I45
EQUINE VETERINARY JOURNAL V P I . J . (1978), 10 (3). 145-147
&uine
The Course of Serum Antibody Development in Two Ponies expe r i menta I Iy infected with Contagious Metritis F. L. M. DAWSON, J. A. BENSON and PATRICIA CROXTON-SMITH MAFF Veterinary Investigation Centre, Madingle y Road, Cambridge CB3 OER
SUMMARY Serum agglutination tests, anti-globulin tests, and complement fixation tests were carried out on sera taken over a period of 98 days from two fillies experimentally infected with the contagious equine metritis organism. The pattern, and significance in diagnosis, of these results is discussed. All 3 tests showed positive titres in the acute phase of experimental disease; reactions in the complement fixation test persisted longest.
INTRODUCTION THE development of antibody in equine sera to the contagious equine metritis organism (CEMO) has been reported. Benson, et at. (1978) described reactions in the serum agglutination and anti-globulin tests on sera from infected horses, suspect subjects and negative controls, while Croxton-Smith, Benson, Dawson and Powell (1978) reported results on additional similar material using a complement fixation test. Most of these results were based on single blood samples from individuals, with only a few sets of 2 or 3 samples taken from individual animals. In order to obtain further data on the applicability of these 3 tests for purposes of field diagnosis, the course of antibody development after experimental infection with CEMO was followed serially in 2 pony fillies. MATERIALS AND METHODS Two ponies, aged about 20 months and weighing approximately 250 kg, were infected to provide a source of CEMO freshly isolated for use in quality control tests. Pony A was infected intracervically from a swab on 7 November 1977 with a liquid-nitrogenstored CEMO culture, which had been isolated from a field case of metritis in the spring of that year. Pony B was infected on 14 November using a cervical swab from Pony A. Blood samples were collected immediately before infection, and after infection weekly or at shorter intervals, and were subjected to the serum agglutination (SAT) and anti-globulin (AGT) tests immediately. Serum from the first 7 samplings was stored at -20°C until the complement fixation test (CFT) could be performed; CFTs were performed on all later samplings without delay. The tests were carried out by the methods previously described (Benson, et at., 1978 and Croxton-Smith, et at., 1978).
Uterine biopsy samples and endometrial smears were taken at the same time as the blood samples by methods described by Ricketts, Samuel and Rossdale (1978) and by Wingfield-Digby (1978) respectively. Swabs for culture were also taken, before infection and twice weekly thereafter, from the cervix, urethra and clitoris. RESULTS A copious discharge from the cervix was present in both ponies by the third day after infective material was introduced, and dense cultures of CEMO were recovered from cervical swabs taken then and at 3 or 4 day intervals up to the seventeenth day from infection in both ponies, by which time the discharge was subsiding. The results from the serial blood samplings are shown in Table 1 and fig. 1. Based on the observations of Benson, et al. (1978), and Croxton-Smith, et al. (1978), minimum positive titres were taken as 1/80 in the SAT and AGT, and 4/4 in the CFT. In both ponies, SAT/AGT titres were first detected at 7 days, and these peaked between 21 and 24 days after infection (i.e. about 5 days after the abatement of clinical signs of the disease and the disappearance of the CEMO from cervical swabs) although infection persisted in the clitoris and intermittently in the urethra, for several months. Evidence of inflammation was present in uterine biopsy samples and cytological smears, from the seventh to the twenty-eighth day in pony A, and from the third to the thirty-first day after infection in pony B. Antibodies showing in the CFT were present by Day 10 in pony A and at positive diagnostic levels in both ponies by Day 15. Both ponies showed positive CFT titres on 8 occasions, between Days 15 and 70 in pony A and between Days 15 and 63 in pony B.
EQUINE VETERINARY JOURNAL TABLE I TITRES O F ANTIBODY TO THE CONTAGIOUS EQUINE METRITIS ORGANISM I N THE SERUM AGGLUTINATION, ANTI-EQUINE GLOBULIN, AND COMPLEMENT FIXATION TESTS
146
Mare B
Mare A Days from infection
AGT
SAT
CFT
SAT
AGT
~
0 7
10 15 18 21 28 35 42 49 57 63 70
I7 85 91 98
1/10 3/10 3/10 ND 1/20 2/10 2/10 1/20 4/10 2/10 3/10 2/10 2/10 2/10 1/20
3/10 1/20 1/40 ND 1/80 1/40 3/20 2/20 4/10 3/10 1/20 2/10 ..-
1/20 2/10 1/20
~~
-
2/80 ND 31640 111280 ]/I280 21320 21160 1/80 1/40 I /40 1/40 2120 2/20 2/20 1/40 3/10
-
314 3/40 ND 3/20 4/10 414 214 1/10 2/10 1/10
414 214 212 114 AC 114 AC
CFT ~
~
-
]/I60 ND 112560 111280 312560 2/640 21320 3/80 3/40 1/80 2/40 2/20 4/20 2/20 1/40
3/20
-
) ) clinical ) signs of ) endometritis ) cervical swabs ) positive
ND 2/20 3/20 3/10 3/10 2/10 1/10 314
) intensified ) clitoral ) infection
1/10
414 314 114 114 AC 312 312
intensification B's clitoris
Expression of titres: In these tables, denominators of the fractions given indicate degree of dilution of the test serum, while numerators indicate the extent of agglutination or fixation at that dilution. Thus 4/10 indicates complete agglutination or fixation at a dilution of 1 in 10: 2/10 indicates 50 per cent agglutination, etc., at that dilution AC anti-complementary activity present ND not done 2560
-
1280 * -
640
-. HAnti-globulin
320.-
test, Mare A
*--aComplement fixation test, Mare A o----O Anti-globulin test, Mare B
160.-
&' +-+I3
c a
Complement fixation test, Mare B
. Q
80
--
40 0 L
.-
. L
k
40 * -
20
20
10
10 4
2
7 10
Fig. 1.
15 10 21
28
35
42
49
57
63
70
77
85
91
98
Antibody titres in the tanti-globulin and complement fixation tests to the contagious equine metritis organism.
147
EQUINE VETERINARY JOURNAL
A minor peak in titres of the AGT and CFT occurred around the period 56 to 63 days after infection, associated with an increase in density of positive cultures from the clitoral fossa, lasting one week. No recovery in culture from the cervix was made at this time. DISCUSSION The 2 ponies showed a marked individual variation in antibody pattern. Pony A was never positive in the SAT and was only positive on one occasion in the AGT. She did, however, reach a rather high titre (3/40) in the CFT. Pony B was positive in the SAT and ACT from Day 7 to Day 42, and was also positive in the AGT on Day 57. Comparing the CFT titres, however, one can see from fig. 1 that the titres reached were very similar in both ponies. Each peaked between Day 14 and Day 21, and thereafter showed a steady decline with only minor deviations over the period of observation. Nevertheless, the CFT reactions persisted at diagnostic levels for longer than those in the other 2 tests. Similar variations in antibody pattern are seen in equine and bovine brucellosis. There is evidence in CEM (Croxton-Smith, et al., 1978) that low titres in the SAT/AGT and higher titres (often “positive”) in the CFT persist indefinitely in cases of chronic infection. CEMO was recovered, in the absence of treatment, intermittently from the clitoris and urethra of both ponies throughout the period of this study. Their antibody titres thus confirm the bacteriological findings. Platt, Atherton, Dawson and Durrant (1978) and Simpson and Eaton-Evans (1978) have indicated that chronic infection often appears confined to the clitoris.
1.
2. 3.
CONCLUSIONS The 2 ponies provided extreme examples of differences in reaction type, emphasising the need to carry out all 3 forms of test for optimal serological diagnosis in the acute phase. Useful information could be obtained were a similar study to be carried out on a larger group of ponies. The CFT on present knowledge appears to be the best single test; it worked well on both ponies and remained positive over a longer period than the other tests. However, as it gives fewer positive reactions early in the acute phase than the other tests, the SAT should be done first in acute cases; an inconclusive result may be resolved by the
AGT, or if not, by the CFT, which is the test of choice in suspect chronic cases. ACKNOWLEDGEMENTS T o Dr. C. E. D. Taylor, Cambridge, for supplying the original culture o f the CEMO. T o Messrs S. W. Ricketts and N. Wingfield Digby for histological results o n biopsy samples. T o Mr. D. J Simpson for instilling the culture into pony A. To Messrs J. R. Simmons and D. S. Durrant for assistance with blood sampling. REFERENCES Benson, J . A,, Powell, D . G . , Dawson, F. L. M., Edwards, P. and Durrant, D . S. (1978). Vet. Rec. 102, 227-280. Croxton-Smith, Patricia, Benson, J . A., Dawson, F. L. M. and Powell, D. G . Vet. Rec. (In press). Platt, H., Atherton, J. G . , Dawson, F. L. M. and Durrant, D. S. (1978). Isolation of the CEM organism from the clitoris of the mare. Vet. Rec. 102, 19. Simpson, D. J. and Eaton-Evans, W. E. (1978). Isolation o f the CEM organism from the clitoris of the mare. Vet. Rec. 102, 19-20. Ricketts, S. W., Samuel, Carole A. and Rossdale, P. D. (1978). Endometrial biopsy studies of mares with contagious equine metritis 1977. Equine vet. J . 10, 160-166. Wingfield Digby, N. J. (1978). The technique and clinical application of endometrial cytology in mares. Equine vet. J . 10, 167-170.
RESUME Des tests de sero-agglutination, anti-globulines, et de fixation du complement ont CtC effectuks sur des serums prelevCs durant une periode de 98jours sur deux pouliches infectees expkrimentalement. Les rtsultats et leur signification diagnostique sont discutb. Les trois tests ont donne des resultats positifs durant la phase aigue de la maladie experimentale. La reaction de fixation du complement a persiste plus longtemps. ZUSAMMENFASSUNG Serum-Agglutinationstests, Anti-Globulin-Tests und Komplementbindungsreaktion wurden an Serumproben durchgefiihrt, die wahrend 98 Tagen nach experimenteller Infektion mit dem CEM-Organismus bei zwei Ponystuten gewonnen wurden. Die Signifikanz der Resultate fur die Diagnose wird diskutiert. Alle drei Tests zeigten positive Titer in der akuten Phase der experimentellen Erkrankung ; die KBR-Titer persistierten am Iangsten.
Accepted for publication 24.4.78
EQUINE VETERINARY JOURNAL V P I . J . (1978), 10 (3). 145-147
&uine
The Course of Serum Antibody Development in Two Ponies expe r i menta I Iy infected with Contagious Metritis F. L. M. DAWSON, J. A. BENSON and PATRICIA CROXTON-SMITH MAFF Veterinary Investigation Centre, Madingle y Road, Cambridge CB3 OER
SUMMARY Serum agglutination tests, anti-globulin tests, and complement fixation tests were carried out on sera taken over a period of 98 days from two fillies experimentally infected with the contagious equine metritis organism. The pattern, and significance in diagnosis, of these results is discussed. All 3 tests showed positive titres in the acute phase of experimental disease; reactions in the complement fixation test persisted longest.
INTRODUCTION THE development of antibody in equine sera to the contagious equine metritis organism (CEMO) has been reported. Benson, et at. (1978) described reactions in the serum agglutination and anti-globulin tests on sera from infected horses, suspect subjects and negative controls, while Croxton-Smith, Benson, Dawson and Powell (1978) reported results on additional similar material using a complement fixation test. Most of these results were based on single blood samples from individuals, with only a few sets of 2 or 3 samples taken from individual animals. In order to obtain further data on the applicability of these 3 tests for purposes of field diagnosis, the course of antibody development after experimental infection with CEMO was followed serially in 2 pony fillies. MATERIALS AND METHODS Two ponies, aged about 20 months and weighing approximately 250 kg, were infected to provide a source of CEMO freshly isolated for use in quality control tests. Pony A was infected intracervically from a swab on 7 November 1977 with a liquid-nitrogenstored CEMO culture, which had been isolated from a field case of metritis in the spring of that year. Pony B was infected on 14 November using a cervical swab from Pony A. Blood samples were collected immediately before infection, and after infection weekly or at shorter intervals, and were subjected to the serum agglutination (SAT) and anti-globulin (AGT) tests immediately. Serum from the first 7 samplings was stored at -20°C until the complement fixation test (CFT) could be performed; CFTs were performed on all later samplings without delay. The tests were carried out by the methods previously described (Benson, et at., 1978 and Croxton-Smith, et at., 1978).
Uterine biopsy samples and endometrial smears were taken at the same time as the blood samples by methods described by Ricketts, Samuel and Rossdale (1978) and by Wingfield-Digby (1978) respectively. Swabs for culture were also taken, before infection and twice weekly thereafter, from the cervix, urethra and clitoris. RESULTS A copious discharge from the cervix was present in both ponies by the third day after infective material was introduced, and dense cultures of CEMO were recovered from cervical swabs taken then and at 3 or 4 day intervals up to the seventeenth day from infection in both ponies, by which time the discharge was subsiding. The results from the serial blood samplings are shown in Table 1 and fig. 1. Based on the observations of Benson, et al. (1978), and Croxton-Smith, et al. (1978), minimum positive titres were taken as 1/80 in the SAT and AGT, and 4/4 in the CFT. In both ponies, SAT/AGT titres were first detected at 7 days, and these peaked between 21 and 24 days after infection (i.e. about 5 days after the abatement of clinical signs of the disease and the disappearance of the CEMO from cervical swabs) although infection persisted in the clitoris and intermittently in the urethra, for several months. Evidence of inflammation was present in uterine biopsy samples and cytological smears, from the seventh to the twenty-eighth day in pony A, and from the third to the thirty-first day after infection in pony B. Antibodies showing in the CFT were present by Day 10 in pony A and at positive diagnostic levels in both ponies by Day 15. Both ponies showed positive CFT titres on 8 occasions, between Days 15 and 70 in pony A and between Days 15 and 63 in pony B.
EQUINE VETERINARY JOURNAL TABLE I TITRES O F ANTIBODY TO THE CONTAGIOUS EQUINE METRITIS ORGANISM I N THE SERUM AGGLUTINATION, ANTI-EQUINE GLOBULIN, AND COMPLEMENT FIXATION TESTS
146
Mare B
Mare A Days from infection
AGT
SAT
CFT
SAT
AGT
~
0 7
10 15 18 21 28 35 42 49 57 63 70
I7 85 91 98
1/10 3/10 3/10 ND 1/20 2/10 2/10 1/20 4/10 2/10 3/10 2/10 2/10 2/10 1/20
3/10 1/20 1/40 ND 1/80 1/40 3/20 2/20 4/10 3/10 1/20 2/10 ..-
1/20 2/10 1/20
~~
-
2/80 ND 31640 111280 ]/I280 21320 21160 1/80 1/40 I /40 1/40 2120 2/20 2/20 1/40 3/10
-
314 3/40 ND 3/20 4/10 414 214 1/10 2/10 1/10
414 214 212 114 AC 114 AC
CFT ~
~
-
]/I60 ND 112560 111280 312560 2/640 21320 3/80 3/40 1/80 2/40 2/20 4/20 2/20 1/40
3/20
-
) ) clinical ) signs of ) endometritis ) cervical swabs ) positive
ND 2/20 3/20 3/10 3/10 2/10 1/10 314
) intensified ) clitoral ) infection
1/10
414 314 114 114 AC 312 312
intensification B's clitoris
Expression of titres: In these tables, denominators of the fractions given indicate degree of dilution of the test serum, while numerators indicate the extent of agglutination or fixation at that dilution. Thus 4/10 indicates complete agglutination or fixation at a dilution of 1 in 10: 2/10 indicates 50 per cent agglutination, etc., at that dilution AC anti-complementary activity present ND not done 2560
-
1280 * -
640
-. HAnti-globulin
320.-
test, Mare A
*--aComplement fixation test, Mare A o----O Anti-globulin test, Mare B
160.-
&' +-+I3
c a
Complement fixation test, Mare B
. Q
80
--
40 0 L
.-
. L
k
40 * -
20
20
10
10 4
2
7 10
Fig. 1.
15 10 21
28
35
42
49
57
63
70
77
85
91
98
Antibody titres in the tanti-globulin and complement fixation tests to the contagious equine metritis organism.
147
EQUINE VETERINARY JOURNAL
A minor peak in titres of the AGT and CFT occurred around the period 56 to 63 days after infection, associated with an increase in density of positive cultures from the clitoral fossa, lasting one week. No recovery in culture from the cervix was made at this time. DISCUSSION The 2 ponies showed a marked individual variation in antibody pattern. Pony A was never positive in the SAT and was only positive on one occasion in the AGT. She did, however, reach a rather high titre (3/40) in the CFT. Pony B was positive in the SAT and ACT from Day 7 to Day 42, and was also positive in the AGT on Day 57. Comparing the CFT titres, however, one can see from fig. 1 that the titres reached were very similar in both ponies. Each peaked between Day 14 and Day 21, and thereafter showed a steady decline with only minor deviations over the period of observation. Nevertheless, the CFT reactions persisted at diagnostic levels for longer than those in the other 2 tests. Similar variations in antibody pattern are seen in equine and bovine brucellosis. There is evidence in CEM (Croxton-Smith, et al., 1978) that low titres in the SAT/AGT and higher titres (often “positive”) in the CFT persist indefinitely in cases of chronic infection. CEMO was recovered, in the absence of treatment, intermittently from the clitoris and urethra of both ponies throughout the period of this study. Their antibody titres thus confirm the bacteriological findings. Platt, Atherton, Dawson and Durrant (1978) and Simpson and Eaton-Evans (1978) have indicated that chronic infection often appears confined to the clitoris.
1.
2. 3.
CONCLUSIONS The 2 ponies provided extreme examples of differences in reaction type, emphasising the need to carry out all 3 forms of test for optimal serological diagnosis in the acute phase. Useful information could be obtained were a similar study to be carried out on a larger group of ponies. The CFT on present knowledge appears to be the best single test; it worked well on both ponies and remained positive over a longer period than the other tests. However, as it gives fewer positive reactions early in the acute phase than the other tests, the SAT should be done first in acute cases; an inconclusive result may be resolved by the
AGT, or if not, by the CFT, which is the test of choice in suspect chronic cases. ACKNOWLEDGEMENTS T o Dr. C. E. D. Taylor, Cambridge, for supplying the original culture o f the CEMO. T o Messrs S. W. Ricketts and N. Wingfield Digby for histological results o n biopsy samples. T o Mr. D. J Simpson for instilling the culture into pony A. To Messrs J. R. Simmons and D. S. Durrant for assistance with blood sampling. REFERENCES Benson, J . A,, Powell, D . G . , Dawson, F. L. M., Edwards, P. and Durrant, D . S. (1978). Vet. Rec. 102, 227-280. Croxton-Smith, Patricia, Benson, J . A., Dawson, F. L. M. and Powell, D. G . Vet. Rec. (In press). Platt, H., Atherton, J. G . , Dawson, F. L. M. and Durrant, D. S. (1978). Isolation of the CEM organism from the clitoris of the mare. Vet. Rec. 102, 19. Simpson, D. J. and Eaton-Evans, W. E. (1978). Isolation o f the CEM organism from the clitoris of the mare. Vet. Rec. 102, 19-20. Ricketts, S. W., Samuel, Carole A. and Rossdale, P. D. (1978). Endometrial biopsy studies of mares with contagious equine metritis 1977. Equine vet. J . 10, 160-166. Wingfield Digby, N. J. (1978). The technique and clinical application of endometrial cytology in mares. Equine vet. J . 10, 167-170.
RESUME Des tests de sero-agglutination, anti-globulines, et de fixation du complement ont CtC effectuks sur des serums prelevCs durant une periode de 98jours sur deux pouliches infectees expkrimentalement. Les rtsultats et leur signification diagnostique sont discutb. Les trois tests ont donne des resultats positifs durant la phase aigue de la maladie experimentale. La reaction de fixation du complement a persiste plus longtemps. ZUSAMMENFASSUNG Serum-Agglutinationstests, Anti-Globulin-Tests und Komplementbindungsreaktion wurden an Serumproben durchgefiihrt, die wahrend 98 Tagen nach experimenteller Infektion mit dem CEM-Organismus bei zwei Ponystuten gewonnen wurden. Die Signifikanz der Resultate fur die Diagnose wird diskutiert. Alle drei Tests zeigten positive Titer in der akuten Phase der experimentellen Erkrankung ; die KBR-Titer persistierten am Iangsten.
Accepted for publication 24.4.78
