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Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1049-1055, Vol. 8, No. 6
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1049-1055.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evaluation of Two Enzyme-Linked Immunosorbent Assays
for Detecting Salmonella enterica subsp.
enterica Serovar Dublin Antibodies in Bulk
Milk
J.
Veling,1,*
F. G.
van Zijderveld,2
A. M.
van Zijderveld-van
Bemmel,2
Y. H.
Schukken,3 and
H.
W.
Barkema1
Animal Health Service, 7400 AA
Deventer,1 and Department of
Bacteriology, Institute for Animal Science and Health, ID Lelystad,
8200 AB, Lelystad,2 The Netherlands, and
Department of Population Medicine and Diagnostic Sciences,
Cornell University, Ithaca, New York 148533
Received 20 February 2001/Returned for modification 24 April
2001/Accepted 23 July 2001
 |
ABSTRACT |
Two enzyme-linked immunosorbent assays (ELISAs) for the detecting
Salmonella enterica subsp. enterica
serovar Dublin antibodies in bulk milk were developed and evaluated for
potential use in control programs. The ELISAs were based on either
lipopolysacharide (LPS ELISA) or flagellar antigen (GP ELISA).
Sensitivity was determined with 79 case herds with a wide range of
clinical signs. Specificity was determined with 125 Dutch and 200 Swedish control herds. The relation between antibodies in bulk milk,
antibodies in serum, and the level of milk production of individual
cows was studied with 61 case herds. The optimal optical density (OD)
values of the LPS ELISA and the GP ELISA were determined to be 0.2 and
0.5, respectively. The sensitivities of the LPS ELISA and the GP ELISA were 54 and 63%, respectively, with a specificity of 98% for both ELISAs with samples from the Dutch control herds. The specificities for
samples from the Swedish herds were 100% for the LPS ELISA and 95%
for the GP ELISA. The sensitivity of the combination of tests was 65%
when samples were run in parallel, and the specificity was 100% when
samples were run in series, irrespective of whether the samples came
from Dutch or Swedish control herds. The variance (R2) in the OD value for bulk milk samples
could be explained by the percentage of seropositive lactating cows in
a herd with the LPS ELISA for 51% of the samples and with the
GP ELISA for 72%. The variance in the OD value was best explained by
the combination of the percentage of seropositive lactating cows in the
herd and the mean log10 serum antibody titer for that herd
(R2 = 62% for the LPS ELISA and
R2 = 75% for the GP ELISA). Case herds
more often tested negative by the ELISA with bulk milk when the
percentage of seropositive lactating cows was less than 5%. It
is concluded that both ELISAs with bulk milk can be used in control
programs to distinguish between infected and noninfected herds.
Specificity can be increased by using the two tests in combination.
Sensitivity was relatively low for both single tests and both
tests combined.
| |
INTRODUCTION |
Salmonellosis occurs
throughout the world and has a great impact on farm economics
and public health (11, 27a). Salmonella enterica subsp. enterica serovar Dublin and serovar
Typhimurium appear to be the commonest serovars isolated from cattle.
Serovar Dublin infections in dairy herds may cause serious problems in calves and adult cows, such as septicemia, diarrhea, and abortion. In
The Netherlands, serovar Dublin is the most frequently isolated serovar
and is the second most common cause of enzootic abortion (27).
Screening to distinguish between infected and noninfected herds is
important in control programs. The use of tests adapted for use
with bulk milk samples is of interest because of the potential cost
savings and the possibility for the automation of testing. Tests
adapted for use with bulk milk have been developed for several bovine diseases, such as those caused by Brucella abortus
(22), bovine leukemia virus (19), bovine
viral diarrhea virus (15), Leptospira hardjo
(1), bovine herpesvirus type 1 (24),
Mycobacterium avium subsp. paratuberculosis
(14), bovine corona virus (23), bovine
respiratory syncytial virus (2), Coxiella
burnetii (16), Ostertagia spp.
(10), and Staphylococcus aureus
(5).
Enzyme-linked immunosorbent assays (ELISAs) based on lipopolysaccharide
(LPS) for serovar Dublin and serovar Typhimurium in milk have been
evaluated by Hoorfar et al. (7) and Hoorfar and Wedderkopp
(8). A study of serovar Dublin indicated the possibility
of identifying serovar Dublin-positive and serovar Dublin-negative
herds by an LPS ELISA with bulk milk (7) with a
sensitivity of 100% and a specificity of 95%. However, in that study
the number of case herds was limited. Wedderkopp (28) evaluated an LPS ELISA on a larger scale and determined a sensitivity of 88% and a specificity of 89%.
Recently, two ELISAs became available for evaluation of bulk milk, one
ELISA based on LPS antigen (LPS ELISA) and one ELISA based on flagellar
antigen (GP ELISA). The GP ELISA has antigenic code "g,p,"
according to the Kauffmann-White scheme for flagellar antigens
(17). The fact that the ELISAs are based on different antigens offers the opportunity to increase the specificity of the LPS
ELISA by using the ELISAs in combination.
The purpose of this study, therefore, was to evaluate the test
characteristics and potential use in control programs of two ELISAs,
ELISAs based on LPS and flagellar antigen, for screening of bulk milk
for serovar Dublin antibodies. Additionally, the relationship between
the detection of antibodies in bulk milk, on the one hand, and the
serology and the level of milk production of individual lactating cows,
on the other, was determined.
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MATERIALS AND METHODS |
Study design. (i) Farms.
The study was performed with
samples from 79 known serovar Dublin-infected herds (case herds) and
325 herds without a history of serovar Dublin infection (control
herds). The 79 serovar Dublin-infected herds were selected between
September 1995 and February 1997 from among herds for which samples or
dead animals had been sent to the Animal Health Service (Drachten, The
Netherlands) for diagnostic reasons. Clinical signs were confirmed by
at least one serovar Dublin-positive culture. All 79 farmers stated
that this was the first known Salmonella infection on the
farm. This statement was confirmed by the veterinary practitioner and
by laboratory information for the farm recorded at the Animal Health
Service for a period of at least 3 years before the outbreak. The time
of the outbreak (day 0 [D0]) was defined for each farm as the day
that the first serovar Dublin-positive culture was sampled. Animals
were individually identified by ear tags provided by the Identification
& Registration system applied in The Netherlands (13). The
mean number of animals per herd was 141 (range, 28 to 370 animals).
The herds without a history of salmonellosis consisted of 125 herds
from The Netherlands and 200 herds from the north of Sweden. Dutch
herds were selected from dairy farms that had sent in bulk milk samples
for bovine herpesvirus type 1 certification. Farmers were asked
to participate if the herd had had no history of clinical salmonellosis
in the past 3 years. The clinical status of the herd was confirmed by
checking laboratory information for the farm recorded at the Animal
Health Service. The 200 control herds from the north of Sweden were
randomly selected. Infections with serovar Dublin are unknown in this
part of Sweden (A. Engvall, personal communication).
(ii) Data collection.
Blood and bulk milk samples from the
case herds were collected on the same day and were immediately
transported to the laboratory of the Animal Health Service. Samples
were processed within 24 h and were stored at 
15°C before
analysis at ID Lelystad (Lelystad, The Netherlands). Samples were
collected 2 to 4 months after D0, with an average time of collection of
87.3 days. The time of sampling was chosen according to the expectation
that a maximum number of blood samples would be seropositive at the
time of sampling. Blood samples were taken from the coccygeal vein.
Information about the lactation status (lactating versus
nonlactating) of cows was recorded on the day of sampling. The
level of milk production of individual animals could be
estimated for animals in 61 of the 79 serovar Dublin-infected herds.
Excluded from this analysis were 12 herds for which the level of milk
production was not regularly recorded and 6 herds for which the
interval between sampling and regular recording of the level of milk
production was longer than 42 days. Data on the level of milk
production were obtained from the Royal Dutch Cattle Syndicate (Arnhem,
The Netherlands). Milk samples from the control herds were collected
without preservatives and were stored at 15°C before analysis at ID Lelystad.
ELISAs. (i) LPS ELISA and GP ELISA for serum antibodies.
Both the indirect ELISA with LPS (LPS ELISA) and the indirect ELISA
with the serovar Dublin flagellin (GP ELISA) for serum antibodies have
been described before (26). Briefly, the wells of
microdilution plates were coated with 100 µl of a solution of
purified LPS or 100 µl of a solution of purified flagellin with antigenic code g,p (hence, GP ELISA) of serovar Dublin, both of
which contained 5 µg of antigen per ml. Serum samples were added in
serial twofold dilutions, starting with a dilution of 1:100. After
incubation for 1 h at 37°C, the plates were washed and the
conjugate, an optimal dilution of horseradish peroxidase-labeled monoclonal antibody against immunoglobulin G1 (IgG1), was added. After
incubation for 1 h at 37°C the substrate solution with
5-aminosalicylic acid as the chromogen was added. The plates were read
after a 2-h incubation at room temperature. Titers are expressed as the reciprocal of the highest dilution or its logarithm that yielded an
A450 that was 50% of the absorbance
value obtained for the positive control serum.
(ii) LPS ELISA or GP ELISA for testing of bulk milk samples.
The LPS ELISA and the GP ELISA were the same as those used for the
serum samples, except that bulk milk samples were tested undiluted and
a ready-to-use substrate solution with 3,3',5,5'-tetramethylbenzidine (Ceditest; ID Lelystad) was used as the chromogen. The enzyme reaction
was stopped after a 15-min incubation at room temperature by adding 100 µl of 1.5 N sulfuric acid per well. The optical density (OD) of each
sample minus the OD of the blank wells (net OD value) was measured at
450 nm with an ELISA reader. Appropriate controls were included on each
test plate. The net OD values were used for analysis.
Analysis and analytical methods.
Bulk milk samples from case
and control herds were used to estimate the sensitivities and the
specificities of the two ELISAs. Sensitivity was defined as the
proportion of bulk milk samples that tested positive (with an OD
value equal to or above the cutoff value) among the case herds.
Specificity was defined as the proportion of bulk milk samples that
tested negative (with an OD value below the cutoff value) among the
control herds (12). Cutoff values were evaluated with
respect to sensitivity, specificity, and differential positivity rate
(DPR) (9). Optimal cutoff values were defined as those
with a specificity of at least 98% because a lower specificity would
lead to an unacceptable number of false-positive reactions in control
programs. Statistical significance was defined at P equal to
0.05. Differences in sensitivities and specificities between the two
ELISAs were tested by McNemar's chi-square test for paired data
(3). The sensitivities and specificities of the two ELISAs
were further evaluated by receiver operating characteristics (ROC)
analysis and by calculation of the area under the ROC curve (AUC)
(4).
The combination of the two ELISAs with bulk milk was studied by
correlation analysis (Pearson), without fitting of a constant.
Tests
were evaluated in parallel (any positive test result was
regarded as a
positive test result for the combination) and serially
(the result for
the test combination was positive when the results
of both tests were
positive) in an attempt to increase the sensitivities
and specificities
of the
tests.
The following variables were obtained for each of the 61 selected herds: percentage of seropositive lactating cows, percentage
of milk produced by seropositive cows, and mean
log
10 serum antibody
titer for the seropositive
lactating cows. The association between
each variable and the OD value
of the ELISA with bulk milk were
tested by linear regression analysis.
Cows were identified as
seropositive by the ELISA if the blood antibody
titer for that
ELISA was equal to or greater than a titer of 100 (
26). The
percentage of milk produced by seropositive cows
was calculated
as the proportion of milk produced by seropositive cows
in the
herd (in kilograms) divided by the total amount of milk produced
by all cows in that herd (in
kilograms).
| |
RESULTS |
Sensitivity and specificity.
The prevalence of serovar Dublin
antibodies in bulk milk for the case and the control herds is presented
in Table 1. More case herds were positive
for serovar Dublin antibodies by the GP ELISA than by the LPS ELISA
when samples with comparable OD values in a range from 0.1 to 0.9 were
tested. However, more Dutch control herds with an OD value of
0.1 were positive for serovar Dublin antibodies by the GP ELISA. No
serovar Dublin antibodies were detected by the LPS ELISA with the
Swedish control herds.
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TABLE 1.
Prevalence of serovar Dublin antibodies determined by two
ELISAs with bulk milk samples of serovar Dublin-infected herds
(n = 79), control herds from The Netherlands
(n = 125), and control herds from the north of Sweden
(n = 200)
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|
ROC curves for the different combinations of tests and control herds
are presented in Fig.
1A (LPS ELISA) and
Fig.
1B (GP
ELISA). The AUCs were 81.8% (95% confidence interval
[CI], 78.3
to 85.2%) for the LPS ELISA and the Dutch control
herds, 84.2%
(95% CI, 80.9 to 87.4%) for the LPS ELISA and the
Swedish control
herds, 92.4% (95% CI, 90.3 to 94.4%) for the GP
ELISA and the
Dutch control herds, and 88.0% (95% CI, 85.6 to 90.4%)
for the
GP ELISA and the Swedish control herds. The AUC for
the GP ELISA
was greater than that for the LPS ELISA with samples from
the
Dutch control herds.
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FIG. 1.
ROC curves of an LPS ELISA (A) and a GP ELISA (B) with
bulk milk samples from Dutch serovar Dublin-infected herds
(n = 79) to determine sensitivity and with bulk
milk samples from control herds from The Netherlands
(n = 125) and from the north of Sweden
(n = 200) to determine specificity.
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The highest DPR by the LPS ELISA was reached with an OD value of 0.1 (DPRs, 59 and 68% for the combination with the Dutch
control herds and
the Swedish control herds, respectively). The
highest DPR by the GP
ELISA was reached with an OD value of 0.4
(DPRs, 71 and 65% for the
combination with the Dutch control herds
and the Swedish control herds,
respectively).
With the specificity set at 98%, the cutoff values of the ELISAs were
analyzed further. The sensitivities of the LPS ELISA
were 54.4% (95%
CI, 43.5 to 65.4%) for the combination of the
Dutch case and the Dutch
control herds (OD = 0.2) and 63.3% (95%
CI, 52.7 to 73.9%) for
the combination of the Dutch case and the
Swedish control herds
(OD = 0.1). The sensitivities of the GP
ELISA were 63.3% (95%
CI, 52.7 to 73.9%) for the combination of
the Dutch case and the Dutch
control herds (OD = 0.5) and 39.2%
(95% CI, 28.5 to 50.0%) for
the combination of the Dutch case
and the Swedish control herds
(OD = 0.8). The sensitivity of the
LPS ELISA was higher than that
of the GP ELISA for samples from
the Swedish control herds when the
specificity was at least 98%.
No difference between the sensitivities
of the two ELISAs was
found when samples from Dutch control herds were
used. OD values
of 0.2 (LPS ELISA) and 0.5 (GP ELISA) were chosen as
optimal cutoff
values for further evaluation of the combination of the
ELISAs
and of the relation between the results of the ELISAs with bulk
milk and the serology of lactating animals. The chosen cutoff
values
resulted in sensitivities of 54.4 and 63.3% for the LPS
ELISA and the
GP ELISA with bulk milk,
respectively.
Combination of LPS and GP ELISA.
The association between the
results of the LPS and GP ELISAs with bulk milk is presented for 79 case herds (Fig. 2A) and 125 Dutch
control herds (Fig. 2B). The correlation coefficient of the two ELISAs
with bulk milk from case herds was 0.94 (P < 0.001). With the cutoff values of the LPS ELISA (OD = 0.2) and the GP ELISA (OD = 0.5) that had been determined, samples from 8.9% of the herds were positive by the GP ELISA but negative by the LPS ELISA
(Fig. 2A). Samples from one herd were positive by the LPS ELISA but
negative by the GP ELISA. The sensitivities of the two tests in
combination for samples from the case herds were 64.6% (95% CI, 53.8 to 75.3%) and 53.1% (95% CI, 41.9 to 64.4%) when parallel testing
and serial testing were applied, respectively. Therefore, parallel
testing did not increase the sensitivity, and serial testing did not
decrease it.
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FIG. 2.
Association between LPS ELISA and GP ELISA with bulk
milk samples from Dutch serovar Dublin-infected herds
(n = 79; A) and Dutch control herds
(n = 125; B). The dashed lines indicate the cutoff
values for the LPS ELISA (OD = 0.2) and the GP ELISA (OD = 0.5).
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The correlation coefficient of the two ELISAs with bulk milk samples
from the Dutch control herds was 0.28 (
P = 0.001).
With
the cutoff values of the LPS ELISA (OD = 0.2) and the
GP ELISA
(OD = 0.5) that had been determined, samples from two
herds tested
positive by each ELISA but not by the two ELISAs in
combination
(Fig.
2B). The specificity of the combination of
the two tests
was 96.8% (95% CI, 93.7 to 100%) when samples
were tested in parallel.
All samples were negative when serial testing
was applied (specificity,
100%).
Association between antibodies in bulk milk and serum of lactating
cows.
The association between OD values for bulk milk and
within-herd seroprevalence (percentage of seropositive lactating cows) for 61 herds is presented in Fig. 3A (LPS
ELISA) and Fig. 3B (GP ELISA). The correlation coefficients for the
bulk milk OD value and the percentage of seropositive lactating cows
were 0.72 and 0.84 for the LPS ELISA and the GP ELISA, respectively.
The variance in the OD value for bulk milk samples could be explained
for 51% (LPS ELISA) and 72% (GP ELISA) by the percentage of
seropositive lactating cows (Table 2).
The mean proportions of seropositive lactating cows per herd were
15.0% (range, 0 to 55.6%; 95% CI, 11.7 to 18.4%) for the LPS ELISA
and 13.7% (range, 0 to 52.8%; 95% CI, 11.0 to 16.3%) for the GP
ELISA. The mean proportions of seropositive lactating cows per herd
were 19.5% (95% CI, 14.8 to 24.2%) and 9.7% (95% CI, 5.3% to
13.5%) for herds that tested positive and negative by the LPS ELISA
with bulk milk, respectively. The mean proportions of seropositive
lactating cows per herd were 16.0% (95% CI, 12.3 to 19.8%) and 9.7%
(95% CI, 6.8 to 12.5%) for herds that tested positive and negative by
the GP ELISA with bulk milk, respectively. Bulk milk samples tested
negative by both ELISAs more often when the percentage of seropositive
cows was lower than 5% than when the seroprevalence was higher (for the LPS ELISA, chi-square = 8.09 and P = 0.004;
for the GP ELISA, chi-square = 3.84 and P = 0.05)
(Fig. 4).
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FIG. 3.
Association between percentage of seropositive lactating
cows and the OD value for bulk milk samples for 61 Dutch serovar
Dublin-infected herds determined by LPS ELISA (A) and GP ELISA (B). A
simple regression line is included.
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TABLE 2.
Univariate linear regression analysis of OD values for
bulk milk and serological status of lactating animals in serovar
Dublin-infected herds (n = 61)
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FIG. 4.
Relationship between antibodies to serovar Dublin in
bulk milk samples and percentage of seropositive lactating cows per
herd (titer,  1:100) determined by an LPS ELISA (OD 0.2) or GP
ELISA (OD 0.5).
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The combination of the percentage of seropositive lactating cows and
the percentage of milk produced by these cows explained
54% (LPS
ELISA) and 72% (GP ELISA) of the variations in the OD
values for bulk
milk samples. The mean level of milk production
per herd was 25.1 kg/cow (standard deviation, 3.7 kg/cow; range,
14.8 to 33.6 kg/cow).
Seropositive cows detected by the LPS ELISA
and the GP ELISA produced
2.8 and 3.5% less milk than seronegative
cows of the same herds,
respectively.
The combination of the percentage of seropositive lactating cows in the
herd and the mean log
10 serum antibody titer of
that
herd explained 62% (LPS ELISA) and 75% (GP ELISA) of the
variations
in the OD values for bulk milk samples. For all seropositive
animals,
the proportions of animals with serum antibody titers higher
than
the cutoff value (>1:100) were 19.7 and 22.3% for the LPS ELISA
and the GP ELISA, respectively. Bulk milk samples tested positive
by
the LPS ELISA more often when one or more lactating animals
in the herd
had an antibody titer of 200 or higher (chi-square
= 13.46 and
P < 0.001). For the GP ELISA, this association tended
to be significant (chi-square = 2.92 and
P = 0.087).
| |
DISCUSSION |
The sensitivities of the LPS ELISA and the GP ELISA were 54 and
63%, respectively. Both values are lower than those reported by
Hoorfar et al. (7), who found a sensitivity of 100% and a
specificity of 95% with 10 case herds and 20 control herds. Wedderkopp
(27) also reported a higher sensitivity of 88% and a
specificity of 89%. The relatively low sensitivities of the ELISAs
used in our study may be the consequence of the selection criteria used
for the case herds, the time of sampling, and the cutoff values chosen
for the tests. The severities of clinical symptoms and the ages of the
diseased animals were not included as selection criteria. The only
selection criterion for the case herds in this study was a single
bacteriological culture positive for serovar Dublin, irrespective of
whether the positive sample came from a lactating animal or a
nonlactating animal. Selection of only those herds with lactating cows
in which clinical symptoms occurred would certainly influence the
test results (26a). Sampling of blood and bulk milk sampling
occurred 2 to 4 months after the first serovar Dublin-positive sample
was detected. It is possible that no antibodies were detected because
antibody titers can decrease rapidly in seropositive cows (21,
26) or because seropositive cows were culled shortly after
infection. The rate of detection of infected herds therefore can
possibly be increased by more frequent sampling (28). The
cutoff values of the LPS ELISA and GP ELISA for bulk milk samples were
determined with a specificity of at least 98%. This specificity is
consistent with the common practice of defining the cutoff value as the
mean plus 2 or 3 standard deviations for the negative control group
(18). A disadvantage of this approach is that it assumes
that the results for the negative control group have a normal
distribution, whereas the results for the control herds in our study
were not normally distributed. Another disadvantage is that the test
results for the case herds are neglected. Selection of the cutoff value
by using the DPR has the advantage that it combines the test results
for the control and case herds. A disadvantage of this approach is that
it aims for the highest value, irrespective of the purpose of the test and of the consequence of false-positive and false-negative reactions. In control programs that emphasize the certification of herds as being
free of serovar Dublin, a higher cutoff value can be chosen to increase
the specificity, resulting in fewer false-positive reactions. In
control programs that emphasize the identification of infected herds, a
lower cutoff value can be chosen to increase the sensitivity, resulting
in fewer false-negative reactions.
At the same specificity, the cutoff value of the GP ELISA was higher
(OD = 0.5) than that of the LPS ELISA (OD = 0.2). The higher
cutoff value of the GP ELISA is probably due to the presence of
flagellar epitopes common to other Salmonella serovars or
other bacteria. The unexpected finding that samples from the Swedish control herds tested positive by the GP ELISA is in line with this
assumption. Clinical infections with serovar Dublin are unknown in the
part of Sweden where the samples were obtained. These observations support the choice of the LPS ELISA for screening of bulk milk for
serovar Dublin.
The LPS ELISA used in the study cannot discriminate between infections
with serovar Dublin and serovar Typhimurium (8). It
has become possible to distinguish between the two serovars in serum by
making use of monoclonal antibodies (6) or modification of
the LPS antigen (11). Further research is needed to
evaluate the possibility of a test based on the flagellar antigen (GP
ELISA) (25, 26) to discriminate between the two serovars
in cattle.
The OD values of the two ELISAs for bulk milk were evaluated in
relation to individual titers in serum rather than individual titers in
milk. The reason for this is that earlier studies found unexpectedly
high titers in milk during the first 14 days after parturition
(20) and more false-positive reactions in control herds
(7). In addition, in control programs for serovar Dublin it is expected that serum will be used more frequently than milk for
the detection of carrier animals because carriers are not always
lactating. Further research is needed to evaluate the use of ELISAs
with bulk milk in relation to the presence of carrier animals.
The variation in the OD values of the two ELISAs with bulk milk could
best be explained by the percentage of seropositive cows and the mean
log10 serum antibody titer for the seropositive animals in the herd. Control programs for Salmonella are
especially important if carriers of Salmonella are present.
The observation that the LPS ELISA with bulk milk detected positive
samples more often when one or more lactating cows had a serum antibody
titer higher than the cutoff value is therefore interesting because carriers of serovar Dublin often have a high serum antibody titer (26). Introduction of the percentage of milk produced by
seropositive cows did not further increase the percentage of the
variance in the OD value that was explained. This may be because the
overall difference in the volume of milk produced between seropositive and seronegative cows was small.
This study underlines the potential use of ELISAs with bulk milk for
distinguishing between serovar Dublin-infected and serovar Dublin-uninfected herds. The actual use of such ELISAs in a
control program depends on the test characteristics of the ELISAs in
combination with the true prevalence of the infection in a country or
region. In The Netherlands, serovar Dublin is estimated to be present in 5 to 10% of the dairy herds (unpublished observations). At this
prevalence, the number of false-positive reactions will be relatively
high compared with the number of true-positive reactions if the
specificity of the test or combination of tests is lower than 100%. We
found a specificity of 98.4% for both ELISAs for the Dutch control
herds with the cutoff values that had been determined. Use of a
combination of the two ELISAs in our study increased the specificity to
100% without a significant decrease in sensitivity, although the
number of samples tested was small. With samples from the Swedish
control herds, the specificity of the LPS ELISA was 100% and that of
the GP ELISA was 94.5%. Use of a combination of the two tests would
give a specificity of 100%. It is possible to increase the rate of
detection of serovar Dublin-infected herds by using an ELISA with bulk
milk in combination with serology for young calves (26a).
In conclusion, an LPS ELISA can best be used to screen bulk milk
samples for serovar Dublin because it has a better specificity than the
GP ELISA. Use of a combination of the LPS and GP ELISAs can be
advantageous to further increase specificity. The OD values for bulk
milk samples can best be predicted by the percentage of seropositive
cows and the mean log10 serum antibody titer for the herd. The amount of milk produced by seropositive cows had no
additional effect on the variation in the OD values of the two ELISAs
with bulk milk.
| |
ACKNOWLEDGMENTS |
This work was supported by "Diergezondheid in Beweging," a
mutual project organization of the Ministry of Agriculture, Nature Management and Fisheries (grant DIB/97858/FP).
We are indebted to A. Engvall from the National Veterinary Institute of
Sweden for providing the bulk milk samples from Sweden. We thank H. J. Stel for collecting the samples and J. v. d. Schans for coordinating
the data collection. We also thank J. Verhoeff for suggestions
regarding the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Animal Health
Service, P.O. Box 9, 7400 AA Deventer, The Netherlands. Phone: 31-570 660343. Fax: 31-570 660345. E-mail:
j.veling{at}gdvdieren.nl.
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Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1049-1055, Vol. 8, No. 6
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1049-1055.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
