Development and biological properties of a new live attenuated mumps vaccine

To develop a new live attenuated mumps vaccine, a wild mumps Y7 strain isolated from a patient who developed mild parotitis was treated with nitrosoguanidine and ultraviolet, followed by selection of a temperature-sensitive clone. The selected clone, Y125, showed stable temperature-sensitivity in Vero cells. Intraspinal inoculation of marmosets with the Y125 produced only minimal histopathological changes, while intracerebral inoculation of neonatal rats revealed that the Y125 did not cause hydrocephalus. Both these effects of the Y125 were similar to those of the non-neurovirulent Jeryl Lynn strain. Furthermore, subcutaneous inoculation of the Y125 induced high levels of neutralizing antibodies in all Cercopithecus monkeys examined. Although the safety and immunogenicity should be confirmed in further field trials in humans, the present results indicate that the Y125 could be a promising vaccine candidate.     

Mycobacterium ulcerans infection in an Indian flap-shelled turtle (Lissemys punctata punctata)

We report an atypical mycobacterial infection in an Indian flap-shelled turtle, Lissemys punctata punctata, that died in an aquarium in Japan. At necropsy, the turtle showed multiple white nodules on the capsular surface and parenchyma of various organs such as the liver, spleen, intestine, and lung. Histologically, granulomatous inflammation surrounding a central zone of necrosis was observed. Sections stained by the Ziehl-Neelsen method revealed numerous acid-fast bacilli in the cytoplasm of macrophages and in the central area of necrosis. The organisms were identified as a mycobacterial species by PCR and nucleotide sequence analysis and revealed 98-100% homology to M. ulcerans. This is, to our knowledge, the first report of mycobacteriosis due to M. ulcerans in a turtle.     

Fucose-containing sulfated polysaccharides from brown seaweeds inhibit proliferation of melanoma cells and induce apoptosis by activation of caspase-3 in vitro

Fucose-containing sulfated polysaccharides (FCSPs) extracted from seaweeds, especially brown macro-algae, are known to possess essential bioactive properties, notably growth inhibitory effects on tumor cells. In this work, we conducted a series of in vitro studies to examine the influence of FCSPs products from Sargassumhenslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively, on proliferation of melanoma B16 cells and to investigate the underlying apoptosis promoting mechanisms. Cell viability analysis showed that both FCSPs products, i.e., FSAR and FVES, decreased the proliferation of the melanoma cells in a dose-response fashion, with FSAR being more potent at lower dosages, and FVES being relatively more anti-proliferative than FSAR at higher dosages. Flow cytometric analysis by Annexin V staining of the melanoma cells exposed to the FCSPs products confirmed that both FSAR and FVES induced apoptosis. The FCSPs-induced apoptosis was evidenced by loss of plasma membrane asymmetry and translocation of the cell membrane phospholipids and was accompanied by the activation of caspase-3. The FCSPs bioactivity is proposed to be attributable to distinct structural features of the FCSPs, particularly the presence of sulfated galactofucans (notably in S.henslowianum) and sulfated fucans (notably in F. vesiculosus). This study thus indicates that unfractionated FCSPs may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3.     

Characterization of Erysipelothrix rhusiopathiae strains isolated from recent swine erysipelas outbreaks in Japan

The objective of the present study was to characterize Erysipelothrix sp. strains from recent erysipelas outbreaks in Japan. Eighty-three (100%) strains were identified as E. rhusiopathiae, based on serotyping and spaA PCR. Fifty (60.3%), 5 (6.0%), and 28 (33.7%) strains were isolated from animals with acute, subacute and chronic outbreaks, respectively, of which 79 (95.2%), 1 (1.2%), and 3 (3.6%) belonged to serotypes 1a, 2a, and untypeable, respectively. Fifteen strains (including 3, 2, and 10 from acute, subacute, and chronic cases, respectively) were sensitive to acriflavine, and showed high levels of virulence in mice; of which strains from acute cases, and from subacute and chronic cases killed 100%, and 80 to 100% mice, respectively at challenge doses of 10(2) CFU per mouse. Based on sequence analysis of a 432-bp hypervariable region in spaA gene, 83 strains could be divided into 3 groups: (i) group 1 (3 strains of serotype 1a) had Ala-195 and Ile-203; (ii) group 2 (76 strains of serotype 1a and 3 of untypeable) had Asp-195 and Met-203; and (iii) group 3 (one strain of serotype 2a) had Asn-195 and Ile-203. The results of the present study suggest that the serotype 1a strains belonging to the group 2 might be widespread in pig populations in Japan.     

Superficial necrolytic dermatitis associated with extrapancreatic glucagonoma in a dog

An 11-year-old Shih Tzu presented with crusting and erythema, mainly on the abdomen and the root of the tail. Based on histopathological findings, blood examinations and necropsy findings, the condition was diagnosed as superficial necrolytic dermatitis associated with a glucagon-secreting extrapancreatic neuroendocrine tumour. Gross necropsy revealed tumour invasion into the spleen, liver, adrenal glands and mesenteric lymph nodes. Immunohistochemical analysis of the neoplastic cells revealed that the tumour was a glucagonoma, consistent with earlier findings of persistent glucagonaemia and hypoaminoacidaemia.     

Middle upper beak fracture in a Red-crowned crane that completely recovered with external skeletal fixation

An adult Red-crowned crane in captivity that had a displaced compound fracture of the middle upper beak caused by an accident was treated by using Type I-a external skeletal fixator (ESF). The ESF that was equipped with a unilateral epoxy putty fixator and with five half-pins was fixed on the premaxilla bone. The crane with the ESF on the beak was able to feed smoothly by itself. The ESF was removed 49 days after the first surgery. Beak malocclusion, which was observed in the latter half period of fixing, spontaneously improved within one month after removal of the ESF. This is the first successful case of repair of an upper beak fracture in a Red-crowned crane.     

Detection and sequence analysis of DNA polymerase and major envelope protein genes in koi herpesviruses derived from Cyprinus carpio in Gunma prefecture, Japan

Koi herpesvirus (KHV), which is believed to be an emerging virus, causes fatal diseases in carps. Since the 1990s, the presence of KHV has been confirmed in several countries. In Japan, from 2003 to 2004, large outbreaks of KHV infection in farmed carps resulted in the death of numerous fishes. From April to May 2004, we collected 43 dead or dying carps exhibiting typical symptoms of KHV infection in Gunma prefecture. To conduct a molecular epidemiologic study of KHV in our prefecture, we amplified DNA polymerase and the major envelope protein genes of KHV derived from carp gills using newly designed primers. We also performed sequence analysis of both genes of KHV. Sensitivity of our PCR method for amplification of DNA polymerase and the major envelope protein genes of KHV was 3 x 10(2) (100 fg) and 3 x 10(3) (1000 fg) copies of KHV genome, respectively. We detected both DNA polymerase and major envelope protein genes in 37 of 43 carps (86%). No mutation was found in both the genes sequenced from 11 strains, which included two foreign strains and one domestic strain. The results suggested that KHV strains derived from carps in our prefecture were closely related genetically to the other KHV strains.     

In vivo characterization of primitive hematopoietic cells in clonal ginbuna crucian carp (Carassius auratus langsdorfii)

Primitive hematopoietic cells in mammalian bone marrow are purified by flow cytometry using Hoechst 33342 (Hoechst) and rhodamine-123 (Rho), because these dyes efflux activities of hematopoietic cells widely conserved in mammals. Hematopoietic stem cells (HSCs) are identified as side population (SP) cells, characterized by specific Hoechst efflux pattern in flow cytometric analysis. We previously demonstrated that SP cells from teleost body kidney (BK) had the HSC activity by a transplantation experiment using clonal ginbuna crucian carp (Carassius auratus langsdorfii). In the present study, to isolate HSCs and hematopoietic progenitor cells (HPCs) from teleosts using Hoechst and Rho, we compared the hematopoietic activity of Rho-negative (Rho(-)) cells with that of SP cells by ginbuna transplantation experiments. Rho(-) cells were clearly identified from ginbuna BK, and the majority of these cells (85%) showed a non-SP phenotype. Transplantation experiments showed that long-term repopulating activity (HSC activity) of Rho(-) cells was lower than that of SP cells, while Rho(-) cells had higher short-term repopulating activity (HPC activity) than SP cells. These results suggest that Rho(-) cells in ginbuna BK contain various stages of hematopoietic cells, while SP cells are highly enriched for HSCs, and that these dyes are useful for purification of HSCs and HPCs in teleosts.     

The extinction of genetic resources of Asian wild rice, Oryza rufipogon Griff.: A case study in Thailand

Natural populations of wild rice, Oryza rufipogon Griff., are now threatened with the disturbance of their natural habitats by various human activities. To obtain basic information on genetic erosion or loss of genetic diversity in wild rice, we investigated how environmental changes of habitat affected the genetic structure of its natural population at a study site in the central plain of Thailand. During 10 years from 1985 to 1994, the wild-rice population at this site was seriously destroyed and fragmented. Using two sets of seed sample collected in 1985 and 1994 from the same population, allozyme variability at 17 loci of 11 enzymes were examined. Isozyme genotypes of mother plants of seed samples were estimated by the segregation in each progeny, and we calculated genetic parameters for the population. Gene diversity severely decreased in the 1994 sample compared with the 1985 sample. It is supposed that declining and fragmentation of the wild rice population, which happened during the 10 years, caused loss of genetic variability and forced the habitually outbreeding plants to inbreed, accelerating a reduction in gene variability. Pgi1-1 allele which was common in Indica rice cultivars of this region was found in the wild rice plants growing at the side of rice fields. Probably, introgression has occurred between wild and cultivated rice plants, and consequently the intrinsic nature of wild rice was gradually blurred by cultivar genes. We must realize that the genetic erosion of wild rice is rapidly proceeding and that an action for their conservation in natural environment, so called in situ conservation, is urgently needed.