An enzyme-linked immunosorbent assay using nuclear antigen for detection of feline herpesvirus 1 antibody.

To detect antibody against feline herpesvirus 1 (FHV-1) in the sera of cats, the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) using nuclear antigen was investigated. The standardized optical density readings (ODs) of the ELISA obtained by the 1-step serum dilution (1:80) method were compared with the serum neutralization test (SNT) results, with a correlation of 0.993, and with the hemagglutination inhibition (HI) test results, with a correlation of 0.851. The ODs for the ELISA titers were obtained using the serial serum dilution method and were compared with the SNT results, with a correlation of 0.933, and with the HI test results, with a correlation of 0.987. In the experimental infection of 4 specific-pathogen-free cats, the results of different serologic tests (SNT and HI) and the ELISA using the serial serum dilution method revealed rapid production of antibodies after inoculation, whereas the ELISA using the one-step serum dilution method indicated that titers increased more slowly. These results indicate that with the present ELISA using nuclear antigen, there are fewer demands on time and labor, making the method convenient for monitoring FHV-1 infection.     

Origin of the diclidophorid monogenean Neoheterobothrium hirame Ogawa, 1999, the causative agent of anemia in olive flounder Paralichthys olivaceus

In the mid-1990s, Neoheterobothrium hirame suddenly appeared as a new species in olive flounder Paralichthys olivaceus in Japanese coastal waters. Anemia caused by the parasite has prevailed in wild and cultured populations of olive flounder since that time. In this study, to clarify the origin of N. hirame, two Neoheterobothrium species, namely unidentified Neoheterobothrium species (tentatively abbreviated as Neoheterobothrium sp. PL) and N. affine, were collected from Paralichthys lethostigma and Paralichthys dentatus, respectively, off the east coast of North America and compared with N. hirame collected in Japan. No substantial differences were detected in the morphology and DNA sequences of ITS1-5.8S rRNA-ITS2 and mitochondrial cytochrome oxidase subunit I (mt COI) regions between N. hirame and Neoheterobothrium sp. PL. On the other hand, the congeneric N. affine was clearly distinguished from both N. hirame and Neoheterobothrium sp. PL in its longer isthmus and the DNA sequences in ITS1 and mt COI. The absence of differences between N. hirame and Neoheterobothrium sp. PL and the clear difference between both of these and N. affine indicate that N. hirame is conspecific with Neoheterobothrium sp. PL infecting P. lethostigma and that N. hirame was recently introduced from North America to Japan.     

Properties of a calicivirus isolated from cats dying in an agitated state

In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCV-SAKURA) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3.0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70.4 per cent nucleotide and 77.3 per cent amino acid homology and FCV-F9 had a 68.6 per cent nucleotide and 73.9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.     

Accumulation of Denitrifying Bacteria and Enhancement of Denitrification Activity in Deep Subsurface Soil in Relation to the Geological Profile

We investigated the denitrification activity and the distribution of the denitrifying bacteria of a boring survey site located on a volcanic plateau, where the geological profiles from surface to deep subsurface soil at the groundwater level had been examined. There were differences between the water quality in the Ito pyroclastic flow deposit (Shirasu) layer (44.2 to 54.5 m) and that in the Osumi pumice fall deposit (Pumice) layer (below 54.5 m) corresponding to the impermeable layer of unconfirmed groundwater: The nitrate concentration was less than 1 mg kg⁻¹ in the Shirasu layer and more than 10 mg kg⁻¹ in the Pumice layer (Kubota et al. 2005). Denitrification activity decreased from the surface to the loam layer and was enhanced in the Shirasu layer and the Haraigawa clay impermeable layer at a depth of 65 m. It was observed that the highest potential denitrification activity (10³ ng-N₂O d⁻¹ g⁻¹) in the impermeable layer was almost equal to that of a Kuroboku surface soil with slurry application. Viable counts of the sonic-samples, which indicated the presence of bacterial group with soil particles attached, increased in the impermeable layer. The ratios of viable or denitrifying bacterial counts in the sonic-samples to those in the wash-samples were significantly higher in the impermeable layer than those in the surface layer. These results suggest that the hydrogeological conditions enhanced the denitrification activity in the impermeable layer, the niches of which might be relatively anaerobic and have a sufficient supply of substrates to enable the denitrifying bacterial populations to multiply.     

Experimental challenges of juvenile and adult Manila clams with the protozoan Perkinsus olseni at different temperatures

Infection with Perkinsus species, primarily P. olseni, is thought to be a major cause of the decline of Manila clam populations in Japan since the 1980s. However, the pathogenicity of the infection has not been sufficiently evaluated to estimate the impact of infection on wild Manila clam populations. We experimentally challenged juvenile (3- to 6-mm shell length) and adult (18- to 22-mm shell length) Manila clams with P. olseni at 18, 23, 28, and 30 °C. Mortality was significantly higher in challenged groups than in control groups. The difference in mean mortality between the challenged and control groups (all life stages and temperatures) was only significant above a threshold of infection intensity ~10⁶ cells/g soft wet tissue (SWT). As temperature increased, the onset of mortality occurred more rapidly. The increase in mortality occurred earlier in juveniles than adults at 28 °C and lower. Our results suggest that the pathogenicity of P. olseni is higher in juveniles than in adults and at higher water temperatures. Given the infection intensities (ca. 10⁶ cells/g SWT) previously reported in wild Manila clams, the parasite likely has considerable impact on wild Manila clam populations, particularly juveniles during periods of high temperature.     

Large-scale survey of adverse reactions to canine non-rabies combined vaccines in Japan

Canine non-rabies combined vaccines are widely used to protect animals from infectious agents, and also play an important role in public health. We performed a large-scale survey to investigate vaccine-associated adverse events (VAAEs), including anaphylaxis, in Japan by distributing questionnaires on VAAEs to veterinary hospitals from April 1, 2006 through May 31, 2007. Valid responses were obtained for 57,300 vaccinated dogs at 573 animal hospitals; we obtained VAAEs information for last 100 vaccinated dogs in each veterinary hospital. We found that of the 57,300, 359 dogs showed VAAEs. Of the 359 dogs, death was observed in 1, anaphylaxis in 41, dermatological signs in 244, gastrointestinal signs in 160, and other signs in 106. Onset of VAAEs was mostly observed within 12h after vaccination (n=299, 83.3%). In this study, anaphylaxis events occurred within 60 min after vaccination, and about half of these events occurred within 5 min (n=19, 46.3%). Furthermore, where anaphylaxis was reported, additional information to support the diagnosis was obtained by reinvestigation. Our resurvey of dogs with anaphylaxis yielded responses on 31 dogs; 27 of these demonstrated collapse (87.1%), 24 demonstrated cyanosis (77.4%), and both signs occurred in 22 (71.0%). Higher rates of animal VAAEs, anaphylaxis, and death were found in Japan than in other countries. Further investigations, including survey studies, will be necessary to elucidate the interaction between death and vaccination and the risk factors for VAAEs, and thus develop safer vaccines. Moreover, it may also be necessary to continually update the data of VAAEs.     

IgE reactivity to a Cry j 3, an allergen of Japanese cedar (Cryptomeria japonica) pollen in dogs with canine atopic dermatitis

The present study investigated IgE reactivity to a new Cryptomeria japonica pollen allergen (Cry j 3) in dogs with atopic dermatitis by using a fluorometric ELISA. Serum samples from 15 dogs that showed IgE sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to each allergen, individually. All 15 dogs had anti-Cry j 1 IgE, 6 (40%) had anti-Cry j 2 IgE, and 11 (73%) had anti-Cry j 3 IgE. Further, we found that these anti-Cry j 3 IgE reacted to Cry j 3 with immunoblotting analysis. These findings indicate that Cry j 3 may be a major allergen in dogs.     

Artificial insemination with canine spermatozoa frozen in a skim milk/glucose-based extender

Due to the recent outbreak of avian influenza, transportation of frozen canine semen with egg yolk has been sharply restricted. Thus, there is urgent need to develop a novel egg yolk-free extender for freezing canine spermatozoa. In the present study, the effect of using skim milk/glucose (SG)-based extender without egg yolk on the motility and fertilizing capacity of canine spermatozoa frozen-thawed in the presence of glycerol was examined. There was a tendency for the proportion of motile spermatozoa exposed to SG-based extender for 3 h to be higher than that exposed for 1 h, but the difference was not significant. The motility and other viability parameters of canine spermatozoa after thawing were similar to those obtained with an egg yolk-based extender. When spermatozoa frozen with SG-based extender containing glycerol after 3 h exposure were transcervically inseminated into 2 recipient bitches, a total of 6 pups were obtained. These results suggest that a simple extender composed of skim milk, glucose and glycerol is useful for cryopreservation of canine spermatozoa, which may contribute to improved exchange of genetic material and efficient production of companion and working dogs, such as guide dogs for the blind.     

Depressed gross mineralization and immobilization of nitrogen and changes in microbial population in soil after heat treatment

Partial sterilization causes a change in N mineralization in soil. An increase in the net rate of N mineralization was reported in soil with chloropicrin applied to it (Rovira 1976), and has been well known in soil fumigated with chloroform to measure the microbial biomass N (Jenkinson and Ladd 1981). The gross rate of N mineralization increased in soil inoculated with fresh soil following fumigation with chloroform (Shen et al. 1984). The increased rate of N mineralization has been attributed to the rapid decomposition of organisms killed by partial sterilization (Jenkinson 1966). On the other hand, Nira et al. (1996) reported that the application of a fumigant in a field depressed the gross rates of N mineralization and immobilization in spite of the increase in the net rate of N mineralization. These results suggested that the increase in the net rate of N mineralization by partial sterilization is presumably due to the change in the ratio of N mineralization to immobilization. However, the residues of a fumigant may depress gross N transformation in the field, because the residues may continue to influence microbial activity long after the original treatment (Jenkinson 1966). Some effects of partial sterilization without residues on gross N mineralization remain to be determined.     

Sex steroid hormones do not enhance the direct stimulatory effect of kisspetin‐10 on the secretion of growth hormone from bovine anterior pituitary cells

The aims of the present study were to clarify the effect of kisspeptin10 (Kp10) on the secretion of growth hormone (GH) from bovine anterior pituitary (AP) cells, and evaluate the ability of sex steroid hormones to enhance the sensitivity of somatotrophic cells to Kp10. AP cells prepared from 8–11‐month‐old castrated calves were incubated for 12 h with estradiol (E₂, 10^?8 mol/L),progesterone (P₄, 10^?8 mol/L), testosterone (T, 10^?8 mol/L), or vehicle only (control), and then for 2 h with Kp10. The amount of GH released in the medium was measured by a time‐resolved fluoroimmunoassay. Kp10 (10^?6 or 10^?5 mol/L) significantly stimulated the secretion of GH from the AP cells regardless of steroid treatments (P < 0.05), and E₂, P₄, and T had no effect on this response. The GH‐releasing response to growth hormone‐releasing hormone (GHRH, 10^?8 mol/L) was significantly greater than that to Kp10 (P < 0.05). The present results suggest that Kp10 directly stimulates the release of GH from somatotrophic cells and sex steroid hormones do not enhance the sensitivity of these cells to Kp10. Furthermore, they suggest that the GH‐releasing effect of Kp10 is less potent than that of GHRH.