Molecular detection of Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos' in cattle in Hokkaido, Japan

Blood samples from 78 cattle were tested for hemoplasma infection using molecular methods. PCR and sequence analysis revealed that 17 cattle were infected with Mycoplasma wenyonii, while 13 were infected with 'Candidatus Mycoplasma haemobos'. Four animals were infected with both species. This is the first study to report hemoplasma species infection among cattle in Japan.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Management and yield prediction of Kunugi (Quercus acutissima) grazing forests

This study deals with grazing in kunugi (Quercus acutissima) forests in the Aso district of Kyushu Island in southwest Japan. These forests are managed for production of bed-logs for shiitake mushrooms and cow-calf farming. One of their characteristics is short-term rotation such as 10–15 years for bed-logs and a year for calf production. A forest grazing experiment was begun in Minamioguni to look at forest growth, vegetation change and grazing intensity. Stem densities dropped in a few years. After sprout cutting, they also dropped gradually, then stabilized. Although grazing caused tree damage and suppressed tree growth, grazing intensity of up to 150 cow-days/ha·year did not harm forest regeneration. Herbage volume decreased as grazing was repeated and trees grew. Another investigation of kunugi grazing forests in Minamioguni and Asaji showed the correlation betweenRy (yield index in Stand density diagram) and grazing capacity could be expressed with a regression equation. The results were also used to design a yield table for kunugi grazing forests. The yield table has items ofRy and grazing capacity in addition to usual yield table items, and can indicate timber yield and grazing capacity at the same time. The table estimates that proper grazing capacity is 60–80 cow-days/ha·year in wild grass sites. In the light of these results, an optimal management plan was proposed as a diagram integrating stem density, forest yield, and forest management.     

Linkage analysis of RFLP markers for clubroot resistance and pigmentation in Chinese cabbage (Brassica rapa ssp. pekinensis)

A restriction fragment length polymorphism (RFLP) – based linkage map of Chinese cabbage (Brassica rapa ssp. pekinensis) (2n=20) including two agronomic traits, clubroot resistance and orange-yellow pigmentation, was constructed using doubled haploid parents. The total linkage distance was 735 cM; 63 loci were distributed into ten linkage groups. Clubroot resistance of the parental line T136-8 to the current pathotype, race 2, was predominantly controlled by a single dominant gene that originated from European turnip. The locus for clubroot resistance by the dominant major gene (CRa) was mapped on linkage group 3, and RFLP loci HC352b and HC181 were located 3 cM and 12 cM from it, respectively. The locus HC352b was identified by a 4.4 Kb Eco R I fragment, which segregated for null allele. The absence of an allelic fragment in HC352b could be interpreted by deletion in the resistance source; homozygotes for CRa could be efficiently selected by detecting null types for the marker. Orange-yellow pigmentation expressed in head inner leaves and petals was governed by a single recessive gene. The locus (Oy) for the pigmentation was mapped on linkage group 1, being located 17–19 cM from three RFLP loci that were closely linked to each other. The linkage analysis for clubroot resistance and unique pigmentation revealed some informative RFLP markers. Identification of molecular markers for clubroot resistance and other agronomically important traits would provide useful information in breeding programs of Chinese cabbage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antibodies produced by mice immunized with recombinant vaccinia viruses expressing two different types of a major Theileria sergenti surface antigen (p32) react with the native surface antigen.

A 32 kDa major surface antigen, p32, of Theileria sergenti at the piroplasm stage is the main target of the host immune response. The immunogenic property of the p32 varies in some strains among the population of Theileria sergenti in Japan where the Chitose type and the Ikeda type are the most common varieties. We have constructed vaccinia virus recombinants vv/p32C and vv/p32I which harbor the Chitose and Ikeda types of p32 gene, respectively. It was found that vv/p32C and vv/p32I produced type-specific p32 which did not cross react with the monoclonal antibodies (mAbs) against the other type of p32. When mice were immunized with vv/p32C and vv/p32I, antibodies against p32 were detectable 2 weeks after the immunization, and these antibodies reacted with the native surface antigen in purified T. sergenti merozoite.     

Examination of a practical method for zinc enrichment of euryhaline rotifers (Brachionus plicatilis)

Larval growth and survival of marine finfish in mass seed production are affected by the nutritional value of live feeds such as rotifers and Artemia. Thus far, many studies have been conducted to develop effective methods for the enrichment of live feeds with essential fatty acids and vitamins. In this study, a practical method for enrichment of rotifers with zinc was investigated. Changes in the concentrations of other minerals when zinc was added to the rotifer-enrichment tanks were also studied. The mineral composition of rotifers and Chlorella after zinc enrichment revealed that the direct addition of zinc to the culture media was not effective because rotifers cannot efficiently accumulate waterborne zinc. The ability of Chlorella to absorb waterborne zinc is much higher than that of rotifers, and hence, zinc was pre-accumulated in Chlorella, which was then fed to the rotifers. The maximum zinc content of the rotifers was 585.0 μg g^? 1 (dry matter) when the rotifers were enriched with zinc alone. This zinc concentration is comparable to that found in natural zooplankton. In rotifers simultaneously enriched with zinc and n?3 highly unsaturated fatty acids (HUFAs), the zinc content increased, but the n?3 HUFA content did not. Therefore, separate enrichment with zinc and fatty acids was adopted. The zinc content of rotifers fed zinc-enriched Chlorella was significantly higher than that of rotifers fed unenriched Chlorella. After zinc enrichment, rotifers were enriched with fatty acids, and the docosahexaenoic acid (DHA) and n?3 HUFA levels in rotifers were higher than the levels obtained after simultaneous enrichment with zinc and fatty acids. With regard to the concentration of other minerals in rotifers after zinc enrichment, the manganese content tended to decrease when the zinc content increased.The results of this study demonstrated that zinc enrichment of rotifers was successfully performed by using microalgae that had accumulated zinc, and the enrichment of rotifers with fatty acids was also achieved after the completion of zinc enrichment and before feeding the larvae. This method could be utilized for the enrichment of zooplankton with other minerals as well.     

Amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> interleukin-10 negatively suppresses host cell-mediated immunity

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10 genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC)?+?rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM⁺ cells among spleen leukocytes in N. seriolae?+?rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.     

Involvement of heme synthesis in the growth stimulation of maize seedlings by 5‐aminolevulinic acid

The growth of maize seedlings was stimulated by shoot‐applied 5‐aminolevulinic acid 2 days after treatment at 90 and 120 μmol L^?1. The effects of 5‐aminolevulinic acid on the activity of nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1) and the chlorophyll, ammonium, heme and total free amino acid content were investigated by using maize seedlings to clarify the involvement of nitrogen metabolism and heme synthesis in the growth stimulation. 5‐Aminolevulinic acid increased the level of nitrate reductase activity at 90 μmol L^?1 and the ammonium and heme content at 90 and 120 μmol L^?1 2 days after treatment. The total amino acid content increased by 90 and 150 μmol L^?1 5‐aminolevulinic acid 2 and 3 days after treatment, respectively. However, no significant change was observed in the activity of nitrite reductase or the chlorophyll content after the 5‐aminolevulinic acid treatment. These results suggest that the enhancement of nitrogen metabolism by nitrate reductase activation is involved in the 5‐aminolevulinic acid‐induced stimulation of maize seedling growth. The activation of nitrate reductase might be related to an increase in the heme content following the 5‐aminolevulinic acid treatment.     

Immunohistochemical analysis of cyclooxygenase-2 induction during wound healing in dog skin

Cyclooxygenase (COX)-2 is an inducible isoform of COX and is expressed under abnormal health conditions. This study elucidated the cutaneous induction of COX-2 during the wound healing processes in dog skin. Dog skin was sutured after punch biopsy and investigated histologically and immunohistochemically on days 0 (normal), 1, 3, 5, 7, 10, and 14 after injury. Histological changes, including infiltration of inflammatory cells and proliferation of fibroblast-like cells, were observed as predicted, and there was a close and significant correlation between these 2 events. COX-2-positive cells were detected in the epidermis between days 1 and 7, and bimodal peaks were observed in the case of the percentage of COX-2-positive cells. In inflammatory cells, COX-positive signals were detected on day 3 only. Here, we clarified the localization and pattern of the induced COX-2 expression during wound healing in dog skin.