Extracellular virulence factors of fish Vibrio: relationships between toxic material, hemolysin, and proteolytic enzyme

Biological activities of cell-free culture filtrate of 3 virulent strains of fish Vibrio were examined to determine the relationship to the pathogenesis of fish vibriosis. Among the 3 strains examined, V anguillarum strains NCMB6 and NCMB571 produced hemolysin and protease, whereas V ordalii strain N7802 did not. Culture filtrate of strain NCMB571 were lethal to rainbow trout and produced a cytotoxic effect on fish cell line. Results revealed that the extracellular products may be involved in the pathogenesis of fish vibriosis.     

Induction of superovulation by immunoneutralization of endogenous inhibin in immature rats

The effects of passive immunoneutralization of endogenous inhibin on ovulation rate in immature rats were investigated. Efficiency of superovulation on production of fertilized oocytes was compared between the inhibin antiserum (inhibin-AS) and equine chorionic gonadotropin (eCG) protocols. Immature female Wistar strain rats were superovulated with a single injection of 100-200 microl inhibin-AS, with and without an injection of human chorionic gonadotropin (hCG). A total of 77.8% of the 26-30-day-old rats treated with a single injection of 100-200 microl inhibin-AS ovulated 72 h after treatment, while rats given normal goat serum (NGS; 200 microl) did not ovulate. At 28 days of age, all of the inhibin-AS treated rats ovulated when additional hCG treatment was given, whereas the number of ovulated oocytes was not affected. The number of ovulated oocytes in the inhibin-AS-hCG treated groups was significantly higher than that of the NGS-hCG treated group. In addition, plasma concentrations of FSH in the inhibin-AS-hCG treated group significantly increased compared with the NGS treated group. While the percentage of mated rats in the 200 microl inhibin-AS-hCG treated group was significantly lower than that of the 15 IU eCG-hCG treated group, the fertilization rate was comparable between the two groups. The number of fertilized oocytes in the 200 microl inhibin-AS-hCG treated group was significantly higher in comparison with the 15 IU eCG-hCG treated group. These results suggest that immunoneutralization of endogenous inhibin could be a reliable method for induction of superovulation to collect a large number of normally fertilized oocytes in immature rats.     

Field trials on a live bovine respiratory syncytial virus vaccine in calves.

Field trials were carried out in calves using a live bovine respiratory syncytial (BRS) virus vaccine prepared from the attenuated BRS virus, strain rs-52. Two hundred seventy-five and 353 calves were vaccinated intranasally and intramuscularly, respectively. No undesirable postvaccinal reactions were observed in the vaccinated calves. Of the serum neutralizing (SN) antibody negative calves 89.7% (26/29) and 92.8% (90/97) developed SN antibody 1 month after intranasal and intramuscular vaccination, respectively. Most of the calves having SN antibody titers of 1:1 or 1:2 at the time of vaccination showed a significant increase in SN antibody titer. About 70% and 90% of the calves vaccinated intranasally and intramuscularly, respectively, maintained SN antibody for 6 months after vaccination. In a field trial, a natural BRS virus infection occurred about 5 months after the start of the trial. Ten of the 16 unvaccinated control calves showed respiratory symptoms due to BRS virus infection. On the contrary, all of the 68 vaccinated calves exhibited no symptoms at all, indicating efficacy of the vaccine.     

Comparison of virulence of various hantaviruses related to hemorrhagic fever with renal syndrome in newborn mouse model

The virulence of hantaviruses that are antigenically related but have different genetic characteristics from the prototype of hantavirus, Hantaan (HTN) virus, was examined in newborn mice. The H5 and B78 strains of the Amur (AMR) genotype, the Bao14 strain of the Far East (FE) genotype, and the 76-118 strain of HTN virus were inoculated subcutaneously (1focus-forming unit; FFU) into newborn mice. All of the AMR and FE genotype viruses inoculated mice were died by 16 days post-infection (dpi) and 21 dpi, respectively, while 50% of the HTN virus inoculated mice survived until 30 dpi. The AMR and FE genotype viruses inoculated mice had high viral titers in the lung (1.3x10(6) to 1.3x10(8) FFU/gram [g] tissue) , brain (2.1x10(7) to 1.2x10(9) FFU/g tissue), and kidney(2.5x10(5) to 1.6x10(7) FFU/g tissue), and showed a detectable level of antibodies (titers 1:16-1:32) at 14 dpi. In contrast, the HTN virus infected mice had viruses only in the lungs at low titers (1.1-5.3x10(5) FFU/g tissue). Observations of body-weight changes revealed that the AMR and FE genotype viruses inoculated mice had lower growth rates than the HTN virus inoculated mice. These data suggest that the AMR and FE genotype viruses are more virulent than the HTN virus in newborn mice.     

Xenografting of bovine secondary follicles into ovariectomized female severe combined immunodeficient mice

Xenografting of ovarian tissue into immunodeficient mice has been used as a model to study the dynamics of follicular development and provides an alternative method for the production of mature oocytes. In a previous experiment, we demonstrated that xenografted bovine secondary follicles developed to the antral stage in severe combined immunodeficient (SCID) mice. In the present study, we examined the development of bovine secondary follicles (140-190 microm in diameter) grafted into ovariectomized mice in comparison with intact female mice as a control. At 4 weeks after grafting, several antral follicles ranging from 350 to 550 microm (457.6 +/- 50.8 microm) in diameter were found in the control mice, while a single large (larger than 2.5 mm) antral follicle and other small follicles were observed in every ovariectomized mouse. At 6 weeks after grafting, the mean diameter of morphologically normal follicles had further increased in the control group (591.8 +/- 132.0 microm). In ovariectomized mice, however, the mean diameter of follicles decreased (4 weeks: 864.2 +/- 988.2 microm; 6 weeks: 496.5 +/- 137.6 microm), since the single large antral follicle observed at 4 weeks had degenerated by 6 weeks. In control mice, more than 70% of follicles were morphologically normal and formed an antrum, and most of the follicles contained morphologically normal oocytes which grew to 122.5 +/- 2.2 microm. In ovariectomized mice, morphologically normal oocytes also grew larger than before grafting, but their survival rate was significantly lower than that in control mice. These results suggest that ovariectomy of host mice alters the developmental pattern of xenografted bovine secondary follicles to accelerate a single follicle to develop in the graft.     

Inhibitory effects of Zingiber officinale Roscoe derived components on aldose reductase activity in vitro and in vivo

Ginger (Zingiber officinale Roscoe) continues to be used as an important cooking spice and herbal medicine around the world. Scientific research has gradually verified the antidiabetic effects of ginger. Especially gingerols, which are the major components of ginger, are known to improve diabetes including the effect of enhancement against insulin-sensitivity. Aldose reductase inhibitors have considerable potential for the treatment of diabetes, without increased risk of hypoglycemia. The assay for aldose reductase inhibitors in ginger led to the isolation of five active compounds including 2-(4-hydroxy-3-methoxyphenyl)ethanol (2) and 2-(4-hydroxy-3-methoxyphenyl)ethanoic acid (3). Compounds 2 and 3 were good inhibitors of recombinant human aldose reductase, with IC50 values of 19.2 +/- 1.9 and 18.5 +/- 1.1 microM, respectively. Furthermore, these compounds significantly suppressed not only sorbitol accumulation in human erythrocytes but also lens galactitol accumulation in 30% of galactose-fed cataract rat model. A structure-activity relationship study revealed that the applicable side alkyl chain length and the presence of a C3 OCH3 group in the aromatic ring are essential features for enzyme recognition and binding. These results suggested that it would contribute to the protection against or improvement of diabetic complications for a dietary supplement of ginger or its extract containing aldose reductase inhibitors.     

Effect of bread baking on the bioavailability of hydrogen-reduced iron powder added to unenriched refined wheat flour

Elemental iron powders are widely used to fortify flour and other cereal products. Our objective was to test the hypothesis that baking enhances the bioavailability of elemental iron powders by oxidizing Fe(0) to Fe(2+) or Fe(3+). An in vitro digestion/Caco-2 cell culture model and a piglet model were used to measure bioavailability. Bread flour, either unfortified or fortified with hydrogen-reduced (HR) iron powder or FeSO(4) (300 mg Fe/kg flour), was baked into bread. For the in vitro studies, bread samples were treated with pepsin at pH 2, 3, 4, 5, 6, or 7 and subsequently incubated with pancreatic enzymes at pH 7 in a chamber positioned above monolayers of cultured Caco-2 cells. Ferritin formation in the cells was used as an index of iron bioavailability. Ferritin formation in cells fed HR Fe bread was similar to cells fed FeSO(4) bread when the peptic digestion was conducted at a pH 2 but lower when the peptic phase was conducted at pH 3, 4, 5, 6, or 7 (P < 0.05). Pig diets containing 35% dried bread were prepared and fed to cross-bred (Hampshire x Landrace x Yorkshire) anemic pigs in two studies. The rate of increase in hemoglobin Fe over the feeding period was used to calculate relative biological value (RBV), an index of iron bioavailability. In the first pig study, RBV of HR Fe added to flour prior to baking was 47.9% when compared to FeSO(4) fortified flour (P < 0.05). In the second pig study, a third treatment consisting of unfortified bread with HR iron added during diet mixing (after bread baking) was included. RBVs of the HR Fe diet (Fe added after baking) and HR Fe diet (Fe added before baking) were 40.1% and 53.5%, respectively, compared to the FeSO(4) diet. Differences in RBV between the HR Fe (before and after baking) and FeSO(4) (before baking) treatment groups were significant, but the difference between the before and after HR treatment groups was not significant. We conclude that bread baking does not enhance the bioavailability of elemental iron powders.     

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.