Invasion and colonization of mature apple fruit by Erwinia amylovora tagged with bioluminescence genes

Invasion and colonization of mature apple fruit by a transformant of Erwinia amylovora tagged with bioluminescence genes from Vibrio fischeri was examined. The transformant was deposited on cut surfaces of fruit stems, wounds on the shoulders and calyces, injured fruit-bearing twigs of harvested apple fruit, and cut fruit flesh. After incubation in closed stainless steel or plastic boxes at 25°C, fruit were periodically observed with a two-dimensional luminometer. The presence of the transformant in luminous areas was confirmed by isolating it on selective media. E. amylovora, when deposited in fruit stems: (1) can invade mature as well as immature apple fruit; (2) vertically and horizontally spreads and colonizes along vascular bundles, increasing its population; (3) reaches the calyx end and the flesh just under the exocarp within 3–4 days after inoculation; (4) when deposited on cut fruit flesh, irrespective of its maturity, can easily increase its population and survive 2–4 weeks or more at 25°C; and (5) even at the time of fruit maturation, can migrate within twigs rapidly and reaches the abscission layers between fruit-bearing twigs and fruit stems.     

Characterization of virulence plasmid types in Rhodococcus equi isolates from foals,pigs, humans and soil in Hungary

Rhodococcus equi isolates (204) obtained from foals (lung abscesses, lymph nodes, nasal discharge, rectal swabs) bred in 15 studs located throughout Hungary, isolates from soil samples, lymph nodes of pigs and from lesions of human patients were examined to determine genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17 kDa virulence-associated protein antigen (VapA) and 20k Da (VapB) genes by polymerase chain reaction (PCR). Plasmid DNAs were isolated and analysed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisms. Of 146 clinical isolates from foals in 15 studs, 129 (88.3%) gave positive results for the VapA gene, showing a 564 bp product of the expected size in the PCR amplification. Of the 129 clinical isolates from foals, 123 contained an 85 kb type I plasmid and the remaining six contained an 87 kb type I plasmid. Of 48 soil isolates from two horse studs, 26 (54.2%) were positive for VapA gene and contained an 85 kb type I plasmid. Of three pig isolates, one was positive for VapA gene and contained an 85 kb type I plasmid, and the remaining two were positive for the VapB gene, showing a 827 bp product of the expected size in the PCR amplification and were R. equi of intermediate virulence which contained a 95 kb type S5 plasmid. Of the seven human isolates, five were positive for VapB gene by PCR, these were R. equi of intermediate virulence, which contained a 95 kb type S5 plasmid. These results revealed that virulent R. equi strains harbouring a virulence plasmid of 85 kb type I or 87 kb type I, which have been found in clinical isolates from Europe and North and South America, are widespread in Hungary. Furthermore, same intermediately virulence plasmid type was found in both human and pig isolates.     

Gonadosomatic index and testis morphology of common carp (Cyprinus carpio) in rivers contaminated with estrogenic chemicals

To study the effect of estrogenic chemicals on fish, the gonadosomatic index (GSI = [testis weight/body weight] x 100) and testis histology of mature common carp (Cyprinus carpio) from 2 contaminated sites (Ishizu and Wada rivers, Osaka) and a control site were examined between June 1998 and March 2001. The concentration of nonylphenol, bisphenol A and 17beta-estradiol in the Ishizu river was 3-4 times higher than in the Wada river. In the pre-breeding and breeding seasons, there were no significant differences in body weight among carp from the 3 sites, the body weight of Ishizu river carp being significantly lower (p<0.05) than that of Wada river fish only in the post-breeding season. The GSI and testis weight in fish from the Ishizu river were significantly lower (p<0.05) than in control fish during all phases of gonadal cycle and lower than in Wada river fish in the pre-breeding and post-breeding season. No histological abnormalities were found in the testes of the males examined. Histological observation of the testes revealed a delay in the onset of spermatogenesis in fish from the Ishizu river compared with those from the other sites. These results clearly imply that the estrogenic chemicals in the Ishizu river adversely affect the testis development of the fish.     

Long-term excretion of Shiga toxin-producing Escherichia coli (STEC) and experimental infection of a sheep with O157

To investigate a long-term shedding of Shiga toxin (Stx)-producing Escherichia coli (STEC) from sheep, a fifteen-month study for STEC isolation from a sheep, which had yielded STEC before, was attempted. The sheep continued to shed STEC and 39 STEC were isolated. The number of STEC in the feces was estimated at 1.7 x 10(3) per gram. In addition, although Stx1-negative O157 and stx2-encoding bacteriophage were experimentally infected to the sheep, Stx-positive O157 or Stx2- producing bacterial cells were not detected. The genetical and biochemical characterization of those 39 STEC strains showed that all STEC strains produced Shiga toxin 1 (Stx1) and were divided into three classes (I to III). From phylogenetic analysis of their amino acid sequences, class-I STEC was classified as group 1 comprising mainly human STEC, and classes II/III were as group 2 comprising sheep STEC. Our results suggest that STEC easily colonized in sheep and that the sheep continued to shed STEC, showing that sheep might be an important reservoir for human STEC infection.     

Histopathological classification of systemic Mycobacterium avium complex infections in slaughtered domestic pigs

The aim of this study was to classify the histopathological features of pigs infected with Mycobacterium avium complex (MAC). We used slaughtered pig organs systemically infected with MAC. The results showed granulomatous lesions which were observed predominantly in the digestive organs and regional lymph nodes rather than respiratory organs. The histological picture showed a wide range of granulomatous stages from exudative to fibrotic reactions to the MAC infection. Eosinophils and giant cells were characteristically observed in the exudative reactions. The histopathological type in primary focus tended to be maintained in the respective organs. Most strains with the same genotype showed pathogenicity for guinea pigs irrespective of the type of granuloma. Although these findings suggest that different stages of a granulomatous lesion originating from the same causative agent might influence histological patterns, other possibilities such as the hereditary background of the host, or the effects of viral infections should be considered.     

Effects of temperature,food availability,and body size on daily growth rate of Japanese anchovy <Emphasis Type="Italic">Engraulis japonicus</Emphasis> larvae in Hiuchi-nada

With data sampled from 2003 to 2006, this study presents the effects of temperature, food availability (C), and body size on the somatic growth rate [^(g)] \hat{g} (mm day⁻¹) and on the size-specific growth rates [^(G)] \hat{G} (day⁻¹) of larval Japanese anchovy Engraulis japonicus caught in Hiuchi-nada, Seto Inland Sea, Japan, a semi-enclosed narrow sea. C was calculated using an individual-based model, which is an encounter model between copepods and anchovy where the anchovy resulted in a successful capture of the copepod. [^(g)] \hat{g} decreased with increasing anchovy length, but increased with increasing C. No significant relationship was found between [^(g)] \hat{g} and temperature. [^(G)] \hat{G} decreased with increasing temperature, but anchovy weight, on the other hand, increased with increasing C. In the literature, the mechanism of growth determination for larvae of pelagic fish species is considered to be primarily determined by sea temperature and secondarily by food availability. However, [^(g)] \hat{g} and [^(G)] \hat{G} were found to be dependent on C rather than temperature. [^(G)] \hat{G} was dependent on anchovy weight rather than C and temperature. For forecasting the number of days required for development from an arbitrary pre-recruitment size to the recruitment size, we address not only temperature but also food availability and the size effect when considering the mechanism of growth determination for larvae.     

Distribution of cells immunopositive for AM-3K, a novel monoclonal antibody recognizing human macrophages, in normal and diseased tissues of dogs, cats, horses, cattle, pigs, and rabbits

The monoclonal antibody AM-3K, which was developed using human pulmonary macrophages as the immunogen, immunocytochemically labels most human macrophages except for blood monocytes and dendritic cell populations. AM-3K also shows cross-reactivity in some animal species. To evaluate the usefulness of AM-3K, the present study investigated the detailed distribution of AM-3K-immunopositive macrophages in normal and diseased tissues of dogs, cats, horses, cattle, pigs, and rabbits. Zamboni's solution-fixed, paraffin-embedded sections were the most available for the immunocytochemistry with AM-3K. In all animal species examined, AM-3K labeled most macrophages in splenic red pulp, lymph node sinuses and thymus, and tissue macrophages in the interstitium of various organs and sites such as the kidneys, lungs, heart, pancreas, intestines, and skin. Alveolar macrophages and perivascular microglial cells were also immunoreactive for AM-3K. Interestingly, Kupffer cells of dogs, cats, and horses were labeled for AM-3K, but those of cattle, pigs, and rabbits were not. Furthermore, in tumor tissues and inflammatory lesions such as liver fibrosis and encephalomalacia that were obtained from dogs, infiltrating macrophages were stained with AM-3K, but not all infiltrating macrophages reacted to AM-3K. In addition, only 30-50% of pulmonary and peritoneal macrophages obtained from cats and dogs were reactive for AM-3K. AM-3K did not react with blood monocytes, dendritic cell populations, and osteoclasts. These observations indicate that AM-3K specifically labels most exudate and tissue macrophages in the animal species examined. However, the expression of antigens recognized by AM-3K on macrophages may be dependent on differential maturation stages or different functions evoked by some conditions. AM-3K immunoreaction products were seen on the cytoplasmic membrane of macrophages by immunoelectron microscopy. AM-3K would be useful for detection of macrophage populations in the animal species examined here.     

Late seeding of soybean after rice transplanting period using variety "Yukihomare" in central area of Hokkaido

(Jpn. J. Soil Sci.Plant Nutr., 77, 293–298, 2006)
"Kitanokaori" is a new variety of wheat for bread use bred at the National Agricultural Research Center for Hokkaido Region. The grain protein content of wheat for bread use should be higher than 120 g kg−1. Much nitrogen application is necessary to obtain high grain protein content. Therefore, it is necessary to determine the optimum amount of nitrogen to obtain the required protein content and to prevent nitrogen from remaining in the soil. The nutrition diagnosis using leaf color was investigated to predict the need and the amount of top-dressing. Field experiments were conducted for four years with nitrogen treatments in Andosol, which has moderate nitrogen fertility, and in Histosol, which is a fertile soil. The leaf color was measured using a chlorophyll meter SPAD502 (CM value) at the middle part of the leaf, avoiding the center rib. The colors of the 10·15 uppermost second leaves were measured in one plot and averaged.
A close relation was found between leaf color at the full heading stage and grain protein content at harvest. Leaf color at the full heading stage is therefore a good index to control the protein content. Considering the effect of top-dressing at the full heading stage in each CM value, the diagnosis criterion was decided. When the CM value is over 52 at the full heading stage, more nitrogen application is not needed. When the CM value is 50·52, 30 kg N ha−1 of top-dressing at the full heading stage is needed, and when the CM value is 45·50, 60 kg N ha−1 of top-dressing is needed to obtain a grain protein content of more than 120 g kg−1.     

Insecticidal effect of phthalides and furanocoumarins from Angelica acutiloba against Drosophila melanogaster

Insecticidal activity of Angelica acutiloba extract and its constituents was investigated and compared with that of rotenone. Bioassay-guided isolation of the chloroform extract of A. acutiloba against larvae of Drosophila melanogaster afforded two phthalides, (Z)-butylidenephthalide (1) and (Z)-ligustilide (2), and two furanocoumarins, xanthotoxin (3) and isopimpinellin (4). The structures of these compounds were established by spectroscopic analysis. The isolated compounds 1, 2, 3, and 4 exhibited LC(50) values of 0.94, 2.54, 3.35, and 0.82 micromol/mL of diet concentration against larvae of D. melanogaster, respectively. Against both sexes (males/females, 1:1) of adults (5-7 days old), compound 1 showed the most potent activity with a LD(50) value of 0.84 microg/adult. Compound 1 is a more active insecticide than rotenone (LD(50) = 3.68 microg/adult) and has potential as a novel insect control agent. However, compound 2 was inactive against adults. The structure-activity relationship of phthalides isolated indicated that the aromaticity appeared to play an important role in the activity of both larvae and adults. To determine the insecticide mode of action for acute adulticidal activity, acetylcholinesterase (AChE) inhibitory activity was also investigated in vitro, and the result indicated that the acute adulticidal activity of compounds 3 and 4 was due to the inhibition of AChE.