Genetic diversity and population structure analyses based on microsatellite DNA of parthenogenetic Fasciola flukes obtained from cattle and sika deer in Japan.

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.

Molecular differentiation of five Sarcocystis species in sika deer (Cervus nippon centralis) in Japan based on mitochondrial cytochrome c oxidase subunit I gene (cox1) sequences.

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.

Phylogenetic relationships between Dicrocoelium chinensis populations in Japan and China based on mitochondrial nad1 gene sequences.

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.

Ascaridia nymphii n. sp. (Nematoda: Ascaridida) from the alimentary tract of a severely emaciated dead cockatiel Nymphicus hollandicus.

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.

Unique genomic sequences in a novel Mycobacterium avium subsp. hominissuis lineage enable fine scale transmission route tracing during pig movement.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct.

Sex-inducing effects toward planarians widely present among parasitic flatworms.

Various parasitic flatworms infect vertebrates for sexual reproduction, often causing devastating diseases in their hosts. Consequently, flatworms are of great socioeconomic and biomedical importance. Although the cessation of parasitic flatworm sexual reproduction is a major target of anti-parasitic drug design, little is known regarding bioactive compounds controlling flatworm sexual maturation. Using the planarian Dugesia ryukyuensis, we observed that sex-inducing substances found in planarians are also widespread in parasitic flatworms, such as monogeneans and flukes (but not in tapeworms). Reverse-phase HPLC analysis revealed the sex-inducing substance(s) eluting around the tryptophan retention time in the fluke Calicophoron calicophorum, consistent with previous studies on the planarian Bipalium nobile, suggesting that the substance(s) is likely conserved among flatworms. Moreover, six of the 18 ovary-inducing substances identified via transcriptome and metabolome analyses are involved in purine metabolism. Our findings provide a basis for understanding and modifying the life cycles of various parasitic flatworms.

The first detection of Dicrocoelium chinensis sporocysts from the land snail Aegista vulgivaga in Gifu Prefecture, Japan.

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.

Genomic features of Mycobacterium avium subsp. hominissuis isolated from pigs in Japan.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in humans and pigs. There have been advances in genome analysis of MAH from human isolates, but studies of isolates from pigs are limited despite its potential source of infection to human. Here, we obtained 30 draft genome sequences of MAH from pigs reared in Japan. The 30 draft genomes were 4,848,678-5,620,788 bp in length, comprising 4652-5388 coding genes and 46-75 (median: 47) tRNAs. All isolates had restriction modification-associated genes and 185-222 predicted virulence genes. Two isolates had tRNA arrays and one isolate had a clustered regularly interspaced short palindromic repeat (CRISPR) region. Our results will be useful for evaluation of the ecology of MAH by providing a foundation for genome-based epidemiological studies.

Morphological and molecular characteristics of seven Sarcocystis species from sika deer (Cervus nippon centralis) in Japan, including three new species.

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.

Hepatozoon apri n. sp. (Adeleorina: Hepatozoidae) from the Japanese wild boar Sus scrofa leucomystax (Mammalia: Cetartiodactyla).

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.

Venison, another source of Paragonimus westermani infection.

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.

Modified sugar centrifugal flotation technique for recovering Echinococcus multilocularis eggs from soil.

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.

Larva migrans by Baylisascaris transfuga: fatal neurological diseases in Mongolian jirds, but not in mice.

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.

Pterygodermatites nycticebi (Nematoda: Rictulariidae): accidental detection of encapsulated third-stage larvae in the tissue of a white-fronted marmoset.

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.

Seroprevalence of Toxoplasma gondii infection in cattle, horses, pigs and chickens in Japan.

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.

Detection and genotype of Encephalitozoon cuniculi DNA from urine and feces of pet rabbits in Japan.

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.