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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.

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.

Aims Insulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice. Main methods To localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2 +/+ mice. IA-2 −/− mice served as a negative control. Key findings Western blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells. Significance The IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.

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.

Cellular prion protein (PrPC) is expressed not only in neuronal cells but also in non-neuronal cells such as astroglial cells. In the present study, the prion protein (PrP) gene (Prnp)-deficient astroglial cell line GpL1 from hippocampal cells of ZrchI Prnp−/− mice were established. Transfection of Prnp suppressed cell death in GpL1 cells under serum-free conditions. The PrP-expressing GpL1 cells showed increased superoxide dismutase activity compared to control GpL1 cells. These results suggest that the anti-oxidative activity of PrPC functions in not only neuronal cells but also astroglial cells possibly due to the increased anti-oxidative activity of astroglial cells.

The neuroprotective function of prion protein (PrP) was revealed first by the fact that reintroduction of the mouse prion protein gene (Prnp) into a mouse Prnp−/− neuronal cell line, HpL3-4, could prevent apoptosis induced by serum deprivation. In the present study, the anti-apoptotic activities of mouse, hamster, and bovine PrP were compared by expressing mouse PrP (MoPrP), hamster PrP (HaPrP), and bovine PrP (BoPrP) in HpL3-4 cells, respectively. Morphological analysis and DNA fragmentation assays demonstrated that HpL3-4 cells expressing HaPrP, BoPrP, and empty vector (EM) showed the typical features of apoptosis with DNA laddering and apoptotic bodies after serum deprivation, whereas HpL3-4 cells expressing MoPrP showed decreased levels of apoptosis in comparison. The levels of histone-associated DNA fragments (mono- and oligonucleosomes) in the cytosol fractions of the cells correlated with the levels of DNA laddering. These results indicate a species-specific anti-apoptotic function of PrP exists, suggesting that the interaction of the mouse PrP with mouse host factors is required for its anti-apoptotic activity.

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 canine immune system undergoes continuous remodeling with advancing age. We measured the Th1/Th2 balance in peripheral blood lymphocytes (PBLs) obtained from 23 Beagles ranging in age from 0.5 to 11.8 years by flow cytometric analysis using intracellular cytokine staining. The percentage of CD4 cells producing interferon-gamma (Th1) increased with age. The percentage of CD4 cells producing interleukin-4 (Th2) increased but much less so. In conclusion, we demonstrated that the Th1/Th2 balance in canine peripheral blood could be measured by this flow cytometry technique and the Th1/Th2 balance inclined to dominance of the Th1 subpopulation in PBLs as the dog matured.

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.

This study was conducted to induce immunotolerance to human chorionic gonadotropin (hCG) as a heterologous protein in neonatal mice. Ovulatory responses after repeated induction of superovulation were also investigated in the animals. Subcutaneous injection of hCG (100 IU) was performed in female mice within 24 hours after birth. Serum anti-hCG antibody titers were assayed and ovulatory responses were observed after the repeated superovulation with pregnant mare serum gonadotropin (PMSG) and hCG.As a result, the production of anti-hCG antibody was suppressed. Thus, tolerance induction to hCG was achieved, although it was incomplete. Compared with the control mice, the ovulatory rate was higher after the induction of immunotolerance to hCG. It was suggested that the production of anti-hCG antibody has a close relation with a decrease in the ovulatory response after the repeated induction of superovulation by gonadotropins.

Using wood mice (Apodemus speciosus) captured in the wild in Niigata, we analysed the proportion of various endocrine cells in pancreatic islets for both immunohistochemical and microscopic characteristics. In both the dorsal and ventral portions of the pancreas, the centre of the pancreatic islets was occupied predominantly by insulin-positive (B) cells, surrounded by glucagon-positive (A), somatostatin-positive (D), and pancreatic-polypeptide-positive (PP) cells. Although the proportions of the various endocrine cells in pancreatic islets varied from one mouse to the next, in most animals B cells accounted for more than half of all endocrine cells. Dorsal and ventral portions of the pancreas differed in the proportions of various endocrine cells, specifically, in the A-to-PP cell ratio: the proportion of PP cells higher in the ventral portion. The same tendency is seen in humans, rats and mice. Microscopic examinations revealed morphologically distinct secretory granules in A, B and D cells. The morphology of these granules was similar that of secretory granules found in rats and mice.

Changes in prolactin (PRL)-, growth hormone (GH)- and PRL/GH-containing cells in the anterior pituitary of pre- and postnatal male and female rats were determined using immunocytochemistry with double fluorescent antibodies. The pituitary glands from a fetus on Day 20 of gestation and pups on Days 0, 1, 4, 7, 12, and 20 of the postnatal period with sex distinction were monodispersed and subjected to immunocytochemistry. Following immunostaining, the three types of cells described above were counted (i.e., only PRL-, only GH-, and both PRL and GH-positive cells in a visual field of microscope). Anterior pituitaries and blood were obtained from pups on Days 0, 1, 4, 7, 12, and 20 after birth and pooled with sex distinction, and bioactive PRL levels in the anterior pituitary and serum were measured using an Nb2 rat lymphoma cell bioassay. Double fluorescent-labeled immunocytochemistry was able to distinguish reliably between PRL-, GH-, and PRL/GH-containing cells. The PRL cells increased rapidly after parturition to 4 days of age and after 12 days of age in both genders. The latter increase was involved in the remarkable increase of PRL/GH-containing cells, which were first found at term and remained at low levels until 12 days of age. The weight and PRL level of the anterior pituitary increased in both sexes throughout the lactational stage, but the levels in female rats at 20 days of age were significantly greater than those of male rats. Serum PRL concentration remained at low levels except on Day 0. The present results demonstrate that the development of PRL-containing cells in postnatal rats during the lactational period is activated with two distinct stages, at term to Day 4 and after Day 12, and the development in the latter stage is involved with sexual dimorphism.

A strain of cytopathic virus, named strain TG/K79, was isolated from the brain of a newborn piglet, pure Hampshire breed, which died shortly after birth. The physicochemical property of virus was considered to be that of the family Adenoviridae. A significant difference between our isolate and 4 reference porcine adenoviruses was demonstrated by cross-seroneutralization test Differences between TG/K79 and other porcine adenoviruses were also seen in electrophoretic patterns of viral DNA in agarose gel after digested by restiction endonucleases. Two SPF pigs, 2-month-old, experimentally infected via intranasal showed a fever and a hemorrhagic enteritis. A serological survey indicates that at least swine in the farm where the virus was isolated have been highly contaminated.

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.

The susceptibility to Leptospira interrogans serovar copenhageni in Mongolian gerbils treated with 10 micrograms of serum thymic factor (FTS) 1 day before infection was examined. Susceptibility of gerbils treated 5 times with 10 micrograms of FTS was also investigated. Mortality of FTS-treated gerbils was significantly lower than that of controls when small challenge doses were used. To analyse the FTS-induced resistance to leptospiral infection, natural killer (NK) cell activity and macrophage activity were studied. Macrophage activity was unaltered but NK cell activity was enhanced in FTS-treated gerbils, with or without leptospiral infection. Since no side-effects of FTS were observed, this compound should be considered for the treatment of leptospirosis.

In Mongolian gerbil, morphological changes with age in the content of endocrine cells in the pancreatic islets were analyzed and their relation evaluated by an oral glucose tolerance test. The glucose level 2 hours after glucose administration was 125 +/- 5 mg./d1 in the young group and 103 +/- 4 mg./d1 in the old group. In the dorsal portion, B cells were mainly observed in the central area of the islets, surrounded by circular layers of A cells in the peripheral area. Between the A cells and B cells, D cells were scattered or present in layers. A few PP cells were present in the peripheral area of the islets. In the ventral portion, only a few A cells were observed in the peripheral area of the islets, and B cells were surrounded by PP cells. Secretory granules of A cells generally had an electron-dense spherical core in the limiting membrane. The halo between the limiting membrane and core in A cells was narrower than that in B cells. Secretory granules of B cells were larger than those in A cells, and the core was less electron-dense, and the halo was wider. Secretory granules of D cells were similar in size to those of A cells; the core showed low electron density, and the halo was very narrow. Granules of PP cells resembled those of A cells, but the electron density of the core was slightly lower. The gerbils showed changes in glucose tolerance, the size of the pancreatic islets, the percentage of B cells, and of A cells in the dorsal portion with age.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the prevalence of avian Plasmodium in 509 mosquitoes of 9 species collected from the Ishigaki and Iriomote islands in the Yaeyama Archipelago, located southwest from the mainland of Japan. Two identical avian Plasmodium lineages were detected from Culex (Culiciomyia) nigropunctatus. Detected lineages were phylogenetically classified into different clade to avian Plasmodium lineages from birds and mosquitoes in the mainland of Japan but identical to a lineage detected from a resident bird, White-breasted Waterken (Amaurornis phoenicurus). This is the first detection of avian Plasmodium DNA from mosquitoes in the Yaeyama Archipelago and suggested that resident birds might have been infected with an avian Plasmodium lineage specific to the studied area and C. nigropunctatus could be the candidate vector mosquito species.

Several species of captive and wild birds have been found to be infected with various avian blood protozoa in Japan. We investigated the prevalence and transmission of avian malaria parasite and determined the bloodmeal hosts of mosquitoes collected in a zoological garden in Tokyo, Japan, by using the polymerase chain reaction. In total, 310 unfed and 140 blood-fed mosquitoes of seven species were collected by using sweep nets and CDC traps. Bloodmeal identification indicated that mosquitoes had fed on 17 avian and five mammalian species, including captive animals. The results of avian malaria parasite detection from mosquitoes with avian bloodmeals indicated that Culex pipiens pallens Coquillet is a main vector of avian Plasmodium in the current study site and that some captive and wild birds could be infected with avian malaria parasites. Furthermore, the distances between the collection site of blood-fed mosquitoes and the locations of their blood-source captive animals were estimated. Most females with fresh bloodmeals were found within 40 m of caged animals, whereas half-gravid and gravid females were found between 10 and 350 m from caged host animals. We demonstrated that blood-fed mosquitoes can provide useful information regarding the mosquito vector species of avian malaria parasites and allows for noninvasive detection of the presence of avian malaria parasites in bird populations.

The infection dynamics of avian haematozoa, which includes the genera Plasmodium, Haemoproteus, and Leucocytozoon, are complicated by a variety of environmental factors and host-parasite interactions. In Japan, the prevalence of haematozoa in wild birds has recently been determined in several local areas. However, no information on the annual prevalence of avian haematozoa in a single study site has been reported. Here, we investigated the long-term infection dynamics of haematozoa in wild birds inhabiting a mountain forest of Japan. Blood samples were collected from 415 wild birds captured in the Chichibu mountains in Saitama Prefecture at an altitude of 1650 m between 2007 and 2010. All obtained samples were examined for haematozoan infection using nested polymerase chain reaction (PCR) of the cytochrome b (cytb) genes of haematozoa. A total of 62 out of 415 (14.9%) forest birds were PCR positive for haematozoa. Relatively high infection rates of Leucocytozoon were found among several bird species (Parus ater, 64.3%; Parus montanus, 81.8%) and may be due to the host preference of vector black flies and host nestling pattern in this forest. Phylogenetic analysis of amplified cytb sequences revealed for the first time that a variety of lineages of avian haematozoa are distributed among wild bird hosts in a high-altitude forest stand in Japan. Notably, significant seasonal changes of the prevalence of avian haematozoa were not observed; however, continuous investigation will likely provide detailed information on host-parasite interactions, including local environmental factors, that influence the dynamics of avian haematozoan infections.

Cellular PrP (PrPC) is necessary for bovine spongiform encephalopathy (BSE) infection. The purpose of the present experiment was the quantification of PrPC in peripheral tissues to assess the risk of BSE infection from these tissues. The tissue distribution of PrPC was examined by a sandwich enzyme-linked immunosorbent assay (sELISA) and histochemical analysis. PrPC-deficient cows were used as a negative control. The sELISA revealed that the brain contained the highest PrPC content (10.7 µg/g tissue), while other organs/tissues harbored lower amounts, in decreasing order as follows: longissimus capitis muscle, iliocostalis thoracis muscle, splenius muscle, biceps femoris muscle, triceps brachii muscle, longissimus thoracis muscle, ileum, jejunum, duodenum, colon, cecum, apex linguae, omotransversarius muscle, posterior part of the corpus linguae, anterior part of the corpus linguae and radix linguae (5.2- to 31-fold less PrPC than the brain). In the tissue/organs of PrP-deficient cows, PrPC levels were under the limit of detection. Histochemical analysis showed that PrPC was expressed in nerve cells in intestinal tissues. The presence of PrPC in the bovine tongue, skeletal muscles and intestines raises the possibility of PrPSc accumulation in these tissues, indicating that these organs/tissues may serve as potential sources of BSE infection.

Prion diseases are a family of neurodegenerative zoonotic foodborne disorders. Although prions can be transmitted orally, the mechanism by which prions are incorporated into the intestine remains unclear. Our previous studies have shown that an abnormal isoform of prion protein (PrP(Sc)), which is the main component of prions, was efficiently incorporated into the intestine in suckling mice but not in weaned mice. Furthermore, suckling SCID mice lacking maternal antibodies showed decreased uptake of PrP(Sc) into the intestine compared with suckling wild-type mice, while the lack of PrP(Sc) uptake into the intestine of suckling SCID mice was rescued by the oral administration of IgG. These findings raise the possibility that the neonatal Fc receptor (nFcR), which contributes to the uptake of maternal antibodies into the intestine, plays a role in PrP(Sc) incorporation into the intestine. The present immunohistochemical study further showed that the FcR blocker Z-ε-aminocaproic acid (ZAA) inhibited PrP(Sc) incorporation into the intestinal villi of suckling mice, supporting the above mentioned concept. Therefore, our findings provide strong evidence that nFcR and maternal antibodies are involved in PrP(Sc) incorporation into the intestine before the weaning period.

Transmissible spongiform encephalopathies (TSE) are caused by dietary oral exposure to infectious prion proteins (PrPSc); however, the mechanism behind the uptake of PrPSc in the intestines is poorly understood. In addition, epidemiological studies of BSE showed that most cattle are exposed to the agents in the first 6 months of life, during the suckling and weaning periods. In the present study, to elucidate the enteric invasion mechanism of prions and to investigate the age-dependent transmission mechanism suggested by epidemiological studies, wild-type and SCID mice were orally administered brain homogenate from scrapie (Tsukuba 1)-infected mice during the suckling and weaning stages, before being analyzed histopathologically. PrPSc was found to be incorporated into the villous columnar epithelial cells and was also detected in the villous lacteal of 15-day-old suckling mice. However, no such uptake of PrPSc was observed in the weaned mice at 25-days-old. Four different strains of mice were tested. There was no mouse strain difference in the frequency of PrPSc positive columnar epithelial cells. In addition, the uptake of PrPSc in suckling SCID mice lacking maternal antibodies was significantly lower than that in the wild-type suckling mice, and the uptake of PrPSc was enhanced by dilution with purified IgG. In the present study, it was suggested that the weaning period and maternal immunoglobulin are important risk factors for the oral transmission of PrPSc.

SUMMARYThe highly pathogenic avian protozoanLeucocytozoon caulleryiinfects host chicken cells, and interference by the host genome results in difficulty in obtaining protozoal DNA for genetic analysis. We used flow cytometry analysis to separate expelledL. caulleryigametocytes from infected chicken blood and to analyse cell populations and sorting by FACS efficiency. Infected blood cells stained with SYTO-24 showed a specific area on 2-dimensional scattergrams compared to uninfected blood. The specific area was sorted, and approximately 85% of the sorted cells were identified asL. caulleryigametocytes by microscopic observation. DNA was also extracted from the sorted fraction, and a clear increase in polymerase chain reaction (PCR) amplification of protozoal DNA was observed compared to infected blood without sorting. Host-derived DNA was also detected by PCR; however, its amplification was decreased compared to that in unsorted infected blood. This is the first report of the separation ofL. caulleryigametocytes from infected host blood using flow cytometry. This method may be applied to further genetic analyses such as studies of the dynamics of stage-specificL. caulleryigene expression.

Nonalcoholic steatohepatitis (NASH) is one of the most frequent causes of abnormal liver dysfunction associated with synthesis and oxidation of fatty acids. Adiponectin receptors (AdipoR1/R2) and insulin receptor substrates (IRS-1/-2) are known as modulators of these fatty acid metabolisms in the liver; however, the regulatory roles of these receptors in the synthesis and oxidation of fatty acids are unclear in the liver of NASH. In this study, we examined the roles of hepatic AdipoR1/R2 and IRS-1/-2 in NASH using an animal model. After feeding a high-fat and high-cholesterol diet to obese fa/fa Zucker rats for 8 weeks, rats showed fatty liver spontaneously with inflammation and fibrosis that are characteristic of NASH. The expression levels of AdipoR1/R2 and IRS-2 were significantly decreased, whereas IRS-1 was significantly increased, in NASH. As a result of the decrease of AdipoR1/R2 expression, the messenger RNA expression levels of genes located downstream of AdipoR1/R2, adenosine monophosphate–activated protein kinase α 1/ α 2, which inhibits fatty acid synthesis, and peroxisome proliferator–activated receptor α , which activates fatty acid oxidation, also decreased. Expression level of sterol regulatory element binding protein–1c was found to be elevated, suggesting the up-regulation of IRS-1, and resulted in increased fatty acid synthesis. Furthermore, increase of forkhead box protein A2 expression was observed, which might be associated with the down-regulation of IRS-2, facilitating fatty acid oxidation. Taken together, increased synthesis and oxidation of fatty acids by up- or down-regulation of AdipoR or IRS may contribute to the progression of NASH. Thus, AdipoR and IRS might be crucially important regulators for the synthesis and oxidation of fatty acids in the liver of NASH.

Chemiluminescence studies on superoxide generation by phagosomes using opsonized zymosan showed the highest fluorescence in murine splenic macrophages among four different kinds of splenic or peritoneal macrophages from mice or gerbils. Murine splenic macrophages phagocytized two to three times more latex particles than gerbil splenic macrophages, but peritoneal macrophages did not show a significant difference in phagocytic activity between mice and gerbils. Phagocytosis by macrophages was determined by a technique based on measurement of the release of hydrogen peroxide and myeloperoxidase from phagosomes using microspheres conjugated with 3-(p-hydroxyphenyl) propionic acid (HPPA-MS). HPPA is a substrate of lysosomal myeloperoxidase. The fluorescence of HPPA-HPPA-MS produced by phagocytized HPPA-MS was measured with an immunoreaction analysis system (IMRAS), and the enzyme activities of the four different kinds of peritoneal or splenic macrophages from mice and gerbils were compared. All four kinds of macrophages produced HPPA-HPPA-MS in their phagosomes during phagocytosis and murine splenic macrophages showed the highest level of enzyme activity.

A cell line was established from cell cultures of kidney cortex of a pantropical spotted dolphin, Stenella attenuate. The replication of 6 strains of herpesviruses was studied in the cells. Five strains of them, herpes simplex virus type I and type II, equine rhinopneumonitis virus, infectious bovine rhinotracheitis virus and Aujeszky's disease virus, were grown fairly well in showing clear cytopathic effects and plaques under agar overlay medium.

Probable arthropod vectors of avian blood protozoa, Leucocytozoon lovati, were collected in the alpine regions of Japan, the habitats of the host birds of Japanese rock ptarmigan (Lagopus mutus japonicus). Seven alpine regions of Japan, Asahidake, Chogatake, Tateyama, Jiigatake, Norikura, Kitadake, and Senjyogatake were investigated for black fly collection during 2004 to 2007. The collected 490 insects were morphologically identified as six species of female black flies, including Prosimulium hirtipes group (n = 59), Prosimulium mutata (n = 13), Prosimulium yezoense (n = 10), Similium japonicum (n = 359), Similium uchidai (n = 39), and Twinnia japonensis (n = 10). Extracted DNAs from individual black fly species were utilized for the amplification of the partial mitochondrial cytochrome b gene sequences of Leucocytozoon lovati previously reported. Four S. japonicum, two S. uchidai, and two P. hirtipes group studied were positive for the nested PCR among 490 black flies collected (1.6%; 8/490). All amplified sequences from the black flies were completely identical to those of L. lovati previously detected from Japanese rock ptarmigan. Our results suggest that at least three species of black flies, S. japonicum, S. uchidai, and P. hirtipes group, studied in this area could be regarded as potential vectors for L. lovati in the rock ptarmigan. This is the first detection case of Leucocytozoon from black flies of Japan.

We analyzed extra chromosomal genomes of avian blood protozoa, Leucocytozoon caulleryi. One of the genomes, the mitochondrial genome was completely sequenced resulting 5,959 bp in length. This genome contained the identical gene organization and contents to that of other avian blood protozoa previously analyzed: three functional genes for cytochrome c oxidase subunit I, III, and cytochrome b with following sets of discontinuous and scrambled 15 ribosomal subunit RNA genes. In addition, the mitochondrial genome was estimated to have the tandem repeated structure as well as previously found in avian Plasmodium species. Furthermore, we found partial gene sequences of apicoplast DNA, another extra chromosomal genome, from L. caulleryi. These sequences were estimated as partial caseinolytic protease C and elongation factor Tu A genes. This report is the first description of two extra chromosomal genomes of L. caulleryi.

A 1.9 kb nucleotide sequence of part of the mitochondrial (mt) genome covering the cox1-trnT-rrnL-trnC-rrnS region, and the order of the remaining mitochondrial protein-coding genes for S. nasale of Sri Lankan origin, has been determined for analysis of the possible placement of this species in the genus Schistosoma. The gene order of this species is similar to that of the African and Indian Schistosoma species, but strikingly different from the East Asian species. Analysis of an alignment of the 1.9 kb sequence with available sequences from other schistosomes indicated affinities with S. spindale (found in Sri Lanka) and African species (in particular S. intercalatum and S. haematobium). Phylogenetic trees inferred from the alignment including 1 kb of RNA (transfer RNA and ribosomal RNA) sequence for 8 other Schistosoma spp. and Fasciola hepatica as an out-group revealed that S. nasale is placed proximally to S. spindale, S. intercalatum, S. haematobium and S. mansoni in the African sub-group while the East Asian species are more distant. S. incognitum lies basal to the combined African/Indian clade. The mtDNA analysis strongly supports the hypothesis that S. nasale is closely affiliated with the African/Indian schistosome group rather than the East Asian Schistosoma species.

The immunological effects of recombinant feline interferon-omega (rFeIFN-omega ; KT-80, Toray) were examined on administration to healthy dogs. The activities of whole blood cells, macrophages, and natural killer cells were enhanced. Moreover, the whole blood activity was examined when KT-80 was administered to dogs which had been diagnosed as having natural canine parvovirus (CPV) infection. Only some cases in which the activity increased until 3 hr post-administration survived. These results suggest that rFeIFN-omega (KT-80) treatment enhanced the cellular immunity of normal dogs, and could exert significant therapeutic effects on only natural CPV infected dogs with induced continuous immunoenhancement.

We analyzed mitochondrial genomes of two avian malaria protozoa, Plasmodium gallinaceum and Plasmodium juxtanucleare. Both mitochondrial genomes were estimated to be 6,002 and 6,014 bp in length, respectively, and to have the identical gene organization and contents to that of other Plasmodium species previously analyzed; three functional genes for cytochrome c oxidase subunit I, III, and cytochrome (cyt b), with following sets of discontinuous and scrambled 15 ribosomal subunit RNA (rRNA) genes. Similarities of the three protein-coding genes showed closer relationship within avian malaria protozoa rather than mammalian Plasmodium species. In addition, we showed the tandem repeated structure of each mitochondrial genome of both P. gallinaceum and P. juxtanucleare as well as previously found in mammalian Plasmodium species. This study revealed the complete sequences and structure of the mitochondrial genomes of avian malaria protozoa for the first time.

Cryptosporidium infection was confirmed by fecal examination for the first time in pet rabbits in a wholesale store located in Kanagawa Prefecture, Japan. Fecal samples were obtained postmortem from juvenile rabbits (n=66), which had died after developing diarrhea. Feces from healthy rabbits (n=30) were also collected and examined as controls. Two types of Cryptosporidium oocysts distinctive in size and shape were found (Type A and B). Types A and B oocysts were detected from 16.7% and 13.6% of the diarrheic, and 3.3% and 0% of the normal feces, respectively. Since Cryptosporidium oocysts were detected at a higher rate in the diarrheic rabbits than in the healthy rabbits, special caution should be taken when handling a pet rabbit presenting with diarrhea.

In an attempt to study the immunological effects on normal and adenocarcinoma (AC) dogs, the natural killer (NK) activity was determined. Augmentation of NK cell activities in responsive normal and AC dogs that indicatedanergy in the phaseolus vulgaris agglutinin (PHA) skin-test was manifested when the animals were accordingly treated with germanium (GN).

The effect of Bacillus subtilis (strain A102) spores on the activation of murine macrophages and natural killer cells (NK) was examined. The macrophage activity and NK activity were enhanced by oral administration of A102 spores, and slightly enhanced by oral administration of culture supernatant. There was no difference in the results of macrophage activity and NK activity using other live or dead spores. The NK activity and macrophage activity were increased with increments of concentration up to 0.1 g per mouse, and both activities were decreased at concentration of more than 0.15 g per mouse. The NK activity was increased 1 and 2 days after oral administration of A102 spores, and the activity level 2 days after administration was about 3-fold higher than the level prior to treatment. Macrophage activity was also increased from 1 to 3 days after oral administration of A102 spores, and the activity level 3 days after administration was about 3-fold higher than the level prior to treatment. The induction of interferons at 1 day after oral administration in mouse serum was 5-fold higher than that in controls. These findings indicate that oral administration of A102 gave rise to the induction of interferons, and it is likely that macrophages and NK cells were activated by interferons.

1 School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan 2 Central Laboratories for Key Technologies, Kirin Company Limited, Fukuura 1-13-5, Yokohama, Kanagawa 236-0004, Japan 3 Laboratory of Biometabolic Chemistry, School of Health Sciences, Faculty of Medicine, University of Ryukyus, 1207 Uehara, Nishihara, Okinawa 903-0215, Japan 4 College of Bioresource Sciences, Nihon University, 1186 Kameino, Fujisawa, Kanagawa 252-0880, Japan

We studied the effects of immune sera, an antibiotic, and a corticosteroid used alone or in combination on leptospira infection in Mongolian gerbils (Meriones unguiculatus). The results suggest that the combined used of immune serum and corticosteroid (pledonizoron: PZ) inhibits the effects of serum and the marked effects of antibiotic (procaine penicillin-G: PC-G) on leptospirosis. PZ had no effects and rather shortened the survival period. PZ did not affect the effects of PC-G when used in combination. These results suggest that treatment of leptospirosis with corticosteroids requires special caution.

Gerbils were inoculated with reovirus type 3 (Abney strain), and the pancreas, brain and other organs histopathologically evaluated. Male Mongolian gerbils (Meriones unguiculatus) aged 5-10 days (newborn, 9 animals), 4 weeks (juvenile, 5 animals), and 12 weeks (adult, 5 animals) were used. Gerbils inoculated intraperitoneally with the virus were sacrificed 3, 5, 7, or 10 days later. Tissues were observed by light and electron microscopy. The pancreas of newborns showed inflammatory edema with infiltrates of neutrophilis and mononuclear round cells, degranulation of acinar cells and dissociation of lobules and acini with necrosis at 3 days or later after inoculation but not degeneration of pancreatic islets. Brains of newborns had necrosis of neurons and aggregation of microglial cells in the brain stem and cerebral hemisphere. Azurophilic inclusion bodies were seen in the cytoplasm of some neurons. In electron micrographs, virus particles (60-80 nm in diameter) were observed around the nucleus of neurons in the brain stem. No changes were observed in the juvenile and adult gerbils.

Mongolian gerbils, Meriones unguiculatus, were shown to be highly susceptible to Leptospira interrogans serovars such as icterohaemorrhagiae, copenhageni, canicola, autumnalis, javanica, pyrogenes and hebdomadis as compared to guinea pigs and hamsters. Mortality with signs of haemorrhage and jaundice was recorded in all experimental rodents after intraperitoneal inoculation with all strains of the serovars indicated. However, Mongolian gerbils were comparatively susceptible to strains which were of low virulence to guinea pigs and hamsters. Use of leptospiral vaccination proved effective in protecting the animals against inoculum challenges. The Mongolian gerbil is a species that may be selectively and preferentially useful for assays on the protective effects of leptospiral vaccination.

Slow-reacting, complement-requiring hemagglutination-inhibiting (HI) antibody was detected in sera from pigs infected with pseudorabies virus; approximately 16 hemolytic units of complement were necessary for the detection of such antibody. Higher HI antibody titers were obtained when antigen and serum were allowed to incubate before addition of complement than when all three components were incubated at the same time. A HI test was developed in which antigen-serum mixtures were incubated at 4 degrees C for 48 h and then with complement at 37 degrees C for 1 h; this gave an improved sensitivity over the previous incubation without complement.

Pseudorabies virus grown in CPK cell cultures was tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, 25°C and 37°C. HA was observed at all temperatures with mouse erythrocytes but not with cattle, sheep, goat, swine, cat, rabbit, guinea pig, rat, mongolian gerbil, chicken, and goose erythrocytes. Mice showed a strain variation in agglutinability of their erythrocytes, requiring selection of mice to obtain erythrocytes for HA. The HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were established. HI antibody titers of individual pig sera showed a significant positive correlation with their neutralizing antibody titers.

Leucocytozoon caulleryi, a haemosporidian parasite of the chicken (Gallus gallus domesticus), can be highly pathogenic and often fatal. Although this parasite is extremely relevant to veterinary science, knowledge of its genomic features is limited. To gain information applicable to developing novel control methods for the parasite, we analyzed the apicoplast genome of L. caulleryi. This extranuclear organellar DNA of 85.1 % A + T and a unit of 34,779 bp was found to encode almost the same set of genes as the plastid genome of Plasmodium falciparum, including 16 tRNA and 30 protein coding genes, and except for one open reading frame, ORF91 absent in L. caulleryi. As in P. falciparum, the L. caulleryi apicoplast DNA contains two sets of a unique inverted repeat (IR), each one 5,253 bp and encoding genes specifying one large and one small rRNA subunit and nine tRNAs but no protein, and separated by a unique 13 bp sequence. Studies of several haemosporidian apicoplast DNA sequences have identified a corresponding IR region; however, none of these studies has looked at the complete sequence, even for well-studied species such as P. falciparum. Phylogenetic studies using a concatenated amino acid sequence based on the open reading frames confirmed the close relationship between L. caulleryi and Plasmodium spp. In this study, we determined the nucleotide sequence of the entire L. caulleryi apicoplast genome, including the region connecting the two IR units. This is the first report of the complete nucleotide sequence of a haemosporidian apicoplast DNA with a canonical IR.