First Report of Mermithidae (Enoplea: Mermithida) Parasitizing Adult Stable Flies in Japan.

Mermithidae is a family of nematodes that parasitize a wide range of invertebrates worldwide. Herein, we report nematodes that were unexpectedly found in three of 486 adult stable flies (Stomoxys calcitrans) captured from three farms (F1, F2, and F3) in different regions of Gifu Prefecture, Japan. We aimed to characterize these nematodes both at the morphological and molecular level. Morphological studies revealed that the nematodes were juveniles of Mermithidae. Phylogenetic analysis based on 18S and 28S rDNA indicated that the mermithids from farms F1 and F2 could be categorized into the same cluster as Ovomermis sinensis and Hexamermis sp., whereas the mermithid from farm F3 clustered with Amphimermis sp. Additionally, these mermithids could be categorized within the same clusters as related mermithids detected in Japan that parasitize various arthropod orders. Our findings suggest that these stable flies may have been parasitized by mermithids already present in the region and that genetically distinct species of mermithids occur across Japan. To the best of our knowledge, this is the first report of mermithids parasitizing adult stable flies in Japan.

An adult cystidicolid nematode (Nematoda: Cystidicolidae) from the subcutaneous tissue around the eye of percupinefish, Diodon nichthemerus Cuvier.

Three adult nematode specimens, all ovigerous females belonging to the family Cystidicolidae Skryabin, 1946, were found for the first time in the subcutaneous tissue around the eye of the captive porcupinefish Diodon nichthemerus Cuvier at a public aquarium in Osaka, Japan. Because no male was available, these could not be identified to the genus and species. This case highlights the risk of parasitism in aquaculture puffer fish, as these may ingest small shrimp, which probably act as intermediate hosts for the nematode.

Echinococcus species from red foxes, corsac foxes, and wolves in Mongolia.

The small intestines of 420 wild canids (111 corsac foxes, 191 red foxes and 118 wolves) from Mongolia, were examined for adult worms of the genus Echinococcus. The Mongolian genotype of Echinococcus multilocularis was found in fifteen red foxes and four wolves, whereas two genotypes (G6/7 and G10) of Echinococcus canadensis were found in two and three wolves, respectively. No adult Echinococcus worms were found in the corsac foxes examined. The genotypes of E. multilocularis and E. canadensis are discussed in terms of host specificity and distribution in Mongolia. The importance of wolves in the completion of the life cycle of Echinococcus spp. is also discussed.

Identification and phylogenetic analysis of Babesia parasites in domestic dogs in Nigeria.

The present study examined the presence of Babesia parasites in 104 domestic dogs in Nigeria. Sequentially, Babesia parasites infecting domestic dogs underwent genetic and phylogenetic analyses. The results of nested PCR based on the Piroplasmida 18S rRNA gene illustrated that 13.5% (14/104) of the samples were positive. The obtained positive samples determined the nucleotide sequences of the 18S rRNA genes. In the genetic and phylogenetic analyses, four of five nucleotide sequences were similar to Babesia canis rossi, and one sample exhibited a close similarity to a Babesia sp. isolated from a raccoon in Hokkaido, Japan. The present study revealed the widespread presence of B. canis rossi among domestic dogs in Nigeria.

Molecular phylogeny of a newfound hantavirus in the Japanese shrew mole (Urotrichus talpoides).

Recent molecular evidence of genetically distinct hantaviruses in shrews, captured in widely separated geographical regions, corroborates decades-old reports of hantavirus antigens in shrew tissues. Apart from challenging the conventional view that rodents are the principal reservoir hosts, the recently identified soricid-borne hantaviruses raise the possibility that other soricomorphs, notably talpids, similarly harbor hantaviruses. In analyzing RNA extracts from lung tissues of the Japanese shrew mole (Urotrichus talpoides), captured in Japan between February and April 2008, a hantavirus genome, designated Asama virus (ASAV), was detected by RT-PCR. Pairwise alignment and comparison of the S-, M-, and L-segment nucleotide and amino acid sequences indicated that ASAV was genetically more similar to hantaviruses harbored by shrews than by rodents. However, the predicted secondary structure of the ASAV nucleocapsid protein was similar to that of rodent- and shrew-borne hantaviruses, exhibiting the same coiled-coil helix at the amino terminus. Phylogenetic analyses, using the maximum-likelihood method and other algorithms, consistently placed ASAV with recently identified soricine shrew-borne hantaviruses, suggesting a possible host-switching event in the distant past. The discovery of a mole-borne hantavirus enlarges our concepts about the complex evolutionary history of hantaviruses.

Notocotylus ikutai n. sp. (Digenea: Notocotylidae) from lymnaeid snails and anatid birds in Hokkaido, Japan.

An unknown species of the genus Notocotylus (Digenea: Notocotylidae) was found as the larval stage from the lymnaeid snail, Radix auricularia, in a static water area of the Chubetsu River, Hokkaido, the northernmost island of Japan. A DNA barcoding identification system was applied to detect the adult stage. Through the inspection of anatid game birds in Hokkaido, Anas crecca, Anas platyrhynchos, Anas zonorhyncha, and Mareca penelope were demonstrated to serve as the definitive hosts. The detailed morphological features of the species were characterized using adults raised experimentally in immunosuppressed mice and naturally developed larvae in R. auricularia. Although the species is morphologically similar to Notocotylus attenuatus and Notocotylus magniovatus in both adult and larval stages, its taxonomic independence was confirmed by a comprehensive study based on molecular phylogeny, morphology, and ecology. Here we propose Notocotylus ikutai n. sp. for this species. The migratory behavior of the anatid hosts and the North-Eurasian distribution of R. auricularia suggest that the new species is widely distributed in the northern Far East.

Clistobothrium sp. (Cestoda: Tetraphyllidea) in oarfish (Regalecus russelii) stranded on the coast of Akita Prefecture, Japan.

Oarfish (Regalecus russelii Cuvier) are mesopelagic fish with little known about their life history. Oarfish live in deep water, making it difficult for researchers to collect specimens; thus, records of their parasitic helminths are limited. Two plerocercoids were found for the first time in an oarfish stranded on the coast of Akita Prefecture, Japan. These plerocercoids were identified as Clistobothrium sp. RR-1 using morphological and molecular analyses. It was revealed that oarfish represent one of the intermediate hosts of the genus Clistobothrium, and large sharks are the definitive hosts for these parasites.

Epidemiologic Survey of Avian Influenza Virus Infection in Shorebirds Captured in Hokkaido, Japan.

There is limited information about virus epidemiology of shorebirds (family Charadriidae and Scolopacidae) in the East Asia-Australasia flyway. We investigated the prevalence of avian influenza viruses (AIVs) in shorebirds in Hokkaido, Japan, the stopover site of the flyway, to understand the ecology of AIV translocation in the flyway from 2006 to 2010. In total, 1,698 shorebirds belonging to 26 species were captured and released into two different sites using mist nets. Tracheal and cloacal swabs were collected from each bird using cotton swabs. The RNA of influenza A viruses was detected using reverse transcription loop-mediated isothermal amplification. One AIV-positive sample was obtained from a Lesser Sand Plover (Charadrius mongolus) captured in September 2010 at Lake Komuke. Full lengths of hemagglutinin (HA), neuraminidase (NA), polymerase acidic protein, nucleoprotein, matrix protein 1, and nuclear export protein genes were successfully amplified from the AIV-positive sample. All sequences showed the highest identity with sequences obtained from virus strains from Anseriformes species. Shorebirds migrated to Japan 1 mo earlier than did Anseriformes species. Therefore, the Lesser Sand Plover could have been infected by the virus from Anseriformes species on the breeding grounds. The HA sequence showed the highest identity with the H10 sequence whereas the NA sequence exhibited the highest identity with the N7 sequence. Phylogenic analysis showed that the detected subtype H10N7 belongs to the Eurasia lineage and the related strain might have widely spread in Asia in 2009.

Preliminary studies on developing a nested PCR assay for molecular diagnosis and identification of nematode (Heterakis isolonche) and trematode (Glaphyrostomum sp.) in Okinawa rail (Gallirallus okinawae).

To investigate and detect internal parasites in Okinawa rail (Gallirallus okinawae), a novel nested polymerase chain reaction with high level of sensitivity and specificity was developed. Specific PCR primers that target partial DNA sequences of mitochondrial Cytochrome C oxidase subunit 1 (cox1) gene were designed to differentiate between nematode (Heterakis isolonche) and trematode (Glaphyrostomum sp.) found in the intestine of Okinawa rail. Nested PCR assay was established based on primers design and applied to seven fecal samples from road killed birds containing nematode and/or trematode eggs. The results indicated this new nested PCR protocol was available for the identification of parasites and of value to clinical application.

Molecular evidence for the presence of new Babesia species in feral raccoons (Procyon lotor) in Hokkaido, Japan.

We recently reported that feral raccoons (Procyon lotor) with splenomegaly native to Japan were carriers of a Babesia microti-like parasite identical to that found in the United States, which was likely introduced to Japan from North America via raccoons imported as pets. Thus, we attempted extensive molecular survey for piroplasma infections of feral raccoon with normal spleen in Hokkaido, Japan using nested PCR that target broadly to 18S ribosomal RNA gene (SSU-rDNA) of all the parasites in the genus Babesia, Theileria, Cytauxzoon and B. microti group. Of the 348 raccoon samples analyzed, 9 gave positive signals. Cloning and phylogenetic analysis on SSU-rDNA sequences revealed that six of nine positives were found to be infected with Babesia and the remaining three with previously unreported Sarcocystis. Babesia sequences were further separated into two distantly related groups, those that reside in a novel phylogenetic group were consisted solely of four parasites found in this study, while those which included one identical sequence found in the three of our specimens were assembled together with both Babesia parasites of tick's in Japan and of raccoon's in U.S. These results may indicate that not only a B. microti-like parasite but also at least two yet undescribed Babesia species are being established in their new life cycles in the feral raccoon populations in Japan.

Borna disease virus in Raccoons (Procyon lotor) in Japan.

We have examined the seroprevalence of BDV in wild Raccoons (Procyon lotor) in Hokkaido, Japan. Serum samples from raccoons were examined using ELISA and Western blot assays to detect the presence of serum antibodies that react specifically to BDV antigens. Among 549 investigated individuals, eleven (2.0%) showed a positive reaction to BDV antigens. Brain tissue samples from five individuals were subjected to RT-PCR, which detected BDV sequences in three of them. Sequence analysis revealed a high degree of genetic conservation between BDV sequences derived from raccoons and previously published sequences derived from other animal species.

Detection of avian haemosporidia from captive musophagid birds at a zoological garden in Japan.

One captive musophagid bird at a zoological garden in Japan showed clinical symptoms and was found to be infected with avian haemosporidia. We subsequently collected blood from all musophagid birds kept in the garden and examined for avian haemosporidia using both microscopic and molecular examination. Only Haemoproteus gametocytes were observed in the blood of two Guinea turaco (Tauraco persa). Three genetic lineages of Haemoproteus were identified from three Guinea turacos and one genetic lineage of Leucocytozoon was identified from a grey plantain-eater (Crinifer piscator). Detected Haemoproteus lineages were all identical and completely different from those previously reported in Japan, suggesting that these birds were infected in their original habitat. This is the first record of Haemoproteus infection in Guinea turacos.

Comparison of the intestinal helminth community of the large Japanese field mouse (Apodemus speciosus) between urban, rural, and natural sites in Hokkaido, Japan.

Anthropogenic ecosystem modification has affected over 80% of the global land cover. Interest in its effects on wildlife has been growing over the past several decades, specifically in regard to biodiversity and ecosystem function. Parasites are of particular interest, because they directly impact animal health, and can be transmitted to humans through the process of zoonosis. However, most studies so far tended to focus on only one or two parasites with few looking at the entire community, thereby limiting our understanding of the effects of ecosystem modification on parasitic organisms. In this study, we estimated the intestinal helminth diversity and species richness of the large Japanese field mouse (Apodemus speciosus), as well as the prevalence and abundance of each species in two modified ecosystems, a rural agricultural area and an urban park. We then compared them to a natural area to see how they have been altered. We found that diversity, prevalence, and abundance were all highly altered within both modified ecosystems, but generally to a greater degree within the urban park. However, there was great variation in the direction and degree of response of each helminth species, suggesting that generalized trends may be difficult to ascertain. Furthermore, it is important to analyze the entire helminth community, because interspecific interactions and the effect that ecosystem modification has on them may help determine what species persist.

Distribution records of three species of Leucochloridium (Trematoda: Leucochloridiidae) in Japan, with comments on their microtaxonomy and ecology.

Insectivorous birds serve as definitive hosts for trematodes of the genus Leucochloridium. The parasites exclusively use amber snails of the family Succineidae as intermediate hosts. A pulsating and colorful display of the larval broodsac in the snail's eyestalk seems to be a caterpillar mimic for attracting birds. A colored design of the broodsac is very useful for parasite identification. In Japan, characteristic broodsacs from amber snails have been recorded from 1980's, but their taxonomic discrimination from Asian, European, and North American species has not been achieved. In this study, old scientific records, sighting information on broodsacs from the general public, and direct molecular evidence by DNA barcoding clearly showed that at least three species of Leucochloridium are distributed in Japan. A vertical-striped broodsac found from Succinea sp. in Okinawa, the subtropical island of Japan, were treated as Leucochloridium sp., but being almost identical to that of Leucochloridium passeri in neighboring Taiwan. The European species of Leucochloridium perturbatum and Leucochloridium paradoxum were frequently detected from Succinea lauta in Hokkaido, the northernmost island of Japan. The former species was common in inland areas of Hokkaido, whereas the latter species was frequently seen in the coastal areas. A possible explanation for the parasite distribution pattern is that principal definitive hosts (migratory or resident birds) differ in each parasite. The conspecificity of Leucochloridium variae in North America and L. perturbatum in Europe and the Far East is also discussed.

Serological survey of Toxoplasma gondii in wild waterfowl in Chukotka, Kamchatka, Russia and Hokkaido, Japan.

Antibodies to Toxoplasma gondii were assayed by ELISA in 22 experimentally inoculated domestic ducks. In addition, a serological assay was carried out at Obihiro, Hokkaido, Japan, in 2004 and 2005, on 221 wild ducks of 5 species: Anas platyrhynchus (n = 111); A. poecilorhyncha (n = 27); A. acuta (n = 58); A. penelope (n = 16); and A. crecca (n = 9). Assays were also conducted using sera from 197 wild geese of 2 species, i.e., Anser albifrons (n = 162) and Ans. fabalis (n = 35). Birds were collected between 2003 and 2005 from 3 different areas: Lake Miyajima-numa, Hokkaido, Japan, regions around Anadyr city of Chukotka autonomous okrug, and Lake Makobetukoe, Kamchatka oblast, Russia. The ELISA cutoff value (OD) was > or =0.395 based on results from uninfected ducks; the final dilution ratio recognized as positive was represented by the end titer. The end titer in the experimentally infected ducks ranged from 1:400 to 1:3,200. Antibodies to T. gondii were found in 49 of the 221 wild duck samples from Japan: A. platyrhynchus (22/74); A. poecilorhyncha (2/15); A. penelope (3/16); A. acuta (4/58); and A. crecca (0/9), all in 2004. In 2005, T. gondii was found in A. platyrhynchus (13/37); and A. poecilorhyncha (5/12). Thirty-two of 197 wild goose samples were seropositive, i.e., Ans. albifrons (7/51) in 2004 and (11/72) in 2005 in Miyajima-numa, Japan and 9/39 in Chukotka, Russia as well as in Ans. fabalis (5/35) in Kamchatka, for which the end titer ranged from 1:100 to 1:3,200. In immunoblotting, the A. platyrhynchus samples showed specific IgG antibody binding to several antigens in the T. gondii lane, i.e., at 30 and 43 kDa, but not in the Neospora caninum lane. No specific bands were noted in samples for which antibody activity was not detected. These results suggest that wild waterfowl inhabiting Hokkaido, Chukotka, and Kamchatka may be exposed to T. gondii.

Borna disease virus RNA detected in Japanese macaques (Macaca fuscata).

We have examined the seroprevalence of BDV in wild Japanese macaques (Macaca fuscata) in the peninsula (Chiba prefecture), Japan. Serum samples from macaques were examined by the ELISA, Western blot and immunofluorescence assays to detect the presence of serum antibodies that react specifically to BDV antigens. Among 49 investigated individuals, 6 (12.2%) showed positive reaction to BDV antigens. RT-PCR studies detected BDV sequences in brain tissue of one case among four seropositive cases examined. Sequence analysis revealed a high degree of genetic conservation between BDV sequences derived from Japanese macaques and those documented for other animal species. Nevertheless, phylogenetic analysis revealed unique differences between macaque and other species derived BDV sequences.

Redescription of Subulura (Murisubulura) andersoni (Cobbold, 1876) (Nematoda: Subuluridae) from Bunomys spp. (Rodentia: Muridae) of Sulawesi, Indonesia, with special reference to S. (M.) suzukii and other related species in the adjacent areas.

Subulura (Murisubulura) andersoni (Cobbold, 1876) (Nematoda: Subuluridae) is redescribed in detail using light and scanning electron microscopy based on materials collected from Bunomys chrysocomus and Bunomys andrewsi (Rodentia: Muridae) of Sulawesi, Indonesia, to provide basic reference data for identification. Comparison was made among congeners from adjacent areas, especially Subulura (Murisubulura) suzukii Yagi and Kamiya, 1981 of Japan. Because morphological differences distinguishing S. (M.) andersoni from S. (M.) suzukii and S. (M.) sipiroki were only the thickness of female tail and length of male tail, respectively, further study using DNA sequence analysis is necessary to prove their distinctness.

Geographical distribution and seasonality of the prevalence of Leucocytozoon lovati in Japanese rock ptarmigans (Lagopus mutus japonicus) found in the alpine regions of Japan.

In this study, we investigated the geographical distribution and seasonality of Leucocytozoon lovati infection in the Japanese rock ptarmigan (Lagopus mutus japonicus); this bird is one of the special natural monuments of Japan that inhabits the Japanese alpine regions. We examined blood samples from birds captured in the Kubiki, Hida, and Akaishi mountain ranges for three years from 2002 to 2005. Seventy-three blood samples from 42 males, 19 females, and 12 birds of unknown sex were used for this study. The rate of infection with L. lovati was 78.1% in the 73 birds examined. We demonstrated that the L. lovati infection was distributed across wide ranges of ptarmigan populations from the northern to the southern alpine zones. There was no sex bias in the prevalence ratio. The prevalence of L. lovati and the level of parasitization of the blood cells tended to increase from spring through summer; in contrast, a decrease was observed from summer through autumn. Although L. lovati infection was observed in a number of local populations inhabiting three mountainous regions, no infected birds were found in Mt. Johnen-dake and Mt. Maejohnen-dake. It is necessary to continue surveying the relationship between the population dynamics of the ptarmigan and the density of the arthropod vector from the perspective of in situ conservation of this endangered species.

Stress assessment using hair cortisol of kangaroos affected by the lumpy jaw disease.

The aim of this study was to objectively assess stress of kangaroos affected by lumpy jaw disease (LJD) using plasma and hair cortisol concentrations. The plasma and hair samples were collected from kangaroos with LJD and healthy controls. Collected hair samples were extracted with methanol after washing with isopropanol, following which they were processed with the cortisol enzyme immunoassay kit. The plasma cortisol concentration of LJD animals tended to be higher than that of the control. Ventral hair cortisol, but not dorsal hair, of LJD animals was significantly higher than that of the control. In conclusion, stress in kangaroos infected with LJD could be assessed by measuring ventral hair cortisol.

Characterizing the temporal patterns of avian influenza virus introduction into Japan by migratory birds.

The objectives of the present study were to observe the temporal pattern of avian influenza virus (AIV) introduction into Japan and to determine which migratory birds play an important role in introducing AIV. In total, 19,407 fecal samples from migratory birds were collected at 52 sites between October 2008 and May 2015. Total nucleic acids extracted from the fecal samples were subjected to reverse transcription loop-mediated isothermal amplification to detect viral RNA. Species identification of host migratory birds was conducted by DNA barcoding for positive fecal samples. The total number of positive samples was 352 (prevalence, 1.8%). The highest prevalence was observed in autumn migration, and a decrease in prevalence was observed. During autumn migration, central to southern Japan showed a prevalence higher than the overall prevalence. Thus, the main AIV entry routes may involve crossing the Sea of Japan and entry through the Korean Peninsula. Species identification was successful in 221 of the 352 positive samples. Two major species sequences were identified: the Mallard/Eastern Spot-billed duck group (115 samples; 52.0%) and the Northern pintail (61 samples; 27.6%). To gain a better understanding of the ecology of AIV in Japan and the introduction pattern of highly pathogenic avian influenza viruses, information regarding AIV prevalence by species, the prevalence of hatch-year migratory birds, migration patterns and viral subtypes in fecal samples using egg inoculation and molecular-based methods in combination is required.