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Avian malaria is a mosquito-borne disease of birds caused by avian Plasmodium spp. in worldwide scale. Some naïve birds show serious symptoms which can result in death. Surveillance of vectors and parasites are important to understand and control this disease. Although avian malaria has been found in Japan, detailed prevalence and dynamics remained understudied. We aimed to observe annual changes in the abundance of mosquitoes and the prevalence of avian Plasmodium parasites in Japan. Mosquitoes were collected using dry ice traps over a 10-year period, at a fixed research area located in Kanagawa prefecture. Collected mosquitoes were investigated for the species composition, population size and prevalence of avian Plasmodium by PCR. Mosquitoes belonging to 13 species in 7 genera were collected (n=8,965). The dominant species were Aedes (Ae.) albopictus and Culex (Cx.) pipiens group (gr.). Seven avian Plasmodium lineages, all of which were previously known, were detected from Cx. pipiens gr., Ae. albopictus, and Tripteroides bambusa. Three genetic lineages were dominant and were probably transmitted by Cx. pipiens gr. whose could be the primary vector of these parasites. Annual variations in the seasonal prevalence of mosquitoes and avian Plasmodium were revealed for the first time during recent 10 years in Japan. Namely, avian Plasmodium occurrence in the vector population peaked often in June to July and September to October when the density of the vector population was presumably high enough for the transmission of avian Plasmodium upon appearance of infected birds.

Widespread surveys of avian haemosporidia (Plasmodium, Haemoproteus, and Leucocytozoon) in wild birds have substantially advanced information on the haemosporidian fauna of Japan. However, many areas and bird species remain insufficiently investigated. Bird carcasses collected for personal specimen collection seldom reach academic audience particularly in the veterinary field. The presence of avian haemosporidia was investigated in these personally collected bird carcasses, in order to better understand the avian haemosporidian fauna in Japan. Bird carcasses were donated through personal contact upon approval of the study. Tissue samples were collected from the birds and examined for haemosporidian parasites using nested-PCR targeting the cytochrome b gene. One hundred and forty-three birds of 85 species were donated, including 34 species and two subspecies that were molecularly or collectively investigated for the first time in Japan. Avian haemosporidian DNA was detected from 37 of the 134 tested birds (27.61%). In 8 bird species, avian haemosporidia was detected for the first time. Twenty-nine lineages were detected, including 8 novel and 9 known lineages detected in Japan for the first time. Furthermore, 16 lineages were detected from novel host species. While information that could be drawn was limited and risk management of zoonotic diseases needs re-consideration, these findings expanded information on the host range and distribution of several lineages. Collectively, this method of investigation using personally collected bird samples can provide important additions to more fully understand the avian haemosporidian fauna of Japan, as well as other areas with limited investigations.

Parasites of the genus Haemoproteus are vector-borne avian haemosporidia commonly found in bird species of the world. Haemoproteus infections are typically considered relatively benign in birds. However, some Haemoproteus species cause severe disease and mortality, especially for captive birds removed from their original habitat. In September 2018, a captive 15-year-old snowy owl (Bubo scandiacus), kept in a zoological garden of Japan, died subacutely after presenting leg dysfunction. This case showed significantly low PCV and elevated AST, ALT, CK, and LDH values. Many megalomeronts with prominent morphological characteristics of Haemoproteus were observed in the left leg muscles. Those megalomeronts exhibited multilocular structures and were internally filled with merozoites. A new lineage of Haemoproteus was detected by subsequent PCR for the cytochrome b (cytb) gene of avian haemosporidia from DNA extracted from several organ tissues. The detected lineage was classified in the subgenus Parahaemoproteus and was similar to those from the wild birds inhabiting the region including the study area, suggesting that this snowy owl likely acquired its infection from wild birds. This is the first report of a fatal case of a captive bird with a locally transmitted Haemoproteus infection in Japan. We considered the pathogenicity of this infection in conjunction with the clinical course and hematology results. We surmise that snowy owls may be particularly susceptible to infection with Haemoproteus parasites, and warming northern temperatures may exacerbate the overall health of these and other high latitude birds. Further research into the prevalence of Haemoproteus in wild birds near zoological gardens and potential biting midge vectors is necessary for the ex situ conservation of introduced birds.

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.

Aim: Macroecological analyses provide valuable insights into factors that influence how parasites are distributed across space and among hosts. Amid large uncertainties that arise when generalizing from local and regional findings, hierarchical approaches applied to global datasets are required to determine whether drivers of parasite infection patterns vary across scales. We assessed global patterns of haemosporidian infections across a broad diversity of avian host clades and zoogeographical realms to depict hotspots of prevalence and to identify possible underlying drivers. Location: Global. Time period: 1994–2019. Major taxa studied: Avian haemosporidian parasites (genera Plasmodium, Haemoproteus, Leucocytozoon and Parahaemoproteus). Methods: We amalgamated infection data from 53,669 individual birds representing 2,445 species world-wide. Spatio-phylogenetic hierarchical Bayesian models were built to disentangle potential landscape, climatic and biotic drivers of infection probability while accounting for spatial context and avian host phylogenetic relationships. Results: Idiosyncratic responses of the three most common haemosporidian genera to climate, habitat, host relatedness and host ecological traits indicated marked variation in host infection rates from local to global scales. Notably, host ecological drivers, such as migration distance for Plasmodium and Parahaemoproteus, exhibited predominantly varying or even opposite effects on infection rates across regions, whereas climatic effects on infection rates were more consistent across realms. Moreover, infections in some low-prevalence realms were disproportionately concentrated in a few local hotspots, suggesting that regional-scale variation in habitat and microclimate might influence transmission, in addition to global drivers. Main conclusions: Our hierarchical global analysis supports regional-scale findings showing the synergistic effects of landscape, climate and host ecological traits on parasite transmission for a cosmopolitan and diverse group of avian parasites. Our results underscore the need to account for such interactions, in addition to possible variation in drivers across regions, to produce the robust inference required to predict changes in infection risk under future scenarios.

Background Captive populations of penguins outside of their natural distributions are often maintained in outdoor facilities, such as zoos and aquariums. Consequently, such penguins in captivity are constantly exposed to mosquito vectors and risk of avian malarial infection during their active period from spring to autumn, which can be lethal to these naïve birds. Previous studies have investigated parasite prevalence in mosquitoes or penguins, but simultaneous investigations, which would be crucial to monitor the transmission dynamics and cycle within a facility, have not been done. To identify dominant lineages and trends, multiple-year surveys are recommended. Methods Avian malaria parasites (Plasmodium spp.) and related haemosporidia were tested in penguins and mosquitoes at an aquarium in Japan through multiple years from 2011 to 2018. Prevalence and dynamics were confirmed, and molecular analyses targeting the protozoal cytb gene were used to reveal the transmission cycle. Blood meals of mosquitoes were also identified using molecular methods. Results Parasite detection in penguins tended to fluctuate within an individual. Two Plasmodium lineages were consistently detected in mosquitoes that had fed on penguins and wild birds observed around the aquarium. Plasmodium lineage CXPIP09 was detected from both mosquitoes and penguins, suggesting active transmission at this facility. However, Plasmodium cathemerium PADOM02 was only detected in mosquitoes, which may be due to host, vector or parasite-related factors, or detection methods and their limits. Additionally, Haemoproteus larae SPMAG12 was detected from penguins, suggesting active transmission via biting midges. Conclusions The mismatch in parasite composition between penguins and mosquitoes shows that multiple aspects such as captive birds, wild birds and vector insects should be monitored in order to better understand and control avian malarial infection within ex-situ conservation facilities. Furthermore, morphological analyses would be needed to confirm competency and infection dynamics of avian malaria parasites.

Additional file 1: Table S1. Information on Plasmodium spp. samples used to prepare mixed-infections.

A 1-month-old brown wood owlet (Strix leptogrammica) purchased from a wholesaler and housed as a companion bird by an individual owner in Japan showed severe dehydration and anorexia following a week of vomiting and severe diarrhea. A great number of approximately 5 × 4-μm-sized Cryptosporidium oocysts were found in the feces by microscopy. The owlet was administered subcutaneous fluid and intragastric tube feeding for 2 weeks, resulting in improvement of the condition with a decreased number of oocysts in the feces. At days 51 and 119, no oocysts were found in the feces by microscope and PCR detection. These results suggested that this parasite was a possible agent of severe diarrhea in the affected bird. Molecular analysis of DNA extracted from oocysts based on the 18SrRNA loci identified C. avium; however, analysis of actin and hsp (heat shock protein) genes identified a novel genotype indicating a mixed infection with C. avium and a novel genotype.

Avian haemosporidia have been reported in various birds of Japan, which is part of the East Asian-Australian flyway and is an important stopover site for migratory birds potentially carrying new pathogens from other areas. We investigated the prevalence of avian malaria in injured wild birds, rescued in Tokyo and surrounding areas. We also evaluated the effects of migration by examining the prevalence of avian malaria for each migratory status. 475 birds of 80 species were sampled from four facilities. All samples were examined for haemosporidian infection via nested polymerase chain reaction (PCR) of the cytochrome b (cytb) gene. 100 birds (21.1%) of 43 species were PCR positive for avian haemosporidia. Prevalence in wintering birds, migratory breeders, and resident birds was 46.0%, 19.3%, 17.3% respectively. There was a bias in wintering birds due to Eurasian coot (Fulica atra) and Anseriformes. In wintering birds, lineages which are likely to be transmitted by Culiseta sp. in Northern Japan and lineages from resident species of Northern Japan or continental Asia were found, suggesting that wintering birds are mainly infected at their breeding sites. Meanwhile, there were numerous lineages found from resident and migratory breeders, suggesting that they are transmitted in Japan, some possibly unique to Japan. Although there are limits in studying rescued birds, rehabilitation facilities make sampling of difficult-to-catch migratory species possible and also allow for long-term monitoring within areas., Graphical abstract Image 1, Highlights • Prevalence of avian malaria in rescued wild birds in Japan has been demonstrated. • Many new lineages have been identified, including possibly unique to Japan. • Rehabilitation facilities allow sampling and monitoring of wild birds possible.

Background Haemosporidian parasites of the genera Plasmodium and Haemoproteus can have detrimental effects on individual birds and populations. Despite recent investigations into the distribution and richness of these parasites and their vertebrate hosts, little is known about their dipteran vectors. The Neotropics has the highest diversity of mosquitoes in the world, but few studies have tried to identify vectors in this area, hampering the understanding of the ecology of avian malaria in the highly diverse Neotropical environments. Methods Shannon traps and active collection were used to capture 27,110 mosquitoes in a Seasonally Dry Tropical Forest in southeastern Brazil, a highly endangered ecosystem. Results We screened 17,619 mosquito abdomens from 12 different species and several unidentified specimens of Culex, grouped into 1,913 pools, for the presence of haemosporidians. Two pools (out of 459) of the mosquito Mansonia titillans and one pool (out of 29) of Mansonia pseudotitillans were positive for Plasmodium parasites, with the detection of a new parasite lineage in the former species. Detected Plasmodium lineages were distributed in three different clades within the phylogenetic tree revealing that Mansonia mosquitoes are potential vectors of genetically distant parasites. Two pools of Culex spp. (out of 43) were positive for Plasmodium gallinaceum and closely related lineages. We found a higher abundance of these putative vectors in pasture areas, but they were also distributed in areas at intermediate and late successional stages. One pool of the mosquito Psorophora discrucians (out of 173) was positive for Haemoproteus. Conclusions The occurrence of different Plasmodium lineages in Mansonia mosquitoes indicates that this genus encompasses potential vectors of avian malaria parasites in Brazil, even though we did not find positive thoraces among the samples tested. Additional evidence is required to assign the role of Mansonia mosquitoes in avian malaria transmission and further studies will add information about evolutionary and ecological aspects of avian haemosporidia and untangle the diversity of their vectors in Brazil.

The heartworm Dirofilaria immitis is the causative agent of dirofilariasis in dogs. Studies have shown that parasite-derived cell-free DNA (cfDNA) can be detected in host blood and may be a promising diagnostic marker for parasitic infections. Thus, our aim was to detect D. immitis-derived cfDNA in host serum by nested PCR. Sera were collected from 12 dogs with natural D. immitis infections; eight were microfilaria (mf)-positive, and the remaining four were mf-negative. Culture fluids derived from single-sex adult D. immitis worms (mf-producing females and males) were also tested for cfDNA. All mf-positive sera were positive by nested PCR, whereas no amplification products were detected in mf-negative sera. The culture fluid of mf-producing females was positive by nested PCR but that of males was negative. All products amplified by nested PCR were sequenced to confirm that the amplicons were those of D. immitis. These results indicate that D. immitis DNA circulates freely in dog serum, except in mf-negative dogs. Additionally, D. immitis cfDNA may primarily be derived from the mf, and adult worms appeared to be minor contributors of cfDNA concentrations in serum; however, the contribution of D. immitis cfDNA derived from larvae of other developmental stages is unclear. An evaluation of the kinetics of D. immitis cfDNA in host serum throughout the parasite life cycle could facilitate the development of early molecular diagnostic techniques. To the best of our knowledge, this is the first report of the detection of mitochondrial DNA from a filarial parasite in host serum.

An orbivirus was isolated from a sample from the ornithophilic mosquito Culex sasai in Japan. The virus, designated Koyama Hill virus (KHV), replicated to high titer in a mosquito cell line and to a low titer in an avian cell line, but the release of progeny viruses was not observed in mammalian cell lines inoculated with KHV. Electron microscopic examination of KHV-infected mosquito cells showed approximately 70-nm virus particles and viral tubules typical of members of the genus Orbivirus, family Reoviridae. KHV efficiently replicated in Cx. sasai mosquitoes, suggesting a potential vector species for KHV transmission in nature. Full-length viral genome sequencing and phylogenetic analysis revealed that KHV is closely related to Umatilla virus (UMAV) and Stretch Lagoon orbivirus (SLOV). This suggests that KHV is a new member of the species Umatilla virus, an orbivirus species not previously observed in East Asia. The KHV genome segment encoding NS1 contains a notable sequence deletion and heterogeneity compared with a prototype UMAV, which may affect its growth properties and pathogenicity in host cells. These results provide new insights into the genetic diversity and geographic distribution of members of the species Umatilla virus.

We investigated for the first time the prevalence of avian haemosporidia of genera Plasmodium, Haemoproteus and Leucocytozoon among birds and mosquitoes on Tsushima Island of Japan, which is located between Japan and the Korean Peninsula. Of 55 wild birds belonging to 33 species, 16 (29.1%) tested positive for haemosporidia as follows: Plasmodium spp. (11/55; 20.0%); Haemoproteus spp. (2/55; 3.6%); and Leucocytozoon spp. (3/55; 5.5%). A genetic lineage isolated from the Eurasian Sparrowhawk (Accipiter nisus) was identical to that of the known avian malaria parasite P. circumflexum. Several genetic lineages were identical or closely related to the parasite lineages that were previously detected in birds and mosquitoes in Japan and Korea. Another single identical genetic lineage was also detected in both migratory and resident birds. A total of 753 mosquitoes from 12 species were collected; and one fully fed Aedes albopictus was positive for avian Plasmodium(1/753; 0.13%) which is identical to a genetic lineage detected in both mosquitoes in Japan and birds in Korea. Blood-meal identifications of blood-fed mosquitoes showed direct contact between the mosquitoes and 4 species of mammals including humans, cattle, rodents and the endangered Tsushima leopard cat (Prionailurus bengalensis euptilura). Migratory birds use Tsushima Island as a site for wintering, breeding and resting, and our results suggest the transmission of avian haematozoa between resident and migratory birds during their stay on Tsushima Island.

In mice, the number of intestinal villous columnar epithelium cells that incorporate abnormal prion protein (PrP(Sc) ) decreases significantly after weaning. In this study, the dynamics of PrP(Sc) uptake during the growth of hamsters were investigated by inoculating scrapie 263K agent orally into suckling and weanling Syrian hamsters and estimating the number of PrP(Sc) -positive villous epithelium cells immunohistochemically. The number of PrP(Sc) -positive cells declined significantly as the hamsters aged. The present results suggest that a tendency toward decline of PrP(Sc) -positive cells with increasing age might be a common phenomenon among the superfamily Muridae.

In Japan, the prevalence of avian Plasmodium in birds and mosquitoes has been partially examined in the temperate and subtropical zones; however, mosquitoes in the Japanese subarctic zone have not been adequately investigated. In this study, mosquito collections and avian Plasmodium detections from the mosquito samples were carried out to demonstrate the avian Plasmodium transmission between vector mosquitoes and birds inhabiting in Kushiro Wetland, subarctic zone of Japan. A total of 5,657 unfed mosquitoes from 18 species and 320 blood-fed mosquitoes from eight species was collected in summer 2008, 2009, and 2010. Three Aedes esoensis that fed on Hokkaido Sika Deer and one unfed Culex pipiens group were found to be positive for avian Plasmodium by polymerase chain reaction. This is the first report of the detection of avian Plasmodium DNA from mosquitoes distributing in the subarctic zone of Japan. The blood meals were successfully identified to captive or wild animals, including seven mammalian species, four bird species, and one amphibian species. These results indicated that infected birds with avian Plasmodium inhabited and direct contacts occurred between the infected birds and mosquitoes in Kushiro Wetland, Hokkaido, Japan.

蚊媒介性感染症のベクターとなる蚊の生息状況解明を目的として，2005年5月から5か月間，横浜市立よこはま動物園内においてドライアイストラップ，グラビッドトラップおよびスウィーピング法を用いた捕集調査を行った。その結果，アカイエカ種群蚊（Culex pipiens group），ヒトスジシマカ（Aedes albopictus），トラフカクイカ（Lutzia vorax）など計9属14種2,623個体が捕集された。アカイエカ種群蚊およびヒトスジシマカが捕集蚊全体の約85％を占め，これらの蚊が園内における優占種であると考えられた。神奈川県内で生息が確認されている蚊26種のうち53.8％にあたる種が捕集されたことから，本動物園およびその周辺地域は，各種蚊が選好する多様な環境で構成されていると考えられた。なお，捕集蚊の10.6％で吸血が認められ，動物園動物を吸血源としている可能性が示唆された。動物園動物の蚊媒介性感染症を制御し，希少種の生息域外保全を行うためには，蚊種の生態に応じた防除対策が必要である。

One of vector-borne avian protozoa, Leucocytozoon lovati, has been found in the Japanese rock ptarmigans (Lagopus mutus japonicus), the endangered bird species distributed in the alpine regions in Japan. Vector arthropod species of L. lovati has also been estimated as Simuliidae black flies distributed in the same habitat of the host bird, however, possible blood meals of the black flies were not identified yet. To reveal host animals of black flies, we estimated the blood resources by using molecular techniques. Black flies were collected at Mt. Chogatake, one of the alpine regions of Japan in which Japanese rock ptarmigans live in June 2005. The analyzed 144 specimens were morphologically identified into five species including Simulium japonicum (n = 87), Prosimulium hirtipes (n = 48), Prosimulium yezoense (n = 3), Twinnia japonensis (n = 3), and Cnephia mutata (n = 3). Individually extracted DNA from the black flies was subjected to polymerase chain reaction (PCR) amplification targeting the partial mitochondrial cytochrome b gene of birds or mammals to identify the blood meals. Of 144 black flies examined, 34 specimens were PCR positive for avian hosts (23.6%). No mammalian-derived bloods were detected from the samples studied through. Sequences amplified from 11 black flies consist of S. japonicum, P. hirtipes, and C. mutata showed high similarity to that of the Japanese rock ptarmigan. Therefore, present results conclusively suggest that these three species of black flies might suck the bloods of Japanese rock ptarmigans and could be the vector for L. lovati infection among this endangered bird species of Japan.

Several species of captive birds at zoological gardens of Japan were found to be infected with avian Plasmodium. However, incriminated vector mosquito species have not been identified yet. To indicate the competent vectors of avian malaria parasite, we collected mosquitoes at a zoological garden in Japan and examined for the avian malaria parasite DNA. Totally, 1,361 mosquitoes of 11 species were collected in the zoological garden of Kanagawa, the south of Tokyo in Japan in 2005. Captured mosquitoes were pooled by each species, date collected, and location and used for DNA extraction. Eight out of 169 DNA samples were positive for the nested PCR of avian Plasmodium cyt b gene. Estimated minimum infection rates of mosquitoes were 5.9 per 1,000. The PCR positive mosquito species were Culex pipiens group and Lutzia vorax. Some DNA sequences amplified from collected mosquitoes were identical to avian Plasmodium lineages detected from captive birds in the same zoological garden studied. Our results suggest that C. pipiens group and L. vorax could be incriminated vectors of avian malaria parasite transmitting in captive birds kept in the zoological garden in Japan.

Here, we describe for the first time the prevalence and genetic properties of Bartonella organisms in wild rodents in Japan. We captured 685 wild rodents throughout Japan (in 12 prefectures) and successfully isolated Bartonella organisms from 176 of the 685 rodents (isolation rate, 25.7%). Those Bartonella isolates were all obtained from the rodents captured in suburban areas (rate, 51.8%), but no organism was isolated from the animals captured in city areas. Sequence analysis of rpoB and gltA revealed that the Bartonella isolates obtained were classified into eight genetic groups, comprising isolates closely related to B. grahamii (A-I group), B. tribocorum and B. elizabethae (B-J group), B. tribocorum and B. rattimassiliensis (C-K group), B. rattimassiliensis (D-L group), B. phoceensis (F-N group), B. taylorii (G-O group), and probably two additional novel Bartonella species groups (E-M and H-P). B. grahamii , which is one of the potential causative agents of human neuroretinitis, was found to be predominant in Japanese rodents. In terms of the relationships between these Bartonella genetic groups and their rodent species, (i) the A-I, E-M, and H-P groups appear to be associated with Apodemus speciosus and Apodemus argenteus ; (ii) the C-K, D-L, and F-N groups are likely implicated in Rattus rattus ; (iii) the B-J group seems to be involved in Apodemus mice and R. rattus ; and (iv) the G-O group is probably associated with A. speciosus and Clethrionomys voles. Furthermore, dual infections with two different genetic groups of bartonellae were found in A. speciosus and R. rattus . These findings suggest that the rodent in Japan might serve as a reservoir of zoonotic Bartonella infection.

Several species of birds in Minami Daito Island, an oceanic island located in the far south from the main islands of Japan, were found to be infected with avian Plasmodium. However, no vector species of the avian malaria in this island have been revealed yet. To speculate potential vectors, we collected mosquitoes there and investigated using a PCR procedure whether the mosquitoes harbor avian malaria or not. Totally 1,264 mosquitoes including 9 species were collected during March 2006 to February 2007. The mosquitoes collected were stored every species, sampled date and location for DNA extraction. Fifteen out of 399 DNA samples showed positive for the partial mtDNA cytb gene of avian Plasmodium. Estimated minimum infection rate among collected mosquitoes was 1.2% in this study. Four species of mosquitoes; Aedes albopictus, Culex quinquefasciatus, Lutzia fuscanus and Mansonia sp. had avian Plasmodium gene sequences. Detected DNA sequences from A. albopictus and L. fuscanus were identical to an avian Plasmodium lineage detected in bull-headed shrike (Lanius bucephalus) captured in the island. Different sequences were detected from C. quinquefasciatus, which were corresponding to an avian Plasmodium from a sparrow (Passer montanus) and Plasmodium gallinaceum. Our results suggest that A. albopictus, Lutzia fuscanus, C. quinquefasciatus, and Mansonia sp. could be potential vectors of avian malaria in Minami Daito Island. This study was the first report of molecular detection of avian Plasmodium from mosquitoes in Japan.

The side effects of hyperbaric oxygen (HBO) treatment, such as oxidative stress and oxygen toxicity, have long been of interest. However, there are no comprehensive studies evaluating such toxic effects in diabetes mellitus (DM). The purpose of this study was to determine the effects of HBO on glucose homeostasis and histological changes in pancreatic beta-cells of experimentally induced diabetic rats. A total of 24 male Wistar rats were randomly divided into 4 groups: 1) Control group, no diabetic induction without HBO treatment; 2) HBO group, exposed to 100% oxygen at 2.8 ATA (atmosphere absolute) for 2 h once daily, for 7 days; 3) DM group, diabetes induced by streptozotocin (STZ) injection; and 4) DM + HBO group, received both STZ injection and HBO exposure. HBO treatment, with clinically recommended pressures and duration of therapy, was started on day 5 after STZ injection, when the blood glucose levels were significantly increased. After the last HBO treatment, the pancreatic tissues were immunostained to measure the areas of insulin immunoreactive beta-cells in the islets of Langerhans. The blood glucose increased significantly following exposure to HBO, with the highest levels achieved in rats, which had been treated with both HBO and diabetic induction. The area populated with insulin immunoreactive beta-cells decreased significantly following diabetic induction and/or HBO exposure, with the smallest area in DM + HBO group. Thus, HBO exposure enhanced the cytotoxic effect of STZ in the beta-cells of the pancreas. HBO should be cautiously employed in diabetic patients.

Tick DNA samples from cattle in Xinjiang Uygur Autonomous Region Area, China, were examined for Rickettsia infection by citrate synthase gene-based PCR and sequencing. Four positive samples were detected from Haemaphysalis danieli and high levels of similarity were found with recently detected 'Candidatus Rickettsia principis.'

Eight species of Japanese birds were found to be infected with Leucocytozoon species using microscopic analysis. We used PCR and sequence analysis of the mitochondrial cytochrome b gene (cyt b) to compare the genetic background among these detected protozoa species. In 20 individuals of 22 samples, a single amplified band was detected from 6 of 8 bird species; 9 Japanese rock ptarmigans (Lagopus mutus japonicus), 4 large-billed crows (Corvus macrorhynchos), 2 carrion crows (C. corone), 2 scops owls (Otus scops), 1 Japanese grosbeak (Eophona personata), and 2 brown-eared bulbuls (Hypsipetes amaurotis), respectively. Phylogenetic analysis based on the partial cyt b sequences revealed that all Leucocytozoon isolates in Japan closely grouped with other Leucocytozoon species previously reported in the literature. Among the Japanese isolates, the phylogenetic tree suggested that L. lovati from the Japanese rock ptarmigan may be basal to the parasites found in other bird species. Our study is the first to identify the molecular relationships among Leucocytozoon parasites in the avifauna 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.

Triploid, parthenogenetic forms of the lungfluke, Paragonimus westermani, occur in Japan, Korea and China. The origin(s) of triploidy has been debated over the years. Sequences of two regions in the mitochondrial DNA, i.e. partial lrRNA (16S), and a portion of the non-coding region, were obtained from natural populations of P. westermani. All triploid individuals (Japan, Korea, China) and a single tetraploid individual (China) had identical sequences in the 16S region studied. Some sequence variation was observed among diploids, with those from Taiwan being distinct from the remainder. Both neighbour joining and parsimony trees using the 16S region placed diploid individuals from southwestern Japan close to the triploids and the tetraploid. The fragment amplified from the mitochondrial non-coding region showed dimorphism. One form (type A) consisted of 239 bp comprising two identical tracts of 70 bp separated by a tract of 93 bp. The second form (Type B) consisted of only a single 70 bp tract. All diploid individuals from Taiwan, China and Korea possessed type A, while those from Japan were polymorphic; individuals from Oita and Hyogo had type B, those from Chiba had type A, but both types were found in Mie. On the other hand, all of the triploid individuals and two tetraploid individuals possessed type B. Both the form present in the non-coding region and the 16S sequence suggest an affinity between a south-eastern group of diploid populations in Japan and the triploid form. A possible mechanism responsible for the origin of the triploid is discussed.

Haemosporidian parasites infection among wild birds inhabiting Minami-daito Island was studied. Blood films from 183 birds representing 4 species of 4 families were examined microscopically. Avian haemosporidian parasites were detected in 3 species with an overall prevalence of 59.6%. None of the 30 Daito scops owls (Otus scops interpositus) examined were infected. Either Haemoproteus sp. or Plasmodium sp. infection was found in 14 of 31 (45.2%) Borodino islands white-eyes (Zosterops japonicus daitoensis). Plasmodium spp. were found in 94 of 102 (92.2%) bull-headed shrikes (Lanius bucephalus) and 1 of 20 (5%) tree sparrows (Passer montanus).

Experimental infection of larval Echinococcus multilocularis in the rodent brain was attempted to establish a murine model for cerebral alveolar echinococcosis. Balb/c mice and jirds were injected intracranially with 10% of a homogenated hydatid cyst mass. Small cystic larvae were observed macroscopically in the cranial cavity 1, 2 and 5 months post-infection in both mice and jirds. Some larval cysts from both rodents contained mature or immature protoscoleces. In mice, the laminated layer was found in the lateral ventricle 2 months post-infection but without protoscoleces. At five months post-infection, larger larval cysts were found in the cranial cavity of a mouse, which also demonstrated partial palsy of the legs. A laminated layer with mature protoscoleces was observed in the third ventricle and the mouse also harboured, in the left lung, a larval cyst containing protoscoleces surrounded by lymphocytes. Jirds were also found to be infected with metacestodes in the cranial cavity, but neither unusual behaviour nor establishment of cysts inside the brain was observed in jirds during the course of infection.

A 64-year-old woman with Lyme disease and manifesting facial nerve palsy had been bitten by a tick on the left frontal scalp 4 weeks previously. Erythema migrans appeared on the left forehead, accompanied by left facial paralysis. Nested polymerase chain reaction-restriction fragment length polymorphism analysis (nested PCR-RFLP) was performed on DNA extracted from a skin biopsy of the erythema on the left forehead. Borrelia flagellin gene DNA was detected and its RFLP pattern indicated that the organism was B. garinii, Five weeks later, B. garinii was isolated by conventional culture from the erythematous skin lesion, but not from the cerebrospinal fluid. After treatment with ceftriaxone intravenously for 10 days and oral administration of minocycline for 7 days, both the erythema and facial nerve palsy improved significantly. Nested PCR and culture taken after the lesion subsided, using skin samples obtained from a site adjacent to the original biopsy, were both negative. We suggest that nested PCR-RFLP analysis might be useful for the rapid diagnosis of Lyme disease and for evaluating therapy.

Fecal samples were collected from 64 Japanese rock ptarmigans (Lagopus mutus japonicus) at 13 locations in the alpine zone of Japan and examined for internal parasites. We found 2 morphologically different types of eimerian oocysts. Based on morphological characteristics, one was identified as Eimeria uekii, which had already been found from Japanese rock ptarmigans in 1981, and the other was likely a new type of Eimeria. The new type of eimerian oocysts required a longer sporulation time than E. uekii. The prevalence of both eimerian oocysts in Japanese rock ptarmigans was 62.5%, while that of E. uekii was 60.9% and the new type 29.7%. Mixed infection of both types was found in 28.1%.

The prevalence of Babesia (Theileria) equi and B. caballi infections in donkeys in western Xinjiang China was investigated. In total, 93 serum samples were randomly taken from donkeys in the Kashi and Ili areas, and examined for B. equi and B. caballi infections by enzyme-linked immunosorbent assays using recombinant antigens. Of the 93 samples, 9 (9.6%) and 36 (38.7%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 2 (2.2%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine babesiosis might be extensively prevalent in donkeys in western Xinjiang.

One-hundred and fifty-seven Borrelia isolated from adult ticks, Ixodes persulcatus, and wild rodents, Clethrionomys rufocanus and Apodemus peninsulae, in the far eastern part of Russia were characterized and identified by restriction fragment length polymorphism (RFLP) of the 5S-23S rRNA intergenic spacer. Some isolates showed unique RFLP patterns and were determined as Borrelia garinii on the basis of a sequence analysis of the intergenic spacer amplicon and reactivity with species-specific monoclonal antibodies (MAbs). 86.5 and 12.7% of the tick isolates, and 74.2 and 12.9% of the rodent isolates were determined as Borrelia garinii and Borrelia afzelii, respectively, but not Borrelia burgdorferi sensu stricto was detected. This finding is similar to the results obtained from Borrelia surveys of I. persulcatus and wild rodents in Hokkaido, Japan.

Based on a near-complete mitochondrial DNA sequence, the phylogenetic status of a lung fluke collected in the Philippines was evaluated. The lung fluke from Leyte Island, Philippines, resembles Paragonimus westermani morphologically and is sometimes regarded as a subspecies, P westermani filipinus. In this study, all mitochondrial genes of the Leyte form were sequenced : 12 subunits of mitochondrial enzymes, 2 ribosomal RNA genes and 21 transfer RNAs. The gene order is the same as that of the previously-published P. westermani from Korea. All genes are transcribed in the same direction. The sequence from Leyte was 88.8% identical to the previously-published sequence (accession No. AF219379). This is further evidence that the Philippine form should be regarded as specifically distinct from the form in East Asia (China, Japan and Korea). This conclusion is strengthened by the observation that the molluscan host of the Philippine form is of a different family from that of the East Asian form.

During a mosquito collection, a female of the pigeon louse fly, Pseudolynchia canariensis (Diptera: Hippoboscidae), was collected by a mosquito trap baited with dry ice in Ishigaki-jima, Yaeyama Islands, Japan. This is the 1st record of P. canariensis from Yaeyama Islands.