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.     

Echinococcus multilocularis in Grisons: distribution in foxes and presence of potential intermediate hosts

The southern border of the European endemic area of Echinococcus multilocularis runs along the alpine crest. This endemic transition area was analysed in the canton Grisons on a small spatial scale. A total of 543 foxes originating from 10 areas north and 4 areas south of the main alpine divide were investigated. Parasites were isolated using the sedimentation and counting technique on intestinal contents. The mean prevalence of E. multilocularis was 6.4% with significant differences between different areas. In the southern valleys, only foxes from the Val Müstair were infected (14.3%). On the northern side of the main alpine divide, prevalences varied between 0 and 40%. The predation habits of foxes on potential intermediate hosts was investigated by means of stomach content analyses (n=530). Rodents of the genera Microtus/Pitymys were found in 19.6% of fox stomachs, Clethrionomys glareolus in 8.0% and Arvicola terrestris in 0.4%. A small scale analysis based on a 10 x 10 km grid suggested that the predation rate on the genera Microtus/Pitymys significantly correlated with the prevalence of E. multilocularis in foxes (Spearman's r = 0.51). Hence, E. multilocularis appears to occur in the alpine study area on a very small spatial scale. These local sources of infection may persist for decades. This may partly explains why, during the last 34 years, some human cases of alveolar echinococcosis occurred in areas of the Canton Grisons where the parasite is presently endemic in foxes. No such human cases have been recorded in other areas free of E. multilocularis.     

Evaluation of coproantigen diagnosis for natural Echinococcus multilocularis infection in red foxes.

The validity of a coproantigen ELISA for Echinococcus multilocularis was evaluated by comparison of three diagnostic methods; autopsy, egg examination and the ELISA. Of 71 foxes, 39 were found to be infected with the cestode at autopsy. The overall mean of worm burdens was 3,451, but the number varied (1-34,522). The ELISA could detect 94.9% (37/39) of the worm positives and there were no false-positives. Two false-negatives were infected with 1 and 4 cestodes, whereas 3 cases with similar worm burdens (2, 4 and 6 worms) were diagnosed as positives. This indicates the detection limit of the assay may be equivalent to less than 10 (in the worm burden). On the other hand, egg examination showed low sensitivity (43.6%, 17/39). These results suggest the ELISA has a potential to replace for the conventional methods.     

Field evaluation of an intravital diagnostic test of Echinococcus multilocularis infection in red foxes

Echinococcus multilocularis parasitizes the small intestine of red foxes (Vulpes vulpes) and other carnivores, and has a wide distribution throughout the northern hemisphere. This cestode is the causative agent of human alveolar echinococcosis, a life-threatening helminth zoonosis. In 2000-2002, 2130 red foxes were examined for its presence in Slovakia, with a total prevalence of 30.7%. The data on occurrence were obtained by the combination of necropsy of small intestines from red foxes and coproantigen detection in faecal samples. The correlation between the number of detected specimens and the value of optical density of copro-ELISA test was found. When worm burdens were low (1-25 specimens) the sensitivity of the method was 31.3+/-8.64%, when worm burdens were >50 specimens, 81.8+/-0.66%, and with high worm burdens (>1000 specimens) the sensitivity reached 100+/-0.34%. E. multilocularis presence was detected using the nested PCR method from the eggs in the faecal samples with a 100% specificity. In epidemiological surveys of this zoonosis, it is of crucial importance to detect animals with a high level of infection, which are responsible for the bulk of environmental contamination. The advantage of copro-ELISA test lies in allowing the intravital diagnostics to be employed within the epidemiological survey of E. multilocularis occurrence in the protected and urban areas.     

Increase in the prevalence of Echinococcus multilocularis infection in red foxes in Lower Saxony.]

Echinococcus multilocularis is a tapeworm having carnivores as final hosts, the red fox in particular, dog and cat less frequent. Its two host life cycle consists of a larval cycle which predominantly takes place in the liver of rodents such as mice but it can also develop in musk rats as intermediate hosts. Man can also be infected and serves as a wrong intermediate host. He develops an alveolar echinococcosis which usually ends lethal without medical treatment. The prevalence of E. multilocularis among 5.365 red foxes in Lower Saxony was monitored from 1991 to 1997. The data were analysed using spatial epidemiological methods. This evaluation is based on a significance test which was applied to the parameters of spatial autoregressive regression models (CAR) fitting to the data of two successive sampling periods from 1991-1994 and 1994-1997. The mean prevalence (spatial median) increased from 6% to 11%. The results of this epidemiological study which was restricted to Lower Saxony support for the first time the earlier reported assumption that the prevalence of foxes infected with E. multilocularis has risen in Europe in the last decade. The reasons for this development are still unknown.     

Echinococcus multilocularis and Toxocara canis in urban red foxes (Vulpes vulpes) in Brussels, Belgium

During the last decades, European red foxes (Vulpes vulpes) have been implicated in the transmission of several viral or parasitic pathogenic agents to domestic animals and humans. In urban areas, risks of zoonoses transmission are likely to increase as a result of a higher rate of intra- and inter-species contacts. Foxes occur on 35% of the Brussels-Capital Region area and local densities reach up to 4 family groups per square kilometre. According to the directive 2003/99/ECC, a first survey for the presence in foxes of Echinococcus multilocularis and Toxocara canis was conducted in Brussels from 2001 to 2004. None of 160 foxes were found to be infected with E. multilocularis and 24 of 134 foxes were found to be infected with T. canis. Considering numbers of examined foxes, the sensitivity and the specificity of tests used for diagnosis, the 95% credibility intervals for the true prevalence of E. multilocularis and T. canis were estimated in a Bayesian framework to be 0 to 1.87% (median value of 0%) and 12.7 to 26% (median value of 18.7%), respectively. For T. canis, a significantly higher risk to be a carrier occurs in cubs and a significantly lower risk in adults.     

Detection of Echinococcus multilocularis in foxes: evaluation of a protocol of the intestinal scraping technique

The intestinal scraping technique (IST) is widely used for the detection of definitive hosts infected with Echinococcus multilocularis. The sensitivity of the method has been questioned in recent years. Several variations of the technique are used that may differ in their performance. We therefore estimated the diagnostic sensitivity and specificity of the IST protocol used in our lab by examining the small intestines of 210 E. multilocularis-infected foxes and 294 foxes that had tested negative for this parasite. To this end, IST was first performed on 48 and 294 intestines from infected and uninfected foxes respectively, followed by an examination of the entire remaining mucosa of the small intestine to discover any infections with E. multilocularis that had not been detected by IST. The point estimate for diagnostic sensitivity was 100%. Five different classes of infection intensities were formed. The lower limits of the 95% confidence intervals ranged between 76.8% and 92.6% depending on the number of samples analysed per class. When the small intestines of another 162 infected foxes were examined by IST and the results recorded separately for the anterior, middle and posterior third of the small intestine, a strong preference of E. multilocularis for the posterior third was observed. For epidemiological purposes, it may thus be possible to restrict routine investigations to the posterior portion of the small intestine. By contrast, a reduction of the number of slides examined per infected animal may lead to a considerable loss in sensitivity in animals with a low intensity of infection.     

Reduced egg production of Echinococcus multilocularis in experimentally infected and re-infected red foxes (Vulpes vulpes)

Ingestion of eggs of the small fox tapeworm, Echinococcus multilocularis, causes the severe human disease alveolar echinococcosis. Previously, the dynamics of the egg excretion from infected carnivores have been studied only where the host animals have been exposed to a single experimental infection. In nature, foxes are most likely repeatedly infected. To study the effect of repeated exposure, twenty-one foxes were inoculated with a high dose of E. multilocularis protoscoleces three times over a 1-month period. For comparative purposes, three groups of twenty-one foxes were respectively inoculated with low, medium, or high single dose of protoscoleces. For each group, worm number and morphology were analyzed after necropsy of seven foxes at 1, 2, and 4 months after last inoculation. The establishment of intestinal worms was very low in all foxes, and surprisingly, most of the worms did not produce eggs. Although most reproductive structures were detectable, the genital pore and the cirrus pouch often had abnormal enlargements that spread internally, most likely preventing the reproductive function. The reason for this abnormality could not be determined, but the preparation and storage conditions of the inoculated protoscoleces may have contributed to the stunted development. Physical stress of E. multilocularis at the larval stage in rodents may later adversely affect the reproductive success of the adult tapeworm in the carnivore definitive host; as in the present study where a worm establishment in the definitive host was only followed by a neglectable egg production.     

Choropleth mapping of regional count data of Echinococcus multilocularis among red foxes in Lower Saxony, Germany.

In this paper, choropleth maps display the geographical distribution of Echinococcus multilocularis in red foxes in Lower Saxony. Areas of high prevalence of the infection in foxes and with high fox population density pose high risk for the human population for alveolar echinococcosis. Spatial statistical methods were used to analyse regional count data obtained from 5365 hunted or found-dead foxes. Spatial smoothing (of raw estimates of regional prevalences based on count data before mapping) was used because raw estimates can give an erratic impression of the spatial pattern of the infection. For smoothing, empirical Bayesian methods are used as an explorative spatial epidemiological tool. The resulting map showed the geographical variation of the estimated prevalences around a median at 9% and indicated the presence of spatial-trend effects. Based on this finding, conditional autoregressive spatial modelling for Freeman-Tukey transformed data was used as an inferential spatial epidemiological tool. There were significant additive linear and quadratic spatial-trend effects with elevated prevalences in the north and south and extreme values (prevalences>38%) for the south of Lower Saxony.     

Spatial spreading of Echinococcus multilocularis in Red foxes (Vulpes vulpes) across nation borders in Western Europe

The occurrence of the fox tapeworm Echinococcus multilocularis in Red foxes was studied in Belgium and a neighbouring region in The Netherlands. A total number of 1202 foxes were analysed (1018 in Belgium and 184 in The Netherlands) of which 179 were infected with E. multilocularis (164 in Belgium and 15 in The Netherlands). Further, the spatial distribution of infection among sampled foxes was analysed with an ellipsoidal gradient, demonstrating a decreasing prevalence in northwestern direction. Using this gradient, we showed that the spatial patterns of infection in Belgium and the neighbouring region in The Netherlands correspond, indicating a continuous distribution of E. multilocularis across the nation borders. Part of the Belgian data allowed investigating temporal changes in the spatial distribution of E. multilocularis. This revealed a northwestern spread of E. multilocularis.     

Efficacy of a combined paste formulation of praziquantel/febantel against immature Echinococcus granulosus and immature Echinococcus multilocularis

A combined paste formulation of praziquantel (1 mg/kg of body wt)/febantel (10 mg/kg) given for 3 consecutive days gave 100% clearance of immature Echinococcus granulosus and E multilocularis in experimentally infected dogs. The formulation was extremely convenient to administer. Adverse reactions were not noted in the treated animals.     

Echinococcus multilocularis in Belgium: prevalence in red foxes (Vulpes vulpes) and in different species of potential intermediate hosts

Echinococcus multilocularis causes a rare but potentially lethal zoonotic infection in humans. This tapeworm is known to be endemic in foxes in several countries of Western and Central Europe. In Western Europe, the common vole (Microtus arvalis) and the water vole (Arvicola terrestris) are considered to be the most important intermediate host species of this cestode whereas the red fox is by far the most important final host. The purpose of this study was to provide data on the prevalences in Wallonia (Southern part of Belgium) both in the red fox and in different potential intermediate hosts. A total of 990 red foxes were examined between January 2003 and December 2004 for the presence of E. multilocularis. The average prevalence was 24.55% (22.38-27.87). Out of 1249 rodents or insectivores belonging to the species Apodemus sylvaticus, Arvicola terrestris, Clethrionomys glareolus, Microtus arvalis, Microtus agrestris and Sorex araneus, only one M. arvalis (out of 914-0.11% (0.003-0.61) and one C. glareolus (out of 23-4.3% (0.1-21.9) were found to be infected. However, the muskrat (Ondatra zibethicus) seems to be a good intermediate host as 11.18% (9.72-12.76) of the animals (n=1718) were found to be infected. A positive correlation was found between the prevalences in foxes and in muskrats in each of the different geological regions. This study indicates that the muskrat is highly sensitive to this zoonotic tapeworm and could perhaps represent a good bioindicator when studying the epidemiology of this parasitic infection in Belgium and in other countries where the muskrat is present.     

The occurrence of Echinococcus multilocularis in red foxes in lower Saxony: identification of a high risk area by spatial epidemiological cluster analysis

There is considerable interest in the spatial distribution of Echinococcus multilocularis in red foxes (Vulpes vulpes L.), because this parasite causes the zoonoses of alveolar echinococcosis which is potentially of high fatality rate. High risk areas are known from France, Switzerland and the Swabian Alb in Germany for a long time. In this work, the spatial scan statistic is introduced as an instrument for identification and localisation of high risk areas, so called disease clusters in spatial epidemiology. The use of the spatial scan statistic along with data about the distribution of the parasite in 5365 red foxes in Lower Saxony, that were collected during 1991 to 1997, led to the identification of another high risk area. The relative risk for this disease cluster is approximated by RR = 5.03 (CI0.95(RR) = [4.27; 6.58]) for the period of 1991 to 1994 and by RR = 4.45 (CI0.95(RR) = [3.53; 5.59]) for the period of 1994 to 1997, respectively.     

The anthelmintic efficacy of netobimin against naturally acquired gastrointestinal nematodes in sheep

The broad-spectrum anthelmintic efficacy of netobimin (SCH 32481, Schering Corporation) was evaluated using 30 cross-bred spring lambs with naturally acquired infections of gastrointestinal nematodes. Three groups of 10 animals each were allotted into either control (given a tap water drench as a placebo) or 7.5 and 20 mg kg-1 dosage groups (given the netobimin as an oral drench). Seven to fourteen days post-treatment, animals were necropsied and nematodes recovered by standard techniques. Examination of fecal samples taken on dates of necropsy showed median egg production was reduced in treated animals (61.98% with 7.5 mg kg-1 and 100% with 20 mg kg-1). The compound was highly effective in removal of adult nematodes representing a number of genera and species of trichostrongyloids at the 7.5 and 20 mg kg-1 dose levels (shown, respectively, below). These included Ostertagia spp., with O. circumcincta, O. trifurcata, O. ostertagi and Teladorsagia davtiani (96.20%; 100%), Trichostrongylus spp., with T. axei, T. vitrinus and T. colubriformis (100%; 98.72%), Nematodirus spp., with N. spathiger, N. filicollis and N. battus (100% both levels) and Haemonchus contortus (100% both levels). High efficacies against other species of nematodes (at both dose levels) were not statistically significant (Cooperia spp., Chabertia ovina and Oesophagostomum venulosum). At 20 mg kg-1, netobimin significantly reduced populations of early and late fourth stage larvae of Ostertagia spp. by 100%. The overall efficacy (all life stages included) was 90.16% at 7.5 mg kg-1 and 98.77% at 20 mg kg-1 dose levels. No adverse reactions or signs of toxicosis were observed.     

Detection of Echinococcus multilocularis in wild boars in France using PCR techniques against larval form

Recently, new data have been collected on the distribution and ecology of Echinococcus multilocularis in European countries. Different ungulates species such as pig, goat, sheep, cattle and horse are known to host incomplete development of larval E. multilocularis. We report a case of E. multilocularis portage in two wild boars from a high endemic area in France (Department of Jura). Histological examination was performed and the DNA was isolated from hepatic lesions then amplified by using three PCR methods in two distinct institutes. Molecular characterisation of PCR products revealed 99% nucleotide sequence homology with the specific sequence of the U1 sn RNA gene of E. multilocularis, 99 and 99.9% nucleotide sequence homology with the specific sequence of the cytochrome oxydase gene of Echinococcus genus and 99.9% nucleotide sequence homology with a genomic DNA sequence of Echinococcus genus for the first and the second wild boar, respectively.     

The first detection of Echinococcus multilocularis in slaughtered pigs in Poland

Alveolar echinococcosis is a serious zoonosis caused by the larval stage of Echinococcus multilocularis. Pigs, as humans, can play the role of non-specific intermediate hosts of this parasite. Detection of E. multilocularis in swine can be taken as an indicator of infection risk for humans. This paper presents the first cases where E. multilocularis larval forms have been detected in pigs in Poland. A total of 256 pig livers with different lesions were collected from slaughterhouses in southern Poland and examined by nested PCR method. Three of them were identified as E. multilocularis positive. They contained noticeable spherical nodular whitish forms on the surface of the liver, recessed partially into the liver tissue. In two cases only single lesions (3mm and 6mm in diameter) were found, while in one case numerous (eight) lesions (2mm in diameter) were found. PCR E. multilocularis identification was confirmed by sequencing and comparison with the GenBank database.     

The anthelmintic efficacy of netobimin against experimental infections of Fasciola hepatica in sheep

Netobimin (coded SCH 32481, Schering Corporation), a new broad-spectrum anthelmintic having both fasciolicidal and nematocidal properties was evaluated for efficacy against mature Fasciola hepatica infections in sheep. The trial was conducted with 30 cross-bred spring lambs, each experimentally infected with 250 F. hepatica metacercariae. A single treatment of netobimin was administered at 17 weeks post-infection (PI) by oral drench at 7.5 or 20 mg kg-1 body weight while 10 animals remained as untreated controls. At necropsy (either 1 or 2 weeks post-treatment), the mean number of adult flukes recovered from the control, 7.5 and 20 mg kg-1 groups were 94.7, 35.9 and 8.8, respectively. The resulting efficacies were 62% (P less than or equal to 0.05) and 90.7% (P less than or equal to 0.01), respectively. No clinical signs of fascioliasis were noted in any sheep during the trial. No signs of toxicosis nor any adverse reactions to the drug were observed.     

The anthelmintic efficacy of thiabendazole and levamisole against inhibited Haemonchus contortus larvae in sheep

Fifty-five dogs, 6 to 18 months old, from 11 areas of New Zealand were examined for gastro-intestinal helminths. Thirty-eight (69.1%) were infected. Twenty-one (38.2%) had Toxocara canis, 20 (36.4%) Trichuris vulpis, 20 (36.4%) Uncinaria stenocephala, 3 (5.5%) Toxuscaris leonina, 3 (5.5%) Dipylidium caninum and 1 (1.8%) Taenia sp. Helminth infections were less frequent and populations were smaller in females than in males.     

The anthelmintic efficacy of fenbendazole against a mixed nematode infection in sheep.

Fenbendazole (methyl-5-(phenylthio)-2-benzimidazole carbamate) at dose rates of 5 mg/kg and above was 100 per cent effective in eliminating a naturally acquired Dictyocaulus filaria infection in sheep. The drug was 100 per cent effective in eliminating concurrent infections of adult Trichostrongylus axei, Haemonchus contortus, Haemonchus placei, Ostertagia circumcincta, Ostertagia ostertagii, Cooperia oncophora, Cooperia mcmasterii, Nematodirus spathiger, Neumatodirus filcollis, Oesophagostomum venulosum and Chabertia ovina. Fenbendazole was 93 per cent and 97 per cent effective at doses of 5 and 10 mg/kg respectively in removing infection with adult T colubriformis, and post-treatment worm-egg production was completely suppressed in surviving female worms. No adverse side-effects were observed in treated sheep at either of the two dose rates used.