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.

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.

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.

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.

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.

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.

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.

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.

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.

Plasma lead, silicon and titanium concentrations are considerably higher in green sea turtle from the suburban coast than in those from the rural coast in Okinawa, Japan.

The purpose of this study was to compare the concentration of trace elements in the plasma of sea turtles that inhabited the suburban (Okinawa Main Island, n=8) and the rural coast (Yaeyama Island, n=57) in Okinawa, Japan. Particle induced X-ray emission allowed detection of 20 trace and major elements. The wild sea turtles in the suburban coast in Okinawa were found to have high concentrations of Pb, Si and Ti in the plasma when compared to the rural area but there were no significant changes in the Al, As and Hg concentrations. These results may help to suggest the status of some elements in a marine environment. Further, monitoring the plasma trace and major element status in sea turtles can be used as a bio-monitoring approach by which specific types of elements found here could indicate effects that are related to human activities.

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.

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.

Plasma endotoxin activity in Eastern grey kangaroos (Macropus giganteus) with lumpy jaw disease.

Progressive pyogranulomatous osteomyelitis involving the mandible or maxilla of captive macropods, referred to as "Lumpy jaw disease (LJD)", is one of the most significant causes of illness and death in captive macropods. The aim of the present study was to evaluate the relationship between the severity of LJD and plasma endotoxin activity in kangaroos. Plasma samples obtained from moderate (n=24) and severe LJD (n=12), and healthy kangaroos (n=46), were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. Plasma endotoxin activity was measured using the Limulus amebocyte lysate (LAL)-kinetic turbidimetric (KT) assay. Plasma endotoxin activity was higher in kangaroos with severe LJD (0.199 ± 0.157 EU/ml) than in those with moderate LJD (0.051 ± 0.012 EU/ml, P<0.001) and healthy controls (0.057 ± 0.028 EU/ml, P<0.001). Our results suggest that the severity of LJD in captive macropods may be related to the plasma endotoxin activity.

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.

Brachylaima ezohelicis sp. nov. (Trematoda: Brachylaimidae) found from the land snail Ezohelix gainesi, with a note of an unidentified Brachylaima species in Hokkaido, Japan.

In the Japanese Archipelago, Ezohelix gainesi, a member of bradybaenid land snails, is endemic mainly to the island of Hokkaido. During July to August of 2016, a survey to detect trematode infections from E. gainesi was carried out at a forest city park in Asahikawa, Hokkaido. Systemic infections of the snails with sporocysts containing short-tailed cercariae were found in 5.3% of 94 individuals examined. Furthermore, most of them (90.4%) harbored non-encysted metacercariae within their kidneys. A DNA sequence identification revealed that both of the sporocyst and the metacercaria belong to an unknown species of the family Brachylaimidae. The metacercariae showed a genetic diversity with 6 haplotypes of mitochondrial DNA (mtDNA) even in the limited sampling area. A definitive host of the unknown species could not be determined, although 34 field mice (Apodemus speciosus) and 21 voles (Myodes rufocanus) from the city park were examined for intestinal parasites. To examine the adult stage, the metacercariae were perorally administrated to mice, together with anti-inflammatory treatment with methylprednisolone. Fully matured adult worms were recovered from the intestinal ileum 8 and 14days postinfection. The gravid adults showed typical features of the genus Brachylaima. A morphological and biogeographical evaluation prompted us to propose Brachylaima ezohelicis sp. nov. for the parasite from E. gainesi. The autochthony of the first intermediate host and the spatial heterogeneity of mtDNA suggest that the new species found in the city park is not a recently expanded population of immigrant origin.

Plasma endotoxin activity in kangaroos with oral necrobacillosis (lumpy jaw disease) using an automated handheld testing system.

The aim of the present study was to evaluate the reliability and effectiveness of directly determining endotoxin activity in plasma samples from kangaroos with lumpy jaw disease (LJD, n=15) and healthy controls (n=12). Prior to the present study, the ability of the commercially available automated handheld portable test system (PTS(TM)) to detect endotoxin activity in kangaroo plasma was compared with that of the traditional LAL-kinetic turbidimetric (KT) assay. Plasma samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS(TM) was not significantly different from that of the traditional LAL-based assay. The data obtained using PTS(TM) correlated with those using KT (r(2)=0.963, P<0.001). These findings indicated that the PTS(TM) is applicable as a simplified system to assess endotoxin activity in macropods. In the present study, we demonstrated the diagnostic value of plasma endotoxin activity in kangaroos with systemic inflammation caused by oral necrobacillosis and identified plasma endotoxin activity as a sensitive marker of systemic inflammation in kangaroos with LJD. Based on ROC curves, we proposed a diagnostic cut-off point for endotoxin activity of >0.22 EU/ml for the identification of LJD. Our results indicate that the assessment of plasma endotoxin activity is a promising diagnostic tool for determining the outcome of LJD in captive macropods.

Syphacia (Syphacia) maxomyos sp. n. (Nematoda: Oxyuridae) from Maxomys spp. (Rodentia: Muridae) from Sulawesi and Sumatra, Indonesia.

The present report describes Syphacia (Syphacia) maxomyos sp. n. (Nematoda: Oxyuridae) from two species of spiny rats, Maxomys musschenbroekii from Sulawesi and M. whiteheadi from Sumatra. It is characterized by a cephalic plate extending laterally with dorsoventral constriction and stumpy eggs with an operculum rim reaching pole. It is readily distinguishable by the former feature from all of hitherto known representatives of this genus in Indonesia, but it resembles parasites in Murini and Hydromyni rodents in continental Asia and Sahul. This is the first Syphacia species distributed in both the Sunda Shelf and Sulawesi with the exception of Syphacia muris, a cosmopolitan pinworm found in rodents of the of genus Rattus. It is surmised that S. maxomyos is specific to Maxomys and that it was introduced to Sulawesi by dispersal of some Maxomys from the Sunda Shelf.

Musserakis sulawesiensis gen. et sp. n. (Nematoda: Heterakidae) collected from Echiothrix centrosa (Rodentia: Muridae), an old endemic rat of Sulawesi, Indonesia.

Musserakis sulawesiensis gen. et sp. n. (Nematoda: Heterakidae) is described from the large-bodied shrew rat, Echiothrix centrosa, one of the old endemic rats of Sulawesi, Indonesia. Musserakis is readily distinguished from other heterakid genera by having non-recurrent and non-anastomosing cephalic cordons, by lacking papillae between papillae groups around precloacal sucker and cloacal aperture and by lacking teeth in the pharyngeal portion. The spicules are equal but with marked dimorphism among individuals. Heterakids collected from other old endemic murids examined, i.e., Crunomys celebensis, Tateomys macrocercus and Tateomys rhinogradoides, and the new endemic rats of Sulawesi, were Heterakis spumosa Schneider, 1866, a cosmopolitan nematode of various murids. It is suggested that M. sulawesiensis is specific to Echiothrix.

Mass mortality of Eurasian Tree Sparrows (Passer montanus) from Salmonella Typhimurium dt40 in Japan, winter 2008-09.

An outbreak of salmonellosis in wild passerines caused mass mortality of Eurasian Tree Sparrows (Passer montanus) in Hokkaido, Japan, 2005-06; however, the etiology was poorly understood. In winter 2008-09, sparrow mortality again occurred in Hokkaido, and 202 deaths in 100 incidents at 94 sites were reported. We conducted a comprehensive investigation to evaluate the cause and impact on sparrow populations. We collected 26 carcasses at 13 sites, including a zoological park. In addition, Salmonella screening of zoo animals was conducted as a biosecurity measure. Salmonella Typhimurium was isolated from multiple organs in all examined sparrows; they were diagnosed with septicemic salmonellosis. Eleven sites (85%) were related to wild bird feeding and six of eight sparrow fecal samples, including from the zoo, were S. Typhimurium-positive. No infection was detected in zoo animals. Isolates belonged to three phage types: DT40 (88%), DT110 (8%), and DT120 (4%). Pulsed-field gel electrophoresis patterns were the same in all isolates, regardless of phage type. Biochemical characteristics and antibiotic-resistance profiles of DT40 were similar in all isolates, indicating a single origin. The mortality was likely associated with that in 2005-06 because the isolates had the same profiles. Tissue levels of sodium, calcium, and magnesium (the main components of chemical deicer suspected to be the major cause of poisoning deaths in 2005-06 mortality) were not higher in the affected sparrows. We conclude that an emerging epidemic infection with S. Typhimurium DT40 related to bird feeding was the cause of sparrow mortality in 2008-09 and suggest that this causative strain is host-adapted to sparrows in Japan. The mortality might have had some impact on the local population, but its influence was limited.

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.