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Safety evaluation and experiments to study effects of live vaccine
against equine rhinopneumonitis: Experimental research necessary
when applying for approval to manufacture live vaccine (2006-2008)
(Joint research conducted with Nippon Institute for Biological
Science and Research Institute for Animal Science in Biochemistry)
Research
necessary when applying for approval to manufacture live vaccine
against equine rhinopneumonitis was conducted in this study.
The research involved tests on the cohabitation infectivity,
pathogenic reversion potential and reactivation potential of
live vaccine, and tests on the comparative effects of live vaccine
with existing commercial inactive vaccines. In every case, significant
results were obtained with a view to applying for approval to
manufacture the vaccine. Toxicology and storage stability tests
were conducted by the Nippon Institute for Biological Science,
while safety tests and field tests on the vaccine were conducted
by the Research Institute for Animal Science in Biochemistry.
In each case, significant results were obtained with a view to
applying for approval to manufacture the vaccine.
Development of serological
diagnosis methods for equine coronavirus infection (2006-2008)
(Joint research conducted with National Institute of Animal Health)
The
aim of this study was to develop a neutralization test and an
ELISA method for equine coronavirus. It was confirmed that the
equine coronavirus NC99 strain propagates well in Vero cells,
accompanied by CPE. As a result, a neutralization test for equine
coronavirus using Vero cells was established. Meanwhile, equine
coronavirus NP genes were detected from the fecal samples of
febrile horses at a draft-horse racecourse in Hokkaido, where
a mass outbreak had occurred. The expressed protein obtained
by combining these genes with E. coli showed specific
reaction to equine coronavirus immune rabbit serum. Therefore,
it is expected that an ELISA method for detecting antibodies
in infected horses can be developed by using this expressed protein
as an antigen.
Investigation of gene regions
useful for genetic diagnosis of vesicular stomatitis virus and
their application to diagnosis (2006-2008)
(Joint research conducted with National Institute of Animal
Health)
The
aim of this study was to develop a new method of genetic diagnosis
for vesicular stomatitis and to develop a quick method of testing
antibodies allowing a large number of samples to be processed.
PCR-ELOSA was developed as a method that enables genes of the
two serotypes of vesicular stomatitis virus (the New Jersey type
and the Indiana type) to be detected quickly and efficiently.
In addition, monoclonal antibodies were used to develop a competitive
ELISA method for detecting serum antibodies against New Jersey
type virus, irrespective of the species of animal.
Establishment of IgM-captured
ELISA test on Japanese encephalitis (2005-2006)
Purpose
Ever
since it entered the USA in 1999, the danger of West Nile Virus
(WNV) being introduced into Japan has been increasing. In a previous
research project, we established WNV-IgM captured ELISA, using
WNV as an antigen, as a diagnostic method for WNV infected horses,
and incorporated this in a pathological appraisal manual based
on the ﾒPrevention Manual
of WNV infection in Japanﾓ.
However, although WNV has similar antigenicity to Japanese Encephalitis
Virus (JEV), its cross-reactivity with IgM ELISA has yet to be
studied. In this study, therefore, we first established a technique
for IgM ELISA using JEV as an antigen (JEV- IgM captured ELISA),
then studied cross-reactivity with JEV and WNV-IgM ELISA using
the serum of horses infected by both viruses.
Results
1.
We studied the reproductivity of JEV and WNV in Vero cells and
C6/36 cells. As a result, both viruses showed high reproductivity
in C6/36 cells, and it was indicated that the virus titers would
peak on the 4th to 5th day after inoculation. In view of these
results, we decided to retrieve and inactivate the supernatant
in this period and prepare an antigen for IgM ELISA.
2.
Using the post-serum of two horses experimentally infected with
WNV, we studied cross-reactivity with JEV and WNV-IgM ELISA.
As a result, horse No.1, which showed high reactivity to WNV
antigens, also showed positivity to JEV antigens due to cross-reaction
on the 14th day, when antibody titers reach their peak. In horse
No.2, which had weak reactivity to WNV, reactivity to JEV remained
negative, however.
3.
We studied cross-reactivity with JEV and WNV-IgM ELISA using
the serum of JEV experimentally infected horses and eight naturally
infected horses. As a result, antibody titers against the homogenous
JEV antigens showed higher values than those against WNV antigens
in all serum samples.
4.
We studied reactivity with JEV and WNV-IgM captured ELISA in
the serum of yearlings in the Hidaka region both before and after
inoculation with JEV vaccine (140 samples and 30 samples, respectively).
As a result, reactivity to both types of ELISA was negative in
all serum samples. From this result, it was shown that, as with
WNV-IgM, JEV-IgM captured ELISA also does not detect antibodies
in vaccinated horses. Meanwhile, the rate of appearance of non-specific
reactions was estimated to be no more than 1% (as 0 of 140 samples
tested positive). Next, to study the status of JEV infection
among outdoor horses, we studied JEV and WNV-IgM captured ELISA
reactivity of racehorse serum sampled at the Ritto Training Center
during the periodic testing in November 2005 (70 samples obtained
from horses inoculated regularly with JEV vaccine). As a result,
all of the samples tested this time were negative for both types
of ELISA.
Epidemiological surveillance
of equine infectious disease in breeding areas (2004-2006)
Purpose
As
horseracing becomes progressively more global, we carry out continuous
epidemiological surveillance in breeding areas in response to
contagions of equine infectious diseases not only overseas but
also in Japan.
Results
1.
Equine arteritis; As a result of antibody tests conducted on
a total of 499 horses, it was confirmed that unvaccinated horses
were antibody negative and that there was no rise in the antibody
titers of vaccinated horses.
2.
Influenza: On conducting antibody tests for a total of 497 yearlings,
all except one vaccinated imported horse were negative.
3.
Equine rhinopneumonitis; Cohabitant yearlings in farms that had
suffered a spate of EHV-1 miscarriages had a high rate of antibody
positivity towards equine herpes virus type 1 (EHV-1). The possibility
was suggested that pregnant mares and cohabitant yearlings play
a part in the transmission and propagation of EHV-1 inside farms.
4.
Rotavirus infection; The rotavirus positive rate of diarrheic
stools over three years was 56%, and the majority of diarrhea
over several months after birth was due to rotavirus infection.
5.
Rhodococcus equi infection; We showed that cytological
diagnosis using bronchoalveolar lavage is useful as a test method
for this disease.
6.
Strangles, equine protozoal myeloencephalitis (EPM); In both
cases, no incidences or antibody positive horses were observed
during the study period.
Conclusions
Equine
arteritis and equine influenza have not been introduced. Equine
rhinopneumonitis, rotavirus infection and Rhodococcus equi
infection occurred continuously, but there was no incidence of
strangles, and no horses were found to be antibody positive to
EPM.
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