Preparation and characterization of polyclonal antibody against resting spores of Olpidium virulentus, fungal vector of lettuce big-vein disease

Research on Olpidium virulentus, the vector of the serious lettuce big-vein disease, is difficult because its resting spores persist in soil and retain virus for years and the fungus is not culturable. We developed protocols to obtain purified, viable resting spores from infected roots using an enzymatic treatment and two-step density gradient centrifugation and to generate a polyclonal antibody against these spores. The antibody was species-specific in a direct immunostaining assay and in western blots produced two specific bands (ca. 30.5, 29.0 kDa) against resting spores. The detection limit for resting spores in an indirect ELISA was 200 spores/100 μL.     

Mammary lesions associated with bovine herpesvirus type 4 in a cow with clinical mastitis

Intranuclear eosinophilic inclusion bodies were seen in the lactiferous duct and sinus epithelium of mammary tissues collected from a cow with clinical mastitis. Transmission electron microscopy revealed herpesvirus particles in these cells. Immunolabeling against anti bovine herpesvirus type 4 (BHV-4) rabbit serum was detected in nuclei that had intranuclear inclusion bodies. In addition, BHV-4 was isolated from the mammary tissue. The viral DNA was detected by nested PCR from the same tissue. This is the first report to describe mammary lesions in association with BHV-4.     

Sequence and polymerase chain reaction–restriction fragment length polymorphism analysis of the large subunit rRNA gene of bivalve: Simple and widely applicable technique for multiple species identification of bivalve larva

ABSTRACT:   One of the biggest and long-standing difficulties in investigation of larval ecology in the field is species-level identification. In the present study, we developed polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis based on the large subunit (LSU) rRNA gene (rDNA) D1/D2/D3 region for identification of multiple species of bivalve larvae using 14 species of bivalve collected from Maizuru Bay. The LSU rDNA D1/D2/D3 region of all analyzed species could be amplified by PCR using bvD1f/bvD3r primers, and RFLP analysis by Hae III digestion on the PCR products showed species-specific fragment patterns. Furthermore, this analysis applied to single bivalve larvae in Maizuru Bay revealed efficient amplification of the target region and the species-specific pattern from 80% of the larvae, 75% of which showed a pattern that matched a certain pattern of the adult bivalves. In addition, the analysis of inter- and intraspecies variation of the LSU rDNA D1/D2/D3 region using the sequence data of the genus Crassostrea from the DDBJ database showed high applicability of this RFLP analysis on closely related species. Because of the wide applicability and technical simplicity, this method can become the standard for the identification of bivalve larvae species once the sequence data of the LSU rDNA D1/D2/D3 region of many bivalve species have been accumulated.     

Oogenesis and relevant changes in egg quality of abalone Haliotis discus hannai during a single spawning season

Oogenesis of abalone Haliotis discus hannai was examined histologically during a single spawning season using broodstock of various maturation conditions, which were controlled by effective accumulative temperature (EAT). The quality of eggs spawned was determined in relation to oogenesis. For histological examinations, three to five females were sacrificed at 300, 600, 850, 1050, and 1150 °C days EAT, without induction of artificial spawning. Other females were successfully induced to spawn at 700 °C days EAT and were reared following spawning. Three of these females were then sacrificed every 200 °C days EAT until 1300 °C days EAT. Gonad histology showed that two oocyte cohorts matured in H. discus hannai ovaries during a single spawning season. One mature oocyte cohort could be spawned in multiple times. The second oocyte cohort started developing after the first oocyte cohort had been spawned or reabsorbed, and became fully mature 400 °C days EAT after the first cohort was depleted. For egg quality measurements, three to five females were successfully induced to spawn at 850, 1050, 1150, 1900, and 2350 °C days EAT (Experiment 1). Three females were induced to spawn twice, at 700 and 1500 °C days EAT, resulting in two batches of eggs from the same individuals (Experiment 2). Total lipid and protein content of eggs were measured and were greater in eggs from the second cohort than in eggs from the first cohort. No carbohydrates were detected in eggs and there was no difference in cytoplasm volume between the two cohorts. In hatcheries producing H. discus hannai, it is important to increase post-larval starvation tolerance by increasing the quality of eggs, to yield higher and more consistent survival. The results of this study suggest that H. discus hannai hatcheries should use eggs from the second oocyte cohort, which are of higher quality, rather than eggs from the first oocyte cohort.     

Isolation and identification of indigestible pyroglutamyl peptides in an enzymatic hydrolysate of wheat gluten prepared on an industrial scale

An enzymatic hydrolysate of wheat gluten was further digested in vitro with porcine pepsin and pancreatin to obtain an indigestible peptide. Indigestible pyroglutamyl peptide was isolated from the digest by strong cation-exchange, size-exclusion, and reversed-phase chromatographies. The pyroglutamyl peptide was digested with pyroglutamate aminopeptidase, and the digest was reacted with phenyl isothiocyanate. The resultant phenylthiocarbamyl (PTC) peptides were purified by reversed-phase HPLC by using binary gradient elution with ammonium acetate buffer, pH 6.0, and acetonitrile. The PTC peptides were analyzed with an automatic peptide sequencer on the basis of the Edman degradation method with a modified program. Some pyroglutamyl peptides were also analyzed by fast-atom bombardment ionization mass spectrometry without the pyroglutamate amino peptidase digestion. Consequently, pyroGlu-Asn-Pro-Gln, pyroGlu-Gln-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gly-Gln-Gly-Gln, pyroGlu-Gln, pyroGlu-Gln-Pro, pyroGlu-Ile-Pro-Gln, pyroGlu-Ile-Pro, pyroGlu-Gln-Pro-Leu, pyroGlu-Gln-Phe-Pro-Gln, pyroGlu-Ser-Phe-Pro-Gln, pyroGlu-Phe-Pro-Gln, and pyroGlu-Gln-Pro-Pro-Phe-Ser were identified.     

Compositions of fatty acid hydroperoxide positional isomers in sweet smelt, yellowtail and rainbow trout livers phosphatidylcholine

ABSTRACT: Hydroxy fatty acid isomers derived from phosphatidylcholine hydroperoxides (PC-OOH) in the livers of three fish species, including sweet smelt, yellowtail and rainbow trout, were determined by selected ion monitoring of gas chromatography/mass spectrometry. Total molar amounts of all hydroxy fatty acids determined in the present study coincided well with those of PC-OOH reported previously, suggesting that hydroxy fatty acid composition reflects hydroperoxide composition. The total amount of hydroperoxide isomers accumulated in the livers of sweet smelt was much higher than those of yellowtail and rainbow trout. The amounts of certain isomers, including 10-hydroperoxy octadecanoic acid, 12-hydroperoxy eicosanoic acid and 14-hydroperoxy docosanoic acid, were significantly higher than those of rainbow trout and yellowtail. These results suggest that differences in the contents and compositions of certain hydroperoxide isomers, which are possible precursors of a watermelon-like or cucumber-like aroma, result in differences of fresh fish aroma between aromatic fish and non-aromatic fish.