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Historically, pork was the most prominent food source which contributed towards the transmission of Trichinella spp. and T. gondii infections. To prevent Trichinella transmission via pork an EU regulation specifies that 1) every pig carcass is systematically sampled and examined for Trichinella and infected carcasses are considered unfit for human consumption or alternatively, 2) pigs are kept according the specifications of a controlled housing system (CHS). For T. gondii, no regulation exists which prevents transmission via the consumption of pork. Based on historical information in several pig populations, the T. gondii infection prevalence is higher than that of Trichinella. Trichinella spp. and T. gondii transmission towards pigs run partly via a concurrent route, namely, the consumption of (parts of) an infected animal. Due to the partial mutual infection route of Trichinella and T. gondii to pigs a correlation between the two parasitic infestations in these hosts may be expected. In case a correlation is present and T. gondii prevalence is higher as compared to Trichinella spp., the presence or absence of Trichinella spp. may be correlated to the presence or absence of T. gondii, respectively. As a consequence, given that the T. gondii prevalence is higher than that of Trichinella spp., the absence of T. gondii may predict the absence of Trichinella spp. The subject of this thesis is to test the applicability of a system which predicts Trichinella absence from the absence of T. gondii. The method is intended as an alternative and/or addition to the present EU Trichinella monitoring and control program. Information from our studies indicate that serological tests can be used to indicate the Trichinella and T. gondii infection status of pigs. However, due to their imperfect test accuracy, use of these serological tests will lead to false interpretation of infection status of the individual animal. Alternatively, the infection status can be qualified on population level. In this approach, the level of confidence that an infected population is qualified as infected increases with each additional tested animal. The correlation between the two parasitic infections in pigs was tested in animals from an endemic area. The results show that there is an association between the two parasitic infections in the studied pigs. Because the samples of animals used within this study were preselected from farms with Trichinella cases, the sampling method may have been biased. From the study it remains unclear whether the infections are always correlated. Assuming that in CHS populations the transmission of both parasites are not correlated, the probability of Trichinella absence is equal in T. gondii infected and non‑infected animals. Assuming that the transmission of both parasites are correlated, the probability of Trichinella absence is higher in the T. gondii uninfected animals. Therefore, Trichinella free predicted pork from pig from CHS has a similar to lower Trichinella infection risk for human consumers as compared to the current situation of Trichinella monitoring. Testing the T. gondii infection status generates information on prevalence within that population.

Nematodes of the genus Trichinella are parasites of mammals, birds and reptiles. Twelve taxa are recognised in this genus. Humans may develop trichinellosis through consumption of undercooked meat from Trichinella-infected wildlife and domestic animals. The severity of the disease depends on the dose and species ingested. Meat originating from wild boar and pigs that were kept under non-controlled housing is the most important source for trichinellosis outbreaks in humans in Europe. The overall aim of this thesis was to study prevalence of Trichinella spp. in wildlife host species in the Netherlands, and to assess the risks for humans by using a quantitative microbial risk analysis (QMRA) approach. A QMRA approach allows quantification of hazards in the meat production chain, including attribution of factors as carcass control, pig husbandry system, types of meat and meat products to support risk-based methods for Trichinella control. We studied three wildlife host species that play a role in the sylvatic lifecycle of Trichinella. We show that the Trichinella prevalence in Dutch red fox (0.27%) decreased significantly in the study area, compared to fifteen years ago (3.9%, p = 0.0006). In contrast, other helminth fauna in Dutch foxes increased in biodiversity over the last three decades. Trichinella seroprevalence in Dutch wild boar during the period 2007 – 2015 (0.16%) was shown to be significantly lower than in 2004 - 2005 (0.61%, p = 0.0311), using a validated in-house ELISA. However, we were not able to demonstrate Trichinella in wild rats using artificial digestion. Using molecular techniques, we demonstrate hybridisation between T. spiralis and T. britovi from wild boar and red fox in a highly endemic area in Europe (Poland), where both Trichinella species co-exist. In view of proven absence of Trichinella from slaughter pigs reared under controlled housing during the last decades, and the large sums that are spent on individual carcass control, a risk-based control approach has been approved for Trichinella control in Europe, but the scientific background is lacking. We developed a QMRA model for Trichinella, which quantifies distribution of Trichinella ML in and among swine, test sensitivity at carcass control, partitioning of edible pork parts, Trichinella ML distribution in edible muscle types, heat inactivation by cooking and portion size. This model was combined with a dose response model for Trichinella species that was previously developed in our lab, to estimate the incidence of human illness after consumption of infected meat. The QMRA model was validated with Polish data and we estimated an average incidence of 1.56 trichinellosis cases per million persons per year (Mpy) from consumption of pigs that were reared under non-controlled housing, and 1.97 cases per Mpy from consumption of wild boar. The total estimated incidence is similar to the observed incidence of human trichinellosis (1.15 Mpy) in Poland. Overall, in the European Union, we estimated less than one predicted case of trichinellosis every 39.4 years from pork produced under controlled housing. Therefore, Trichinella testing of pigs reared under controlled housing is not adding any value to protect human health.

Human norovirus is a frequent cause of gastroenteritis worldwide, and viewed as the most common cause of foodborne disease. Numerous norovirus outbreaks associated with fresh produce, especially soft-berries and lettuce are described. Risk management strategies need to be improved in order to reduce the foodborne norovirus disease burden from consumption of fresh produce. For this purpose, data on virus persistence, the relevance of norovirus contamination sources, and the efficacy of mitigation measures were obtained. Norovirus infectivity cannot be determined, and our results are based on the detection of norovirus genomes and infectivity estimates derived from murine norovirus (MNV-1), a commonly used surrogate to study human norovirus persistence. Norovirus GI.4 and GII.4, two strains commonly associated to foodborne norovirus disease, and MNV-1, were found to persist during post-harvest conditions of fresh raspberries and strawberries. Thus post-harvest contamination of berries and contamination during primary production, at least shortly before harvest, likely pose a public health risk. Avoiding norovirus contamination, rather than reducing the presence of persistent viruses on produce, is the preferred approach for ensuring food safety. Infected food handlers, contaminated surfaces and water, are considered to be relevant sources for norovirus contamination of fresh produce. To simulate the spread of noroviruses by food handler, quantitative data on virus transfer proportions from hands to fresh produce and vice versa were determined. The simulation showed that a single infected food handler picking raspberries may spread norovirus to up to 3 kg of raspberries, which may result in large number of infected consumers, given the model assumptions. In addition, the established farm-to-fork risk assessment confirmed food handlers not practicing good hand hygiene as one of the dominant sources of contamination, given the data and model used. To prevent norovirus cross-contamination between contaminated surfaces and fresh produce, the effect of wiping surfaces in reducing norovirus loads was studied. Cleaning wet spots with dry wipes was determined to efficiently reduce spot contamination on surfaces (removal of 104 noroviruses), however, spreading of noroviruses on surfaces by wiping may result in an increased public health risk at high initial virus loads. Noroviruses are found in various water sources. To investigate the potential of norovirus introduction by application of in water diluted pesticides, the persistence of noroviruses in such dilutions was studied. Noroviruses were found to frequently persist in pesticides, and their application may therefore pose a microbiological risk to public health if contaminated water is used for pesticides dilution. Current guidelines for fresh produce production fail to provide specific criteria on the required water quality. The established quantitative farm-to-fork risk assessment model provides a basis for food safety regulations, for example, by presenting a framework for deriving performance objectives for norovirus concentrations in water used to spray irrigate lettuce. A mean concentration of around 0.4 norovirus particles per liter was estimated to realize with 95% certainty an annual health target of less than 1 infection per 10,000 consumers of lettuce. These calculations are specific to the described lettuce chain and need to be reevaluated for other scenarios.

Water in urban areas may pose a public health risk when people are exposed to urban water, because it may contain pathogens. These pathogens may originate from fecal bird droppings, runoff from paved surfaces (including e.g. dog feces), growth of micro-organisms in water and in some cases discharges of combined sewer overflows. Since the extent to which exposure to urban water poses a risk for public health was unknown, this thesis aimed to investigate health risks associated with urban water systems and to determine ways to minimize those risks. Chapter 1 provides an overview of pathogens that have caused outbreaks of waterborne illness. (The lack of ) Water quality guidelines, regulations and policies for urban water management are also addressed. Exposure to contaminated aerosols and water originating from water features may pose public health risks. In chapter 2, endotoxins (in air and water) and fecal bacteria (in water) of water features were measured as markers for exposure to microbial cell debris and enteric pathogens. Exposure to air and water near water features was shown to result in exposure to endotoxins and fecal bacteria, which may lead to respiratory health effects and gastrointestinal health complaints. Regression analyses showed that the endotoxin concentration in air was significantly influenced by the concentration of endotoxin in water, the distance to the water feature and the tangibility of water spray. This study provides estimates for aerosolisation ratios that were used as input for a quantitative microbial risk assessment in chapter 3 to quantify infection risks for exposure to splash parks. In the Netherlands, rainwater becomes more and more popular as an economic and environmentally sustainable water source for splash parks.The associated public health risk, however, and underlying risk factors were unknown. Therefore, in chapter 3, a quantitative microbial risk assessment was performed using Legionella pneumophila as a target pathogen to quantify the risk of infection for exposure due to inhalation and Campylobacter jejuni by ingestion. The risk of infection for a mean exposure duration of 3.5 minutes was 9.3*10-5 for inhalation of L. pneumophila and 3.6*10-2 for ingestion of C. jejuni. The results of the QMRA showed that using rainwater as source water for splash parks may pose a health risk. This study provided a methodology to quantify exposure volumes using observations on site. Furthermore, it gives insight into the effect of setting water quality standards, which may limit infection risks from exposure at splash parks. Splash parks have been associated with infectious disease outbreaks as a result of human exposure to poor water quality. To be able to protect public health, in chapter 4, risk factors were identified that determine poor water quality. Samples were taken at seven splash parks and were analyzed for E.coli. Higher concentrations of E. coli were measured in water of splash parks filled with rainwater or surface water as compared with sites filled with tap water. Inspection intervals and employed disinfection has no significant additional effect on the fecal contamination of the water. Management practices to prevent fecal contamination and guarantee maintaining good water quality at splash parks should include selection of acceptable source water quality and application of disinfection Flooding and heavy rainfall have been associated with waterborne infectious disease outbreaks. It is unclear to which extent they pose a risk for public health upon exposure. In chapter 5, risks of infection from exposure to urban floodwater were assessed using quantitative microbial risk assessment. To that aim, urban flood waters were sampled in the Netherlands. The water contained Campylobacter jejuni, Giardia spp., Cryptosporidium spp.,noroviruses and enteroviruses. Exposure data were collected at flood sites by questionnaires. The mean risk of infection for children who were exposed to floodwater originating from combined sewers, storm sewers and rainfall generated surface runoff was 33%, 23% and 3.5% per event, respectively, and for adults it was 3.9%, 0.58% and 0.039% per event. Warm, wet weather has been associated with cases of Legionellosis, the source of the majority of the infections, however, remains unknown. Therefore, in chapter 6, urban waters and water of wastewater treatment plants were analyzed for Legionella. Legionella was present in 3 of 6 samples of urban floodwater originating from rainfall generated surface runoff and in 5 of 24 samples originating from wastewater treatment plants. Several isolated Legionella strains belonged to sequence types that have been previously identified in patients. The presence of Legionella in urban floodwater indicates a possible transmission route for this pathogen. In chapter 7 the results of the study are discussed. The risks of infection from exposure to splash parks and floodwater were compared with the risk of infection after swimming in bathing water of good water quality according to the European Bathing Water Directive. This comparison showed that the risk of infection per case of exposure was higher for exposure to floodwater and splash parks than for swimming. The yearly infection risks are, however, dependent on the presence of pathogens in water and the frequency and the extent of exposure per year. Furthermore, new scenario’s for urban water management, like climate change and new sanitation, which may change the health risks associated with urban watermanagement were discussed. Subsequently, recommendations for the prevention of health risks associated with water features, splash parks and urban floodwater were discussed.

Clostridium difficile is an anaerobic spore forming gram-positive bacterium. Infection with C. difficile may lead in humans to symptomless carriership, but may also lead to diarrhea varying in severity from mild to life-threatening pseudomembraneous colitis. C. difficile spores can survive for long periods of time in the environment and are resistant to extreme temperatures, drought, radiation, and chemicals including disinfectants. C. difficile was considered a classical nosocomial pathogen, with a risk group consisting of elderly patients with comorbidities, treated with antimicrobials. However, since the beginning of the 21th century C. difficile infection (CDI) is increasing in incidence and severity, and is also reported in younger patients, not receiving antimicrobials, and in patients in the community. Ribotype 078 is associated with this change in epidemiology and has increased in prevalence, to the 3rd most common isolated type in human patients in Europe. The finding of the same 078 ribotype in diarrheal piglets led to the concern that interspecies transmission could occur. The goal of this PhD research was to investigate the risk of transmission of C. difficile from pigs to humans. C. difficile was commonly found in fecal samples of neonatal piglets with diarrhea. The use of a more sensitive detection method for the bacterium resulted in isolating C. difficile in fecal samples of almost all piglets, regardless their disease status. With this method, samples from sows, piglets and the environment were investigated to determine how piglets become infected with C. difficile. Since C. difficile was found in all piglets within 48 hours after birth, while all sows became positive within 113 hours after parturition, contamination of the environment with C. difficile is the most logical source for infection. Indeed, C. difficile was present in environmental samples in the farm. C. difficile was also found in the air of the farrowing ward. Follow-up research on all pig wards at the same pig farm revealed a widespread dissemination of C. difficile. Furthermore, C. difficile was present in air samples 20 meter outside the farm. Consumption of pork meat is considered a possible infection route, because C. difficile was found in retail pork samples. To determine if the source could be slaughter pigs colonized with the bacterium, rectal samples of slaughter pigs were taken at the abattoir. A prevalence of around 10% was found. However, whether C. difficile can subsequently contaminate the carcass depends on the slaughter hygiene. Human and pig C.difficile ribotype 078 seem to be genetically related because a comparison of C. difficile isolates from humans with CDI and piglets with diarrhea revealed a high similarity in antimicrobial susceptibility patterns. Samples from farmers, employees, family members and the pigs at the same farms were investigated for the presence of C. difficile. Colonization rates of C. difficile in pig farmers and their employees were higher then the colonization rates described in healthy adults. C. difficile isolates from pig farmers and their pigs are highly similar, which indicates that working with pigs is an occupational risk for colonization with C. difficile.

Biotraceability is defined as the ability to use down stream information to point to materials, processes or actions within a particular food chain that can be identified as the source of undesirable agents. We propose a practical framework in which a Monte Carlo model, where sequential events in the chain are simulated, is converted into a Bayesian Belief Network (BBN). A BBN represents the full joint probability of the model which allows interaction between the data and the model parameters and, hence, inferential queries. This makes a BBN a convenient structure for answering biotraceability questions. We discuss a model for biotracing the sources of Salmonella contamination for individual positive carcasses at the re-work station. The model is a BBN, in which Salmonella concentration information at different locations in the slaughterhouse are used in combination with prior knowledge about the dynamics of Salmonella through the slaughter line. Microbial samples were taken in a Dutch slaughterhouse from 118 carcasses directly after exsanguination, from the same carcasses after meat inspection and from the carcass splitter and belly opener. Prevalence and concentration data were used to specify prior beliefs about the model inputs and to iteratively refine the distributions of the parameters in the model so that it optimally describes that specific slaughterhouse. The model results indicate that house flora on or in the carcass splitter was the causative source of contamination for many carcasses, especially for those that carried contamination on the cutting side. Furthermore, the model indicates that the parameter values of the model may be subject to temporal variation and can be used as a tool to provide estimates of such trends. Sensitivity analysis of the model showed that the results of the model were sensitive to the true values of the parameters describing the transfer of Salmonella from the equipment, which could only be estimated with limited accuracy. More realistic estimates of the cross-contamination rates were obtained by a laboratory experiment in which the transfers of Salmonella from contaminated pork meat to a butcher’s knife and from the knife back to pork meat were determined. Recovery efficiency of bacteria from both surfaces was also determined and accounted for in the analysis. A BBN was proposed which allows the combination of uncertainty within one experiment and variability over multiple experiments. The transfer ratio probability distributions were shown to have a large variability, with a mean value of 0.19 for the transfer of Salmonella from pork to the knife and 0.58 for the transfer of Salmonella from the knife to pork. The results of the biotracing model were compared to the results of an epidemiological approach in which Salmonella serotyping data from our sampling experiment were used. The results of both approaches were similar for most Salmonella-positive carcasses at the re-work station, yet, some some differences were observed. The biotracing model can be seen as a step in the process towards an operational biotracing system, by which a stakeholder can initiate immediate responses to hazards imposed in the pork slaughterhouse.

Animal forensic DNA analysis is being used for human criminal investigations (e.g traces from cats and dogs), wildlife management, breeding and food safety. The most common DNA markers used for such forensic casework are short tandem repeats (STR). Rules and guidelines concerning quality assurance (QA) and quality control (QC) have been well established for human forensic STR DNA testing, which is most crucial in order to be able to defend the results from forensic DNA analysis in court. However, for several animal species the current laboratory practices do not meet those rules and guidelines. Consequently, QA and QC in animal genetic forensic analysis are important for laboratories, authorities, suspects, victims, the legal justice system and also for public health. In this thesis forensic STR-based DNA analysis in cattle and horse is further improved and harmonized by standardizing the commonly used STR assays for forensic purposes. The conversion tables of the ISAG to the repeat-based nomenclature system, published in this thesis, allow an unambiguous nomenclature, thereby increasing the forensic usefulness of existing datasets. By carrying out large-scale population studies, the quality and statistical power of these marker sets have been assessed. We elaborate on the general considerations that apply to the validation of forensic analysis of human and animal STR markers and in this context, evaluate our contribution to the validation of cattle and horse forensic typing. We discuss how the same data allow a reconstruction of the phylogeny of breeds, survey the state-of-the-art of forensic analysis for other animal species and finally describe the future prospects of DNA forensic analysis. Although STR is still the favourite type of marker for both human and animal forensic identity and parentage testing, there is an increasing interest in the forensic use of single nucleotide polymorphism (SNP) typing. The use of autosomal SNPs in forensic genetic casework has been widely discussed. Although much effort has been done to implement human SNPs for forensic parentage and identity testing further optimization is still required. In human it is not likely that forensic SNP typing will replace STRs, but used rather as an alternative for challenging samples that defy STR analysis. We envisage that in the near future STRs will remain the favourite type of marker for animal forensic DNA typing. However, other current or future non-forensic applications will require more modern approaches as high-throughput SNP genotyping and genomic sequencing. Ideally, the recommendations concerning forensic genetic investigations are identical for human and non-human DNA, respectively. Thanks to the rapid progress, as described in this thesis for cattle and horse, this is being realized.

Toxoplasma gondii is an important zoonotic parasite that is best known as a cause of abortion and abnormalities in newborns if a woman is primary infected during pregnancy, although ocular toxoplasmosis from acquired infection may contribute substantially to the disease burden. Infectious parasites are present as tissue cysts in undercooked meat, and as oocysts in cat faeces, contaminated soil, or water. In The Netherlands prevention focuses on educating pregnant women about the risks for infection. However, inclusion of other measures that also target the total population may be more effective. To set up effective prevention strategies the importance of the different sources needs to be known. Therefore this thesis focuses on the prevalence of infection in various animal reservoirs. First a method for sensitive PCR-based detection of tissue cysts (MC-PCR) was developed. There was a strong correlation between detection of antibodies by ELISA and parasites by MC-PCR in sheep. Therefore the high seroprevalence in sheep (27.8%) indicates that the consumption of undercooked mutton or lamb poses a risk of infection. It was difficult to develop a sufficiently discriminatory serological assay for cattle, and only two cattle, which were negative by ELISA, tested positive by PCR. This demonstrates that the seroprevalence in cattle does not give an indication of the risk of infection from beef. Whether the PCR prevalence gives an indication remains to be studied, as dead or non-infective parasites may have been detected. The seroprevalence in sheep, PCR prevalence in cattle, previously published seroprevalence data for pigs, meat consumption and preparation data, and literature data on T. gondii survival during processing were integrated in a quantitative microbial risk assessment (QMRA) model. Although the predicted number of infections was too high, the results demonstrate that beef can be an important source of infection even if the prevalence is low, and raw products contributed 40% of the total number of predicted infections. This thesis also includes a study on the seroprevalence of T. gondii in wild boar, and based on fitting compartmental infection models to the observed age-seroprevalence relation, we hypothesized that antibodies, and possibly the infection itself, do not persist lifelong in wild boar. Unfortunately no specific consumption data for wild boar were available; therefore these data could not be included in the QMRA. The last study focuses on T. gondii infection in the definitive host, the cat. Because domestic cats are the only definitive host of importance in The Netherlands, prevention of infection in cats (and thereby prevention of oocyst shedding) can be highly effective in decreasing both oocyst- and tissue cyst acquired infections in humans. In this study risk factors were identified by logistic regression analysis on serological results and questionnaire data. Hunting and feeding of raw meat were identified as risk factors that could be targeted by prevention measures. Finally, an overview of the various options for intervention in T. gondii transmission to humans is given and it is discussed how the presented results and the QMRA model can aid deciding between those options.

The use of recreational water of poor microbiological quality may negatively impact on the health of people that are exposed during a diversity of leisure activities. Microbiological contamination of recreational waters may arise from various human and animal faecal sources, but the microbiological quality of bathing water may also deteriorate due to proliferation of micro-organisms of non-faecal origin, such as Trichobilharzia and Vibrio. Long-term surveillance of recreational water related illness in The Netherlands showed that most recreational waterborne outbreaks comprised of skin conditions (48%) and gastroenteritis (31%). The number of outbreaks in a bathing season was strongly associated with weather conditions, with higher numbers in warm summers, but did not decline over the years, while compliance with European bathing water legislation increased. Current European bathing water quality standards are guided by faecal contamination events, but do not directly address waterborne pathogens of either faecal or non-faecal origin. The presence of Trichobilharzia, Vibrio, Cryptosporidium and Giardia was demonstrated in Dutch bathing waters compliant with European bathing water legislation, suggesting that monitoring of faecal indicator parameters and striving for compliance with required water quality standards may not sufficiently protect bathers from exposure to faecal and non-faecal waterborne pathogens. Cases of swimmers’ itch, caused by larvae of the parasite Trichobilharzia, are one of the most frequently reported health conditions resulting from bathing water exposure in The Netherlands. Epidemiological data from outbreaks of swimmers’ itch suggested that longer and more frequent exposure to suspected water resulted in increased reporting of symptoms. The detection of the parasite in the suspected bathing water guides interventions to prevent further cases, but is often difficult. A novel approach of simultaneously testing of collected snails and water samples from implicated bathing waters enhances the chance of parasite detection. Vibrio infections through exposure to Dutch bathing waters seem rare, but may be underestimated. Vibrio was present at four official Dutch bathing sites, albeit in low numbers, and more samples were positive at elevated water temperatures. The common occurrence of Vibrio spp. stresses the need to provide information on this micro-organism particularly to risk groups, such as people with underlying illness or open wounds. In addition, low numbers of Cryptosporidium and Giardia in recreational lakes in Amsterdam, compliant with European bathing water legislation, posed infection risks ranging between 0.01% and 0.05% for Cryptosporidium and between 0.0004% and 0.01% for Giardia, with highest infection risks for children. Infection risk assessment was improved by using newly generated exposure data collected in a study among swimmers in freshwater, seawater and swimming pools through questionnaires administered to approximately 19000 persons representing the general Dutch population. Monitoring of faecal indicator parameters and striving for compliance with standards for these parameters in European bathing water legislation does not prevent all outbreaks of recreational waterborne disease. A more effective approach for adequate protection of bathers may be a combination of the use of information available from bathing water profiles, risk assessment and high frequency monitoring using rapid molecular detection methods, addressing both faecal and non-faecal waterborne pathogens

Salmonella is one of the major causes of bacterial gastro-enteritis of humans, and the consumption of eggs contaminated with Salmonella Enteritidis (S. Enteritidis) is known as an important source of these infections. Because consumers want to use raw eggs, public health organizations and retailers want to reduce the risk of human salmonellosis without treatment of the raw product. Consequently, control measures should be taken on primary farm level. Total eradication of Salmonella spp. from the environment is not to be expected, however, and the risk for introduction of the bacteria on laying hen farms most likely cannot be reduced to zero. Therefore, it is important to detect a colonized laying hen flock as soon as possible by adequate surveillance. The probability of detection of a colonized flock depends, amongst others, on the characteristics of the tests used for detection of S. Enteritidis in samples from laying hens. In this thesis, the quality of an immunological test using egg was examined. This diagnostic test offers the opportunity to perform multi-analyte analysis, i.e. to combine Salmonella surveillance with other surveillance objectives, like avian influenza, in a single run. Moreover, because sampling can be done automatically at egg-packing stations semi-continuous surveillance of laying hen farms could be performed. The efficacy of a surveillance programme also depends on the within-flock prevalence, which changes over time since introduction of the bacteria into the flock. For development or optimization of surveillance knowledge is required of how long it takes to reach a certain prevalence which in turn depends on the within-flock dynamics of S. Enteritidis. Transmission dynamics were studied in experiments with pairs and groups of laying hens, the latter being more representative for the practical situation. Control measures on primary farm level, like vaccination and hygiene measures, in combination with targeted testing have been associated with a lower reported incidence of S. Enteritidis in egg-laying flocks as well as in humans. By combining knowledge of transmission within flocks and characteristics of detection in surveillance, a further reduction of laying hen-related human exposure to S. Enteritidis may be obtained.

Natural sausage casings are produced from the intestines of various species and used as edible containers for many different types of sausage around the world. Casings must therefore be fit for human consumption and must meet all food safety and hygiene requirements that apply to food of animal origin. As a hazard analysis of sausages would either focus on the final product or on the meat batter inside the casing, it becomes clear that many studies were done on sausages and only a handful on casings. Salt has been in use for centuries as the main preservative agent of casings and a study was done to determine whether this technique can meet the current microbiological criteria. Results indicate that the antimicrobial properties of salt used for the preservation of casings are sufficient to reduce the bacterial contamination (except for Clostridium spores) well below acceptable levels at a water activity level of 0.85 or lower during a 30-day storage period. To prevent the outgrowth of bacterial spores, additional preservation measures should be implemented. In the experiments described the use of the bacteriocin nisin was evaluated to reduce outgrowth of spores in desalinated casings. Additionally, the binding of nisin to casings, using 14C-labeled nisin Z and subsequent bioavailability of nisin were evaluated. Results demonstrate that nisin is bound to casings but if sufficient nisin was present the outgrowth of Clostridium spores in this model was reduced. Neural and lymphoid tissue can be regarded as marker tissues for the potential presence of Bovine Spongiform Encephalopathy (BSE) disease-specific prion protein (PrPPScP). These tissue types can also be present in cleaned beef casings. Based on the results from a quantitative histological analysis it was calculated that a standard 200 g sausage produced in beef casings contains 0.09 g lymphoid and 0.02 g neural tissue originating from the casing. Using these quantitative histological results a BSE infectivity risk - consumer exposure assessment was made, indicating that the potential risk when consuming a sausage produced in beef casings is far less than consuming a T-bone steak from the same animal. Casings are produced and shipped all over the world and this trans-global movement of casings means that restrictive measures, implemented to prevent the spread of contagious animal diseases, are also applicable. Two studies describe the efficacy of FMD and CSF virus inactivation in cleaned casings, either by salt (NaCl) or by using phosphate supplemented salt. After storage at about 20 C for 30 days, no remaining FMDV infectivity was found after either treatment, whereas casings stored at 4 oC still contained infectivity. CSFV infectivity was no longer present after treatment with phosphate supplemented salt and storage for 30 days at either 4 oC or 20 oC. To verify that phosphate supplemented salt was used, a commercially available phosphate assay kit was validated for the qualitative determination of phosphate present in treated casings without interference of naturally occurring phosphate in salt used for brines in which casings are preserved.