Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi

Twelve Heterodera species are of major economic significance in wheat and barley. Of these, H. avenae, H. filipjevi and H. latipons are among the most important ones, and sometimes coexist. The identification of Heterodera species using morphological characteristics is time consuming, requires specialized skill and can be imprecise, especially when they occur mixed in field populations. Molecular techniques can provide a more accurate way for nematode identification. This study reports the results of experiments targeting the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop species-specific primers that could be used for the identification of H. avenae and H. filipjevi. The COI gene of 9 Heterodera spp. and Punctodera punctata was partially sequenced and the resultant sequences were aligned to find unique sites suitable for the design of primers. The alignment showed variability between H. avenae, H. filipjevi and other Heterodera species. Two sets of species-specific primers were identified for the identification of both species and the conditions for their use in PCR were optimised. The specificity of the designed primers was checked by comparison with one population of P. punctata and populations of 14 other Heterodera species, nine populations of H. avenae and 10 populations of H. filipjevi originating from different countries. To test the sensitivity, the PCR was run with DNA extracted from five second-stage juveniles (J2) of H. avenae or five J2 of H. filipjevi mixed with DNA extracted from varying numbers of J2 of H. latipons. It was possible to detect as few as five J2 of H. avenae or H. filipjevi among 100 J2 of H. latipons. The two primers sets allow the detection of H. avenae and H. filipjevi where they occur in mixed populations with other Heterodera spp.     

Ingestion of proteoglycan fraction from shark cartilage increases serum inhibitory activity against matrix metalloproteinase-9 and suppresses development of N-nitrosobis(2-oxopropyl)amine-induced pancreatic duct carcinogenesis in hamster

A water extract of shark cartilage was fractionated into acidic and basic fractions by preparative isoelectric focusing on the basis of the amphoteric nature of samples. The acidic fraction was further fractionated into ethanol-soluble and -precipitate fractions. After the carcinogenesis treatment using N-nitrosobis(2-oxopropyl)amine, hamsters received a diet containing each fraction or purified chondroichin sulfate to give 0.4% (w/w) for 50 days. Only administration of the acidic ethanol-precipitate-fraction-containing diet significantly increased serum inhibitory activity against matrix metalloproteinase (MMP)-9 and reduced the number of adenocarcinomas in the pancreatic duct. The active fraction predominantly consisted of chondroichin sulfate-containing proteoglycan. However, the purified chondroichin sulfate had no significant activity. These results suggest that the protein moiety of the proteoglycan might be involved in the increase of serum inhibitory activity against MMP-9 and suppression of pancreatic carcinogenesis in hamster.     

Heritability estimates of fractures in Japanese Thoroughbred racehorses using a non‐linear model

Thoroughbred racehorses are produced by mating small numbers of Arabian stallions and native British mares, and have been improved by selection of horseracing performance for about 300 years. While these improvements led to good performance as racehorses, they exposed horses to numerous medical disorders, aggravated by extensive exercise. Fractures are frequent medical disorders in Thoroughbred racehorses. In this study, fracture heritability was estimated using 3,927 Japanese Thoroughbred racehorses to elucidate the risk of racehorse fractures. The heritability estimates of all examined fractures were low (h² = 0.06), while those of fractures in carpal bone and carpus (carpal bone plus distal radius) were moderate (h² = 0.37, 0.24, respectively). Fracture occurrence age for carpal bone and distal radius was both 3.3 years old and was younger than that for other fractures. These results indicated that a larger proportion of the variation in the studied population was due to genetic factors for carpal fractures than for other fractures, while the fractures at other bones were largely affected by environmental factors, correlated with the athlete period (number year in racing). These findings contribute to develop a management plan for suppressing racehorse fractures and improving horseracing safety.     

Composition of inorganic elements and the leaching behavior of biomass combustion ashes discharged from wood pellet boilers in Japan

To evaluate the characteristics of wood ash as fertilizer, composition of inorganic elements and the leaching behavior were studied. The leaching behavior was evaluated by three leaching methods using different solvents (water, hydrochloric acid, and acetic acid: Japanese leaching test Nos. 18 and 19 and modified toxicity characteristic leaching procedure prescribed by the US Environmental Protection Agency). The ash composition varied according to raw materials for fuel and ash collection systems. Concentration of Na, Al, Si, and V became higher in bark ashes while that of K became higher in wood ashes. Pb, Cd, Se, and Zn were more volatile and enriched in fly ashes. Of the nutrient elements, K showed high water solubility and its phytoavailability also appeared high, although the amount of water-soluble K was low in bark ash. Ca and Mg had intermediate solubility while P was less soluble in both water and acetic acid. The amount of hazardous substances having leached was low for most, but not all the ashes, in terms of Pb, Cr and As. Nevertheless, the phytoavailability of these hazardous substances after their application to the forest appeared low, due to their amphoteric leaching behavior and the acidity of Japanese forest soils.     

Establishing a laboratory animal model from a transgenic animal: RasH2 mice as a model for carcinogenicity studies in regulatory science

Transgenic animal models have been used in small numbers in gene function studies in vivo for a period of time, but more recently, the use of a single transgenic animal model has been approved as a second species, 6-month alternative (to the routine 2-year, 2-animal model) used in short-term carcinogenicity studies for generating regulatory application data of new drugs. This article addresses many of the issues associated with the creation and use of one of these transgenic models, the rasH2 mouse, for regulatory science. The discussion includes strategies for mass producing mice with the same stable phenotype, including constructing the transgene, choosing a founder mouse, and controlling both the transgene and background genes; strategies for developing the model for regulatory science, including measurements of carcinogen susceptibility, stability of a large-scale production system, and monitoring for uniform carcinogenicity responses; and finally, efficient use of the transgenic animal model on study. Approximately 20% of mouse carcinogenicity studies for new drug applications in the United States currently use transgenic models, typically the rasH2 mouse. The rasH2 mouse could contribute to animal welfare by reducing the numbers of animals used as well as reducing the cost of carcinogenicity studies. A better understanding of the advantages and disadvantages of the transgenic rasH2 mouse will result in greater and more efficient use of this animal model in the future.     

Immuno-magnetic separation and agar layer methods for the isolation of freeze-injured Yersinia enterocolitica O:8 from water

To develop an effective method to isolate an injured pathogenic Yersinia enterocolitica O:8 organism from environmental samples, we compared the isolation of freeze-injured and non-injured Y. enterocolitica O:8 and found that the isolation was more successful when immuno-magnetic separation (IMS) with anti-Y. enterocolitica O:8 antibody was used. Plating onto cefsulodin-irgasan-novobiocin (CIN) agar and Virulent Yersinia enterocolitica (VYE) agar by means of the agar layer method was found to be effective in isolating the injured cells. The alkali treatment which is generally used for selective detection of Yersinia organism failed to isolate freeze-injured pathogenic Y. enterocolitica O:8 cells. Recovery methods without using the alkali treatment were superior for detecting freeze-injured Y. enterocolitica O:8. Our results demonstrate that the IMS and the agar layer methods should be used to isolate injured pathogenic Yersinia organisms from environmental samples such as water.     

Enterocolitis associated with dual infection by Clostridium piliforme and feline panleukopenia virus in three kittens

Dual infection by Clostridium piliforme and feline panleukopenia virus (FPLV) was found in three kittens. In all cases, we found focal necrosis and desquamation of epithelial cells with occasional neutrophil infiltration in the large intestine. Large filamentous bacilli and spores were observed in the epithelium by using the Warthin-Starry method. Electron microscopy revealed the vegetative forms with characteristic peritrichous flagella and spore forms. Immunohistochemically, these bacilli showed a positive reaction with mouse antisera against the RT and MSK C. piliforme strains. Polymerase chain reaction (PCR) using cecum specimens demonstrated the 196-bp band specific to C. piliforme 16S rRNA. All three kittens were also diagnosed as FPLV-infected on the basis of the characteristic mucosal lesions, including intranuclear inclusions and PCR study for the FPLV genomic DNA. The PCR techniques are useful for confirming the C. piliforme and FPLV infection in spontaneous cases.     

Development of chloroplast DNA markers in Japanese Imperata cylindrica

Imperata cylindrica (cogongrass) is one of the most invasive grass weeds found worldwide. In Japan, this grass grows as three climate types: cool temperate (CT), common (CM) and subtropical (ST). An early flowering (E) type has been also reported. Among them, the CM type is large and most invasive. Recent global warming may cause the extension of distribution of the CM type to northern parts of Japan and may allow hybrids to form between the CM and CT or E types. The hybrids sometimes show heterosis and this leads to new weed problems. Distinguishing between the hybrids and parents on a morphological basis is difficult. We investigated polymorphisms of eight cpDNAs to enable distinction between the ecotypes and the hybrids. Fourteen haplotypes were detected in 33 Japanese clones, based on variations in eight cpDNA regions. Based on three substitutions in the psbA–matK spacer, the ORF170 intron1, the trnL(UAA) intron–trnF(GAA) spacer and indels in the trnL(UAA) intron–trnF(GAA) spacer, the haplotypes were clustered into three phylogenetic groups: I, IIa and IIb, which corresponded with the CM and ST, E and CT types, respectively. The polymorphism in the cpDNA therefore enables us to distinguish among the ecotypes and is useful for prediction of the range expansion of the CM type.