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This article was published in 1969-70
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GLENFIELD COMMENTS ON THE R.W.B.T. TECHNIQUE AND RESULTS OBTAINED TO DATE
J. C. KEAST, B.V.Sc., Director of Veterinary Research, Glenfield
1. Experience with the application of the W.B.A.T. in pullorum disease control emphasises the necessity of conducting the tests in a thorough manner with accurate measurement and adequate mixing of reactants. This is considered so important in pullorum testing that a special section has been drafted in a recent Pullorum Report relative to the test in poultry.
2. The preliminary results from the three herds indicate that the 11 per cent and 8 per cent cell suspension antigens gave better agreement with the serum agglutination tube test than did the 4 per cent antigen, when read at one plus or greater (≥1).
3. The results with the 11 per cent and 8 per cent cell antigen are considered particularly promising with agreements of 92 per cent and 90 per cent respectively with the serum tube test results. (In pullorum disease where the W.B.A.T. is extensively used throughout the world for control and eradication purposes the percentage agreement between the W.B.A.T. and actual bird infection varies between 92 per cent and 96 per cent.)
4. The slight superiority of the 11 per cent cell suspension over the 8 per cent suspension (92 per cent and 90 per cent at reading of ≥ 1) was due to the 8 per cent reacting with one additional cow negative to the S.A.T. Both antigens detected 36 of the 38 cows reacting to the S.A.T.
5. The 11 per cent antigen appeared somewhat pale giving inconsistency in the size of the agglutinated clumps in several cases causing lower values of agglutination to be read compared with the 8 per cent antigen. In other words, there were more cases of less conspicuous reactions than with the 8 per cent antigen, and for this reason the 8 per cent antigen was considered preferable in the observations to date.
6. Readings of ≥ Tr. in the W.B.A.T. detected more of the S.A.T. reacting animals (37 of 38 with all three antigens) than readings of ≥ 1 but also falsely detected a larger number of the animals negative to the S.A.T. In practice, in an eradication programme where the removal of as many infected animals as possible could be desirable, if may be possible to remove all frank reactors (≥ 1) and segregate the minimal reactors (Tr.) until the results of the tube test are available.
7. Clotting occurred in six animals of 121 but was less of a problem when the tests were conducted out of the wind. Mr. Jackson is of the opinion that clotting may not be any greater problem than with pullorum when tests are read at two minutes. Recent work on pullorum in Australia is aimed at reducing the reading time to even 30 seconds as is done in Canada. To avoid problems in clotting in cattle testing it may be better to attempt producing a more sensitive antigen allowing tests to be read at 60 or 90 seconds rather than using an anticoagulant separately on the slides or test plate. Failing this the inclusion of an anticoagulant in the antigen may be possible.
8. Possibly consideration of the points mentioned in (7) above might best be deferred pending results and experience of laboratory workers in other States with the W.B.A.T.
TABLE 1
HERD "A" - TEST RESULTS
| Rapid Whole Blood Agg. Test | Standard Tube Agg. Test Reciprocal of titre ** | Milk Ring Test | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Operator 1 | Operator 2 | |||||||||||
| No. | Age | Date Calved | History | 11% | 8% | 4% | 11% | 8% | 4% | Bulk | Dil Bulk | |
| 1 | 3 | Dec., '67 | — | — | — | — | — | — | — | — | — | |
| 2 | 5 | Feb., '68 | — | — | — | — | — | — | — | *S | — | |
| 3 | 3 | Nov., '67 | — | — | — | — | — | — | — | — | — | |
| 4 | 2 | Jan., '68 | — | — | — | — | — | — | — | *S | — | |
| 5 | 6 | Sep., '67 | — | — | — | — | — | — | — | + | — | |
| 6 | 9 | Aug., '67 | Aborted '64 | Tr | 1 | 1 | Tr | 1 | 1 | — | ++ | — |
| 7 | 3 | Nov, '67 | — | — | — | — | — | — | — | — | — | |
| 8 | 5 | Dec., '67 | — | — | — | — | — | — | — | — | — | |
| 9 | 5 | Mar., '68 | — | — | — | — | — | — | — | — | — | |
| 10 | 3 | Dec., '67 | — | — | — | — | — | — | — | *S | — | |
| 11 | 5 | Oct., '67 | Aborted '67 | 2 | 2 | 3- | 3 | 3 | 3 | 320 | ++ | S |
| 12 | 3 | Jan., '67 | — | — | — | — | — | — | — | *+ | — | |
| 13 | 6 | Aug., '67 | — | — | — | — | — | — | — | *S | — | |
| 14 | 7 | Sep., '67 | 2+ | 2+ | 2 | 2 | 2+ | 2 | 160 | ++ | ++ | |
| 15 | 6 | Jun., '65 | 3 | 3 | 3 | 3 | 3 | 3 | 1280 | +++ | ++ | |
| 16 | 5 | Oct., '67 | — | — | — | — | — | — | — | — | — | |
| 17 | 9 | Oct., '67 | — | — | — | — | — | — | — | *S | — | |
| 18 | 3 | Nov., '67 | — | — | — | — | — | — | — | *S | — | |
| 19 | 3 | Nov., '67 | — | — | — | — | — | — | — | *S | — | |
| 20 | 8 | Aug., '67 | — | — | — | — | — | — | — | + | — | |
| 21 | 4 | Jun., '67 | Dystokia '67 | — | — | — | — | — | — | — | *S | — |
| 22 | 7 | Sep., '67 | — | — | — | — | — | — | — | *+ | — | |
| 23 | 4 | Mar., '68 | — | — | — | — | — | — | — | — | — | |
| 24 | 10 | Sep., '67 | — | — | — | — | — | — | — | — | — | |
| 25 | 4 | Mar., '68 | — | — | — | — | — | — | — | — | — | |
| 26 | 4 | ? | Tr | — | — | — | — | — | — | — | — | |
| 27 | 4 | Mar., '68 | — | — | — | — | — | — | — | — | — | |
| 28 | 4 | Feb., '67 | 2+ | 2+ | 2+ | 2 | 2+ | 2+ | 1280 | +++ | ++ | |
| 29 | 5 | Jan., '68 | — | — | — | — | — | — | — | — | — | |
| 30 | 3 | Nov., '67 | — | — | — | — | — | — | — | — | — | |
| 31 | 5 | Aug., '67 | — | — | — | — | — | — | — | — | — | |
| 32 | 5 | Feb., '68 | — | — | — | — | — | — | — | *S | — | |
| 33 | 3 | Feb., '68 | — | — | — | — | — | — | — | *S | — | |
| 34 | 5 | Sep., '67 | — | — | 1 | — | — | Tr | — | + | — | |
| 35 | 4 | Jan., '68 | — | — | — | — | — | — | — | + | — | |
| 36 | 4? | Apr., '68 | — | — | — | — | — | &mdash | — | &mdash | — | |
| 37 | 9 | Sep., '67 | 2 | 1 | 2 | 2 | 1 | 2 | 10 | + | — | |
| 38 | 5 | Aug., '67 | Aborted '66 | 2 | 2 | 2 | 2 | 2 | 2 | 20 | ++ | S |
| 39 | 7 | Sep., '67 | Prem. calf '67 | 3- | 2 | 2 | 2 | 2 | 2 | 40 | +++ | +++ |
| 40 | 6 | Feb., '68 | Aborted 5 mth. | 3 | 3 | 3 | 4 | 4 | 4 | 640 | ++ | + |
Nos. 5, 6, 15, 17, 37 purchased as "springers" - Vaccination History Unknown.
* Excessive reactions in some bulk milk samples possibly due to delay in reading.
** Tube Test Titre
40n = 100 i.u. antibody and interpreted as positive.
20 = 50 i.u. antibody and interpreted as doubtful.
< 10 = < 50 iu, antibody and interpreted as negative.
KEY:
Bulk - Pooled Quarter Samples.
Dil Bulk - Bulk diluted approx. 1/25 with neg. milk.
S- Suspicious M.R.T.
Tr - Trace agglutination.
NT - Not tested.
— - Negative.
TABLE 2
HERD "B" - TEST RESULTS
| Rapid Whole Blood Agg. Test | Standard Tube Agg. Test Reciprocal of titre ** | Milk Ring Test | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Operator 1 | Operator 2 | |||||||||||
| No. | Age | Date Calved | History | 11% | 8% | 4% | 11% | 8% | 4% | Bulk | Dil Bulk | |
| 1 | 10 | Oct., '67 | — | — | — | — | — | — | — | + | + | |
| 2 | 7 | Nov., '67 | 4 | 4 | 4 | 4 | 4 | 4 | 160 | +++ | ++ | |
| 3 | 7 | Feb., '68 | Aborted '67, '68 | 1 | Tr | Tr | 1 | Tr | Tr | 20 | ++ | + |
| 4 | 4 | Jan., '68 | — | — | — | — | — | — | — | — | — | |
| 5 | 8 | Oct., '67 | 3- | 3 | 3- | 3 | 3 | 2 | 320 | ++ | ++ | |
| 6 | 8 | Oct., '67 | 2- | 2 | 2 | 2 | 2 | 2 | 20 | ++ | S | |
| 7 | 5 | Jan, '68 | Aborted '68 | 3- | 3 | 3- | 2 | 3 | 2 | 1280 | +++ | ++ |
| 8 | 4 | Nov., '67 | — | — | — | — | — | — | — | — | — | |
| 9 | 6 | Oct., '67 | Vag. Disch. | — | — | — | — | — | — | — | — | — |
| 10 | 8 | Nov., '67 | — | — | — | — | — | — | — | — | — | |
| 11 | 4 | Oct., '67 | — | — | — | — | — | — | — | — | — | |
| 12 | 8 | Aug., '67 | Aborted '67 | 2 | 2 | 2 | 2 | 2 | 2 | 160 | + | — |
| 13 | 5 | Feb., '68 | Aborted '68 | 4 | 4 | 4 | 4 | 4 | 4 | 640 | ++ | + |
| 14 | 9 | Jan., '68 | 3 | 2 | 2 | 2 | 2 | 2 | 80 | ++ | + | |
| 15 | 7 | Aug., '67 | 1 | 1 | 2 | 1 | 1 | 2 | 20 | + | — | |
| 16 | 9 | Aug., '67 | 2 | 2 | 2 | 2 | 2+ | 2 | 640 | ++ | + | |
| 17 | 13 | Sep., '67 | Small calves | 2 | 2 | 3 | 2 | 2 | 3 | 320 | +++ | ++ |
| 18 | 4 | Nov., '67 | — | — | Tr | — | — | — | — | — | — | |
| 19 | 4 | Sep., '67 | 3 | 3 | 3 | 2 | 2 | 2 | 640 | +++ | ++ | |
| 20 | 4 | Oct., '67 | — | — | — | — | — | — | — | — | — | |
| 21 | 7 | Oct., '67 | Clot | 1 | 1 | Clot | 1 | 1 | 10 | + | — | |
| 22 | 7 | Jan., '68 | — | — | — | — | — | — | — | — | — | |
| 23 | 4 | Mar., '68 | Aborted '68 | 2- | 2 | 2- | 2- | 2 | 2- | 40 | + | — |
| 24 | 5 | Sep., '67 | 2- | 2 | Tr | 2- | 2 | Tr | 80 | +++ | + | |
| 25 | 5 | Dec., '67 | — | — | — | — | — | — | — | — | S | |
| 26 | 7 | Mar., '68 | — | — | — | — | — | — | — | — | S | |
| 27 | 10 | Aug., '67 | — | — | — | — | — | — | — | — | — | |
| 28 | 4 | Apr., '68 | Tr | Clot | Tr | Tr | Clot | — | 10 | — | — | |
| 29 | 4 | Oct., '67 | — | — | — | — | — | — | — | — | — | |
| 30 | 8 | Oct., '67 | Clot | — | — | Clot | — | — | — | — | — | |
| 31 | 6 | Feb., '68 | Clot | Clot | 2 | Clot | Clot | 1 | — | — | — | |
Nos. 21, 28, 30, 31 rejected from analysis because of clotting in W.B.A.T.
TABLE 3
HERD "C" - TEST RESULTS
| Rapid Whole Blood Agg. Test | Standard Tube Agg. Test Reciprocal of titre ** | Milk Ring Test | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Operator 1 | Operator 2 | ||||||||||
| No. | Date Calved | History | 11% | 8% | 4% | 11% | 8% | 4% | Bulk | Dil Bulk | |
| 1 | Feb., '68 | — | — | — | — | — | — | — | |||
| 2 | Jun., '67 | Aborted '65, '67 | 3+ | 3+ | 3 | 3 | 3 | 2 | 1280 | ||
| 3 | Nov., '67 | 3 | 3 | 2 | 3 | 2 | 2 | 320 | |||
| 4 | May., '67 | — | — | — | — | — | — | — | |||
| 5 | Aug., '67 | Clot | Clot | 1 | Clot | Clot | 1 | 80 | |||
| 6 | Aug., '67 | — | — | — | Tr | Tr | Tr | — | |||
| 7 | Jul., '67 | Aborted '65, '66 | 2 | 2+ | 2 | 2+ | 2+ | 2+ | 1280 | ||
| 8 | May., '67 | Aborted '64 | — | — | Tr | — | — | Tr | — | ||
| 9 | Dec., '67 | 3- | 3 | 2 | 3 | 3 | 2+ | 320 | |||
| 10 | Jan., '68 | 1 | 1 | Tr | 1 | 1 | Tr | 40 | |||
| 11 | Aug., '67 | — | — | — | — | — | — | — | |||
| 12 | Oct., '67 | 2 | 2 | 2 | 2 | 2 | 2 | 80 | |||
| 13 | Sep., '67 | — | — | — | — | — | — | 80 | |||
| 14 | Apr., '68 | — | — | — | — | — | — | — | |||
| 15 | May., '67 | — | — | — | — | — | — | — | |||
| 16 | Jan., '68 | 2 | 2 | Tr | 2 | 2 | Tr | 40 | |||
| 17 | Nov., '67 | — | — | — | — | — | — | — | |||
| 18 | Nov., '67 | 1+ | 1+ | Tr | 1 | 1 | Tr | 160 | |||
| 19 | Feb., '68 | — | — | — | — | — | — | — | |||
| 20 | Feb., '68 | — | — | — | — | — | — | — | |||
| 21 | Dec., '67 | — | — | — | — | — | — | — | |||
| 22 | Feb., '68 | — | — | — | — | — | — | — | |||
| 23 | Aug., '67 | — | — | — | — | — | — | — | |||
| 24 | Aug., '67 | — | — | — | — | — | — | — | |||
| 25 | Feb., '68 | 2 | 2+ | 1 | 2 | 2+ | 1 | 160 | |||
| 26 | Feb., '68 | — | — | — | — | — | — | — | — | — | |
| 27 | Feb., '68 | — | — | — | — | — | — | — | — | — | |
| 28 | Aug., '67 | — | — | — | — | — | — | — | — | — | |
| 29 | Nov., '67 | — | — | — | — | — | — | — | — | — | |
| 30 | Nov., '67 | — | — | — | — | — | — | — | — | — | |
| 31 | Feb., '68 | — | — | — | — | — | — | — | — | — | |
| 32 | Sep., '67 | — | — | — | — | — | — | — | — | — | |
| 33 | Jan., '68 | Tr | Tr | Tr | — | — | — | — | — | — | |
| 34 | Nov., '67 | — | — | — | — | — | — | — | — | — | |
| 35 | Aug., '67 | — | — | — | — | — | — | — | — | — | |
| 36 | Jun., '67 | — | — | — | — | — | — | — | — | — | |
| 37 | Jun., '67 | — | — | — | — | — | — | — | + | — | |
| 38 | May., '67 | — | Tr | Tr | — | Tr | Tr | — | |||
| 39 | Jan., '68 | — | — | — | — | — | — | 10 | + | S | |
| 40 | Feb., '68 | 2+ | 3 | 3 | 3 | 3+ | 3+ | 160 | ++ | + | |
| 41 | May., '67 | 4 | 4 | 4 | 4 | 4 | 4 | 1280 | ++ | ++ | |
| 42 | Jan., '68 | 3 | 3 | 3 | 3 | 3 | 3 | 1280 | ++ | + | |
| 43 | Feb., '68 | — | — | Tr | — | — | Tr | — | — | — | |
| 44 | Aug., '67 | Tr | 1 | Tr | Tr | 1 | Tr | 40 | — | — | |
| 45 | Mar., '68 | 1 | 1+ | 1 | 1 | 1+ | 1 | 20 | |||
| 46 | Dec., '687 | 2 | 2 | 1 | 2 | 2 | 1 | 1280 | +++ | ++ | |
| 47 | Jun., '67 | — | — | Tr | — | — | Tr | — | — | — | |
| 48 | Apr., '68 | Aborted '67, '68 | 1 | 2 | 1+ | 1 | 2 | 2 | 320 | ||
| 49 | Feb., '68 | — | — | Tr | — | — | Tr | — | — | — | |
| 50 | Feb., '68 | Clot | Clot | Clot | Clot | Clot | Clot | ||||
Nos. 5 and 50 rejected from analysis because of clotting in W.A.B.T.
TABLE 4
HERD "A" - Summary of Test Results (excluding Milk Tests)
No. of animals in the herd - 40.
No. reacting (positive plus doubtful) to Tube Agglutination Test - 7.
No. negative to the Tube Agg. Test - 33.
Rapid Whole Blood Test results with three antigens and two operators on the 40 animals.
| Grade of Reaction to R.W.B. Test | 11% Cells | 8% Cells | 4 % Cells | ||||
|---|---|---|---|---|---|---|---|
| Op. 1 | Op. 2 | Op. 1 | Op. 2 | Op. 1 | Op. 2 | ||
| 4 | 0 | 1 | 0 | 1 | 0 | 1 | |
| 3 | 3 | 2 | 2 | 2 | 2 | 2 | |
| 2 | 5 | 5 | 5 | 4 | 5 | 5 | |
| 1 | 0 | 0 | 2 | 2 | 2 | 1 | |
| Tr | 2 | 1 | 0 | 0 | 0 | 1 | |
| Total Reacting ≥ 1 | 8 | 8 | 9 | 9 | 10 | 9 | |
| Total Reacting ≥ Tr | 10 | 9 | 9 | 9 | 10 | 10 | |
| No. of Tube Test Reactors detected | 7 | 7 | 7 | 7 | 7 | 7 | |
| No. of Negatives to Tube Test Reacting to R.W.B.T.: | |||||||
| when read at ≥ 1 | 1 | 1 | 2 | 2 | 3 | 2 | |
| when read at ≥ Tr | 3 | 2 | 2 | 2 | 3 | 3 | |
TABLE 5
HERD "B" - Summary of Test Results (excluding Milk Tests)
No. of animals in the herd - 31.
No. animals excluded because of clotting in the R.W.B. Test - 4.
No. reacting (positive plus doubtful) to Tube Agglutination Test - 14.
No. negative to the Tube Agg. Test - 13.
Rapid Whole Blood Test results with three antigens and two operators on the 27 animals.
| Grade of Reaction to R.W.B. Test | 11% Cells | 8% Cells | 4 % Cells | ||||
|---|---|---|---|---|---|---|---|
| Op. 1 | Op. 2 | Op. 1 | Op. 2 | Op. 1 | Op. 2 | ||
| 4 | 2 | 2 | 2 | 2 | 2 | 2 | |
| 3 | 4 | 1 | 3 | 2 | 4 | 1 | |
| 2 | 6 | 9 | 7 | 8 | 6 | 9 | |
| 1 | 2 | 2 | 1 | 1 | 0 | 0 | |
| Tr | 0 | 0 | 1 | 1 | 3 | 2 | |
| Total Reacting ≥ 1 | 14 | 14 | 13 | 13 | 12 | 12 | |
| Total Reacting ≥ Tr | 14 | 14 | 14 | 14 | 15 | 14 | |
| No. of Tube Test Reactors detected: | |||||||
| when read at ≥ 1 | 14 | 14 | 13 | 13 | 12 | 12 | |
| when read at ≥ Tr | 14 | 14 | 14 | 14 | 14 | 14 | |
| No. of Negatives to Tube Test Reacting to R.W.B.T.: | |||||||
| when read at ≥ 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
| when read at ≥ Tr | 0 | 0 | 0 | 0 | 1 | 0 | |
TABLE 6
HERD "C" - Summary of Test Results (excluding Milk Tests)
No. of animals in the herd - 50.
No. animals excluded because of clotting in the R.W.B. Test - 2.
No. reacting (positive plus doubtful) to Tube Agglutination Test - 17.
No. negative to the Tube Agg. Test - 31.
Rapid Whole Blood Test results with three antigens and two operators on the 48 animals.
| Grade of Reaction to R.W.B. Test | 11% Cells | 8% Cells | 4 % Cells | ||||
|---|---|---|---|---|---|---|---|
| Op. 1 | Op. 2 | Op. 1 | Op. 2 | Op. 1 | Op. 2 | ||
| 4 | 1 | 1 | 1 | 1 | 1 | 1 | |
| 3 | 4 | 5 | 5 | 4 | 3 | 2 | |
| 2 | 6 | 5 | 6 | 7 | 4 | 6 | |
| 1 | 4 | 4 | 4 | 4 | 4 | 3 | |
| Tr | 2 | 2 | 2 | 2 | 10 | 10 | |
| Total Reacting ≥ 1 | 15 | 15 | 15 | 15 | 12 | 12 | |
| Total Reacting ≥ Tr | 17 | 17 | 18 | 18 | 22 | 22 | |
| No. of Tube Test Reactors detected: | |||||||
| when read at ≥ 1 | 15 | 15 | 16 | 16 | 12 | 12 | |
| when read at ≥ Tr | 16 | 16 | 16 | 16 | 16 | 16 | |
| No. of Negatives to Tube Test Reacting to R.W.B.T.: | |||||||
| when read at ≥ 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
| when read at ≥ Tr | 1 | 1 | 2 | 2 | 6 | 6 | |
TABLE 7
Percentage of 38 Tube Test Reactors detected by R.W.B.A.T.
121 cattle samples but results cover only 115 due to blood from six clotting on slide.
| Reading of R.W.B.A.T. | Rapid Whole Blood Antigen | ||
|---|---|---|---|
| 11% Cells | 8% Cells | 4% Cells | |
| 4 | 9 | 9 | 9 |
| ≥3 | 34* | 33 | 29 |
| ≥2 | 79 | 81 | 72 |
| ≥1 | 95 | 95 | 81 |
| ≥Tr | 97 | 97 | 97 |
* Indicated 34 animals gave 3 or higher reading.
TABLE 8
Percentage of 77 Non-Reactors to Tube Test giving reactions to the R.W.B.A.T.
| Reading of R.W.B.A.T. | Rapid Whole Blood Antigen | ||
|---|---|---|---|
| 11% Cells | 8% Cells | 4% Cells | |
| 4 | 0 | 0 | 0 |
| ≥3 | 0 | 0 | 0 |
| ≥2 | 1 | 0 | 1 |
| ≥1 | 1 | 3 | 3 |
| ≥Tr | 6 | 6 | 12 |
TABLE 9
The Percentage Correlation or Agreement between Rapid Whole Blood Agglutination Tests and Serological Tube Tests on 38 reactors (positive plus doubtful) and 77 non-reactors to the Tube Test
| Reading of R.W.B.A.T. | Rapid Whole Blood Antigen | ||
|---|---|---|---|
| 11% Cells | 8% Cells | 4% Cells | |
| 4 | 9.21 | 9.21 | 9.21 |
| ≥3 | 34.21 | 32.89 | 28.95 |
| ≥2 | 76.92 | 81.58 | 70.52 |
| ≥1 | 92.31 | 90.00 | 76.55 |
| ≥Tr | 88.15 | 88.09 | 77.90 |
92% correlation (if Trace reactions are excluded)
66% correlation (excluding Trace reactions) ( sic )