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Evaluation of Five Screening Tests Used for Estimating Leucocyte Counts in Bulk Milk1
Evaluation of Five Screening Tests Used for Estimating Leucocyte Counts in Bulk Milk 1 J. J. JANZEN
Department of Dairy Science, Clemson University, Clemson, South Carolina 29631 Abstract
l~ive screening tests, Direct Microscopic Leucocyte Count, Wisconsin Mastitis Test, California Mastitis Test, Clemson Catalase Test, Wisconsin Catalase Test, used in estimating leucocyte concentrations were evaluated using 1,308 producer milk samples and 97 tank-delivered samples. These milk samples were representative of the South Carolina milk supply. The Wisconsin test was the most reliable method for estimating leucocytes in milk, using the microscopic count for comparison. This applied to both producer and tank-delivered samples. The Clemson and Wisconsin Catalase tests and the California test were not as reliable predictive tests of leucocyte concentration when compared with microscopic count. The leucocyte data were classified into three groups: less than 500,000, 500,000 to 1,000,000, and more than 1,000,000. The microscopic leucocyte count distributions of producer milk samples were 46.6, 42.4, and 11.0%, respectively, and for tank-delivered samples they were 47.4, 43.3, and 9.3%, respectively. Abnormal milk, as indicated by mastitis, has been a problem for many years. Regulations require that milk offered for sale shall be normal. Normal milk has been defined as having less than 100,000 leucocytes per milliliter; whereas, numbers in excess of 500,000 leucocytes pe r milliliter are indicative of abnormal milk (10). Screening tests have been and are being used to detect abnormal milk. Most of these tests measure the numbers of leucocytes, either directly or indirectly, in the milk supply. The action by the Interstate Milk Shippers Conference (April 3-6, 1967) has emphasized the need for more research. By July 1, 1970, each health jurisdiction must have in operation Received for publicatio~ April 29, 1968. 1 Technical Contribution no. 720, South Carolina Agricultural Experiment Station. Published by permission of the Director.
a mastitis and abnormal milk control program. The enforcement efforts will be based on results obtained from screening tests. A high leucocyte count in three out of five samplings of producers' milk shall be grounds for loss of permit to sell milk. Because screening tests will play a major role in control programs, more information is needed to evaluate existing tests for estimating leucocyte numbers. The purpose of this study was to evaluate some of the more common screening procedures that might be used in an abnormal milk control program. Our major objective was concerned with screening procedures that could be applied to bulk milk samples. The Public Health Service (8) has presented five screening procedures that have received widespread recognition and use. They are the California. Mastitis Test (CMT), Catalase Test (CT), Direct Microscopic Leucocyte Count (DMLC), Modified Whiteside Test (MWT), and the Wisconsin Mastitis Test (WMT). Postle (7) compared the California, Brabant mastitis reaction, modified Whiteside, Catalase, total cell count, Hotis test, chloride determination, electric conductivity measurement, and Wisconsin procedures as screening tests for mastitis in bulk milk. The Wisconsin test had the highest correlation with leucocyte counts, while electric conductivity measurement and chloride determination had the lowest. Reproducibility studies indicated the electric conductivity measurement having the lowest coefficient of variability, and the microscopic count the highest. A comparison of the Wisconsin, Whiteside, and the microscopic procedures (5) indicated considerable variation, especially between the Wisconsin and microscopic methods. Thompson and Postle (11) reported very good correlation (correlation coefficient 0.91) between the Wisconsin test and the square root of the microscopic count. Natzke et al. (4) reported a significant correlation (0.577) between the Catalase test and the microscopic test. North (6) stated that more research is needed to evaluate the presently used screening tests to determine which tests best measure the cell count in bulk milk samples. The micro-
89.9
330
JANZEN
scopic count has been used as a standard basis of comparison, however, and North also states that it needs more complete standardization to assure more uniformity of results both within and between operators and laboratories.
Experimental Procedures The following abnormal milk screening tests were evaluated: Wisconsin, California, Wisconsin Catalase test ( W C T ) , Clemson Catalase test (CCT), and the Direct Microscopic Leucocyte count. The procedures for Wisconsin and California Mastitis test were those described in Public Health publication 1306 (8), except for two minor modifications. The Wisconsin test results were recorded in leucocytes per milliliter using the measuring square ~ calibrated to read directly in leucocytes per milliliter. Recording Wisconsin test results as leucocytes per milliliter permitted a convenient and direct comparison with results for the microscopic count. In determining the California test results, all samples having a reaction considerably more positive than three were assigned a value of four. This represents a modification of the California test described in the Public Health publication (8). The Wisconsin Catalase test standardized procedure (9) used 9 ml milk
and 1 ml of 3% H20_~ solution in a 12-ml Roehr a monoject plastic syringe and a 3-hr incubation period at 22.2 C. As gas is liberated, the milkperoxide mixture is allowed to drip from the syringe tip. The liberated gas collects in the barrel and the amount is read directly in the calibrated syringe and converted to per cent gas. Varying amounts of milk drip from these syringes during incubation which, in turn, affect the accuracy of this test procedure. The Clemson Catalase test (3) was designed to be simple, yet permit the collection and measurement of all the oxygen released by the action of catalase on hydrogen peroxide. Standard Methods (1) procedure was followed in the slide preparation for the direct microscopic examination. A standardized loop was used to transfer 0.01 ml of milk to the slide. The Levowitz-Weber modification of the Newman-Lampe~ stain was used for all staining procedures. Microscopic examinations of stained milk films were made using the oil immersion objective. The following procedure for the counting of leucocytes was used: a) slide centered under the objective, b) slide moved to right to focus on edge of smear, c) slide then moved five fields to the left and counting begun. Thirty fields were observed and counted passing
2 Z. D. Roundy, 5130 Pepin Place, Madison, Wisconsin 53716.
:~Roehr Products Co., Inc., Deland, Florida.
TABLE 1. Relationship of four screening tests on producer samples, a
Leucocyte classification (ml) 500,000
Direct microscopic leucocyte counts
Wisconsin mastitis test
Samples
Samples
(no.) 380
(%) 45.5
(no.) 390
California b mastitis test Units
(%) 46.7
~1 2 3
Subtotal 500,000 to 1 million
354
42.4
345
41.3
101
12.1
100
12.0 Subtotal
Total
835
835
(no.) 352 28 0
(%) 92.6 7.4 0.0
(%) ~30 30-50 ~50
~1 2 ~3
125 185 44
35.3 52.3 12.4
~30 30-50 ~50
354 ~1
4
2 ~3
21 76
Samples (no.) 229 141 10
(%) 60.3 37.1 2.6
380 89 195 70
25.1 55.1 19.8
354 4.0 20.8 75.2
~30 30-50 ~50
11 35 55
101
101
835
835
May to September data. b Ranges of readings encountered for each group classification of leucocytes. J . DAIRY SOIEI~OE VOW. 52, NO. 3
02
380
Subtotal ~ 1 million
Samples
Clemson b ca.talase test
10.9
34.7 54.4
LEUCOCYTES
through the center plane moving from right to left. Bulk tank milk samples were obtained at individual farms by the milk hauler. All samples were collected either in Jeb tubes or plastic sampling bags and kept iced until delivered. Milk samples were received in the laboratory within 24 hr of pick-up and analyzed within 1 to 3 hr thereafter. Bulk milk produced on farms supplying all major milk plants within the state were sampled. All milk tested was sampled at the farm tank at least twice, some more frequently. Samplings were staggered so as to represent late spring and summer (May-September) and late fall (October-December). Approximately 1,300 producer samples were analyzed using the screening procedures. Tankdelivered milk samples (commingled milk) were also obtained from participating milk plants. A total of 97 tanker samples was obtained and analyzed. Results and Discussion
The data of 835 producer milk samples, obtained during May through September, were summarized using the microscopic count as the standard for comparison. Three major groupings were used to classify the data; namely, less than 500,000, 500,000 to 1,000,000, and more than 1,000,000 leucocytes per milliliter of
331
IN ]~ILK
milk. For each microscopic count leucocyte grouping, the corresponding Wisconsin, California, and Clemson test distributions are presented in Table 1. Wisconsin catalase data are not included because of sample loss and the resultant lower readings of this procedure. I t should be noted that a wide range of California mastitis test and Clemson catalase values occurred for each microscopic grouping. Similar data for 473 producer milk samples, obtained during October through December, are summarized in Table 2. I t is worthy of note that this group of samples showed an over-all improvement based on the microscopic count. I n addition to producer milk samples, tankdelivered milk samples also were obtained during the same time periods. The tank-delivered milk data were smnmarized in the same manner as producer samples. Table 3 represents 50 tank-milk samples obtained during May through September and Table 4 represents 47 samples obtained during October through December. A major improvement in the leucocyte picture is evident in the October to December samples. The reason for this improvement is not known, although it may be attributed to the season of the year, stage of lactation, or the producers may have become more conscious of their problem following receipt of the earlier results. The data obtained for the Microscopic, Wisconsin Mastitis, Wisconsin Catalase, and Call-
TABLE2. Relationship of four screening tests on producer samples. ~
Leucocyte classification
Direct microscopic leucocyte count
Wisconsin mastitis test
Samples
Samples
(ml)
(no.)
(%)
(11o.)
(%)
~500,000
230
48.6
225
47.6
California ~ mastltis test Units ~1 2 ~3
Samples (no.) 199 31 O
Clemson b catalase test Oe
(%) 86.5 13.5 0.0
(%) ~30 30-50 ~50
230
Samples (no.) 121 102 7
(%) 52.6 44.3 3.1
230
Subtota~ 500,000 to 1 million
200
42.3
201
42.5
~1 2 ~3
34 126 40
17.0 63.0 20.0
~30 30-50 ~50
20O Subtotal ~ 1 ml]]ion
43
9.1
47
9.9 Subtotal
Total
473
473
~1 2 ~3
0 0 43
20 133 47
10.0 66.5 23.5
200 0.O 0.0 100.0
~30 30-50 ~50
0 10 33
43
43
473
473
0.0 23.3 76.7
October to December data. b Ranges of readings encountered for each group classification of leucocytes. J , DAIRY SCIENCE VOL. 52, NO. 3
332
JANZEN
TABLE 3. Relationship of four screening tests on tank-delivered samples.~
Leucocyte classification
(ml) <500,000
Direct microscopic leucocyte count
Wisconsin mastitis ~est
Samples
Samples
(no.) 13
(%) 26.0
(no.) 14
Units
(%) 28.0
28
56.0
27
54.0
9
18.0
9
18.0
<1 2 >3
50
(%)
(%)
1O0.0 0.0 0.0
<30 30-50 >50
4 22 2
<1 2 >3
50
0 1 8
Samples
(no.)
(%)
4 9 O
30.8 69.2 0.0
13 14.3 78.6
7.1
<30 30-50 >50
28
Subtotal Total
13 0 0
02
13
Subtotal > 1 million
catalase test
Samples
(no.) <1 2 >3
Subtotal 500,000 to 1 million
Clemson b
California b mastitis test
0 20 8
0.0 71.4 28.6
28 0.0 11.1 88.9
<30 30-50 >50
0 1 8
9
9
50
50
0.0 11.1 88.9
May to September data. b Ranges of readings encountered for each group classification of leucocytes. f o r n i a p r o c e d u r e s were analyzed statistically u s i n g the simple linear regression analysis. The average, m a x i m u m , m i n i m u m values a n d the s t a n d a r d deviations are s u m m a r i z e d in Table 5. The W i s c o n s i n Catalase p e r cent oxygen values
were consistently lower t h a n the Clemson Catalase values. This would a p p e a r to be due to s a m p l e loss in the W i s c o n s i n procedure. The simple linear regressions based on five variables (mastitis screening tests) are pre-
TABLE 4. Rela£ionship of four screening tests on tank-delivered samples.~
Leucocyte classification (ml) <500,000
Direct microscopic leucocyte count
Wisconsin mastitis test
Samples
Samples
(no.) 33
(%) 70.2
(no.) 32
California b mastitis test Units
(%) 68.1
<1 2 >3
Subtotal 500,000 to 1 million
14
29.8
15
31.9
0
0.0
0
0.0 Subtotal
Total
47
47
Samples (no.) 28 5 o
O2
(%) 84.8 15.2 o.o
(%) <30 30-50 >50
33 <1 2 >3
Subtotal > 1 million
Clemson b catalase test
2 10 2
0 0 0
14.3 71.4 14.3
<30 30-50 >50
(%) 27.3 72.7 o.o
1 12 1
7.1 85.8 7.1
14 0.0 0.0 0.0
<30 30-50 >50
0 47
October to December data. b Ranges of readings encountered for each group classification of leucocytes. J. DAIRY SCIEI~CE ~OL. 52, NO. 3
(no.) 9 24 o 33
14 <1 2 >3
Samples
0 0 0 0 47
0.0 0.0 0.0
333
LEUCOCYTES IN MILK
TABLE 5. Averages, maximums, minimums, and standard deviations for five mastitis screening tests." Tests
Maximum
Minimum
Average
DMLC (leucocytes/ml) WMT (leucocytes/ml) CCT (% 02) WCT (% 03) CMT (score) b
3,130,000 3,000,000 156.0 84.0 5.0
Producer milk, 835 observations 19,000 614,000 20,000 588,000 8.0 36.3 4.0 30.5 0.0 2.2
DMLG (leucocytes/ml) WMT (leucocytes/ml) CCT (% 02) WCT (% 02) CMT (score) b
2,449,000 2,400,000 116.0 72.0 5.0
Tank-delivered milk, 50 observations 316,000 758,000 300,000 738,000 20.0 49.5 20.0 38.3 1.0 2.9
SD 407,000 384,000 17.2 10.9 1.2 470,000 473,000 20.8 11.8 1.1
May to September data. b Coded values, 0, 1, 2, 3, 4, 5 represent actual values of N, Trace, 1, 2, 3, 4. sented in Table 6. I t should be noted that the regressions of microscopic on Wisconsin and Clemson test on Wisconsin Catalase test were judged the same for both producer and tanker milks. The remaining four equations were judged to be significantly different at the 1% level for producer versus tanker milk. The close agreement between microscopic and Wisconsin mastitis procedures for both producer and tanker milks is very encouraging. Both tests measure the leucocyte numbers and show a good correlation (0.97 and 0.99, respectively). Good agreement between Clemson and Wisconsin catalase procedures for both producer and tanker milks is because the same unit o£ measurement is used in each test, the per cent oxygen released during a standardized time and temperature o£ incubation. This TABLE 6. Prediction equations based on five variables (mastitis screening tests) for producer and tank-delivered samples. Variables Y
X
Equation "
r
DMLC DMLC DMLC DMLC CCT CCT
Producer, 835 samples WMT ¥ = 11.6 -b 1.0 X CCT Y = 194.6 ÷ 11.5 X WCT Y = 95.1 -t- 17.0 X CMT ¥ = 48.2 + 252.0 X WCT ¥ : -- 7.6 + 1.4 X CMT Y : 22.5 + 6.2 X
0.97 0.49 0.46 0.76 0.92 0.44
DMLC DMLC DMLC DMLC CCT CCT
Tank-dellvered, 50 samples WMT Y ----31.6 -~ 1.0 X CCT Y -----266.9 -b 20.7 X WCT Y = --603.8 -b 35.5 X CMT Y ------299.0 -I- 364.5 X WCT Y = --11.1 -t- 1.6 X CMT Y = 3.1 + 16.0 X
0.99 0.92 0.90 0.83 0.90 0.83
a DMLC, WMT values expressed as leucocytes per milliliter (000). CCT, WCT values expressed as per cent oxygen. CMT values expressed as numerical s c o r e .
correlation was lower, however, than that of microscopic and Wisconsin mastitis test procedures. The reasons for significant differences between the other equations when comparing producer and tanker milks is not as obvious and may require additional study. A suggested explanation for equations: Microscopic on Clemson test and Miscroscopic on Wisconsin catalase test may be because different units of measurement were used to determine abnormal milk. Both catalase tests measure the amount of oxygen released. The microscopic procedure is a measure of intact leucocytes only, whereas the catalase tests are affected by leucocytes, bacteria, and other sources of catalase. Additional handling involved in the tank milk may also contribute to the variation, because of break-up of leucocytes and bacterial clumps. The lack of agreement for the microscopic and California test between producer and tankmilk may be attributed mostly to the rigorous treatment involved (in tank-milk) with the resultant rupturing of leucocytes that, in turn, affect the California test reaction. I n addition, the California test readings are subjective, thus resulting in greater variability than a standardized objective test such as the microscopic count. The poor relationship between producer and tank-delivered samples for the Clemson on California tests can be attributed to the different units of measurement employed and the subjective nature of the California test. Upon examination of the sample correlation coefficients (r), it can be seen that a high percentage o£ the variation in the dependent variable is accounted for by the variation in the independent variable except for microscopic on Wisconsin catalase, microscopic on Clemson catalase, and Clemson eatalase on California test, for producer samples. J . DAIRY SOIENCE VOL. 52, NO. 3
334
JA~ZEN
I n conclusion, it can be stated t h a t the W i s consin mastitis test a p p e a r s to be the most reliable m e t h o d f o r the estimation o f leucocyte concentration in bulk milk using the microscopic test as a basis f o r comparison, a n d t h a t all regressions were significant at the one p e r cent level.
(5)
Acknowledgments Appreciation is expressed to Mrs. Florence Crawford for her untiring efforts in the performance of tests and collection of data, and to Dr. W. P. Byrd, Experimental Statistics, for the statistical ana]yses.
References (1) American Public Health Association. 1960. Standard Methods for the Examination of Dairy Products. American Publ. tIealth Ass., Inc., New York, New York. (2) Burch, C. W. 1966. Catalase test procedure. Personal communicatiorL received from Dr. Burch, Sept. 5, 1966, via Dr. H. S. Powell, Clemson Livestock Laboratory, Columbia, South Carolina. (3) Janzen, J. J., and W. C. Cook. 1967. A simple quantitative cata]ase procedure for abnormal milk. J. Milk Food Technol., 30 : 205. (4) Natzke, 1~. P., L. It. Schultz, G. R. Barr, and W. B. Boltmann. 1965. Variation in mastitis screening tests and milk composition of udder quarters under normal c o n -
J . D~IRY SOIENOE "~OL. 52, NO. 3
(6)
(7)
(8)
(9)
(10)
(11)
ditions and following omission of a milking. J. Dairy Set., 48: 1295. New York Mastitis Control Laboratory. 1966. A comparison of the Wisconsin mastitls test, the modified Whiteside test, and the direct microscopic leucocyte count. Personal communication from Dr. tI. G. Hedges (January 5, 1967), of data compiled at New York Mastitis Control Laboratory, Ithaca, New York. North, R. H. 1967. Statement of t~obert It. North to the Subcommittee on Agriculture, House Appropriations Committee, May 11. Washington, D.C. Pestle, D. S. ]964. Comparisons of bulk scremdng procedures for mastitis. Proceedings of U. S. Livestock Sanitary Association. Public Health Service. 1965. Screening tests for the detection of abnormal milk. Public Health Serv. Publ. 1306. U. S. Dept. of Health, Education, and Welfare, Bureau of State Services (Environmental Health), Washington, D. C. Spencer, G. R., and J. Simon. 1960. The catalase, California mid cell count tests for detecting abnormalities in mi]k. Amer. J. Vet. Res., 21: 578. The National Mastitis Council, Inc. 1963. Current Concepts of Bovine Mastitis. Published by The National Mastitis Council, Inc., 910 17th Street, N. W., Washington, D.C. Thompson, D. I., and D. S. Pestle. 1964. The Wisconsin mastitis t e s t - - A n indirect estimation of leucocytes in milk. J. Milk Food Teehno]., 27: 271.
Department of Dairy Science, Clemson University, Clemson, South Carolina 29631 Abstract
l~ive screening tests, Direct Microscopic Leucocyte Count, Wisconsin Mastitis Test, California Mastitis Test, Clemson Catalase Test, Wisconsin Catalase Test, used in estimating leucocyte concentrations were evaluated using 1,308 producer milk samples and 97 tank-delivered samples. These milk samples were representative of the South Carolina milk supply. The Wisconsin test was the most reliable method for estimating leucocytes in milk, using the microscopic count for comparison. This applied to both producer and tank-delivered samples. The Clemson and Wisconsin Catalase tests and the California test were not as reliable predictive tests of leucocyte concentration when compared with microscopic count. The leucocyte data were classified into three groups: less than 500,000, 500,000 to 1,000,000, and more than 1,000,000. The microscopic leucocyte count distributions of producer milk samples were 46.6, 42.4, and 11.0%, respectively, and for tank-delivered samples they were 47.4, 43.3, and 9.3%, respectively. Abnormal milk, as indicated by mastitis, has been a problem for many years. Regulations require that milk offered for sale shall be normal. Normal milk has been defined as having less than 100,000 leucocytes per milliliter; whereas, numbers in excess of 500,000 leucocytes pe r milliliter are indicative of abnormal milk (10). Screening tests have been and are being used to detect abnormal milk. Most of these tests measure the numbers of leucocytes, either directly or indirectly, in the milk supply. The action by the Interstate Milk Shippers Conference (April 3-6, 1967) has emphasized the need for more research. By July 1, 1970, each health jurisdiction must have in operation Received for publicatio~ April 29, 1968. 1 Technical Contribution no. 720, South Carolina Agricultural Experiment Station. Published by permission of the Director.
a mastitis and abnormal milk control program. The enforcement efforts will be based on results obtained from screening tests. A high leucocyte count in three out of five samplings of producers' milk shall be grounds for loss of permit to sell milk. Because screening tests will play a major role in control programs, more information is needed to evaluate existing tests for estimating leucocyte numbers. The purpose of this study was to evaluate some of the more common screening procedures that might be used in an abnormal milk control program. Our major objective was concerned with screening procedures that could be applied to bulk milk samples. The Public Health Service (8) has presented five screening procedures that have received widespread recognition and use. They are the California. Mastitis Test (CMT), Catalase Test (CT), Direct Microscopic Leucocyte Count (DMLC), Modified Whiteside Test (MWT), and the Wisconsin Mastitis Test (WMT). Postle (7) compared the California, Brabant mastitis reaction, modified Whiteside, Catalase, total cell count, Hotis test, chloride determination, electric conductivity measurement, and Wisconsin procedures as screening tests for mastitis in bulk milk. The Wisconsin test had the highest correlation with leucocyte counts, while electric conductivity measurement and chloride determination had the lowest. Reproducibility studies indicated the electric conductivity measurement having the lowest coefficient of variability, and the microscopic count the highest. A comparison of the Wisconsin, Whiteside, and the microscopic procedures (5) indicated considerable variation, especially between the Wisconsin and microscopic methods. Thompson and Postle (11) reported very good correlation (correlation coefficient 0.91) between the Wisconsin test and the square root of the microscopic count. Natzke et al. (4) reported a significant correlation (0.577) between the Catalase test and the microscopic test. North (6) stated that more research is needed to evaluate the presently used screening tests to determine which tests best measure the cell count in bulk milk samples. The micro-
89.9
330
JANZEN
scopic count has been used as a standard basis of comparison, however, and North also states that it needs more complete standardization to assure more uniformity of results both within and between operators and laboratories.
Experimental Procedures The following abnormal milk screening tests were evaluated: Wisconsin, California, Wisconsin Catalase test ( W C T ) , Clemson Catalase test (CCT), and the Direct Microscopic Leucocyte count. The procedures for Wisconsin and California Mastitis test were those described in Public Health publication 1306 (8), except for two minor modifications. The Wisconsin test results were recorded in leucocytes per milliliter using the measuring square ~ calibrated to read directly in leucocytes per milliliter. Recording Wisconsin test results as leucocytes per milliliter permitted a convenient and direct comparison with results for the microscopic count. In determining the California test results, all samples having a reaction considerably more positive than three were assigned a value of four. This represents a modification of the California test described in the Public Health publication (8). The Wisconsin Catalase test standardized procedure (9) used 9 ml milk
and 1 ml of 3% H20_~ solution in a 12-ml Roehr a monoject plastic syringe and a 3-hr incubation period at 22.2 C. As gas is liberated, the milkperoxide mixture is allowed to drip from the syringe tip. The liberated gas collects in the barrel and the amount is read directly in the calibrated syringe and converted to per cent gas. Varying amounts of milk drip from these syringes during incubation which, in turn, affect the accuracy of this test procedure. The Clemson Catalase test (3) was designed to be simple, yet permit the collection and measurement of all the oxygen released by the action of catalase on hydrogen peroxide. Standard Methods (1) procedure was followed in the slide preparation for the direct microscopic examination. A standardized loop was used to transfer 0.01 ml of milk to the slide. The Levowitz-Weber modification of the Newman-Lampe~ stain was used for all staining procedures. Microscopic examinations of stained milk films were made using the oil immersion objective. The following procedure for the counting of leucocytes was used: a) slide centered under the objective, b) slide moved to right to focus on edge of smear, c) slide then moved five fields to the left and counting begun. Thirty fields were observed and counted passing
2 Z. D. Roundy, 5130 Pepin Place, Madison, Wisconsin 53716.
:~Roehr Products Co., Inc., Deland, Florida.
TABLE 1. Relationship of four screening tests on producer samples, a
Leucocyte classification (ml) 500,000
Direct microscopic leucocyte counts
Wisconsin mastitis test
Samples
Samples
(no.) 380
(%) 45.5
(no.) 390
California b mastitis test Units
(%) 46.7
~1 2 3
Subtotal 500,000 to 1 million
354
42.4
345
41.3
101
12.1
100
12.0 Subtotal
Total
835
835
(no.) 352 28 0
(%) 92.6 7.4 0.0
(%) ~30 30-50 ~50
~1 2 ~3
125 185 44
35.3 52.3 12.4
~30 30-50 ~50
354 ~1
4
2 ~3
21 76
Samples (no.) 229 141 10
(%) 60.3 37.1 2.6
380 89 195 70
25.1 55.1 19.8
354 4.0 20.8 75.2
~30 30-50 ~50
11 35 55
101
101
835
835
May to September data. b Ranges of readings encountered for each group classification of leucocytes. J . DAIRY SOIEI~OE VOW. 52, NO. 3
02
380
Subtotal ~ 1 million
Samples
Clemson b ca.talase test
10.9
34.7 54.4
LEUCOCYTES
through the center plane moving from right to left. Bulk tank milk samples were obtained at individual farms by the milk hauler. All samples were collected either in Jeb tubes or plastic sampling bags and kept iced until delivered. Milk samples were received in the laboratory within 24 hr of pick-up and analyzed within 1 to 3 hr thereafter. Bulk milk produced on farms supplying all major milk plants within the state were sampled. All milk tested was sampled at the farm tank at least twice, some more frequently. Samplings were staggered so as to represent late spring and summer (May-September) and late fall (October-December). Approximately 1,300 producer samples were analyzed using the screening procedures. Tankdelivered milk samples (commingled milk) were also obtained from participating milk plants. A total of 97 tanker samples was obtained and analyzed. Results and Discussion
The data of 835 producer milk samples, obtained during May through September, were summarized using the microscopic count as the standard for comparison. Three major groupings were used to classify the data; namely, less than 500,000, 500,000 to 1,000,000, and more than 1,000,000 leucocytes per milliliter of
331
IN ]~ILK
milk. For each microscopic count leucocyte grouping, the corresponding Wisconsin, California, and Clemson test distributions are presented in Table 1. Wisconsin catalase data are not included because of sample loss and the resultant lower readings of this procedure. I t should be noted that a wide range of California mastitis test and Clemson catalase values occurred for each microscopic grouping. Similar data for 473 producer milk samples, obtained during October through December, are summarized in Table 2. I t is worthy of note that this group of samples showed an over-all improvement based on the microscopic count. I n addition to producer milk samples, tankdelivered milk samples also were obtained during the same time periods. The tank-delivered milk data were smnmarized in the same manner as producer samples. Table 3 represents 50 tank-milk samples obtained during May through September and Table 4 represents 47 samples obtained during October through December. A major improvement in the leucocyte picture is evident in the October to December samples. The reason for this improvement is not known, although it may be attributed to the season of the year, stage of lactation, or the producers may have become more conscious of their problem following receipt of the earlier results. The data obtained for the Microscopic, Wisconsin Mastitis, Wisconsin Catalase, and Call-
TABLE2. Relationship of four screening tests on producer samples. ~
Leucocyte classification
Direct microscopic leucocyte count
Wisconsin mastitis test
Samples
Samples
(ml)
(no.)
(%)
(11o.)
(%)
~500,000
230
48.6
225
47.6
California ~ mastltis test Units ~1 2 ~3
Samples (no.) 199 31 O
Clemson b catalase test Oe
(%) 86.5 13.5 0.0
(%) ~30 30-50 ~50
230
Samples (no.) 121 102 7
(%) 52.6 44.3 3.1
230
Subtota~ 500,000 to 1 million
200
42.3
201
42.5
~1 2 ~3
34 126 40
17.0 63.0 20.0
~30 30-50 ~50
20O Subtotal ~ 1 ml]]ion
43
9.1
47
9.9 Subtotal
Total
473
473
~1 2 ~3
0 0 43
20 133 47
10.0 66.5 23.5
200 0.O 0.0 100.0
~30 30-50 ~50
0 10 33
43
43
473
473
0.0 23.3 76.7
October to December data. b Ranges of readings encountered for each group classification of leucocytes. J , DAIRY SCIENCE VOL. 52, NO. 3
332
JANZEN
TABLE 3. Relationship of four screening tests on tank-delivered samples.~
Leucocyte classification
(ml) <500,000
Direct microscopic leucocyte count
Wisconsin mastitis ~est
Samples
Samples
(no.) 13
(%) 26.0
(no.) 14
Units
(%) 28.0
28
56.0
27
54.0
9
18.0
9
18.0
<1 2 >3
50
(%)
(%)
1O0.0 0.0 0.0
<30 30-50 >50
4 22 2
<1 2 >3
50
0 1 8
Samples
(no.)
(%)
4 9 O
30.8 69.2 0.0
13 14.3 78.6
7.1
<30 30-50 >50
28
Subtotal Total
13 0 0
02
13
Subtotal > 1 million
catalase test
Samples
(no.) <1 2 >3
Subtotal 500,000 to 1 million
Clemson b
California b mastitis test
0 20 8
0.0 71.4 28.6
28 0.0 11.1 88.9
<30 30-50 >50
0 1 8
9
9
50
50
0.0 11.1 88.9
May to September data. b Ranges of readings encountered for each group classification of leucocytes. f o r n i a p r o c e d u r e s were analyzed statistically u s i n g the simple linear regression analysis. The average, m a x i m u m , m i n i m u m values a n d the s t a n d a r d deviations are s u m m a r i z e d in Table 5. The W i s c o n s i n Catalase p e r cent oxygen values
were consistently lower t h a n the Clemson Catalase values. This would a p p e a r to be due to s a m p l e loss in the W i s c o n s i n procedure. The simple linear regressions based on five variables (mastitis screening tests) are pre-
TABLE 4. Rela£ionship of four screening tests on tank-delivered samples.~
Leucocyte classification (ml) <500,000
Direct microscopic leucocyte count
Wisconsin mastitis test
Samples
Samples
(no.) 33
(%) 70.2
(no.) 32
California b mastitis test Units
(%) 68.1
<1 2 >3
Subtotal 500,000 to 1 million
14
29.8
15
31.9
0
0.0
0
0.0 Subtotal
Total
47
47
Samples (no.) 28 5 o
O2
(%) 84.8 15.2 o.o
(%) <30 30-50 >50
33 <1 2 >3
Subtotal > 1 million
Clemson b catalase test
2 10 2
0 0 0
14.3 71.4 14.3
<30 30-50 >50
(%) 27.3 72.7 o.o
1 12 1
7.1 85.8 7.1
14 0.0 0.0 0.0
<30 30-50 >50
0 47
October to December data. b Ranges of readings encountered for each group classification of leucocytes. J. DAIRY SCIEI~CE ~OL. 52, NO. 3
(no.) 9 24 o 33
14 <1 2 >3
Samples
0 0 0 0 47
0.0 0.0 0.0
333
LEUCOCYTES IN MILK
TABLE 5. Averages, maximums, minimums, and standard deviations for five mastitis screening tests." Tests
Maximum
Minimum
Average
DMLC (leucocytes/ml) WMT (leucocytes/ml) CCT (% 02) WCT (% 03) CMT (score) b
3,130,000 3,000,000 156.0 84.0 5.0
Producer milk, 835 observations 19,000 614,000 20,000 588,000 8.0 36.3 4.0 30.5 0.0 2.2
DMLG (leucocytes/ml) WMT (leucocytes/ml) CCT (% 02) WCT (% 02) CMT (score) b
2,449,000 2,400,000 116.0 72.0 5.0
Tank-delivered milk, 50 observations 316,000 758,000 300,000 738,000 20.0 49.5 20.0 38.3 1.0 2.9
SD 407,000 384,000 17.2 10.9 1.2 470,000 473,000 20.8 11.8 1.1
May to September data. b Coded values, 0, 1, 2, 3, 4, 5 represent actual values of N, Trace, 1, 2, 3, 4. sented in Table 6. I t should be noted that the regressions of microscopic on Wisconsin and Clemson test on Wisconsin Catalase test were judged the same for both producer and tanker milks. The remaining four equations were judged to be significantly different at the 1% level for producer versus tanker milk. The close agreement between microscopic and Wisconsin mastitis procedures for both producer and tanker milks is very encouraging. Both tests measure the leucocyte numbers and show a good correlation (0.97 and 0.99, respectively). Good agreement between Clemson and Wisconsin catalase procedures for both producer and tanker milks is because the same unit o£ measurement is used in each test, the per cent oxygen released during a standardized time and temperature o£ incubation. This TABLE 6. Prediction equations based on five variables (mastitis screening tests) for producer and tank-delivered samples. Variables Y
X
Equation "
r
DMLC DMLC DMLC DMLC CCT CCT
Producer, 835 samples WMT ¥ = 11.6 -b 1.0 X CCT Y = 194.6 ÷ 11.5 X WCT Y = 95.1 -t- 17.0 X CMT ¥ = 48.2 + 252.0 X WCT ¥ : -- 7.6 + 1.4 X CMT Y : 22.5 + 6.2 X
0.97 0.49 0.46 0.76 0.92 0.44
DMLC DMLC DMLC DMLC CCT CCT
Tank-dellvered, 50 samples WMT Y ----31.6 -~ 1.0 X CCT Y -----266.9 -b 20.7 X WCT Y = --603.8 -b 35.5 X CMT Y ------299.0 -I- 364.5 X WCT Y = --11.1 -t- 1.6 X CMT Y = 3.1 + 16.0 X
0.99 0.92 0.90 0.83 0.90 0.83
a DMLC, WMT values expressed as leucocytes per milliliter (000). CCT, WCT values expressed as per cent oxygen. CMT values expressed as numerical s c o r e .
correlation was lower, however, than that of microscopic and Wisconsin mastitis test procedures. The reasons for significant differences between the other equations when comparing producer and tanker milks is not as obvious and may require additional study. A suggested explanation for equations: Microscopic on Clemson test and Miscroscopic on Wisconsin catalase test may be because different units of measurement were used to determine abnormal milk. Both catalase tests measure the amount of oxygen released. The microscopic procedure is a measure of intact leucocytes only, whereas the catalase tests are affected by leucocytes, bacteria, and other sources of catalase. Additional handling involved in the tank milk may also contribute to the variation, because of break-up of leucocytes and bacterial clumps. The lack of agreement for the microscopic and California test between producer and tankmilk may be attributed mostly to the rigorous treatment involved (in tank-milk) with the resultant rupturing of leucocytes that, in turn, affect the California test reaction. I n addition, the California test readings are subjective, thus resulting in greater variability than a standardized objective test such as the microscopic count. The poor relationship between producer and tank-delivered samples for the Clemson on California tests can be attributed to the different units of measurement employed and the subjective nature of the California test. Upon examination of the sample correlation coefficients (r), it can be seen that a high percentage o£ the variation in the dependent variable is accounted for by the variation in the independent variable except for microscopic on Wisconsin catalase, microscopic on Clemson catalase, and Clemson eatalase on California test, for producer samples. J . DAIRY SOIENCE VOL. 52, NO. 3
334
JA~ZEN
I n conclusion, it can be stated t h a t the W i s consin mastitis test a p p e a r s to be the most reliable m e t h o d f o r the estimation o f leucocyte concentration in bulk milk using the microscopic test as a basis f o r comparison, a n d t h a t all regressions were significant at the one p e r cent level.
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Acknowledgments Appreciation is expressed to Mrs. Florence Crawford for her untiring efforts in the performance of tests and collection of data, and to Dr. W. P. Byrd, Experimental Statistics, for the statistical ana]yses.
References (1) American Public Health Association. 1960. Standard Methods for the Examination of Dairy Products. American Publ. tIealth Ass., Inc., New York, New York. (2) Burch, C. W. 1966. Catalase test procedure. Personal communicatiorL received from Dr. Burch, Sept. 5, 1966, via Dr. H. S. Powell, Clemson Livestock Laboratory, Columbia, South Carolina. (3) Janzen, J. J., and W. C. Cook. 1967. A simple quantitative cata]ase procedure for abnormal milk. J. Milk Food Technol., 30 : 205. (4) Natzke, 1~. P., L. It. Schultz, G. R. Barr, and W. B. Boltmann. 1965. Variation in mastitis screening tests and milk composition of udder quarters under normal c o n -
J . D~IRY SOIENOE "~OL. 52, NO. 3
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(7)
(8)
(9)
(10)
(11)
ditions and following omission of a milking. J. Dairy Set., 48: 1295. New York Mastitis Control Laboratory. 1966. A comparison of the Wisconsin mastitls test, the modified Whiteside test, and the direct microscopic leucocyte count. Personal communication from Dr. tI. G. Hedges (January 5, 1967), of data compiled at New York Mastitis Control Laboratory, Ithaca, New York. North, R. H. 1967. Statement of t~obert It. North to the Subcommittee on Agriculture, House Appropriations Committee, May 11. Washington, D.C. Pestle, D. S. ]964. Comparisons of bulk scremdng procedures for mastitis. Proceedings of U. S. Livestock Sanitary Association. Public Health Service. 1965. Screening tests for the detection of abnormal milk. Public Health Serv. Publ. 1306. U. S. Dept. of Health, Education, and Welfare, Bureau of State Services (Environmental Health), Washington, D. C. Spencer, G. R., and J. Simon. 1960. The catalase, California mid cell count tests for detecting abnormalities in mi]k. Amer. J. Vet. Res., 21: 578. The National Mastitis Council, Inc. 1963. Current Concepts of Bovine Mastitis. Published by The National Mastitis Council, Inc., 910 17th Street, N. W., Washington, D.C. Thompson, D. I., and D. S. Pestle. 1964. The Wisconsin mastitis t e s t - - A n indirect estimation of leucocytes in milk. J. Milk Food Teehno]., 27: 271.