[Show abstract][Hide abstract] ABSTRACT: Helminths and their products can suppress the host immune response which may benefit parasite survival. Trichinella spiralis can establish chronic infections in a wide range of mammalian hosts including humans and mice. Here, we aim at studying the effect of T. spiralis muscle larvae excretory/secretory products (TspES) on the functionality of DC and T cell activation. We found that TspES suppress in vitro DC maturation induced by both S- and R-form lipopolysaccharide(LPS) from enterobacteria. Using different toll-like receptor (TLR) agonists, we show that the suppressive effect of TspES on DC maturation is restricted to TLR4. These helminth products also interfere with the expression of several genes related to the TLR-mediated signal transduction pathways. To investigate the effect of TspES on T cell activation, we used splenocytes derived from OVA-TCR transgenic D011.10 that were incubated with OVA and TspES-pulsed DC. Results indicate that the presence of TspES resulted in the expansion of CD4(+) CD25(+) Foxp3+ T cells. These regulatory T (Treg) cells were shown to have suppressive activity and to produce TGF-β. Together these results suggest that T. spiralis secretion products can suppress DC maturation and induce the expansion of functional Treg cells in vitro.
[Show abstract][Hide abstract] ABSTRACT: Q fever has emerged as an important human and veterinary public health problem in the Netherlands with major outbreaks in three consecutive years. Goat farms are probably the prime source from which Coxiella burnetii have spread throughout the environment, infecting people living in the vicinity. Coxiella burnetii infection not only spilled over from animal husbandry to humans but could also have spread to neighbouring wildlife and pets forming novel reservoirs and consequently posing another and lingering threat to humans, companion animals and livestock. In these cases, transmission routes other than airborne spread of contaminated aerosols may become significant. Therefore, the role of ticks in the transmission of Coxiella burnetii in the current situation was investigated. A total of 1891 questing Ixodes ricinus ticks and 1086 ticks feeding on pets, wildlife and livestock were tested by a recently developed multiplex Q-PCR. All ticks were negative, except for a few ticks feeding on a herd of recently vaccinated sheep. Coxiella-positive ticks were not detected after resampling this particular herd three months later. Based on these data we conclude that the current risk of acquiring Q fever from questing ticks in the Netherlands is negligible. However, for future risk assessments, it might be relevant to sample more ticks in the vicinity of previously C. burnetii infected goat farms and to assess whether C. burnetii can be transmitted transovarially and transstadially in I. ricinus ticks.
Zoonoses and Public Health 06/2011; 59(1):69-75. · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
International journal for parasitology 12/2010; 41(3-4):343-54. · 3.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Preventive Veterinary Medicine 09/2010; 96(3-4):232-40. · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
International journal of food microbiology 03/2010; · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To support the development of early warning and surveillance systems of emerging zoonoses, we present a general method to prioritize pathogens using a quantitative, stochastic multi-criteria model, parameterized for the Netherlands.
A risk score was based on seven criteria, reflecting assessments of the epidemiology and impact of these pathogens on society. Criteria were weighed, based on the preferences of a panel of judges with a background in infectious disease control.
Pathogens with the highest risk for the Netherlands included pathogens in the livestock reservoir with a high actual human disease burden (e.g. Campylobacter spp., Toxoplasma gondii, Coxiella burnetii) or a low current but higher historic burden (e.g. Mycobacterium bovis), rare zoonotic pathogens in domestic animals with severe disease manifestations in humans (e.g. BSE prion, Capnocytophaga canimorsus) as well as arthropod-borne and wildlife associated pathogens which may pose a severe risk in future (e.g. Japanese encephalitis virus and West-Nile virus). These agents are key targets for development of early warning and surveillance.
PLoS ONE 01/2010; 5(11):e13965. · 3.73 Impact Factor
[Show abstract][Hide abstract] 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.
International journal of food microbiology 01/2010; 139 Suppl 1:S3-15. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Evidence from experimental studies indicates that during chronic infections with certain helminth species a regulatory network is induced that can down-modulate not only parasite-induced inflammation but also reduce other immunopathologies such as allergies and autoimmune diseases. The mechanisms however, and the molecules involved in this immunomodulation are unknown. Here, we focus on the effect of Trichinella spiralis excretory/secretory antigens (TspES) on the innate immune response by studying the effect of TspES on DC maturation in vitro. Bone marrow-derived DC from BALB/c mice were incubated with TspES either alone or in combination with LPS derived from two different bacteria. As indicators of DC maturation, the cytokine production (IL-1alpha, IL-6, IL-10, IL-12p70 and TNF-alpha) and the expression of various surface molecules (MHC-II, CD40, CD80 and CD86) were measured. Results indicate that while TspES alone did not change the expression of the different surface molecules or the cytokine production, it completely inhibited DC maturation induced by Escherichia coli LPS (E. coli LPS). In contrast, DC maturation induced by LPS from another bacterium, Neisseria meningitidis, was not affected by TspES. These results were confirmed using TLR4/MD2/CD14 transfected HEK 293 cells. In conclusion, T. spiralis ES antigens lead to suppression of DC maturation but this effect depends on the type of LPS used to activate these cells.
[Show abstract][Hide abstract] ABSTRACT: Prevalences of parasitic infections in pigs from different housing systems may vary, due to their contact with the environment, and this might have consequences for food safety. In this study, 40 organic, 9 free-range and 24 intensive farms were selected and a total of 845 serum samples were tested for antibodies specific for Toxoplasma and Trichinella using ELISA assays. The overall seroprevalence of Toxoplasma in the total number of 845 serum samples tested is 2.6%, ranging from 0.38% in intensively raised pigs to 5.62% in free-range pigs. Of the housing systems tested, 4% (intensive farms) to 33% (free-range farms) is infected with Toxoplasma gondii. The risk of detecting Toxoplasma antibodies in a free-range farm are statistically higher (almost 16 times higher) than in an intensive farm. We observed that the risk of detecting specific antibodies is twice as high as in free-range compared with organic farms. Seropositivity of Trichinella spiralis antibodies was 0.12-0.35% (depending on the cut-off value at the 99.5% or 97.5% level). There was a tendency that Trichinella seropositivity was higher in organic pig farming (0.24%), but this was not significant. This serological study in pigs from different farming systems shows that the seroprevalence of antibodies specific for T. gondii is higher and for Trichinella equivalent in pigs raised in systems where there is contact with the environment than in pigs raised in intensive, indoor farming systems. This indicates that the prevalence of parasitic infections is higher in outdoor farming systems than in indoor farming systems. The possible consequences for food safety are discussed.
[Show abstract][Hide abstract] ABSTRACT: Paratuberculosis is a chronic granulomatous inflammation of the small intestine of cattle and other ruminants, caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). The disease can be found in ruminant herds worldwide, causing substantial economic losses at farm level due to premature culling and production losses. In previous studies, it has been shown that immune responses to recombinant MAP Hsp70 proteins were predominantly cell mediated. As protective immunity to the intracellular mycobacterial pathogens is thought to be cell-mediated in origin, we have studied the use of a recombinant MAP Hsp70 as a subunit vaccine in cattle experimentally infected with MAP. The results of the current study demonstrate that recombinant MAP Hsp70 can be successfully used as a subunit vaccine against bovine paratuberculosis, significantly reducing shedding of bacteria in feces during the first 2 years following experimental infection.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium avium paratuberculosis (M.a.p.) resides and replicates in macrophages. Many of the of immune mechanisms aiding M.a.p. survival in the host's cells are known. However, little is known about interactions of M.a.p. with dendritic cells (DC). As DC are important for the induction of protective immunity against infectious diseases, we investigated the interaction of M.a.p. with these cells. Quantitative real-time PCR (RT-PCR) was used to analyse differential expression of cytokine genes after 6 h and 24 h of incubation by immature DC that phagocytosed either M.a.p. or Escherichia coli (E. coli). We hypothesized that phagocytosis of E. coli would induce pro-inflammatory cytokines due to abundant presence of lipopolysaccharide (LPS) and that the cytokine expression profile induced by phagocytosis of live M.a.p. would differ. In addition we hypothesized that incubation of immature DC with rHsp70, an immunodominant antigen of M.a.p., would induce a similar profile of cytokine gene expression as phagocytosis of intact M.a.p. However, phagocytosis of both E. coli and M.a.p. resulted in a cytokine gene expression pattern representative of a (pro-)inflammatory reaction, dominated by strong induction of IL-12 gene expression, that was higher after 24 h than after 6 h of incubation, although the response to M.a.p. was less vigorous than to E. coli. Incubation with rHsp70 resulted in a more inhibitory type of cytokine gene expression, with delayed IL-12 gene expression and downregulation of the genes for IL-1beta and IL-6 after 24 h of incubation. We conclude that bovine DC produce an immuno-stimulatory, anti-mycobacterial response to infection with M.a.p., while Hsp70 potentially contributes to pathogen virulence by allowing the bacteria to invade the host cell.
Veterinary Immunology and Immunopathology 09/2005; 107(1-2):153-61. · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Heat shock proteins (Hsp) can deliver antigen into the major histocompatibility complex class I presentation pathway of antigen-presenting cells (APC), a process called cross priming, thus stimulating antigen-specific CD8+ T-cell reactions. Hsp were shown to elicit proinflammatory responses in APC. Both processes require interaction of Hsp with APC via specific receptors. This study describes the interaction of recombinant Hsp70 (rHsp70) of Mycobacterium avium subspecies paratuberculosis with bovine peripheral blood mononuclear cells that was restricted to CD14+ cells. Characterized monocyte-derived macrophages, monocyte-derived dendritic cells (DC) and BoMac, an immortalized bovine macrophage cell line, were used to investigate the interaction of rHsp70 with different bovine APC. Saturation of immature DC with high concentrations of rHsp70 is demonstrated, and it was found that interaction of rHsp70 with DC was related to the maturation stage of the DC. Involvement of CD91 as a cellular receptor for rHsp70 was demonstrated; however, competition studies with immature DC demonstrated that other receptors exist on bovine APC. These data suggest that rHsp70-based vaccines may be useful for the successful immunization of cattle.
Scandinavian Journal of Immunology 04/2005; 61(3):242-50. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Paratuberculosis in cattle is a chronic intestinal disease in which a distinctive cellular reactivity of a Th1-type preceeds the phase in which antibody titers are easily detectable and the animal becomes clinically ill. During infection with Mycobacterium avium ssp. paratuberculosis (M.a.p), a decrease in CD4 T-helper cells has been observed in the clinical phase. Our ultimate aim is to elicit a cytotoxic reaction against infected macrophages, using recombinant Hsp70 (rHsp70) of M.a.p. as a tool to shuttle antigen into the MHC class I antigen presentation pathway. To investigate the mechanism of rHsp70 as a carrier for antigen into the cell, we studied the interaction between APC and Fitc-labelled rHsp70, using FACS analysis and confocal microscopy. Interaction of rHsp70 with the cell surface of bovine APC, presumably via a receptor, was shown on monocytes, monocyte derived macrophages and dendritic cell (DC). The interaction is detectable on the complete population of freshly derived monocytes, although peak intensity of fluorescence is lower on these cells than on macrophages and DCs. DCs show interaction on a high percentage of the cells, with high intensity, while in the case of macrophages only a subpopulation interacts with rHsp70. Efficient uptake of rHsp70 as compared to OVA is shown. Preincubation of DC with unlabelled rHsp70 leads to a decreased interaction with rHsp70-FITC. DC interacting with rHsp70 in addition showed high expression of MHC I, MHC II, Myd-1 (CD172a) and CD40. Further research will focus on loading of the rHsp70 with M.a.p. antigen for presentation in MHC class I.
Veterinary Immunology and Immunopathology 10/2002; 87(3-4):239-44. · 1.88 Impact Factor