Prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma

Currently, antimicrobial-resistant staphylococci, particularly methicillin-resistant Staphylococcus pseudintermedius (MRSP), are frequently isolated from canine superficial pyoderma in Japan. However, little is known regarding the nasal prevalence of MRSP in pet dogs. Here, we determined the prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma. Of the 125 nares and 108 affected sites of pet dogs with superficial pyoderma, 107 (13 species) and 110 (eight species) staphylococci strains, respectively, were isolated. The isolation rate of S. pseudintermedius from pyoderma sites (82/110 strains, 74.5%) was significantly higher than that from nares (57/107 strains, 53.3%) (P<0.01). Notably, the prevalence of MRSP (18/57 strains, 31.6%) in nares was equivalent to that in pyoderma sites (28/82 strains, 34.1%). Furthermore, the phenotypes and genotypes of antimicrobial resistance in MRSP strains from nares were similar to those from pyoderma sites. Our findings revealed that the prevalence of antimicrobial-resistant staphylococci in the nares of pet dogs with superficial pyoderma is the same level as that in affected sites. Therefore, considerable attention should be paid to the antimicrobial resistance of commensal staphylococci in companion animals.     

Antifungal properties of terpenoids in Picea abies against Heterobasidion parviporum

The antifungal properties of 14 major oleoresin‐constitutive terpenoids of Norway spruce (Picea abies) against Heterobasidion parviporum were evaluated in vitro at three gradient concentrations, 0.1, 0.2 and 0.4 μmol/cm², on potato dextrose agar medium. Eight monoterpene hydrocarbons (+)‐ and (?)‐α‐pinene, (?)‐β‐pinene, (+)‐3‐carene, myrcene, (+)‐ and (?)‐limonene, terpinolene; four oxygenated monoterpenes α‐terpineol, terpinen‐4‐ol, 1,8‐cineole, bornyl acetate; and two resin acids abietic acid and dehydroabietic acid were selected. Abietic and dehydroabietic acids showed the highest antifungal activities at all concentrations. Among oxygenated monoterpenes, bornyl acetate and α‐terpineol showed antifungal activity at the highest concentration. Among monoterpene hydrocarbons, (+)‐α‐pinene showed similar activity to terpinen‐4‐ol and 1,8‐cineole at the highest concentration; however, it was lower than α‐terpineol and bornyl acetate. Other monoterpene hydrocarbons inhibited mycelial growth by <10%. Re‐extraction of medium surfaces after the test period revealed that most α‐terpineol and terpinen‐4‐ol remained unchanged on the surface but monoterpene hydrocarbons completely disappeared. However, notable fungal transformed products were observed on surfaces applied with 1,8‐cineole and bornyl acetate. Thus, mycelial growth inhibition of monoterpenoids might be caused by complex functions such as biodegradation and/or detoxification by interaction between mycelium and compound. These results provide a basis for future studies considering the role of chemodiversity in the comprehensive chemical defence of P. abies.     

Interannual variability of the ecosystem of the Kii Channel, the Inland Sea of Japan, as influenced by bottom intrusion of cold and nutrient-rich water from the Pacific Ocean, and a recent trend of warming and oligotrophication

Interannual variability of the ecosystem of the Kii Channel, productive shelf water on the Pacific side of south‐western Japan, was analysed based on physicochemical environmental variables and abundance of major zooplankton taxa collected monthly for 12 yr from 1987 to 1999. The Kii Channel experienced both short‐term (i.e. 3–4 yr) cyclical changes and a long‐term (i.e. decadal) environmental trend. The short‐term variability was primarily associated with year‐to‐year differences in intrusion of subsurface, cold nutrient‐rich water along the bottom of the Kii Channel from the Pacific Ocean. When this bottom intrusion was intense, the Kii Channel experienced a cold, new production‐dominated ecosystem. The bottom intrusion, however, has become less intense in recent years, because of the closer proximity of the Kuroshio flow axis to the Kii Channel. Hence, there was a consistent trend towards warming and a regenerated production‐dominated ecosystem. In accordance with such environmental changes, the zooplankton community shifted towards more oceanic conditions; major herbivorous calanoids (i.e. Calanus sinicus, Paracalanus parvus (s.l.), Clausocalanus spp. and Acartia omorii) decreased, while carnivores (i.e. hydromedusae and Sagitta spp.) increased. The amount of total fish catch also decreased, while the catch of subtropical species increased. These findings lead us to conclude that the bottom intrusion from the Pacific Ocean plays a key role in determining the biological production in the Kii Channel.     

Identification of a novel cytotoxic protein, Cry45Aa, from Bacillus thuringiensis A1470 and its selective cytotoxic activity against various mammalian cell lines

Parasporal inclusion proteins produced by Bacillus thuringiensis strain A1470 exhibit strong cytotoxicity against human leukemic T cells when activated by protease treatment. One of the cytotoxic proteins was separated by anion exchange chromatography and gel filtration chromatography and designated Cry45Aa. Its gene was then expressed in recombinant Escherichia coli, in which the Cry45Aa precursor was accumulated in an inclusion body. It was solubilized in sodium carbonate buffer and processed with proteinase K, and cytotoxic activities of the protein against various mammalian cell lines were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay. The protein exhibited high cytotoxic activity against CACO-2, Sawano, MOLT-4, TCS, and HL60 cells and moderate activity against U-937 DE-4, PC12, and HepG2 cells. On the other hand, the EC50 values against Jurkat, K562, HeLa, A549, Vero, COS-7, NIH3T3, CHO, and four normal tissue cells (human primary hepatocyte cells, UtSMC, MRC-5, and normal T cells) were >2 microg/mL.     

Growth inhibition activities of Sugi bark components against Heterosigma akashiwo

In our ongoing efforts to develop new uses for wood-based waste streams, the growth inhibition activities of extracts obtained from Sugi (Cryptomeria japonica) bark were examined against Heterosigma akashiwo, otherwise known as red tide plankton. The Sugi bark was separated into its outer and inner barks and then extracted sequentially with hexane, ethyl acetate, and methanol. Strong inhibitory activities against H. akashiwo were observed in the tests involving the hexane extract from the inner and outer barks, as well as the ethyl acetate extract from the inner bark. Gas chromatography mass spectroscopy (GC–MS) analysis revealed that cubebol, phyllocladanol, 6,7-dehydroferruginol, ferruginol, and sugiol were the main components in the active extracts. These components themselves were then tested for their growth inhibition activities against H. akashiwo. Cubebol and ferruginol showed potent inhibitory activities, whereas phyllocladanol, 6,7-dehydroferruginol, and sugiol were only weakly active. Taken together, these results suggested that the Sugi bark extracts could be used as inhibition reagents against red tide plankton.     

N -Methyl- N -nitrosourea-induced Renal Tumors in Rats: Immunohistochemical Comparison to Human Wilms Tumors

N-Methyl-N-nitrosourea (MNU)-induced renal tumors in rats and Wilms tumors in humans were compared. Renal mesenchymal tumors (RMTs) and nephroblastomas (blastemal and epithelial components) in female Lewis rats treated with a single intraperitoneal injection of 50 mg/kg MNU at birth and Wilms tumors (blastemal, epithelial and mesenchymal components) in humans were analyzed for the expression of pancytokeratin (CK), vimentin, p63, α-smooth muscle actin (SMA), desmin, S-100, CD57, CD117/c-kit, Wilms tumor 1 protein (WT1) and β-catenin. The mesenchymal components of rat RMTs and human Wilms tumors expressed vimentin, SMA and β-catenin. The blastemal components of rat nephroblastomas and human Wilms tumors expressed vimentin, CD117/c-kit and β-catenin. The epithelial components of rat nephroblastomas and human Wilms tumors expressed vimentin and β-catenin. WT1 was expressed in different cellular components of rat tumors as compared with human Wilms tumors; the expression was seen in mesenchymal tumors and blastemal components of nephroblastomas in rats and epithelial components in human Wilms tumors. CK, p63 and CD57 were not expressed in rat RMTs or nephroblastomas, while CK and WT1 were expressed in epithelial components and CD57 was expressed in blastemal and epithelial components of human Wilms tumors. Rat and human tumors were universally negative for the expression of desmin and S-100. The immunohistochemical characteristics of rat renal tumors and human Wilms tumors may provide valuable information on the differences in renal oncogenesis and biology between the two species.     

Overwintering of <Emphasis Type="Italic">Taphrina wiesneri</Emphasis> within cherry shoots monitored with species-specific PCR

Taphrina wiesneri, the pathogen of witches’ broom of cherry, is highly pathogenic to Cerasus × yedoensis, the most widely planted ornamental cherry species in Japan. For adequate control of this disease, it is necessary to understand the life history of T. wiesneri. However, sites inhabited by T. wiesneri within infected trees are little understood, except during flowering and leafing periods in spring. Therefore, we attempted to detect the location of T. wiesneri in shoots of witches’ broom before flowering and leafing in spring using PCR with a T. wiesneri-specific primer pair that was designed from 69 sequences in rDNA-internal transcribed spacer region of 32 Taphrina species. DNA extracted from symptomatic and asymptomatic C. × yedoensis sampled before leafing was amplified by PCR. T. wiesneri was detected in every bud and 5-mm stem segment of symptomatic shoots, except for one stem segment, and locally inside buds and the inner bark of stem segments. These results indicate that T. wiesneri overwinters inside symptomatic shoots. Fungal hyphae were observed with an epifluorescence microscope in intercellular spaces of young leaves in symptomatic buds but not in asymptomatic ones in thin sections stained with fluorescein-isothiocyanate-conjugated concanavalin A. This observation supports the results of PCR detection.     

In vitro development and postvitrification survival of cloned feline embryos derived from preadipocytes

The aim of the present study was to optimize the conditions for in vitro development and postvitrification survival of somatic cell cloned feline embryos. To determine the effects of cell cycle synchronization of the nuclear donor cells, we cultured preadipocytes under serum starvation or conventional conditions. After two days in serum starvation culture, the proportion of synchronized donor cells at the G0/G1 phase was 91.6%. This was significantly higher than the proportion of non-synchronized cells in the proliferative phase (72.6%, P<0.05). The in vitro development of somatic cell nuclear transfer (SCNT) embryos reconstructed using donor cells treated under serum starvation conditions (normal cleavage rate of 65.7%, 46/70, and blastocyst formation rate of 20.0%, 14/70) was comparable to that of the serum supplemented group (52.5%, 31/59, and 20.3%, 12/59). Use of in vitro or in vivo matured oocytes as recipient cytoplasts equally supported development of the SCNT embryos to the blastocyst stage (11.9%, 5/42, vs. 9.5%, 2/21). SCNT-derived blastocysts were vitrified using the original minimum volume cooling (MVC) or the modified (stepwise) MVC method. Although none (n=10) of the SCNT blastocysts survived following vitrification by the original MVC method, the stepwise MVC method resulted in 100% survival after rewarming (n=11). In conclusion, we demonstrated that feline somatic cell cloned embryos with a high developmental ability can be produced irrespective of cell cycle synchronization of donor cells using either in vivo or in vitro matured oocytes. Furthermore, by utilizing a stepwise vitrification method, we showed that it is possible to cryopreserve cloned feline blastocysts.     

Contribution of a liana species, <Emphasis Type="Italic">Mucuna macrocarpa</Emphasis> Wall., to litterfall production and nitrogen input in a subtropical evergreen broad-leaved forest

Annual amounts of litterfall and nitrogen input by litterfall were measured in a subtropical evergreen broad-leaved forest to examine the contribution of a liana species, Mucuna macrocarpa Wall., to the spatial heterogeneity of litterfall production and nitrogen input. The total litterfall in the study plot was 7.1 t ha⁻¹ year⁻¹. The amount of litterfall varied with topography and was greatest at the valley bottom and decreased toward the ridges. Macuna macrocarpa litterfall was absent on the ridges although it accounted for the largest percentage, 32%, of total leaf litter production in the valley. Nitrogen input by litterfall was 69 kg ha⁻¹ year⁻¹ in the plot. Nitrogen input by litterfall was also largest at the valley bottom and decreased toward the ridges. Leaf litter of M. macrocarpa had approximately twice the nitrogen concentration of litterfall of other species. Macuna macrocarpa accounted for 42% of nitrogen input by leaf litter in the valley. The abundance and the high nitrogen concentration of M. macrocarpa intensified differences in the amount of litterfall and nitrogen input by litterfall between valleys and ridges. It was concluded that a liana species, M. macrocarpa, can contribute to the spatial heterogeneity of litterfall and may subsequently affect nutrient cycling in a subtropical evergreen broad-leaved forest on Okinawa Island.