Molecular identification targeting cox1 and 18S genes confirms the high prevalence of Sarcocystis spp. in cattle in the Netherlands.

The reported prevalence of Sarcocystis infection in cattle in Europe ranges between 66 and 94%. Although in the Netherlands a prevalence of 100% was reported in 1993, this study aimed to develop a method for sensitive and specific molecular detection and species identification of Sarcocystis spp., in order to provide more recent data on the prevalence and identification of these protozoa in cattle meat intended for human consumption in the Netherlands. For this purpose, 104 cattle samples were obtained from Dutch slaughterhouses. Genomic DNA was extracted, and analysed by 18S and cox1 PCR. Magnetic capture was used to extract and amplify 18S-specific DNA. Sarcocystis DNA was detected in 82.7% of the samples. PCR amplicons of both targets were sequenced, and sequence identities of ≥97% were observed for Sarcocystis cruzi (65.4%), Sarcocystis hominis (12.5%), Sarcocystis bovifelis (8.7%), Sarcocystis hirsuta and Sarcocystis heydorni (both 1.0%). Mixed infections were observed in 17.3% of the samples. The magnetic capture was not significantly more sensitive compared with standard DNA extraction, but magnetic capture did add to the overall sensitivity. Using cox1 sequencing, all species are clearly distinguished, whereas for 18S the variation between species is limited, which particularly hampers reliable identification of thick walled Sarcocystis spp. Furthermore, the detection of 12.5% S. hominis and 1% S. heydorni points towards an established transmission route between cattle and humans in the Netherlands. The availability of four additional well-identified and well-referenced S. hominis cox1 sequences in public databases enables development of species-specific diagnostic PCRs targeting cox1, which in combination with magnetic capture could provide the means to determine the prevalence of human sarcocystosis.

The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries.

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.

The design of a Social Cost-Benefit Analysis of preventive interventions for toxoplasmosis: An example of the One Health approach.

Toxoplasma gondii infections cause a large disease burden in the Netherlands, with an estimated health loss of 1,900 Disability Adjusted Life Years and a cost-of-illness estimated at €44 million annually. Infections in humans occur via exposure to oocysts in the environment and after eating undercooked meat containing tissue cysts, leading to asymptomatic or mild symptoms, but potentially leading to the development of ocular toxoplasmosis. Infection in pregnant women can lead to stillbirth and disorders in newborns. At present, prevention is only targeted at pregnant women. Cat vaccination, freezing of meat destined for undercooked consumption and enhancing biosecurity in pig husbandries are possible interventions to prevent toxoplasmosis. As these interventions bear costs for sectors in society that differ from those profiting from the benefits, we perform a social cost-benefit analysis (SCBA). In an SCBA, costs and benefits of societal domains affected by the interventions are identified, making explicit which stakeholder pays and who benefits. Using an epidemiological model, we consider transmission of T. gondii after vaccination of all owned cats or cats at livestock farms. To identify relevant high-risk meat products that will be eaten undercooked, a quantitative microbial risk assessment model developed to attribute predicted T. gondii infections to specific meat products will be used. In addition, we evaluate serological monitoring of pigs at slaughter followed by an audit and tailor made advice for farmers in case positive results were found. The benefits will be modelled stochastically as reduction in DALYs and monetized in Euro's following reference prices for DALYs. If the balance of total costs and benefits is positive, this will lend support to implementation of these preventive interventions at the societal level. Ultimately, the SCBA will provide guidance to policy makers on the most optimal intervention measures to reduce the disease burden of T. gondii in the Netherlands.

Influence of adjuvant formulation on inducing immune response in mice immunized with a recombinant serpin from Trichinella spiralis.

Nematodes of the genus Trichinella are one of the most widespread zoonotic pathogens on the world, and they can still cause major public health problems in many parts of the world. Vaccination against the helminth nematode Trichinella could be a good strategy to reduce the risk of human and animal infection. It was our aim to evaluate three adjuvants, which could be used as an efficient vaccine for animals in combination with rTs-Serpin antigen. In this study, BALB/c mice were vaccinated by an intramuscular route with rTs-Serpin antigen from the parasite Trichinella spiralis in combination with three different adjuvant formulations: Montanide ISA201, Montanide IMS 1313 N PR VG and Freund's complete adjuvant/Freund's incomplete adjuvant (FCA/FIA). The dynamics of IgG, IgM, IgE and cytokine production from spleen cells and worm reduction rate of the vaccinated mice were analysed. The results showed that rTs-serpin can induce partial protection against Trichinella larvae challenge in mice, when compared to the FCA-/FIA-formulated vaccination, the IMS1313 plus rTs-serpin mixture showed higher humoral immunity and similar levels of cellular immunity and worm reduction rate. The study suggested that Montanide IMS nanoparticles 1313 are as effective as FCA but less toxic; thus, Montanide IMS nanoparticles 1313 can be used as a good candidate of adjuvant for developing vaccine against Trichinella spiralis.

Characterization of Puumala hantavirus in bank voles from two regions in the Netherlands where human cases occurred.

Puumala hantavirus (PUUV) is the most common and widespread hantavirus in Europe and is associated with a mild form of haemorrhagic fever with renal syndrome in humans, called nephropathia epidemica. This study presents the molecular characterization of PUUV circulating in bank voles in two regions of the Netherlands. Most human cases of hantavirus infection are from these two regions. Phylogenetic analysis of the (partial) S, M and L-segments indicated that the Dutch strains belong to the CE lineage, which includes PUUV strains from France, Germany and Belgium. We have identified two distinct groups of PUUV, corresponding with their geographic origin and with adjoining regions in neighbouring countries.

Implementing wildlife disease surveillance in the Netherlands, a One Health approach.

The surveillance of (emerging) wildlife diseases can provide important, objective evidence of the circulation of pathogens of interest for veterinary and/or public health. The involvement of multiple research institutions in wildlife disease surveillance can ensure the best use of existing knowledge and expertise, but can also complicate or add challenges to the integration of wildlife disease surveillance components into a national programme. Documenting the existing efforts in a country's surveillance of wildlife diseases, including the institutes in which it takes place, provides a basis for policy-makers and authorities to identify gaps and priorities in their current surveillance programmes. This paper describes the wildlife disease surveillance activities taking place in the Netherlands. The authors recommend that, in addition to funding these current activities, surveillance resources should be allocated with the flexibility to allow for additional targeted surveillance, to detect and adequately respond to newly introduced or emerging pathogens. Similar structured overviews of wildlife disease surveillance in other countries would be very useful to facilitate international collaboration.

La surveillance exercée sur les maladies (émergentes) de la faune sauvage permet de réunir des données déterminantes, objectives et probantes sur la présence d'agents pathogènes importants pour la santé animale et/ou publique. La participation de plusieurs instituts de recherche dans les activités de surveillance des maladies de la faune sauvage permet de tirer le meilleur parti des connaissances et de l'expertise disponibles mais, dans certains cas, elle peut aussi se traduire par une complexité ou des difficultés supplémentaires qui compromettent l'intégration des composantes axées sur les maladies de la faune sauvage dans les programmes nationaux de surveillance. La collecte d'informations sur les efforts déployés au niveau national pour surveiller les maladies des animaux sauvages ainsi que sur les institutions chargées de cette surveillance constitue une première étape essentielle pour que les responsables des politiques sanitaires et les autorités puissent identifier les lacunes et les priorités des programmes de surveillance en vigueur. Les auteurs décrivent les activités de surveillance des maladies de la faune sauvage conduites actuellement aux Pays-Bas. Ils recommandent que parallèlement au financement des activités en cours, les ressources destinées à la surveillance soient allouées de manière plus souple afin de couvrir de nouvelles activités ciblées, de détecter les agents pathogènes émergents ou d'introduction récente et de préparer une réponse adéquate. Ils préconisent de réaliser dans d'autres pays des études structurées similaires sur la surveillance des maladies de la faune sauvage afin de faciliter la collaboration internationale.

La vigilancia de enfermedades (emergentes) de la fauna silvestre puede proporcionar importantes elementos de prueba objetivos sobre la circulación de patógenos de interés para la salud pública y/o veterinaria. La participación de numerosos establecimientos de investigación en estas actividades de vigilancia puede garantizar que se haga un uso idóneo de los conocimientos teóricos y técnicos existentes, pero a veces también complica o dificulta la integración en un programa nacional de las tareas de vigilancia de las enfermedades de la fauna silvestre. El hecho de repertoriar las actividades en la materia que se llevan a cabo en un país, incluidos los establecimientos donde tienen lugar, sienta las bases para que las autoridades e instancias de planificación de políticas puedan determinar las carencias y prioridades de los programas de vigilancia que ya tengan en marcha. Tras describir las actividades de vigilancia sanitaria de la fauna silvestre que se llevan a cabo en los Países Bajos, los autores recomiendan que los recursos para fines de vigilancia se asignen de manera flexible para que, además de costear las actividades ya en curso, sirvan para financiar otras labores de vigilancia selectiva que permitan detectar patógenos emergentes o recién introducidos en el país y responder debidamente a ellos. Para facilitar la colaboración internacional sería muy útil contar con estudios estructurados similares, que ofrezcan una visión de conjunto de la vigilancia sanitaria de la fauna silvestre en otros países.

Seoul hantavirus in brown rats in the Netherlands: implications for physicians--Epidemiology, clinical aspects, treatment and diagnostics.

The recent discovery of Seoul hantavirus (SEOV) presence in wild rat populations in the Netherlands has direct implications for Dutch clinicians and hantavirus diagnostics. SEOV is amongst the Old World hantaviruses which cause haemorrhagic fever and renal syndrome (HFRS) in humans. HFRS is characterised by a classical triad of fever, acute kidney injury and haemorrhage, but can show different signs and symptoms in specific cases. SEOV is transmitted from infected rats to humans by inhalation of aerosolised excreta. When compared with the known circulating hantaviruses in the Netherlands, Puumala (PUUV) and Tula (TULV), SEOV causes a more severe form of HFRS. Data from cohort studies undertaken in China and Northern Europe show differences in signs and symptoms at onset of disease, (haemorrhagic) complications and mortality. Furthermore, routine diagnostics currently available for hantavirus diagnosis in the Netherlands are not optimised for SEOV detection. The clinical outcome of an SEOV and PUUV infection will greatly benefit from an early diagnosis which will reduce the costs of unnecessary tests and treatments as well. The discovery of SEOV circulation in the Netherlands follows recent findings of SEOV infections in both rodents and humans in England, Wales, France, Belgium and Sweden, indicating the emerging character of SEOV and a high importance of this hantavirus for Public Health in large areas of Europe. Here, we review the current knowledge on the clinical manifestation of SEOV versus PUUV infections in humans, the treatment of clinical cases and diagnostics.

Significant increase of Echinococcus multilocularis prevalence in foxes, but no increased predicted risk for humans.

The emergence of the zoonotic tapeworm Echinococcus multilocularis, causative agent of alveolar echinococcosis (AE), poses a public health risk. A previously designed risk map model predicted a spread of E. multilocularis and increasing numbers of alveolar echinococcosis patients in the province of Limburg, The Netherlands. This study was designed to determine trends in the prevalence and worm burden of E. multilocularis in foxes in a popular recreational area in the southern part of Limburg to assess the risk of infection for humans and to study the prevalence of E. multilocularis in dogs in the adjacent city of Maastricht. Thirty-seven hunted red foxes were tested by the intestinal scraping technique and nested PCR on colon content. Additionally, 142 fecal samples of domestic dogs from Maastricht were analyzed by qPCR for the presence of E. multilocularis. In foxes, a significantly increased prevalence of 59% (95% confidence interval 43-74%) was found, compared to the prevalence of 11% (95% CI 7-18%) in 2005-2006. Average worm burden increased to 37 worms per fox, the highest since the first detection, but consistent with the prediction about the parasite population for this region. Updated prediction on the number of AE cases did not lead to an increase in previous estimates of human AE cases up to 2018. No dogs in the city of Maastricht tested positive, but results of questionnaires showed that deworming schemes were inadequate, especially in dogs that were considered at risk for infection.

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%).

Specific serum antibody responses following a Toxoplasma gondii and Trichinella spiralis co-infection in swine.

The aim of this study was to examine the dynamics of parasite specific antibody development in Trichinella spiralis and Toxoplasma gondii co-infections in pigs and to compare these with antibody dynamics in T. spiralis and T. gondii single infections. In this experiment, fifty-four pigs were divided into five inoculated groups of ten animals, and one control group of four animals. Two groups were inoculated with a single dose of either T. gondii tissue cysts or T. spiralis muscle larvae, one group was inoculated simultaneously with both parasites and two groups were successively inoculated at an interval of four weeks. Specific IgG responses to the parasites were measured by ELISA. T. gondii burden was determined by MC-PCR carried out on heart muscle and T. spiralis burden by artificial digestion of diaphragm samples. Specific IgG responses to T. gondii and T. spiralis in single and simultaneously inoculated animals showed a respective T. gondii and T. spiralis inoculation effect but no significant interaction of these parasites to the development of specific antibodies with the serum dilutions used. Moreover, our data showed that the specific IgG response levels in groups of animals successively or simultaneously co-infected were independent of a respective previous or simultaneous infection with the other parasite. Additionally, no differences in parasite burden were found within groups inoculated with T. gondii and within groups inoculated with T. spiralis. Conclusively, for the infection doses tested in this experiment, the dynamics of specific antibody development does not differ between single and simultaneous or successive infection with T. gondii and T. spiralis. However, lower parasitic doses and other ratios of doses, like low-low, low-high and high-low of T. gondii and T. spiralis in co-infection, in combination with other time intervals between successive infections may have different outcomes and should therefore be studied in further detail.

Human beings are highly susceptible to low doses of Trichinella spp.

Trichinella is an important foodborne pathogen causing considerable morbidity and mortality. To prevent human trichinellosis, meat inspection for Trichinella spp. at slaughter is a key instrument. Current testing is based on minimal infectious dose in humans, but a scientific basis for this approach is lacking. To this end, a dose-response model must be developed, allowing translation of exposure into disease burden at the population level. We developed novel methods for dose-response assessment using outbreak data incorporating sexual reproduction of the parasite. A selection of suitable outbreak studies, reporting numbers exposed and infected, as well as estimated doses, was collated from a literature study. Humans appear to be highly susceptible: exposure to low doses (few larvae) is associated with a considerable risk of infection. As a consequence, levels of Trichinella in meat must be low to maintain acceptable health risks.

Prevalence of antibodies against Ascaris suum and its association with allergic manifestations in 4-year-old children in The Netherlands: the PIAMA birth cohort study.

The association between helminth infections and childhood atopic diseases remains controversial. The majority of studies have been carried out in tropical areas, whereas less information is available from western countries with low intensity of helminth infections. In the Netherlands, the infection of pigs with Ascaris suum is very common, particularly on pig farms with outdoor facilities. This helminth can also infect humans, causing visceral larva migrans. This study aims at determining the prevalence of antibodies against A. suum and its association with allergic symptoms and sensitisation in a population of 4-year-old children living in The Netherlands. Blood samples from 629 children from the prospective birth cohort Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study were examined for Ascaris antibodies. Data on allergic symptoms and sensitisation were collected using questionnaires and radioallergosorbent tests (RAST). A total of 45 out of 629 (7%) were found to be Ascaris-seropositive. In addition, a positive association between Ascaris seropositivity and wheeze in the last year, doctor-diagnosed asthma and food and aero-allergen sensitisation was found. These results support the hypothesis that low-level or transient infection with helminths enhances allergic reactivity.

[Risk of Echinococcus granulosus becoming endemic in Dutch cattle]. / Het risico van het endemisch worden van Echinococcus granulosus in rundvee in Nederland.

Echinococcus granulosus is rare in The Netherlands and most human patients originate from southern Europe and Africa, where E. granulosus is still endemic in sheep, cattle, and pigs. Since the accession of some south-eastern European countries to the European Union, a large number of cattle have been imported from this area, according to national import data. The objective of this study was to determine the risk of re-introduction of E. granulosus in The Netherlands via the import of cattle from these endemic areas. The number of infected imported cattle was determined by correcting the number of imported cattle with the national animal prevalence of E. granulosus in the country of origin. In 2007, the number of imported E. granulosus-infected cattle varied from 0 (Cyprus) to 4,934 (Romania, accounting for 90% of all positive cattle). The likelihood of detecting E. granulosus at slaughter is low--we assumed, based on confirmed cases, that only 10% of infected cattle will be detected during visual inspection at slaughter. In 2007, 542 infected cattle were probably culled in The Netherlands (assuming that cattle younger than 3 months were not infected). Since the lungs and livers of cattle approved for human consumption may be processed into dog food, there is a risk that dogs that eat E. granulosus-containing dog food may become infected and in turn infect humans. On the basis of a model that assumed that only cattle older than 3 months at the moment of importation were a risk, 23 dogs may have been exposed to E. granulosus in 2007. To reduce the risk of importing E. granulosus, measures should be taken, such as declaring the lungs and livers of Romanian cattle unfit for human consumption and banning the use of infected raw lung and liver in dog food.

Public health risk analysis of European bat lyssavirus infection in The Netherlands.

We present the frequency and the nature of contact incidents of the Serotine bat, Eptesicus serotinus, with humans and with companion animals (specifically cats and dogs), in The Netherlands between 2000 and 2005. Out of 17 bats in bite contact with humans, five tested positive for European bat lyssavirus (EBLV) type 1a. Cats had the most numerous contacts with bats (49 times) but a relatively low number of these bats were EBLV positive (six times). We estimated that the average incidence of human bat rabies infection might be between once per year and once per 700 years, depending mainly on the number of infectious viral particles in bat saliva. The risk of bat rabies is higher between April and October, and in the northern half of the country. This is the first study in Europe describing the risk of human bat rabies after bat contact incidents.

Usefulness of sero-surveillance for Trichinella infections in animal populations.

In this paper we evaluate serology as a tool to monitor Trichinella-free pig herds. Indoor, industrial-raised fattening pigs in the Netherlands are practically Trichinella-free, and were used as a negative reference cohort. A positive cohort was not available but we used sera from an endemic region in Argentina to model a plausible distribution of serological responses (as OD levels) in positive sera, employing the difference between the endemic sera and the negative Dutch sera. We describe a method for correcting for variation among ELISA plates using on-plate reference sera, and demonstrate how to apply these corrections to a collection of test sera from pig farms. The positive and negative reference distributions can be used to estimate fractions true and false positives, necessary for defining appropriate cutoffs to be used for classifying positive and negative animals. Based on this analysis, the serological test was shown to lack the predictive power required for its large scale deployment. The properties of the serological test were also compared to the conventional digestion assay, which is highly specific but considerably less sensitive.

Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates.

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a protozoan organism that can infect the intestinal tract of many animal species including mammals. Genetic heterogeneity of G. duodenalis is well described but the zoonotic potential is still not clear. In this study, we analysed 100 Giardia DNA samples directly isolated from human stool specimens, to get more insight in the different G. duodenalis assemblages present in the Dutch human population. Results showed that these human isolates could be divided into two main Assemblages A and B within the G. duodenalis group on the basis of PCR assays specific for the Assemblages A and B and the DNA sequences of 18S ribosomal RNA and the glutamate dehydrogenase (gdh) genes. Genotyping results showed that G. duodenalis isolates originating from Dutch human patients belonged in 35% of the cases to Assemblage A (34/98) and in 65% of the cases to Assemblage B (64/98) whereas two human cases remained negative in all assays tested. In addition, we compared these human samples with animal samples from the Netherlands and human and animal samples from other countries. A phylogenetic analysis was carried out on the DNA sequences obtained from these Giardia and those available in GenBank. Using gdh DNA sequence analysis, human and animal Assemblage A and B Giardia isolates could be identified. However, phylogenetic analysis revealed different sub-clustering for human and animal isolates where host-species-specific assemblages (C, D, E, F and G) could be identified. The geographic origin of the human and animal samples was not a discriminating factor.

Phylogenetic analysis of encapsulated and non-encapsulated Trichinella species by studying the 5S rDNA tandemly repeated intergenic region.

The identification of sequence regions in the genomes of pathogens which can be useful to distinguish among species and genotypes, is of great importance for epidemiological, molecular, and phylogenetic studies. The 5S ribosomal DNA intergenic spacer region has been identified as a good target to distinguish among eight Trichinella species and genotypes. The recent discovery of two non-encapsulated species in this genus, Trichinella papuae and Trichinella zimbabwensis, which can infect both mammals and reptiles, has suggested analyzing their 5S rDNA. Amplification of the tandem repeats of the 5S rDNA intergenic region of encapsulated species of Trichinella shows a 751bp fragment, whereas the three non-encapsulated species show a fragment of 800bp with T. pseudospiralis showing an additional fragment of 522bp. Although the size of the 800bp PCR fragments of T. papuae and T. zimbabwensis are similar to that of T. pseudospiralis, there are differences in the 5S rDNA intergenic regions among the three non-encapsulated species. Phylogenetic analysis of the 5S rDNA intergenic regions shows a clustering together of the three non-encapsulated Trichinella species that is well separated from the encapsulated ones. In addition, a single PCR-based method allows distinguishing non-encapsulated and encapsulated species.

[Echinococcus multilocularis: the prevalence of the fox tapeworm with possible serious consequences for humans]. / Echinococcus multilocularis: de kleine lintworm van de vos met mogelijk grote gevolgen voor de mens.

The prevalence of the fox tapeworm in foxes (final host) and muskrats (one of the intermediate hosts) in the Netherlands and Europe has been discussed. The tapeworm was found in 9.4% of the investigated foxes from the province of Groningen and in 0.2% of the muskrats from the same region. Also in the province of Limburg positive foxes were found, but no positive muskrats. Possible ways of infection for humans are described together with methods for prevention. It is concluded that at this moment risks for humans to become infected are minimal, but vigilance and monitoring of foxes and muskrats remains needed.