First demonstration of bovine herpesvirus 2 infection among cattle by neutralization test in Japan

Seroepidemiological surveys were performed on neutralizing antibody to bovine herpesvirus 2 (BoHV-2) among cattle in Japan. A total of 1,819 sera were collected from cattle in 27 prefectures from 1997 to 1998. Antibodies were detected in only 18 sera collected from 4 prefectures. However, the most prevalent areas of the infection were found in two islands located in the subtropical zone. Additional 353 sera were collected in three including these islands in 1999-2001.The antibody-positive rates in the farms in these islands ranged from 10% to 81.1%. It was confirmed that BoHV-2 was prevalent in these areas. However, the infection seemed to be latent, because no diseases have been noticed. This is the first report showing the presence of BoHV-2 infection among cattle in Japan.     

Study of migration of neural crest cells to adrenal medulla by three-dimensional reconstruction

Adrenal medullary cells are derived from the neural crest. To study the formation process of the adrenal medulla in the embryonic period, we visualized chromaffin cells of rat embryos at 13 to 17 days of gestation using anti-tyrosine hydroxylase (TH) antiserum, and created three-dimensional images from serial tissue sections. Between 13 and 15 days of gestation, TH-positive cells (chromaffin cells) migrated from a group of TH-positive cells present dorsal to the adrenal primordium via the medial cranial end of the adrenal primordium into the adrenal primordium. At or after 16 days of gestation, the adrenal capsule was formed except on the ventral aspect of the cranial end of the adrenal gland, from which TH-positive cells penetrated into the adrenal gland. The reconstructed images showed that TH-positive cells were present contiguously from the sympathetic chain ganglia through a group of TH-positive cells ventral to the adrenal gland into the adrenal cortex, and that the group of TH-positive cells ventral to the adrenal gland communicated with the preaortic ganglion present ventral and caudal to the adrenal gland. These results suggest that neural crest cells use the same pathway to migrate to the sympathetic chain ganglia dorsal to the adrenal gland, to the adrenal gland, and to the preaortic ganglion.     

Detection of in vivo DNA damage induced by ethanol in multiple organs of pregnant mice using the alkaline single cell gel electrophoresis (Comet) assay

Ethanol is principal ingredient of alcohol beverage, but considered as human carcinogen, and has neurotoxicity. Alcohol consumption during pregnancy often causes fetal alcohol syndrome. The DNA damage is one of the important factors in carcinogenicity or teratogenicity. To detect the DNA damage induced by ethanol, we used an in vivo alkaline single cell gel electrophoresis (Comet) assay in pregnant mice organs and embryos. Pregnant ICR mice on Day 7 of gestation were treated with 2, 4 or 8 g/kg ethanol, and maternal organs/tissues and embryos were subjected to the Comet assay at 4, 8, 12 and 24 hr after ethanol treatment. Four and 8 g/kg ethanol induced DNA damage in brain, lung and embryos at 4 or 8 hr after the treatment. Two g/kg ethanol did not cause any DNA damage, and 8 g/kg ethanol only increased the duration of DNA damage without distinct increase in the degree of the damage. No significant DNA damage was observed in the liver. To detect the effect of acetaldehyde, disulfiram, acetaldehyde dehydrogenase inhibitor, was administered before 4 g/kg ethanol treatment. No significant increase of DNA damage was observed in the disulfiram pre-treated group. These data indicate that ethanol induces DNA damage, which might be related to ethanol toxicity. Since pre-treatment of disulfiram did not increase DNA damage, DNA damage observed in this study might not be the effect of acetaldehyde.     

Effects of option mitigating ammonia volatilization on CH4 and N2O emissions from a paddy field fertilized with anaerobically digested cattle slurry

A lysimeter experiment was carried out to evaluate the effects of the NH₃ volatilization mitigation by adding anaerobically digested cattle slurry (ADCS) alone, with wood vinegar (WV) or with a higher level of floodwater (HFW), on emissions of CH₄ and N₂O from a paddy soil planted with fodder rice. We have carried out the following treatments: (1) chemical fertilizer, (2) ADCS, (3) ADCS + WV, and (4) ADCS + HFW; the height of floodwater was 10 cm in the latter treatment, and it was 3 to 4 cm in the other treatments just before fertilizer applications. Nitrogen fertilizer rate added to soil in each treatment was 30 g NH₄⁺–N m⁻² (split in one basal and two top-dressing additions). Ammonia volatilization in the ADCS treatment was 2.7 g NH₃–N m⁻² throughout the growing season, and it was significantly reduced by 79% and 55% in the ADCS + WV and ADCS + HFW treatments, respectively. The total amount of CH₄ emitted in the ADCS treatment in the growing season was not significantly enhanced by the mitigation of NH₃ volatilization either by adding wood vinegar or by increasing the height of the floodwater. Negligible N₂O emissions were observed in all treatments during the growing period.     

Evaluation of genetic diversity of bread wheat landraces from Pakistan by AFLP and implications for a future collection strategy

We used amplified-fragment-length polymorphism (AFLP) markers to evaluate genetic variation in a set of bread wheat (Triticum aestivum L.) landraces and improved materials. Landraces collected from different geographic and agro-ecological zones in Pakistan in 1987, 1989 and 1991 were separated into two groups based on their geographic origins: northern (Himalaya) and south-western (Balochistan) Pakistan. Six AFLP primer combinations detected 453 AFLP markers in the 43 landrace accessions and four high-yield varieties (HYVs). Of these, 225 (49.67%) were rare (shared with < 5% of all accessions). Among these rare alleles, 23 (10.22%) were common in the Himalaya (shared with > 10% of accessions collected there) but were not found in Balochistan. We conclude that there is a higher probability of collecting rare alleles at overall, but which are in contrast locally common ones in the Himalayan region. Gene diversity was 0.17 in the Himalayan group and 0.15 in the Balochistan group. Considerable genetic variability was found in both groups. Accessions from different agro-ecological zones were indistinguishable by cluster analysis, indicating intensive seed trading within the country. Cluster analysis indicated that the landraces and the HYVs are genetically distinct; suggesting that genetic erosion of wheat landraces has been unlikely taken in place. This study provides an example of how analysis of existing materials and data, can serve as a basis for future collection planning and conservation policies.     

Genetic diversity and population structure of ‘Khao Kai Noi’, a Lao rice (Oryza sativa L.) landrace,revealed by microsatellite DNA markers

Rice (Oryza sativa L.) is the main food for people in Laos, where it has been grown and eaten since prehistory. Diverse landraces are grown in Laos. ‘Khao Kai Noi’, a landrace favored for its eating quality, is held in the nationwide collection of traditional landraces in the Lao national genebank. Genetic diversity is crucial for sustainable use of genetic resources and conservation. To investigate the genetic diversity of ‘Khao Kai Noi’ for conservation, we genotyped 70 accessions by using 23 polymorphic simple sequence repeat markers. The markers generated 2 to 17 alleles (132 in total), with an average of 5.7 per locus. The total expected heterozygosity over all ‘Khao Kai Noi’ accessions was 0.271. Genetic variation was largest among accessions and smallest within accessions. Khao Kai Noi accessions were classified into three different genetic backgrounds, but there was unclear association between the three inferred population and name subgroups and geographical distribution. Most of the accessions were clustered with temperate japonica and showed genetic relatedness to rice from neighboring provinces of Vietnam, suggesting a Vietnamese origin. The results of this study will contribute to the conservation, core collection and future breeding of the Khao Kai Noi population.     

Occurrence of equine coital exanthema in pastured draft horses and isolation of equine herpesvirus 3 from progenital lesions

During the period from 2001 to the following year, progenital diseases had been epidemic among the draft stallions and mares pastured together in Iwate Prefecture, the northeastern district of Japan. A stallion and 8 of 31 mares were affected in 2001, and 1 of 2 stallions and 10 of 36 mares in 2002. The clinical symptoms consisted of the formation of papules, pustules, ulcers and scabs on the progenital skin and mucosa in stallions and mares. In 2002, Equine herpesvirus 3 (EHV3) was isolated from 2 mares and the glycoprotein G gene of the virus detected from a stallion and 4 mares by polymerase chain reaction. Serum neutralizing tests showed that 12 of 38 horses, 10 clinically and 2 subclinically affected, changed to be positive for the EHV3 antibody. The results suggest that the horses were affected with equine coital exanthema (ECE) through coitus. Five mares with the antibody at the pre-pastured period may have been the possible origins of EHV3 infection in 2002, although the exact origin in 2001 remains unknown. The artificial insemination was performed for the prevention of ECE spreading through coitus on the pasture in 2003. There was no epidemic of the disease in 31 mares, although 3 mares with the antibody at the pre-pastured period showed the significant increase in the titers during the pastured period.     

Isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the NC99 strain

A new equine coronavirus was isolated from the feces of adult horses with pyrogenic and enteric disease. The disease outbreak was mainly observed among 2- to 4-year-old horses living in stables of a draft-horse racetrack in Japan. On comparing the isolated virus (isolate Tokachi09) with the equine coronavirus NC99 strain, no significant differences were observed in several biological properties such as hemagglutinating activity, antigenicity (in indirect immunofluorescence and neutralization tests), and one-step growth (in cell culture). The sequences of the nucleocapsid and spike genes of isolate Tokachi09 showed identical size (1341 and 4092 nucleotides, 446 and 1363 amino acids, respectively) and high similarity (98.0% and 99.0% at the nucleotides, 97.3% and 99.0% at the amino acids, respectively) to those of strain NC99. However, the isolate had a 185-nucleotide deletion from four bases after the 3'-terminal end of the spike gene, resulting in the absence of the open reading frame predicted to encode a 4.7-kDa nonstructural protein in strain NC99. These results suggest that the 4.7-kDa nonstructural protein is not essential for viral replication, at least in cell culture, and that the Japanese strain probably originated from a different lineage to the North American strain. This is the first equine coronavirus to be isolated from adult horses with pyrogenic and enteric disease.     

Changes in the aroma compounds of sake during aging

Changes in the aroma of sake during aging were investigated by aroma extract dilution analysis (AEDA) and quantitative analysis using the stir bar sorptive extraction method. In AEDA, more odor zones were detected in aged sake than in fresh sake. The dilution factors of aldehydes, polysulfides, and some esters were greater in the aged sake, and their increase during aging was confirmed through a quantitative analysis of sake stored for 0-35 years. Among these compounds, 3-methylbutanal, methional, and dimethyltrisulfide (DMTS) were present in aged sake at concentrations exceeding their odor thresholds, and the highest odor active value was observed for DMTS. Sensory tests showed that supplementation with DMTS contributed to both the total odor intensity and the sulfury odor of aged sake aroma.     

Effect of antimicrobial agents on the production and release of shiga toxin by enterotoxaemic Escherichia coli isolates from pigs

Edema disease (ED) of pigs is an enterotoxaemic disease caused by enterotoxaemic Escherichia coli (ETEEC) infection. Antimicrobial therapy for pigs with ED is controversial because it may induce death of sickish piglets. In this study, we investigated the effects in vitro of 7 antimicrobial agents, ampicillin, gentamicin, colistin, bicozamycin, fosfomycin, sulfamethoxazole-trimethoprim and enrofloxacin, on the release and production of shiga toxin (Stx) 2e by ETEEC strains. We found that more Stx 2e accumulated in the bacterial cells than was released into supernatant. Associated with inhibition of cell wall synthesis, the exposure to ampicillin or fosfomycin increased the release of Stx 2e. The production levels of Stx 2e in all antimicrobial-treated cultures were equal to the level in the control or less than in the control. These results suggest that cell wall synthesis inhibitors, such as ampicillin and fosfomycin, may change for the worse in the signs in ETEEC infectious pigs. On the other hand, gentamicin, colistin, bicozamycin and enrofloxacin may be useful for the treatment of pigs with ED.