Veterinary Microbiology, 24 ( 1 9 9 0 ) 2 0 5 - 2 0 9 Elsevier Science Publishers B.V., A m s t e r d a m
205
Short Communication The development and evaluation of an enzymelinked immunosorbent assay for the detection of
Mycobacterium bovis B.J. Duffield Queensland Department of Primary Industries, Oonoonba Veterinary Laboratory, P.O. Box 1085, Townsville 4810, Queensland (Australia) (Accepted 25 January 1990)
ABSTRACT Duffield, B.J., 1990. The development and evaluation of an enzyme-linked immunosorbent assay for the detection of Mycobacterium boris. Vet. Microbiol., 24: 205-209. A double antibody sandwich layer enzyme-linked immunosorbent assay (ELISA) was used to detect Mycobacterium bovis. The ELISA detected M. bovis is pure culture at concentrations of 1 X 105 colony-forming units ( C F U ) m1-1 and greater, compared to a m i n i m u m detection level of 1 × 10 6 CFU m l - ~ for isolation techniques. Neither technique detected M. bovis at 1 x 104 CFU m l - 1. The ELISA did not cross-react with common mycobacterial contaminants such as Mycobacterium avium intracellulare-scrofulaceum complex serotypes 18 and 42, M. terrae, M. fortuitum, M. flavescens and with Escherichia coli or Rhodococcus equi. Further work is needed to evaluate this assay in detecting M. bovis in tissues and the environment.
INTRODUCTION
Alkaline solutions are routinely used to eliminate contaminating non-mycobacterial organisms from specimens submitted to the laboratory for isolation ofMycobacterium bovis (Vestal, 1981 ). However, these decontaminants may also destroy a proportion of M. bovis (Duffield, 1988) as they do M. tuberculosis (Krasnow and Wayne, 1966; Mitchison et al., 1972) and other mycobacteria (Hedgecock and Faucher, 1961 ) thus reducing sensitivity of isolation techniques. Modifications of the enzyme-linked immunosorbent assay (ELISA) (Engvall and Perlmann, 1971 ) have been used to detect viral antigens (Yolken and Torsch, 1982 ) and surface antigens of Staphylococcus sp. (Lentino and Rytel, 1982 ), Escherichia coli (Mills et al., 1982 ) and Streptococcus sp. (Elder et al., 1982). The work described here compared a modified double antibody sandwich 0378-1135/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
206
KJ. DUFF1ELD
layer ELISA (Voller et al., 1979) and isolation for sensitivity in detecting M. bovis, and examined specificity of the ELISA against other mycobacteria and common contaminants. MATERIALS AND METHODS
Test organisms M. boris, M. avium intracellulare-scrofulaceum (MAIS) complex serotypes 18 and 42, M. terrae, M. fortuitum, M. flavescens, E. coli and Rhodococcus equi were examined. These organisms were isolated from samples submitted to the Oonoonba Veterinary Laboratory for tuberculosis diagnosis (Duffield, unpublished data). The mycobacteria were isolated from bovine lymph nodes and identified as described by Rogers et al. (1980). E. coli and R. equi were isolated from bovine samples on sheep blood agar and identified as described by Cowan ( 1981 ). ELISA Disposable ABS C-BE41 beads (Sekisui Chemical Co. Ltd., Japan) were used as the solid phase support. The beads were coated with the first antibody diluted to 10 #g ml -~ in carbonate-bicarbonate buffer (pH 9.6) by shaking overnight at 4 ° C. The first antibody was a monospecific polyclonal antibody prepared from hyperimmune rabbit serum affinity purified to purified protein derivatives ofM. bovis (Radford and Duffield, 1988 ). For each test, one bead was placed in each well of a 20-well disposable plastic reaction tray (Abbott Laboratories, Chicago ). Subsequent reactions and washings were carried out in these trays. The coated beads were washed four times with 5 ml phosphate buffered saline containing 0.05% Tween 20 (PBST) using a Pentawash II Controller (Abbott Laboratories, Chicago). Two hundred/zl of each control or test organism, diluted in phosphate buffered saline containing 0.05% Tween 20 and 0.5% casein (PBSTC) were dispensed into appropriate wells of the reaction trays and incubated at 37°C for 3 h. The beads were washed with PBST then 200 #1 of second antibody, diluted to 5/tg ml-~ in phosphate buffered saline (PBS) were added. The second antibody was a murine monoclonal antibody (Product code 4C3/17) specific to M. bovis (Agen, Acacia Ridge, Australia). The beads were incubated at 37°C for 1 h, and then washed as above. Two hundred #1 of affinity purified goat anti-mouse immunoglobulin-G (light and heavy chain) conjugated to horseradish peroxidase (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD) diluted 1 : 800 in PBS were added to each well. Trays were incubated for 1 h at 37°C. After a final wash, the beads were transferred to disposable 5 ml assay tubes (Abbott Laboratories, Chicago).
AN ELISA FOR THE DETECTION OF M. BOV1S
207
Three hundred #1 of freshly prepared o-phenylenediamine-2 HC1 ( O P D ) substrate (Voller et al., 1979) were added to each tube and to two empty tubes (substrate blanks). The tubes were incubated in the dark at 25 °C for 30 min. One ml of sulphuric acid (1N) was added to each tube to stop the reaction. The optical density (OD) was read at 492 n m using a Q u a n t u m II Analyser (Abbott Laboratories, Chicago) set in mode 0. For reading of the test, a cut-off point was established from OD readings obtained with negative controls (uninoculated sterile saline). Specimens with OD readings equal to or greater than the cut-off point were considered positive for M. boris antigen.
Experimental procedure One ml of concentrations at 1 × 10 4, 1 × 105, 1 × 10 6, 1 × l0 7 and 1 × 108 colony forming units ( C F U ) ml-1 of each of the eight organisms suspended in sterile saline, and negative controls was centrifuged at 3000 g for 20 min at 4°C. Following the m e t h o d of Postgate (1969), plate counts were done to determine the number of CFU. The pellets were suspended in 200/A of PBSTC then added to coated beads in the reaction trays. The ELISA was carried out as described above. Duplicate centrifuged pellets were cultured after decontamination with 2% sodium TABLE 1 E n z y m e - l i n k e d i m m u n o s o r b e n t assay a n d isolation results for different c o n c e n t r a t i o n s o f Mycobacterium boris a n d s e v e n o t h e r o r g a n i s m s in pure culture Organisms
Number of organisms 0
1X 10 4
1 × 10 5
1X 10 6
1X 10 7
1 × l0 s
Mycobacterium boris
0.052 _3
0.06 _
0.15 _
0.20 +
0.40 +
1.0 +
M A I S 181
0.04
0.05
0.04
0.04
0.07
0.06
_
_
+
+
+
+
MAIS 42
0.05
0.04
0.04
0.04
0.07
0.07
_
_
+
+
+
+
Mycobacterium terrae
0.04
0.06
0.04
0.05
0.06
0.08
_
_
+
+
+
+
Mycobacteriumfortuitum
0.06
0.05
0.05
0.05
0.06
0.08
_
_
+
+
+
+
Mycobacteriumflavescens
0.05
0.05
0.05
0.04
0.05
0.06
--
+
+
+
+
+
Escherichia coli
0.05
0.04
0.06
0.08
0.08
0.07
-
+
+
+
+
+
Rhodococcus equi
0.06 .
0.05 .
0.05
0.07
0.08 +
0.08 +
.
.
~MAIS = M. avium intracellulare-scrofulaceum c o m p l e x serotype. 2Average o f triplicate e n z y m e - l i n k e d i m m u n o s o r b e n t assay optical density readings at 492 n m . 3Culture results: + = o r g a n i s m s isolated; - = no o r g a n i s m s isolated.
208
B.J.DUFFIELD
hydroxide for 20 min (Rogers et al., 1980). The ELISA and isolation technique were performed in triplicate for each sample. RESULTS The OD values for the negative controls ranged from 0.04 to 0.06 with a mean of 0.05. The cut-off point for a positive reaction was chosen as twice the mean OD value of the negative controls. Thus a test result was considered positive if the OD value was equal to or greater than 0.10. Table 1 shows the ELISA and isolation results for the eight test organisms. The ELISA detected M. bovis as concentrations of 1 X 105 CFU m l - ~ to 1 X 108 CFU m l - 1. The ELISA did not detect the five other mycobacteria, E. coli or R. equi at any concentration. M. bovis was isolated at 1 X 106 to 1 X 108 CFU m l - 1. The other mycobacteria were isolated at lower concentrations. E. coli was isolated at all five cell concentrations examined and R. equi at the two higher concentrations only. DISCUSSION
M. bovis in pure culture was detected by ELISA at a minimum concentration of 1 X 105 CFU m l - 1 but not isolated at this concentration. As only 14% ofM. bovis survive exposure to 2% sodium hydroxide for 20 min (Duffield, 1988 ), the failure to isolate at the lower concentration was probably due to the decontamination procedure. The ELISA was specific for M. bovis within the selection of other organisms examined. Slight nonspecific reactivity at high concentrations of other organisms may have been due to residual attachment ofnonspecific protein. However, the values were clearly less than the cut-off point for the test in all cases. The ELISA has distinct advantages compared to isolation techniques in that it is not affected by contamination and results are obtained within 48 h compared to 8 weeks for isolation. Further work will ascertain the value of the ELISA for detecting M. bovis in tissues and measuring its survival in the environment. However, further development of DNA-probes specific for M. bovis may prove more sensitive than ELISA technology for detecting M. bovis in tissues (Radford, personal communication ). ACKNOWLEDGEMENTS Funds for this work were provided by the Australian National Tuberculosis and Brucellosis Eradication Scheme.
AN ELISA FOR THE DETECTION OF M. BOVIS
209
REFERENCES Cowan, S.T., 1981. Cowan and Steel's manual for the identification of medical bacteria. Cambridge University Press, London, pp. 45-122. Duffield, B.J., 1988. Immunological and microbiological studies ofMycobacterium bovis. Ph.D. thesis. James Cook University of North Queensland. Elder, B.L., Boraker, D.R. and Fivestayler, P.M., 1982. Whole-bacterial cell ELISA for Streptococcussanguis fimbrial antigens. J. Clin. Microbiol., 16: 141-144. Engvall, E. and Perlmann, P., 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobin G. Immunochemistry, 8:871-874. Hedgecock, L.W. and Faucher, I.O., 1961. Isolation of increased numbers of unclassified mycobacteria following decontamination of sputum with malachite green and crystal violet. Am. Rev. Resp. Dis., 84: 710-714. Krasnow, I. and Wayne, L.G., 1966. Sputum digestion. I. The mortality rate of tubercle bacilli in various digestion systems. Am. J. Clin. Pathol., 45: 352-355. Lentino, J.R. and Rytel, M.J., 1982. Detection of circulating free and complexed staphylococcal antigens by enzyme-linked immunosorbent assays. J. Clin. Microbiol., 16: 1019-1024. Mills, K.W., Phillips. R.M., Kelly, B.L. and Baughman, D.L., 1982. Using enzyme-linked immunosorbent assay to detect Escherichia coli K88 pili antigens from clinical isolates. Am. J. Vet. Res., 43: 365-367. Mitchison, D.A., Allen, B.W., Cassal, L., Dickinson, J.M. and Aber, V.R., 1972. A selective oleic acid albumin agar medium for tubercle bacilli. J. Med. Microbiol., 5:165-175. Postgate, J.R., 1969. Viable counts and viability. In: J.T. Norris and D.W. Ribbons (Editors), Methods in Microbiology Vol. 1. Academic Press, New York, pp. 611-628. Radford, A.J. and Duffield, B.J., 1988. Cloning of species specific antigen of Mycobacterium bovis. Infect. Immun., 56(4): 921-925. Rogers, R.J., Donald, B.A. and Schultz, K., 1980. The distribution of Mycobacteriurn bovis in Queensland cattle herds with observations on the laboratory diagnosis of tuberculosis. Aust. Vet. J., 56: 542-546. Vestal, A.L., 1981. Procedures for the isolation and identification of mycobacteria. Centre for Disease Control. U.S. Dept. of Health and Human Services, Public Health Service, Atlanta, Georgia. Voller, A., Bidwell, D. and Bartlett, A., 1979. The enzyme-linked immunosorbent assay (ELISA). Dynatech, Europe, Guernsey. Yolken, R.H. and Torsch, V.M., 1982. Enzyme-linked immunosorbent assay for detection and identification of coxsackie-viruses A. Infect. Immun., 31: 742-750.
205
Short Communication The development and evaluation of an enzymelinked immunosorbent assay for the detection of
Mycobacterium bovis B.J. Duffield Queensland Department of Primary Industries, Oonoonba Veterinary Laboratory, P.O. Box 1085, Townsville 4810, Queensland (Australia) (Accepted 25 January 1990)
ABSTRACT Duffield, B.J., 1990. The development and evaluation of an enzyme-linked immunosorbent assay for the detection of Mycobacterium boris. Vet. Microbiol., 24: 205-209. A double antibody sandwich layer enzyme-linked immunosorbent assay (ELISA) was used to detect Mycobacterium bovis. The ELISA detected M. bovis is pure culture at concentrations of 1 X 105 colony-forming units ( C F U ) m1-1 and greater, compared to a m i n i m u m detection level of 1 × 10 6 CFU m l - ~ for isolation techniques. Neither technique detected M. bovis at 1 x 104 CFU m l - 1. The ELISA did not cross-react with common mycobacterial contaminants such as Mycobacterium avium intracellulare-scrofulaceum complex serotypes 18 and 42, M. terrae, M. fortuitum, M. flavescens and with Escherichia coli or Rhodococcus equi. Further work is needed to evaluate this assay in detecting M. bovis in tissues and the environment.
INTRODUCTION
Alkaline solutions are routinely used to eliminate contaminating non-mycobacterial organisms from specimens submitted to the laboratory for isolation ofMycobacterium bovis (Vestal, 1981 ). However, these decontaminants may also destroy a proportion of M. bovis (Duffield, 1988) as they do M. tuberculosis (Krasnow and Wayne, 1966; Mitchison et al., 1972) and other mycobacteria (Hedgecock and Faucher, 1961 ) thus reducing sensitivity of isolation techniques. Modifications of the enzyme-linked immunosorbent assay (ELISA) (Engvall and Perlmann, 1971 ) have been used to detect viral antigens (Yolken and Torsch, 1982 ) and surface antigens of Staphylococcus sp. (Lentino and Rytel, 1982 ), Escherichia coli (Mills et al., 1982 ) and Streptococcus sp. (Elder et al., 1982). The work described here compared a modified double antibody sandwich 0378-1135/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
206
KJ. DUFF1ELD
layer ELISA (Voller et al., 1979) and isolation for sensitivity in detecting M. bovis, and examined specificity of the ELISA against other mycobacteria and common contaminants. MATERIALS AND METHODS
Test organisms M. boris, M. avium intracellulare-scrofulaceum (MAIS) complex serotypes 18 and 42, M. terrae, M. fortuitum, M. flavescens, E. coli and Rhodococcus equi were examined. These organisms were isolated from samples submitted to the Oonoonba Veterinary Laboratory for tuberculosis diagnosis (Duffield, unpublished data). The mycobacteria were isolated from bovine lymph nodes and identified as described by Rogers et al. (1980). E. coli and R. equi were isolated from bovine samples on sheep blood agar and identified as described by Cowan ( 1981 ). ELISA Disposable ABS C-BE41 beads (Sekisui Chemical Co. Ltd., Japan) were used as the solid phase support. The beads were coated with the first antibody diluted to 10 #g ml -~ in carbonate-bicarbonate buffer (pH 9.6) by shaking overnight at 4 ° C. The first antibody was a monospecific polyclonal antibody prepared from hyperimmune rabbit serum affinity purified to purified protein derivatives ofM. bovis (Radford and Duffield, 1988 ). For each test, one bead was placed in each well of a 20-well disposable plastic reaction tray (Abbott Laboratories, Chicago ). Subsequent reactions and washings were carried out in these trays. The coated beads were washed four times with 5 ml phosphate buffered saline containing 0.05% Tween 20 (PBST) using a Pentawash II Controller (Abbott Laboratories, Chicago). Two hundred/zl of each control or test organism, diluted in phosphate buffered saline containing 0.05% Tween 20 and 0.5% casein (PBSTC) were dispensed into appropriate wells of the reaction trays and incubated at 37°C for 3 h. The beads were washed with PBST then 200 #1 of second antibody, diluted to 5/tg ml-~ in phosphate buffered saline (PBS) were added. The second antibody was a murine monoclonal antibody (Product code 4C3/17) specific to M. bovis (Agen, Acacia Ridge, Australia). The beads were incubated at 37°C for 1 h, and then washed as above. Two hundred #1 of affinity purified goat anti-mouse immunoglobulin-G (light and heavy chain) conjugated to horseradish peroxidase (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD) diluted 1 : 800 in PBS were added to each well. Trays were incubated for 1 h at 37°C. After a final wash, the beads were transferred to disposable 5 ml assay tubes (Abbott Laboratories, Chicago).
AN ELISA FOR THE DETECTION OF M. BOV1S
207
Three hundred #1 of freshly prepared o-phenylenediamine-2 HC1 ( O P D ) substrate (Voller et al., 1979) were added to each tube and to two empty tubes (substrate blanks). The tubes were incubated in the dark at 25 °C for 30 min. One ml of sulphuric acid (1N) was added to each tube to stop the reaction. The optical density (OD) was read at 492 n m using a Q u a n t u m II Analyser (Abbott Laboratories, Chicago) set in mode 0. For reading of the test, a cut-off point was established from OD readings obtained with negative controls (uninoculated sterile saline). Specimens with OD readings equal to or greater than the cut-off point were considered positive for M. boris antigen.
Experimental procedure One ml of concentrations at 1 × 10 4, 1 × 105, 1 × 10 6, 1 × l0 7 and 1 × 108 colony forming units ( C F U ) ml-1 of each of the eight organisms suspended in sterile saline, and negative controls was centrifuged at 3000 g for 20 min at 4°C. Following the m e t h o d of Postgate (1969), plate counts were done to determine the number of CFU. The pellets were suspended in 200/A of PBSTC then added to coated beads in the reaction trays. The ELISA was carried out as described above. Duplicate centrifuged pellets were cultured after decontamination with 2% sodium TABLE 1 E n z y m e - l i n k e d i m m u n o s o r b e n t assay a n d isolation results for different c o n c e n t r a t i o n s o f Mycobacterium boris a n d s e v e n o t h e r o r g a n i s m s in pure culture Organisms
Number of organisms 0
1X 10 4
1 × 10 5
1X 10 6
1X 10 7
1 × l0 s
Mycobacterium boris
0.052 _3
0.06 _
0.15 _
0.20 +
0.40 +
1.0 +
M A I S 181
0.04
0.05
0.04
0.04
0.07
0.06
_
_
+
+
+
+
MAIS 42
0.05
0.04
0.04
0.04
0.07
0.07
_
_
+
+
+
+
Mycobacterium terrae
0.04
0.06
0.04
0.05
0.06
0.08
_
_
+
+
+
+
Mycobacteriumfortuitum
0.06
0.05
0.05
0.05
0.06
0.08
_
_
+
+
+
+
Mycobacteriumflavescens
0.05
0.05
0.05
0.04
0.05
0.06
--
+
+
+
+
+
Escherichia coli
0.05
0.04
0.06
0.08
0.08
0.07
-
+
+
+
+
+
Rhodococcus equi
0.06 .
0.05 .
0.05
0.07
0.08 +
0.08 +
.
.
~MAIS = M. avium intracellulare-scrofulaceum c o m p l e x serotype. 2Average o f triplicate e n z y m e - l i n k e d i m m u n o s o r b e n t assay optical density readings at 492 n m . 3Culture results: + = o r g a n i s m s isolated; - = no o r g a n i s m s isolated.
208
B.J.DUFFIELD
hydroxide for 20 min (Rogers et al., 1980). The ELISA and isolation technique were performed in triplicate for each sample. RESULTS The OD values for the negative controls ranged from 0.04 to 0.06 with a mean of 0.05. The cut-off point for a positive reaction was chosen as twice the mean OD value of the negative controls. Thus a test result was considered positive if the OD value was equal to or greater than 0.10. Table 1 shows the ELISA and isolation results for the eight test organisms. The ELISA detected M. bovis as concentrations of 1 X 105 CFU m l - ~ to 1 X 108 CFU m l - 1. The ELISA did not detect the five other mycobacteria, E. coli or R. equi at any concentration. M. bovis was isolated at 1 X 106 to 1 X 108 CFU m l - 1. The other mycobacteria were isolated at lower concentrations. E. coli was isolated at all five cell concentrations examined and R. equi at the two higher concentrations only. DISCUSSION
M. bovis in pure culture was detected by ELISA at a minimum concentration of 1 X 105 CFU m l - 1 but not isolated at this concentration. As only 14% ofM. bovis survive exposure to 2% sodium hydroxide for 20 min (Duffield, 1988 ), the failure to isolate at the lower concentration was probably due to the decontamination procedure. The ELISA was specific for M. bovis within the selection of other organisms examined. Slight nonspecific reactivity at high concentrations of other organisms may have been due to residual attachment ofnonspecific protein. However, the values were clearly less than the cut-off point for the test in all cases. The ELISA has distinct advantages compared to isolation techniques in that it is not affected by contamination and results are obtained within 48 h compared to 8 weeks for isolation. Further work will ascertain the value of the ELISA for detecting M. bovis in tissues and measuring its survival in the environment. However, further development of DNA-probes specific for M. bovis may prove more sensitive than ELISA technology for detecting M. bovis in tissues (Radford, personal communication ). ACKNOWLEDGEMENTS Funds for this work were provided by the Australian National Tuberculosis and Brucellosis Eradication Scheme.
AN ELISA FOR THE DETECTION OF M. BOVIS
209
REFERENCES Cowan, S.T., 1981. Cowan and Steel's manual for the identification of medical bacteria. Cambridge University Press, London, pp. 45-122. Duffield, B.J., 1988. Immunological and microbiological studies ofMycobacterium bovis. Ph.D. thesis. James Cook University of North Queensland. Elder, B.L., Boraker, D.R. and Fivestayler, P.M., 1982. Whole-bacterial cell ELISA for Streptococcussanguis fimbrial antigens. J. Clin. Microbiol., 16: 141-144. Engvall, E. and Perlmann, P., 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobin G. Immunochemistry, 8:871-874. Hedgecock, L.W. and Faucher, I.O., 1961. Isolation of increased numbers of unclassified mycobacteria following decontamination of sputum with malachite green and crystal violet. Am. Rev. Resp. Dis., 84: 710-714. Krasnow, I. and Wayne, L.G., 1966. Sputum digestion. I. The mortality rate of tubercle bacilli in various digestion systems. Am. J. Clin. Pathol., 45: 352-355. Lentino, J.R. and Rytel, M.J., 1982. Detection of circulating free and complexed staphylococcal antigens by enzyme-linked immunosorbent assays. J. Clin. Microbiol., 16: 1019-1024. Mills, K.W., Phillips. R.M., Kelly, B.L. and Baughman, D.L., 1982. Using enzyme-linked immunosorbent assay to detect Escherichia coli K88 pili antigens from clinical isolates. Am. J. Vet. Res., 43: 365-367. Mitchison, D.A., Allen, B.W., Cassal, L., Dickinson, J.M. and Aber, V.R., 1972. A selective oleic acid albumin agar medium for tubercle bacilli. J. Med. Microbiol., 5:165-175. Postgate, J.R., 1969. Viable counts and viability. In: J.T. Norris and D.W. Ribbons (Editors), Methods in Microbiology Vol. 1. Academic Press, New York, pp. 611-628. Radford, A.J. and Duffield, B.J., 1988. Cloning of species specific antigen of Mycobacterium bovis. Infect. Immun., 56(4): 921-925. Rogers, R.J., Donald, B.A. and Schultz, K., 1980. The distribution of Mycobacteriurn bovis in Queensland cattle herds with observations on the laboratory diagnosis of tuberculosis. Aust. Vet. J., 56: 542-546. Vestal, A.L., 1981. Procedures for the isolation and identification of mycobacteria. Centre for Disease Control. U.S. Dept. of Health and Human Services, Public Health Service, Atlanta, Georgia. Voller, A., Bidwell, D. and Bartlett, A., 1979. The enzyme-linked immunosorbent assay (ELISA). Dynatech, Europe, Guernsey. Yolken, R.H. and Torsch, V.M., 1982. Enzyme-linked immunosorbent assay for detection and identification of coxsackie-viruses A. Infect. Immun., 31: 742-750.
