Location, size and age at onset of metamorphosis in the Japanese eel Anguilla japonica.

This study clarifies the location, size and age at the onset of metamorphosis in Japanese eels Anguilla japonica through oceanic surveys, rearing experiments and analyses of the morphology and otoliths of leptocephali and glass eels. Twenty-eight metamorphosing leptocephali were collected in the mesoscale eddy region to the east of Taiwan during research expeditions in 2004. Rearing experiments showed that the total length (LT ) of leptocephali decreased by an average of 12·5% during metamorphosis and 13·9% during the 2-12 h after death. Thus, the mean back-calculated LT at the onset of metamorphosis for 630 glass eels from Taiwan and Japan was estimated at 67·8 ± 2·7 mm (mean ± S.D.). The estimated mean ante-mortem size of the fully grown pre-metamorphic leptocephali collected in 2004 was 64·6 ± 3·4 mm, which was consistent with the LT estimate for glass eels. Otolith analysis showed that the mean age at the onset of metamorphosis was 137 ± 15 days and indicated that Japanese eels may have a recruitment route through the mesoscale eddies to the east of Taiwan in addition to the direct transfer route from the North Equatorial Current to the Kuroshio Current.

Reproductive isolation between sympatric Anguilla japonica and Anguilla marmorata.

Species-specific restriction fragment length polymorphism in the intron of the androgen receptor gene (ar5) was found in glass to silver-stage individuals of Anguilla japonica (n = 51) and A. marmorata (n = 21). The sequence analysis of 16S rDNA from 328 anguillid leptocephali collected in the North Equatorial Current of the western North Pacific Ocean revealed the specimens to be A. japonica (n = 194), A. marmorata (n = 128), A. bicolor pacifica (n = 5) and A. luzonensis (n = 1). All leptocephali of A. japonica and A. marmorata were monomorphic and did not share an allele at the ar5 locus, indicating that the two species are reproductively isolated.

A giant anguilliform leptocephalus Thalassenchelys foliaceus Castle & Raju is a junior synonym of Congriscus maldivensis (Norman 1939).

A single specimen of giant leptocephalus Thalassenchelys foliaceus Castle & Raju 1975 was caught in subtropical waters of the western North Pacific Ocean. Mitochondrial coI gene sequence divergence between T. foliaceus and Congriscus maldivensis (Norman 1939) was 0·64 ± 0·27% (mean ± s.e.), and the myomere and vertebral counts of these species were similar, indicating T. foliaceus is a junior synonym of C. maldivensis.

Isolation and preliminary characterization of an orbivirus of the Palyam serogroup from biting midge Culicoides oxystoma in Japan.

An orbivirus of the Palyam serogroup was isolated from Culicoides oxystoma collected in a cowshed in Kagoshima, Southern Kyushu Island, Japan. This is the first isolation of an orbivirus of the Palyam serogroup in Japan. The virus was a spherical non-enveloped RNA virus, approximately 60 nm in diameter. The virus was resistant to ethyl ether, sodium deoxycholate and freezing-thawing, but readily inactivated by trypsin. The virus was not stabilized by 1 M MgCl2, was labile at pH 3.0 and was not precipitated by protamine sulfate. Indirect immunofluorescent staining of infected Vero cells indicated the virus to be antigenically related to D'Aguilar and Bunyip Creek viruses of the Palyam serogroup. Neutralization tests showed the virus to have no relationship with D'Aguilar virus, but to have a one-way cross-reaction with Bunyip Creek virus. The virus was tentatively designated as Kagoshima virus. A serological survey indicated dissemination of the virus in cattle populations in Kagoshima Prefecture.

Detection of antibody to pseudorabies virus in swine sera by indirect immunoperoxidase plaque staining.

A new method for the detection of pseudorabies antibody in swine sera was developed by use of the indirect immunoperoxidase plaque staining test. The indirect immunoperoxidase plaque staining test demonstrated serologic responses in pigs that were subcutaneously infected with pseudorabies virus. The test had a sensitivity and a specificity comparable to those of the complement-dependent neutralization test and appeared to be somewhat more sensitive than the neutralization test and the enzyme-linked immunosorbent assay. The test was simple to perform, and the results could be read with the unaided eye under ordinary light, making the test particularly suitable for the routine testing of large numbers of serum samples.

Isolation of Akabane virus from the biting midge Culicoides oxystoma in Japan.

Akabane virus was isolated from the biting midge, Culicoides oxystoma, collected in a cowshed in Kagoshima on Kyushu Island of Japan. This is the first report on the isolation of Akabane virus from biting midges of the genus Culicoides in Japan. Two calves kept as bait in the cowshed seroconverted to Akabane virus. These results strongly suggest that C. oxystoma may be a vector of Akabane virus.

Sequential isolation of rotavirus from individual calves.

An outbreak of neonatal calf diarrhea was studied on a breeding farm of Japanese indigenous beef cattle. During the breeding season of 1982, 43 calves were born over the period 27 February-28 April. All but one of the calves suffered from neonatal diarrhea and 5 died. Bovine rotavirus was isolated in cell cultures from fecal specimens of 39 (90.7%) of the 43 calves during the outbreak, strongly suggesting that this was the causative agent; the virus was readily isolated from 81 (83.5%) of 97 specimens of diarrhea. Rotavirus was subsequently isolated from the feces of 7 of the calves in early May, more than one month after the initial virus isolation in early March. Two of these calves were again rotavirus-positive in early June, 41 days after the second virus isolation. Diarrhea had ceased in all 7 calves in March. Some antigenic differences were shown by the neutralization test between the early and later isolates from one of these calves, suggesting either re-infection with a serologically different virus, or persistent infection with the original virus following antigenic modulation.

Electron microscopy of Akabane virus.

Electron microscopy of negatively stained purified virus and of thin sections of infected cells and tissues showed Akabane virus being similar in morphology and morphogenesis to members of the family Bunyaviridae.

An attenuated strain of Akabane virus: a candidate for live virus vaccine.

An attempt was made to attenuate the high virulent OBE-1 strain of Akabane virus by adaptation to low temperature. In it the virus was subjected to passage through HmLu-1 cell cultures at 30 degrees C. Cloning was carried out on the virus which had undergone 20 passages through these cultures to select a strain adapted to low temperature. Finally, ten clones were obtained. As a result, nine strains of clone in which virus replication was poor in HmLu-1 cell cultures at 40 degrees C were obtained. Of them, five strains of clone produced uniform plaques. Of these strains, one, or the TS-C2 strain, was selected. It was considerably lower both in peripheral infectivity to suckling mice and in intracerebral infectivity to 3-week-old mice than the OBE-1 strain. Calves and pregnant cows inoculated with the TS-C2 strain by the intracerebral, intravenous, or subcutaneous route were free from pyrexia, leukopenia, and viremia. Virus recovery was negative from various organs and fetuses. All the animals inoculated, however, were found to have neutralizing antibody produced. The results mentioned above suggested that the TS-C2 strain might have been so attenuated as to be available as a candidate strain for a live virus vaccine.

Immune response of various animals to Akabane disease live virus vaccine.

When various animals and routes of inoculation were examined for antibody response to Akabane disease live virus vaccine, the intracerebral (ic) inoculation of mice induced a better antibody response than the subcutaneous (sc) inoculation of calves, guinea pigs, hamsters, mice, or rats. Immunogenicity was compared among lots of this vaccine by performing ic inoculation of mice and sc inoculation of calves and guinea pigs. As a result, there was no distinct significant difference between any two lots of the vaccine, regardless of the animal species used. There was a tendency that the larger the dose of inoculation of the virus, the earlier the production of neutralizing (NT) antibody took place in calves inoculated with the vaccine, and the higher the antibody titer and the rate of taking a turn for positivity for antibody became in these calves. When calves immunized with the vaccine and cows in the field possessing NT antibody were given booster inoculation with the vaccine, the antibody titer showed a significant increase in almost all the calves and cows that exhibited an NT antibody titer of 4 or less at the time of booster inoculation. There were, however, no changes in antibody titer in such calves and cows as presenting an NT antibody titer of 8 or more. Calves and pregnant cows immunized with the vaccine were prevented from viremia and fetal infection when challenged by inoculation with virulent virus.

Development of inactivated vaccine for Akabane disease.

Virus inactivated by formalin or beta-propiolactone was superior to that inactivated by ether and heating in immunogenicity to mice. There were no significant differences in the antibody response of mice among such adjuvants as aluminum phosphate gel, aluminum hydroxide gel, and sodium arginate gel. When cattle were immunized with vaccine inactivated by formalin and adsorbed to aluminum phosphate gel, an excellent effect was obtained by injection with two doses of 3 ml each given at a 4-week interval. It was also suggested that mice and guinea pigs might be available for the potency test of vaccine. When calves and pregnant goats were injected with vaccine in the same manner as mentioned above, they were prevented from viremia and fetal infection caused by challenge virus. Even when stored at 4 degrees C for 12 months, vaccine was found to retain its stabilized immunogenicity. When pregnant cows were injected with vaccine in the field, the positive rate of neutralizing antibody was 88.5% in the 2 months after the first injection. When other pregnant cows were injected twice with vaccine, this rate was 34.6, 100, 65.4, and 45.8% in the 1, 2, 6, and 10 months, respectively, after the first injection. In another experiment, the antibody levels attained declined rather rapidly in several months. A single dose of vaccine given one year later provoked a rapid antibody response. The vaccination caused no clinical symptoms, abnormal birth, or decrease in milk yield in these cows.