Br. vet.]. (1976) 132. 621
A COMPARATIVE STUDY ON THE USE OF WHEY AND SERUM FOR MICROSCOPIC AGGLUTINATION TEST FOR THE DETECTION OF LEPTOSPIRA ANTIBODIES BY
S. N .
HussAINI
Bacteriology Department, Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, Wrybridge, Surrry SUMMARY
Whey and serum from I 23 animals in three herds were tested using a microscopic agglutination test. A close relationship between the whey and serum agglutination titres indicates that whey agglutination test is a dependable test which can be employed in testing individual or bulk milk samples. A titre of I :4, giving so% agglutination, using whey is suggested as a diagnostic titre as it compares well with the diagnostic titre of I :I oo for serum. INTRODUCTION
As early as 1893 Breiger & Ehrlich demonstrated that goats when inoculated with tetanus toxin secreted antibodies lasting the entire period of lactation and that these antibodies could also be detected in their whey. This knowledge has since been applied by several workers in detecting brucella infection in cattle (Seddon, I915; Cooledge, I9I6; Smith, Orcutt & Little, I923). It was Fleischauer (I937) however who eventually developed the milk ring test (MRT) using a stained brucella antigen. The use of milk and whey for the detection of brucella infection prompted van der Hoeden (I952, I955) to utilize whey agglutination test (WAT) in the investigation of several outbreaks of leptospirosis in cattle, due to Leptospira grippotyphosa and L. pomona, and in goats, due to L. grippotyphosa. He concluded that the test was always specific and recommended it as a screening test and as a confirmatory test after serum agglutination testing (SAT). Morse, Allen, Pope & Krohn (1955) observed that whey agglutinins to L. pomona in cows could be demonstrated for as long as 29 months after the onset ofleptospirosis in the herd. The highest whey reactions were usually observed in cows which had recently experienced infection. Carbrey & Packer (I96 I) noted that the whey titre of the individual cow varied in relation to the serum titre. They further observed the absence of a significant difference between the quarter titres of the same cow and regarded this as evidence that localized udder infection does not occur in leptospirosis. They were also able to detect antibodies against L. pomona in a
BRITISH VETERINARY JOURNAL, 132, 6
herd using composite herd milk sample from the bulk-tank or milk-can. However, attempts at evolving a comparable MRT for leptospirosis met with failure because effective staining destroyed the antigenicity of the leptospirae and well defined rings appeared in negative milk samples (United States Department of Agriculture, I 953). The test most commonly used for the diagnosis of leptospirosis is the microscopic agglutination test (MAT) employing blood serum (World Health Organization, I967). This paper includes an evaluation of a test using milk whey as an alternative or as an adjunct to serum in the diagnosis ofleptospirosis, since milk is easier to obtain. The detection of leptospira antibodies in a herd milk sample, taken from a bulk-tank or milk-can, is a convenient way of screening a herd for leptospira infection. MATERIALS AND METHODS
Selection of herds The selection of farms A and B was based on the previous serological examination of the herds which revealed a high number of reactors to the hebdomadis group, and in the case of farm B one of the cowmen developed leptospirosis caused by leptospira of the hebdomadis group. Farm C was selected as a known serologically negative herd. Whry preparation Milk collected in Universal bottles (23 ml) was defatted by centrifugation at 2500 revjmin (Boo g) for 15 min and removal of the fat layer. Rennet was added at the rate of I drop to 2 ml of the defatted milk and the mixture was incubated for I h at 37°C and the whey was used for microscopic agglutination test (MAT). It was sometimes necessary to clarify the whey by passing it through a Millipore* prefilter and a filter of o·22 fl. pore diameter. Bulk milk Aliquots of milk from individual animals were pooled. Serum Blood collected from the coccygeal vem m I o ml Vacu tainert tubes was allowed to clot and serum separated. Microscopic agglutination test Microscopic agglutination tests (MAT) using whey and serum samples, with living antigen suspensions, were performed as described by Wolff (1954). The antigens were L. wolffi and L. mini belonging to the hebdomadis serogroup, grown for 7-Io days at 30°C in Korthof's medium. For the whey agglutination test (WAT) the lowest dilution examined was prepared by mixing equal amounts of whey and antigen, resulting in a final * MilJipore (UK) Limited, MilJipore House, Abbey Road, London NWw 7SP. t Becton, Dickinson UK Limited, York House, Empire Way, Wembley, Middlesex HAgoPS.
MICROSCOPIC AGGLUTINATION TESTS
whey dilution of 1 :2 and subsequent twofold dilutions of whey were prepared giving dilutions of 1 :2, 1 :4, 1 :8, 1 :16, 1 :32 and 1 :64. The final serum dilutions used for the serum agglutination test (SAT) were 1 :roo, 1 :400 and 1 :r6oo.
Number of samples One hundred and twenty-three pairs of serum and whey samples were tested from cattle from farms A (64 pairs), B (44 pairs) and C ( 15 pairs). TABLE I COMPARATIVE FINDINGS ON THE USE OF BOVINE SERUM AND MILK WHEY SAMPLES BY THE MICROSCOPIC AGGLUTINATION TEST IN THE DIAGNOSIS OF LEPTOSPIROSIS
Microscopic agglutination test (MAT ) * Location and total No. of samples
Dilutions Serum (I/IOo, Ij4oo, I/I6oo)
L. wolffi
Farm A 64
tr/IOO I/Ioo 2/100 2/400 2/ I6oo
(I8) ( s) ( 6) (23 ) ( Io) ( 2)
L. mini
(20) tr/ Ioo ( 2) I / 100
(I I )
2/IOO (I8) 2/400 (I2) 2/I6oo ( I )
Pooled milk (positives only) Pooled milk (including negatives)
FarmB 44
I/Ioo 2/IOO 3/IOO 2/400 3/400 2/I6oo
Pooled milk (including negatives) FarmC IS
( s) ( I) (Ig) ( 8) ( 7) ( I) ( 3)
Ij iOO 2/ IOO 3/ Ioo 2/400 3/400 2jr6oo
Dilutions Whey (I/2, I/4, I/8, Iji6, I/32, I/64)
L. wolffi
tr/ 2 I/2 2/2 3/2 2/4 3/4 2/8 3/8 2/ I6 3/ I6 2/32 2/64
(10)
( o) ( 6) ( I) (I ) ( 2)
tr/2 I/2 2/2 3/2 2/4 3/4 2/8 3/8 2/I6 3/I6 2/32 2/64
2/I6
2/I6
3/8
2/8
( s) ( I) (22) ( 5) ( 7) ( 4) ( o)
(I8) ( o) ( 3) ( 8) ( 2) (I2) ( I)
L. mini
( s)
I /2 2/2 3/2 4/2 2/4 3/4 2/8 2/ I6 2j32
( o) ( 6) ( 3) ( o) (II) ( g) (II) ( 5) ( o)
(16) ( I) ( o) ( g) ( 2) ( IO) ( 2) ( g) ( 1) (I2) ( o) ( I) ( I)
( s)
I/ 2 2/2 3/2 4/2 2/4 3/4 2/8 2/ I6 2/32
( I) ( 4) ( 6) ( I) (II) ( s)
( 6) ( 4) ( 1)
2/8
2/8
( 6) 2/2 ( 4) 2/4 ( s)
( I4) 2/2 ( o) 2/4 ( I)
* Results are expressed as degree of reaction/dilution of serum or whey. Figures in brackets indicate number of samples. tr = trace.
BRITISH VETERINARY JOURNAL, 132, 6 RESULTS
The serum and whey titres obtained from the samples are given in Table I. These results show that the whey agglutination test (WAT) correctly classified the serologically positive and negative animals, that is, the presence or absence of agglutinins in the serum was equally demonstrated in whey. A comparison of the titres obtained for the Leptospira serotypes woljfi and mini for serum and whey samples is shown in Table II. A summary of the agreement of SAT and WAT, assuming that a titre of 2 + at I: I oo is a positive SAT result, is given in T a ble III. The results ofWAT on pooled milk samples given in Table IV show a clear cut positive titre even when negative samples are included. TAB LE II SE RU M AND WHEY LEPTOSPIRA AG GL UTI NIN LEV ELS. NU MBER OF PAIRS OF SER UM AND WHEY SAMPLES AND THEIR COMPARATIVE TITR ES IN TH E TWO TESTS
L. wolffi
Serum dilution < I/IOO
1/ IOO
dilutions I/2 I /4: Iji6 I/32 I/64:
-1ve I + 2+ 3+ 2+ 3+ 2+ 2+ 3+ 2+ 2+
Trace
I + 2 2
5 5
2+
3+
2+
2
5 2 6 2
9 3 I4: 2 8 3
Iji6oo
I /4:00
Whey
4:
3+
2+
3+
3
2
L.mini
Serum dilution
A COMPARATIVE STUDY ON THE USE OF WHEY AND SERUM FOR MICROSCOPIC AGGLUTINATION TEST FOR THE DETECTION OF LEPTOSPIRA ANTIBODIES BY
S. N .
HussAINI
Bacteriology Department, Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, Wrybridge, Surrry SUMMARY
Whey and serum from I 23 animals in three herds were tested using a microscopic agglutination test. A close relationship between the whey and serum agglutination titres indicates that whey agglutination test is a dependable test which can be employed in testing individual or bulk milk samples. A titre of I :4, giving so% agglutination, using whey is suggested as a diagnostic titre as it compares well with the diagnostic titre of I :I oo for serum. INTRODUCTION
As early as 1893 Breiger & Ehrlich demonstrated that goats when inoculated with tetanus toxin secreted antibodies lasting the entire period of lactation and that these antibodies could also be detected in their whey. This knowledge has since been applied by several workers in detecting brucella infection in cattle (Seddon, I915; Cooledge, I9I6; Smith, Orcutt & Little, I923). It was Fleischauer (I937) however who eventually developed the milk ring test (MRT) using a stained brucella antigen. The use of milk and whey for the detection of brucella infection prompted van der Hoeden (I952, I955) to utilize whey agglutination test (WAT) in the investigation of several outbreaks of leptospirosis in cattle, due to Leptospira grippotyphosa and L. pomona, and in goats, due to L. grippotyphosa. He concluded that the test was always specific and recommended it as a screening test and as a confirmatory test after serum agglutination testing (SAT). Morse, Allen, Pope & Krohn (1955) observed that whey agglutinins to L. pomona in cows could be demonstrated for as long as 29 months after the onset ofleptospirosis in the herd. The highest whey reactions were usually observed in cows which had recently experienced infection. Carbrey & Packer (I96 I) noted that the whey titre of the individual cow varied in relation to the serum titre. They further observed the absence of a significant difference between the quarter titres of the same cow and regarded this as evidence that localized udder infection does not occur in leptospirosis. They were also able to detect antibodies against L. pomona in a
BRITISH VETERINARY JOURNAL, 132, 6
herd using composite herd milk sample from the bulk-tank or milk-can. However, attempts at evolving a comparable MRT for leptospirosis met with failure because effective staining destroyed the antigenicity of the leptospirae and well defined rings appeared in negative milk samples (United States Department of Agriculture, I 953). The test most commonly used for the diagnosis of leptospirosis is the microscopic agglutination test (MAT) employing blood serum (World Health Organization, I967). This paper includes an evaluation of a test using milk whey as an alternative or as an adjunct to serum in the diagnosis ofleptospirosis, since milk is easier to obtain. The detection of leptospira antibodies in a herd milk sample, taken from a bulk-tank or milk-can, is a convenient way of screening a herd for leptospira infection. MATERIALS AND METHODS
Selection of herds The selection of farms A and B was based on the previous serological examination of the herds which revealed a high number of reactors to the hebdomadis group, and in the case of farm B one of the cowmen developed leptospirosis caused by leptospira of the hebdomadis group. Farm C was selected as a known serologically negative herd. Whry preparation Milk collected in Universal bottles (23 ml) was defatted by centrifugation at 2500 revjmin (Boo g) for 15 min and removal of the fat layer. Rennet was added at the rate of I drop to 2 ml of the defatted milk and the mixture was incubated for I h at 37°C and the whey was used for microscopic agglutination test (MAT). It was sometimes necessary to clarify the whey by passing it through a Millipore* prefilter and a filter of o·22 fl. pore diameter. Bulk milk Aliquots of milk from individual animals were pooled. Serum Blood collected from the coccygeal vem m I o ml Vacu tainert tubes was allowed to clot and serum separated. Microscopic agglutination test Microscopic agglutination tests (MAT) using whey and serum samples, with living antigen suspensions, were performed as described by Wolff (1954). The antigens were L. wolffi and L. mini belonging to the hebdomadis serogroup, grown for 7-Io days at 30°C in Korthof's medium. For the whey agglutination test (WAT) the lowest dilution examined was prepared by mixing equal amounts of whey and antigen, resulting in a final * MilJipore (UK) Limited, MilJipore House, Abbey Road, London NWw 7SP. t Becton, Dickinson UK Limited, York House, Empire Way, Wembley, Middlesex HAgoPS.
MICROSCOPIC AGGLUTINATION TESTS
whey dilution of 1 :2 and subsequent twofold dilutions of whey were prepared giving dilutions of 1 :2, 1 :4, 1 :8, 1 :16, 1 :32 and 1 :64. The final serum dilutions used for the serum agglutination test (SAT) were 1 :roo, 1 :400 and 1 :r6oo.
Number of samples One hundred and twenty-three pairs of serum and whey samples were tested from cattle from farms A (64 pairs), B (44 pairs) and C ( 15 pairs). TABLE I COMPARATIVE FINDINGS ON THE USE OF BOVINE SERUM AND MILK WHEY SAMPLES BY THE MICROSCOPIC AGGLUTINATION TEST IN THE DIAGNOSIS OF LEPTOSPIROSIS
Microscopic agglutination test (MAT ) * Location and total No. of samples
Dilutions Serum (I/IOo, Ij4oo, I/I6oo)
L. wolffi
Farm A 64
tr/IOO I/Ioo 2/100 2/400 2/ I6oo
(I8) ( s) ( 6) (23 ) ( Io) ( 2)
L. mini
(20) tr/ Ioo ( 2) I / 100
(I I )
2/IOO (I8) 2/400 (I2) 2/I6oo ( I )
Pooled milk (positives only) Pooled milk (including negatives)
FarmB 44
I/Ioo 2/IOO 3/IOO 2/400 3/400 2/I6oo
Pooled milk (including negatives) FarmC IS
( s) ( I) (Ig) ( 8) ( 7) ( I) ( 3)
Ij iOO 2/ IOO 3/ Ioo 2/400 3/400 2jr6oo
Dilutions Whey (I/2, I/4, I/8, Iji6, I/32, I/64)
L. wolffi
tr/ 2 I/2 2/2 3/2 2/4 3/4 2/8 3/8 2/ I6 3/ I6 2/32 2/64
(10)
( o) ( 6) ( I) (I ) ( 2)
tr/2 I/2 2/2 3/2 2/4 3/4 2/8 3/8 2/I6 3/I6 2/32 2/64
2/I6
2/I6
3/8
2/8
( s) ( I) (22) ( 5) ( 7) ( 4) ( o)
(I8) ( o) ( 3) ( 8) ( 2) (I2) ( I)
L. mini
( s)
I /2 2/2 3/2 4/2 2/4 3/4 2/8 2/ I6 2j32
( o) ( 6) ( 3) ( o) (II) ( g) (II) ( 5) ( o)
(16) ( I) ( o) ( g) ( 2) ( IO) ( 2) ( g) ( 1) (I2) ( o) ( I) ( I)
( s)
I/ 2 2/2 3/2 4/2 2/4 3/4 2/8 2/ I6 2/32
( I) ( 4) ( 6) ( I) (II) ( s)
( 6) ( 4) ( 1)
2/8
2/8
( 6) 2/2 ( 4) 2/4 ( s)
( I4) 2/2 ( o) 2/4 ( I)
* Results are expressed as degree of reaction/dilution of serum or whey. Figures in brackets indicate number of samples. tr = trace.
BRITISH VETERINARY JOURNAL, 132, 6 RESULTS
The serum and whey titres obtained from the samples are given in Table I. These results show that the whey agglutination test (WAT) correctly classified the serologically positive and negative animals, that is, the presence or absence of agglutinins in the serum was equally demonstrated in whey. A comparison of the titres obtained for the Leptospira serotypes woljfi and mini for serum and whey samples is shown in Table II. A summary of the agreement of SAT and WAT, assuming that a titre of 2 + at I: I oo is a positive SAT result, is given in T a ble III. The results ofWAT on pooled milk samples given in Table IV show a clear cut positive titre even when negative samples are included. TAB LE II SE RU M AND WHEY LEPTOSPIRA AG GL UTI NIN LEV ELS. NU MBER OF PAIRS OF SER UM AND WHEY SAMPLES AND THEIR COMPARATIVE TITR ES IN TH E TWO TESTS
L. wolffi
Serum dilution < I/IOO
1/ IOO
dilutions I/2 I /4: Iji6 I/32 I/64:
-1ve I + 2+ 3+ 2+ 3+ 2+ 2+ 3+ 2+ 2+
Trace
I + 2 2
5 5
2+
3+
2+
2
5 2 6 2
9 3 I4: 2 8 3
Iji6oo
I /4:00
Whey
4:
3+
2+
3+
3
2
L.mini
Serum dilution
