Quantitative tyramine analysis method for Apis mellifera larvae infected with Melissococcus plutonius, the causative agent of European foulbrood.

Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus plutonius that causes European foulbrood in honey bee brood. In the present study, we developed a method for quantitative analysis of tyramine in culture medium and honey bee larvae with a limit of quantitation of 3 ng/mL and a recovery rate of >97% using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry and deuterium-labeled tyramine, demonstrating for the first time that a highly virulent M. plutonius strain actually produces tyramine in infected larvae. This method will be an indispensable tool to elucidate the role of tyramine in European foulbrood pathogenesis in combination with exposure bioassays using artificially reared bee larvae.

Occurrence and the potential ecological risk of veterinary antimicrobials in swine farm wastewaters in Japan: Seasonal changes, relation to purchased quantity and after termination of oxytetracycline usage.

This study provides the first comprehensive investigation of the residual concentrations of eight classes of antimicrobial agents (AMs, 20 compounds) in 13 swine wastewater treatment facilities in Japan. These facilities implemented the aerobic activated sludge (AS) or its alternative methods. The maximum concentrations before treatment were found at the level of 7100, 6900, 6000, 3600, 3400, and 1400 µg/L for tilmicosin, oxytetracycline (OTC), chlortetracycline, lincomycin , sulfamethoxazole, and trimethoprim, respectively. The highest detection rate (96.3%) in influents was noted for the morantel, which was a feed additive. The seasonal difference in residual concentration was much greater for tetracyclines (TCs) and macrolides (MLs) when their residual concentrations were high, especially in the cold season. There was a positive correlation between the purchased quantity of TCs and fluoroquinolones (FQs) and their residue levels detected in the effluents (p < 0.01). The estimated removal rate of AMs was greater than 80%. In contrast, on a few occasions, it was diminished due to failing operating conditions, such as water temperature and AS rate in the aeration tank. The estimated ecological risks of AMs in effluents based on risk quotients (RQs) considered to enhance the selection pressure for drug resistance (RQs-AMR) were high for TCs and FQs, whereas ecotoxicological effects (RQs-ENV) to aquatic organisms were higher for sulfonamides and MLs. When OTC usage ceased, its concentration in wastewater decreased rapidly; however, it remained longer period in the effluents, probably due to OTC desorption from the AS. The concentrations (and respective RQs) of TCs were decreased by >99.8% and >92% in the influents and effluents, respectively. This data suggested that it is essential to reduce the amount used and introduce more efficient methods and operating conditions to constantly remove AMs during the treatment to reduce the risk of AM discharge from swine farms.

De novo transcriptome assembly associated with fumonisin production by the rice pathogen Fusarium fujikuroi.

The present study employed a next-generation sequencing method to assemble a de novo transcriptome database designed to distinguish gene expression changes exhibited by the fumonisin-producing fungus Fusarium fujikuroi when grown under 'fumonisin-producing' compared to 'non-fumonisin-producing' conditions. The raw data of this study have been deposited at DNA Data Bank of Japan (DDBJ) under the accession ID DRA006146.

Mycotoxin Contamination in Japanese Domestic Feed.

This study summarizes the levels of the mycotoxins aflatoxin B1, deoxynivalenol, zearalenone, ochratoxin A, and fumonisin B1 in domestic feed produced in Japan. We collected samples of Japanese domestic feed used in livestock farming establishments or by small farmers from April 2012 to March 2014, and measured mycotoxin concentrations in each sample. Regarding corn, deoxynivalenol had the highest detection rate (84%), maximum value (2370 µg/kg), and mean value (400 µg/kg). These results suggest that deoxynivalenol is a major mycotoxin contaminant in Japanese-produced domestic feed. Zearalenone and fumonisin B1 presented the second highest detection rates. The maximum concentration of zearalenone was 1200 µg/kg in grass samples, but its median was under 25 µg/kg, and, overall, it occurred at low concentrations. The detection rate of fumonisin B1 was about 30%, but its maximum concentration in corn was 2400 µg/kg, slightly higher than deoxynivalenol. Overall, mycotoxin concentrations were lower in grass than in corn. Although deoxynivalenol was detected in samples collected from all regions, concentrations in samples from Hokkaido were particularly high. Fumonisin B1 was mainly distributed in Kanto and the southern regions. Concentrations of ochratoxin A and aflatoxin B1 were low; however, the maximum concentration of aflatoxin B1 was 22 µg/kg in corn. Although aflatoxin B1 and ochratoxin A were rarely detected, they occasionally presented high levels, therefore requiring attention. Overall, mycotoxins produced by Fusarium sp. fungi require attention in Japanese-produced domestic feed.

Effect of Different Silage Storing Conditions on the Oxygen Concentration in the Silo and Fermentation Quality of Rice.

We investigated the effects of different silage storing conditions on the oxygen concentration in the silo and fermentation quality of rice (Oryza sativa L.). Forage rice was ensiled in bottles (with or without space at the bottlemouth, with solid or pinhole cap, and with oxygen scavenger, ethanol transpiration agent, oxygen scavenger and ethanol transpiration agent, or no adjuvant) and stored for 57 days. The oxygen concentration decreased with the addition of the oxygen scavenger and increased with that of the ethanol transpiration agent. The oxygen scavenger facilitated silage fermentation and fungus generation, whereas the ethanol transpiration agent suppressed silage fermentation and fungus generation. However, the combined use of the oxygen scavenger and ethanol transpiration agent facilitated silage fermentation and also suppressed fungus generation. Overall, this study revealed the negative effects of oxygen on the internal silo and the positive effects of the combined use of the oxygen scavenger and ethanol transpiration agent on silage fermentation quality.

A single nucleotide polymorphism in the translation elongation factor 1α gene correlates with the ability to produce fumonisin in Japanese Fusarium fujikuroi.

PCR-RFLP based on the translation elongation factor 1α (TEF) gene was developed to identify Fusarium fujikuroi in the Fusarium (Gibberella) fujikuroi species complex. Ninety-three strains, most of which were obtained from various sources in Japan, were identified as F. fujikuroi and their capability to produce fumonisin was investigated using an in vitro assay. Fumonisin production was detected in 50 strains isolated from maize, strawberry, wheat, and rice, whereas it was undetectable in 43 strains derived from rice seeds and rice seedlings carrying the bakanae disease, and from unknown sources. A single nucleotide polymorphism in the TEF gene (T618G) correlated with the ability to synthesize fumonisin.

Changes in the concentration of fumonisins in forage rice during the growing period, differences among cultivars and sites, and identification of the causal fungus.

To clarify the changes in rice fumonisin (FUM) concentrations, we conducted field cultivation of 10 forage rice cultivars and inoculation with fumonisin-producing fungal isolates. We cultivated 10 forage rice cultivars at the NARO Institute of Livestock and Grassland Science and one cultivar at two additional farmland sites in Japan in 2011 and 2012. Fusarium fujikuroi, which primarily infects plants shortly after heading, was inoculated on rice just after heading, and we sampled heads at the yellow-ripe and full-ripe stages to assess FUM concentrations. We found differences among cultivars in the FUM concentration and differences among the sites for the same cultivar, but no cultivar had high levels in leaves and stems. Fusarium fujikuroi was the main fumonisin producer. The FUM concentration in heads increases from <1 to 4760 µg/kg DM after the yellow-ripe stage. To control FUM levels, it is necessary to select low-FUM cultivars and manage the cultivation environment.

Lactobacillus silagei sp. nov., isolated from orchardgrass silage.

A Gram-reaction-positive, facultatively anaerobic, non-spore-forming and catalase-negative rod-shaped bacterial strain, designated IWT126(T), was isolated from orchardgrass (Dactylis glomerata L.) silage preserved in Hachimantai, Iwate, Japan. The isolate showed growth at 15-45 °C, pH 3.5-7.5 and with 4.0 % (w/v) NaCl. The cell wall peptidoglycan did not contain meso-diaminopimelic acid, and the DNA G+C content was 45.6 mol%. The major cellular fatty acids were C16 : 0 and C19 : 1 cyclo 9,10. Based on 16S rRNA gene sequence similarity, strain IWT126(T) was classified as a member of the genus Lactobacillus and was most closely related to Lactobacillus odoratitofui YIT 11304(T) (98.7 %), Lactobacillus similis JCM 2765(T) (98.5 %), Lactobacillus collinoides JCM 1123(T) (97.6 %), Lactobacillus paracollinoides DSM 15502(T) (97.6 %) and Lactobacillus kimchicus DCY51(T) (96.9 %). Based on sequence analysis of the phenylalanyl-tRNA synthase α-subunit (pheS) gene, strain IWT126(T) was well separated from its phylogenetic neighbours in the genus Lactobacillus. Based on physiological, biochemical and genotypic results, as well as low DNA-DNA relatedness to recognized phylogenetic relatives in the genus Lactobacillus, classification of strain IWT126(T) as a representive of a novel species named Lactobacillus silagei sp. nov. is proposed. The type strain is IWT126(T) ( = JCM 19001(T) = DSM 27022(T)).

Description of Lactobacillus iwatensis sp. nov., isolated from orchardgrass (Dactylis glomerata L.) silage, and Lactobacillus backii sp. nov.

Two bacterial strains, designated IWT246(T) and IWT248, were isolated from orchardgrass (Dactylis glomerata L.) silage from Iwate prefecture, Japan, and examined for a taxonomic study. Both organisms were rod-shaped, Gram-stain-positive, catalase-negative, facultatively anaerobic and homofermentative. The cell wall did not contain meso-diaminopimelic acid and the major fatty acids were C18 : 1ω9c and C19 cyclo 9,10/:1. Comparative analyses of 16S rRNA, pheS and rpoA gene sequences revealed that these strains were novel and belonged to the genus Lactobacillus. Based on 16S rRNA gene sequence similarity, the isolates were most closely related to the type strains of the following members of the genus Lactobacillus: Lactobacillus coryniformis subsp. coryniformis (96.7 % similarity), L. coryniformis subsp. torquens (96.6 %), L. bifermentans (95.5 %) and L. rennini (94.1 %). However, the 16S rRNA gene sequences of both IWT246(T) and IWT248 were 99.7 % similar to that of 'Lactobacillus backi' JCM 18665; this name has not been validly published. Genotypic, phenotypic and chemotaxonomic analyses confirmed that these novel strains occupy a unique taxonomic position. DNA-DNA hybridization experiments demonstrated genotypic separation of the novel isolates from related Lactobacillus species. The name Lactobacillus iwatensis sp. nov. is proposed for the novel isolates, with strain IWT246(T) ( = JCM 18838(T) = DSM 26942(T)) as the type strain. Our results also suggest that 'L. backi' does represent a novel Lactobacillus species. The cells did not contain meso-diaminopimelic acid in their cell-wall peptidoglycan and the major fatty acids were C16 : 0, C19 cyclo 9,10/:1 and summed feature 10 (one or more of C18 : 1ω11c, C18 : 1ω9t, C18 : 1ω6t and unknown ECL 17.834). We therefore propose the corrected name Lactobacillus backii sp. nov., with the type strain JCM 18665(T) ( = LMG 23555(T) = DSM 18080(T) = L1062(T)).

Changes in the concentrations of fumonisin, deoxynivalenol and zearalenone in corn silage during ensilage.

We assessed the production of the mycotoxins fumonisin, deoxynivalenol and zearalenone during the ensiling of corn. Corn was harvested at yellow-ripe or full-ripe stage and separated into the stem and leaf parts and the ear parts, including bracts. Each material was ensiled under five conditions: (1) no fungus added, anaerobic conditions; (2) no fungus added, aerobic conditions; (3) mycotoxin-producing fungus added, anaerobic conditions; (4) mycotoxin-producing fungus added, aerobic conditions; and (5) mycotoxin-producing fungus added to autoclaved material, aerobic conditions. After 40 days of ensilage, we analyzed the silage fermentative quality and mycotoxin concentration. The fermentative quality of all materials was good in treatments (1) and (3), because the pH < 4 increased the lactic acid content preventing mycotoxin levels from increasing. In treatments (2) and (4), fermentative quality of all materials was poor, and mycotoxin levels were slightly increased. In treatment (5), fermentative quality was poor, and mycotoxin levels were increased remarkably. These results indicate that mycotoxins are not produced under anaerobic conditions and are hardly produced under aerobic condition during the ensiling of corn. Our findings suggest that almost all mycotoxins in corn silage are produced pre-harvest.

Lactobacillus oryzae sp. nov., isolated from fermented rice grain (Oryza sativa L. subsp. japonica).

The taxonomic position of three Lactobacillus-like micro-organisms (strains SG293(T), SG296 and SG310) isolated from fermented rice grain (Oryza sativa L. subsp. japonica) in Japan was investigated. These heterofermentative lactic acid bacteria were Gram-stain-positive, rod-shaped, facultatively anaerobic, non-motile, non-spore-forming and did not show catalase activity. 16S rRNA gene sequence analysis of strain SG293(T) revealed that the type strains of Lactobacillus malefermentans (98.3 %), Lactobacillus odoratitofui (96.2 %), Lactobacillus similis (96.1 %), Lactobacillus kimchicus (96.1 %), Lactobacillus paracollinoides (95.9 %) and Lactobacillus collinoides (95.7 %) were the closest neighbours. Additional phylogenetic analysis on the basis of pheS and rpoA gene sequences, as well as biochemical and physiological characteristics, indicated that these three strains were members of the genus Lactobacillus and that the novel isolates had a unique taxonomic position. The predominant cellular fatty acids were C18 : 1ω9c and C19 : 1 cyclo 9,10. Because low DNA-DNA hybridization values among the isolates and Lactobacillus malefermentans JCM 12497(T) were observed, it is proposed that these unidentified isolates be classified as a novel species of the genus Lactobacillus, Lactobacillus oryzae sp. nov. The type strain is SG293(T) (= JCM 18671(T) = DSM 26518(T)).

Changes of fumonisin production in rice grain during ensiling.

We assessed fumonisin production during the ensiling of rice grain. Rice grain was harvested at the full-ripe stage and prepared as rough rice, crushed rough rice, brown rice or crushed brown rice. Each material was ensiled under six conditions: (1) no fungus, anaerobic; (2) no fungus, aerobic; (3) water added, anaerobic; (4) water and fumonisin-producing fungus added, anaerobic; (5) water and fumonisin-producing fungus added, aerobic; or (6) fumonisin-producing fungus added to autoclaved material, aerobic. After 40 days of ensilage, we analyzed the silage fermentative quality and fumonisin concentration. The fermentative quality of all materials was good in treatments (3) and (4) (pH < 4), reasonable in treatment (5) (pH = 5∼6) and unacceptable in treatments (1) and (2) (pH > 6.5). The fumonisin concentration was low in all materials in treatments (1) to (4), slightly increased in the three materials other than rough rice in treatment (5), and enormously increased in all materials in treatment (6). The results indicate that the fumonisin-producing fungus does not produce fumonisin in anaerobic conditions. It is important that an anaerobic condition be maintained during ensiling in order to reduce the fumonisin content in rice grain silage.

Weissella oryzae sp. nov., isolated from fermented rice grains.

A taxonomic study was conducted on two Gram-reaction-positive, catalase-negative, irregular short-rod-shaped or coccoid lactic acid bacteria, designated strains SG25(T) and SG23, that were isolated from grains of fermented Japanese rice (Oryza sativa L. subsp. japonica). A phylogenetic analysis based on 16S rRNA gene sequence data clearly showed that the strains belonged to the genus Weissella and were most closely related to Weissella soli LMG 20113(T) (with a sequence similarity of 96.9 % for each novel strain). The peptidoglycan of each strain contained the amino acids glutamic acid, lysine, serine and alanine in a molar ratio of 1.0 : 1.2 : 0.5 : 3.0, respectively. On the basis of the unusual phenotypic characteristics of the novel strains and the low levels of DNA-DNA relatedness recorded between each novel strain and Weissella soli JCM 12536(T), strains SG25(T) and SG23 represent a single novel species in the genus Weissella, for which the name Weissella oryzae sp. nov. is proposed. The type strain is SG25(T) ( = JCM 18191(T)  = DSM 25784(T)).

Lactobacillus hokkaidonensis sp. nov., isolated from subarctic timothy grass (Phleum pratense L.) silage.

Four strains of Gram-positive, non-spore-forming, rod-shaped, catalase-negative and non-motile lactic acid bacteria, LOOC260(T), LOOC253, LOOC273 and LOOC279, were isolated from timothy grass (Phleum pratense L.) silage produced in Hokkaido, a subarctic region of Japan. These isolates grew at 4-37 °C, indicating the psychrotolerant nature of these strains. Phylogenetic analysis on the basis of 16S rRNA and pheS gene sequences, as well as biochemical and physiological characteristics, indicated that these four strains were members of the genus Lactobacillus. 16S rRNA gene sequence analysis of strain LOOC260(T) demonstrated that the closest neighbours were the type strains of Lactobacillus suebicus (97.7 %), Lactobacillus oligofermentans (96.7 %) and Lactobacillus vaccinostercus (96.7 %). Strain LOOC260(T) showed low levels of DNA-DNA association with Lactobacillus suebicus JCM 9504(T) (14.7 ± 3.5 %), Lactobacillus oligofermentans JCM 16175(T) (15.1 ± 4.8 %) and Lactobacillus vaccinostercus JCM 1716(T) (10.7 ± 3.0 %). The cell wall contained meso-diaminopimelic acid and the major fatty acids were C18 : 1ω9c and C19 : 1 cyclo 9,10. On the basis of phenotypic, physiological and phylogenetic evidence, these isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus hokkaidonensis sp. nov. is proposed. The type strain is LOOC260(T) ( = JCM 18461(T) = DSM 26202(T)).

Strain-dependent effects of inoculation of Lactobacillus plantarum subsp. plantarum on fermentation quality of paddy rice (Oryza sativa L. subsp. japonica) silage.

Paddy rice has been of particular interest as a forage crop in Japan. In this study, the isolated strains TO1000, TO1001, TO1002, and TO1003 were characterized by phenotypic and genotypic approaches. These strains were identified as Lactobacillus plantarum subsp. plantarum by species-specific PCR. Phenotypic characteristics varied among different strains of the same subspecies, and the strains represented unique and diverse phenotypes related to fermentation factors, such as carbohydrate assimilation and range of pH and temperature allowing growth. PCR analysis revealed that the patterns of presence/absence of known plantaricin genes differed in a strain-specific manner. Using these strains as inoculants for preparation of whole crop paddy rice silage, fermentation quality was significantly improved, as shown by lower pH, higher lactic acid content, and inhibition of the growth of undesirable microorganisms such as molds, coliform bacteria, and clostridia, after 30 and 60 days of storage, with effectiveness differing from strain to strain. These observations suggest that suitable candidates for bacterial inoculants in silage preparation should be screened at the strain level. Strain TO1002 may be useful for producing silage inoculants for the production of well-preserved whole crop paddy rice silage.

Identification and characterization of lactic acid bacteria isolated from mixed pasture of timothy and orchardgrass, and its badly preserved silage.

In order to understand the relationship between lactic acid bacteria (LAB) species and silage fermentation, a total of 65 LAB strains isolated from mixed pasture of timothy (Phleum pratense L.) and orchardgrass (Dactylis glomerata L.), and its badly preserved silages were subjected to phenotypic and genetic analysis. According to these analyses, the isolates were divided into 13 groups, including Enterococcus gallinarum, Lactobacillus acidipiscis, L. coryniformis subsp. coryniformis, L. coryniformis subsp. torquens, L. curvatus, L. paraplantarum, L. plantarum subsp. argentoratensis, L. plantarum subsp. plantarum, L. sakei subsp. carnosus, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weissella hellenica, Weissella paramesenteroides and Carnobacterium divergens. This is the first report to document that C. divergens, L. acidipiscis, L. sakei subsp. carnosus, L. garvieae, phenotypically novel L. lactis subsp. cremoris, E. gallinarum and W. hellenica are present in vegetative forage crops. L. plantarum group strains were most frequently isolated from the badly preserved silages. Some isolates showed a wide range of growth preferences for carbohydrate utilization, optimal growth pH and temperature in vitro, indicating that they have a high growth potential. These results are useful in understanding the diversity of LAB associated with decayed silage of timothy and orchardgrass.

Genotypic and phenotypic characterization of lactic acid bacteria isolated from Italian ryegrass silage.

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.

Polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in rice plants: possible contaminated pathways.

Uptake of dioxins was confirmed in rice plants. We determined (i) dioxin concentrations and isomer profiles in leaf+stem samples of rice plants grown in three soils with different dioxin concentrations and isomer profiles and (ii) temporal changes in dioxin concentrations and isomer profiles in rice grown in a paddy field on which agricultural chemicals containing dioxin impurities had been used in previous years. Principal-component and chemical mass-balance analyses of the data showed that dioxin concentrations and isomer profiles in the rice grown in the different soils were similar, even though the dioxin concentrations and isomer profiles of the soils were different. However, the dioxin concentrations in the rice were influenced by dioxin levels in the atmospheric gas phase. Changes in dioxin levels during the growing period of the rice were also strongly influenced by adhesion of the soil to plant components during the early stage of growth, but gradually the influence of the atmospheric gas phase became predominant. Calculated dietary uptake estimates indicated that, even in rice grown in Japan on soil contaminated with high concentrations of dioxins, the amount of dioxins ingested by humans from rice is extremely small, and the amount of dioxins ingested by livestock from forage rice is similar to that for other forage crops.