Genetic diversity of cervid Trypanosoma theileri in Honshu sika deer (Cervus nippon) in Japan.

The taxonomy of ruminant Trypanosoma theileri and its relatives (Kinetoplastida: Trypanosomatidae) is controversial, with recent phylogenetic studies segregating T. theileri in cattle and other ruminants worldwide into two major genetic lineages (the TthI and TthII clades) based on genetic markers. In the present study, T. theileri-like trypanosomes isolated from Honshu sika deer (Cervus nippon) in the western Japan (YMG isolate) were genetically characterized using a number of genetic markers. Sika deer trypanosomes of the YMG isolate were genetically different from the Trypanosoma sp. TSD1 isolate previously recorded from Hokkaido sika deer in northern Japan, with the former trypanosome isolate being genetically closer to European cervid trypanosomes and the bovine T. theileri TthII lineage. In contrast, the latter isolate exhibited greater relatedness to North American cervid trypanosomes and the bovine T. theileri TthI lineage, although a clear genetic distinction between these was apparent. Furthermore, trypanosomes in Honshu sika deer from the central part of Japan harboured additional genetic diversity and were closer to either TSD1 or YMG isolates, while distinct from known T. theileri-related genotypes. Importantly, cervids and wild ruminants worldwide might harbour divergent descendants of a T. theileri ancestor, which exhibit rigid host specificity to either bovines or cervid species.

Morphological and molecular genetic characterization of Kudoa konishiae n. sp. (Myxosporea: Multivalvulida) in the muscle of Japanese Spanish mackerel (Scomberomorus niphonius).

The Japanese Spanish mackerel (Scomberomorus niphonius; Perciformes: Scombridae) is widely distributed in the continental shelves of the northwestern Pacific Ocean around Japan, Sea of Japan, and East China Sea. In the present study, small, spindle-shaped myxosporean plasmodia (0.15-0.53 mm by 0.04-0.13 mm) were frequently encountered in the myofiber of trunk muscles of two Japanese Spanish mackerels; one fished in the Sea of Japan off western Japan and the other in the northwestern Pacific Ocean off southern Japan in the autumn of 2016. Isolated myxospores of Kudoa konishiae n. sp. (Myxosporea: Multivalvulida) from these two fish were stellate with six equal shell valves and polar capsules, 8.1-9.7 µm in width, 7.1-8.8 µm in thickness, and 7.1-8.8 µm in length. The polar capsules were teardrop-shaped, 2.7-4.7 µm by 1.2-2.5 µm. The lateral view of spores revealed a drawstring-pouch shape. The nucleotide sequences of the 18S and 28S ribosomal RNA gene (rDNA) were distinct from any recorded species. Phylogenetic trees demonstrated a close relationship of the present new species with Kudoa spp. with stellate spores with five or more shell valves/polar capsules, recorded in scombrid fishes. To clarify the phylogenetic relationships between three closely related species, i.e., Kudoa konishiae n. sp., Kudoa hexapunctata, and Kudoa neothunni, three mitochondrial DNA genes (cytochrome c oxidase subunit 1 gene (cox-1) and the small and large subunits of the ribosomal RNA gene (rns-rnl)) of two isolates of the new species, six isolates of K. hexapunctata, and 13 isolates of K. neothunni were sequenced. The interspecific and intraspecific variations of the newly obtained cox-1 and rns-rnl nucleotide sequences of K. hexapunctata, K. neothunni, and K. konishiae n. sp. were clarified for the first time.

An unexpected case of a Japanese wild boar (Sus scrofa leucomystax) infected with the giant thorny-headed worm (Macracanthorhynchus hirudinaceus) on the mainland of Japan (Honshu).

The giant thorny-headed worm Macracanthorhynchus hirudinaceus is a zoonotic acanthocephalan species with a worldwide distribution. Its natural definitive hosts are primarily pigs and wild boars (Sus scrofa), and scarabaeid beetles serve as the intermediate host. To date, there has only been one record of this acanthocephalan in Japan: a Ryukyu wild boar (Sus scrofa riukiuanus) hunted in 1973 on Amami Island, faraway from the Japanese mainland. The present study reports the second case of this acanthocephalan in Japan: a Japanese wild boar (Sus scrofa leucomystax) hunted in May 2017 in Yamaguchi Prefecture, the westernmost part of the Japanese mainland (Honshu). More than a dozen acanthocephalans (123 to 233 mm in length and 5 to 6 mm in width) were found with their proboscides inserted deeply into the intestinal wall, forming grossly visible nodules on the external surface. Isolated worms underwent a molecular genetic characterization of the ribosomal RNA gene (rDNA) and mitochondrial cytochrome c oxidase subunit 1 gene (cox-1). Long rDNA nucleotide sequences (5870 to 5890 bp) spanning the beginning of the 18S rDNA through to the 28S rDNA, including the internal transcribed spacer regions, and 1384-bp cox-1 nucleotide sequences were obtained. In the future, in conjunction with the accumulation of molecular genetic data of multiple M. hirudinaceus isolates from different endemic localities abroad, our data may help to postulate the origin or present transmission status of this extremely rarely encountered acanthocephalan in Japan.

Molecular genetic diversity of Gongylonema neoplasticum (Fibiger & Ditlevsen, 1914) (Spirurida: Gongylonematidae) from rodents in Southeast Asia.

More than a dozen Gongylonema spp. (Spirurida: Spiruroidea: Gongylonematidae) have been described from a variety of rodent hosts worldwide. Gongylonema neoplasticum (Fibiger & Ditlevsen, 1914), which dwells in the gastric mucosa of rats such as Rattus norvegicus (Berkenhout) and Rattus rattus (Linnaeus), is currently regarded as a cosmopolitan nematode in accordance with global dispersion of its definitive hosts beyond Asia. To facilitate the reliable specific differentiation of local rodent Gongylonema spp. from the cosmopolitan congener, the genetic characterisation of G. neoplasticum from Asian Rattus spp. in the original endemic area should be considered since the morphological identification of Gongylonema spp. is often difficult due to variations of critical phenotypical characters, e.g. spicule lengths and numbers of caudal papillae. In the present study, morphologically identified G. neoplasticum from 114 rats of seven species from Southeast Asia were selected from archived survey materials from almost 4,500 rodents: Thailand (58 rats), Cambodia (52 rats), Laos (three rats) and Philippines (one rat). In addition, several specimens from four rats in Indonesia were used in the study. Nucleotide sequences of the ribosomal RNA gene (rDNA) (5,649 bp) and the cytochrome c oxidase subunit 1 gene (cox1) (818 bp) were characterised. The rDNA showed little nucleotide variation, including the internal transcribed spacer (ITS) regions. The cox1 showed 24 haplotypes, with up to 15 (1.83%) nucleotide substitutions regardless of parasite origin. Considering that Rattus spp. have been shown to originate from the southern region of Asia and G. neoplasticum is their endogenous parasite, it is reasonable to propose that the present study covers a wide spectrum of the genetic diversity of G. neoplasticum, useful for both the molecular genetic speculation of the species and the molecular genetic differentiation of other local rodent Gongylonema spp. from the cosmopolitan congener.

Morphological and genetic characterization of Kudoa whippsi (Myxosporea: Multivalvulida) from Cheilodactylus zonatus in the western Pacific Ocean off Japan, and two new Kudoa spp. (K. akihitoi n. sp. and K. empressmichikoae n. sp.) from Acanthogobius hasta in the Sea of Ariake, Japan.

Molecular genetic characterization using the ribosomal RNA (rDNA) gene accrues a wealth of knowledge regarding the true nature of species diversity of Kudoa Meglitsch, 1947 (Myxozoa: Myxosporea: Multivalvulida) and the biogeographical relationships of isolates from different host fish and sea areas. In the present study, we characterized morphologically and genetically three Kudoa spp. with four shell valves and polar capsules (SV/PC), forming pseudocysts in the myofiber of trunk muscles of Cheilodactylus zonatus or Acanthogobius hasta in the natural seawater around Japan. Myxospores from C. zonatus fished in the western Pacific Ocean off Kochi, Japan, were unequal quadrangular pyramids with one large and three smaller SV/PC, morphologically closest to Kudoa whippsi recorded in various pomacentrid and apogonid fish from the Australian Coral Sea. The 18S and 28S rDNA nucleotide sequences of the Japanese isolate were highly similar to some Australian K. whippsi isolates, but also displayed less similarity to other K. whippsi isolates from the same sea mainly due to instability of nucleotides at certain base positions and/or segments of different isolates. All the K. whippsi isolates including the present Japanese isolate, however, were distinct from Kudoa gunterae, K. whippsi's closest kudoid species in morphology, molecular phylogeny, and biogeography. Our detection of K. whippsi from C. zonatus in the natural seawater around Japan is a new host and geographical record. Kudoid myxospores from A. hasta from the Sea of Ariake, a deep bay of the western part of Japan, exhibited two morphotypes, one resembling K. whippsi and the other Kudoa quadricornis with distinct posteriolateral SV projections. However, rDNA nucleotide sequencing revealed that these two Kudoa spp. were distinct from any known congeners; thus, Kudoa akihitoi n. sp. and Kudoa empressmichikoae n. sp. were erected. The morphological differentiation of K. akihitoi n. sp. from multiple Kudoa spp. with scalene stellate myxospores containing one large and three smaller SV/PC was difficult, whereas K. empressmichikoae n. sp. with spherical spore bodies extending small posteriolateral SV projections was distinct from known congeners with similar but elongated spore bodies and PC, i.e., K. quadricornis and Kudoa paraquadricornis, found in the trunk muscle of carangid fish from the Australian Coral Sea.

Enteromyxum leei (Myxosporea: Bivalvulida) as the cause of myxosporean emaciation disease of farmed olive flounders (Paralichthys olivaceus) and a turbot (Scophthalmus maximus) on Jeju Island, Korea.

A life-threatening emaciation disease of unknown cause(s) is affecting the farming of olive flounders (Paralichthys olivaceus) and turbots (Scophthalmus maximus) on Jeju Island, Korea. As this is one of the major industries in the region, it is of great concern to local farmers trying to develop successful and sustainable aquaculture. We examined 16 olive flounders and one turbot cultured at three farms located in the southern part of Jeju Island, which manifested moderate to severe emaciation such as thinning of the body with notable appearance of bony ridges of the skull on heads. Fresh mucosal scrapings of the intestinal mucosa contained many myxosporean vegetative stages at various developments but not fully grown spores. Histological examination of gastrointestinal and other visceral organs revealed striking changes in the intestinal mucosa such as detachment and loss of the epithelium due to intensive parasitism of the myxosporean vegetative stages, accompanied by considerable leukocyte infiltration in the lamina propria, and at the final stage villus atrophy with no epithelial lining. Specific polymerase chain reaction using a pair of primers targeting a fragment of the 18S ribosomal RNA gene (rDNA) of Enteromyxum leei, a known pathogen causing myxosporean emaciation disease in a variety of cultured fish in Mediterranean countries and Japan, amplified 433-bp products in almost all diseased fish samples, particularly the gastrointestinal tract. Nearly the whole length of the 18S rDNA, 1672-bp long excluding primer-aligning sequences, of the present Korean isolate was comparable to those of E. leei isolates from Japan and Europe, particularly those from the former region. Taking the heavy load of various developmental stages of E. leei in the gastrointestinal mucosa into account, we ascribe the emaciation disease of the fish examined in the present study to this well-known myxosporean species and not to another unknown pathogen(s).

Intraspecific and interspecific genetic variation of Gongylonema pulchrum and two rodent Gongylonema spp. (G. aegypti and G. neoplasticum), with the proposal of G. nepalensis n. sp. for the isolate in water buffaloes from Nepal.

The gullet worm (Gongylonema pulchrum) has been recorded from a variety of mammals worldwide. In an earlier study, we demonstrated two separate transmission cycles in cattle (Bos taurus) and wild mammals in Japan based on nucleotide sequences of the ribosomal RNA gene (rDNA) and cytochrome c oxidase subunit I (cox-1) region of mitochondrial DNA of multiple isolates of different origins. Our earlier study additionally demonstrated two major cox-1 haplotypes of G. pulchrum prevalent in cattle in Japan. In the present study, we collected G. pulchrum from cattle and goats (Capra hircus) in Alashan League, Inner Mongolia, China; Gongylonema aegypti from spiny mice (Acomys dimidiatus) in the Sinai Peninsula, Egypt; and Gongylonema neoplasticum from a black rat (Rattus rattus) in Okinawa Island, Japan, to analyze their genetic relationships with G. pulchrum in Japan. The gullet worms from Alashan League had almost identical rDNA nucleotide sequences and two cox-1 haplotypes as seen in G. pulchrum from the cattle in Japan. The two rodent Gongylonema spp. had distinct rDNA nucleotide sequences compared with those of G. pulchrum; only the 18S and 5.8S rDNA sequences showed high identities at 97.2-98.7%, while the remaining sequences were less than 75% identical. The 18S, 5.8S, and 28S rDNA sequences of the two rodent Gongylonema spp. showed nucleotide identities of 99.8% (1811/1814), 100% (158/158), and 98.9% (3550/3590), respectively. The cox-1 regions showed 91.6% (338/369)-92.1% (340/369) identities, with completely identical amino acid sequences. The genetic diversities of three distinct Gongylonema spp. and their possible intraspecific genetic variation may allow us to resolve the taxonomic position of Gongylonema spp. which display few obvious morphological differences from their congeners. Consequently, the Gongylonema isolate from water buffaloes (Bubalus bubalis) in Nepal reported in our previous study is concluded to be a new species, and Gongylonema nepalensis n. sp. is erected for it.