Novel Genetic Variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a Novel Ehrlichia sp. in Wild Deer and Ticks on Two Major Islands in Japan

Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL. DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum, a novel Ehrlichia sp., Anaplasma centrale, and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis, and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.     

Differentiation ofMelampsora rust species on willows in Japan using PCR-RFLP analysis of ITS regions of ribosomal DNA

To develop a reliable method for identifyingMelampsora species parasitic on willows in Japan, we differentiated 10Melampsora species by PCR-RFLP analysis. Internal transcribed, spacer (ITS) regions, including 5.8S ribosomal DNA, of 63 collections of 10Melampsora species and 4 collections of unidentified species were amplified by PCR. The fragments from the 67 collections varied in size (approximately 880 bp, 860 bp and 840 bp). The restriction sites in the amplified DNA fragments were mapped after the RFLP analysis using four restriction enzymes,Dra I,EcoRI,SspI andTaqI. All the collections were divided into 11 RFLP types. In the 6 species,M. caprearum, M. epiphylla, M. kamikotica, M. larici-urbaniana, M. microsora andM. yezoensis, the RFLP type was species-specific. The RFLP type ofM. chelidonii-pierotii andM. coleosporioides was identical. The collections ofM. epitea were separated into three RFLP types. One of these three types was identical with the type ofM. humilis. It is suggested that the PCR-RFLP analysis of ITS regions is a useful and reliable method for species identification ofMelampsora. Contribution No. 131, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Influences of microhabitat use and foraging mode similarities on intra- and interspecific aggressive interactions in a size-structured stream fish assemblage

Aggressive interactions, foraging behaviour and microhabitat use were observed among four sympatric stream fishes inhabiting the water column: ayu (Plecoglossus altivelis), white-spotted charr (Salvelinus leucomaenis), masu salmon (Oncorhynchus masou) and Japanese dace (Tribolodon hakonensis), each species being categorised into five body-size classes (species-size groups; SSG's). Aggressive interactions were observed between most pairs of SSG's, an almost linear dominance order being apparent throughout the three-month study period. Ayu were relatively subordinate in June, but became the second most dominant in July and the most dominant in August, as a consequence of a reversal in dominance order with salmon. In contrast, smaller-sized dace, which continually suffered from intra- and interspecific aggression, occupied the most subordinate ranks throughout the study period. Intensive aggression was observed among various SSG's, exhibiting same microhabitat propensity throughout the three months. The direction and frequency of aggressive interactions varied month by month due to a reversal in dominance order between ayu and masu salmon, and/or changes in density, body size and resource use of the component members. Opponent selectivity was higher within SSG's, where resource use was assumed to be highly overlapping, rather than among SSG's throughout the study period. Correlation analysis indicated that opponent selectivity in aggressive interactions among SSG's was positively correlated with similarity in microhabitat selectivity in June, but not in other months or with that in foraging habits, suggesting that intensive aggressive behaviour reflected overlapping habitat use among assemblage members during a certain period.     

Nonsense mutations at Arg-1947 in two cases of familial neurofibromatosis type 1 in Japanese

We report two familial cases of NF1 presenting as C to T transitions changing an Arg-1947 codon to a stop codon. In one of the two families, cosegregation of the mutation with NF1 was demonstrated, indicating this mutation causes the disease in this family. As the same mutation at Arg-1947 has been reported previously in three cases of unrelated Caucasians (two are sporadic; the origin of the other is not reported), the codon at Arg-1947 (CGA) in the NF1 gene is considered to be a hotspot common among different ethnic groups and also among familial and sporadic cases.     

Characterization of Immunological Activity of a Low Toxicity Antitumor Lipopolysaccharide from Bordetella pertussis

Immunological properties of a low toxicity lipopolysaccharide (BP-LPS) extracted from Bordetella pertussis (Tohama strain) which was reported to have high antitumor activity against murine tumors were examined and compared with those of LPS extracted from other enterobacteria. The activation or stimulation of murine macrophages and lymphocytes by these LPS, including TNF induction, was found to be similar. However, BP-LPS was clearly less active in its stimulation of murine and human neutrophils as estimated by neutrophil-adherence assay and by their TNF production than E. coli LPS. Furthermore, BP-LPS also suppressed the activation of human neutrophils by Escherichia coli LPS. A comparative study with 7 LPS preparations indicated that their toxicity in terms of animal body weight loss correlated with their ability to induce human neutrophil adherence. The inability of BP-LPS to activate neutrophils may thus have some bearing on its low toxicity.     

A Rapid Colorimetric Assay for Determination of Leukocyte-Mediated Inhibition of Mycelial Growth of Candida albicans

A staining method with crystal violet (CV) was demonstrated to be useful for a simple, quick and objective assessment of in vitro growth inhibitory activity of leukocytes against Candida albicans cells. Candida cells incubated with murine neutrophils or macrophages for 14 hr in microwells were stained with CV and, after washing with 0.25% sodium dodecyl sulfate (SDS), treated with isopropanol containing HCl (0.04 N) to extract Candida cell-bound CV. Then the absorbance at 590 nm of the isopropanol extract was photometrically measured. The results showed that the photometrical absorbance was proportional to the amount of³H-glucose taken up by C. albicans cells, which reflected the number of viable Candida cells.     

Suppression of Anti-Candida Activity of Murine and Human Neutrophils by Glucocorticoids

Effects of glucocorticoid (GC) compounds on inhibitory activity of neutrophils to mycelial growth of Candida albicans were examined by in vitro crystal violet staining method with 14 hr co-culture. Both GC hormones (hydrocortisone ≥6 × 10^–7 m and corticosterone ≥10^–6 m ) and anti-inflammatory GC agents (prednisolone ≥10^–7 m and dexamethasone ≥10^–8 m ) significantly suppressed anti-Candida activity of murine casein-induced neutrophils. Anti-Candida activity of human neutrophils prepared from peripheral blood was also suppressed by hydrocortisone (≥6 × 10^–7 m ). These GC compounds did not affect the Candida growth in the absence of neutrophils. Steroidal compounds without anti-inflammatory activity, cholesterol, cholic acid, aldosterone did not suppress neutrophil activity. These results suggest that GCs at their physiological or clinical concentration may suppress anti-Candida activity of neutrophils in vivo.     

Respiration for maintenance and growth inReynoutria japonica ecotypes from different altitudes on Mt Fuji

Respiration for maintenance and growth ofReynoutria japonica ecotypes from altitudes of 700 and 2420 m on Mt Fuji were measured in two controlled thermal conditions. The maintenance respiration of the high-altitude ecotype at both 15 and 25°C was significantly (1.7-fold) higher than that of the low-altitude ecotype, whereas growth respiration was independent of both ecotype and temperature. The temperature coefficient (Q₁₀) of the maintenance respiration was about 1.9 in both ecotypes. The results show that there is ecotypic differentiation in the performance of maintenance respiration. It is suggested that the high maintenance respiration of the high-altitudeR. japonica ecotype is advantageous in severe upland environments but disadvantageous in a warm lowland climate in terms of carbon economy.