Marieke Opsteegh

Food-borne diseases - the challenges of 20 years ago still persist while new ones continue to emerge.

Abstract The burden of diseases caused by food-borne pathogens remains largely unknown. Importantly data indicating trends in food-borne infectious intestinal disease is limited to a few industrialised countries, and even fewer pathogens. It has been predicted that the importance of diarrhoeal disease, mainly due to contaminated food and water, as a cause of death will decline worldwide. Evidence for such a downward trend is limited. This prediction presumes that improvements in the production and retail of microbiologically safe food will be sustained in the developed world and, moreover, will be rolled out to those countries of the developing world increasingly producing food for a global market. In this review evidence is presented to indicate that the microbiological safety of food remains a dynamic situation heavily influenced by multiple factors along the food chain from farm to fork. Sustaining food safety standards will depend on constant vigilance maintained by monitoring and surveillance but, with the rising importance of other food-related issues, such as food security, obesity and climate change, competition for resources in the future to enable this may be fierce. In addition the pathogen populations relevant to food safety are not static. Food is an excellent vehicle by which many pathogens (bacteria, viruses/prions and parasites) can reach an appropriate colonisation site in a new host. Although food production practices change, the well-recognised food-borne pathogens, such as Salmonella spp. and Escherichia coli, seem able to evolve to exploit novel opportunities, for example fresh produce, and even generate new public health challenges, for example antimicrobial resistance. In addition, previously unknown food-borne pathogens, many of which are zoonotic, are constantly emerging. Current understanding of the trends in food-borne diseases for bacterial, viral and parasitic pathogens has been reviewed. The bacterial pathogens are exemplified by those well-recognized by policy makers; i.e. Salmonella, Campylobacter, E. coli and Listeria monocytogenes. Antimicrobial resistance in several bacterial food-borne pathogens (Salmonella, Campylobacter, Shigella and Vibrio spp., methicillin resistant Staphylcoccus aureas, E. coli and Enterococci) has been discussed as a separate topic because of its relative importance to policy issues. Awareness and surveillance of viral food-borne pathogens is generally poor but emphasis is placed on Norovirus, Hepatitis A, rotaviruses and newly emerging viruses such as SARS. Many food-borne parasitic pathogens are known (for example Ascaris, Cryptosporidia and Trichinella) but few of these are effectively monitored in foods, livestock and wildlife and their epidemiology through the food-chain is poorly understood. The lessons learned and future challenges in each topic are debated. It is clear that one overall challenge is the generation and maintenance of constructive dialogue and collaboration between public health, veterinary and food safety experts, bringing together multidisciplinary skills and multi-pathogen expertise. Such collaboration is essential to monitor changing trends in the well-recognised diseases and detect emerging pathogens. It will also be necessary understand the multiple interactions these pathogens have with their environments during transmission along the food chain in order to develop effective prevention and control strategies.

Direct detection and genotyping of Toxoplasma gondii in meat samples using magnetic capture and PCR.

Different transmission routes, including the ingestion of undercooked meat, can result in Toxoplasma gondii infection in humans. The development of effective prevention strategies is hampered by a lack of quantitative information on the contamination level of different types of meat. Therefore, we developed a method for detection and quantification of T. gondii. The method involved preparation of crude DNA extract from hundred gram samples of meat, magnetic capture of T. gondii DNA and, quantitative real-time PCR targeting the T. gondii 529-bp repeat element. The detection limit of this assay was approximately 230 tachyzoites per 100 g of meat sample. There was a linear relation between the number of parasites added to the samples and Cp-values. Results obtained with the PCR method were comparable to bioassay results for experimentally infected pigs, and to serological findings for sheep. In addition, the T. gondii in 50% of the positive sheep samples could be genotyped by sequencing of the GRA6 gene, after isolation of the gene by magnetic capture. Two subtypes of GRA6 type II were identified in the 16 samples from sheep. For seven samples, the identification of T. gondii as type II was confirmed by microsatellite typing. The PCR method can be used as an alternative to bioassay for detection and genotyping of T. gondii, and to quantify the organism in meat samples of various sources.

Evaluation of ELISA test characteristics and estimation of Toxoplasma gondii seroprevalence in Dutch sheep using mixture models.

Lamb and mutton are considered important sources of human Toxoplasma gondii infections, but actual data on the prevalence of T. gondii in sheep in The Netherlands is lacking. The aim of this study was to investigate the prevalence of T. gondii in slaughtered sheep to get more insight in the importance of sheep as a source of human infection. In addition, regional variation in prevalence was studied, as this may indicate differences in environmental contamination. An in-house ELISA that detects antibodies against T. gondii was developed and used to test 1179 sera collected from sheep presented at 11 Dutch slaughterhouses between October and December 2007. Since validation of the serological assay was hampered by a lack of appropriate reference sera, the diagnostic performance and seroprevalence were estimated by fitting a binormal mixture model. ROC-curve analysis on the fitted distributions showed high discriminatory power (AUC=0.995), and high sensitivity and specificity of the ELISA. The overall prevalence was estimated at 27.8% (25.6-29.9%), but was significantly higher in sheep over 1 year old, and in sheep from the central provinces. The high sensitivity and specificity of the in-house ELISA were confirmed by Bayesian analysis together with three commercially available assays: Toxo-Screen DA (bioMérieux), Chekit Toxotest Antibody ELISA (IDEXX), and Toxoplasmosis serum screening ELISA (Institut Pourquier). In conclusion, the binormal mixture model proved a useful method to obtain estimates of diagnostic performance and seroprevalence without use of reference sera. The seroprevalence in sheep was high, and as sheep with antibodies usually carry tissue cysts, this indicates that undercooked lamb and mutton may indeed be important sources of human toxoplasmosis in The Netherlands.

A quantitative microbial risk assessment for meatborne Toxoplasma gondii infection in The Netherlands.

Toxoplasma gondii is an important foodborne pathogen, and the cause of a high disease burden due to congenital toxoplasmosis in The Netherlands. The aim of this study was to quantify the relative contribution of sheep, beef and pork products to human T. gondii infections by Quantitative Microbial Risk Assessment (QMRA). Bradyzoite concentration and portion size data were used to estimate the bradyzoite number in infected unprocessed portions for human consumption. The reduction factors for salting, freezing and heating as estimated based on published experiments in mice, were subsequently used to estimate the bradyzoite number in processed portions. A dose-response relation for T. gondii infection in mice was used to estimate the human probability of infection due to consumption of these originally infected processed portions. By multiplying these probabilities with the prevalence of T. gondii per livestock species and the number of portions consumed per year, the number of infections per year was calculated for the susceptible Dutch population and the subpopulation of susceptible pregnant women. QMRA results predict high numbers of infections per year with beef as the most important source. Although many uncertainties were present in the data and the number of congenital infections predicted by the model was almost twenty times higher than the number estimated based on the incidence in newborns, the usefulness of the advice to thoroughly heat meat is confirmed by our results. Forty percent of all predicted infections is due to the consumption of unheated meat products, and sensitivity analysis indicates that heating temperature has the strongest influence on the predicted number of infections. The results also demonstrate that, even with a low prevalence of infection in cattle, consumption of beef remains an important source of infection. Developing this QMRA model has helped identify important gaps of knowledge and resulted in the following recommendations for future research: collect processing-effect data in line with consumer style processing and acquire product specific heating temperatures, investigate the presence and concentration of viable bradyzoites in cattle, determine the effect of mincing meat on bradyzoite concentrations using actual batch sizes, and obtain an estimate of the fraction of meat that has been frozen prior to purchase. With more accurate data this QMRA model will aid science-based decision-making on intervention strategies to reduce the disease burden from meatborne T. gondii infections in The Netherlands.

Age-Related Toxoplasma gondii Seroprevalence in Dutch Wild Boar Inconsistent with Lifelong Persistence of Antibodies

Toxoplasma gondii is an important zoonotic pathogen that is best known as a cause of abortion or abnormalities in the newborn after primary infection during pregnancy. Our aim was to determine the prevalence of T. gondii in wild boar to investigate the possible role of their meat in human infection and to get an indication of the environmental contamination with T. gondii. The presence of anti-T. gondii antibodies was determined by in-house ELISA in 509 wild boar shot in 2002/2003 and 464 wild boar shot in 2007. Most of the boar originated from the “Roerstreek” (n = 673) or the “Veluwe” (n = 241). A binormal mixture model was fitted to the log-transformed optical density values for wild boar up to 20 months old to estimate the optimal cut-off value (−0.685) and accompanying sensitivity (90.6%) and specificity (93.6%). The overall seroprevalence was estimated at 24.4% (95% CI: 21.1–27.7%). The prevalence did not show variation between sampling years or regions, indicating a stable and homogeneous infection pressure from the environment. The relation between age and seroprevalence was studied in two stages. Firstly, seroprevalence by age group was determined by fitting the binary mixture model to 200 animals per age category. The prevalence showed a steep increase until approximately 10 months of age but stabilized at approximately 35% thereafter. Secondly, we fitted the age-dependent seroprevalence data to several SIR-type models, with seropositives as infected (I) and seronegatives as either susceptible (S) or resistant (R). A model with a recovery rate (SIS) was superior to a model without a recovery rate (SI). This finding is not consistent with the traditional view of lifelong persistence of T. gondii infections. The high seroprevalence suggests that eating undercooked wild boar meat may pose a risk of infection with T. gondii.

Low predictive value of seroprevalence of Toxoplasma gondii in cattle for detection of parasite DNA

The role of beef in human infections with Toxoplasma gondii is not clear. To get a better understanding of the value of seroprevalence as an indication of the role of beef in human infections with T. gondii we studied the seroprevalence of T. gondii in Dutch cattle and analysed the correlation between detection of antibodies and parasitic DNA. An indirect ELISA was developed and used to test a sample of the Dutch cattle population. Since validation of the ELISA was hampered by a lack of sufficient bovine reference sera, the results were analysed in two different ways: using a cut-off value that was based on the course of the OD in 27 calves followed from birth until 16 months of age, and by fitting a mixture of two normal distributions (binormal mixture model) to the log-transformed ODs observed for the different groups of cattle in the study population. Using the cut-off value, the seroprevalence was estimated at 0.5% for white veal, 6.4% for rosé veal and 25.0% for cattle. However, using the frequency distributions the prevalences were higher: 1.9% for white veal, 15.6% for rosé veal and 54.5% for cattle. Next, for 100 cattle the results with two different serological assays (ELISA and Toxo-Screen DA) were compared with detection of parasites by our recently developed sensitive magnetic capture PCR. Toxoplasma gondii DNA was detected in only two seronegative cattle. This discordance demonstrates that seroprevalence cannot be used as an indicator of the number of cattle carrying infectious parasites. Demonstrating parasitic DNA in seronegative cattle and not in seropositive cattle suggests that only recent infections are detectable. Whether beef from these PCR-positive cattle is infectious to humans remains to be studied.

Quantification of Toxoplasma gondii in tissue samples of experimentally infected goats by magnetic capture and real-time PCR.

Undercooked meat containing tissue cysts is one of the most common sources of Toxoplasma gondii infection in humans. Goats are very susceptible to clinical toxoplasmosis, and especially kids are common food animals, thereby representing a risk for human infection. A sequence-specific magnetic capture method was used for isolation of T. gondii DNA from tissue samples from experimentally infected goat-kids and real-time PCR for the 529 bp repeat element allowed quantification of T. gondii DNA. The contamination level in different types of tissue and in two groups of goats euthanized 30 and 90 dpi was compared. The highest concentration of T. gondii DNA in both groups of goats was found in lung tissue, but only the higher parasite count in lung tissue compared to other organs in group A (euthanized 30 dpi) was statistically significant. T. gondii concentrations were higher in liver and dorsal muscle samples from goats euthanized 90 dpi than in goats euthanized at 30 dpi, while the T. gondii concentration in hearts decreased. This study describes for the first time distribution of T. gondii parasites in post-weaned goat kids. New information about T. gondii predilection sites in goats and about the progression of infection between 30 and 90 dpi was achieved.

Seroprevalence and risk factors for Toxoplasma gondii infection in domestic cats in The Netherlands.

Cats, as definitive hosts, play an important role in the transmission of Toxoplasma gondii. To determine the seroprevalence and risk factors for T. gondii infection in Dutch domestic cats, serum samples of 450 cats were tested for T. gondii antibodies by indirect ELISA. Binary mixture analysis was used to estimate the seroprevalence, the optimal cut-off value and the probability of being positive for each cat. The seroprevalence was estimated at 18.2% (95% CI: 16.6-20.0%) and showed a decrease with age in very young cats, an increase up to about 4 years old and ranged between 20 and 30% thereafter. Hunting (OR 4.1), presence of a dog in the household (OR 2.1), former stray cat (OR 3.3) and feeding of raw meat (OR 2.7) were identified as risk factors by multivariable logistic regression analysis. Prevalence differences were estimated by linear regression on the probabilities of being positive and used to calculate the population attributable fractions for each risk factor. Hunting contributed most to the T. gondii seroprevalence in the sampled population (35%).

Toxoplasma gondii in Romanian household cats: evaluation of serological tests, epidemiology and risk factors.

Felines are the key species in the epidemiology of Toxoplasma gondii infection, as they are the definitive host of the parasite and are the only species that can shed resistant oocysts in the environment. Different assays are in use for the detection of antibodies against T. gondii in cats. However, assay validation studies are limited. For that reason it was our aim to first evaluate 6 serological tests (one commercial and 2 in-house ELISAs, ImmunoComb, IFAT and MAT) for antibodies (IgG) against T. gondii in cats by Bayesian modeling. Factors associated with seropositivity were evaluated by bivariable and multivariable methods. The test evaluation indicated the commercial ELISA had the highest Youden Index. The estimated sensitivity ranged between 95.7% and 97.1% and the specificity between 97.3% and 97.6%. Using this commercial ELISA 111 out of 236 cats (47%) were positive for T. gondii antibodies. Two peaks in the percentage of strong positive samples (S/P≥200) were observed, around 10-months-old and 8-years-old. In bivariable analysis the seroprevalence was significantly higher in adult cats, cats with mixed diet, with outdoor access, in cats from a rural area and in cats from centre and north-western Romania. Adult age (adults: OR 6.98; 95% CI: 2.02-24.14 and geriatrics (cats older than 10-years): OR 12.01; 95% CI: 1.60-90.15) and outdoor access (OR 6.38; 95% CI: 2.32-17.53) remained significant risk factors in the multivariable logistic regression analysis. Our results suggest that T. gondii infection is common in household cats in Romania, and especially in those with outdoor access.

Intervention Strategies to Reduce Human Toxoplasma gondii Disease Burden

Infection with Toxoplasma gondii is acquired through consumption of undercooked infected meat, or by uptake of cat-shed oocysts. Although congenital toxoplasmosis is generally considered to contribute most to the disease burden of T. gondii, ocular disease from acquired infection was recently shown to add substantially to the burden. In addition, toxoplasmosis in immune-compromised individuals usually results from reactivation of an infection acquired earlier in life. Nevertheless, prevention of toxoplasmosis commonly targets mainly pregnant women. We summarize current prevention strategies of congenital toxoplasmosis and evaluate options to improve protection of the general population (including pregnant women). To protect the general population, freezing of meat destined for raw or undercooked consumption is the most readily applicable option, especially when limited to meat from animals originating from nonbiosecure husbandry systems. In the long term, more health benefits are expected from cat vaccination; therefore, development of a cat vaccine and evaluation of its implementation is a research priority.