Joke van der Giessen

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.

Identification of Zoonotic Genotypes of Giardia duodenalis

Giardia duodenalis, originally regarded as a commensal organism, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. Giardiasis causes major public and veterinary health concerns worldwide. Transmission is either direct, through the faecal-oral route, or indirect, through ingestion of contaminated water or food. Genetic characterization of G. duodenalis isolates has revealed the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals, but the role of animals in the epidemiology of human infection is still unclear, despite the fact that the zoonotic potential of Giardia was recognised by the WHO some 30 years ago. Here, we performed an extensive genetic characterization of 978 human and 1440 animal isolates, which together comprise 3886 sequences from 4 genetic loci. The data were assembled into a molecular epidemiological database developed by a European network of public and veterinary health Institutions. Genotyping was performed at different levels of resolution (single and multiple loci on the same dataset). The zoonotic potential of both assemblages A and B is evident when studied at the level of assemblages, sub-assemblages, and even at each single locus. However, when genotypes are defined using a multi-locus sequence typing scheme, only 2 multi-locus genotypes (MLG) of assemblage A and none of assemblage B appear to have a zoonotic potential. Surprisingly, mixtures of genotypes in individual isolates were repeatedly observed. Possible explanations are the uptake of genetically different Giardia cysts by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia species, which may cause disease. Other explanations for mixed genotypes, particularly for assemblage B, are substantial allelic sequence heterogeneity and/or genetic recombination. Although the zoonotic potential of G. duodenalis is evident, evidence on the contribution and frequency is (still) lacking. This newly developed molecular database has the potential to tackle intricate epidemiological questions concerning protozoan diseases.

Longitudinal Analysis of Tick Densities and Borrelia, Anaplasma, and Ehrlichia Infections of Ixodes ricinus Ticks in Different Habitat Areas in The Netherlands

ABSTRACT From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m2). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m2. The highest tick density was found in the dune area (139 to 551/100 m2). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.

Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry flea-borne Rickettsia species

BackgroundHard ticks have been identified as important vectors of rickettsiae causing the spotted fever syndrome. Tick-borne rickettsiae are considered to be emerging, but only limited data are available about their presence in Western Europe, their natural life cycle and their reservoir hosts. Ixodes ricinus, the most prevalent tick species, were collected and tested from different vegetation types and from potential reservoir hosts. In one biotope area, the annual and seasonal variability of rickettsiae infections of the different tick stages were determined for 9 years.ResultsThe DNA of the human pathogen R. conorii as well as R. helvetica, R. sp. IRS and R. bellii-like were found. Unexpectedly, the DNA of the highly pathogenic R. typhi and R. prowazekii and 4 other uncharacterized Rickettsia spp. related to the typhus group were also detected in I. ricinus. The presence of R. helvetica in fleas isolated from small rodents supported our hypothesis that cross-infection can occur under natural conditions, since R. typhi/prowazekii and R. helvetica as well as their vectors share rodents as reservoir hosts. In one biotope, the infection rate with R. helvetica was ~66% for 9 years, and was comparable between larvae, nymphs, and adults. Larvae caught by flagging generally have not yet taken a blood meal from a vertebrate host. The simplest explanation for the comparable prevalence of R. helvetica between the defined tick stages is, that R. helvetica is vertically transmitted through the next generation with high efficiency. The DNA of R. helvetica was also present in whole blood from mice, deer and wild boar.ConclusionBesides R. helvetica, unexpected rickettsiae are found in I. ricinus ticks. We propose that I. ricinus is a major reservoir host for R. helvetica, and that vertebrate hosts play important roles in the further geographical dispersion of rickettsiae.

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.

Circulation of four Anaplasma phagocytophilum ecotypes in Europe

BackgroundAnaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of A. phagocytophilum have been characterized genetically, but their host range, zoonotic potential and transmission dynamics has only incompletely been resolved.MethodsThe presence of A. phagocytophilum DNA was determined in more than 6000 ixodid ticks collected from the vegetation and wildlife, in 289 tissue samples from wild and domestic animals, and 69 keds collected from deer, originating from various geographic locations in The Netherlands and Belgium. From the qPCR-positive lysates, a fragment of the groEL-gene was amplified and sequenced. Additional groEL sequences from ticks and animals from Europe were obtained from GenBank, and sequences from human cases were obtained through literature searches. Statistical analyses were performed to identify A. phagocytophilum ecotypes, to assess their host range and their zoonotic potential. The population dynamics of A. phagocytophilum ecotypes was investigated using population genetic analyses.ResultsDNA of A. phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, feeding I. hexagonus, I. frontalis, I. trianguliceps, and deer keds, but was absent in questing I. arboricola and Dermacentor reticulatus. DNA of A. phagocytophilum was present in feeding ticks and tissues from many vertebrates, including roe deer, mouflon, red foxes, wild boar, sheep and hedgehogs but was rarely found in rodents and birds and was absent in badgers and lizards. Four geographically dispersed A. phagocytophilum ecotypes were identified, that had significantly different host ranges. All sequences from human cases belonged to only one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant expansion.ConclusionFour ecotypes of A. phagocytophilum with differential enzootic cycles were identified. So far, all human cases clustered in only one of these ecotypes. The zoonotic ecotype has the broadest range of wildlife hosts. The expansion of the zoonotic A. phagocytophilum ecotype indicates a recent increase of the acarological risk of exposure of humans and animals.

Impacts of globalisation on foodborne parasites

Globalisation is a manmade phenomenon encompassing the spread and movement of everything, animate and inanimate, material and intangible, around the planet. The intentions of globalisation may be worthy--but may also have unintended consequences. Pathogens may also be spread, enabling their establishment in new niches and exposing new human and animal populations to infection. The plethora of foodborne parasites that could be distributed by globalisation has only recently been acknowledged and will provide challenges for clinicians, veterinarians, diagnosticians, and everyone concerned with food safety. Globalisation may also provide the resources to overcome some of these challenges. It will facilitate sharing of methods and approaches, and establishment of systems and databases that enable control of parasites entering the global food chain.

Spatiotemporal dynamics of emerging pathogens in questing Ixodes ricinus.

Ixodes ricinus transmits Borrelia burgdorferi sensu lato, the etiological agent of Lyme disease. Previous studies have also detected Rickettsia helvetica, Anaplasma phagocytophilum, Neoehrlichia mikurensis, and several Babesia species in questing ticks in The Netherlands. In this study, we assessed the acarological risk of exposure to several tick-borne pathogens (TBPs), in The Netherlands. Questing ticks were collected monthly between 2006 and 2010 at 21 sites and between 2000 and 2009 at one other site. Nymphs and adults were analysed individually for the presence of TBPs using an array-approach. Collated data of this and previous studies were used to generate, for each pathogen, a presence/absence map and to further analyse their spatiotemporal variation. R. helvetica (31.1%) and B. burgdorferi sensu lato (11.8%) had the highest overall prevalence and were detected in all areas. N. mikurensis (5.6%), A. phagocytophilum (0.8%), and Babesia spp. (1.7%) were detected in most, but not all areas. The prevalences of pathogens varied among the study areas from 0 to 64%, while the density of questing ticks varied from 1 to 179/100 m2. Overall, 37% of the ticks were infected with at least one pathogen and 6.3% with more than one pathogen. One-third of the Borrelia-positive ticks were infected with at least one other pathogen. Coinfection of B. afzelii with N. mikurensis and with Babesia spp. occurred significantly more often than single infections, indicating the existence of mutual reservoir hosts. Alternatively, coinfection of R. helvetica with either B. afzelii or N. mikurensis occurred significantly less frequent. The diversity of TBPs detected in I. ricinus in this study and the frequency of their coinfections with B. burgdorferi s.l., underline the need to consider them when evaluating the risks of infection and subsequently the risk of disease following a tick bite.

Have foodborne parasites finally become a global concern

The report on the ranking of the foodborne parasites is the result of an international effort, coordinated and organized by FAO and WHO. The following experts participated in the ranking exercise: Pascal Boireau, Laboratory for Animal Health Maisons Alfort, France; Jorge E. Bolpe, Ministerio de Salud de la Provincia de Buenos Aires, Argentina; Allal Dakkak, Institut Agronomique et Veterinaire Hassan II, Morocco; Brent Dixon (co-chair of meeting), Health Canada, Canada; Ronald Fayer, United States Department of Agriculture, USA; Jorge E. Gomez Marin, Centro de Investigaciones Biomedicas de la Universidad del Quindio, Colombia; Erastus Kang’ethe, University of Nairobi, Kenya; Malcolm Kennedy, University of Glasgow, UK; Samson Mukaratirwa, University of KwaZulu-Natal, South Africa; K. Darwin Murrell, University of Copenhagen, Denmark; Tomoyoshi Nozaki, National Institute of Infectious Diseases, Japan; Ynes Ortega, University of Georgia, USA; Subhash C. Parija, Jawaharlal Institute of Postgraduate Medical Education and Research, India; Lucy Robertson, Norwegian School of Veterinary Science, Norway; Mohammad Bagher Rokni, Tehran University of Medical Sciences, Iran; Patrizia Rossi, Instituto Superiore di Sanita, Italy; Said Shalaby, National Research Center, Egypt; Paiboon Sithithaworn, Khon Kaen University, Thailand; Rebecca Traub (rapporteur of meeting), University of Queensland, Australia; Nguyen van De, Hanoi Medical University, Vietnam; Joke W.B. van der Giessen (Chair of meeting), National Institute of Public Health and the Environment, The Netherlands.Resources persons for this exercise were: Michael Batz, University of Florida, USA; Annamaria Bruno, Joint FAO/WHO Food Standards Programme; Verna Carolissen, Joint FAO/WHO Food Standards Programme; Steve Hathaway, Science and Risk Assessment Standards Branch, New Zealand; Iddya Karunasagar, FAO; Gillian Mylrea, World Organisation for Animal Health (OIE); Patrick Otto, FAO; Edoardo Pozio, Instituto Superiore di Sanita, Italy; and Andrijana Rajic, FAO.The secretariat was composed of: Sarah Cahill, FAO; Marisa Caipo, FAO; Mina Kojima, WHO; Simone Magnino, WHO; and Kaye Wachsmuth, International Public Health Consultant, USA.

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.