Alveolar echinococcosis is a worldwide zoonosis of great public health concern. Analysis of genome data for Echinococcus multilocularis has identified antigen families that can be used in diagnostic assays and vaccine development. However, little gene expression data is available for antigens of the egg and early larval stages. To address this information gap, we used a Next-Generation Sequencing approach to investigate three different stages (non-activated and activated oncospheres, and early stage metacestodes) of E. multilocularis (Nemuro strain). Transcriptome data analysis revealed that some diagnostic antigen gp50 isoforms and the antigen Eg95 family dominated in activated oncospheres, and the antigen B family dominated in early stage metacestodes. Furthermore, heat shock proteins and antigen II/3 are constantly expressed in the three stages. The expression pattern of various known antigens in E. multilocularis may give fundamental information for choosing candidate genes used in diagnosis and vaccine development. Humans become infected with E. multilocularis through oral ingestion of the eggs. Untreated alveolar echinococcosis has a fatality rate of >90% in humans, causing great public health concern in the northern hemisphere. The genome databases of E. multilocularis have been recently published, but still there is no available data about transcriptomic profile datasets of the early larval stages (non-activated and activated oncospheres and immature metacestodes). In this study, the gene expression profile of E. multilocularis oncospheres and metacestodes were tested using the Next-Generation Sequencing (NGS) approach. The expression level of hundreds genes was significantly different between oncospheres and metacestodes. Some known tapeworm-specific antigen family like diagnostic antigen gp50, antigen Eg95, and antigen B are predominated in oncospheres and metacestodes, respectively. Furthermore, within the tapeworm-specific antigen family, some antigen subunits or variations were stage-specific expression. The identification of genes expression profile during the parasite development stages, especially the stage of oncospheres, will give fundamental information for choosing candidate genes used in vaccination and early diagnosis.

The intestinal helminth fauna of Tibetan sand foxes (Vulpes ferrilata) and red foxes (Vulpes vulpes) inhabiting in Qinghai, China, was evaluated by conducting necropsy of hunted foxes and fecal egg examination of field-collected feces. In northeast and south Qinghai, 36 foxes were necropsied, and the species of foxes and the parasites detected were identified by the DNA barcoding. In 27 red foxes and 9 Tibetan sand foxes examined, Mesocestoides litteratus (total prevalence: 64%), Toxascaris leonina (50%), Taenia pisiformis (8%) and Taenia crassiceps (8%) were found in both species of foxes. Echinococcus shiquicus (8%) and Taenia multiceps (6%) were found only in Tibetan sand foxes. Echinococcus multilocularis (3%) and Alaria alata (8%) were found only in red foxes. In the fecal egg examination of the rectal feces, 100% of taeniid cestodes, 73% of Toxascaris and 27% of Mesocestoides worm-positive samples showed egg-positive, indicating that coprological survey for parasite eggs could only provide partial information of intestinal parasite fauna. For field-collected feces, molecular identification of feces origins and fecal egg examination were performed. In 15 Tibetan sand fox and 30 red fox feces, we found E. multilocularis eggs in one feces of Tibetan sand fox. The present study indicated that the upper intestinal helminth fauna of the two fox species in Qinghai does not differ significantly and both species would play an important role in the maintenance of taeniid cestodes.

Echinococcus multilocularis employs aerobic and anaerobic respiration pathways for its survival in the specialized environment of the host. Under anaerobic conditions, fumarate respiration has been identified as a promising target for drug development against E. multilocularis larvae, although the relevance of oxidative phosphorylation in its survival remains unclear. Here, we focused on the inhibition of mitochondrial cytochrome bc1 complex (complex III) and evaluated aerobic respiratory activity using mitochondrial fractions from E. multilocularis protoscoleces. An enzymatic assay revealed that the mitochondrial fractions possessed NADH-cytochrome c reductase (mitochondrial complexes I and III) and succinate-cytochrome c reductase (mitochondrial complexes II and III) activities in the aerobic pathway. Enzymatic analysis showed that atovaquone, a commercially available anti-malarial drug, inhibited mitochondrial complex III at 1.5 nM (IC50). In addition, culture experiments revealed the ability of atovaquone to kill protoscoleces under aerobic conditions, but not under anaerobic conditions, indicating that protoscoleces altered their respiration system to oxidative phosphorylation or fumarate respiration depending on the oxygen supply. Furthermore, combined administration of atovaquone with atpenin A5, a quinone binding site inhibitor of complex II, completely killed protoscoleces in the culture. Thus, inhibition of both complex II and complex III was essential for strong antiparasitic effect on E. multilocularis. Additionally, we demonstrated that oral administration of atovaquone significantly reduced primary alveolar hydatid cyst development in the mouse liver, compared with the untreated control, indicating that complex III is a promising target for development of anti-echinococcal drug.

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.

Infection with Moniezia benedeni is sometimes found in confined cattle in Japan. Between October 2011 and January 2013, we monitored the fecal egg prevalence at a confined cattle farm in Miyazaki prefecture where continuous M. benedeni infection has been recognized for years to evaluate the possible infection routes. Fecal egg prevalence changed seasonally with the highest in October 2011 (27.3%: 9/33). This was followed by a gradual decrease until July 2012 (9.4%: 3/32) and then an increase between August to December 2012 when new egg-excreting cases were observed. The pattern of seasonal changes was similar to that reported previously for cattle kept in a barn with an outside playing yard. Although M. benedeni-infected mites were not found, we constantly detected an oribatid mite, Oribatula sakamorii Aoki, 1970, in the litter of cattle bedding from May to October 2012. This species belongs to a genus which has been reported to be a suitable intermediate host for M. benedeni, suggesting that M. benedeni infection may have been autonomously maintained at the farm via oribatid mites living in the cowshed. When infected cattle were treated with praziquantel, it was found that a single oral inoculation with a dose of 5 mg/kg was effective for deworming.

Samples of diaphragm were collected from 53 sika deer from Gifu Prefecture, Japan; 220 sarcocysts were isolated, examined in wet mounts and classified according to their cyst wall protrusions. The sarcocysts were then examined molecularly in order to assign them to different species. All but 11 of the 220 sarcocysts were initially identified by means of a multiplex PCR assay targeting cox1 of five species, whereas the remaining 11 sarcocysts were identified by standard PCR and sequencing. DNA from selected sarcocysts was used for PCR amplification and sequencing of cox1 (59 sequences) and 18S rDNA (23 sequences). The 220 sarcocysts comprised seven major cox1 sequence types or species. Types 4 and 7 were assigned to the known species Sarcocystis pilosa and Sarcocystis ovalis, whereas types 1, 3 and 5 were considered to represent three new species, for which the names Sarcocystis japonica, Sarcocystis matsuoae and Sarcocystis gjerdei have been proposed. Types 2 and 6 were most similar to Sarcocystis tarandi and Sarcocystis taeniata, respectively, but could not be unequivocally assigned to these species. Sarcocysts belonging to S. japonica were macroscopic with fairly thick finger-like protrusions, whereas most sarcocysts of the six other species were microscopic. Sarcocysts of S. cf. tarandi and S. matsuoae were spindle-shaped and possessed thin finger-like cyst-wall protrusions. Sarcocysts of S. pilosa and S. gjerdei had similar hair-like protrusions, whereas those of S. cf. taeniata had a smooth surface. Sarcocysts of S. japonica, S. pilosa, S. cf. tarandi, S. gjerdei, S. matsuoae, S. cf. taeniata and S. ovalis were found in 50 (94.3%), 29 (54.7%), 22 (41.5%), 10 (18.9%), 8 (15.1%), 6 (11.3%) and 1 (1.9%) of the 53 sika deer examined, respectively. An improved multiplex PCR assay targeting cox1 was developed, through which the seven Sarcocystis spp. found in the present study could be identified.