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Evaluation of MPB70, bovine PPD and lipoarabinomannan as antigens in ELISA for the serodiagnosis of bovine tuberculosis
PREVENTIVE VETERINARY MEDICINE
Evaluation of MPB70, bovine PPD and lipoarabinomannan as antigens in ELISA for the serodiagnosis of bovine tuberculosis Jorge Hembdez De Anda ay*,Michael Monaghan b, John D. Collins, b, Patrick J. Brennan ‘, MO D. Salman
C
a Institute de Inuestigociones en Ciencias Veterinarias, Universidad Aut&oma de Baja California, Mexicali, BC 21100, Me!xico b Faculty of Veterinary Medicine, University College Dublin, Dublin 4, Ireland ’ Department of Microbiology and Department of Environmental Health, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA
Accepted 6 October 1995
Abstract The performance of the secretory protein MPB70 of Mycobacterium bovis, bovine PPD. and Iipoarabinomannan (LAM) were evaluated as antigens in ELISA for detection of tuberculosis (TB) infected cattle. Sera were from 120 M. bouis infected cattle and 223 cattle from a TB free herd. ELBA results were analyzed using receiver operating characteristic (ROC) curves in relation to
culture results. The areas under the three ROC curves were 71 + 49% SE (MPB701, 71 + 27% SE (bovine: PPD), and 56 f 4% SE (LAM).
1. Introduction
Several ELISA methods have been investigated to evaluate their performance in the serodiagnosis of tuberculosis (TB) in cattle. Auer (1987) reported that an indirect ELISA using unpurified antigen was considered to be unsuitable as an alternative to tuberculin testing for detection of Mycobacterium bouis infected animals; the sensitivity and specificity of the ELISA test were 88% and 52%, respectively. Work with TB infected
* Correqonding author. 0167-5877/96/$15.00
0 1996 Elsevier Science B.V. All rights reserved
SSDI 0167.5877(95)00572-2
212
J. Herncindez De Andu et d/Preventive
Veterinary Medicine 27 (1996) 21 l-215
and non-infected cattle reported by Ritacco et al. (19871, Ritacco et al. (19901, Ritacco et al. (1991) determined sensitivity and specificity estimates, using bovine PPD as antigen in ELISA, which ranged from 90% to 35% and 94% to 89%, respectively. The MPB70 secretory protein of M. bovis was reported to be highly species-specific for M. bovis (Harboe et al., 1990 and Nagai et al., 1981). However, when MPB70 was used as antigen in ELISA, Harboe et al. (1990) concluded that the assay had insufficient sensitivity to provide important diagnostic information for cattle and other animal species with natural M. bovis infections. Wood et al. (1992) reported a sensitivity of 18% and a specificity of 94%, using MPB70 as antigen in ELISA, in a field evaluation to test the serological response to TB infection in cattle. Lipoarabinomannan (LAM) is a cross-reactive antigen among mycobacterial species; it has been used with some success for the serodiagnosis of paratuberculosis in cattle (McNab et al., 1991) and tuberculosis in humans (Sada et al., 1990). For tuberculosis in cattle, however, the performance of LAM in ELISA has not yet been investigated. The purpose of the present work was to evaluate the performance of MPB70, bovine PPD, and LAM as antigens in ELISA for discrimination of M. bovis infected and non-infected cattle.
2. Materials
and methods
2.1. Serum samples Three-hundred and forty-three blood samples were obtained from two groups of cattle in Ireland. In Ireland, all herds in which TB is detected (by tuberculin testing or during routine post-mortem examination of carcasses in the abattoir) are placed under movement restriction. These herds are tuberculin tested at 60-day intervals and the restrictions remain until the herd has two consecutive clear tests. Group 1 consisted of 120 dairy cattle, from 15 dairy herds, which were bovine-PPD-positive, with tuberculous lesions in bronchial and retropharyngeal lymph nodes where M. bovis infection was confirmed by histopathologic and bacteriologic procedures. Animals in this group were defined as TB-infected cattle. However, serum samples in this group were not representative of the infection/disease spectrum to be expected in infected subclinical or clinical cattle. Group 2 consisted of 223 cattle from a dairy herd free of M. bovis since 1982. Consequently, they only represented one specific environment capable of producing or not producing non-specific reactions. 2.2. Antigens The MPB70 protein as the recombinant product was provided by A.J. Radford et al. (1990) and bovine PPD (used for the Comparative-Cervical Skin Test) was from the Central Veterinary Institute, Lelystad, The Netherlands. LAM has been described by Hunter and Brennan (1990).
J. Herncinder De Andu et al./Preventive
Veterinary Medicine 27 (1996) 211-215
213
2.3. ELISA procedures The assay was performed using rabbit anti-bovine IgG as the secondary antibody conjugated to peroxidase. Dilutions of antigen, serum, and conjugate were chosen to give maximum separation of positive and negative serum controls. For purposes of coating plates with antigen, MPB70 (0.5 pg ml ? - l}), bovine PPD (10 pg ml < - I}), or LAM (0.5 pg ml /f - 1)) in standard carbonate-bicarbonate coating buffer (pH 9.6) were added to each well of U-bottomed 96-well polystyrene plates. Plates were incubated at 37°C overnight in a humid chamber. One-hundred microliters of egg albumin in PBS with 0.05% Tween 80 (PBST) were added to each well as blocking solution and incubated for 15 min at 37°C. Single serum dilutions (1:200) in PBS diluent (PBST with rabbit normal serum 10%) were added in 50-~1 volumes to duplicate wells and incubated for 1 h at 37°C in a humid chamber. Plates then were emptied and manually washed five times with PBS washing solution (NaH,PO, monobasic, Na,HPO, dibasic, :NaCl). One positive and one negative serum control were added in duplicate wells in each plate. Sera were kept stored at -20°C before testing. Rabbit anti-bovine IgG peramxidase-conjugated diluted 1:4000 in PBS diluent was added in volumes of 50 ~1 to each well. Plates were incubated for 30 min at 37°C and washed as above. Tetramethylbenzidine (TMB) substrate (50 ~1) was added to each well. TMB diluted in DMSO was mixed with a TMB buffer (citric acid/sodium acetate, and 30% hydrogen peroxide;) to make the substrate color indicator. After 10 min, the reaction was stopped with 50 ill of 2 M H,SO,. ELISA adsorbance values at 450 nm were determined using a reader instrument. 2.4. Analysis Receiver operating characteristic (ROC) curve analysis was used to evaluate the MPB70, bovine PPD, and LAM in ELISA relative to culture results, as described by Hanley and McNeil (1982). Sensitivity and specificity were estimated for the three assays along the entire optical density (normal log (In) values) scale by dichotomizing (positive and negative) the ELISA results at 0.5 In increment values.
3. Resulls
and discussion
Fig. 1 shows the ROC curves for the MPB70, bovine PPD, and LAM in ELISA relative to culture and isolation of M. bovis from tissues. The areas under the three ROC curves were 71 f 49% SE (MPB70), 71 + 27% SE (bovine PPD), and 56 + 4% SE (LAM). These areas describe the ability of the tests to discriminate between TB-infected and non-infected cattle. A perfect test has an area of lOO%, while a non-informative test has an area of 50%. A perfect test allows for a diseased animal to always be detected; a non-informative test is ‘no better than flipping a coin’ (Centor, 1991). In conclusion, the ELISA MPB70 and bovine PPD methods performed better than the ELISA LAM for detection of TB infection status in animals tested in this study. The variation of antibody response against MPB70 and bovine PPD, among TB infected
J. Hermklez
214
De Ada
et al./Preuentioe
Veterinary Medicine 27 (1996) 211-215
S
e n 8
t i V
Y
%
60
60
(100 - Specificity) Fig. 1. Receiver operating characteristic culture and isolation of Mycobucterium
MPB70
+
PPD
+
LAM
curves for MPB70, bovine PPD, and lipoarabinomammn
relative to
houis from tissues.
animals, can be explained by the fact that samples selected from infected animals were not representatives of the infection/disease spectrum to be expected in infected subclinical or clinical cattle. Previous investigations related with the serological diagnosis of TB in cattle (Auer, 1987; Plackett et al., 1989; Wood et al., 1992) suggested that in the field the level of cross-reactivity of MPB70 antigen may be higher than originally thought. Also, the lack of detectable antibody responses in M. bovis infected cattle is in line with previous reports on both the humoral and cellular responses in bovine TB (Wood et al., 1992; Ritacco et al., 1990; Harboe et al., 1990; Plackett et al., 1989).
Acknowledgements Supported in part by a grant from CONACyT Mexico (93/8083).
(143M9107)
and FOMES
SEP in
References Auer, L.A., 1987. Assessment of an enzyme linked immunosorbent assay for the detection of cattle infected with Mycobacterium bouis, 1987. Aust. Vet. J., 64: 172-176. Centor, R.M., 1991. Signal detectability: the use of ROC curves and their analyses. Med. Decis. Making; II: 102-106. Hanley, J.A. and McNeil, B.J., 1982. The meaning and use of the arca under a receiver operating characteristic (ROC) curve. Radiology, 143: 29-36. Harboe, M., Wiker, H.G., Duncan, J.R., Garcia, M.M., Dukes, T.W., Brooks, B.W., Turcotte, C. and Nagai, S., 1990. Protein G-based enzyme-linked immunosorbent assay for anti-MPB70 antibodies in bovine tuberculosis. J. Clm. Microbial., 28: 913-921.
J. Herna’ndet De Anda et al. / Preuentiue Veterinary Medicine 27 (1996) 21 I -2/S
215
Hunter, S.W. and Brennan, P.J., 1990. Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobucterium tubercuhis. J. Biol. Chem., 265: 9272-9279. McNab, W.B., Meek, A.H., Duncan, J.R., Brooks, B.W., van Dreumel, A.A., Martin, SW., Nielsen, K.H., Sugden, E.A. and Turcotte, C., 1991. An evaluation of selected screening tests for bovine paratuberculosis. Can. I. Vet. Res., 55: 252-259. Nagai, S., Matsumoto, J. and Nagasuga, T., 1981. Specific skin-reactive protein from culture filtrate of Mycobocterium bovis BCG. Infect. Immun., 31: 1152- 1160. .~ Placket&P., Ripper, J., Comer, L.A., Small, K., de Witte K., Melville, L., Hides, S. and Wood, P.R., 1989. An ELISA for detection of anergic tuberculous cattle. Aust. Vet. J., 66: 15- 19. Radford, A.J., Wood, P.R., Billmat-Jacobe, H., Geysen, H.M., Mason, T.J. and Gordon, T., 1990. Epitope mappin,; of the Mycobacterium bouis secretory protein MPB70 using overlapping peptide analysis. J. Gen. lMicrobiol., 136: 265-272. Ritacco, V., Kantor, I.N., Barrera, L., Nader, A., Bemardelli, A., Torrea, G., Errico, F. and Fliess, E., 1987. Assessment of the sensitivity and specificity of enzyme-linked immunosorbent assay (ELISA) for the detection of mycobacterial antibodies in bovine tuberculosis. J. Vet. Med., 34: 119- 125. Ritacco, V., Lbpez, B., Barrera, L., Nader, A., FIiess, E. and Kantor, I.N., 1990. Further evaluation of an indirect enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis. J. Vet. Med., 37: 19-27. Ritacco, V., L6pez, B., Kantor, I.N., Barrera, L., Errico, F. and Nader, A., 1991. Reciprocal cellular and humoral immune responses in bovine tuberculosis. Res. Vet. Sci., 50: 365-367. Sada, E., Brennan, P.J., Herrera, T. and Torres, M., 1990. Evaluation of Iipoarabinomannan for the serological diagnosis of tuberculosis. J. Clin. Microbial., 28: 2587-2590. Wood, P.R , Comer, L.A., Rothel, J.S., Ripper, J.L., Fifis, T., McCormick, B.S., Francis, B., Melville, L., Small, K., De Witte, K., Tolson, J., Ryan, T.J., De Lisle, Cox, J.C. and Jones, S.L., 1992. A field evaluation of serological and cellular diagnostic tests for bovine tuberculosis. Vet. Microbial., 3 I: 71-79.
Evaluation of MPB70, bovine PPD and lipoarabinomannan as antigens in ELISA for the serodiagnosis of bovine tuberculosis Jorge Hembdez De Anda ay*,Michael Monaghan b, John D. Collins, b, Patrick J. Brennan ‘, MO D. Salman
C
a Institute de Inuestigociones en Ciencias Veterinarias, Universidad Aut&oma de Baja California, Mexicali, BC 21100, Me!xico b Faculty of Veterinary Medicine, University College Dublin, Dublin 4, Ireland ’ Department of Microbiology and Department of Environmental Health, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA
Accepted 6 October 1995
Abstract The performance of the secretory protein MPB70 of Mycobacterium bovis, bovine PPD. and Iipoarabinomannan (LAM) were evaluated as antigens in ELISA for detection of tuberculosis (TB) infected cattle. Sera were from 120 M. bouis infected cattle and 223 cattle from a TB free herd. ELBA results were analyzed using receiver operating characteristic (ROC) curves in relation to
culture results. The areas under the three ROC curves were 71 + 49% SE (MPB701, 71 + 27% SE (bovine: PPD), and 56 f 4% SE (LAM).
1. Introduction
Several ELISA methods have been investigated to evaluate their performance in the serodiagnosis of tuberculosis (TB) in cattle. Auer (1987) reported that an indirect ELISA using unpurified antigen was considered to be unsuitable as an alternative to tuberculin testing for detection of Mycobacterium bouis infected animals; the sensitivity and specificity of the ELISA test were 88% and 52%, respectively. Work with TB infected
* Correqonding author. 0167-5877/96/$15.00
0 1996 Elsevier Science B.V. All rights reserved
SSDI 0167.5877(95)00572-2
212
J. Herncindez De Andu et d/Preventive
Veterinary Medicine 27 (1996) 21 l-215
and non-infected cattle reported by Ritacco et al. (19871, Ritacco et al. (19901, Ritacco et al. (1991) determined sensitivity and specificity estimates, using bovine PPD as antigen in ELISA, which ranged from 90% to 35% and 94% to 89%, respectively. The MPB70 secretory protein of M. bovis was reported to be highly species-specific for M. bovis (Harboe et al., 1990 and Nagai et al., 1981). However, when MPB70 was used as antigen in ELISA, Harboe et al. (1990) concluded that the assay had insufficient sensitivity to provide important diagnostic information for cattle and other animal species with natural M. bovis infections. Wood et al. (1992) reported a sensitivity of 18% and a specificity of 94%, using MPB70 as antigen in ELISA, in a field evaluation to test the serological response to TB infection in cattle. Lipoarabinomannan (LAM) is a cross-reactive antigen among mycobacterial species; it has been used with some success for the serodiagnosis of paratuberculosis in cattle (McNab et al., 1991) and tuberculosis in humans (Sada et al., 1990). For tuberculosis in cattle, however, the performance of LAM in ELISA has not yet been investigated. The purpose of the present work was to evaluate the performance of MPB70, bovine PPD, and LAM as antigens in ELISA for discrimination of M. bovis infected and non-infected cattle.
2. Materials
and methods
2.1. Serum samples Three-hundred and forty-three blood samples were obtained from two groups of cattle in Ireland. In Ireland, all herds in which TB is detected (by tuberculin testing or during routine post-mortem examination of carcasses in the abattoir) are placed under movement restriction. These herds are tuberculin tested at 60-day intervals and the restrictions remain until the herd has two consecutive clear tests. Group 1 consisted of 120 dairy cattle, from 15 dairy herds, which were bovine-PPD-positive, with tuberculous lesions in bronchial and retropharyngeal lymph nodes where M. bovis infection was confirmed by histopathologic and bacteriologic procedures. Animals in this group were defined as TB-infected cattle. However, serum samples in this group were not representative of the infection/disease spectrum to be expected in infected subclinical or clinical cattle. Group 2 consisted of 223 cattle from a dairy herd free of M. bovis since 1982. Consequently, they only represented one specific environment capable of producing or not producing non-specific reactions. 2.2. Antigens The MPB70 protein as the recombinant product was provided by A.J. Radford et al. (1990) and bovine PPD (used for the Comparative-Cervical Skin Test) was from the Central Veterinary Institute, Lelystad, The Netherlands. LAM has been described by Hunter and Brennan (1990).
J. Herncinder De Andu et al./Preventive
Veterinary Medicine 27 (1996) 211-215
213
2.3. ELISA procedures The assay was performed using rabbit anti-bovine IgG as the secondary antibody conjugated to peroxidase. Dilutions of antigen, serum, and conjugate were chosen to give maximum separation of positive and negative serum controls. For purposes of coating plates with antigen, MPB70 (0.5 pg ml ? - l}), bovine PPD (10 pg ml < - I}), or LAM (0.5 pg ml /f - 1)) in standard carbonate-bicarbonate coating buffer (pH 9.6) were added to each well of U-bottomed 96-well polystyrene plates. Plates were incubated at 37°C overnight in a humid chamber. One-hundred microliters of egg albumin in PBS with 0.05% Tween 80 (PBST) were added to each well as blocking solution and incubated for 15 min at 37°C. Single serum dilutions (1:200) in PBS diluent (PBST with rabbit normal serum 10%) were added in 50-~1 volumes to duplicate wells and incubated for 1 h at 37°C in a humid chamber. Plates then were emptied and manually washed five times with PBS washing solution (NaH,PO, monobasic, Na,HPO, dibasic, :NaCl). One positive and one negative serum control were added in duplicate wells in each plate. Sera were kept stored at -20°C before testing. Rabbit anti-bovine IgG peramxidase-conjugated diluted 1:4000 in PBS diluent was added in volumes of 50 ~1 to each well. Plates were incubated for 30 min at 37°C and washed as above. Tetramethylbenzidine (TMB) substrate (50 ~1) was added to each well. TMB diluted in DMSO was mixed with a TMB buffer (citric acid/sodium acetate, and 30% hydrogen peroxide;) to make the substrate color indicator. After 10 min, the reaction was stopped with 50 ill of 2 M H,SO,. ELISA adsorbance values at 450 nm were determined using a reader instrument. 2.4. Analysis Receiver operating characteristic (ROC) curve analysis was used to evaluate the MPB70, bovine PPD, and LAM in ELISA relative to culture results, as described by Hanley and McNeil (1982). Sensitivity and specificity were estimated for the three assays along the entire optical density (normal log (In) values) scale by dichotomizing (positive and negative) the ELISA results at 0.5 In increment values.
3. Resulls
and discussion
Fig. 1 shows the ROC curves for the MPB70, bovine PPD, and LAM in ELISA relative to culture and isolation of M. bovis from tissues. The areas under the three ROC curves were 71 f 49% SE (MPB70), 71 + 27% SE (bovine PPD), and 56 + 4% SE (LAM). These areas describe the ability of the tests to discriminate between TB-infected and non-infected cattle. A perfect test has an area of lOO%, while a non-informative test has an area of 50%. A perfect test allows for a diseased animal to always be detected; a non-informative test is ‘no better than flipping a coin’ (Centor, 1991). In conclusion, the ELISA MPB70 and bovine PPD methods performed better than the ELISA LAM for detection of TB infection status in animals tested in this study. The variation of antibody response against MPB70 and bovine PPD, among TB infected
J. Hermklez
214
De Ada
et al./Preuentioe
Veterinary Medicine 27 (1996) 211-215
S
e n 8
t i V
Y
%
60
60
(100 - Specificity) Fig. 1. Receiver operating characteristic culture and isolation of Mycobucterium
MPB70
+
PPD
+
LAM
curves for MPB70, bovine PPD, and lipoarabinomammn
relative to
houis from tissues.
animals, can be explained by the fact that samples selected from infected animals were not representatives of the infection/disease spectrum to be expected in infected subclinical or clinical cattle. Previous investigations related with the serological diagnosis of TB in cattle (Auer, 1987; Plackett et al., 1989; Wood et al., 1992) suggested that in the field the level of cross-reactivity of MPB70 antigen may be higher than originally thought. Also, the lack of detectable antibody responses in M. bovis infected cattle is in line with previous reports on both the humoral and cellular responses in bovine TB (Wood et al., 1992; Ritacco et al., 1990; Harboe et al., 1990; Plackett et al., 1989).
Acknowledgements Supported in part by a grant from CONACyT Mexico (93/8083).
(143M9107)
and FOMES
SEP in
References Auer, L.A., 1987. Assessment of an enzyme linked immunosorbent assay for the detection of cattle infected with Mycobacterium bouis, 1987. Aust. Vet. J., 64: 172-176. Centor, R.M., 1991. Signal detectability: the use of ROC curves and their analyses. Med. Decis. Making; II: 102-106. Hanley, J.A. and McNeil, B.J., 1982. The meaning and use of the arca under a receiver operating characteristic (ROC) curve. Radiology, 143: 29-36. Harboe, M., Wiker, H.G., Duncan, J.R., Garcia, M.M., Dukes, T.W., Brooks, B.W., Turcotte, C. and Nagai, S., 1990. Protein G-based enzyme-linked immunosorbent assay for anti-MPB70 antibodies in bovine tuberculosis. J. Clm. Microbial., 28: 913-921.
J. Herna’ndet De Anda et al. / Preuentiue Veterinary Medicine 27 (1996) 21 I -2/S
215
Hunter, S.W. and Brennan, P.J., 1990. Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobucterium tubercuhis. J. Biol. Chem., 265: 9272-9279. McNab, W.B., Meek, A.H., Duncan, J.R., Brooks, B.W., van Dreumel, A.A., Martin, SW., Nielsen, K.H., Sugden, E.A. and Turcotte, C., 1991. An evaluation of selected screening tests for bovine paratuberculosis. Can. I. Vet. Res., 55: 252-259. Nagai, S., Matsumoto, J. and Nagasuga, T., 1981. Specific skin-reactive protein from culture filtrate of Mycobocterium bovis BCG. Infect. Immun., 31: 1152- 1160. .~ Placket&P., Ripper, J., Comer, L.A., Small, K., de Witte K., Melville, L., Hides, S. and Wood, P.R., 1989. An ELISA for detection of anergic tuberculous cattle. Aust. Vet. J., 66: 15- 19. Radford, A.J., Wood, P.R., Billmat-Jacobe, H., Geysen, H.M., Mason, T.J. and Gordon, T., 1990. Epitope mappin,; of the Mycobacterium bouis secretory protein MPB70 using overlapping peptide analysis. J. Gen. lMicrobiol., 136: 265-272. Ritacco, V., Kantor, I.N., Barrera, L., Nader, A., Bemardelli, A., Torrea, G., Errico, F. and Fliess, E., 1987. Assessment of the sensitivity and specificity of enzyme-linked immunosorbent assay (ELISA) for the detection of mycobacterial antibodies in bovine tuberculosis. J. Vet. Med., 34: 119- 125. Ritacco, V., Lbpez, B., Barrera, L., Nader, A., FIiess, E. and Kantor, I.N., 1990. Further evaluation of an indirect enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis. J. Vet. Med., 37: 19-27. Ritacco, V., L6pez, B., Kantor, I.N., Barrera, L., Errico, F. and Nader, A., 1991. Reciprocal cellular and humoral immune responses in bovine tuberculosis. Res. Vet. Sci., 50: 365-367. Sada, E., Brennan, P.J., Herrera, T. and Torres, M., 1990. Evaluation of Iipoarabinomannan for the serological diagnosis of tuberculosis. J. Clin. Microbial., 28: 2587-2590. Wood, P.R , Comer, L.A., Rothel, J.S., Ripper, J.L., Fifis, T., McCormick, B.S., Francis, B., Melville, L., Small, K., De Witte, K., Tolson, J., Ryan, T.J., De Lisle, Cox, J.C. and Jones, S.L., 1992. A field evaluation of serological and cellular diagnostic tests for bovine tuberculosis. Vet. Microbial., 3 I: 71-79.