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Use of ppd and phosphatide antigens in an elisa to detect the serological response in experimental bovine tuberculosis
Research in Veterinary Science 1989, 47, 43-47
Use of PPD and phosphatide antigens in an ELISA to detect the serological response in experimental bovine tuberculosis J. HANNA, S. D. NEILL, J. J. O'BRIEN, Department of Agriculture, Veterinary Research Laboratories, Stormont, Belfast BT4 3SD
Five calves from tuberculosis free herds were each Materials and methods inoculated intranasally with 1()6 viable organisms of a field isolate of Mycobacterium bovis. Four of the Animals calves developed acute tuberculosis. ELiSAs employing Eleven crossbred calves of 16 weeks of age were protein and phosphatide extracts of M bovis as obtained from tuberculosis free farms which had no antigens were used to monitor the humoral response history of M bovis infection for the previous seven of the infected calves. Fourteen days after infection years. The calves were separated into two groups. there was a dramatic increase in the level of antibodies Group A consisted of six animals included as undemonstrated by the phosphatide antigen. This infected controls which were housed and fed under increase coincided with the first appearance of signs normal farm conditions. Group B consisted of five of the disease. The results suggest that the experimentally infected animals housed in high phosphatide antigen may be of potential value in security isolation but fed normal rations of hay and detecting a humoral response, if present, in cattle concentrates. infected with M bovis. The tests employing the protein antigen demonstrated a humoral response in only one of the infected calves and emphasises the importance of antigen selection to detect antibodies in Experimental infection tuberculous animals. The inoculum for group B calves was prepared THE comparative intradermal tuberculin test is from a field strain of M bovis which had three employed in the field to identify infection in cattle passages on Lowe'hstein-Jensen medium following caused by Mycobacterium bovis. Although widely isolation from bovine nasal secretions by the bioused this test may suffer from a lack of specificity due logical method, using a guinea pig. The organism, in to antigens cross reacting with those of other myco- 5 ml 7B9 Middlebrook broth medium (Difco Laborabacteria (Chaparas and Maloney 1978) and other tories), was inoculated into Middlebrook broth microorganisms (Minden et al 1971). Attempts to (10 ml) overlying Middlebrook 7H9 agar (100 ml) develop a serodiagnostic test for tuberculosis in man contained in a flat 800 ml flask (Nunc). The culture and animals have included the use of complement was harvested after 14 days incubation in air at 37°C fixation tests (Vardaman and Larsen 1964, Ellis et al and the appropriate dilutions were prepared in 1965), haemagglutination tests (Takahashi and Ono normal saline. To break up any clumps of organisms 1961a, Reggiardo and Middlebrook 1975) and ELISA before inoculation, the culture was subjected to (Nassau et al 1976, Reggiardo et al 1980, Daniel et al ultrasonic vibration for 20 to 30 seconds in an 1986). However, a meaningful interpretation of any . immersion-type sonic bath (Brown 1983). The serodiagnostic test for tuberculosis should be made number of viable organisms in the inoculum was with a full understanding of the humoral response estimated from a subsamp1e according to the method during the course of the disease and this knowledge of Miles and Misra (1938)using 7H9 agar incubated at remains incomplete. This paper reports on the 37°C in 10 per cent carbon dioxide in air. The humoral response produced in a group of calves inoculum was delivered from a syringe through fine challenged with a field isolate of M bovis. An ELISA bore silicone rubber tubing inserted into the upper employing protein and phosphatide antigens was used nasal cavity and two doses, each containing 1()6 to monitor the profile of the immune response colony forming units in 5 ml normal saline were produced. administered on consecutive days to each calf. 43
J. Hanna, S. D. Neill, J. J. O'Brien
44
60
§j
.... ~
Q)
.'" ::l
"iii
40
>
iJ
20
o
o
20
60
40
80
100
Days
FIG 1: ELISAvalues of control calves against lal PPD antigen, (b) phosphatide antigen. 0 calf 3990, l!. calf 786, • calf 972, * calf 484, • calf 970, D calf 622
Specimen collection Blood samples were taken from a jugular vein at weekly intervals from group A animals throughout the duration of the experiment and from group B animals before infection, at days 3, 5, 7, 10, 14 after inoculation and at weekly intervals thereafter. Serum samples were stored at - 20oe.
Mycobacterial antigens The mammalian and avian purified protein derivative (ppo) tuberculins (Weybridge Laboratories) were dialysed in 0·01 M phosphate buffered saline (PBS) pH 7· 2 and stored at - 20°e. For use in the assay each ppo was diluted in 0·1 M carbonate buffer (pH 9'6) to give final protein concentrations of 0·01 mg
rnl: I. A saline emulsion of a phosphatide extract of M bovis (Takahashi and Ono 1961b) was diluted in O' I M carbonate buffer (pH 9' 6) to give a final phosphatide concentration of O' 02 mg ml ' I • ELISA
Serum antibodies to the PPO and phosphatide antigens were determined by ELISA (Kiran et al 1985). Wells of microtitre plates (Dynatech 129A) were coated with 0'1 ml of the selected dilution of each antigen and incubated at 37°e overnight in a moist chamber to prevent drying. After washing the plate six times in 0'02 M PBS containing 0'02 per cent Tween 20, 0·1 ml of test serum diluted 1:100 in 0'01 M PBS (pH 7'2) containing 0'02 per cent Tween
ELISA
80 was added to each of the appropriate plates which were then incubated at 37°C for one hour. The plates were again washed six times and 0·1 ml of immunoconjugate (0'015 ml rabbit anti-bovine IgG [H + Ll conjugated with horseradish peroxidase in 75 ml 0·01 M PBS containing 0'02 per cent Tween 80+ 4 per cent horse serum) was added to each well and incubated at 37°C for one hour. The plates were washed a further six times and 0'1 ml substrate (24 mg orthophenylenediamine + 60 ml citrate/ phosphate buffer pH 5'0+0·024 ml30 per cent w/v hydrogen peroxide) was added to each well and incubated at 37°C for exactly 20 minutes. The absorbance value in each was determined by using a Titertek Multiscan at 492 nM. Positive and negative serum controls and reagent blanks were included in each test plate. Each sample was tested in triplicate and the results expressed as a mean value. The ELISA value was calculated using the following formula: (Mean reading of sample mean reading of blank) 100 _ ELISA va I ue - (M ' . - x ean rea di109 0 f posiuve mean reading of blank) Results
Clinical manifestations oj injection No clinical signs were evident until 18 to 20 days after infection when reduced appetite, a progressive loss of condition and a productive cough was noted in all animals. Calf 1356 died on day 30. Calves 1357, 1300 and 1299 which were severely affected were killed on humane grounds on days 37, 57 and 67, respectively. Calf 1338, although less severely affected than the others, was killed on day 125. All control animals remained healthy during the course of this study. ELISA
45
in serology oj bovine tuberculosis
reaching a maximum on day 21, which was maintained until the animal was killed on day 67. Increases in antibody levels to the phosphatide antigen (Fig 2b) were demonstrated in animals 1299, 1300 and 1338 at day 14. In calf 1299,· the antibody level to the phosphatide antigen continued to increase until the animal was killed on day 67. Calf 1300 produced its maximum antibody level by day 40 after which the level decreased. The antibody responses of calves 1356 and 1357 were much lower and more gradual in onset than the others of the infected group. Discussion This preliminary study has shown that an intranasal inoculation of I (f' viable organisms of M bovis caused a severe illness ranging from peracute to chronic and produced serological responses in a group of five calves. These findings are very similar to those described by Lepper et al (1977) following the intravenous inoculation of calves with 0'02 mg M bovis. 100
i
Q)
:l
~ -c en
::; w
80
60 40
20
o'
1/
-10
120
30
50
I
70
Days after infection
90
110
i
130
b
100
results
In the group A control animals, the anti-ern antibodies remained at a constantly low level throughout the experiment (Fig la). The results using the mammalian and avian antigens were almost identical and therefore only the mammalian results are presented. The results presented in Fig 1b show that in the early samples taken, two of the control animals had much higher levels of antibody to the phosphatide antigen than did the others in the group. However, over a period of three weeks these values decreased to a common level which was maintained until the experiment was terminated. Only one of the infected animals (1299) developed a significant increase in the level of antibody to the PPO antigen (Fig 2a). The onset of this response was on day 14
a
N
S'f
~
80
Q)
:l
iii > -c en ::; w
60 40
20
O'----r--,---,---,-----,--r--...-10
10
30
50
70
90
110
130
Days after infection FIG 2: ELISA values of infected calves against (a) PPD antigen, (b) phosphatide antigen. 0 calf 1338, !J. calf 1299, • calf 1300, * calf 1356, • calf 1357
46
J. Hanna, S. D. Neill, J. J. O'Brien
As these latter workers did not ascertain the number of viable organisms in their inocula it is difficult to compare the effectiveness of the routes of transmission. It is generally accepted that in experimental tuberculosis the progression of the disease is most rapid when using the intravenous route of challenge (Takahashi et al 1961). The results of the present study demonstrate that experimental bovine tuberculosis may be produced rapidly using intranasal inoculation which may be the more natural route of infection. Francis (1947) concluded from field and experimental studies that bovine tuberculosis is usually transmitted via the respiratory tract. The appearance of the first clinical signs of the disease in the infected animals coincided with a substantial increase in the level of antibodies to the phosphatide antigen in each of the three animals which survived longer than 35 days after infection. Only one of these animals produced an increased level of antibody to the protein antigens. This is in agreement with the suggestion of Takahashi et al (1961) that in tuberculous animals only the antibody levels to phosphatide antigen provide meaningful information on the activity and extent of the disease. The two animals which died of the peracute disease at 30 and 32 days after infection showed a much lower and more delayed antibody response than the other calves in this group. This may have been due to their immune system being overwhelmed and unable to control the infection. Takahashi et al (1961) have suggested that antibodies to the phosphatide component of mycobacteria are not produced until the bacilli are destroyed in vivo. The presence of antibodies to the phosphatide antigen in sera from some of the control animals does, however, warrant caution when interpreting the test. It also highlights the need to ascertain the level of such antibodies in a field cattle population. It is difficult to explain why only one of the infected animals produced an increase in antibodies to the protein antigen as other workers have found this antigen useful for diagnosing human tuberculosis. Kalish et al (1983) found that the multiple antigens present in PPD made the ELISA a very useful screening test for human tuberculosis. Kiran et al (1985) also used PPD in an ELISA and reported that 80 per cent of cases with pulmonary tuberculosis were detected. The inability to demonstrate the presence of circulating antibodies in tuberculosis serum has been attributed in some cases to the suppression of the activity of the B lymphocytes by an increased number of T suppressor cells (Katz et al 1979). Other workers have suggested that the absence of a detectable humoral response may be due to antibodies being bound to circulating immune complexes (Carr et al 1980). The results of this study have shown the inability of the PPD antigen to detect a humoral response in a number
of infected animals in which seroconversion was clearly demonstrated by the phosphatide antigen. These findings emphasise the importance of antigen selection to detect a humoral response in tuberculous animals in which antibodies to the multiple components of the infecting organism may be presented at various stages during the disease. The aim of this study was to establish infection in calves with a field isolate of M bovis in order to monitor the development of the disease and the duration of any humoral response produced. The speed and severity of the disease condition produced resulted in a marked reduction of the time over which the present authors had anticipated monitoring the infected animals. These findings have shown the necessity for further experiments using a reduced number of organisms in the inocula which should possibly extend the period of time over which the humoral response may be observed. Acknowledgements The authors wish to acknowledge the excellent technical assistance of Mr E. Walton, Mr S. McBride and Mrs F. McBride. References BROWN, I. N. (1983) The Biology of the Mycobacteria, Vol 2. London, Academic Press. pp 173-234 CARR, R. I., CHAKRABORTY, A. K., BRENDA, M. J., DAVIDSON, P. T., DAMLE, P. B., HARDTKE, M. A., GILBRIDE, K. J. & MINDEN, P. (1980) Clinical and Experimental Immunology 39, 562-569 CHAPARAS, S. D. & MALONEY, C. J. (1978) American Review of Respiratory Disease 117, 897-902 DANIEL, T. M., DE MURILLO, G. L., SAWYER, J. A., GRIFFEN, A. MeL., PINTO, E., DEBAUNE, S. M., ESPINOSA, P. & CESPIDIS, E. (1986) American Review of Respiratory Disease 134, 662-665 ELLIS, E. M., WRIGHT, H. S. & r.ICHARDS, W. D. (1%5) American Review of Respiratory Disease 93, 749-753 FRANCIS, J. (1947) BovineTuberculosis, London, Staples Press. pp 86-92 KALISH, S. B., RADIN, R. C., PHAIR, J. P., TEVITY, D., ZEISS, C. R. & METZGER, E. (1983) Journal of Infectious Disease 147, 523-530 KATZ, P., GOLSTEIN, R. A. & FAUCI, A. S. (1979) Journal of Infectious Diseases 140, 12-21 KIRAN, U., SHRINIWAS, KUMAR, R. & SHARMA, A. (1985) European Journal of Respiratory Diseases 66, 187-195 LEPPER, A. W. D., CORNER, L. A. & PEARSON, C. W. (1977) Australian Veterinary JournalS3, 301-305 MILES, A. A. & MISRA, S. S. (1938) Journal of Hygiene, Cambridge 38, 732-749 . MINDEN, P., McCLATCHY, J. K., COOPER, R., BARDANA, E. J. & FARR, R. S. (1971) Science 176, 57-58 NASSAU, E., PARSONS, E. R. & JOHNSTON, G. D. (1976) Tubercle 57, 67- 70 REGGIARDO, Z. & MIDDLEBROOK, G. (1975) American Journal of Epidemiology lOll, 469-476 REGGIARDO, Z., VASQUEZ, E. & SMAEPER, L. (1980) Journal of Immunological Methods 34, 55-60
ELISA
in serology of bovine tuberculosis
TAKAHASHI, Y. & ONO, K. (l96la) American Review of Respiratory Disease 83,381-385 TAKAHASHI, Y. & ONO, K. (l96lb) American Review of Respiratory Disease 83, 386-393 TAKAHASHI, Y., ONDERA, T. & YAMAMOTO, K. (1961) Journal of Experimental Medicine 114, 555-567
47
VARDAMAN, T. H. & LARSEN, A. B. (1964) American Journal of Veterinary Research 25, 690-692
Received June /3, /988 Accepted August I, /988
Use of PPD and phosphatide antigens in an ELISA to detect the serological response in experimental bovine tuberculosis J. HANNA, S. D. NEILL, J. J. O'BRIEN, Department of Agriculture, Veterinary Research Laboratories, Stormont, Belfast BT4 3SD
Five calves from tuberculosis free herds were each Materials and methods inoculated intranasally with 1()6 viable organisms of a field isolate of Mycobacterium bovis. Four of the Animals calves developed acute tuberculosis. ELiSAs employing Eleven crossbred calves of 16 weeks of age were protein and phosphatide extracts of M bovis as obtained from tuberculosis free farms which had no antigens were used to monitor the humoral response history of M bovis infection for the previous seven of the infected calves. Fourteen days after infection years. The calves were separated into two groups. there was a dramatic increase in the level of antibodies Group A consisted of six animals included as undemonstrated by the phosphatide antigen. This infected controls which were housed and fed under increase coincided with the first appearance of signs normal farm conditions. Group B consisted of five of the disease. The results suggest that the experimentally infected animals housed in high phosphatide antigen may be of potential value in security isolation but fed normal rations of hay and detecting a humoral response, if present, in cattle concentrates. infected with M bovis. The tests employing the protein antigen demonstrated a humoral response in only one of the infected calves and emphasises the importance of antigen selection to detect antibodies in Experimental infection tuberculous animals. The inoculum for group B calves was prepared THE comparative intradermal tuberculin test is from a field strain of M bovis which had three employed in the field to identify infection in cattle passages on Lowe'hstein-Jensen medium following caused by Mycobacterium bovis. Although widely isolation from bovine nasal secretions by the bioused this test may suffer from a lack of specificity due logical method, using a guinea pig. The organism, in to antigens cross reacting with those of other myco- 5 ml 7B9 Middlebrook broth medium (Difco Laborabacteria (Chaparas and Maloney 1978) and other tories), was inoculated into Middlebrook broth microorganisms (Minden et al 1971). Attempts to (10 ml) overlying Middlebrook 7H9 agar (100 ml) develop a serodiagnostic test for tuberculosis in man contained in a flat 800 ml flask (Nunc). The culture and animals have included the use of complement was harvested after 14 days incubation in air at 37°C fixation tests (Vardaman and Larsen 1964, Ellis et al and the appropriate dilutions were prepared in 1965), haemagglutination tests (Takahashi and Ono normal saline. To break up any clumps of organisms 1961a, Reggiardo and Middlebrook 1975) and ELISA before inoculation, the culture was subjected to (Nassau et al 1976, Reggiardo et al 1980, Daniel et al ultrasonic vibration for 20 to 30 seconds in an 1986). However, a meaningful interpretation of any . immersion-type sonic bath (Brown 1983). The serodiagnostic test for tuberculosis should be made number of viable organisms in the inoculum was with a full understanding of the humoral response estimated from a subsamp1e according to the method during the course of the disease and this knowledge of Miles and Misra (1938)using 7H9 agar incubated at remains incomplete. This paper reports on the 37°C in 10 per cent carbon dioxide in air. The humoral response produced in a group of calves inoculum was delivered from a syringe through fine challenged with a field isolate of M bovis. An ELISA bore silicone rubber tubing inserted into the upper employing protein and phosphatide antigens was used nasal cavity and two doses, each containing 1()6 to monitor the profile of the immune response colony forming units in 5 ml normal saline were produced. administered on consecutive days to each calf. 43
J. Hanna, S. D. Neill, J. J. O'Brien
44
60
§j
.... ~
Q)
.'" ::l
"iii
40
>
iJ
20
o
o
20
60
40
80
100
Days
FIG 1: ELISAvalues of control calves against lal PPD antigen, (b) phosphatide antigen. 0 calf 3990, l!. calf 786, • calf 972, * calf 484, • calf 970, D calf 622
Specimen collection Blood samples were taken from a jugular vein at weekly intervals from group A animals throughout the duration of the experiment and from group B animals before infection, at days 3, 5, 7, 10, 14 after inoculation and at weekly intervals thereafter. Serum samples were stored at - 20oe.
Mycobacterial antigens The mammalian and avian purified protein derivative (ppo) tuberculins (Weybridge Laboratories) were dialysed in 0·01 M phosphate buffered saline (PBS) pH 7· 2 and stored at - 20°e. For use in the assay each ppo was diluted in 0·1 M carbonate buffer (pH 9'6) to give final protein concentrations of 0·01 mg
rnl: I. A saline emulsion of a phosphatide extract of M bovis (Takahashi and Ono 1961b) was diluted in O' I M carbonate buffer (pH 9' 6) to give a final phosphatide concentration of O' 02 mg ml ' I • ELISA
Serum antibodies to the PPO and phosphatide antigens were determined by ELISA (Kiran et al 1985). Wells of microtitre plates (Dynatech 129A) were coated with 0'1 ml of the selected dilution of each antigen and incubated at 37°e overnight in a moist chamber to prevent drying. After washing the plate six times in 0'02 M PBS containing 0'02 per cent Tween 20, 0·1 ml of test serum diluted 1:100 in 0'01 M PBS (pH 7'2) containing 0'02 per cent Tween
ELISA
80 was added to each of the appropriate plates which were then incubated at 37°C for one hour. The plates were again washed six times and 0·1 ml of immunoconjugate (0'015 ml rabbit anti-bovine IgG [H + Ll conjugated with horseradish peroxidase in 75 ml 0·01 M PBS containing 0'02 per cent Tween 80+ 4 per cent horse serum) was added to each well and incubated at 37°C for one hour. The plates were washed a further six times and 0'1 ml substrate (24 mg orthophenylenediamine + 60 ml citrate/ phosphate buffer pH 5'0+0·024 ml30 per cent w/v hydrogen peroxide) was added to each well and incubated at 37°C for exactly 20 minutes. The absorbance value in each was determined by using a Titertek Multiscan at 492 nM. Positive and negative serum controls and reagent blanks were included in each test plate. Each sample was tested in triplicate and the results expressed as a mean value. The ELISA value was calculated using the following formula: (Mean reading of sample mean reading of blank) 100 _ ELISA va I ue - (M ' . - x ean rea di109 0 f posiuve mean reading of blank) Results
Clinical manifestations oj injection No clinical signs were evident until 18 to 20 days after infection when reduced appetite, a progressive loss of condition and a productive cough was noted in all animals. Calf 1356 died on day 30. Calves 1357, 1300 and 1299 which were severely affected were killed on humane grounds on days 37, 57 and 67, respectively. Calf 1338, although less severely affected than the others, was killed on day 125. All control animals remained healthy during the course of this study. ELISA
45
in serology oj bovine tuberculosis
reaching a maximum on day 21, which was maintained until the animal was killed on day 67. Increases in antibody levels to the phosphatide antigen (Fig 2b) were demonstrated in animals 1299, 1300 and 1338 at day 14. In calf 1299,· the antibody level to the phosphatide antigen continued to increase until the animal was killed on day 67. Calf 1300 produced its maximum antibody level by day 40 after which the level decreased. The antibody responses of calves 1356 and 1357 were much lower and more gradual in onset than the others of the infected group. Discussion This preliminary study has shown that an intranasal inoculation of I (f' viable organisms of M bovis caused a severe illness ranging from peracute to chronic and produced serological responses in a group of five calves. These findings are very similar to those described by Lepper et al (1977) following the intravenous inoculation of calves with 0'02 mg M bovis. 100
i
Q)
:l
~ -c en
::; w
80
60 40
20
o'
1/
-10
120
30
50
I
70
Days after infection
90
110
i
130
b
100
results
In the group A control animals, the anti-ern antibodies remained at a constantly low level throughout the experiment (Fig la). The results using the mammalian and avian antigens were almost identical and therefore only the mammalian results are presented. The results presented in Fig 1b show that in the early samples taken, two of the control animals had much higher levels of antibody to the phosphatide antigen than did the others in the group. However, over a period of three weeks these values decreased to a common level which was maintained until the experiment was terminated. Only one of the infected animals (1299) developed a significant increase in the level of antibody to the PPO antigen (Fig 2a). The onset of this response was on day 14
a
N
S'f
~
80
Q)
:l
iii > -c en ::; w
60 40
20
O'----r--,---,---,-----,--r--...-10
10
30
50
70
90
110
130
Days after infection FIG 2: ELISA values of infected calves against (a) PPD antigen, (b) phosphatide antigen. 0 calf 1338, !J. calf 1299, • calf 1300, * calf 1356, • calf 1357
46
J. Hanna, S. D. Neill, J. J. O'Brien
As these latter workers did not ascertain the number of viable organisms in their inocula it is difficult to compare the effectiveness of the routes of transmission. It is generally accepted that in experimental tuberculosis the progression of the disease is most rapid when using the intravenous route of challenge (Takahashi et al 1961). The results of the present study demonstrate that experimental bovine tuberculosis may be produced rapidly using intranasal inoculation which may be the more natural route of infection. Francis (1947) concluded from field and experimental studies that bovine tuberculosis is usually transmitted via the respiratory tract. The appearance of the first clinical signs of the disease in the infected animals coincided with a substantial increase in the level of antibodies to the phosphatide antigen in each of the three animals which survived longer than 35 days after infection. Only one of these animals produced an increased level of antibody to the protein antigens. This is in agreement with the suggestion of Takahashi et al (1961) that in tuberculous animals only the antibody levels to phosphatide antigen provide meaningful information on the activity and extent of the disease. The two animals which died of the peracute disease at 30 and 32 days after infection showed a much lower and more delayed antibody response than the other calves in this group. This may have been due to their immune system being overwhelmed and unable to control the infection. Takahashi et al (1961) have suggested that antibodies to the phosphatide component of mycobacteria are not produced until the bacilli are destroyed in vivo. The presence of antibodies to the phosphatide antigen in sera from some of the control animals does, however, warrant caution when interpreting the test. It also highlights the need to ascertain the level of such antibodies in a field cattle population. It is difficult to explain why only one of the infected animals produced an increase in antibodies to the protein antigen as other workers have found this antigen useful for diagnosing human tuberculosis. Kalish et al (1983) found that the multiple antigens present in PPD made the ELISA a very useful screening test for human tuberculosis. Kiran et al (1985) also used PPD in an ELISA and reported that 80 per cent of cases with pulmonary tuberculosis were detected. The inability to demonstrate the presence of circulating antibodies in tuberculosis serum has been attributed in some cases to the suppression of the activity of the B lymphocytes by an increased number of T suppressor cells (Katz et al 1979). Other workers have suggested that the absence of a detectable humoral response may be due to antibodies being bound to circulating immune complexes (Carr et al 1980). The results of this study have shown the inability of the PPD antigen to detect a humoral response in a number
of infected animals in which seroconversion was clearly demonstrated by the phosphatide antigen. These findings emphasise the importance of antigen selection to detect a humoral response in tuberculous animals in which antibodies to the multiple components of the infecting organism may be presented at various stages during the disease. The aim of this study was to establish infection in calves with a field isolate of M bovis in order to monitor the development of the disease and the duration of any humoral response produced. The speed and severity of the disease condition produced resulted in a marked reduction of the time over which the present authors had anticipated monitoring the infected animals. These findings have shown the necessity for further experiments using a reduced number of organisms in the inocula which should possibly extend the period of time over which the humoral response may be observed. Acknowledgements The authors wish to acknowledge the excellent technical assistance of Mr E. Walton, Mr S. McBride and Mrs F. McBride. References BROWN, I. N. (1983) The Biology of the Mycobacteria, Vol 2. London, Academic Press. pp 173-234 CARR, R. I., CHAKRABORTY, A. K., BRENDA, M. J., DAVIDSON, P. T., DAMLE, P. B., HARDTKE, M. A., GILBRIDE, K. J. & MINDEN, P. (1980) Clinical and Experimental Immunology 39, 562-569 CHAPARAS, S. D. & MALONEY, C. J. (1978) American Review of Respiratory Disease 117, 897-902 DANIEL, T. M., DE MURILLO, G. L., SAWYER, J. A., GRIFFEN, A. MeL., PINTO, E., DEBAUNE, S. M., ESPINOSA, P. & CESPIDIS, E. (1986) American Review of Respiratory Disease 134, 662-665 ELLIS, E. M., WRIGHT, H. S. & r.ICHARDS, W. D. (1%5) American Review of Respiratory Disease 93, 749-753 FRANCIS, J. (1947) BovineTuberculosis, London, Staples Press. pp 86-92 KALISH, S. B., RADIN, R. C., PHAIR, J. P., TEVITY, D., ZEISS, C. R. & METZGER, E. (1983) Journal of Infectious Disease 147, 523-530 KATZ, P., GOLSTEIN, R. A. & FAUCI, A. S. (1979) Journal of Infectious Diseases 140, 12-21 KIRAN, U., SHRINIWAS, KUMAR, R. & SHARMA, A. (1985) European Journal of Respiratory Diseases 66, 187-195 LEPPER, A. W. D., CORNER, L. A. & PEARSON, C. W. (1977) Australian Veterinary JournalS3, 301-305 MILES, A. A. & MISRA, S. S. (1938) Journal of Hygiene, Cambridge 38, 732-749 . MINDEN, P., McCLATCHY, J. K., COOPER, R., BARDANA, E. J. & FARR, R. S. (1971) Science 176, 57-58 NASSAU, E., PARSONS, E. R. & JOHNSTON, G. D. (1976) Tubercle 57, 67- 70 REGGIARDO, Z. & MIDDLEBROOK, G. (1975) American Journal of Epidemiology lOll, 469-476 REGGIARDO, Z., VASQUEZ, E. & SMAEPER, L. (1980) Journal of Immunological Methods 34, 55-60
ELISA
in serology of bovine tuberculosis
TAKAHASHI, Y. & ONO, K. (l96la) American Review of Respiratory Disease 83,381-385 TAKAHASHI, Y. & ONO, K. (l96lb) American Review of Respiratory Disease 83, 386-393 TAKAHASHI, Y., ONDERA, T. & YAMAMOTO, K. (1961) Journal of Experimental Medicine 114, 555-567
47
VARDAMAN, T. H. & LARSEN, A. B. (1964) American Journal of Veterinary Research 25, 690-692
Received June /3, /988 Accepted August I, /988