Dog Ownership and Risk for Alveolar Echinococcosis, Germany.

Human alveolar echinococcosis is caused by the parasite Echinococcus multilocularis, and dog ownership has been identified as a risk factor. We sought to specify the factors of dog ownership underlying this risk by conducting a case-control study among dog owners in Germany. The analysis revealed an increased odds ratio of ≈7-fold for dog owners whose dogs roam unattended in fields, 13-fold for dog owners who feed their dogs organic waste daily, 4-fold for dog owners who take their dog to a veterinarian only in case of illness, and 10-fold for dog owners who have never been informed by a veterinarian about the risk for infection. The results highlight the risk for infection associated with various factors of dog ownership and the value of veterinarians informing owners about prevention.

Red foxes harbor two genetically distinct, spatially separated Echinococcus multilocularis clusters in Brandenburg, Germany.

BACKGROUND: Alveolar echinococcosis (AE) is a clinically serious zoonosis caused by the fox tapeworm Echinococcus multilocularis. We studied the diversity and the distribution of genotypes of E. multilocularis isolated from foxes in Brandenburg, Germany, and in comparison to a hunting ground in North Rhine-Westphalia. METHODS: Echinococcus multilocularis specimens from 101 foxes, 91 derived from Brandenburg and 10 derived from North Rhine-Westphalia, were examined. To detect potential mixed infections with different genotypes of E. multilocularis, five worms per fox were analyzed. For genotyping, three mitochondrial markers, namely cytochrome c oxidase subunit 1 (Cox1), NADH dehydrogenase subunit 1 (Nad1), and ATP synthase subunit 6 (ATP6), and the nuclear microsatellite marker EmsB were used. To identify nucleotide polymorphisms, the mitochondrial markers were sequenced and the data were compared, including with published sequences from other regions. EmsB fragment length profiles were determined and confirmed by Kohonen network analysis and grouping of Sammon's nonlinear mapping with k-means clustering. The spatial distribution of genotypes was analyzed by SaTScan for the EmsB profiles found in Brandenburg. RESULTS: With both the mitochondrial makers and the EmsB microsatellite fragment length profile analyses, mixed infections with different E. multilocularis genotypes were detected in foxes from Brandenburg and North Rhine-Westphalia. Genotyping using the mitochondrial markers showed that the examined parasite specimens belong to the European haplotype of E. multilocularis, but a detailed spatial analysis was not possible due to the limited heterogeneity of these markers in the parasite population. Four (D, E, G, and H) out of the five EmsB profiles described in Europe so far were detected in the samples from Brandenburg and North Rhine-Westphalia. The EmsB profile G was the most common. A spatial cluster of the E. multilocularis genotype with the EmsB profile G was found in northeastern Brandenburg, and a cluster of profile D was found in southern parts of this state. CONCLUSIONS: Genotyping of E. multilocularis showed that individual foxes may harbor different genotypes of the parasite. EmsB profiles allowed the identification of spatial clusters, which may help in understanding the distribution and spread of the infection in wildlife, and in relatively small endemic areas.

Stability of African Swine Fever Virus in Carcasses of Domestic Pigs and Wild Boar Experimentally Infected with the ASFV "Estonia 2014" Isolate.

Europe is currently experiencing a long-lasting African swine fever (ASF) epidemic, both in domestic pigs and wild boar. There is great concern that carcasses of infected wild boar may act as long-term virus reservoirs in the environment. We evaluated the tenacity of ASF virus (ASFV) in tissues and body fluids from experimentally infected domestic pigs and wild boar, which were stored on different matrices and at different temperatures. Samples were analysed at regular intervals for viral genome and infectious virus. ASFV was most stable in spleen or muscles stored at -20 °C and in blood stored at 4 °C. In bones stored at -20 °C, infectious virus was detected for up to three months, and at 4 °C for up to one month, while at room temperature (RT), no infectious virus could be recovered after one week. Skin stored at -20 °C, 4 °C and RT remained infectious for up to three, six and three months, respectively. In urine and faeces, no infectious virus was recovered after one week, irrespective of the matrix. In conclusion, tissues and organs from decomposing carcasses that persist in the environment for a long time can be a source of infection for several months, especially at low temperatures.

Species Detection within the Echinococcus granulosus sensu lato Complex by Novel Probe-Based Real-Time PCRs.

Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed. We developed quantitative real-time polymerase chain reactions (qPCRs) to target polymorphic regions in the mitochondrial genome of E. granulosus s.l. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto, E. equinus, E. ortleppi and the E. canadensis cluster. The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces.

Besnoitia tarandi in Canadian woodland caribou - Isolation, characterization and suitability for serological tests.

In the present study, we report the first in vitro isolation of Besnoitia tarandi from North America and the second of B. tarandi at all. The parasite was isolated directly from the skin of a Canadian woodland caribou from the migratory ecotype. The animal belonged to the Leaf River Herd, in Northern Quebec, Canada. The isolate was designated Bt-CA-Quebec1. Sequencing of the 3'-end of the 18S rRNA gene, the complete sequence of the ITS1 and the 5'-end of the 5.8S rRNA gene of Bt-CA-Quebec1 revealed only minor differences to rDNA gene fragments of B. besnoiti. In contrast, the patterns for the microsatellite loci Bt-20 and Bt-21 varied substantially from those reported for B. besnoiti and B. bennetti. Surprisingly, the typing results in the loci Bt-6 and Bt-7 differed between Bt-CA-Quebec1 and results obtained for skin samples from caribou of the Canadian regions of Nunavut and the Northwest Territories reported by other investigators. This indicates that differences might exist among B. tarandi in caribou from different regions in Canada. Mice (γ-interferon knockout) intraperitoneally inoculated with 1.2 × 106 or 1.5 × 106 bradyzoites mechanically released from skin tissue cysts fell ill 8, 9 or 18 days post inoculation. GKO mice inoculated with 3.0 × 104 tachyzoites isolated from the peritoneal cavity of a bradyzoites-inoculated mouse became ill earlier, i.e. 5 days post inoculation. Lung was the predilection site in all mice. Bt-CA-Quebec1 tachyzoites rapidly grew in MARC-145 cells and were used for antigen production. Comparative Western blot analyses revealed only a few differences between B. tarandi Bt-CA-Quebec1 and B. besnoiti Evora antigen when probed with sera collected from chronically infected caribou. Due to its fast growth in vitro, the Bt-CA-Quebec1 isolate may represent an interesting antigen source to establish B. tarandi-specific serological tools and to study the biology of this parasite species further.

Potential risk factors associated with human alveolar echinococcosis: Systematic review and meta-analysis.

BACKGROUND: Human alveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode stage of Echinococcus multilocularis. AE is commonly associated with a long incubation period that may last for more than ten years. The objective of this systematic literature review was to identify and summarize the current knowledge on statistically relevant potential risk factors (PRFs) associated with AE in humans. METHODOLOGY/PRINCIPAL FINDINGS: Six bibliographic databases were searched, generating a total of 1,009 publications. Following the removal of duplicate records and the exclusion of papers that failed to meet the criteria of a previously agreed a priori protocol, 23 publications were retained; however, 6 of these did not contain data in a format that allowed their inclusion in the meta-analysis. The remaining 17 publications (6 case-control and 11 cross-sectional studies) were meta-analysed to investigate associations between AE and PRFs. Pooled odds ratios (OR) were used as a measure of effect and separately analysed for case-control and cross-sectional studies. In the case-control studies, the following PRFs for human AE showed higher odds of outcome: "dog ownership", "cat ownership", "have a kitchen garden", "occupation: farmer", "haymaking in meadows not adjacent to water", "went to forests for vocational reasons", "chewed grass" and "hunting / handling foxes". In the cross-sectional studies, the following PRFs showed higher odds of outcome: "dog ownership", "play with dogs", "gender: female", "age over 20 years", "ethnic group: Tibetan", "low income", "source of drinking water other than well or tap", "occupation: herding" and "low education". Our meta-analysis confirmed that the chance of AE transmission through ingestion of food and water contaminated with E. multilocularis eggs exists, but showed also that food- and water-borne PRFs do not significantly increase the risk of infection. CONCLUSIONS/SIGNIFICANCE: This systematic review analysed international peer-reviewed articles that have over the years contributed to our current understanding of the epidemiology of human AE. The identification of potential risk factors may help researchers and decision makers improve surveillance and/or preventive measures that aim at decreasing human infection with E. multilocularis. More primary studies are needed to confirm potential risk factors and their role in the epidemiology of human AE.

Comparison of different commercial DNA extraction kits and PCR protocols for the detection of Echinococcus multilocularis eggs in faecal samples from foxes.

Effective and sensitive methods for the molecular detection of Echinococcus multilocularis in faecal samples of final hosts are crucial for the prevention and control of human alveolar echinococcosis and for studies on the epidemiology of the parasite. Little is known about the suitability of commercial test kits for isolating DNA of E. multilocularis from fox faeces and the performance of standard Polymerase Chain Reaction (PCR) protocols in relation to the quality of DNA extracted by these kits. We compared four different kits: ZR Faecal DNA MiniPrep™ (Zymo Research), FastDNA® SPIN Kit for Soil (MP Biomedicals), QIAamp® Fast DNA Stool Mini Kit (QIAGEN) and NucleoSpin® Soil Kit (Macherey-Nagel) for the extraction of DNA from E. multilocularis eggs present in faeces of foxes. Negative faecal samples were spiked with 600, 300, 150, 75, 37, 18, 9, 5 or 2 E. multilocularis eggs, and each egg concentration was tested 10 times with each of the DNA extraction kits. Each extracted DNA sample was amplified using three PCR protocols: i. conventional PCR (cPCR, Platinum®Taq, Invitrogen), ii. qPCR with the iQ™ Supermix (Bio-Rad) and iii. qPCR with the QuantiTect® Multiplex-Master Mix (QIAGEN). The highest analytical sensitivities for molecular detection of E. multilocularis eggs in spiked fox faeces were observed when combining either the QIAamp® Fast DNA Stool Mini Kit or the ZR Faecal DNA MiniPrep™ kit with the qPCR using the QuantiTect® Multiplex-Master Mix (Sensitivities 97% and 94%, respectively). Combinations including the remaining test kits (NucleoSpin® Soil Kit and FastDNA® SPIN Kit for Soil) showed a markedly lower analytical sensitivity for PCR examinations. The results of the present study indicate that it is of utmost importance to select suitable DNA extraction kits in combination with robust PCR methods or reagents to achieve acceptable analytical sensitivity in the molecular detection of E. multilocularis eggs in fox faecal samples.

Harmonizing methods for wildlife abundance estimation and pathogen detection in Europe-a questionnaire survey on three selected host-pathogen combinations.

BACKGROUND: The need for wildlife health surveillance as part of disease control in wildlife, domestic animals and humans on the global level is widely recognized. However, the objectives, methods and intensity of existing wildlife health surveillance programs vary greatly among European countries, resulting in a patchwork of data that are difficult to merge and compare. This survey aimed at evaluating the need and potential for data harmonization in wildlife health in Europe. The specific objective was to collect information on methods currently used to estimate host abundance and pathogen prevalence. Questionnaires were designed to gather detailed information for three host-pathogen combinations: (1) wild boar and Aujeszky's disease virus, (2) red fox and Echinococcus multilocularis, and (3) common vole and Francisella tularensis. RESULTS: We received a total of 70 responses from 19 European countries. Regarding host abundance, hunting bags are currently the most widely accessible data source for widely distributed mid-sized and larger mammals such as red fox and wild boar, but we observed large differences in hunting strategies among countries as well as among different regions within countries. For small rodents, trapping is the method of choice, but practical applications vary among study sites. Laboratory procedures are already largely harmonized but information on the sampled animals is not systematically collected. CONCLUSIONS: The answers revealed that a large amount of information is available for the selected host-pathogen pairs and that in theory methods are already largely harmonized. However, the comparability of the data remains strongly compromised by local differences in the way, the methods are applied in practice. While these issues may easily be overcome for prevalence estimation, there is an urgent need to develop tools for the routine collection of host abundance data in a harmonized way. Wildlife health experts are encouraged to apply the harmonized APHAEA protocols in epidemiological studies in wildlife and to increase cooperation.

Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii.

Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and Besnoitia besnoiti were not recognized by mAb K8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii.

Echinococcus multilocularis: Epidemiology, surveillance and state-of-the-art diagnostics from a veterinary public health perspective.

Alveolar echinococcosis (AE), caused by the larval (metacestode) stage of Echinococcus multilocularis, is considered one of the most serious parasitic zoonoses in Central and Eastern Europe and is emerging also in large parts of Asia and in North America. The red fox represents the main definitive host of E. multilocularis in Europe, but the raccoon dog, the domestic dog and to a much lesser extent the domestic cat also represent potential definitive hosts. The natural intermediate hosts of E. multilocularis are mainly voles. The spectrum of accidental hosts is broad and includes many species of monkeys, pigs, dogs and humans which get infected by oral uptake of the viable eggs. Yet, human AE is a very rare disease in Europe; incidences have increased in recent years, while the infection is widely distributed in foxes with high prevalences reaching up to 70% in some areas. Generally, infected foxes represent a zoonotic risk, which may be particularly relevant in urban areas. Furthermore, there is concern that the risk for humans to acquire AE may rise due to the suspected geographical spread of the parasite as assessed by infections in its definitive hosts and the high prevalences in some regions. Monitoring and surveillance activities have therefore been initiated in a few European countries. Several diagnostic strategies have been developed and validated in recent years, applying classical worm detection by microscopy, but also immunological (ELISA for coproantigen detection) and molecular tests (copro-DNA detection by PCR). However, there is an urgent need for defining minimal requirements and harmonised approaches for these activities to allow for a reliable assessment of the epidemiological situation in Europe and comparable results from different countries.

[Lethal alveolar echinococcosis in a dog: clinical symptoms and pathology]. / Letale alveoläre Echinokokkose bei einem Hund: Klinik und Pathologie.

Epidemiological data indicate a progressing spread of the fox tapeworm in Germany. Here we report on a case of lethal alveolar echinococcosis in a dog from Brandenburg. The patient was clinically presented with abdominal distension. Ultrasonic examination revealed severe structural alterations of the liver and in a fine needle aspiration cytology larval tape worm fragments were suspected. Explorative laparotomy suggested inoperable lesions and the animal was euthanized with unfavorable prognosis. Pathology confirmed the diagnosis of hepatic echinococcosis. PCR analysis of the liver identified Echinococcus multilocularis, the so called "small fox tapeworm". The infection, reportable in Germany, is an important zoonotic disease that is transmitted by accidentally ingested tapeworm eggs shed by foxes or dogs. The prevalence between 7.6% and 16.7% in the fox population of Brandenburg is significantly lower than in the endemic regions of South and Southwest Germany, however, it is suspected to increase. This underlines the importance of a regional monitoring in domestic animals living in close contact to humans. In this regard, especially dogs should be taken into consideration as a potential definitive host and source of infection for people.

Serotyping of Toxoplasma gondii in cats (Felis domesticus) reveals predominance of type II infections in Germany.

BACKGROUND: Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany. METHODOLOGY: To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7). FINDINGS: Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III. CONCLUSIONS: Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.

Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle.

Cattle are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis hominis which use canids, felids or primates as definitive hosts (DH), respectively, and in addition of Sarcocystis sinensis from which the DH is unknown. The aims of the present study were to develop and optimize a multiplex real time PCR for a sensitive and specific differentiation of Sarcocystis spp. affecting cattle and to estimate the prevalence of Sarcocystis spp. in Argentinean cattle. The 18S rRNA genes from individual sarcocysts were amplified and cloned to serve as controls. For the amplification of bovine Sarcocystis spp. a total of 3 primers were used in combination with specific individual probes. Each assay was evaluated and optimized individually and subsequently combined in a multiplex assay (BovSarcoMultiplex real time PCR). The analytical specificity of the multiplex assay was assessed using 5 ng of DNA of heterologous Sarcocystis spp. and other apicomplexan parasites, and no positive reactions were observed other than for the species the PCR targeted. The analytical sensitivity ranged between 0.0125 and 0.125 fg of plasmid DNA (equivalent to the DNA of 2-20 plasmid DNA copies) or resembling DNA of 0.1-0.3 bradyzoites. A total of 380 DNA loin samples from Argentina were tested and 313, 29, 14 and 2 were positive for S. cruzi, S. sinensis, S. hirsuta and S. hominis, respectively. S. sinensis was the most prevalent species among thick walled Sarcocystis spp. in Argentinean cattle. Mixed infections were detected in 8.9% of all samples. Diagnostic sensitivity and specificity for the BovSarcoMultiplex real time PCR relative to previous microscopic examination for thin and thick-walled cyst were 91.5% and 41.7%, 36.3% and 95.9% respectively. Improved DNA extraction methods may allow to further increase the specific and sensitive detection of Sarcocystis spp. in meat samples.

Analysis of clonal type-specific antibody reactions in Toxoplasma gondii seropositive humans from Germany by peptide-microarray.

BACKGROUND: Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals. METHODOLOGY: A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100). FINDINGS: The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II-III, type I-III or type I-II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers. CONCLUSIONS: Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution.

Bayesian space-time analysis of Echinococcus multilocularis-infections in foxes.

A total of 26,220 foxes that were hunted or found dead in Thuringia, Germany, between 1990 and 2009 were examined for infection with Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, and 6853 animals were found infected. The available data on the foxes including the location (local community; district) and the date of hunting/death were analyzed using a hierarchical Bayesian space-time model. The distribution of the model parameters and their variability was estimated on the basis of the sample size, the number of cases per spatial unit and time interval, and an adjacency matrix of the municipalities using a Markov Chain Monte Carlo simulation technique to assess the spatial and temporal changes in the distribution of the parasite. The model used to evaluate the data is widely applicable and can be applied to analyse data sets with gaps and variable sample sizes per spatial and temporal unit. In the study area, the prevalence of E. multilocularis increased from 11.9% (95% confidence interval 9.9-14.0%) in 1990 to 42.0% (39.1-44.1%) in 2005. While the infection was present in foxes only in the north-western parts of Thuringia in 1990, it had spread over the entire state by 2004. These results demand increased vigilance for human alveolar echinococcosis in Thuringia.

Alveolar echinococcosis in a captive red-necked wallaby (Macropus rufogriseus).

A 2.5-year old male red-necked wallaby (Macropodus rufogriseus) kept privately in an outdoor enclosure in Germany died with severe jaundice and abdominal enlargement. Post mortem examination revealed ascites, and multiple nodular lesions in liver, diaphragm, omentum, mesentery, spleen, lung, hepatic and thoracic lymph nodes. Histologically, the nodules consisted of predominantly fertile larval tissue of a taeniid cestode, necrosis and granulomatous inflammation. Echinococcus multilocularis infection was confirmed by PCR. Macropodids have therefore to be added to the list of intermediate hosts which can develop alveolar echinococcosis.

Statistics and sample design in epidemiological studies of Echinococcus multilocularis in fox populations.

In this paper possible sampling strategies for estimating the prevalence of Echinococcus multilocularis infections in foxes are discussed. To draw valid conclusions from the analysis of fractions of a total fox population, each member of the total population must have the same chance of being selected for the investigation (random sampling), the sample must be representative with respect to all epidemiologically relevant conditions in the population (e.g. age, endemic status, seasonal effects, population density), and it must be large enough to obtain results with the required precision. For detection/exclusion of infections at a pre-specified prevalence threshold and confidence level (e.g. 99%), the required sample is rather small, but the information obtained from the data is limited. For prevalence estimates, the required sample sizes depend on the expected prevalence, the desired precision of the estimate and the chosen confidence level (e.g. 90, 95, or 99%). The samples need to be taken in spatial units where the variation of the conditions potentially influencing the infection can be neglected. A first impression of the spatial distribution of E. multilocularis infections in foxes can also be obtained by mapping the investigated sample (infected and uninfected animals) using the municipalities where they were shot or found as a spatial grid. To analyse the local influence of environmental factors, data on the geographical positions where the animals were sampled need to be collected and analysed in the context of a Geographic Information System (GIS).

Cloning and expression analysis of two mucin-like genes encoding microfilarial sheath surface proteins of the parasitic nematodes Brugia and Litomosoides.

In several filarial genera the first stage larvae (microfilariae) are enclosed by an eggshell-derived sheath that provides a major interface between the parasite and the host immune system. Analysis of the polypeptide constituents of the microfilarial sheath from the cotton rat filaria Litomosoides sigmodontis identified two abundant surface glycoproteins: Shp3a and Shp3. The corresponding genes and the orthologues of the human parasite Brugia malayi and the rodent filaria Brugia pahangi were cloned and sequenced. They encode secreted, mucin-like proteins with N-terminal Ser/Thr-rich repeats and a C-terminal anchor domain rich in aromatic amino acids. About 75% of the protein molecular masses result from post-translational modifications. The Ser/Thr-rich motifs are supposed to serve as targets for dimethylaminoethanol-phosphate substitutions. These modifications were detected only on the sheaths of the late developmental stage of stretched microfilariae, corresponding with the expression of the proteins in the epithelium of the distal part of the uterus and the specific transcription of shp3 and shp3a in the anterior female worm segment. Genomic analysis of all three species demonstrated a conserved linkage of the two genes. Their transcripts undergo cis- and trans-splicing. The transcription start sites of the primary transcripts were determined for the L. sigmodontis genes. The core promoter regions are remarkably conserved between the paralogue genes Ls-shp3a and Ls-shp3 and their orthologues in Brugia, implicating conserved regulatory elements.