Biologic&
(1990) 18, 19-23
Detection of Bovine Leukemia Virus Antibodies in Bulk Tank Milk Using an ELISA Test: Improvement of the Predictive Value of Results by Repeated Testing M. Eloit,*/l
F. Lamy,t
M. Glevarec,$ F. Galaup,+ R. Turmel,$ J. J. Benet* and B. Toma*
A. Vigouroux,§
*Ecole V&&inaire, Service des Maladies Contagieuses, 94704 Maisons-Alfort, France tlJnion des Centres lnterprofessionnels Laitiers, Zone artisanale, 29270 Carhaix, France $G. D. S. 29, 3 Al/Be Sully, Kerfeunteun 2900 Quimper, France SLaboratoire V6tBrinaire Departmental, Ty Nay Quimper, 7 rue Aim6 Robert Jacques Turgot, B. P 528, 29107 Quimper Cedex, France
Abstract.
In 9457 dairy farms located in an area with low prevalence of bovine leukemia virus (BLV) infection, bulk tank milk was examined to detect for the presence of antibodies using an ELISA test. If the result was positive or doubtful, serum of all animals in the farm was tested and bulk tank milk was tested again five times every 8-l 2 days. The results were used to establish decision rules in the event of a positive or doubtful result during mass screening.
Introduction To detect BLV antibodies in bulk tank milk, the ELISA test is increasingly used in dairy farms because of its low cost associated with good sensitivity. l-7 If the result is positive, serological examination of all animals in the herd helps identify infected cattle. In a recent study undertaken in an area with low prevalence of BLV infection we identified 34 positive or doubtful herds out of 933. Among those 34 herds, 19 had infected animals. The specificity of the test (near 98%) was satisfactory. Because of the low prevalence of infection, the predictive value of a positive or doubtful result was, however, only fair (19134 = 56%). Half of the positive herds had no animal with detectable antibodies in their serum and, therefore, individual testing, which is costly, was of no benefit and could discredit the ELISA test. To avoid unnecessary testing we wanted to see whether we could improve the predictive value of a positive or doubtful result by repeated examination of bulk tank milk when the first result was positive or doubtful. To achieve this objective, 9457 dairy herds in Finistere (France), an area with low prevalence of BLV infection, were studied. Each time bulk tank milk was 1)To whom correspondence should be addressed. 1045-1056190/010019+05
$03.00/O
either positive or doubtful, serum of all animals in the herd was examined for the presence of antibodies. In addition, bulk tank milk was tested five times, 8-12 days apart. Results were used to establish decision rules whenever a positive or doubtful result was obtained during mass screening campaigns. Material
and Methods
Milk and blood sample collection
Bulk tank milk samples were obtained from 9457 dairy herds. If the result was positive, bulk tank milk samples were tested again five times, 8-12 days apart. In addition, all animals over 1 year of age were bled and their serum examined using the agargel immunodiffusion test. Bleeding was done around the time of the last bulk tank milk testing. ELISA test
Flow ELISA test (No. 270 457) was used. Positive levels were described previously.5 The index, I, was computed for each milk sample, where: I=
optical density of milk sample optical density of kit positive milk control
Results were interpreted as follows: I 2 1 indicated a positive result; O-5 d I < 1 a doubtful result; and I < O-5 a negative result. @ 1990 The International Association of Biological Standardization
20
M. Eloit
et
Results
al.
Specificity, initial test
sensitivity
and predictive
values of the
Table 1 shows the results from the first test and the five subsequent tests. Herds are divided into four groups. Group 1 represents herds found positive during all tests, group 2 represents all other herds found positive during the first test, group 3 represents herds with first test doubtful and subsequent tests doubtful or positive, and group 4 represents herds with first test doubtful and subsequent tests negative.
Table 2 presents the contingency table for the results of the initial test. Out of 9457 herds, 39 gave a positive or doubtful result. Of the 20 herds with a positive result (Table 1, groups 1 and 2), 15 were infected, with infection ranging between 3 and 44%. Of the 19 herds with a doubtful result (Table 1, groups 3 and 4), three had
Table
positive or doubtful
Group 1
2
3 4
1. Results obtained
No. Herd 30 32 26 48 10 25 22 44 24 42 11 27 47 14 17 2 5 19 21 29 46 36 1 3 4 8 9 13 16 18 20 23 33 37 38 39 41 45
from 38 herds with an initial
Total Total number of number of cattle infected (cows) cattle (cows) 63 21 67 30 60 33 32 101 69 14 108 25 24 35 33 30 11 14 17 38 83 40 33 57 11 11 21 58 69 54 18 41 2’7 24 29 69 17 58
(37) (17) (40) (19) (35) (23) (23) (60) (28) (14) (67) (25) (23) (24) * * * * * * (58) (28) * * * * * * * * * * * * * * * *
28 9 15 6 7 3 5 10 6 1 5 1 l(l) l(1) 1* 0 0 0 0 0 7 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
(17) ( 8) (11) ( 6) (7) (3) (5) ( 9) (5) (1) (3) (1)
* * * * * (6) ( 2) * * * * * * * * * * * * * * * *
% of infected cattle (cows) 44 (45) 43 (47) 22 (27) 20 (31) 12 (20) 9 (13) 16 (22) 10 (15) 9 (18) 7 ( 7) 5 ( 4) 4 ( 4) 4 ( 4) 3 (4) 3*2 0 * 0 * 0 * 0 * 0 * 8 (10) 7.5 ( 7) 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 *
Initial test ___ I Result 8.1 5 3.4 4.4 5.6 1.3 1.9 2.3 2.4 1.5 2.4 2.5 1.1 1.21 1.2 2.3 1.1 1.4 1.3 0.8 0.9 0.7 0.5 0.8 0.6 0.7 0.7 0.6 0.6 0.6 0.7 0.6 0.5 0.6 0,8 0.6 0.6
+ + + + + + + + + + + + + + + + + + + + D D D D D D D D D D D D D D D D D D
1st test ____ Z Result
2nd test ____ I Result
1.5 1.9 1.8 1.2 2.7 1.7 0.7 0.8 0.3 0.53 0.48 0 0.2 0.49 0.47 0 0.1 0 0 0 1 0.8 0.1 0 0 0 0.1 0 0 0 0.2 0 0 0 0 0 0 0
1.9 1.9 2.1 1.4 2.3 1 1.9 0.8 0.4 0.7 0.8 0 0.3 0 0.42 0 0.3 0 0 0 1.3 0.6 0 0 0.2 0 0.1 0 0 0 0 0 0.4 0.1 0 0 0 0
+ + + + + + D D D + D -
+ = positive Result; - = negative Result; D = doubtful Result; * = not determined; prevalence of BLV infection in each group.
+ + + + + + + D D D + D -
test 3rd test ____ Z Result 2.2 1.4 1.1 1.8 2.4 1.5 2.3 0.3 0.4 0.4 0.3 0 0.6 0.9 1.3 0 0.3 0.1 0 0 0.5 0.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
+ + + + + + + D D + D D -
4th test
5th test
Z Result
Z Result
2 2.1 1.4 2.2 3.2 2.3 1 0.3 0.7 0.4 1 0 0.7 1.4 1.1 0.1 0 0 0.1 0 0.5 0.96 0 0 0 0 0 0 0 0.3 0 0 0 0 0 0 0 0
1.6 1.2 2.7 1.7 2.1 1.8 1.9 0.2 0.96 0.9 0.8
+ + + + + + + D + -0 D + + -0 -0 D D -0 -0 -0 -0 -0 -0 -0 -0 -0 -0 -0 -0 -0 -0
0.7 0 0 0 0.6 0 0.6 1
0
0.3
i + + + + + + D D D D D D + -
Z = index. Herds were ordered by decreasing
21
Bulk tank milk and ELISA
Table
2. Results of initial Infected herds
Positive result Doubtful result Negative result Total
On the other hand, because of the low prevalence of BLV infection in the studied area, predictive values of positive and doubtful results were fair and poor (15/20 = 75% and 3119 = 16%, respectively). Sensitivity of the test could not be computed without knowing the infection status of all cattle in the 9418 herds with an initial negative result. We noticed, however, that 12 out of 18 infected herds had a percentage of infection less than or equal to 10.
test Non-infected herds
15 3 a* c*
5 16 b* d”
Total 20 19 9418 9457
* Values a-d are unknown because cattle of the 9418 herds with a negative initial test were not tested individually.
infected animals, with infection ranging between 1.5 and 8%. Specificity of positive results, as well as doubtful ones, can be calculated precisely if it is assumed that the number of false negative herds (Table 2 ‘a’ value) is low compared to the number of true negative herds (Table 2 ‘b’ value). It can be considered, then that the value ‘b’ (Table 2) is close to 9418. Specificity of positive and doubtful results was greater or equal to 99% (94181423 and 941819434, respectively). Table
3. Performances
of the initial
Results of subsequent tests on bulk tank milk samples
Results obtained from the five subsequent tests following an initial positive or doubtful result are reported in Table 1. We noticed that the most infected herds usually gave positive or doubtful results in subsequent tests. Table 3 presents results as a function of various variables (number of infected and
uninfected herds specificity, predictive values, number of subsequent tests). In all cases, an initial positive or doubtful result was validated if at least one
subsequent
test was positive or doubtful.
Control of positive results obtained during initial test. After the second test, specificity and predictive
test as a function
of the results of subsequent
tests
Initial test validated after:
Initial
test
Control 1 Control 2 Control 3 Control 4 Control 5
Number of identified herds with
Positive results Doubtful results Total Number of infected herds among identified herds: Positive results Doubtful results
Total
20 19 39
9 2
10
13 2 15
14 2 16
15 2 1.7
14 2 16
11
2 12
9
10
2
11
2 12
13 2 15
14 2 16
3 99% 3 99%
100% 100% 100%
100% 100% 100%
100% 100% 100%
100% 100% 100%
3 99% 100% z 99%
75% 16% 46%
100% 100% 100%
100% 100% 100%
100% 100% 100%
100% 100% 100%
93% 100% 94%
6
5
2
1 1
1 1
ii
i
2
2
15 3 18
Specificity:
Positive results Doubtful results Positive and doubtful Predictive value of:
2 99%
results
Positive results
Doubtful results Positive and doubtful results Number of infected herds not identified in the sample: Positive results Doubtful results
Total
0 0 0
1 7
In all casesthe initial result (positive or doubtful) was validated if at least onesubsequenttest gave a positive or doubtful result.
22
M. Eloit
value of positive results were 100% because all false positive herds at the initial test gave a negative result. On the other hand, there was an important loss in sensitivity because six infected herds out of 15 did not have their initial positive result confirmed (No. 11, 14,17,24,27,47). All subsequent tests did not allow the identification of these herds as infected, except for one (No. 27). In addition, the last test gave a doubtful result for one non-infected herd which had a positive initial test (No. 5). Control of doubtful results obtained during the initial test. All 16 herds having a doubtful result for the initial test gave negative results for all subsequent tests. Specificity and predictive value of doubtful results became, therefore, 100%. Loss in sensitivity resulting from subsequent testing could not be evaluated since only three herds were actually infected (No. 34, 36, 46). However, one of these three herds (No. 34) did not give a positive or doubtful result after an initial doubtful result (herd infection rate = 25%). Discussion In the area studied infection, specificity
with its low prevalence of BLV of positive or doubtful results of
Table 4. Benefit-cost
Initial
test
Subsequent control on five bulk tank milk samples Individual
et
al.
the ELISA test on bulk tank milk was excellent (> 99%). Sensitivity was also satisfactory; all herds with an infection rate greater than or equal to 10% gave one or many positive results (No. 10,25,26,30,32,48, 22,44,46). Moreover, five herds with an infection rate less than or equal to 5% (No. 11, 14,27,47,34) gave, at times, positive or doubtful results. However, the predictive value could be improved, especially for doubtful results. Individual testing of herds that are non-infected would be too costly in a screening scheme. Predictive value of positive results, after at least one subsequent test, was close to 100%. It was necessary, however, to make four control tests in order not to have a decrease in sensitivity. Thus, after the second test, predictive value of doubtful results became lOO%, while one out of three infected herds did not have its initial result confirmed. In contrast, there was no improvement of sensitivity after subsequent testing. However, this result should be interpreted with caution because of the small number of infected herds with an initial doubtful test. Table 4 gives results of the cost of two different screening schemes on the 9457 herds, which are as follows: (1) individual testing of all animals from herds with an initial positive or doubtful result (1610
analysis for two schemes to identify
infected herds
Scheme 1
Scheme 2
9.457 x 8 FF = 75.656 FF
9.457 x 8 FF = 75.656 FF
0
39x5x8FF= 1.560 FF
control
Total Number of infected herds identified Number of infected cattle identified Cost of detection of an infected herd Cost of detection of an infected animal
1.610 x 25 FF = 40.250 FF 115-906 FF
948 x 25 FF = 23.700 FF
18
17
110
109
6.439 FF
5.936 FF
1.054 FF
926 FF
100.916 FF
Scheme 1: individual testing of herds with an initial positive or doubtful result. Scheme2: individual testing of herds with an initial positive result, or of herds with an initial doubtful result confirmed by at least one subsequenttest among five. ELISA: 8 FF-Blood collection 25 FF.
Bulk tank milk and ELBA
animals); and (2) individual testing of all animals from herds with an initial positive result or with an initial doubtful result confirmed by five subsequent tests (948 animals). Costs associated with detection of an infected herd for schemes 1 and 2 are 6438 FF and 5935 FF, respectively. Cost of detection of herd No. 34, which had one infected animal identified by scheme 1 only, was 14 950 FF (115 900-100 910). The first scheme implied unnecessary interventions in 21 herds, more than half of identified herds. The second scheme had only one unnecessary intervention. In areas with low prevalence of BLV infection, the second scheme, therefore, should be preferred. However, other techniques such as milk concentration5 could improve our results. We are in the process of evaluating this method. Decisions that can be proposed using results of the present study depend on the objective of the screening scheme as well as on available funds. If it is financially possible to bleed all animals from herds with an initial positive or doubtful result, sensitivity of the test will be optimal but unnecessary testing will be done in many herds (21/39 in our study, or 54%). If we need to optimize our resources and if we accept the risk of not testing individual animals in some infected herds, then we need to differentiate herds with an initial positive result from herds with a doubtful one. All animals from herds with an initial positive result should be tested individually because subsequent tests do not improve markedly the predictive value and decrease the sensitivity, unless tests are repeated three to five times (Table 3). In the case of doubtful results, another test should be done to eliminate doubtful results given by noninfected herds. It can be seen that even when sensitivity of the test was not improved by repeated testing, this result was not necessarily confirmed by a larger sample (Table 2). There is no reason to suspect that herds with an initial doubtful result will have constant results on subsequent tests more often than herds with an initial positive result, which we showed
23
needed sometimes many additional tests to be confirmed. Since our results were obtained with a single kit of which the positivity level was previously defined, one should be careful in extending our conclusions to other kits, even if the described procedure may have general application. Acknowledgements
We would like to thank Dr Lassauzet for the English translation of the manuscript. This work was funded by the Ministry of Agriculture, Direction g&r&ale de 1’Enseignement et de la Recherche, actions experimentation et developpement. References 1. Eloit M, Hillion E, Argente G, Daniel L, Toma B. Depis-
2.
3.
4.
5. 6.
7.
tage de la leucosebovine enzootique par le test ELISA sur lait de melange dans un departement a faible prevalence de l’infection leucosique. Ret Med Vet 1988: 139: 293-299. Eloit M, Toma B, Vuillaume A, Duret C, Parodi AL. Depistage de la leucosebovine enzootique par le test ELISA applique au lait de melange: etude d’un modele theorique. J Biol Stand 1986; 14: 67-74. Forschner E, Bunger I. Detection of IBWIPV, EBL and brucellosis antibodies in samplesof bulk milk using a simplemethod for concentration of the antibodies. Dtsch Tierarztl Wochenschr 1986: 93: 112-l 15. Manz D, Wiegand D, Behrens F. Studies on the concentration of specific antibodies to the virus of enzootic bovine leucosis in individual and pooled milks. Dtsch Tierarztl Worchenschr 1986; 93: 105-144. Prevost P, Eloit M, Toma B. Depistage de la leucose bovine enzootique applique au lactoserum concentre de tank. J Biol Stand 1988; 16: 91-97. Toma B, Vuillaume A, Manet G, Duret C, Eloit M, Crespeau F, Chappuis G, Parodi AL. Depistage de la leucosebovine enzootiquepar application du test ELISA sur le lait. Ret Med Vet 1984; 160: 53-60. Toma B, Vuillaume A, Prevost P, Duret C, Eloit M,
Chappuis G, Parodi AL. Depistage de la leucose bovine enzootique a l’aide du test ELISA applique au lait de melange et au lait individuel. Ann Rech Vet 1986; 17: 75-83.
Received for publication 20 April accepted 15 June 1989.
1989;
(1990) 18, 19-23
Detection of Bovine Leukemia Virus Antibodies in Bulk Tank Milk Using an ELISA Test: Improvement of the Predictive Value of Results by Repeated Testing M. Eloit,*/l
F. Lamy,t
M. Glevarec,$ F. Galaup,+ R. Turmel,$ J. J. Benet* and B. Toma*
A. Vigouroux,§
*Ecole V&&inaire, Service des Maladies Contagieuses, 94704 Maisons-Alfort, France tlJnion des Centres lnterprofessionnels Laitiers, Zone artisanale, 29270 Carhaix, France $G. D. S. 29, 3 Al/Be Sully, Kerfeunteun 2900 Quimper, France SLaboratoire V6tBrinaire Departmental, Ty Nay Quimper, 7 rue Aim6 Robert Jacques Turgot, B. P 528, 29107 Quimper Cedex, France
Abstract.
In 9457 dairy farms located in an area with low prevalence of bovine leukemia virus (BLV) infection, bulk tank milk was examined to detect for the presence of antibodies using an ELISA test. If the result was positive or doubtful, serum of all animals in the farm was tested and bulk tank milk was tested again five times every 8-l 2 days. The results were used to establish decision rules in the event of a positive or doubtful result during mass screening.
Introduction To detect BLV antibodies in bulk tank milk, the ELISA test is increasingly used in dairy farms because of its low cost associated with good sensitivity. l-7 If the result is positive, serological examination of all animals in the herd helps identify infected cattle. In a recent study undertaken in an area with low prevalence of BLV infection we identified 34 positive or doubtful herds out of 933. Among those 34 herds, 19 had infected animals. The specificity of the test (near 98%) was satisfactory. Because of the low prevalence of infection, the predictive value of a positive or doubtful result was, however, only fair (19134 = 56%). Half of the positive herds had no animal with detectable antibodies in their serum and, therefore, individual testing, which is costly, was of no benefit and could discredit the ELISA test. To avoid unnecessary testing we wanted to see whether we could improve the predictive value of a positive or doubtful result by repeated examination of bulk tank milk when the first result was positive or doubtful. To achieve this objective, 9457 dairy herds in Finistere (France), an area with low prevalence of BLV infection, were studied. Each time bulk tank milk was 1)To whom correspondence should be addressed. 1045-1056190/010019+05
$03.00/O
either positive or doubtful, serum of all animals in the herd was examined for the presence of antibodies. In addition, bulk tank milk was tested five times, 8-12 days apart. Results were used to establish decision rules whenever a positive or doubtful result was obtained during mass screening campaigns. Material
and Methods
Milk and blood sample collection
Bulk tank milk samples were obtained from 9457 dairy herds. If the result was positive, bulk tank milk samples were tested again five times, 8-12 days apart. In addition, all animals over 1 year of age were bled and their serum examined using the agargel immunodiffusion test. Bleeding was done around the time of the last bulk tank milk testing. ELISA test
Flow ELISA test (No. 270 457) was used. Positive levels were described previously.5 The index, I, was computed for each milk sample, where: I=
optical density of milk sample optical density of kit positive milk control
Results were interpreted as follows: I 2 1 indicated a positive result; O-5 d I < 1 a doubtful result; and I < O-5 a negative result. @ 1990 The International Association of Biological Standardization
20
M. Eloit
et
Results
al.
Specificity, initial test
sensitivity
and predictive
values of the
Table 1 shows the results from the first test and the five subsequent tests. Herds are divided into four groups. Group 1 represents herds found positive during all tests, group 2 represents all other herds found positive during the first test, group 3 represents herds with first test doubtful and subsequent tests doubtful or positive, and group 4 represents herds with first test doubtful and subsequent tests negative.
Table 2 presents the contingency table for the results of the initial test. Out of 9457 herds, 39 gave a positive or doubtful result. Of the 20 herds with a positive result (Table 1, groups 1 and 2), 15 were infected, with infection ranging between 3 and 44%. Of the 19 herds with a doubtful result (Table 1, groups 3 and 4), three had
Table
positive or doubtful
Group 1
2
3 4
1. Results obtained
No. Herd 30 32 26 48 10 25 22 44 24 42 11 27 47 14 17 2 5 19 21 29 46 36 1 3 4 8 9 13 16 18 20 23 33 37 38 39 41 45
from 38 herds with an initial
Total Total number of number of cattle infected (cows) cattle (cows) 63 21 67 30 60 33 32 101 69 14 108 25 24 35 33 30 11 14 17 38 83 40 33 57 11 11 21 58 69 54 18 41 2’7 24 29 69 17 58
(37) (17) (40) (19) (35) (23) (23) (60) (28) (14) (67) (25) (23) (24) * * * * * * (58) (28) * * * * * * * * * * * * * * * *
28 9 15 6 7 3 5 10 6 1 5 1 l(l) l(1) 1* 0 0 0 0 0 7 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
(17) ( 8) (11) ( 6) (7) (3) (5) ( 9) (5) (1) (3) (1)
* * * * * (6) ( 2) * * * * * * * * * * * * * * * *
% of infected cattle (cows) 44 (45) 43 (47) 22 (27) 20 (31) 12 (20) 9 (13) 16 (22) 10 (15) 9 (18) 7 ( 7) 5 ( 4) 4 ( 4) 4 ( 4) 3 (4) 3*2 0 * 0 * 0 * 0 * 0 * 8 (10) 7.5 ( 7) 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 * 0 *
Initial test ___ I Result 8.1 5 3.4 4.4 5.6 1.3 1.9 2.3 2.4 1.5 2.4 2.5 1.1 1.21 1.2 2.3 1.1 1.4 1.3 0.8 0.9 0.7 0.5 0.8 0.6 0.7 0.7 0.6 0.6 0.6 0.7 0.6 0.5 0.6 0,8 0.6 0.6
+ + + + + + + + + + + + + + + + + + + + D D D D D D D D D D D D D D D D D D
1st test ____ Z Result
2nd test ____ I Result
1.5 1.9 1.8 1.2 2.7 1.7 0.7 0.8 0.3 0.53 0.48 0 0.2 0.49 0.47 0 0.1 0 0 0 1 0.8 0.1 0 0 0 0.1 0 0 0 0.2 0 0 0 0 0 0 0
1.9 1.9 2.1 1.4 2.3 1 1.9 0.8 0.4 0.7 0.8 0 0.3 0 0.42 0 0.3 0 0 0 1.3 0.6 0 0 0.2 0 0.1 0 0 0 0 0 0.4 0.1 0 0 0 0
+ + + + + + D D D + D -
+ = positive Result; - = negative Result; D = doubtful Result; * = not determined; prevalence of BLV infection in each group.
+ + + + + + + D D D + D -
test 3rd test ____ Z Result 2.2 1.4 1.1 1.8 2.4 1.5 2.3 0.3 0.4 0.4 0.3 0 0.6 0.9 1.3 0 0.3 0.1 0 0 0.5 0.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
+ + + + + + + D D + D D -
4th test
5th test
Z Result
Z Result
2 2.1 1.4 2.2 3.2 2.3 1 0.3 0.7 0.4 1 0 0.7 1.4 1.1 0.1 0 0 0.1 0 0.5 0.96 0 0 0 0 0 0 0 0.3 0 0 0 0 0 0 0 0
1.6 1.2 2.7 1.7 2.1 1.8 1.9 0.2 0.96 0.9 0.8
+ + + + + + + D + -0 D + + -0 -0 D D -0 -0 -0 -0 -0 -0 -0 -0 -0 -0 -0 -0 -0 -0
0.7 0 0 0 0.6 0 0.6 1
0
0.3
i + + + + + + D D D D D D + -
Z = index. Herds were ordered by decreasing
21
Bulk tank milk and ELISA
Table
2. Results of initial Infected herds
Positive result Doubtful result Negative result Total
On the other hand, because of the low prevalence of BLV infection in the studied area, predictive values of positive and doubtful results were fair and poor (15/20 = 75% and 3119 = 16%, respectively). Sensitivity of the test could not be computed without knowing the infection status of all cattle in the 9418 herds with an initial negative result. We noticed, however, that 12 out of 18 infected herds had a percentage of infection less than or equal to 10.
test Non-infected herds
15 3 a* c*
5 16 b* d”
Total 20 19 9418 9457
* Values a-d are unknown because cattle of the 9418 herds with a negative initial test were not tested individually.
infected animals, with infection ranging between 1.5 and 8%. Specificity of positive results, as well as doubtful ones, can be calculated precisely if it is assumed that the number of false negative herds (Table 2 ‘a’ value) is low compared to the number of true negative herds (Table 2 ‘b’ value). It can be considered, then that the value ‘b’ (Table 2) is close to 9418. Specificity of positive and doubtful results was greater or equal to 99% (94181423 and 941819434, respectively). Table
3. Performances
of the initial
Results of subsequent tests on bulk tank milk samples
Results obtained from the five subsequent tests following an initial positive or doubtful result are reported in Table 1. We noticed that the most infected herds usually gave positive or doubtful results in subsequent tests. Table 3 presents results as a function of various variables (number of infected and
uninfected herds specificity, predictive values, number of subsequent tests). In all cases, an initial positive or doubtful result was validated if at least one
subsequent
test was positive or doubtful.
Control of positive results obtained during initial test. After the second test, specificity and predictive
test as a function
of the results of subsequent
tests
Initial test validated after:
Initial
test
Control 1 Control 2 Control 3 Control 4 Control 5
Number of identified herds with
Positive results Doubtful results Total Number of infected herds among identified herds: Positive results Doubtful results
Total
20 19 39
9 2
10
13 2 15
14 2 16
15 2 1.7
14 2 16
11
2 12
9
10
2
11
2 12
13 2 15
14 2 16
3 99% 3 99%
100% 100% 100%
100% 100% 100%
100% 100% 100%
100% 100% 100%
3 99% 100% z 99%
75% 16% 46%
100% 100% 100%
100% 100% 100%
100% 100% 100%
100% 100% 100%
93% 100% 94%
6
5
2
1 1
1 1
ii
i
2
2
15 3 18
Specificity:
Positive results Doubtful results Positive and doubtful Predictive value of:
2 99%
results
Positive results
Doubtful results Positive and doubtful results Number of infected herds not identified in the sample: Positive results Doubtful results
Total
0 0 0
1 7
In all casesthe initial result (positive or doubtful) was validated if at least onesubsequenttest gave a positive or doubtful result.
22
M. Eloit
value of positive results were 100% because all false positive herds at the initial test gave a negative result. On the other hand, there was an important loss in sensitivity because six infected herds out of 15 did not have their initial positive result confirmed (No. 11, 14,17,24,27,47). All subsequent tests did not allow the identification of these herds as infected, except for one (No. 27). In addition, the last test gave a doubtful result for one non-infected herd which had a positive initial test (No. 5). Control of doubtful results obtained during the initial test. All 16 herds having a doubtful result for the initial test gave negative results for all subsequent tests. Specificity and predictive value of doubtful results became, therefore, 100%. Loss in sensitivity resulting from subsequent testing could not be evaluated since only three herds were actually infected (No. 34, 36, 46). However, one of these three herds (No. 34) did not give a positive or doubtful result after an initial doubtful result (herd infection rate = 25%). Discussion In the area studied infection, specificity
with its low prevalence of BLV of positive or doubtful results of
Table 4. Benefit-cost
Initial
test
Subsequent control on five bulk tank milk samples Individual
et
al.
the ELISA test on bulk tank milk was excellent (> 99%). Sensitivity was also satisfactory; all herds with an infection rate greater than or equal to 10% gave one or many positive results (No. 10,25,26,30,32,48, 22,44,46). Moreover, five herds with an infection rate less than or equal to 5% (No. 11, 14,27,47,34) gave, at times, positive or doubtful results. However, the predictive value could be improved, especially for doubtful results. Individual testing of herds that are non-infected would be too costly in a screening scheme. Predictive value of positive results, after at least one subsequent test, was close to 100%. It was necessary, however, to make four control tests in order not to have a decrease in sensitivity. Thus, after the second test, predictive value of doubtful results became lOO%, while one out of three infected herds did not have its initial result confirmed. In contrast, there was no improvement of sensitivity after subsequent testing. However, this result should be interpreted with caution because of the small number of infected herds with an initial doubtful test. Table 4 gives results of the cost of two different screening schemes on the 9457 herds, which are as follows: (1) individual testing of all animals from herds with an initial positive or doubtful result (1610
analysis for two schemes to identify
infected herds
Scheme 1
Scheme 2
9.457 x 8 FF = 75.656 FF
9.457 x 8 FF = 75.656 FF
0
39x5x8FF= 1.560 FF
control
Total Number of infected herds identified Number of infected cattle identified Cost of detection of an infected herd Cost of detection of an infected animal
1.610 x 25 FF = 40.250 FF 115-906 FF
948 x 25 FF = 23.700 FF
18
17
110
109
6.439 FF
5.936 FF
1.054 FF
926 FF
100.916 FF
Scheme 1: individual testing of herds with an initial positive or doubtful result. Scheme2: individual testing of herds with an initial positive result, or of herds with an initial doubtful result confirmed by at least one subsequenttest among five. ELISA: 8 FF-Blood collection 25 FF.
Bulk tank milk and ELBA
animals); and (2) individual testing of all animals from herds with an initial positive result or with an initial doubtful result confirmed by five subsequent tests (948 animals). Costs associated with detection of an infected herd for schemes 1 and 2 are 6438 FF and 5935 FF, respectively. Cost of detection of herd No. 34, which had one infected animal identified by scheme 1 only, was 14 950 FF (115 900-100 910). The first scheme implied unnecessary interventions in 21 herds, more than half of identified herds. The second scheme had only one unnecessary intervention. In areas with low prevalence of BLV infection, the second scheme, therefore, should be preferred. However, other techniques such as milk concentration5 could improve our results. We are in the process of evaluating this method. Decisions that can be proposed using results of the present study depend on the objective of the screening scheme as well as on available funds. If it is financially possible to bleed all animals from herds with an initial positive or doubtful result, sensitivity of the test will be optimal but unnecessary testing will be done in many herds (21/39 in our study, or 54%). If we need to optimize our resources and if we accept the risk of not testing individual animals in some infected herds, then we need to differentiate herds with an initial positive result from herds with a doubtful one. All animals from herds with an initial positive result should be tested individually because subsequent tests do not improve markedly the predictive value and decrease the sensitivity, unless tests are repeated three to five times (Table 3). In the case of doubtful results, another test should be done to eliminate doubtful results given by noninfected herds. It can be seen that even when sensitivity of the test was not improved by repeated testing, this result was not necessarily confirmed by a larger sample (Table 2). There is no reason to suspect that herds with an initial doubtful result will have constant results on subsequent tests more often than herds with an initial positive result, which we showed
23
needed sometimes many additional tests to be confirmed. Since our results were obtained with a single kit of which the positivity level was previously defined, one should be careful in extending our conclusions to other kits, even if the described procedure may have general application. Acknowledgements
We would like to thank Dr Lassauzet for the English translation of the manuscript. This work was funded by the Ministry of Agriculture, Direction g&r&ale de 1’Enseignement et de la Recherche, actions experimentation et developpement. References 1. Eloit M, Hillion E, Argente G, Daniel L, Toma B. Depis-
2.
3.
4.
5. 6.
7.
tage de la leucosebovine enzootique par le test ELISA sur lait de melange dans un departement a faible prevalence de l’infection leucosique. Ret Med Vet 1988: 139: 293-299. Eloit M, Toma B, Vuillaume A, Duret C, Parodi AL. Depistage de la leucosebovine enzootique par le test ELISA applique au lait de melange: etude d’un modele theorique. J Biol Stand 1986; 14: 67-74. Forschner E, Bunger I. Detection of IBWIPV, EBL and brucellosis antibodies in samplesof bulk milk using a simplemethod for concentration of the antibodies. Dtsch Tierarztl Wochenschr 1986: 93: 112-l 15. Manz D, Wiegand D, Behrens F. Studies on the concentration of specific antibodies to the virus of enzootic bovine leucosis in individual and pooled milks. Dtsch Tierarztl Worchenschr 1986; 93: 105-144. Prevost P, Eloit M, Toma B. Depistage de la leucose bovine enzootique applique au lactoserum concentre de tank. J Biol Stand 1988; 16: 91-97. Toma B, Vuillaume A, Manet G, Duret C, Eloit M, Crespeau F, Chappuis G, Parodi AL. Depistage de la leucosebovine enzootiquepar application du test ELISA sur le lait. Ret Med Vet 1984; 160: 53-60. Toma B, Vuillaume A, Prevost P, Duret C, Eloit M,
Chappuis G, Parodi AL. Depistage de la leucose bovine enzootique a l’aide du test ELISA applique au lait de melange et au lait individuel. Ann Rech Vet 1986; 17: 75-83.
Received for publication 20 April accepted 15 June 1989.
1989;
