Development and evaluation of an indirect enzyme-linked immunosorbent assay for detection of foot-and-mouth disease virus nonstructural protein antibody using a chemically synthesized 2B peptide as antigen.

Forty peptides were synthesized corresponding to hydrophilic clusters of amino acids within the sequences of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP). Six peptides were studied in more detail and the most promising, a 2B peptide, was evaluated in enzyme-linked immunosorbent assay (ELISA) using sera from naive, vaccinated, and vaccinated-and-challenged cattle as well as bovine sera from field outbreaks. The performance of the new NSP peptide ELISA was compared to that of 4 commercial NSP ELISA kits. Antibody to 2B was detectable from the end of the first week to the second week after infection in most of the nonvaccinated animals and by the second to third week in vaccinated-and-challenged animals. The sensitivity of the 2B peptide ELISA was comparable to the 3ABC Ceditest (Ceditest FMDV-NS, Cedi Diagnostics B.V.; Chung et al., 2002). With some modification and further validation, this 2B test could be useful as a screening or conformational NSP test in postvaccination surveillance for FMD.     

The effects of traffic on the raccoon dog (Nyctereutes procyonoides viverrinus) and other mammals in Japan

We investigated the extent of raccoon dog roadkills, and the factors influencing them, using data from the National Expressways in Japan for 1987-1998. A high rate of raccoon dog roadkills occurred in western Japan, whereas a relatively low rate was typical of the central region and of Hokkaido. Kills on the National Expressways increased as traffic and the length of the road network increased between 1987 and 1998. The relationship between mortality and traffic volume was scale-dependent. The number of roadkills was positively correlated with traffic volume at national level, but negatively correlated with the amount of traffic along sections of an individual major road between exits. There were spring and autumn peaks, and possibly early morning and evening peaks, of mortality. Mortality was higher for roads in a cutting, where the road was closer to water, and where the roadside habitat was broad-leaved woodland. Coniferous forest was negatively associated with roadkills. The total numbers of roadkills on four types of roads (National Expressways, National, Prefectural, and Municipal roads), was estimated to be between 110,000 and 370,000 in 1998. We suggest some preventative measures which might mitigate road casualties (while remaining attentive to human safety). Roadkill data, after controlling for traffic volume, can be useful as a population trend index. Patterns for racoon dogs are compared with those for other species.     

Stability and emulsion-forming ability of water-soluble fish myofibrillar protein prepared by conjugation with alginate oligosaccharide

Carp myofibrillar protein (Mf) was conjugated with alginate oligosaccharide (AO) through the Maillard reaction under low relative humidity, and the functional properties of the Mf-AO conjugate were investigated under different NaCl concentrations and pH levels. Mf became highly solubilized at lower NaCl concentrations by conjugation with AO, with a slight loss of available lysine. The thermal stability of Mf was effectively improved by conjugation with AO. Heat treatment at 80 degrees C for 2 h had no effect on the solubility of the Mf-AO conjugate attached to 227 microg/mg of AO regardless of the NaCl concentration and pH. Furthermore, the Mf-AO conjugate showed excellent emulsion-forming ability regardless of NaCl concentration. The improved functionalities of Mf by conjugation with AO remained even at a nearly isoelectric point. These results indicate that conjugation with AO through the Maillard reaction is an effective way to prepare high-functional food material from fish muscle protein.     

Effect of bensulfuron-methyl (a sulfonylurea herbicide) on the soil bacterial community of a paddy soil microcosm

The effect of bensulfuron-methyl (BSM) on a soil microbial community in a model paddy microcosm was studied. Total bacterial numbers in the overlying water and surface soil were monitored for 2 months after the application of BSM at the field rate and a ten-fold field rate. Pentachlorophenol (PCP) was used for comparison. Neither chemical affected the total bacterial numbers remarkably, either in the overlying water or in the surface soil. In contrast, the nitrification potential was significantly suppressed by the BSM application. The bacterial community structure, as evaluated by the denaturing gradient gel electrophoresis (DGGE) of PCR amplification products from bacterial 16S rDNA, was unaffected by the BSM treatments over 8 weeks in the surface soil, compared with the control (no pesticide). In contrast, the surface soil exposed to PCP at a ten-fold field rate showed different patterns from the controls at 4 weeks and 8 weeks after application. The DGGE patterns of the overlying water were much more variable than those of the surface soil in any treatments. Cluster analysis showed that the BSM plots were classified within the same group as the control at 1 week after application and that the BSM and PCP plots from 2 weeks onward after application were grouped differently from the control. Of 22 clones excised from the DGGE gels, 20 clones belonged to the Proteobacteria and two belonged to the Verrucomicrobia. It was considered that the impact of BSM on the overall microbial community (total numbers, community structure of soil) was negligible, although BSM had an impact on some specific functions of the soil microbial community (nitrification) and a part of the community (overlying water).     

Artificial infection by transplantation of juvenile Fasciola worms into the abdominal cavity of rats

Rats were successfully infected with Japanese Fasciola sp. by transplantation of juvenile worms (JW) or metacercariae (MC) into the abdominal cavity. Moreover, the rat was investigated on its suitability for different experiments with liver flukes. JWs or MCs transplanted intraperitoneally (IP) matured in the bile duct of rats. Moreover, more stable infections were established by inoculation of JWs than MCs. About 3 of 10-15 JWs transplanted into the abdominal cavity of a rat matured and laid eggs in the bile duct. The mean prepatent period was 63.5 days in the JW inoculated group. EPG values were kept constant at a level of 10(2)-10(3) about 100 to 230 days after the transplantation of JWs. The life span of Japanese liver flukes was estimated to be about 400 days in rats. From these results, it was concluded that the rat is suitable for various experiments with Fasciola sp.     

Fine surface structure of bovine acrosome-intact and reacted spermatozoa observed by atomic force microscopy

The atomic force microscope (AFM) provides nanometer resolution, topographic data of the natural surface structure of materials. We studied the topology of the surface structure of bovine sperm heads during the acrosome reaction by AFM. In addition, we numerically analyzed the areas of the median sagittal plane of the sperm heads. Bovine frozen-thawed spermatozoa were washed, capacitated by heparin, and incubated with lysophosphatidylcholine (LPC) to induce the acrosome reaction, smeared on a cover glass, air-dried, and observed with AFM using the dynamic force (tapping) mode. AFM analysis of spermatozoa showed the clear surface structure of acrosomes, equatorial segments, postacrosomal regions and necks. Although AFM images of spermatozoa capacitated by heparin had complete acrosomes, most spermatozoa treated with LPC had no acrosomal caps as shown by AFM. These observations coincided with those obtained by light microscopy after staining with naphthol yellow S and erythrosin B. Furthermore, numerical analysis of AFM images indicated that areas of the median sagittal plane of the anterior portions of acrosome-reacted sperm heads (2679 +/- 616 pixels) were approximately 40% less than those of intact heads (4535 +/- 174 pixels, P<0.05). These results indicate that AFM can usefully observe and numerically analyze the fine surface structures of bovine spermatozoa.     

Prevalence of Giardia intestinalis infection in dogs of breeding kennels in Japan

Giardia intestinalis antigen in fecal samples was examined in 361 dogs of 14 breeding kennels located at various areas in Japan, using a commercial enzyme-linked immunosorbent assay (ELISA) kit. G. intestinalis antigen was detected in 37.4% of the fecal specimens. All of the 14 breeding kennels were positive for G. intestinalis antigen with the range from 6.7 to 59.3%. The prevalence in puppies (54.5%) was significantly (p < 0.01) higher than that in adults (30.9%). There was no difference in prevalence between males and females, and between the puppies from the mother dogs positive and negative for Giardia antigen. In conclusion, G. intestinalis widely invaded the breeding kennels in Japan.     

Changes in allergenicity and digestibility of squid tropomyosin during the Maillard reaction with ribose

The effect of the Maillard reaction on the allergenicity of squid tropomyosin (TM) was investigated. When TM was reacted with ribose (TM-ribose), its human-specific IgE-binding ability decreased markedly and alpha-chymotryptic digestibility of TM was also altered at the early stage of the Maillard reaction. On the other hand, the modification of the lysine residues in TM using 2,4,6-trinitrobenzenesulfonic acid had no effect on the allergenicity and alpha-chymotryptic digestibility of TM. Therefore, the structural change in TM induced by the Maillard reaction would cause the reduction of the allergenicity, rather than the block of lysine residues. Although peptic digestion diminished the specific IgE-binding ability of TM, the reduction of the allergenicity by the Maillard reaction remained after peptic digestion. These results suggest that hypersensitive reaction of TM-ribose in the human body might be lower than that of native TM.     

Expression and subcellular localization of GSE protein in germ cells and preimplantation embryos

We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.