tartare preleves aupres d’une chaine de supermarches ou la plupart des patients (67%) ont vraisemblablement achete leur viande, etaient tous negatifs pour STEC O157. Cependant, les echantillons ont ete pris trois jours apres l’apparition des symptomes chez les derniers cas rapportes. La maladie etant survenue chez 88% des cas sur une periode de deux semaines, une contamination ponctuelle de la source peut expliquer ces resultats negatifs. Il a ete conclu que le steak tartare etait la cause la plus probable de cette premiere epidemie a STEC O157 associee aux aliments aux Pays-Bas.

Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods have been described. Use of many of these results in the isolation of non-pathogenic Yersinia strains. At present no single isolation procedure is available for the recovery of all pathogenic strains of Y. enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of Y. enterocolitica serotypes. Enrichment in irgasan ticarcillin chlorate (ITC) broth is the most efficient method for recovery of strains of serotype O:3, the most prevalent clinical serotype of Y. enterocolitica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most frequently used plating media. For the recovery of serotype O:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN agar seems the most efficient procedure. Selection of the proper isolation procedure will depend on the bio/serogroups of Yersinia spp. sought and on the type of food to be examined. Use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Serotyping, biotyping and virulence testing is essential for differentiation between pathogenic and environmental Yersinia strains. The International Standard Organization method for the detection of presumptive pathogenic Y. enterocolitica includes parallel use of the following two isolation procedures: (1) Enrichment in peptone, sorbitol and bile salts (PSB) broth for 2–3 days at 22–25°C with agitation or 5 days without agitation; plating on CIN agar directly and after alkaline treatment and incubation for 24 h at 30°C. (2) Enrichment in ITC for 2 days at 24°C; plating on SSDC agar and incubation for 2 days at 30°C.

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.

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.

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in childhood, can be caused by different serotypes of vero cytotoxin (VT; i.e., Shiga toxin)-producing Escherichia coli (VTEC). Recently, VT was shown to bind to polymorphonuclear leukocytes (PMNL) in the systemic circulation of patients with HUS. This study investigated whether VT bound to PMNL could be detected in persons in households with patients with HUS. Serum antibodies against E. coli O157 and, when available, fecal samples from patients with HUS and household members were studied for the presence of VTEC infection. The circulating PMNL of 82% of the household members were positive for VT, whereas stool and/or serum examination showed only 21% positivity. Thus, current methods underestimate the number of infected persons in households with patients with HUS.

The chilling process of pig carcasses in slaughter houses reduced the rate of culturable campylobacters on the carcass surface significantly.Genotyping of C. coli revealed heterogeneous patterns among the human and porcine C. coli pool.It shows that different sources of infection in humans are most probable.In minced meat we did not detect C. coli bacteriologically, but by the use of paramagnetic beads combined with PCR-technique we detected C. coli positive samples.This shows, that the meat was contaminated with C. coli.It is not clear what importance the presence of C. coli DNA in minced meat has for human infection, even though the presence of viable and culturable C. coli cells could be ruled out by bacteriological investigation.We could not distinguish between dead cells and VBNC forms of C. coli cells.The role of VBNC form of C. coli, a specific phenomen of campylobacters (Lazaro et al., 1999) and certain other bacteria, has to be investigated further. Conclusion:Porcine strains as sources of human C. coli infection can not be ruled out.Further research is needed to evaluate the C. coli findings in minced meat and the role of VBNC for human infection.

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.

SummaryThe report Salmonella in Eggs by the Advisory Committee on the Microbiological Safety of Foods (ACMSF) considered the scientific and epidemiological evidence up to 1992. Salmonella enteritidis phage type 4 is the most prevalent type of salmonella causing food poisoning in the UK. Microbiological and epidemiological evidence in the UK and abroad has shown a strong association between food poisoning caused by Salmonella enteritidis and the consumption of eggs and poultry.The ACMSF made recommendations on methods available to the poultry industry, the catering industry and the consumer on how to prevent food poisoning resulting from the consumption of eggs. These include eating eggs within three weeks, and storing them, whether in the home or by caterers, in a refrigerator at 8°C. Eggs must be stored at, and transported within, a system not exceeding 20°C which avoids excessive temperature fluctuations. The use of pasteurised liquid egg should be promoted as a replacement for raw eggs in dishes which traditionally have included raw egg, particularly in those not subject to further cooking prior to consumption. The Committee recommended improving hygiene in the poultry industry and that the industry (including those distributing and retailing egg products) should draw up a code of practice for the handling and storage of eggs. Once more the need for education and training was stressed.

In 1996 and 1997, 2,941 fresh and processed meat products obtained from supermarkets and butcher shops in The Netherlands were examined for the presence of verocytotoxin-producing Escherichia coli of serogroup O157 (O157 VTEC). Additionally, the fate of O157 VTEC in raw meat products stored at low temperatures and the effect of different additives were evaluated. O157 VTEC strains were isolated from 6 (1.1%) of 571 samples of raw minced beef, 2 (0.5%) of 402 samples of raw minced mixed beef and pork, 1 (1.3%) of 76 samples of raw minced pork, 1 (0.3%) of 393 samples of other raw pork products, and 1 (0.3%) of 328 samples of cooked or fermented ready-to-eat meats. Other raw beef products (n = 223) and meat samples originating from poultry (n = 819), sheep or lamb (n = 46), or wild animals (n = 83) were all found to be negative for O157 VTEC. For the survival experiments we used tartaar (minced beef with a fat content of less than 10%) and filet americain (tartaar mixed with a mayonnaise-based sauce [80 to 20%]). The O157 VTEC strain tested was able to survive in tartaar and filet americain stored at -20, 0, 5, or 7 degrees C for 3 days. At both 7 and at 15 degrees C, O157 VTEC counts in tartaar and filet americain remained virtually unchanged throughout a storage period of 5 days. Addition of acetic acid (to pH 4.0), sodium lactate (1 and 2% [wt/wt]), or components of the lactoperoxidase-thiocyanate-hydrogen peroxide system to filet americain did not result in a reduction of viable O157 VTEC cells during storage at 7 or 15 degrees C. It was concluded that raw meat contaminated with O157 VTEC will remain a hazard even if the meat is held at low or freezing temperatures.