A vague understanding of the biology and epidemiology of echinococcosis by dog owners in Hokkaido, an endemic island for Echinococcus multilocularis in Japan.

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.

The first instance of a cat excreting Echinococcus multilocularis eggs in Japan.

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.

Taenia taeniaeformis: colonic hyperplasia in heavily infected rats.

Only one study previously mentioned the involvement of colon during Taenia taeniaeformis larvae infection in rats with inconsistent occurrence of lesions. Present study aimed to determine the consistency of histopathologic changes in colonic epithelia, and the proliferation of mucosal cells through BrdU and PCNA immunohistochemistry. Results demonstrated that crypt hyperplasia of the colon was found in all infected rats, although variable in degree even in a single tissue section. Cystic cavities were frequently seen in severely hyperplastic mucosa. Proliferative zone lengths were significantly increased and PCNA positive cells were observed throughout the colonic crypt lengths at 9 but not at 6 weeks post infection. Cell proliferation involving the major types of cells in the epithelial colon was also increased in infected rats at 9 weeks post infection, with labeling indices significantly greater than the control rats throughout the BrdU time course labeling. Findings suggested that massive increases in epithelial cells and depth of colonic crypts were due to a remarkable increase in cell proliferation. The study concluded that enteropathy in the colon during T. taeniaeformis infection could be consistently observed in heavily infected rats.

A latex agglutination test for the detection of Echinococcus multilocularis coproantigen in the definitive hosts.

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.

Research on targeting sources of alveolar echinococcosis in Japan.

Echinococcus multilocularis is a fatal zoonotic parasite in the Northern Hemisphere. Recently, it has become endemic in many countries in Asia, especially in the northern island of Hokkaido in Japan. The increasing threat of public health due to alveolar echinococcosis has compelled researches for sensitive diagnosis and effective control. This paper reviews on the epidemiology, diagnosis and control of echinococcosis specifically in Japan. International collaborative responses by researchers and government initiatives such as mandatory reporting system for veterinarians who diagnose echinococcosis in dogs are presented. Successful control measures in Japan using anthelmintic fortified baits for foxes are described. Assessment of prevalence rates during control campaigns is analyzed favoring the use of intravital diagnosis rather than the traditional necropsy method from hunting or trapping activities of wild foxes. The novel concept of "endogenous development" by local resident volunteers towards sustainable control of echinococcosis is stressed.

The use of tetracycline in anthelmintic baits to assess baiting rate and drug efficacy against Echinococcus multilocularis in foxes.

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.

Genetic uniformity of Echinococcus multilocularis collected from different intermediate host species in Hokkaido, Japan.

DNA from several isolates of Taenia taeniaeformis and Echinococcus multilocularis were digested with restriction enzymes and hybridized with digoxigenated oligonucleotide probe (CAC)5. Within the six wild isolates of Taenia taeniaeformis from Norway rats in Hokkaido, although several bands were common among isolates, fingerprinting patterns were specific to each isolate. In the case of E. multilocularis, regardless of hosts from which each isolate has been isolated, the five isolates collected from Hokkaido, showed the same fingerprinting pattern. These results indicate that there was very little genetic difference among these isolates. Although the fingerprinting pattern of E. multilocularis from St. Lawrence Is. was similar to that of the Hokkaido isolates, some bands were different from those in the Hokkaido isolates. Echinococcus multilocularis in Hokkaido seems to be closely-related genetically to that from St. Lawrence Is.

Collaborative control initiatives targeting zoonotic agents of alveolar echinococcosis in the northern hemisphere.

Alveolar echinococcosis is one of the most important lethal zoonotic helminth infections in the northern hemisphere. Currently, the threat to public health is increasing, as evidenced by the rising prevalence rate of alveolar echinococcosis, as well as the invasion of urban areas by infected wild foxes. This threat is further increased due to the involvement of pet dogs, and probably cats, as emerging sources of infection. These increased threats to public health also have associated economic risks; therefore, there is a need for effective and sustainable methods of control. In this paper, initiatives to control alveolar echinococcosis by targeting its definitive hosts through anthelmintic baiting campaigns initiated by local residents who used local resources for bait production, distribution and collection of fecal samples for diagnosis are described. Further, when such distribution programs are coupled with the use of GIS-based maps, the optimum distribution of bait was obtained. These programs have also included the use of intravital diagnostic analyses of infection rates, which have been overseen by the Forum on Environment and Animals (FEA), and also allowed a nationwide monitoring of echinococcosis in difinitive hosts. In addition, a government initiative requiring mandatory reporting of echinococcosis in dogs to health authorities was recently initiated in Japan. Overall, the results of this study have shown that use of collaborative control initiatives targeting zoonotic agents of alveolar echinococcosis can be an effective method for reducing the threat of lethal echinococcosis in the northern hemisphere.

Towards the control of Echinococcus multilocularis in the definitive host in Japan.

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.

Production and characterization of monoclonal antibodies against excretory/secretory products of adult Echinococcus granulosus, and their application to coproantigen detection.

Two IgM murine monoclonal antibodies (MAbs), EgC1 and EgC3, were produced against the excretory/secretory (E/S) products of Echinococcus granulosus adult worms. Immunoblotting revealed that both predominantly recognized a 50 kDa antigen in the somatic extract and an 85 kDa component in the E/S products. Immunolocalization showed that both MAbs reacted with the tegument of the parasite, and additionally EgC3 reacted with parenchyma and the tegument lining the external surface of the reproductive organs. A coproantigen capture ELISA was developed using a rabbit polyclonal antibody against E/S products from adult tapeworms as catching antibodies, and each one of MAbs as detecting antibody. The assays detected seven out of eight (EgC1), and eight out of eight (EgC3) experimentally infected dogs (worm burdens ranging from 61 to 57,500), using heat-treated samples obtained at prepatent period, and none (n=8) of helminth-free samples. Time course analysis showed that, after a 12-25 days lag, coproantigen levels rose above cut off O.D. values and typically peaked around 30 days post-infection (DPI) at the end of the experiment. One dog experimentally infected with Taenia hydatigena metacestodes was slightly detected as positive at different time points after 30 DPI. Both MAbs showed a similar pattern of recognition, but T. hydatigena antigens were undetectable for a longer period, and reached lower O.D. values with EgC1. Interestingly, fecal samples from two experimentally infected dogs with Echinococcus multilocularis were not recognized by the EgC1 assay, suggesting a potential value as species-specific diagnostic tool.

Gastric hyperplasia and parietal cell loss in Taenia taeniaeformis inoculated immunodeficient mice.

Immunodeficient mice were studied to determine their suitability as models in investigating the role of Taenia taeniaeformis larval products in the development of gastric hyperplasia. Recombinant active gene 2 (RAG2)-deficient and severe combined immune-deficient (SCID) mice were studied as candidate animal models. RAG2-deficient mice inoculated orally with T. taeniaeformis eggs developed gastric hyperplasia with alcian blue-periodic acid-Schiff-positive cell proliferation similar to those of rats. SCID mice inoculated with different doses and routes of T. taeniaeformis in vitro-hatched oncospheres and those orally inoculated with eggs resulted also in different degrees of gastric hyperplasia. Influence of inoculation forms of parasite, doses and routes of inoculation on initiation of hyperplastic gastropathy was suggested to be dependent on number and size of developed larvae. Both RAG2-deficient and SCID mice with hyperplastic mucosa were observed with significant loss of parietal cells. Apparent decrease in parietal cell number was observed in SCID mice at 2 weeks after intraperitoneal inoculation with oncospheres before hyperplastic lesions developed. Earliest occurrence of gastric hyperplasia in SCID mice was observed at 3 weeks after oral inoculation of in vitro-hatched oncospheres, sooner than orally inoculated rats. The results suggested that these immunodeficient mice could be used as animal models to study factors involved in T. taeniaeformis-induced gastric mucous cell hyperplasia.

Taenia taeniaeformis larval product induces gastric mucosal hyperplasia in SCID mice.

The effects of intraperitoneal implantation of Taenia taeniaeformis larvae and inoculation of in vitro larval products on gastric mucosa of SCID mice were investigated in this study. Mice surgically implanted with T. taeniaeformis larvae developed slight and moderate gastric hyperplasia. When in vitro cultured T. taeniaeformis larval excretory-secretory (TtLES) products containing 1 mg of protein were injected daily into mice, they caused gastropathy after 5-7 days. Mice injected daily with 0.5 mg of TtLES products also showed slight gastric hyperplasia after day 14 and 28. The gastropathy was characterized by reduction of both parietal and zymogenic cell number and increased number of alcian blue-periodic acid Schiff (AB-PAS)-positive cells and by two-fold extension of proliferative zone of gastric units. Larval implantation demonstrated a more potent effect in inducing gastropathy than did in vitro larval culture products. Significant decrease in number of parietal cells with concomitant increase of proliferative zone and AB-PAS-positive cell number indicated their important roles in inducing the hyperplastic lesion. Similarities with other gastropathies indicated that there is a common fundamental regulatory mechanism involved, and that the host response may not be specific to parasites. Present study validated the induction of gastric mucosal hyperplasia by larval ES products of T. taeniaeformis. This proved the hypothesis of previous studies suggesting the role of larvae-derived products in inducing gastric mucosal hyperplasia in T. taeniaeformis-infected rats.

Prevalence and intensity of Echinococcus multilocularis in red foxes (Vulpes vulpes schrencki) and raccoon dogs (Nyctereutes procyonoides albus) in Otaru City, Hokkaido, Japan.

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.

[How can we prevent alveolar echinococcosis? Ecosystem and risk management viewpoints].

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.

[Possibility of invasion of Echinococcus into Honshu with pet dogs from Hokkaido and overseas].

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.

Endocytosis of the aspartic acid/glutamic acid transporter Dip5 is triggered by substrate-dependent recruitment of the Rsp5 ubiquitin ligase via the arrestin-like protein Aly2.

Endocytosis of nutrient transporters is stimulated under various conditions, such as elevated nutrient availability. In Saccharomyces cerevisiae, endocytosis is triggered by ubiquitination of transporters catalyzed by the E3 ubiquitin ligase Rsp5. However, how the ubiquitination is accelerated under certain conditions remains obscure. Here we demonstrate that closely related proteins Aly2/Art3 and Aly1/Art6, which are poorly characterized members of the arrestin-like protein family, mediate endocytosis of the aspartic acid/glutamic acid transporter Dip5. In aly2Δ cells, Dip5 is stabilized at the plasma membrane and is not endocytosed efficiently. Efficient ubiquitination of Dip5 is dependent on Aly2. aly1Δ cells also show deficiency in Dip5 endocytosis, although less remarkably than aly2Δ cells. Aly2 physically interacts in vivo with Rsp5 at its PY motif and also with Dip5, thus serving as an adaptor linking Rsp5 with Dip5 to achieve Dip5 ubiquitination. Importantly, the interaction between Aly2 and Dip5 is accelerated in response to elevated aspartic acid availability. This result indicates that the regulation of Dip5 endocytosis is accomplished by dynamic recruitment of Rsp5 via Aly2.

Monitoring of environmental contamination by Echinococcus multilocularis in an urban fringe forest park in Hokkaido, Japan.

OBJECTIVES: The aim of this study was to determine the prevalence of Echinococcus multilocularis environmental contamination in an urban fringe-the Nopporo forest park of Sapporo city, Hokkaido, Japan. A secondary aim was to determine possible transmission risks areas by associating percentage occurrence of E. multilocularis-positive faeces with the different land-use classes. METHODS: Wild fox faeces collected from the environment were examined by intravital methods, such as the taeniid egg sucrose floatation technique, E. multilocularis coproantigen enzyme-linked immunosorbent analysis and DNA test of taeniid eggs by PCR. Geospatial maps produced by the Global Positioning System and Landsat data were analysed using geographic information system software to determine the association between percentage occurrences of E. multilocularis-positive fox faeces and land-use classes. RESULTS: Our findings showed high prevalence rates in both E. multilocularis egg and coproantigen-positive faeces (16 and 49%, respectively) in the investigated urban fringe forest park. Data revealed that percentage occurrence of E. multilocularis-positive fox faeces was associated with land-use classes, such as forest and open field (P < 0.05). CONCLUSIONS: We conclude that Nopporo forest park in the urban fringe of Sapporo city, Hokkaido is a reservoir with a high prevalence of zoonotic infective agents for alveolar echinococcosis. Our findings suggest that interface habitats between forests or woodlands and open fields are indispensable for continued maintenance of the life-cycle of E. multilocularis and, as such, constitute high risk areas for echinococcosis transmission.

Echinococcus multilocularis infection in pet dogs in Japan.

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.

Immune responses to oral infection with Echinococcus multilocularis protoscoleces in gerbils: modified lymphocyte responses due to the parasite antigen.

Immune responses to oral infection with Echinococcus multilocularis protoscoleces in Mongolian gerbils were investigated. Gerbils not treated with prednisolone expelled most of the parasites within 3 days post-infection and induced parasite-specific intestinal IgA secretion after the oral inoculation with protoscoleces. In contrast, prednisolone-treated gerbils harbored notable numbers of parasites, and the parasite-specific intestinal IgA secretion was lower. In gerbils not treated and orally inoculated with protoscoleces (infected group), parasite-specific antibody levels in sera and intestinal washings were elevated, but blastogenesis against protoscolex antigens was observed only in cells from Peyer's patches at 14 days post-infection. Concanavalin A-induced proliferative lymphocytes from both infected and naive gerbils were suppressed by adding protoscolex somatic antigen. These data indicate that while parasite-specific antibody responses were observed, lymphocyte proliferative responses were suppressed by parasite infection/antigens in the Mongolian gerbil, prednisolone-untreated rodent definitive host model.