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.

Tularaemia in a brown hare (Lepus europaeus) in 2013: first case in the Netherlands in 60 years.

Tularaemia has not been reported in Dutch wildlife since 1953. To enhance detection, as of July 2011, brown hares (Lepus europaeus) submitted for postmortem examination in the context of non-targeted wildlife disease surveillance, were routinely tested for tularaemia by polymerase chain reaction (PCR). Francisella tularensis subspecies holarctica infection was confirmed in a hare submitted in May 2013. The case occurred in Limburg, near the site of the 1953 case. Further surveillance should clarify the significance of this finding.

Simultaneous increase of Cryptosporidium infections in the Netherlands, the United Kingdom and Germany in late summer season, 2012.

Starting August 2012, an increase in Cryptosporidium infections was reported in the Netherlands, the United Kingdom and Germany. It represented a 1.8 to 4.9-fold increase compared to previous years. Most samples were C. hominis IbA10G2. A case­control study was performed in the Netherlands but did not identify an endemic source. A case­case study in the north of England found travel abroad to be the most common risk factor.

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.

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.

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.

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.

Amplification of 16S rRNA sequences to detect Mycobacterium paratuberculosis.

A probe based on 16S ribosomal RNA (rRNA) sequences was developed to detect Mycobacterium paratuberculosis, the causative agent of Johne's disease in cattle. Three universal primers were used to sequence the amplified fragments of the 16S rRNA gene of various species of mycobacteria. When the nucleotide sequences were analysed, a deletion was detected in the sequence of the fast-growing species. An oligonucleotide probe (P) directed to this region was synthesised and hybridised directly with total RNA of various mycobacterial strains in a dot-spot assay. The probe detected M. paratuberculosis, some other slow-growing mycobacteria of the M. avium-intracellulare (MAI) complex, and one atypical strain, M. gordonae. To increase the sensitivity of the probe, a 413-bp fragment of the 16S rRNA gene of M. paratuberculosis between P and a second oligonucleotide primer was amplified and hybridised with a M. paratuberculosis/M. avium-specific probe. When faecal samples of cattle were tested, all culture-positive samples were positive in the PCR assay.

Epidemiology of Cryptosporidium spp. and Giardia duodenalis on a dairy farm.

Prevalences of Cryptosporidium spp. and Giardia duodenalis in relation to age and season were investigated on a dairy farm in The Netherlands over the course of 1year. The whole herd was sampled five times, whereas calves younger than about 2 months were sampled every 2-3 weeks. Associations between diarrhoea and presence of one or more pathogens (Cryptosporidium spp., G. duodenalis, rotavirus) were investigated. Potential transmission routes of Cryptosporidium spp. were evaluated and positive samples of Cryptosporidium spp. and G. duodenalis were identified to genotype level by PCR microsatellite identification and fingerprinting. Shedding of Cryptosporidium spp. was found in all age categories but peaked in calves 1-3 weeks old (39.1%). Herd prevalence of shedding for Cryptosporidium spp. varied from 2.4% in June to 22.2% in December. Shedding of G. duodenalis was found in all age categories but peaked in animals 4-5 months old (54.5%). Herd prevalence of shedding for G. duodenalis varied from 0.8% in June to 15.5% in February. Cryptosporidium spp. and rotavirus appeared to be significantly associated with diarrhoea in calves. Microsatellite analysis showed two different subtypes (C3 and C1) of Cryptosporidium parvum calf strains. Two genotypes of G. duodenalis were found, one positive by A lineage specific PCR and thus closely related to human genotypes and one genotype, which was negative by A and B lineage specific PCR. The results indicate that cow-to-calf and indirect calf-to-calf transmission both are important routes for acquiring infection with Cryptosporidium spp.

Detection of Echinococcus multilocularis in foxes in The Netherlands.

Echinococcus multilocularis was demonstrated in 5 out of 272 foxes in The Netherlands close to the border with Germany and Belgium. Besides microscopic examination of mucosal scrapings, two different PCR assays were used based on the detection of E. multilocularis DNA in colon content. Two distinct areas in The Netherlands were positive for E. multilocularis. Two positive foxes were found in the northern province of Groningen and three positive foxes were found in the southern province of Limburg. Both PCR assays detected more positive foxes compared to microscopic examination of the intestinal content. This is the first report of E. multilocularis in foxes occurring in The Netherlands.

Trichinella britovi in foxes in The Netherlands.

An overall prevalence of 3.9% of Trichinella infection was observed in the fox population in The Netherlands. Random amplified polymorphic DNA analysis of individual muscle larvae demonstrated the presence of Trichinella britovi. This is the first report of T. britovi, etiological agent of sylvatic trichinellosis, in one of the most densely populated countries in Europe and, consequently, the occurrence of cannibalism and scavenger behavior of foxes in areas with a high human population density. The presence of T. britovi in the fox population in these areas appears to be without consequences for commercial pig farming.

Sero-surveillance of wild boar in The Netherlands, 1996-1999.

From 1996 to 1999, blood samples were collected from wild boar shot during the hunting season in Crown properties, national parks and the free wildlife belt in the Netherlands. Sera were screened for the presence of antibodies against classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), Aujeszky's disease virus (ADV) and Trichinella spiralis. The results of the sero-surveillance system indicate that CSFV, SVDV and ADV are uncommon within the wild boar population. Hence, the wild boar population is not thought to be an important reservoir of these viruses in the Netherlands. Infection with ADV and CSFV is endemic in the wild boar population in Germany. Since contact between the wild boar populations of Germany and the Netherlands cannot be excluded in the southern part of the Netherlands, continuation of the sero-surveillance system seems appropriate. In the decade before 1998, no antibodies to Trichinella spp. were found in the wild boar population of the Netherlands. The detection of some seropositive animals during the hunting season of 1998-1999 corresponds to the previous findings in wild boar before 1988. However, the recent data do not have consequences for the pig industry of the Netherlands, since the country has been considered Trichinella-free for many decades.

Swine brucellosis in Indonesia.

Brucella suis biotype 1 was isolated from 13.1% of the pigs slaughtered in Kapuk Jakarta, West Java and from 15.09% of the pigs slaughtered in Surabaya, East Java. The prevalence of B. suis by means of the Rose Bengal Plate Test, was 22.3% for West Java and 14.9% for East Java. The Rose Bengal Plate Test detected more B. suis infected animals (73% of the infected animals) than did the Complement Fixation Test (41%) and the Serum Agglutination Test (54.5%). The high infection rate is a potential health danger for the abattoir workers.