Abstract Book of EAVLD2012 - eavld congress 2012

S3 - P - 02
LIPOPOLYSACCHARIDE-CAPTURE ELISA FOR THE DETECTION OF LEPTOSPIRA
BORGPETERSENII SEROVAR HARDJO IN SHEEP
Zbigniew Arent, Colm Gilmore, William Ellis
OIE Leptospirosis Reference Laboratory, Veterinary Sciences Division, AFBI, Belfast, Northern Ireland, UK
Leptospira, leptospirosis, sheep, ELISA
Introduction
Endemic infection of sheep with Leptospira borgpetersenii
serovar Hardjo (type Hardjo Bovis) is now recognised in many
countries. Many authors demonstrated that infection in sheep is
economically and clinically significant, particularly with regards to
reproductive problems (1).This type of infection is often attributed
to abortion, stillbirth, agalactia and death of weak newborn lambs.
In addition, Hardjo infection is an occupational zoonosis of those
who work with sheep. All the evidence points to sheep being an
alternative maintenance host for this serovar. The efficacy of
epidemiological or diagnostic studies of leptospirosis in sheep
relies mainly on the correct identification of animals which have
been exposed to infection. However, seroprevalence studies in
sheep are difficult to interpret because the microscopic
agglutination test (MAT) - standard serological test, detects only
short-live immunological responses and many previously
exposed and/or infected animals are seronegative. . Therefore
there is a need for more sensitive test than the MAT as an
indicator of past exposure.
Leptospiral lipopolysaccharide (LPS) has been identified as an
immunodominant antigen, both in response to infection and to
vaccination. It has proved useful for diagnosing previous
exposure of cattle to Hardjo infection as it can be used to detect
specific IgG antibodies to be suitable for diagnostic purposes as it
is more specific and it may give some indication of the immune
status of an animal.
The aim of this study was the development of antibody-capture
ELISA using a monoclonal antibody directly against LPS epitope
and its optimisation for use in sheep
Materials & methods
Plates were coated with monoclonal antibody against Leptospira
borgpetersenii serovar Hardjo lipopolysaccharide produced and
characterized earlier in our laboratory (2).
Carbohydrate (CH) antigen was extracted from culture of
Leptospira borgpetersenii serovar Hardjo type Bovis strains 0294
using the hot-phenol–water method.
Mouse monoclonal anti-ovine IgG antibodies prepared earlier in
our laboratory were used to prepared conjugate. The monoclonal
Ig’s were purified on a Protein G column and covalently attached
to horseradish peroxidase (HRP) using periodate oxidation
method. TMB substrate was used as colorimetric indicator.
After reading the plate at 405 nm results were normalized by
expressing the corrected optical density (Serum OD – Negative
Control OD) as the percentage of positivity (%P) of the positive
control serum. The optimal cut-off value for the ELISA was
determined by receiver operating characteristic (ROC) analysis.
Correlation between MAT and ELISA was evaluated using 560
field sheep serum samples
Results
A cut-off point was calculated using 80 serum samples obtained
from flocks with no history of Leptospirosis and which had no
serological evidence of exposure to serovar Hardjo and 60
Hardjo MAT-positive sheep sera. The ROC analysis estimated
the optimized ELISA cut-off as %P ≥ 4.5, with corresponding
diagnostic sensitivity at 95% (95% Cl: 86.1 - 99.0) and specificity
at 98.75% (95% Cl: 93.2 - 100.0) (Fig1).
Sensitivity (%)
100
80
60
40
20
Fig 1 ROC curve analysis
0
cut-off value: >4.5
0 20 40 60 80 100
100-Specificity (%)
The percentage of positive results obtained by ELISA test and
MAT applied to field sera are presented in Tab. 1. In the MAT,
reactions to serovar Hardjo were with 28 ovine sera (5.0%)
reacting at 1/100 or greater. When using ELISA, 82 (14.6%) of
the sera were positive. Only 5 (0.9%) sera were positive in MAT
and negative in ELISA test.
Tab. 1 Comparison of ELISA and MAT results
ELISA
Positive
ELISA
Negative
MAT
≥ 1:100
MAT
Negative
TOTAL
for ELISA
14.6% 85.4%
TOTAL
for MAT
4.6% 0.4% 5.0%
10.0% 85.0% 95.0%
Discussion & conclusions
The results of the study demonstrated that the ELISA was more
sensitive than MAT. The differences in correlation between the
MAT and ELISA tests was to be expected since the tests
measure different classes of antibody. The MAT detects both IgG
and IgM antibodies whereas the ELISA used in this study detects
only IgG. This shows that our ELISA has some limitation
especially as an individual animal test, but despite this, the higher
sensitivity, when compared with the MAT indicates obvious
advantages in use of the ELISA as a flock test, where the aim is
to estimate exposure and immunity levels in the flock. The
ELISA seems to be a valuable complement to serological
diagnosis of leptospirosis but cannot replace MAT, which
represents the gold standard.
Advantages of LPS-captured ELISA test:
- Leptospira Hardjo specific
- Better indicator of past exposure than MAT
- Safe and easy to use
- Suitable for screening large numbers of sera
References
1. Ellis et al. (1983) Possible involvement of leptospires in abortion,
stillbirths and neonatal deaths in sheep. Vet Rec 26, 291-293.
2. Yan K.-T. et al. 1999. Development of an ELISA to detect antibodies to
a protective lipopolysaccharide fraction of Leptospira borgpetersenii
serovar hardjo in cattle. Vet Microbiol 69, 173-187.