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Immunological analysis of flagella antigens of Salmonella
Abortusequi for development of specific serodiagnosis method
of equine paratyphoid (2007-2010)
In
this study, we evaluated usefulness of specific region of the
flagella protein of S. Abortusequi and S. Typhimurium
as an antigen for discriminately serodiagnosis method between
equine paratyphoid and S. Typhimurium infection by immunological
analysis. For the analysis, we constructed truncated flagella
protein libraries and synthesized peptide libraries according
to the amino acid sequence of the flagella protein of each flagellar
phase in the serotypes. Significant increase of IgM antibody
against the specific region of S. Abortusequi was observed
in successive serum collection obtained from horses infected
by experimental intrauterine administration of S.
Abortusequi. In contrast, increase of specific IgM antibody was
not significantly observed in successive serum collection obtained
from horses infected by experimental oral administration of
S. Abortusequi and S. Typhimurium. Increase of
IgG antibody was not observed in these collections. High-level
IgG antibody corresponding each specific region was detected
in serum obtained from horses intramuscularly immunized by live
or killed organisms. IgM or/and IgG antibody against S.
Abortusequi were observed in serum obtained from horses kept
in endemic region of equine paratyphoid and were not observed
in serum from carrier horses of S. Abortusequi with low
flagella expression. Conclusively, the specific region of the
flagella protein of S. Abortusequi and S. Typhimurium
seemed to be useful as the diagnosis antigen to discriminate
between equine paratyphoid and S. Typhimurium infection.
However, we also have to consider that specific antigens have
a limitation of the application for the diagnosis because of
some factors suppressing the production of the antibody against
the specific region including administration antimicrobial agents
at an initial phase of infection, degree of expression of flagella,
and individual variation of host.
Epidemiological and clinical
microbiological studies on equine infectious
diseases (2004-2008)
Here,
study was mainly conducted on epidemiological surveys about infectious
diseases that have a serious influence on race meetings and diagnostic
methods of equine infectious diseases. The principal research
results were as follows.
1. A febrile disease that occurred as a mass outbreak at a draft-horse
racecourse in Hokkaido in 2005 was surveyed. It was suggested
that the disease might have been caused by equine coronavirus
infection.
2. It was demonstrated that a collective occurrence of Salmonella
Typhimurium infection amongst Thoroughbred foals in the Hokkaido
breeding area in 2005 was caused by the multiple drug resistant
DT104 type.
3. The circumstances of an equine influenza epidemic in JRA facilities
in 2007 were clarified.
4. The neutralization test that is a kind of serological diagnosis
method for
equine viral arteritis was standardized in joint research with
the Epizootic Research Center, JRA, the Animal Quarantine Service
and the National Institute of Animal Health.
5.Two methods of detecting Clostridium difficile genes
(the PCR method and the LAMP method) were applied in the field.
Molecular genetic analysis
of the presentation mechanism of Streptococcus equi subsp.
equi PEPK antigens (2006-2008)
(Joint research conducted with National Institute of Animal Health)
While
Streptococcus equi subsp. equi (S. equi)
has bacterial surface proteins called SzPSe antigens, those of
Streptococcus equi subsp. zooepidemicus (S.
zooepidemicus), which is in a substrain relationship with
the former, are called SzP antigens. Both SzPSe antigens and
SzP antigens have PEPK repeated sequences. However, while PEPK
antigens react to the serum of horses infected with strangles,
they do not react to that of S. zooepidemicus infected
horses. This study was conducted to clarify the reason for this.
In the first year, it was confirmed that knockout mutant strains
of S. equi do not produce SzPSe antigens. In the second
year, it was demonstrated that the SzPSe antigens of S. equi
are expressed only by SzPSe genes. In the final year, it was
confirmed that SzPSe antigens are only secreted in culture supernatant
of S. equi. From these results, it was suggested that
the difference in the secretion of SzPSe or SzP antigens between
S. equi and S. zooepidemicus might be related to the
production of specific anti-PEPK antibodies in horses with strangles.
Development of an ELISA test
for diagnosis of strangles using peptide antigens (2004-2006)
Purpose
Strangles
is a contagious disease peculiar to animals in the genus equus,
and is caused by S.equi. It tends to more readily affect
young horses and can be fatal. While a serum diagnostic method
that can specifically diagnose strangles is thought to be effective
in preventing the introduction of this disease from overseas
and its spread inside Japan, no specific method yet exists anywhere
in the world. Therefore, we established a new serum diagnostic
method using specific PEPK repeated peptide antigens for
strangles, resulting from previous research, and studied serum
diagnostic method for strangles.
Results
1.
As a control for the study, we obtained the necessary serum of
S. zooepidemicus infected horses through an infection experiment.
2.
We proved that PEPK 5-times repeated peptide antigens
are ideal as ELISA antigens, and established an ELISA measurement
method for strangles antibodies using these antigens.
3.
Using the serum of horses infected with S.equi, the serum
obtained from the infected horses with S. zooepidemicus and
those of healthy horses, we investigated and compared antibody
titers against PEPK repeated antigens. As a result, we
could clearly differentiate the serum of horses infected with
the strangles bacillus from the other sera.
4.
After searching for the expression and localization of PEPK
antigens in S.equi, it was confirmed that S. equi
expresses antigens on the surface of the bacteria and in
cultured supernatant.
5.
We inoculated horses with two types of strangles vaccine (component
vaccine: Nos. 1, 2 and 3; live vaccine: Nos. 4, 5 and 6), and
studied their side effects and changes in antibody titers against
the PEPK 5-times-repeated-domain of SzPSe. As a
result, no side effects were seen to be caused by vaccine inoculation.
In the antibody titers against the PEPK 5-times-repeated-domain
of SzPSe, on the other hand, a rise in antibody titers
was recognized in all three horses in the component vaccine inoculated
group, although the degree was relatively mild. In the live vaccine
inoculated group, a mild rise was observed in one of the three
horses, but there was no change in the other two.
6.
When we carried out a challenge test for strangles bacillus on
six yearlings variously inoculated with two types of strangles
vaccine, symptoms of strangles were observed in all six horses.
Meanwhile, antibody titers against PEPK repeated peptide
antigens rose moderately in horses inoculated with component
vaccine and mildly in horses inoculated with live vaccine. We
also carried out experimental infections with S equi using
six yearlings, and investigated changes in their antibody titers
over a period of 8 months. The result was that, although a conspicuous
rise in antibody titers was recognized in three of the six horses,
the titers diminished in a relatively short time after inoculation
with S equi.
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