Appendix 33
A comparison of two 3ABC ELISA’s in an African cattle population
with endemic multiple serotype Foot-and-Mouth disease
B.M.deC. Bronsvoort1, K.J. Sørensen5, J. Anderson3, A. Corteyn3, V.N. Tanya2,
R.P.Kitching4, K.L. Morgan1
1
University of Liverpool, Dept. Veterinary Clinical Sciences and Animal Husbandry, Leahurst,
Neston, Wirral, CH64 7TE, UK.
2
Institute of Agricultural Research for Development, Regional Centre of Wakwa, B.P. 65,
Ngaoundere, Cameroon.
3
Institute of Animal Health, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, UK.
4
National Centre for Foreign Animal Disease Canadian Food Inspection Agency Winnipeg,
Manitoba, Canada.
5
Danish Veterinary Institute, Virology Department, Lindholm, DK4771 Kalvehave, Denmark.
Keywords: 3ABC, CHEKIT, competitive ELISA, ROC
Abstract
The development of a foot-and-mouth disease virus (FMDV) serological test which is
quick and easy to use, which can identify all 7 serotypes and which can differentiate
vaccinated from convalescing or potential carriers would be a major advance in the
epidemiological tool kit for FMDV. The polyprotein 3ABC has recently been proposed
as such an antigen and a number of diagnostic tests are being developed. This paper
compares the performance of the 3ABC FMD CHEKIT bov-ov ELISA (Bommeli) and
a competitive 3ABC ELISA developed in Denmark, in an African cattle population in an
endemic FMDV region of Cameroon with multiple serotypes. The test parameters
(sensitivity, specificity and predictive value) were examined over a range of test cut-off’s.
The results indicate that both tests lack sensitivity though the CHEKIT-ELISA is
particularly low at the recommended cut-off. Their performances at different cut-off’s
and how they might perform at the herd-level are discussed.
Introduction
The non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) have
received considerable attention in recent years with the search for improved serological
tests for FMDV 1-5. The virus neutralisation test (VNT) 6 and liquid phase blocking
ELISA (LPBE) 7-9 are currently the recommended tests by the OIE. However, these tests
require each serotype to be tested separately, are time consuming to perform, require
virus containment facilities and cannot differentiate vaccinated from convalescing
animals. The development of a single, quick to use test that covered all 7 serotypes as
well as differentiating vaccinated from convalescing animals would be a major advance
in the epidemiological tool kit for FMDV.
The NSP’s are only expressed in animals with replicating virus and therefore only
animals that have been infected with live virus should develop antibodies to these
272
proteins 1,3,10,11. The currently used inactivated vaccines have been purified to remove
cellular proteins and NSP’s and should not induce antibodies to these proteins. However,
in practice vaccinated animals do produce antibodies to some of the NSP’s such as 3D 5
and antibodies to others such as 2C were found to rapidly fall below detectable levels
12,13
. The polyproteins 3ABC and 3AB appear to be the most promising protein as
diagnostic antigens 4,14-17. Recent studies of FMDV in Morocco and in Taiwan using
3ABC or 3AB ELISA’s are encouraging 18-20. If these tests are to be useful it is essential
that they are evaluated in a range of populations since the diagnostic sensitivity (Se) and
specificity (Sp) are population parameters that describe test performance for a given
reference population 21 and previous studies have shown that when applied in tropical
veterinary medicine, many diagnostic tests do not perform as well as expected 22. In
addition, the overall test performance can be evaluated and the cut-off optimised for field
use.
The following paper describes a comparison of the 3ABC FMD CHEKIT bov-ov
ELISA1 (CHEKIT-ELISA) and a 3ABC competitive ELISA (C-ELISA) developed in
Denmark. This work forms part of a larger study of the epidemiology of FMDV in the
Adamawa Province of Cameroon 23. Sera from a population based sample of the cattle
population of the Adamawa Province, an endemic FMDV region of Cameroon with
multiple serotypes, was used to evaluate the two tests. The tests were compared to the
VNT which was considered to be the ‘gold standard’. The Se (the conditional probability
that an animal is test positive given it is diseased) and Sp (the conditional probability that
the animal is test negative given that it is not diseased) of the two tests were estimated
and the overall test performances compared using receiver operating characteristics
(ROC). The test parameters are then discussed in terms of their implications when
applied at the herd level.
Materials and Methods
Study Population
A full description of the study area, the livestock population and the study design is given
elsewhere 23. In brief, the study area was the five administrative Divisions of the
64,000km2 Adamawa Province of Cameroon. A sample frame of the cattle herds in the
area was constructed from the rinderpest vaccination lists held by the local Veterinary
Centres. A cross-sectional study design was used with a stratified (by Division), twostage (by Centre and Herd) random sampling strategy. The program ‘Survey Toolbox’2,
calculated a two-stage sample of 54 centres and 3 herds per centre, allowing for a 10%
non-response rate. A questionnaire was administered to each herdsman and 5 juvenile (824 month old) and 5 adult (>24 months old) cattle were cast, examined for lesions and a
serum and oropharyngeal fluid or probang sample (OP) taken from each animal. Five
randomly selected animals gives a 95% probability of detecting at least one positive
animal in a herd of 70 if the test is 100% sensitive and specific. The herds were not
believed to have been vaccinated since no vaccine was available in the country and no
1
2
Bommeli Diagnostics, Stationsstrasse 12, CH-3097, Liebefeld, Bern, Switzerland.
http://www.ausvet.com.au/surveillance/toolbox.htm
273
herdsman reported using vaccine. The study was conducted between April and November
2000 which encompasses the rainy season when herds are close to their home areas.
Tissue culture of OP samples
The OP samples were inoculated onto bovine thyroid cell monolayers following the
OIE/WRL protocol. The cultures were incubated at 370C for up to 72 hours and
examined for any signs of cytopathic effect (cpe). Cultures with positive cpe were then
typed using the WRL sandwich antigen ELISA 24,25.
Virus Neutralisation Test (VNT)
VNT’s were performed following OIE/WRL protocol 6. The serum samples were tested
against FMDV type O Manisa, type A (P59/2000-VBM/153/09) and type SAT2
(P26/2000 – FDL/74/10) respectively. The 100 TCID50 end point was estimated for each
sample 26 and the standard OIE cut-off of ≥1/10-1.56 (1/45) used.
3ABC CHEKIT ELISA
The CHEKIT-ELISA was used according to the manufacturers instructions. Briefly, the
serum was diluted 1/100 and added in duplicate to the wells of a 96 well microtitre plate
pre-coated with the vector expressed viral 3ABC antigen. Antibodies specific to 3ABC
were bound to the antigen forming an antigen/antibody complex on the plate surface.
Unbound antibody was washed away. A horseradish peroxidase labeled guinea pig antibovine IgG conjugate was added which bound to any antibody/antigen complexes.
Unbound conjugate was removed by washing and the chromagen substrate added. The
degree of colour that developed was proportional to the amount of antibody complexed
on the plate surface and read at 405nm with a spectrophotometer. The final reading for
the sample was calculated as follows using the means of the pairs of samples and the
median of the 4 positive and negative controls on each plate:
value% =
ODsample − ODneg
OD pos − ODneg
X 100
The manufacturers recommend interpretation is an %OD of less than 20% is negative,
20-30% is ambiguous and greater than 30% is positive.
FMD-3ABC Competitive ELISA
The C-ELISA was performed as described previously 15 with modifications. Microtiter
plates were prepared by capturing 3ABC protein produced in the Baculovirus expression
system with a monoclonal antibody (MabD5) coated on the plates. Dilutions of the sera
were added followed immediately by the competing antibody, horseradish peroxidase
conjugated MabD5. After washing and addition of chromagen substrate (TMB H2O2) the
colour development was measured and the results expressed as percentage of negative
control (ODp) values.
Analysis
The results of the 3 VNT’s were combined such that an animal that was positive for one
or more serotypes was classed as positive on the combined VNT (cVNT). The cVNT and
274
ELISA results were first compared at the animal level in 2x2 tables. The Se and Sp of
each test was calculated using a range of cut-offs from 10-30% for the CHEKIT-ELISA
and ≤50% for the C-ELISA. The test characteristics were further investigated using the
ROC calculated by using AccuROC3.
The herd-level sensitivity (HSe) (the conditional probability that a herd is test positive
given that it is diseased) and specificity (HSp) (the conditional probability that a herd is
test negative given that the herd is not diseased) of the two tests were examined over a
range of possible within-herd prevalences using the Herdacc4 software in a theoretical
herd of 100 animals using a cut point of one positive animal.
Results
The virus isolation results from the probang samples indicated that there were at least 3
serotypes of virus actively circulating in the population and therefore VNT’s were carried
out for serotypes O, A and SAT2 only. The prevalence of antibodies to these 3 serotypes
are given in Table 1 below.
Table 1. Seroprevalence of serotypes O, A and SAT2 in the Adamawa Province
of Cameroon.
Test
Juvenile
Adult
SAT2
42.9 (±4.7)
67.0 (±4.3)
A
12.4 (±4.4)
55.8 (±7.1)
O
5.1 (±2.7)
15.0 (±3.9)
The results of the C-ELISA (Table 2) and CHEKIT-ELISA (Table 3) were then
compared with the cVNT results using 2x2 tables and the Se, Sp and predictive values
estimated.
Table 2. The Se and Sp of the C-ELISA at 50% compared to combined VNT
tests for O, A, and SAT2.
50% cut-off
cVNT +
cVNTPredictive Value
C-ELISA +
647
48
93.1+ %
C-ELISA 260
422
61.9- %
Se=71.3%
Sp=89.8%
3
4
AccuROC v2.4, www.accumetric.com
Herdacc v3.0 David Jordan, University of Guelph, Guelph, ON, Canada, N1G 2W1.
275
Table 3. The Se and Sp of the 3ABC at 30% (a), 20% (b), and 10% (c) compared
to combined VNT tests for O, A, and SAT2 for cattle in Cameroon.
(a)
30% cut-off
cVNT +
cVNTPredictive Value
CHEKIT +
212
10
+ 95.5%
CHEKIT 695
460
- 39.8%
Se=23.4%
Sp=97.9%
(b)
20% cut-off
CHEKIT +
CHEKIT (c)
10% cut-off
CHEKIT +
CHEKIT -
cVNT +
314
593
Se=34.6%
cVNT22
448
Sp=95.3%
Predictive Value
+ 93.5%
- 43.0%
cVNT +
537
370
Se=59.2%
cVNT74
396
Sp=84.3%
Predictive Value
+ 87.9%
- 51.7%
The results show the C-ELISA to have a low sensitivity and specificity at the
recommended cut-off. In comparison, the sensitivity of the CHEKIT-ELISA is extremely
low at the recommended cut-off of 30% though the specificity is very high. The test cutoff’s were examined further using the ROC shown in Figure 1 below. From the curves it
is clear that the C-ELISA performs better overall though the cut-off of 50% may not be
optimal. The CHEKIT-ELISA though not performing as well overall is not optimised at a
cut-off of 30%. Lowering the cut-off to 15% would greatly increase the sensitivity of the
test without too much loss of specificity. The area under the two curves were compared
using a non-parametric method 27.
276
Figure 1. ROC for 3ABC CHEKIT-ELISA and C-ELISA
The estimates of the HSe and HSp of the C-ELISA and the CHEKIT-ELISA when
applied to a theoretical herd of 100 animals are given in Tables 4a and 4b below. A range
of herd-level prevalences were investigated from 1 to 30% for each of the tests. For the
C-ELISA the low Sp is rapidly compounded with the need to increase the sample size to
increase the HSe. Therefore using a cut-off of 45 or 40% would increase the Sp and
overcome some of the imperfections of the test.
The CHEKIT-ELISA by having a high Sp is less prone to the rapid decline in HSp when
applied to the herd. However, because the Se is also very low many more animals have to
be sampled depending on the underlying herd prevalence.
277
Table 4. The herd-level sensitivity and specificity of the C-ELISA (a) and
CHEKIT-ELISA (b)
(a)
Assumptions: Cut-point = 1, Sensitivity = 0.713, Specificity = 0.898.
Sampling without replacement. Herd size = 100.
______________________________________________________________
Sample HSPEC HSENS:
size: 0
1
3
5
10
15
30
(10)
(11)
(12)
(13)
(16)
(19)
(29)
______________________________________________________________
1
0.900 0.110 0.120 0.130 0.160 0.190 0.290
11 0.294 0.742 0.774 0.802 0.869 0.914 0.982
21 0.083 0.936 0.951 0.963 0.984 0.993 1.000
31 0.020 0.987 0.992 0.995 0.999 1.000 1.000
41 0.004 0.998 0.999 0.999 1.000 1.000 1.000
______________________________________________________________
(b)
Assumptions: Cut-point = 1, Sensitivity = 0.234, Specificity = 0.979.
Sampling without replacement. Herd size = 100.
______________________________________________________________
Sample HSPEC HSENS:
size:
0
1
3
5
10
15
30
(2)
(2)
(3)
(3)
(4)
(5)
(8)
______________________________________________________________
1
0.980 0.020 0.030 0.030 0.040 0.050 0.080
11 0.791 0.209 0.298 0.298 0.377 0.449 0.620
21 0.622 0.378 0.511 0.511 0.617 0.701 0.860
31 0.474 0.526 0.676 0.676 0.780 0.851 0.955
41 0.346 0.654 0.799 0.799 0.884 0.934 0.988
_______________________________________________________________
a
Columns are HSPEC/HSENS followed by no. of infected animals in the herd.
Numbers in brackets are the predicted no. of test positives in the herd
b
Discussion
The results of this analysis indicate that at the individual animal level the C-ELISA
performs better than the CHEKIT-ELISA in an Africa cattle population, though neither
test performs particularly well. From a practical point of view there are advantages to
using a competitive ELISA in that there is only one conjugate needed for all species. The
additional use of a Mab as the competitor should make the standardisation of the test
between laboratories much easier. As mentioned in the introduction, Se and Sp are not
fixed values and will vary between sub-populations and between populations conditional
on the distribution of influential covariates (eg. age, stage of disease). Therefore, it may
be useful to calculate the stratum specific Se and Sp where possible or use logistic
regression models that include the covariates in order to obtain estimates, as these may
278
have greater diagnostic utility and may be applied to other populations
anticipated that such analyses will be carried out.
21,28
. It is
It was not unexpected that the Se and Sp were lower than reported in previous
evaluations15. This is most likely due to there being cross reactions from other
pathogens/antigens which animals are exposed to in the tropics, different breeds (Bos
indicus), and management systems, the multiple serotypes of FMDV that the animals
were exposed to and the much wider range of disease states in a natural population21. In
addition the use of the combined VNT results as a ‘gold standard’ has not been
previously used and the estimates should therefore be interpreted with a degree of
caution.
Although the tests are less than perfect they still may be useful depending on the
circumstances in which they are used. When applying a test to a herd it is important to
realise that the HSe and HSp are dependent on, the individual test Se and Sp, the number
of animals tested, the true prevalence in infected herds, the herd cut-off value used to
classify herds as positive and the variation in Se, Sp and prevalence among herds 29. In
particular, if a test is interpreted at the herd level, as long as the individual test Sp is very
high, lower individual Se’s can be overcome to some degree. So although these results
can not be directly applied to the European setting the basic implications still apply.
There are clearly a number of senarios where a NSP test could be useful. For example, in
endemic areas where vaccination is started as a control measure, being able to monitor
the levels of sub-clinical disease would help decision making. Using the right cut-offs
these tests could be used. However, as the prevalence falls, both tests but in particular the
CHEKIT-ELISA will have increasing problems with low HSe unless most of the herd
are sampled. Another proposed use has been at borders. The main problem with this will
be that the test would need to be interpreted at the individual animal level unless all the
animals were from the same source.
For Europe, which is driving much of this research, the motivation is to be able to screen
vaccinated herds after an outbreak and identify herds with sub-clinical infections that
could become ‘carriers’. Recent changes in OIE regulations will allow countries to
resume trading after 6 months instead of 12 months if they vaccinate and can then screen
and demonstrate freedom from disease. This change assumes that there is a test that will
distinguish vaccinated from sub-clinically infected/convalescing animals. However, it is
unclear what the prevalence of such animals would be in a herd though it is likely to be
low in which case both these tests, even with adjusted cut-off’s, may struggle to achieve
acceptable HSe and HSp.
279
APPENDIX
Apparent prevalence AP
True prevalence
Sample size per herd n
TP
AP=(1-Sp)+(Se+Sp-1)*TP
Hse=1-(1-AP)n
HSp=Spn
Acknowledgments
The authors would like to thank Dr. Charles Nfon and Mr. Hamman Saidou Mustaffa for
their considerable efforts in trying conditions in the field. In addition we thank P.
Hamblin and D. Gibson for working on the VNT and ELISA. Mark Bronsvoort is a
Wellcome Trust Research Training Fellow in Clinical Tropical Epidemiology.
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