Hydrogen peroxide produced by a low‐molecular‐weight compound in milk with high electrical conductivity in dairy cows

In dairy cows, hydrogen peroxide (H₂O₂) produced from a low‐molecular‐weight compound in milk from inflamed quarters was lower than that in milk from un‐inflamed quarters. In milk of delivery grade, characteristics of H₂O₂ production in milk with high electrical conductivity (EC) were examined in this study. Milk samples were collected from a total of 230 cows at 1‐month intervals, and the EC of skimmed milk was determined. Based on the highest and the lowest EC of a cow's quarter milk, the inter‐quarter difference of ≥0.6 mS/cm (mean + t_0.01 SE) was taken as a high EC. Milk with high EC was found in 52 quarters. In cows with milk of high EC, H₂O₂ production in milk with normal EC was higher than that in milk with high EC in the same animal but was lower than that in the control population. In milk with high EC, the decrease of H₂O₂ production correlated with the increase in EC. The production of H₂O₂ decreased in particular when the inter‐quarter difference exceeded 0.8 mS/cm. In milk collected from the same quarter 1 month before, EC changed from normal to high, and H₂O₂ production decreased. In milk from the other three quarters, EC remained normal and H₂O₂ production remained unchanged. We concluded that milk with high EC appeared in low H₂O₂‐producing cows. The results suggest that the degree of decrease in H₂O₂ production reflects the extent of quarter abnormality.     

Epigenetic assessment of environmental chemicals detected in maternal peripheral and cord blood samples

Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4￠,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.     

Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.     

Characterization of CTLA-4, PD-1 and PDL-1 of swamp and riverine type water buffaloes

Characterization of CTLA-4, PD-1 and PDL-1 genes from swamp and riverine type water buffaloes was done by molecular cloning, sequencing and phylogenetic analysis. The cloned cDNA of CTLA-4, PD-1 and PDL-1 contained an open reading frame of 666, 849 and 870 nucleotides, encoding a polypeptide of 221, 282 and 298 amino acids, respectively. Nucleotide sequence homology of both CTLA-4 and PDL-1 had 99.8% in swamp and riverine type, which gives the identical polypeptide. Meanwhile, PD-1 genes of swamp and riverine type water buffaloes had 99.2% of homology in nucleotide sequence, which has substitution of two amino acid residues. The hexapeptide motif, phosphatidylinositol 3′-kinase and potential glycosylation sites were conserved within the tribe Bovinae. Phylogenetic analysis confirmed the degree of relationship between the bubaline species and justify the distinctness of each breeds by the bootstrap value generated.     

Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks

The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmosis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals.     

Protozoan predation of Escherichia coli in hydroponic media of leafy vegetables

Multiple outbreaks of food poisoning associated with fresh vegetable consumptions have occurred in many countries. Numerous reports have described human pathogenic bacteria, such as Escherichia coli O157:H7 and Salmonella spp., that can internalize into fresh vegetables via root or leaf surfaces. While attempting to obtain the threshold concentration of internalization of E. coli inoculated into hydroponic medium during vegetable cultivation, we observed a rapid decrease in E. coli numbers. In the present study, we determined that the rapid decline in E. coli was not due to a physiological change into a viable but non-culturable (VNC) state. The population crash was instead caused by true bacterial death, as the rapid descent was also confirmed by micro-colony fluorescence in situ hybridization, a culture-independent method that can detect VNC cells. We next monitored the number of E. coli inoculated into intact or filter-sterilized hydroponic medium after cultivation of various types of plants. We found that the number of E. coli in intact hydroponic medium decreased markedly, whereas the level in filter-sterilized hydroponic medium was completely unchanged. This result suggests that biotic factors were present that could be eliminated by filtering. Robust predation of E. coli by protozoa (ciliates and flagellates) was observed using fluorescently labeled bacteria incorporated into the hydroponic medium. Finally, morphological identification of flagellates by scanning electron microscopy revealed the presence of a species of Stramenopiles. These findings suggest the importance of protozoa as bacterial feeders in hydroponic systems and hence the use of these organisms as potential control agents of human pathogenic bacteria.     

Effects of hydroxy-aluminum on flocculation and permeability of silt and clay fractions separated from paddy soils

Flocculation tests and permeability measurements were carried out for silt and clay fractions separated from surface and subsurface horizons of four paddy soils. There was not much difference in the flocculating power between A1(OH)_2.2C1_0.8 and CaSO₄. Their flocculation value, 0.3 to 3 mM, was attained by adding the reagents at the rate of 0.24 to 2.4 mmol/100 g of the silt and clay. The flocculation with A1(OH)_2.2C1_0.8 or CaSO₄ did not affect the sediment volume of the silt and clay. This sediment volume was smaller for the surface than for the subsurface horizons of each soil, suggesting that soil organic matter and repetition of wetting and drying contribute to the stable aggregation of the silt and clay in the surface horizon. The permeability was higher for the silt and clay fractions separated from the surface horizon than that from the subsurface horizon, and the addition of 2.5 to 4.2 mmol of A1(OH)_2.2C1_0.8 or CaSO₄/100g of the silt and clay increased its permeability by 1.5 to 4 times. The effect of A1(OH)_2.2C1_0.8 was more lasting than that of CaSO₄ and was recognized even after 200 ml of water passed through 1 g of the silt and clay. The effect of the flocculating reagent on the permeability was more or less enhanced by air-drying the treated silt and clay.     

Effect of free-air CO2 enrichment on the storage of carbohydrate fixed at different stages in rice (Oryza sativa L.)

Increasing global atmospheric CO₂ concentrations are expected to influence crop production. To investigate the effect on rice (Oryza sativa L.), plants were grown under ambient CO₂ (AMB) or free-air CO₂-enrichment (FACE) at CO₂ concentrations ranged from 275 to 365 μmol mol⁻¹ above AMB. We supplied ¹³CO₂ to the plants at different growth stages so we could examine the contribution of carbohydrate stored during the vegetative stage or newly fixed carbohydrate produced during the grain-filling stage to ear weight at grain maturity. In plants supplied with ¹³C at the panicle-initiation or pre-heading stages, plants grown under FACE had more starch in the stems at heading, but there was no difference in stem ¹³C content. Furthermore, there were no differences between treatments in whole-plant ¹³C contents at heading and grain maturity. In contrast, plants supplied with ¹³C at the grain-filling stage had more ¹³C in the whole plant and the ears at grain maturity under FACE than under AMB, indicating that the increased amount of photosynthate produced at the grain-filling stage under CO₂ enrichment might be effectively stored in the grains. Furthermore, regardless of when the ¹³C was supplied, plants had more ¹³C in starch in the ears at grain maturity under FACE than under AMB. Therefore, CO₂ enrichment appears to promote partitioning of photosynthate produced during both vegetative and grain-filling stages to the grains.     

Raccoon dogs (Nyctereutes procyonoides) are the new natural definitive hosts of Metagonimus hakubaensis

The definitive hosts of Metagonimus hakubaensis are reported to be hamsters, rats, mice, dogs, cats, chickens, and quails in experimental infection and Japanese water shrews in natural infection. Here we report that raccoon dogs are new natural definitive hosts of M. hakubaensis, based on morphological and molecular analyses of Metagonimus flukes collected from the host species from Aomori Prefecture, Japan. Moreover, M. hakubaensis recovered from raccoon dogs showed higher fecundity than those recovered from Japanese water shrews. Therefore, raccoon dogs were considered as a more suitable natural definitive host of M. hakubaensis than Japanese water shrews.