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Experimental Echinococcus multilocularis infection and deworming was repeated three or five times in nine dogs at various re-infection schedules. The mean number of worms decreased more than 91% in dogs with repeated infection, compared to first infection controls (n= 6). The copro-antigen assay and the egg count in the faeces suggested that the worm burden gradually decreased each time the dogs were re-infected. To examine whether such worm exclusion was a non-specific response, five dogs were sequentially infected with the parasite four times and subsequently fed freely for 6 months. Even after the 6-month interval, the five dogs that were infected five times with the parasite were still able largely to exclude the adult worms. The results suggested that the ability of worm exclusion in dogs that developed a resistance did not become rapidly extinct. Observation of the condition of faeces and the excretion of hooks in the faeces of repeatedly infected dogs revealed that the exclusion of worms started at the first week after the re-infection, and it continued during the patent period. Serum antibodies specific to the parasite antigen increased gradually until the third infection and significantly decreased during the 6-month interval. There was little enhancement of serum antibodies after the fifth infection in most dogs, although no clear correlation was observed between the antibody response and the worm burden. These findings suggested the possibility of developing a vaccine.

Background: Deworming wild foxes by baiting with the anthelmintic praziquantel is being established as a preventive technique against environmental contamination with Echinococcus multilocularis eggs. Improvement of the cost-benefit performance of baiting treatment is required urgently to raise and maintain the efficacy of deworming. We established a spatial model of den site selection by urban red foxes, the definitive host, to specify the optimal micro-habitats for delivering baits in a new modeling approach modified for urban fox populations., Methods: The model was established for two cities (Obihiro and Sapporo) in Hokkaido, Japan, in which a sylvatic cycle of E. multilocularis is maintained. The two cities have different degrees of urbanization. The modeling process was designed to detect the best combination of key environmental factors and spatial scale that foxes pay attention to most (here named 'heeding range') when they select den sites. All possible models were generated using logistic regression analysis, with "presence" or "absence" of fox den as the objective variable, and nine landscape categories customized for urban environments as predictor variables to detect the best subset of predictors. This procedure was conducted for each of ten sizes of concentric circles from dens and control points to detect the best circle size. Out of all models generated, the most parsimonious model was selected using Akaike's Information Criterion (AIC) inspection., Results: Our models suggest that fox dens in Obihiro are located at the center of a circle with 500 m radius including low percentages of wide roads, narrow roads, and occupied buildings, but high percentages of green covered areas; the dens in Sapporo within 300 m radius with low percentages of wide roads, occupied buildings, but high percentages of riverbeds and green covered areas. The variation of the models suggests the necessity of accumulating models for various types of cities in order to reveal the patterns of the model., Conclusions: Our denning models indicating suitable sites for delivering baits will improve the cost-benefit performance of the campaign. Our modeling protocol is suitable for the urban landscapes, and for extracting the heeding range when they select the den sites.

Faecal samples were collected from the rectum of 540 domestic dogs from four districts (Lusaka, Katete, Petauke and Luangwa) in Zambia between 2005 and 2006 and prevalences of canine alimentary tract parasites were determined by coprological examination. Thirteen different ova and parasites including strongyle (43.3%), Spirocerca lupi (18.7%), taeniid (13.1%), Toxocara canis (7.6%), Sarcocystis sp.* (7.5%), Isospora sp.* (5.7%), Physaloptera sp.* (4.6%), Capillaria sp.* (2.8%), Dipylidium caninum (2.2%), Mesocestoides sp.* (2.0%), Ascaris sp.* (1.7%), Trichuris vulpis* (0.4%) and Schistosoma mansoni* (0.4%) were detected, Ascaris and Schistosoma probably originating from coprophagy. The species with asterisks and later-described Taenia multiceps are for the first time reported from dogs in Zambia. A coproantigen enzyme-linked immunosorbent assay (CoproAg-ELISA) developed for Echinococcus spp. revealed 43 positive dogs and 37 of these harboured taeniid eggs. From 63 of the 71 taeniid egg-positive samples, eggs and DNA thereof were isolated and subjected to a multiplex polymerase chain reaction for differentiating E. granulosus sensu lato, E. multilocularis and Taenia spp. Amplicons indicative for Taenia spp. were obtained from 60 samples. Sequencing of amplicons spanning part of the mitochondrial cytochrome c oxidase subunit 1 gene, which was possible with 38 samples, revealed 35 infections with T. hydatigena and 3 with T. multiceps. Therefore, the CoproAg-ELISA showed some positives, but concrete evidence for the existence of canine E. granulosus infection could not be established. Comparison of the results of the CoproAg-ELISA and Taenia species identification indicated that the CoproAg-ELISA cross-reacts with patent infections of T. hydatigena (57%) and T. multiceps (33%).

We show that a conventionally purified glycoprotein component of Echinococcus multilocularis protoscolex, designated as Emgp-89, may be useful as a serodiagnostic antigen for detecting E. multilocularis infection in dogs domesticated in endemic areas. Emgp-89 was obtained from the parasite material by a simple procedure using Con A-agarose and subsequent gel filtration chromatography. The purified fraction showed a molecular weight of >4000kDa upon gel filtration and reacted with a series of lectins that specifically bind to mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Subsequently, serodiagnostic performance of Emgp-89 was evaluated through enzyme-linked immunosorbent assays (ELISAs) by using sera from normal, domestic dogs and dogs infected with other helminths. Emgp-89 positively reacted with all 16 serum samples from E. multilocularis-infected dogs, thus showing that this antigen is highly sensitive. On the other hand, the specificity of Emgp-89-based ELISA, determined using 41 serum samples from dogs infected with other helminths, was relatively low (83%). As an attempt to improve the specificity of Emgp-89-based ELISA, we pretreated Emgp-89 with proteinase K or sodium periodate, expecting that these treatments would enable discrimination of true positives from false positives. The ELISA value increased after treatment with sodium periodate in most false-positive samples, whereas significant decreases were observed in sera from all dogs infected with E. multilocularis. Further evaluation of this antigen should be performed using sera from dogs infected with closely-related parasites, including taeniid cestodes, which are expected to prove that this serodiagnostic system is sufficiently specific for clinical and field applications., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Domesticated dogs are an important potential source of Echinococcus multilocularis infection in humans; therefore, new molecular approaches for the prevention of the parasite infection in dogs need to be developed. Here, we identified and characterized an immunogenic protein of the parasite by using a proteome-based approach. The total protein extracted from protoscoleces was subjected to two-dimensional Western blotting with sera from dogs experimentally infected with E. multilocularis. Two protein spots showed major reactivity to the sera from infected dogs. The N-terminal amino acid sequences of these spots were identical to the deduced amino acid sequence of the product of the putative hsp20 gene. RT-PCR and Western blot analyses revealed that the putative hsp20 gene and its products were expressed in almost all stages of the parasite life cycle. Furthermore, recombinant hsp20 showed specific reactivity to the sera from infected dogs, suggesting that this molecule may facilitate the development of a practical vaccine., (Copyright 2009 Elsevier Inc. All rights reserved.)

A multiplex PCR system was developed to identify the carnivore origins of faeces collected in Hokkaido, Japan, for epidemiological studies on Echinococcus multilocularis. Primers were designed against the D-loop region of mitochondrial DNA. Two separate primer mixtures (mix 1, specific forward primers to fox, raccoon dog and dog, and a universal reverse primer [prH]; and mix 2, specific forward primers to cat, raccoon and weasels and prH) were used so that the PCR products (160 bp, fox and cat; 240 bp, raccoon dog and raccoon; and 330 bp, dog and weasel) were distinguished by size. The multiplex PCR exhibited no cross-reactivity between carnivore species and did not amplify DNA from rodent prey. When 270 field-collected faeces were examined, 250 showed single PCR products belonging to specific target sizes, suggesting successful carnivore identification for 92.6% of samples. Taeniid eggs were detected in 11.1% of samples and coproantigen in 30.4%; whereas the prevalences of taeniid eggs and coproantigen were 12.9% and 34.0% in fox faeces, and 0% and 26.3% in cat faeces, respectively. These results suggest that the prevalence in different target animals can be evaluated individually and precisely using multiplex PCR system.

A survey of Echinococcus multilocularis infections in pet dogs in Japan from 1997 to 2007 was conducted by testing for coproantigen reactivity, fecal taeniid eggs, and egg DNA. In Hokkaido, the only island where E. multilocularis is endemic in Japan, 18 of 4768 dogs (0.4%) excreted taeniid eggs that were positive for E. multilocularis DNA by polymerase chain reaction (PCR). Most of the dogs testing positive for egg DNA were kept free-range, but three dogs had been kept inside their owners' houses. In addition, 15 dogs were suspected to be infected based on the results of a coproantigen test. One dog, which was transported from Hokkaido to Honshu, the main island of Japan, was excreting taeniid eggs that were positive for E. multilocularis DNA by PCR. These results suggest the importance of proper pet management in disease prevention, even for dogs kept indoors, and they point out a possible means by which the parasite may be introduced into non-endemic areas through transport of infected dogs.

A questionnaire survey was conducted by giving 14 statements about echinococcosis to 2,070 dog owners residing in Hokkaido in order to evaluate their understanding about the biology and epidemiology of Echinococcus multilocularis. Analysis of the answers revealed that dog owners understood the disease superficially, and there were several points of confusion in their understanding, especially regarding differences in the modes of transmission and disease development in dogs and humans. The results suggest the need for the proper education of dog owners to perform proper prophylactic measures against the disease.

A cat excreting Echinococcus multilocularis eggs was recently identified in Hokkaido, representing the first such observation in Japan. The cat was raised free-range and frequently ate rodents. Fecal egg examination revealed eggs of taeniids (EPG: 440) and Spirometra spp. (EPG: >1000). PCR targeting part of the mitochondrial cytochrome c oxidase subunit I gene of E. multilocularis was positive with DNA from 3 single isolated taeniid eggs, and sequence analysis of one amplicon confirmed E. multilocularis. The results indicated that the eggs of E. multilocularis distributed in Hokkaido can be excreted in cat feces, and suggested the necessity of further studies to clarify whether the eggs excreted in cat feces are infective and thus whether cats can serve as infectious source to humans in Japan.

A latex agglutination test for detecting Echinococcus multilocularis coproantigen in definitive hosts was developed using latex beads sensitized with EmA9 monoclonal antibody raised against somatic antigens of adult E. multilocularis. A primary test (LA 1) was performed on 82 fecal samples of necropsied foxes, of which 46 were infected, and resulted in 61% sensitivity and 86% specificity. To increase the sensitivity, 4 ng/mL of excretory/secretory antigens of adult worms was added to the samples in a secondary test (LA 2), resulting in 91% sensitivity and 61% specificity. The positive predictive value of the LA 1 test and the negative predictive value of the LA 2 test were both 85%. The combination of the LA 1 and LA 2 tests is applicable and practical for use in situations that require quick diagnosis or screening based on the following interpretation: the samples that are positive in the LA 1 test are positive; the samples that are negative in the LA 2 test are negative; and the samples that are negative in the LA 1 test and positive in the LA 2 test are classified as suspicious.

Alveolar echinococcosis is caused by infection with the larval stage of Echinococcus multilocularis. We recently identified a cDNA clone, designated as emy162, that encodes a putative secreted protein. EMY162 shares structural features with the EM95 antigen, which is a host-protective antigen. The amino acid sequence of EMY162 shows 31.4% identity to EM95 whereas these antigens are distinguishable with respect to their predicted secondary structure and antigenicity on Western blot analysis. RT-PCR analysis revealed that the gene expression of emy162 was significantly higher than that of em95 at each life-cycle stage. Recombinant EMY162 antigen induced a significant level of host-protection (74.3%) in experimental infection with E. multilocularis eggs in mice. Notably, recombinant EMY162 antigen showed significant reactivity to the sera from alveolar echinococcosis patients. These results may help in the development of a practical vaccine to reduce the level of alveolar echinococcosis in humans.

Anthelmintic (praziquantel) baiting of wild red foxes against Echinococcus multilocularis infection was studied in a highly epizootic suburban area of Otaru, Hokkaido (the northern island of Japan) during the summer and autumn in the years 1999-2004. Acceptance of baits containing the biomarker tetracycline (TC) was evaluated. The prevalence of E. multilocularis infection in foxes before baiting (1999-2000) was 58% (88/153), whereas in the fourth year of bait distribution year (2004), it decreased to 11% (5/45). Analysis of TC marking in the teeth of foxes showed that 39% (77/195) of those captured after baiting were estimated to have consumed baits in the year of capture. Importantly, more juvenile (56%, 49/87) than adult foxes (26%, 28/108) were marked, indicating efficient baiting of juveniles, which tended to have a higher worm burden of E. multilocularis. Of 77 marked foxes, E. multilocularis and Alaria alata (monitored as the second indicator species of deworming) were not detected in 70 (90%) and 76 (99%) foxes, respectively. The results suggest effective deworming by bait consumption. However, it was also demonstrated that 9% of the marked foxes were infected or re-infected after bait consumption, suggesting high infection pressure and the importance of frequent baiting.

The authors describe the current control strategies targeting definitive hosts of the most important zoonotic parasite in Japan, Echinococcus multilocularis. A dramatic increase in the prevalence of echinococcosis in foxes in Hokkaido (the second largest of Japan's islands), the invasion of wild foxes into urban areas, infection among pet and stray dogs, and the possibility of spreading the disease to the main island of Japan (Honshu)--all these pose significant threats to public health. Previous research findings and current strategies such as control measures against infections in wild foxes, suggest that it will be possible to eliminate echinococcosis in the future. The enforcement of a national reporting system for veterinarians, international collaboration, and the establishment of a Forum on Environment and Animals (FEA) give further reason to believe that success is possible. This is the first report of a multifaceted control strategy against echinococcosis in definitive hosts that includes collaborative efforts with local residents. This model might provide new ideas for Veterinary Services worldwide in their efforts to control other related zoonotic diseases.

Echinococcus multilocularis is distributed all over Hokkaido, the northern island of Japan. The prevalence in foxes has been around 40% in the last decade. Three trials of anthelmintic bait distribution have been conducted in Hokkaido to reduce the prevalence in foxes. In those trials, bait distribution was done along roads in the study area using cars and/or around fox breeding dens by hand. Changes in the prevalence in foxes were evaluated either by necropsy of captured foxes or by coproantigen and egg detection of faeces collected in field. All of the trials showed bait distribution was effective for the reduction of the prevalence in foxes; however, it was also suggested that a frequent and continuous baiting program is necessary for effective and stable control of the prevalence in foxes. As observed in some cities in Europe, urban foxes infected with the parasite were also recognized in Sapporo. A survey of pet dogs showed that 0.4% of surveyed dogs were determined infected. In addition, a dog which was transported from Hokkaido to the main island of Japan was found excreting E. multilocularis eggs. The results raised the public recognition of canine infections, which in turn lead to the modification of a Japanese law for infectious diseases and to the enforcement of a national reporting system of dogs infected with E. multilocularis by veterinarians.

Parasite-specific antigen responses and lymphocyte blastogenesis in dogs orally inoculated with Echinococcus multilocuralis metacestodes were examined. Serum IgG1 (Th2-oriented) and IgG2 (Th 1-oriented) levels against somatic and excretory-secretory (ES) antigens of protoscoleces and adult worms increased from 7 days post-infection (DPI), with the highest responses against protoscolex excretory-secretory antigen (PES). Specific blastogenesis of peripheral blood mononuclear cells (PBMC) against the parasite antigens was not observed during the 21-day infection period, but Peyer's patches cells from one out of two dogs at 21 DPI showed blastogenesis against PES (stimulation index: 4.65). Interestingly, only at 7 DPI were concanavalin A (ConA)-induce proliferative responses of PBMC reduced. Moreover, ConA-induced proliferative responses of lymphocytes from various origins were suppressed by the addition of parasite antigens, especially with PES. These data suggest that although both Th1- and Th2-oriented humoral immune responses were observed in E. multilocularis infected dogs, the parasite antigens, especially PES, may have incompletely suppressed lymphocyte responses in these dogs.

Purpose: The number of pet dogs moving with their owners to Honshu, the main island of Japan, from Hokkaido, and the number of dogs imported from overseas were examined, and the possibility of invasion of Echinococcus multilocularis and E. granulosus with these dogs was discussed., Methods: The number of pet dogs moving to Honshu-island from Hokkaido was examined with the movement notifications based on the Rabies Prevention Act in 29 prefectures during the period from 1996 to 2001. The number of pets was also examined by questionnaire targeting 3 aviation and 3 ferry companies. The number of dogs imported from overseas was examined with the Annual Reports of the Animal Quarantine Service of Japan, The sanitary conditions of hotels for pet owners were also examined with a questionnaire., Results: Approximately 140 pet dogs were found to have officially moved annually from Hokkaido to Honshu during the study period. However, the actual number might be two to three times this estimate, because many dogs moved without notification. Nearly ten thousand pet dogs were transported a year to and from Honshu and Hokkaido by planes and ferries. A value of three thousand would be expected if people from Hokkaido were accompanying their pets at the rate of the registered dogs per population, one animal per 23 Hokkaido residents. Up to 30 pet dogs infested with E. multilocularis would probably be included per year, according to the infestation rate of 1% in Hokkaido. The number of imported dogs from overseas was assessed at approximately 15 thousand a year, but these dogs were not obligated to receive animal quarantine with respect to echinococcal infestation in Japan. Hotels for pet owners were considered to be managed rather sanitarily, though certain administrative guidance is necessary to prevent hydatid disease infection of travelers and hotel workers. The authors consider that dogs from Hokkaido and also from echinococcosis endemic countries should undergo fecal examination for parasite eggs to prevent invasion of the parasite into Honshu., Conclusion: The authors propose the fecal examination of the dogs from Hokkaido and also from overseas for preventing invasion of E. multilocularis and E. granulosus into Honshu, Japan.

The effect of bait-delivered anthelmintic to reduce the prevalence of Echinococcus multilocularis in wild red foxes was evaluated in Koshimizu, in the eastern part of Hokkaido, Japan. The study area (200 km2) was divided into baited and non-baited sections. The anthelmintic baits were distributed around fox den sites in the baited section every month for 13 months. After 1 year of the anthelmintic bait distribution, the prevalence of E. multilocularis in foxes, evaluated either by the parasite egg examination (from 27.1 to 5.6%) or coproantigen ELISA (from 59.6 to 29.7%), decreased in the baited section contrasting to that in the non-baited section (parasite egg: from 18.8 to 24.2%; ELISA: from 41.9 to 45.8%). The prevalence of E. multilocularis in grey red-backed vole Clethrionomys rufocanus, caught around fox dens, born after bait distribution also decreased and was significantly lower than that in non-baited section. However, within the study periods, the coproantigen-positive rate in fox faeces sporadically increased, while egg-positive rate constantly decreased. Since coproantigen ELISA can detect pre-patent infection, this observation indicates that reinfection pressure in the baited section was still high even after the 13 months of anthelmintic bait distribution. Therefore, the bait distribution longer than our study period is required for the efficient control of E. multilocularis in wild red fox population.

A survey was done in an attempt to investigate the epidemiological status of Echinococcus multilocularis in red foxes and raccoon dogs in Otaru city from June to September 1999. Sixty-seven red foxes (Vulpes vulpes schrencki) and 13 raccoon dogs (Nyctereutes procyonoides albus) were captured, and postmortem examinations were conducted with them. Thirty-eight red foxes (56.7%) and 3 raccoon dogs (23.1%) were found to be infected with E. multilocularis. The total biomass of E. multilocularis in all infected red foxes and raccoon dogs were 2,817,000 and 1,515 worms, respectively. Nine of the infected red foxes harboring more than 100,000 worms accounted for 90.6% of the total biomass. No significant differences in the prevalence were observed between male and female, and juvenile and adult. However, the worm burden was higher in juvenile than in adult foxes. In one of the infected raccoon dogs, mature worms and eggs of E. multilocularis were found in the intestine and fecal sample, respectively. This result suggested that the raccoon dogs are probably playing a small role in the egg contamination of the environment. The validity of coproantigen ELISA for diagnosis of foxes was confirmed by comparing the results of autopsy, egg examination and coproantigen ELISA using rectal fecal samples.

Purpose: This article focuses on understanding epidemiological features of alveolar echinococcosis and discussing its prevention and control, especially from a viewpoint of the ecosystem and risk management., Method: Publications on alveolar echinococcosis throughout the world were systematically reviewed with special reference to ecology, epidemiology and countermeasures., Results: Alveolar echinococcosis, caused by accidental infection with larva of the parasite Echinococcus multilocularis is fatal to humans unless diagnosed at an early stage. No effective control measures have been identified so far because it is difficult to fully understand the ecology of the parasite and its intermediate and definitive hosts. It is also not easy to determine the precise infection route to humans mainly because of the long latent period. In Hokkaido, infection rates among red foxes have recently risen even in low endemic districts. Not only stray and domestic dogs but also some pigs in Hokkaido have been found to be infected. While the number of reported human cases is still small, around 10 cases per year, local residents seem to be threatened with the risk of infection., Discussion and Conclusions: We predict that the incidence of alveolar echinococcosis among humans in Japan will increase in the near future if no effective preventive measures are conducted. In addition, Echinococcus multilocularis infection has the potential to affect the economy of Hokkaido because of its impact on the agricultural and tourist industries. Well-designed epidemiological surveys are therefore urgently required, in the context of ecosystem and risk management prior to large outbreaks. International collaboration is also desired.

Few studies have been carried out for the prevalence of canine echinococcosis in Mongolia. This study was designed to elucidate a preliminary information of the prevalence from feces collected in the field. Sixty-seven fecal samples from dogs and 2 red foxes in Altai town were collected and examined for Echinococcus coproantigen and eggs. Coproantigen detection was performed by a sandwich ELISA using a monoclonal antibody EmA9 raised against Echinococcus multilocularis somatic antigen. Of the dog samples examined, 17 (25.4%) were positive by the ELISA. One out of two foxes was positive, too. Taeniid egg-positive feces were recognized in 12 dog feces. Only 6 samples were both coproantigen and egg positive. Eggs of Ancylostoma sp., Trichuris sp.; and Capillaria sp.; were also registered.

Mongolian gerbils (Meriones unguiculatus) were orally inoculated with 10,000 protoscoleces of Echinococcus multilocularis per head after being divided into five groups (A E). Each group was dosed with prednisolone tertiary-butylacetate (PTBA) as follows: A, 0 mg; B, 0.5 mg; C, 2 mg; D, 5 mg; and E, 10 mg/head. All animals were injected subcutaneously with control solvent or PTBA every other day from 6 days pre- to 6 days post-infection. Autopsy was performed at 7 days post-infection. Doses of PTBA and the number of worms recovered showed a positive correlation (r = 0.929, P < 0.0001). In groups A, B and C, the predilection site of the worms in the small intestine could not be determined, while in group D the worms were found more in the anterior part. In group E, the predilection site was the anterior part, followed by the middle and the posterior parts of the small intestine (Fisher's test: P < 0.01). The number of worms recovered from the anterior and the middle part of the small intestine also correlated positively with PTBA dose (anterior part: r = 0.930, P < 0.0001, middle part: r = 0.917, P < 0.0001). All groups of the PTBA-treated animals showed significant loss of weight compared to the nontreated animals (P < 0.01).

An alternative definitive host model for Echinococcus vogeli was developed using Mongolian gerbils. The animals treated with prednisolone tert-butylacetate were orally inoculated with protoscoleces of E. vogeli and the development of worms in the small intestine was observed until 50 days post infection (DPI). The parasites were recovered most frequently from the anterior part of the small intestine. The band formation (the sign of proglottisation) of the worm was observed at 7 DPI, the second proglottid and tests formation at 14 DPI, spermatozoa in seminal receptacle and cleavage of ova in uterus at 21 DPI, the hook formation of oncospheres at 28 DPI and the embryophore formation at 34 DPI. Eggs in the feces were first detected at 35 DPI. The coproantigen detection method developed for E. multilocularis could detect the E. vogeli coproantigen early in the infection, at 7 DPI during the prepatent period. Therefore, this technique could be used for the diagnosis of E. vogeli, replacing ordinary fecal egg examination.

This study revealed the dose dependency of prednisolone tertiary-butylacetate (PTBA) treatment on the establishment of Echinococcus multilocularis in the small intestine of Mongolian gerbil (Meriones unguiculatus) and that some of the physiological parameters of host were correlated with the doses of PTBA and establishment of the worm. Twenty Mongolian gerbils were divided into 5 groups, according to the doses of PTBA; 0 mg, 0.5 mg, 2 mg, 5 mg and 10 mg per head. All animals were injected intraperitoneally with PTBA every other day from 6 days before to 6 days after infection. Doses of PTBA and the number of worms recovered at 7 days post-infection showed a positive correlation (r = 0.929, P < 0.0001). The increase of total protein (TP) and the decrease of the percentage of lymphocytes in the peripheral leukocytes were dependent on doses of PTBA (TP: r = 0.811, P < 0.0001, percentage of lymphocyte: r = -0.92, P < 0.0001). The TP and the percentage of lymphocyte also correlated with the number of worms recovered (TP: r = 0.617, P = 0.0049; percentage of lymphocyte: r = -0.800, P < 0.0001).

An epidemiological survey was conducted on the seasonal variation of Echinococcus multilocularis prevalence in red foxes from 1997 to 1998, using a monoclonal antibody-based detection of the tapeworm coproantigen. Thirty-six breeding dens of reproductive fox families were identified in the endemic area of Koshimizu, eastern Hokkaido, Japan. Fecal samples from each site were examined by coproantigen detection assay and fecal egg examination. Whereas the prevalence of coproantigen positive feces showed no seasonal fluctuation (51.6-66.7%), variation was found in the prevalence of egg positive feces in which a higher prevalence was observed in the summer and winter (31.1 and 38.7%) than spring and autumn (13.3 and 13.5%). Significant differences were observed between juveniles and adult foxes in both examinations. Samples from juvenile foxes gave higher coproantigen positive results and taeniid egg intensity. Those results suggest more juveniles infected with the cestode than adults in the same period. The practical use of coproantigen assay as a survey tool and factors which affect the prevalence and host age-related difference are discussed.

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.

The study was conducted to evaluate a mAb-based coproantigen detection assay for monitoring fox prevalence of Echinococcus multilocularis infections in the Shiretoko National Park in Hokkaido, Japan. Eight fox families, each consisting of 2-4 adults and their pups, were selected because their territories have been determined. Faeces of each family (total n = 537) were collected monthly from April to October, 1994. Detection of coproantigen and taeniid eggs was recognized in 58 and 27 faeces, respectively, but mostly restricted in 1 family, in which coproantigen ELISA OD values had dual peaks, one in June and the other in October, whereas taeniid eggs were detected only from April to July. Fox pup faeces (n = 51) collected around the dens used by the positive family were all coproantigen and taeniid egg negative except for 1 faeces. These results suggest that the prevalence and infection pressure in the study area were low and the infected family terminated the infection in the summer and acquired a new one in the early autumn, 1994. This monitoring method for fox infection will be a practical management tool for E. multilocularis infection, especially at the tourist spots in Hokkaido where close contact of fox with humans is frequently observed.

Detection of Echinococcus coproantigen using sandwich enzyme-linked immunosorbent assay (sELISA) was performed on fecal samples of red foxes in Hokkaido, Japan. Fecal samples were collected around fox dens in 1990 and 1992. The antibodies used for sELISA recognize heat-resistant antigens, thus all fecal samples were heated to render it safe for handling before examination. Detection of taeniid egg in fox feces collected was considered as an indication of E. multilocularis infection. In fecal samples collected in 1990 and 1992, coproantigen positive results out of taeniid-egg positive cases were 38/40 (95.0%) and 95/97 (97.9%), respectively. In addition, coproantigen was detected regardless of fecal condition when collected from the field, suggesting that the antigens detected by this method are quite stable. These results suggest that detection of coproantigen is useful for field surveys of foxes naturally infected with E. multilocularis.

A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotinylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67,700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, showing a sensitivity of 100% and a specificity of 96%.

Coproantigen excretion during experimental infections of Echinococcus multilocularis in foxes and an alternative definitive host, golden hamsters, was evaluated by a sandwich ELISA using a monoclonal antibody. A sigmoidal increase of antigen excretion from the developing parasites was observed in in vitro incubation of the parasites collected on different days during the first 21 days post-infection (DPI). In hamsters, the ELISA O.D. value of faeces became positive at 4 DPI. Thereafter, the O.D. value increased in semi-sigmoidal fashion in the first 42 DPI, probably reflecting the development of the parasites. In foxes, the O.D. value became positive at 6 DPI. However, contrary to that in hamsters, after the initial steep rise, the O.D. value suddenly decreased to 1/2 the level during 15-17 DPI, indicating that a large number of worms might have been expelled. The parasite eggs were detected by the sugar centrifugal-flotation technique (Ito, Yagi & Ishige, 1989) from 29 to 84 DPI but not thereafter to 125 DPI, although mature parasites were detected at 125 DPI. In contrast, positive O.D. values were obtained almost constantly until 125 DPI, indicating that the coproantigen detection assay was more sensitive than the egg detection assay. The detection limit of the coproantigen assay was roughly estimated to be around 100 worms. These observations, along with the fact that the assay was designed to detect a heat-resistant coproantigen in heat-sterilized fecal samples, indicate that the coproantigen detection assay is a safe and useful method, not only for diagnosis in the definitive host of E. multilocularis, but also for monitoring parasite development and change in parasite burden during an experimental infection.

Cystic hydatidosis (CH) caused by Echinococcus granulosus is an important zoonosis worldwide. Several serodiagnostic and ultrasound tests are currently used for screening asymptomatic CH. The sensitivity of the serodiagnostic tests has been demonstrated by several groups to be lower than ultrasonography (US). To explain the mechanism of the difference in sensitivity, a large-scale US survey combined with an IgG4-enzyme-linked immunosorbent assay test was conducted in Uruguay. Our results suggest that certain cyst images, e.g., multivesicular cysts, may be closely associated with a high antibody response regardless of the cyst size, whereas other hydatid lesions, e.g., solid images with/without rolled parasite membranes, have lower antibody responses with a close relationship to cyst size. Seropositivity of subjects who had been treated surgically for removal of hydatid cysts in the last 5 yr was closest to that of actual CH patients, albeit free from CH by US. These findings are important for the proper use of serodiagnostic tests and US for community-based epidemiological studies.

Golden hamsters as alternative definitive hosts of Echinococcus multilocularis were used for coproantigen detection by means of sandwich ELISA. The test was performed in hamsters infected with approximately 20,000, 4,000, 500, 0 (control) and 100,000 (i.e., group I, II, III, IV and V respectively) protoscoleces. Comparison of mean OD values of each group showed significant differences depending on the number of protoscoleces administered and days postinfection. There was also a relatively high statistical correlation between the number of recovered worms and ELISA OD values (correlation coefficient = 0.699, P < 0.05), although accurate comparison of worm burdens among individual animals was difficult when numbers of infecting worms fell within the same range.

Parenteral strobilation of Echinococcus multilocularis was observed in acid (severe combined immuno-deficient) mice after intracerebral, subcutaneous and intraperitoneal inoculation with protoscoleces. Evaginated protoscoleces and segmented worms were obtained at the inoculated sites. Most worms recovered from peritoneal cavity of scid mice were encapsulated by connective tissue and granulocytes but showed a maximum of 3 proglottids, elongation of genital primordia and vesiculation. Viability of worms recovered from the subcutaneous tissue and peritoneal cavity of scid mice were higher (69.1-91.4%) than those from the immunologically normal C.B-17 (4.0-48.0%) control mice. However, viabilities of worms from the cerebrum of both scid and C.B-17 were almost the same (87.7-94.4%). Worms recovered from scid mice showed further development of reproductive organs when transplanted into the small intestine of prednisolone treated golden hamster. These findings suggest that the parenteral milieu of scid mice allows adult development of E. multilocularis protoscoleces.

Alveolar hydatidosis by Echinococcus multilocularis (Em) infection occurred on a 22-year-old (approx.) male gorilla (Gorilla gorilla) and a 4-year-old female ring-tailed lemur (Lemur catta) in a zoo, in Hokkaido, Japan. Case 1: The gorilla presented neurologic signs in course of nine months and died. Pathologically, alveolar hydatid lesions were found in the liver, the liver-associated lymph nodes, the cerebrum, and the lungs. A protoscolex was found only in one hepatic cyst. Case 2: In the lemur, large masses of hydatide cysts were found on the liver and at the lung-associated lymph nodes. Cysts contained numerous calcareous corpuscles and protoscolices. The lemur appears a favorable intermediate-host for Em. The identification of Em in both cases were confirmed by PCR.

In AKR/J mice inoculated intraperitoneally with protoscoleces of Echinococcus multilocularis, the oral administration of alpha-difluoromethylornithine (DFMO) in drinking water had no obvious effect on the development of hydatid cysts. The DFMO treatment also caused no apparent depletion of three major polyamines (putrescine, spermidine, and spermine) in the cysts, although a slight reduction in the putrescine level was detected. The failure of DFMO treatment to reduce the growth of cysts in mice may result from the absence of ornithine decarboxylase in the parasite.

E. multilocularis infection was suppressed in C.B-17 mice after intraperitoneal inoculation of protoscoleces, with larval cysts weighing no more than 1.0 g. In scid mice, which are genetically identical to C.B-17 except for a deficiency in functional lymphocytes, infection progressed and larval cysts reached a mass of 17.5 g at 15 weeks post-infection. The growth of the larval cyst mass in scid mice was similar to that in other susceptible mouse strains, with a biphasic pattern. Histological observations revealed giant cells and granulomatous inflammation in the C.B-17, but not in the scid mice. These results led to the conclusion that suppression of the growth of the larval cyst mass in the initial stage of infection in susceptible mice strains is caused by factors other than the host's lymphocytic immune response.

Forty-two rats, Rattus norvegicus, captured at a garbage dump in southern Hokkaido, Japan, were examined, and one was found to be infected with Echinococcus multilocularis. The lesions were found in the liver, lung, mesenteric lymph nodes, greater omentum and also free in the abdominal cavity. No necrosis was observed in any of the lesions, and inflammatory reactions were mild. Protoscoleces were observed in the large liver cysts. A homogenate of these cysts, when transplanted into the abdominal cavity of three Mongolian gerbils and a rat, yielded numerous fully developed protoscoleces at 4-7 months post-inoculation. Judging from this, it is postulated that the rat could become a natural intermediate host for E. multilocularis in this area.

Cystic hydatidosis/echinococcosis is an important zoonosis caused by the tapeworm Echinococcus granulosus. Hydatidosis is a serious parasitic disease in Uruguay, and in 1991 a new national control program was implemented by the national commission against hydatidosis (CHLCH). In 1991 (before the control program), farm and town dogs were examined for the prevalence of the parasite in Tacuarembó, Uruguay, using fecal samples of farm dogs after an arecoline purgation and samples of small intestine of town dogs obtained by necropsy. The prevalence of E. granulosus was 23 and 4% in farm and town dogs, respectively. In order to evaluate the impact of the control program, two surveys on ovine hydatidosis were carried out in Tacuarembó before and during the national control program. Sheep were examined in 1991–1992 (before the control program) and 1999 (during the control program). Both prevalence and intensity of E. granulosus infection increased with age in both 1991–1992 and 1999. The prevalence of ovine hydatidosis was 41.6 and 8.5% in 1991–1992 and 1999, respectively. The prevalence of fertile cysts in sheep more than 4 years old was 7.3% and 2.3% in 1991–1992 and 1999, respectively. The remarkable decreased prevalence of ovine hydatidosis suggested the successful control program by CHLCH in Uruguay. [Copyright &y& Elsevier]