This article was downloaded by: [Washington State University Libraries ] On: 24 October 2014, At: 22:08 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
An evaluation of the semi-automated complement fixation test and the microscopic agglutination test for the serological diagnosis of bovine leptospirosis a
a
b
b
a
a
R.T. Hodges , M.E. Carter , K.B. Almand , W. Weddell , J.T.S. Holland , S.F. Lewis & D.E. Lake a
a
Ruakura Animal Health Laboratory , Hamilton
b
Animal Health Reference Laboratory , Upper Hutt Published online: 23 Feb 2011.
To cite this article: R.T. Hodges , M.E. Carter , K.B. Almand , W. Weddell , J.T.S. Holland , S.F. Lewis & D.E. Lake (1979) An evaluation of the semi-automated complement fixation test and the microscopic agglutination test for the serological diagnosis of bovine leptospirosis, New Zealand Veterinary Journal, 27:5, 101-102, DOI: 10.1080/00480169.1979.34609 To link to this article: http://dx.doi.org/10.1080/00480169.1979.34609
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
101
NEW ZEALAND VETERINARY JOURNAL
1979
An evaluation of the semi-automated complement fixation test and the microscopic agglutination test for the serological diagnosis of bovine leptospirosis R. T. Hodges U N.z.
"I!/ )17:
,
M. E. CarterO, K. B. Almandt, W. Weddellt, J. T. S. Hollando, S. F. Lewiso, D. E. Lakeo.
IQI·1
Downloaded by [Washington State University Libraries ] at 22:08 24 October 2014
ABSTRACT
Groups of cows were selected from each 0122 herds which had recent evidence of leptospirosis or abortions. Blood and urine samples were collected from each animal on a number of occa sions over a period of II months. Attempts were made to detect leptospirurla by microscopic and cultural procedures. Sera were examined by the semi-automated complement fixation test (CFT) and the microscopic agglutination test (MAT) using Leptospira interrogan.t serotypes pomona. hardjo. copenhageni and bal/um as antigens. Leptospiruria was detected in 107 cows. Pomona was cultured from 43 animals. hardjo from 13. The identity of the organism was not determined in the remaining 51 cases. Highest CFT and MAT titres occurred with serotypes pomona and hardjo. There was no bacteriological or serological evidence of infection with ('openhageni or bal/um. There were no significant differences between the number of CFT or MAT reactions in cattle with leptospiruria. The CFT gave fewer reactions in cattle that did not show leptospiruria and, in most cases, CFT antibodies were detectable for a shorter time than agglutinins. Both tests gave reactions with the same number of aborting cows that showed leptospiruria near the time of abortion. Our results Indicate that the CFT would be useful in Animal Health Laboratories for the serological diagnosis of bovine leptospirosis. INTRODUCTION
Studies involving a small number of cattle, that had been experimentally infected with various serotypes of leptospirae, showed that complement-fixing (CF) antibodies were detecta ble for a shorter time after infection, and were more closely associated with the period of leptospiruria. than agglutinating antibodies.'l) Because of the longer persistence of the agglutin ation response. the microscopic agglutination test (MAT) does not distinguish between recent and long-standing infections. Therefore. the presence of agglutinating antibodies may not be relevant to current clinical problems. The MAT is more labour-intensive and time-consuming than the CFT which can be adapted to a semi-automated method.C' ) In addition, polyvalent antigens can be used in the CFT so that, if necessary. sera can be screened against a number ofleptospiral serotypes in a single CF procedure(2). Similar screening procedures cannot be used in agglutination tests on bovine seraP) These factors suggest that the CFT may have a number of advantages for the serological diagnosis of bovine leptospirosis. Before incorporating the CFT as a standard procedure into the Animal Health Division Laboratories. it was considered that further comparisons should be made in the field, so that results could be compared in a larger number of cattle under conditions of natural infection. • Ruakura Animal Heahh Laboratory. Hamilton
t Animal Heahh Reference Laboratory. Upper Hutt
MATERIALS AND METHODS
Selection o.fherds Twenty-two herds with histories of leptospirosiS were select ed. and each herd was visited four to six times during a period of II months. A varying number of cows were sampled from each herd. but the same cows were sampled at each visit whenever possible. One hundred and sixty ofthe 553 cows sampled at the first visit were culled for various reasons during the period of investigations.
Serological and microbiological examinations The serological procedures have been described in detail elsewhere.'" 14) The same recently isolated strains of Leptospira interrogalls. se roty pes pomona. hardjo. copenhagen; and bal/um were used as antigens in each test. The CFT was carried out in a Cooke Model 300 "Dynaiiter"tt. Urine was collected from each cow at every visit; formalin was added to give a final concentration of approximately 1%. These samples were centrifuged at 2000g for 15-20 minutes and smears of the deposits were examined by dark ground microscopy. Attempts were made to culture leptospirae from a proportion of urine samples: drops orrreshly voided urine were inoculated on to semi-solid medium (0.1% agar) containing 5-ftuoro-uracil to suppress bacterial contamination.'s, 16) Cultures were incubated at 30°C for up to 6 weeks and examined at intervals for growth of leptospirae. Leptospiral isolates were subcultured several times in liquid medium unlil dense growth was obtained and traces of agar were removed. The serological identity of the isolates was determined as described previously.O) RESULTS
General serological findings
Highest titres (MAT up to I :3000 and CF up to 1:2560 occurred with serotypes pomona or hardjov. In most cases. in dividuals with a high titre to one of these serotypes had a slightly lower titre to the other. In all instances in which the serological identity of the isolate was determined. the highest MAT and CF titres were recorded using the homologous antigens. MAT titres (range I: to to 1:300) or CF litres (range 1:20 to I: 80) to copenhageni or bal/um were recorded in less than 10% and 30% respectively of all these cows. In all cases, these cows had higher titres to pomona or hardjo. The titres to hal/um and copenhageni were considered to be due to cross-reactivity. For the purposes of comparison. a MAT titre producing 50% agglutination at a dilution of I: 100 and a CF titre producing 50% fixation at a dilution of I :20 were taken as the lowest significant litres. COII'S with leptospiruria leptospiruria was observed in 107 cows, and was detected on more than one occasion in 25 of these animals.
tt Division of Dynatech Laboratories Incorporated. Alexandria. Vir· ginia 22314. USA.
102
Serotype pomona was isolated from 43 and hardjo from 13. When isolates were obtained from the same animal on more than one occasion. the same serotype was isolated on each occasion. Concurrent infections with pomona and hardjo were found in three herds. but in no case were both serotypes identified in one animal. Agglutination titres of I: 100 and greater were recorded in 93.9% of serum samples collected at the time when cows had leptospiruria. Complement fixing titres of I :20 or greater were recorded in 89.4% of these samples.
Downloaded by [Washington State University Libraries ] at 22:08 24 October 2014
VOL 27
NEW ZEALAND VETERINARY JOURNAL
Persistence ofantibody titres There were 43 cows. sampled at least four times during the investigations. in which leptospiruria was detected at the first visit but not at subsequent visits. These cows had MAT titres greater than I: 100 and CFT titres greater than I :20 at the first visit. Only 9 cows had CFT titres greater than I :20 at the end of the period. 37-44 weeks later. whereas 42 had MAT titres greater than I: 100. A further 248 clinically normal cows were sampled in which leptospiruria was not detected. At the first visits, 181 (73%) had MAT titres of I: 100 or greater. CF titres of I :20 or greater were recorded in 51 cows. At the final visits. 164 (66%) had MAT titres of I: 100 or greater. and only 4 animals (under 2% ) still had CF titresof 1:20 or greater. Cows which aborted Leptospiruria was detected in 25 cows within 4 weeks after abortion. All animals had MAT titres of I: 100 or greater and CFT titres of 1:20 or greater. Serotype pomona was cultured from the urine of 14 of these animals. No other leptospirae were cultured. Highest titres for serotype pomona were measured in 23 animals and for hardjo in the remainder. DISCUSSION
A numberofherds were selected for this study because the aim was to sample as many freshly infected cattle as possible, and a wide range of infecting serotypes could be studied. Leptospira interrogans serotypes pomona, hardjo, copenhageni and ballum were used as antigens because they are the serotypes so far isolated from cattle in New Zealand. Isolations of serotypes pomona and hardjo were made, and significant serological reactions were obtained with these sero types only. There was no serological or bacteriological evidence of any animals being infected with copenhagen; or ballum in this investigation. Infection of cattle with members of the Hebdomadis sero group other than hardjo has not been recorded in this country, but the possibility that the isolates identified as hardjo may have been other, closely related, serotypes cannot be discounted. The differences in the numberof cattle with leptospiruria that had MAT tit res of I: 100 or greater and CFT titres of I :20 or greater were not statistically significant. Therefore, the MAT and the CFT can be considered equally reliable for detecting cattle with leplospiruria. Asdemonstrated previously,13I CF antibodies did not persist as long as agglutinating antibodies in cattle known to have had leptospiruria. In those animals in which leptospiruria was not demonstrated. there were fewer with significant CF titres compared with agglutinating titres. This difference can most likely be ascribed to the shorter persistence of the CF response, as many of these animals may have been infected at some time before the investigation began. These results indicate that, in any random survey of cattle. 20-50% fewer animals are likely to have CF antibodies than agglutinating antibodies.
The semi-automated CFT is quicker to carry out than the MAT and. at present. the examination of 288 sera per ma chine-hour is possible. This number could be increased by examining sera over a smaller range of dilutions, and the use of a polyvalent CF antigen in a single CF test would provide a rapid method of screening sera for evidence of infection caused by a variety of serotypes. In addition. the reliability of the CFT compared with the MAT in caule with leptospiruria, the shorter period of CF antibody response and its closer association with the period of leptospiruria and recent infection, suggests that the CFT would be a valuable routine serological test for bovine leptospirosis. ACKNOWLEDGEMENTS
The authors thank the farmers involved in this work for their co-operation. Dr M. H. Blunt is thanked for his help and advice, and the assistance is gratefully acknowledged of: N. S. Beatson, R. Blake. I. Doe. D. Lomie. J. F. Montgomery and E. L. Owens; also M.A.F. Livestock Officers, and laboratory staff of the Pal merston North and Ruakura Animal Health Laboratories. and the Animal Health Reference Laboratory. REFERENCES (II Hud~c•• R. T. ( 1973): A ~omplemenl lixalion lesl for Ihe serological diagnosis of leplospirosi, in pig.' experimenlally infccled wilh s.;rolype pomona. N.Z. vel: J. 21: 1·6. R. T.(.~914)~ T~e useof~ polyvalenlanllgen oncomplemenl Ii.allon leslsfor "'-"me lepl'''plnlSIS. N.Z. \('1. J. 21. 21·4. (31 Hodges. R. T.: Ris. D. R. (1914): Complemem fi.ing and agglulinaling anlibody r.'pons., and leplospiruria in calves inocula led wilh Lep,ospira serolypes pomona. l//lrd/". ",,!"'nhoKeIli or bollum. N.Z. vel. J. 12: 25-30. (4) Hodges. R. T.: Weddell. W. ( 1971): Adaplalion ofa complemenl fixalion lesl for large >
New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
An evaluation of the semi-automated complement fixation test and the microscopic agglutination test for the serological diagnosis of bovine leptospirosis a
a
b
b
a
a
R.T. Hodges , M.E. Carter , K.B. Almand , W. Weddell , J.T.S. Holland , S.F. Lewis & D.E. Lake a
a
Ruakura Animal Health Laboratory , Hamilton
b
Animal Health Reference Laboratory , Upper Hutt Published online: 23 Feb 2011.
To cite this article: R.T. Hodges , M.E. Carter , K.B. Almand , W. Weddell , J.T.S. Holland , S.F. Lewis & D.E. Lake (1979) An evaluation of the semi-automated complement fixation test and the microscopic agglutination test for the serological diagnosis of bovine leptospirosis, New Zealand Veterinary Journal, 27:5, 101-102, DOI: 10.1080/00480169.1979.34609 To link to this article: http://dx.doi.org/10.1080/00480169.1979.34609
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
101
NEW ZEALAND VETERINARY JOURNAL
1979
An evaluation of the semi-automated complement fixation test and the microscopic agglutination test for the serological diagnosis of bovine leptospirosis R. T. Hodges U N.z.
"I!/ )17:
,
M. E. CarterO, K. B. Almandt, W. Weddellt, J. T. S. Hollando, S. F. Lewiso, D. E. Lakeo.
IQI·1
Downloaded by [Washington State University Libraries ] at 22:08 24 October 2014
ABSTRACT
Groups of cows were selected from each 0122 herds which had recent evidence of leptospirosis or abortions. Blood and urine samples were collected from each animal on a number of occa sions over a period of II months. Attempts were made to detect leptospirurla by microscopic and cultural procedures. Sera were examined by the semi-automated complement fixation test (CFT) and the microscopic agglutination test (MAT) using Leptospira interrogan.t serotypes pomona. hardjo. copenhageni and bal/um as antigens. Leptospiruria was detected in 107 cows. Pomona was cultured from 43 animals. hardjo from 13. The identity of the organism was not determined in the remaining 51 cases. Highest CFT and MAT titres occurred with serotypes pomona and hardjo. There was no bacteriological or serological evidence of infection with ('openhageni or bal/um. There were no significant differences between the number of CFT or MAT reactions in cattle with leptospiruria. The CFT gave fewer reactions in cattle that did not show leptospiruria and, in most cases, CFT antibodies were detectable for a shorter time than agglutinins. Both tests gave reactions with the same number of aborting cows that showed leptospiruria near the time of abortion. Our results Indicate that the CFT would be useful in Animal Health Laboratories for the serological diagnosis of bovine leptospirosis. INTRODUCTION
Studies involving a small number of cattle, that had been experimentally infected with various serotypes of leptospirae, showed that complement-fixing (CF) antibodies were detecta ble for a shorter time after infection, and were more closely associated with the period of leptospiruria. than agglutinating antibodies.'l) Because of the longer persistence of the agglutin ation response. the microscopic agglutination test (MAT) does not distinguish between recent and long-standing infections. Therefore. the presence of agglutinating antibodies may not be relevant to current clinical problems. The MAT is more labour-intensive and time-consuming than the CFT which can be adapted to a semi-automated method.C' ) In addition, polyvalent antigens can be used in the CFT so that, if necessary. sera can be screened against a number ofleptospiral serotypes in a single CF procedure(2). Similar screening procedures cannot be used in agglutination tests on bovine seraP) These factors suggest that the CFT may have a number of advantages for the serological diagnosis of bovine leptospirosis. Before incorporating the CFT as a standard procedure into the Animal Health Division Laboratories. it was considered that further comparisons should be made in the field, so that results could be compared in a larger number of cattle under conditions of natural infection. • Ruakura Animal Heahh Laboratory. Hamilton
t Animal Heahh Reference Laboratory. Upper Hutt
MATERIALS AND METHODS
Selection o.fherds Twenty-two herds with histories of leptospirosiS were select ed. and each herd was visited four to six times during a period of II months. A varying number of cows were sampled from each herd. but the same cows were sampled at each visit whenever possible. One hundred and sixty ofthe 553 cows sampled at the first visit were culled for various reasons during the period of investigations.
Serological and microbiological examinations The serological procedures have been described in detail elsewhere.'" 14) The same recently isolated strains of Leptospira interrogalls. se roty pes pomona. hardjo. copenhagen; and bal/um were used as antigens in each test. The CFT was carried out in a Cooke Model 300 "Dynaiiter"tt. Urine was collected from each cow at every visit; formalin was added to give a final concentration of approximately 1%. These samples were centrifuged at 2000g for 15-20 minutes and smears of the deposits were examined by dark ground microscopy. Attempts were made to culture leptospirae from a proportion of urine samples: drops orrreshly voided urine were inoculated on to semi-solid medium (0.1% agar) containing 5-ftuoro-uracil to suppress bacterial contamination.'s, 16) Cultures were incubated at 30°C for up to 6 weeks and examined at intervals for growth of leptospirae. Leptospiral isolates were subcultured several times in liquid medium unlil dense growth was obtained and traces of agar were removed. The serological identity of the isolates was determined as described previously.O) RESULTS
General serological findings
Highest titres (MAT up to I :3000 and CF up to 1:2560 occurred with serotypes pomona or hardjov. In most cases. in dividuals with a high titre to one of these serotypes had a slightly lower titre to the other. In all instances in which the serological identity of the isolate was determined. the highest MAT and CF titres were recorded using the homologous antigens. MAT titres (range I: to to 1:300) or CF litres (range 1:20 to I: 80) to copenhageni or bal/um were recorded in less than 10% and 30% respectively of all these cows. In all cases, these cows had higher titres to pomona or hardjo. The titres to hal/um and copenhageni were considered to be due to cross-reactivity. For the purposes of comparison. a MAT titre producing 50% agglutination at a dilution of I: 100 and a CF titre producing 50% fixation at a dilution of I :20 were taken as the lowest significant litres. COII'S with leptospiruria leptospiruria was observed in 107 cows, and was detected on more than one occasion in 25 of these animals.
tt Division of Dynatech Laboratories Incorporated. Alexandria. Vir· ginia 22314. USA.
102
Serotype pomona was isolated from 43 and hardjo from 13. When isolates were obtained from the same animal on more than one occasion. the same serotype was isolated on each occasion. Concurrent infections with pomona and hardjo were found in three herds. but in no case were both serotypes identified in one animal. Agglutination titres of I: 100 and greater were recorded in 93.9% of serum samples collected at the time when cows had leptospiruria. Complement fixing titres of I :20 or greater were recorded in 89.4% of these samples.
Downloaded by [Washington State University Libraries ] at 22:08 24 October 2014
VOL 27
NEW ZEALAND VETERINARY JOURNAL
Persistence ofantibody titres There were 43 cows. sampled at least four times during the investigations. in which leptospiruria was detected at the first visit but not at subsequent visits. These cows had MAT titres greater than I: 100 and CFT titres greater than I :20 at the first visit. Only 9 cows had CFT titres greater than I :20 at the end of the period. 37-44 weeks later. whereas 42 had MAT titres greater than I: 100. A further 248 clinically normal cows were sampled in which leptospiruria was not detected. At the first visits, 181 (73%) had MAT titres of I: 100 or greater. CF titres of I :20 or greater were recorded in 51 cows. At the final visits. 164 (66%) had MAT titres of I: 100 or greater. and only 4 animals (under 2% ) still had CF titresof 1:20 or greater. Cows which aborted Leptospiruria was detected in 25 cows within 4 weeks after abortion. All animals had MAT titres of I: 100 or greater and CFT titres of 1:20 or greater. Serotype pomona was cultured from the urine of 14 of these animals. No other leptospirae were cultured. Highest titres for serotype pomona were measured in 23 animals and for hardjo in the remainder. DISCUSSION
A numberofherds were selected for this study because the aim was to sample as many freshly infected cattle as possible, and a wide range of infecting serotypes could be studied. Leptospira interrogans serotypes pomona, hardjo, copenhageni and ballum were used as antigens because they are the serotypes so far isolated from cattle in New Zealand. Isolations of serotypes pomona and hardjo were made, and significant serological reactions were obtained with these sero types only. There was no serological or bacteriological evidence of any animals being infected with copenhagen; or ballum in this investigation. Infection of cattle with members of the Hebdomadis sero group other than hardjo has not been recorded in this country, but the possibility that the isolates identified as hardjo may have been other, closely related, serotypes cannot be discounted. The differences in the numberof cattle with leptospiruria that had MAT tit res of I: 100 or greater and CFT titres of I :20 or greater were not statistically significant. Therefore, the MAT and the CFT can be considered equally reliable for detecting cattle with leplospiruria. Asdemonstrated previously,13I CF antibodies did not persist as long as agglutinating antibodies in cattle known to have had leptospiruria. In those animals in which leptospiruria was not demonstrated. there were fewer with significant CF titres compared with agglutinating titres. This difference can most likely be ascribed to the shorter persistence of the CF response, as many of these animals may have been infected at some time before the investigation began. These results indicate that, in any random survey of cattle. 20-50% fewer animals are likely to have CF antibodies than agglutinating antibodies.
The semi-automated CFT is quicker to carry out than the MAT and. at present. the examination of 288 sera per ma chine-hour is possible. This number could be increased by examining sera over a smaller range of dilutions, and the use of a polyvalent CF antigen in a single CF test would provide a rapid method of screening sera for evidence of infection caused by a variety of serotypes. In addition. the reliability of the CFT compared with the MAT in caule with leptospiruria, the shorter period of CF antibody response and its closer association with the period of leptospiruria and recent infection, suggests that the CFT would be a valuable routine serological test for bovine leptospirosis. ACKNOWLEDGEMENTS
The authors thank the farmers involved in this work for their co-operation. Dr M. H. Blunt is thanked for his help and advice, and the assistance is gratefully acknowledged of: N. S. Beatson, R. Blake. I. Doe. D. Lomie. J. F. Montgomery and E. L. Owens; also M.A.F. Livestock Officers, and laboratory staff of the Pal merston North and Ruakura Animal Health Laboratories. and the Animal Health Reference Laboratory. REFERENCES (II Hud~c•• R. T. ( 1973): A ~omplemenl lixalion lesl for Ihe serological diagnosis of leplospirosi, in pig.' experimenlally infccled wilh s.;rolype pomona. N.Z. vel: J. 21: 1·6. R. T.(.~914)~ T~e useof~ polyvalenlanllgen oncomplemenl Ii.allon leslsfor "'-"me lepl'''plnlSIS. N.Z. \('1. J. 21. 21·4. (31 Hodges. R. T.: Ris. D. R. (1914): Complemem fi.ing and agglulinaling anlibody r.'pons., and leplospiruria in calves inocula led wilh Lep,ospira serolypes pomona. l//lrd/". ",,!"'nhoKeIli or bollum. N.Z. vel. J. 12: 25-30. (4) Hodges. R. T.: Weddell. W. ( 1971): Adaplalion ofa complemenl fixalion lesl for large >
