Establishment of a potency test by ELISA for a rabies vaccine for animal use in Japan

The ELISA we developed was able to determine the antigen content and was suitable for a potency test, and we described a relative potency assay method which determines the potency of test vaccines by comparing the ELISA value of a test vaccine to that of a reference vaccine. In the present study, we standardized the reference vaccine used for determining the potencies of test vaccines, and established a potency test by ELISA. We evaluated the proposed reference vaccine by the neutralizing antibody responses in dogs after vaccination, by the challenge protection test in guinea pigs (GP potency test), which is the earlier official potency test used in Japan, and by the NIH potency test, which is widely used throughout the world. The results showed that a 4-fold dilution of the proposed reference vaccine induced sufficient immunity in dogs. A 3-fold dilution of the proposed reference vaccine passed the GP potency test. The international units (IU) calibrated by the NIH potency test were 3.7 IU/dose. From the results and the WHO recommendation that veterinary rabies vaccines should have a potency of at least 1.0 IU/dose, we determined to dilute the proposed reference vaccine by 3 fold and regarded it as the reference vaccine. Finally, we confirmed that there is a good agreement between the results of the potency test by ELISA and the results of the GP potency test. The establishment of the potency test by ELISA has made it possible to monitor the potency in the production process and has contributed to the stable production of the vaccine.     

Quantitative analysis of microfilarial periodicity of Dirofilaria immitis in cats

Microfilarial periodicity of Dirofilaria immitis in the venous blood of infected cats was analyzed by a trigonometric model. Cats were infected by subcutaneous transplantation with 120-day-old juvenile D. immitis. Microfilariae in the blood were first observed 98 days after transplantation. Blood was collected at 4h intervals for a 24h period, and examinations were repeated five times in two cats. The calculated periodicity index was 75.1 and 50.3 in these two cats. The estimated hour of peak microfilarial density ranged from 1.00 to 2.84h. Thus, the periodicity of microfilariae of D. immitis in the blood of cats was characterized as nocturnally sub-periodic.     

Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant

Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutant det3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.     

Forecast of Sulfur Deposition in Japan for Various Energy Supply and Emission Control Scenarios

In order to contribute to the analysis and solution of regional scale environmental problems in East Asia, we developed a tool for the comprehensive assessment of alternative policy options to improve air quality. This tool projects the future regional energy supply, calculates the emission levels of sulfur dioxide and estimates the geographical pattern of sulfur deposition resulting from emissions. Sulfur deposition in Japan through 2030 was forecasted for various energy supply and emission control scenarios using the analysis tool. Future sulfur depositions were calculated from the source-receptor matrix for 1995 and the growth rate of emission for the source subregion. In the case of the current legislation scenario, anthropogenic SO₂ emissions in East Asia would grow by 34 percent and sulfur deposition in Japan would increase by approximately 20 percent between 1995 and 2030. This increase in sulfur deposition over these 35 years is sligthly less than the contribution from volcanic emission to sulfur deposition in Japan. In the case of the hypothetical dirty scenario for China, sulfur deposition in several grids which face the Sea of Japan would double by 2030.     

Molecular cloning and sequence analysis of feline interferon-stimulated gene 15

The interferon-stimulated gene 15 (ISG15) is induced by type I interferon (IFN). Recent studies have revealed that like ubiquitin, ISG15 is conjugated with target proteins. In this study, the feline ISG15 (FeISG15) gene was cloned from feline IFNomega (FeIFNomega)-stimulated feline kidney epithelial (CRFK) cells. According to gene sequence results, cDNA was 474bp long and encoded a protein of 157 amino acids. The putative amino acid sequences showed 62.5-72.1% identity with those of other mammalian ISG15s. Similar to human and mouse ISG15, FeISG15 included tandem ubiquitin-like domains; its homology with feline ubiquitin was 36.3-39.5%. The LRLRGG conjugating motif was located only in the carboxyl terminal ubiquitin-like domain. FeISG15 also lacked the carboxyl terminal extension after the LRLRGG motif, which is present in mouse and human ISG15. Recombinant FeISG15 protein was expressed as a His-tagged fusion protein in Escherichia coli and purified by ion-exchange chromatography followed by affinity chromatography. Monoclonal anti-FeISG15 antibodies revealed free FeISG15 and FeISG15 conjugated with target proteins in cells after IFNomega stimulation by Western blotting analysis. Furthermore, mRNA of IFNgamma was detected from peripheral blood mononuclear cells (PBMCs) after stimulation with rFeISG15 extracellularly by RT-PCR. Taken together, these results suggested that FeISG15 had ubiquitin- and cytokine-like activity, as in other species.     

Improved porcine model for Shiga toxin‐producing Escherichia coli infection by deprivation of colostrum feeding in newborn piglets

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)‐producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 10¹¹ colony‐forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum‐deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.     

Comparison of the immunosuppressive effects of dexamethasone, flunixin meglumine and meloxicam on the in vitro response of calf peripheral blood mononuclear cells

This study compared the immunosuppressive effects of dexamethasone (DEX), flunixin meglumine (FLU) and meloxicam (MEL) on the peripheral blood mononuclear cells (PBMCs) of seven healthy Holstein calves in vitro. DEX significantly inhibited lymphocyte proliferation and expression of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 messenger RNA (mRNA) in comparison with FLU and MEL. FLU and MEL dose-dependently inhibited lymphocyte proliferation, but did not significantly reduce mRNA expression. Our in vitro study indicates that steroidal anti-inflammatory drugs (SAIDs) as well as nonsteroidal anti-inflammatory drugs (NSAIDs) have immunosuppressive effects on calf PBMCs. These findings are important for assessing the indications and complications of NSAIDs in calves.     

Antimicrobial resistance of Staphylococcus pseudintermedius isolates from healthy dogs and dogs affected with pyoderma in Japan

Staphylococcus pseudintermedius strains were isolated from healthy dogs and dogs with pyoderma in 2000-2002 and 2009. All the isolates from dogs with pyoderma in 1999-2000 and from healthy dogs in 2000-2002 and 2009 were susceptible to cefalexin and/or other cephalosporins and oxacillin. However, 7.1-12.5 and 11.4% of S. pseudintermedius isolates from dogs with pyoderma in 2009 were resistant to cephalosporins and oxacillin, respectively. All S. pseudintermedius isolates from dogs with pyoderma in 1999-2000 and those from healthy dogs in 2000-2002 were susceptible to fluoroquinolones; however, 50% of the S. pseudintermedius strains isolated from dogs with pyoderma in 2009 and 30% of the S. pseudintermedius strains isolated from healthy dogs in 2009 were resistant to fluoroquinolones. Of the 21 oxacillin-resistant S. pseudintermedius (MRSP) isolates, 11 carried SCCmec type V and 10 carried hybrid SCCmec types II-III. Staphylococcus pseudintermedius strains that were resistant to only one of three fluoroquinolones had a mutation in the quinolone resistance determination region of grlA, whereas S. pseudintermedius strains that were resistant to two or more fluoroquinolones had mutations in the quinolone resistance determination regions of both grlA and gyrA.     

Identification and characterization of feline UBE1L gene

Interferon-stimulated gene 15 (ISG15) is one of the type I interferon-inducible proteins. Addition of ISG15 known as ISGylation is an ubiquitin-like posttranslational modification. Coexpression of ISG15 and ubiquitin-activating enzyme E1-like protein (UBE1L) is required to induce ISGylation. Previously, we identified feline ISG15 gene and found that the capsid protein of feline immunodeficiency virus was ISGylated in vitro by treatment with feline interferon-ω. In this study, we cloned feline UBE1L (FeUBE1L) gene to further study the mechanism of the antiviral activities induced by ISGylation. Sequencing analysis revealed that active sites of FeUBE1L were highly conserved. These data suggest that FeUBE1L has an enzymatic activity. Further, expression of FeUBE1L was induced in feline cell lines by treatment with feline interferon-ω and ovine interferon-τ.