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Understanding the population structure of Fasciola flukes in domestic and wild animals is important for determining the extent of cross-infection between them. Although the parthenogenetic Fasciola flukes in Japan have been shown to comprise five genetic types based on the ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1) regions, these genetic regions are not suitable for analyzing their population structure. In the present study, the genetic diversity and population structure of the parthenogenetic Fasciola flukes in Japan were studied using microsatellite DNA, ITS1, and nad1 regions. A total of 144 parthenogenetic Fasciola flukes, obtained from cattle and sika deer in 16 localities, were individually analyzed using PCR-RFLP for ITS1, PCR-direct sequence analysis for nad1, and post-labeling PCR and capillary electrophoresis for microsatellite DNA regions. The flukes showed higher genetic diversity in the microsatellite DNA regions than ITS1 and nad1. The population structures of parthenogenetic Fasciola flukes were unclear, however, it was suggested that the flukes are more diverse populations. We hypothesized that their distribution throughout Japan is closely related to livestock movement dependent on human activity. Moreover, it is considered that cross-infection of the flukes between cattle and sika deer possibly has occurred in the past.

Trematodes of the genus Dicrocoelium are one of the most common parasites in ruminant animals; however, their life cycles in Japan are unclear. To find the sporocysts of D. chinensis in the natural field, we sampled 269 land snails (14 species) at a location with high level infection of sika deer in Gifu Prefecture, Honshu Island, Japan in autumn between 2017 and 2019. During the sampling period, we found mother sporocysts in the hepatopancreas of Aegista vulgivaga and Cyclophorus herklotsi. DNA barcoding based on the sequences of cytochrome c oxidase subunit 1 showed that the sporocysts from A. vulgivaga belonged to D. chinensis, indicating that this snail has potential as the first intermediate host of D. chinensis at this location.

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. Keywords: Sarcocystis japonica, Sarcocystis matsuoae, Sarcocystis gjerdei, Cervus nippon centralis, 18S ribosomal RNA gene, Cytochrome c oxidase subunit I gene, Japan

Several surveys of Sarcocystis infection in sika deer in Japan have shown a high prevalence, but the identification has been unclear because molecular data have been lacking or have been limited to 18S ribosomal RNA gene sequences. Thus, in our previous study based on such sequences, some Sarcocystis isolates from sika deer were not clearly separated from other species in the phylogenetic analysis. In the present study, we therefore characterized sarcocyst isolates from sika deer (Cervus nippon centralis) at the mitochondrial cytochrome c oxidase subunit I gene (cox1). Moreover, we developed a multiplex PCR based on cox1 sequences of all species found, so that we could rapidly identify sarcocysts of these species. Twenty-one sarcocysts from nine sika deer were examined. Five distinct cox1 sequence types, each with a high sequence identity (> 99%), were found, and the sarcocysts could thus be classified into five species. Based on the sequence comparisons and the phylogeny, Sarcocystis spp. of types 1, 3, and 5 are considered to represent three new species, which were most closely related to Sarcocystis silva/Sarcocystis truncata, Sarcocystis entzerothi, and Sarcocystis iberica/Sarcocystis venatoria, respectively. There was a slight uncertainly whether Sarcocystis sp. with type 2 sequences represented a new species or was identical to Sarcocystis tarandi. Type 4 sequences showed 99% identity with those of Sarcocystis pilosa from sika deer in Lithuania and have therefore been assigned to this species. In the multiplex PCR, type-specific fragments were successfully amplified for all five Sarcocystis spp., indicating that this assay may be useful for a rapid identification of sarcocysts of these species.

Hepatozoon apri n. sp. is described from Japanese wild boars Sus scrofa leucomystax in Japan. The gamonts in the peripheral blood leukocytes were 11.6 ± 1.4 × 6.7 ± 1.3 μm in size. The meronts in the muscle tissues were 35.0–47.5 μm in length and 26.5–30 μm in width. A high rate (53.0%) of infection was found by nested PCR using muscle specimens from 181 wild boars captured in Tokushima, Japan. A phylogenetic analysis based on 18S rRNA gene sequences revealed that H. apri n. sp. detected in wild boars is closely related to Hepatozoon spp. isolated from carnivores. This is the first description of a species belonging to the genus Hepatozoon detected in ungulates., Graphical abstract Image 1, Highlights • Hepatozoon apri n. sp. from Japanese wild boars was described. • The newly described isolates were closely related to Hepatozoon from carnivores. • H. apri n. sp. is widely distributed among wild boars (infection rate, 53%).

This report describes Ascaridia nymphii n. sp., a new species isolated from the alimentary tract of cockatiel Nymphicus hollandicus in Japan. More than 63 nematodes were found in the formalin-fixed small intestine, ventriculus, proventriculus and crop of a 48-day-old young cockatiel that died after exhibiting severe emaciation. No nematode eggs were observed in the faecal examination performed while the cockatiel was alive, but Cryptosporidium oocysts were found. The intestinal mucosa was damaged considerably. Male worms had two alate spicules, well-developed precloacal sucker and a tail with ventrolateral caudal alae and mainly 11 pairs of papillae. Nuclear partial (813 bp) 18S ribosomal RNA gene (18S rDNA) sequences obtained from two female samples were mutually identical. They respectively showed 99.1 and 98.6% identities to those from Ascaridia numidae and Ascaridia galli. Phylogenetic analysis using this locus indicated the present nematode as Ascaridia species. The mitochondrial NADH dehydrogenase subunit 2 gene (nad2) sequences obtained for four samples were mutually identical. They respectively showed 98.7, 85.7 and 82.2% identities with those from Ascaridia columbae, Ascaridia sp. and A. galli. Combining the morphological and sequencing data from two loci, the present nematode was identified as A. nymphii n. sp., which is closely related with A. columbae. This report is the first of a study examining the distribution of Ascaridia species in captive parrots in Japan. This study also identified the trachea and cloaca, like Cryptosporidium baileyi, as the possible location of Cryptosporidium avian genotype V in avian hosts.

Most published data on the physical and physiological profiles of karate athletes is based on studies of male athletes and data on female athletes is rare. The purposes of this study were: 1) to collect baseline data on nutrient intake in order to advise athletes regarding nutrition practices that may enhance performance, and 2) to compare nutrient intake of elite and collegiate karate athletes. Thirtyfive female black belt karate athletes volunteered to participate in this study. They were divided into 2 groups: 20 athletes who were members of the national team (elite athletes) and 15 collegiate karate athletes (collegiate athletes). The elite athletes showed significantly higher mean lean body mass and significantly lower body fat and fat mass than the collegiate athletes. The elite athletes showed significantly higher energy intake and more nutrient intakes than the collegiate athletes. There were no significant differences in diet compositions between the elite and collegiate athletes. The elite athletes showed all micronutrient intakes were above 100% of the Estimated Average Requirement (EAR) or Adequate Intake (AIs), whereas the collegiate athletes showed micronutrient intakes below 100% of the EAR or AIs for potassium, calcium, magnesium, phosphorus, iron, vitamins A and C. Thus, we advised collegiate athletes to consume adequate amounts of carbohydrates and protein. To attain these goals, it is desirable to increase the amount of meals by increasing the intake levels of grains, vegetables, fruit, milk and dairy products, lean meat and fish.

We carried out phylogenetic analyses of the relationships between Dicrocoelium chinensis populations in Japan and China using molecular markers. One hundred nine lancet flukes collected from Japan and China were identified as D. chinensis based on their testis orientation and the nucleotide sequences of their ribosomal ITS2. These flukes were analyzed phylogenetically using mitochondrial nad1 gene sequences. An analysis of molecular variance found that the percentage of variation between the countries was extremely high, indicating that the D. chinensis populations in Japan and China are differentiated genetically. D. chinensis mainly parasitizes wild sika deer, which is thought to originate in northeast Asia and to have colonized into Japan from the Eurasia continent in the Pleistocene glaciations. In addition, phylogenic analyses indicated that Japanese sika deer is genetically differentiated from Chinese population; therefore, we hypothesize that D. chinensis might have been introduced into Japan along with the migration of infected wild ruminants in the Pleistocene, and then the population became differentiated from the Chinese population. This study provides the nucleotide sequences of the nad1 gene of D. chinensis in Japan for the first time and shows that these sequences are useful for elucidating the phylogenetic relationships of the Dicrocoelium species prevalent in Asia.

Much of the published data on the lipid profiles of athletes are based on studies of endurance athletes. Data on female volleyball players are rare. The purpose of this study was thus to examine serum high-density-lipoprotein cholesterol (HDL-C) subfractions in collegiate female volleyball players. Twenty-six female collegiate volleyball players were compared with 26 age- and body mass index-matched control subjects. Dietary information was obtained with a food frequency questionnaire. The subjects were all nonsmokers and were not taking any drug known to affect lipid and lipoprotein metabolism. The volleyball players showed significantly higher mean HDL2-C than the control group. There were no significant differences in HDL-C and HDL3-C between the 2 groups. The results indicate that favorable lipid and lipoprotein profiles could be obtained by vigorous volleyball training and significantly higher HDL2-C could be obtained without observing significant differences in HDL-C and HDL3-C.

A newly developed nested polymerase chain reaction (PCR) assay targeting the internal transcribed spacer (ITS) region of the ribosomal DNA was applied to detect and characterize Encephalitozoon cuniculi DNA from pet rabbits in Japan. The analysis was carried out using 257 urinary samples and 314 fecal samples collected from 307 pet rabbits in the age group of 1 month to 12 years from 30 different prefectures of Japan and 107 fecal samples and 3 urinary samples collected from 1-month-old rabbits from 3 breeding facilities in Japan. We detected 840-bp amplicons in 20 urinary samples (7.78%) from the pet rabbits of the 13 prefectures and in 1 urinary (33.3%) and 6 fecal (5.6%) samples from the rabbits of the 2 breeding facilities. The sequences (803 bp) of the 27 amplicons had no variations and completely coincided with the sequence of E. cuniculi genotype I. To the best of our knowledge, this is the first report on the detection and genotype characterization of E. cuniculi DNA from pet rabbits in Japan.

Paragonimiasis is a typical food-borne parasitic disease, endemic in most parts of Asia, with sporadic case reports from American and African countries. The major source of infection is undercooked freshwater crab or crayfish, though consumption of wild boar meat is also responsible for the infection in Japan, because wild boar is a paratenic host for Paragonimus westermani. Recently, living juveniles of P. westermani were isolated from muscle of a sika deer, Cervus nippon, in Japan, raising the possibility that venison has been another source of infection. In order to clarify the potential contribution of venison consumption to the occurrence of paragonimiasis, we analysed dietary histories of those paragonimiasis patients in whose diagnoses we were involved between 2001 and 2015. Among 380 patients, freshwater crab had been consumed by 208 patients, wild boar meat by 190, and wild deer meat by 76 patients before the onset of the disease. Overall contribution of wild deer meat was estimated to be 6.8% to 20.0%, although in Oita and Gifu Prefectures, where a substantial proportion of patients had consumed raw venison, the contribution of venison consumption was much higher (27.5 to 62.1% and 42.1 to 78.9% in Oita and Gifu Prefectures, respectively). We demonstrated P. westermani-specific antibodies in the sera of 4 out of 160 sika deer from Gifu Prefecture, strongly suggesting that these deer were infected with P. westermani.

Enterobius (Colobenterobius) serratus sp. nov. (Nematoda: Oxyuridae) is described from females collected from a proboscis monkey, Nasalis larvatus (Primates: Cercopithecidae: Colobinae), in Sarawak, Borneo, Malaysia. This species is readily distinguished from other members of the subgenus by the serrated inner margins of lips, lamellated superstructures of the esophageal teeth forming denticles, larger body, and larger eggs.

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.

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.

The presence of antibodies to Toxoplasma gondii in livestock and poultry was investigated by latex agglutination tests; samples that agglutinated at dilutions of 1:64 or higher were regarded as positive. Sera were collected from fattening beef cattle (102 Japanese black, 105 crossbreeds and 114 castrated Holstein), culled dairy cattle (101 Holstein), 100 horses, 115 fattening pigs and 235 chickens (163 free-range and 72 broilers) at abattoirs in Gifu Prefecture, Japan, from August 2012 to August 2013. Antibodies to T. gondii were found in 7.3% (31/422) in cattle, 5.2% (8/155) in pigs, but not in horses or chickens. These results suggest that toxoplasmosis may be transmitted to humans via consumption of T. gondii-infected raw beef in Japan.

Raccoon roundworms (Baylisascaris procyonis) and other Baylisascaris species cause patent or latent larva migrans (LM) in a variety of mammals and birds, including humans. It is not clear whether LM by Baylisascaris transfuga, roundworms of bears, is associated with clinical neurological disorders. To clarify this issue, ICR and BALB/c mice as well as Mongolian jirds (Meriones unguiculatus) were orally inoculated with 2,000-5,000 embryonated eggs of B. transfuga. In mice, the ascarid caused symptomatic LM of limited extent and duration, whereas the infection was fatal in jirds; i.e., they exhibited general signs such as severe depression and emaciation on days 8-11 postinfection (PI) and died, or they developed progressive and fatal neurological disorders after day 14 PI. Histological examination showed B. transfuga larvae in the brain of all mice and jirds examined, and the larvae collected from them developed to a size comparable with that of B. procyonis. There existed, however, critical differences in host reactions against larvae localized in the brain of mice and jirds; B. transfuga larvae found in mice were surrounded by granulomatous reactions and immobilized, whereas larvae found in jirds were free from any host reaction and mobile, causing extensive malacia.

Twin, white-fronted marmosets (Callithrix geoffroyi) born and raised in a zoo in Japan died at 7 mo of age. Several encapsulated nematode larvae were detected in the intestinal wall, as well as a few in the mesenteric lymph nodes of 1 of the twins. In the other marmoset, no encapsulated nematode larva was detected in the organs, but many adult Pterygodermatites nycticebi were found in the intestinal lumen. In the past 5 yr, 5 primates kept in the same zoo, i.e., 1 squirrel monkey (Saimiri sciureus), 2 Pygmy marmosets (Cebuella pygmaea), 1 Senegal galago (Galago senegalensis), and 1 cotton-top tamarin (Saguinus oedipus), died from heavy infestation with the same nematode. A few migrating larvae of the rictulariid were also identified histologically in the intestinal wall and liver of the cotton-top tamarin. Although no other primate currently held in the same zoo was infected with the rictulariid, German cockroaches (Blattella germanica) collected with traps near marmoset cages had encapsulated P. nycticebi larvae, indicating latent perpetuation of the life cycle of this rictulariid species in the zoo premises. Our results indicated that encapsulation or migration of third-stage larvae of P. nycticebi might occur accidentally in the organs of callithrichid primates.

Forty-three red foxes (Vulpes vulpes schrencki) and nine raccoon dogs (Nyctereutes procyonoides viverrinus) were captured in Otaru City, Hokkaido, Japan and examined by muscle digestion for the presence of Trichinella sp. larvae. Of the foxes, five (11.6%) were positive for larvae of Trichinella nativa while none of the raccoon dogs were found to be positive. This finding suggests that the red foxes are important reservoir hosts of sylvatic trichinellosis in Otaru, Hokkaido. This is the first report on Trichinella nativa in wild red foxes of Hokkaido, Japan.

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 non-treated animals (P < 0.01).

Among soil-transmitted parasitic diseases, alveolar hydatidosis due to the ingestion of Echinococcus multilocularis eggs is becoming a serious problem in Hokkaido, the northern most island of Japan. Dissemination of the infection far from the endemic areas can occur if motor vehicles transmit soil contaminated with eggs. No appropriate and validated method for recovering the taeniid eggs from soil is available. A modified sugar centrifugal flotation technique, using a sucrose solution of specific gravity 1.27 and 0.05% Tween-80, was evaluated as a method to successfully recover eggs from soil. Contamination levels as low as 10 eggs per gram could be detected. This method may be useful to determine the prevalence of E. multilocularis, its transmission, and the potential for by monitoring soil contamination with eggs.