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Immunological cross-reactivity of pathogenic Nocardia and related organisms
0147 9571/79/0501-0069502.00/0
Comp. lmmun. Microbiol. infect. Dis., Vol. 2, pp. 69-74. © Pergamon Press Ltd., 1979. Printed in Great Britain
IMMUNOLOGICAL CROSS-REACTIVITY OF PATHOGENIC NOCARDIA A N D RELATED ORGANISMS VISWANATH P. KURUP Department of Medicine, The Medical College of Wisconsin, Milwaukee and Research Service, Veterans Administration Center, Wood (Milwaukee), Wl 53193, U.S.A. Abstract--Aerobic actinomycetes associated with various clinical conditions in man and animals were studied for their antigenic relationships along with related organisms. Culture filtrate antigens obtained from 16 species were tested by antigen-antibody crossed-immunoelectrophoresis and by agar gel double diffusion against rabbit antibodies to Nocardia asteroides, N. brasiliensis and N. caviae. Weak cross-reactivity was detected among the three, Nocardia species and Micropolyspora brevicatena, but no cross-reactivity was noted with antigens from any of the other species studied.
Key words: Immunological cross-reactivity, Nocardia, Micropolyspora, crossed-immunoelectrophoresis, agar gel diffusion R E A C T I O N I M M U N O L O G I Q U E C R O I S E E E N T R E LES N O C A R D I A P A T H O G E N E S E T LES O R G A N I S M E S A P P A R E N T E S Resume----Des actinomyc&es atrobies associts dans difftrents syndromes cliniques chez l'homme et chez l'animal ont 6t6 ~tudits en ce qui concerne leurs relations antigtniques avec les microorganismes indiquts. Les antigenes obtenus par filtrat de culture /t partir de 16 esptces sont testts par immuno~lectrophortse croiste et par double diffusion en gtlose contre des anticorps de lapin antiNocardia asteroides, N. brasiliensis et N. caviae. Une faible rtaction croiste a 6t6 raise en 6vidence chez les trois esptces de Nocardia et Micropolyspora brevicatena, mais nous n'avons pas remarqu6 de rtaction croiste avec les antig~nes d'aucune des autres esptces observtes.
Mots-clefs: Reaction immunologique croiste, Nocardia, Micropolyspora, immunotlectrophortse croisee, diffusion en g~lose
INTRODUCTION Several members o f the aerobic actinomycetes are associated with h u m a n and animal diseases [ 1 - 5 ] . The frequently recognized species in h u m a n diseases include Nocardia
asteroides, N. brasiliensis, N. caviae, Actinomadura madurae, A. pelletieri, Nocardiopsis dassonvillei, Micropolyspora faeni, Saccharomonospora viridis, Thermoactinomyces candidus, T. sacchari and T. vulgaris. O f the 11 species, the first 6 cause invasive or localized infections, while the remainder are associated with hypersensitivity pneumonitis (HP) [1, 2, 4]. Although serological techniques are available in the diagnosis of nocardiosis and actinomycetic m y c e t o m a s , they are not widely used [ 6 - 8 ] . O n the contrary, demonstration o f circulating antibodies in the sera of patients is a valuable criterion for the diagnosis o f H P [5]. In recent years, serological tests using antigen extracts of Nocardia species have been evaluated in detecting specific antibodies in the sera o f patients. Some o f these antigen preparations were found to be specific while others showed cross-reactivity with mycobacterial antigens [7, 8]. M o s t o f the actinomycetes species mentioned a b o v e were isolated from soil or other natural environments. These actinomycetes species are taxonomically-related, but little is known regarding the antigenic relationship o f these 69
70
VISWANATH P. KURUP
organisms. Hence, knowledge of the antigenic cross-reactivity among the frequently occurring aerobic actinomycetes associated with h u m a n diseases m a y be beneficial in evaluating the significance and specificity o f circulating antibodies in the sera o f patients. The present investigation was therefore designed to study the antigenic cross-reactivity among various pathogenic aerobic actinomycetes and related organisms by means o f an a n t i g e n - a n tibody crossed-immunoelectrophoresis technique.
MATERIALS A N D METHODS Aetinomycetes strains The various strains o f actinomycetes used in the study are listed in Table 1. The identity of the Nocardia, Actinomadura and Nocardiopsis species was determined b y the method o f G o r d o n and H o r a n [9] and those o f Micropolyspora, Saccharomonospora and Thermoactinomyces by the method o f K u r u p and Fink [10]. The identity o f all other isolates was ascertained by comparing their characteristics with the original descriptions. All cultures were grown in appropriate media and one set of each was maintained at 4 ° C and at r o o m temperature. They were subcultured every 3 months. Preparation o f antigens F o r the extraction o f antigens, organisms were grown in the dialysates o f appropriate complex media. The method used was a modification o f that described by Ajello et al. [ 11 ] and is briefly as follows: dialysis bags (Arthur T h o m a s Co., 3787-F47, mol. wt cut off 8000)
Table 1. Organisms used in the extraction of antigens Accession number N-801 N-806 N-805 N-816 N-821 N-819 T-150 T-250 T-253 T-249 T-248 T-182 T-283 T-106 T-145 T-101
Source REG*- IMRU-859 REG - IMRU-731 REG - IMRU-1315 REG - IMRU-816 REG - IMRU-513 REG - IMRU-780 ATCC:~- 1 5 3 4 7 HAL§ - 1325 HAL - N-3805 HAL - 1086 HAL - 19125 HAL - 4047 ATCC - 1 9 2 8 5 ATCC - 2 7 8 6 8 ATCC - 27349 Marshfield Clinicl[
Identification Nocardia asteroides N. brasiliensis N. caviae Noeardiopsis dassonvillei Actinomadurapelletieri A. madurae Micropolysporafaeni M. rectivirgula M. viridinigra M. brevicatena M. caesia Saccharomonospora viridis Pseudonocardia thermophilia Thermoactinomyces candidus 7".sacchari T. vulgaris
* REG IMRU, Dr. Ruth E. Gordon, Institute of Microbiology, Rutgers, The State University, New Brunswick, N.J. $ ATCC, American Type Culture Collections, Rockville, Md. § HAL, Dr. H. A. Lechevalier, Institute of Microbiology, Rutgers, The State University, New Brunswick, N.J. [j Marshfield Clinic, Marshfield, Wis.
Immunologicalcross-reactivityof pathogenicNocardia and related organisms were filled with appropriate medium and placed in flasks containing 0. I M glycine in 0.075 M sodium chloride. The dialysis bags with the medium were barely submerged in the outer phases. The flasks with the contents were then sterilized by autoclaving at 121 °C for 15 min. The system was then allowed to equilibrate at room temperature for 48 hr. The outer phase of the medium was separated and asceptically transferred into sterile flasks, and was used to grow the various actinomycetes. The thermophilic actinomycetes (T-150, T-250, T-253, T248, T-283, T-106 and T-101), with the exception of T. sacchari (T-145), were grown in the dialysate fluid from tryptic soy broth (TSB), while the latter was grown in the dialysate of nutrient broth. The remaining isolates were grown in the dialysate from TSB, supplemented with 1% glucose. The thermophilic actinomycetes M. faeni, M. caesia, M. viridinigra, S. viridis, T. vulgaris, T. candidus, T. sacchari and Pseudonocardia thermophilia were grown in stationary cultures at 55°C for 2 weeks, while the Nocardia species, Nocardiopsis species and Micropolyspora brevicatena were grown at 37°C for 2-3 weeks. Both Actinomadura madurae and A. pelletieri were grown for 4 weeks at 28°C before harvesting. At the end of the incubation period the growth was killed by adding phenol to the final concentration of 1% (v/v). The sterility of the culture was determined after 2 4 - 4 8 hr, and the broth was separated from the mycelium by filtration. The filtrate was extensively dialyzed against distilled water for 2 - 3 days at 4°C, and the retentate was passed through a 450 nm membrane filter and freeze-dried. These antigens were used to raise antisera in rabbits as well as for the antigen-antibody crossed-immunoelectrophoresis.
Immunization of rabbits White rabbits, weighing 3 - 4 kg, were injected subcutaneously with N. asteroides, N. brasiliensis and N. caviae antigens. Ten milligrams of antigens in 1 ml of sterile physiological saline were mixed with an equal amount of incomplete Freund's adjuvant. 6 - 8 injections were given at weekly intervals. The rabbits were checked at intervals for antibody production by the agar gel double diffusion method [12] and bled when strong precipitating antibodies were detected. Homologous antisera were pooled and used in the crossed-immunoelectrophoresis studies.
Agar gel double diffusion The 3 antisera were studied for precipitating antibodies using various antigen preparations by the agar gel diffusion method, as previously described [12]. The antigens were used at concentrations of 30 mg/ml for the immunodiffusion studies.
Crossed-immunoelectrophoresis The cross-reactivity of the various actinomycetes was studied by two-dimensional immunoelectrophoresis using antigens from different species of actinomycetes and antibodies of the 3 Nocardia species, as previously described [13, 14]. Approximately 5 - 7 ~1 of antigen (100-125 ~tg protein) were electrophoresed in one direction for 3 hr at 8-10 v/cm. Following electrophoresis, the gel from the plate was removed leaving a 1.5-cm wide antigen band that was replaced with gel containing 10% v/v rabbit antinocardial serum. The gel plates were subjected to electrophoresis at right angles to the first direction at 8 v/cm for 3 hr, and the
72
VISWANATHP. KURUP
plates were stained with Coomassie brilliant blue [ 15]. The number, intensity and peak height of each precipitin arc formed with the various antigen-antibody reactions were studied and compared. RESULTS The agar gel diffusion studies demonstrated that the 3 anfi-Nocardia sera reacted against homologous antigens and produced multiple precipitin arcs. Nocardia asteroides and N. caviae antigens gave strong precipitin arcs with homologous antisera, while N. brasiliensis antigens showed weaker reactions with homologous antiserum. Weak cross-reactivity was invariably detected when nocardial antigens were reacted with the 3 antisera and vice versa. Antigens from M. brevicatena showed weak cross-reactivity with the 3 noeardial antibodies. No other antigens cross-reacted with the nocardial antibodies.
Fig. 1. Crossed-immunoelectrophoresis ofN. asteroides antigens against homologous rabbit antibody. Fig. 2. Crossed-immunoelectrophoresis ofN. caviae antigens against homologous rabbit antibody. Fig. 3. Crossed-immunoelectrophoresis ofN. brasiliensis antigen against homologous rabbit antibody. Fig. 4. Crossed-immunoelectrophoresis of M. brevicatena antigens against anti-N, asteroides rabbit serum (anode at the right and top).
Immunologicalcross-reactivityof pathogenic Nocardia and related organisms
73
Antigens from 3 species of Nocardia: N. asteroides, N. caviae and N. brasiliensis, reacted with the homologous antibodies and demonstrated several precipitin arcs (Figs. 1-3) each by antigen-antibody crossed-immunoelectrophoresis technique. N. asteroides antigens revealed 16 precipitin arcs with the specific antiserum, while it gave only one precipitin arc each with anti-N, caviae and N. brasiliensis rabbit sera. N. caviae demonstrated 7 precipitin arcs against homologous antiserum, 3 - 6 arcs against anti-N, asteroides and 4 against anti-N. brasiliensis sera. N. brasiliensis antigens produced 7 precipitin arcs with homologous antiserum but only one with anti-N, caviae and 4 with anti-N, asteroides sera. None of the antigens from other species, except M. brevicatena, reacted with any of the 3 antisera. M. brevicatena antigens reacted with N. asteroides (Fig. 4), N. brasiliensis and N. caviae antisera and demonstrated 3, 3 and 1 precipitin arcs, respectively.
DISCUSSION The results indicate that N. asteroides, N. brasiliensis and N. caviae share some common antigens. Although several of the actinomycetes species included in the study are taxonomically related to Nocardia, none showed any antigenic cross-reactivity with Nocardia except the species M. brevicatena. M. brevicatena grew well at 37°C and produced colonies morphologically indistinguishable from those of the Nocardia species [ 16]. It is noteworthy that all the known isolates of M. brevicatena have been recovered from clinical materials. Interestingly, no common antigenic determinants were detected in M. brevicatena and the other species of Micropolyspora (unpublished findings). Hence, additional studies are necessary to clarify the pathogenic role and systematic position ofM. brevicatena. Species belonging to Acinornadura and Nocardiopsis were previously included in the genus Nocardia. However, based on differences in cell wall composition, these 2 genera were separated from Nocardia [17, 18]. The present study of antigenic cross-reactivity also supports this conclusion. However, additional studies are needed to determine the relationships between the Actinornadura and Nocardiopsis; the latter genera was described recently to include A. dassonvillei. Antibodies against M. faeni have been detected in the sera of patients with farmer's lung, as well as in a small percentage of apparently normal farmers. None of the nocardial antigens cross-reacted with the sera from 5 patients with farmer's lung or with 2 sera from asymptomatic farmers in whom M. faeni precipitins could be detected. This finding agrees with the antigenic cross-reactivity results that none of the thermophilic actinomycetes crossreacted with Nocardia. M. brevicatena antigens also failed to react with farmer's lung sera, but reacted with all 3 nocardial antibodies. This indicates a stronger antigenic relationship between M. brevicatena and the Nocardia species than M. brevicatena and M.faeni. Among the pathogenic actinomycetes only the antigens from the 3 Nocardia species showed crossreactivity. Acknowledgements - The technical assistance of Mary J. Falk and the editorial assistance of Mavis E. Baurle and Catherine A. Walther are gratefully acknowledged. This investigation was supported by Specialized Center of Research (SCOR) Grant HL-15389 from the National Heart, Lung and Blood Institute. Also supported by the Medical Research Service of the Veterans Administration.
74
VISWANATHP. KURUP REFERENCES
1. Emmons, C. W., Binford, C. H. and Utz J. P., Med. mycology 389-418. Lea & Febiger, Philadelphia. 2. Gordon, M. A., Aerobic pathogenic actinomycetaceae. In Manual o f Clinical Microbiology, Lennette, E. H., Spaulding, E. H. and Truant, J. P. (Eds), pp. 175-188. American Society for Microbiology, Washington, D.C. (1974). 3. Kurup, V. P., Randhawa, H. S. and Gupta, N. P., Nocardiosis: A review, Mycopath. Mycol. AppL 40, 193-219 (1970). 4. Murray, J. F., Finegold, S. M., Froman, S. and Will, D. W., The changing spectrum of nocardiosis, Am. Rev. Resp. Dis. 83, 315-330 (1961). 5. Pepys, J., Jenkins, P. A., Festenstein, G. N., Gregory, P. H., Lacey, M. E. and Skinner, F. A., Farmer's lung. Thermophilic actinomycetes as a source of farmer's lung hay antigens, Lancet ii, 607-611 (1963). 6. Gordon, M. A. and Mahgouh, [] S., Immune responses to aerobic pathogenic actinomycetaceae. In Manual of Clinical Immunology, Rose, N. R. and Friedman, H. (Eds)pp. 338-340. American Society for Microbiology, Washington, D.C. (1976). 7. Pier, A. C., Thurston, J. R. and Larsan, A. B., A diagnostic antigen for nocardiosis: comparative tests in cattle with nocardiosis and mycobacteriosis, Am. J. vet. Res. 29, 397-403 (1968). 8. Thurston, J. R., Phillips, M. and Pier, A. C., Extra cellular antigens ofNocardia asteroides. IlL Immunologic relationships demonstrated by erythrocyte sensitizing antigens, Am. Rev. Resp. Dis. 97, 240-247 (1968). 9. Gordon, R. E. and Horan, A. C., Nocardia dassonvillei, a macroscopic replica of Streptomyces griseus, J. gen. MicrobioL 50, 235-240 (1968). 10. Kurup, V. P. and Fink, J. N., A scheme for the identification of thermophilic actinomycetes associated with hypersensitivity pneumonitis, J. Clin. Microbiol. 2, 55-61 (1975). 11. Ajello, L., Walls, K., Moore, J. C. and Falcone, R., Rapid production of complement fixation antigens for systemic mycotic diseases. I. Coccidioidin: influence of media and mechanical agitation on its development, J." Bact. 77, 753-756 (1959). 12. Fink, J, N., Diseases of the lung. In Manual of Clinical Immunology, Rose, N. R. and Friedman, H. (Eds) pp. 616-619. American Society for Microbiology, Washington, D.C. (1976). 13. Kurup, V. P. and Fink, J. N., Extracellular antigens of Micropolysporafaeni grown in synthetic medium, Infect. Immun. 15, 608-613 (1977). 14. Kurup, V. P., Fink, J. N., Scribner, G. and Falk, M., Variability ofAspergillusfumigatus, Microbios. 19, 191-204 (1978)." 15. Axelsen, N. H. and Bock, E., Identification of antigens and antibodies by means of quantitative immunoelectrophoresis. A survey of methods, J. immun. Methods 1, 109-121 (1972). 16. Lechevalier, H. A., Solotorvsky, M. and McDurmont, C. I., a new genus of actinomycetales: Micropolyspora gen. nov., J. gen. Microbiol. 26, 11-18 (1961). 17. Lechevalier, H. A., Lechevalier, M. P. and Becker, B., Comparison of the chemical composition of cell walls of Nocardiae with that of other aerobic actinomycetes, Int. J. system. Bact. 16, 151-166 (1966). 18. Meyer, J., A new genus of the order of actinomycetales, Int. J. system. Bact. 26, 487-493 (1976).
Comp. lmmun. Microbiol. infect. Dis., Vol. 2, pp. 69-74. © Pergamon Press Ltd., 1979. Printed in Great Britain
IMMUNOLOGICAL CROSS-REACTIVITY OF PATHOGENIC NOCARDIA A N D RELATED ORGANISMS VISWANATH P. KURUP Department of Medicine, The Medical College of Wisconsin, Milwaukee and Research Service, Veterans Administration Center, Wood (Milwaukee), Wl 53193, U.S.A. Abstract--Aerobic actinomycetes associated with various clinical conditions in man and animals were studied for their antigenic relationships along with related organisms. Culture filtrate antigens obtained from 16 species were tested by antigen-antibody crossed-immunoelectrophoresis and by agar gel double diffusion against rabbit antibodies to Nocardia asteroides, N. brasiliensis and N. caviae. Weak cross-reactivity was detected among the three, Nocardia species and Micropolyspora brevicatena, but no cross-reactivity was noted with antigens from any of the other species studied.
Key words: Immunological cross-reactivity, Nocardia, Micropolyspora, crossed-immunoelectrophoresis, agar gel diffusion R E A C T I O N I M M U N O L O G I Q U E C R O I S E E E N T R E LES N O C A R D I A P A T H O G E N E S E T LES O R G A N I S M E S A P P A R E N T E S Resume----Des actinomyc&es atrobies associts dans difftrents syndromes cliniques chez l'homme et chez l'animal ont 6t6 ~tudits en ce qui concerne leurs relations antigtniques avec les microorganismes indiquts. Les antigenes obtenus par filtrat de culture /t partir de 16 esptces sont testts par immuno~lectrophortse croiste et par double diffusion en gtlose contre des anticorps de lapin antiNocardia asteroides, N. brasiliensis et N. caviae. Une faible rtaction croiste a 6t6 raise en 6vidence chez les trois esptces de Nocardia et Micropolyspora brevicatena, mais nous n'avons pas remarqu6 de rtaction croiste avec les antig~nes d'aucune des autres esptces observtes.
Mots-clefs: Reaction immunologique croiste, Nocardia, Micropolyspora, immunotlectrophortse croisee, diffusion en g~lose
INTRODUCTION Several members o f the aerobic actinomycetes are associated with h u m a n and animal diseases [ 1 - 5 ] . The frequently recognized species in h u m a n diseases include Nocardia
asteroides, N. brasiliensis, N. caviae, Actinomadura madurae, A. pelletieri, Nocardiopsis dassonvillei, Micropolyspora faeni, Saccharomonospora viridis, Thermoactinomyces candidus, T. sacchari and T. vulgaris. O f the 11 species, the first 6 cause invasive or localized infections, while the remainder are associated with hypersensitivity pneumonitis (HP) [1, 2, 4]. Although serological techniques are available in the diagnosis of nocardiosis and actinomycetic m y c e t o m a s , they are not widely used [ 6 - 8 ] . O n the contrary, demonstration o f circulating antibodies in the sera of patients is a valuable criterion for the diagnosis o f H P [5]. In recent years, serological tests using antigen extracts of Nocardia species have been evaluated in detecting specific antibodies in the sera o f patients. Some o f these antigen preparations were found to be specific while others showed cross-reactivity with mycobacterial antigens [7, 8]. M o s t o f the actinomycetes species mentioned a b o v e were isolated from soil or other natural environments. These actinomycetes species are taxonomically-related, but little is known regarding the antigenic relationship o f these 69
70
VISWANATH P. KURUP
organisms. Hence, knowledge of the antigenic cross-reactivity among the frequently occurring aerobic actinomycetes associated with h u m a n diseases m a y be beneficial in evaluating the significance and specificity o f circulating antibodies in the sera o f patients. The present investigation was therefore designed to study the antigenic cross-reactivity among various pathogenic aerobic actinomycetes and related organisms by means o f an a n t i g e n - a n tibody crossed-immunoelectrophoresis technique.
MATERIALS A N D METHODS Aetinomycetes strains The various strains o f actinomycetes used in the study are listed in Table 1. The identity of the Nocardia, Actinomadura and Nocardiopsis species was determined b y the method o f G o r d o n and H o r a n [9] and those o f Micropolyspora, Saccharomonospora and Thermoactinomyces by the method o f K u r u p and Fink [10]. The identity o f all other isolates was ascertained by comparing their characteristics with the original descriptions. All cultures were grown in appropriate media and one set of each was maintained at 4 ° C and at r o o m temperature. They were subcultured every 3 months. Preparation o f antigens F o r the extraction o f antigens, organisms were grown in the dialysates o f appropriate complex media. The method used was a modification o f that described by Ajello et al. [ 11 ] and is briefly as follows: dialysis bags (Arthur T h o m a s Co., 3787-F47, mol. wt cut off 8000)
Table 1. Organisms used in the extraction of antigens Accession number N-801 N-806 N-805 N-816 N-821 N-819 T-150 T-250 T-253 T-249 T-248 T-182 T-283 T-106 T-145 T-101
Source REG*- IMRU-859 REG - IMRU-731 REG - IMRU-1315 REG - IMRU-816 REG - IMRU-513 REG - IMRU-780 ATCC:~- 1 5 3 4 7 HAL§ - 1325 HAL - N-3805 HAL - 1086 HAL - 19125 HAL - 4047 ATCC - 1 9 2 8 5 ATCC - 2 7 8 6 8 ATCC - 27349 Marshfield Clinicl[
Identification Nocardia asteroides N. brasiliensis N. caviae Noeardiopsis dassonvillei Actinomadurapelletieri A. madurae Micropolysporafaeni M. rectivirgula M. viridinigra M. brevicatena M. caesia Saccharomonospora viridis Pseudonocardia thermophilia Thermoactinomyces candidus 7".sacchari T. vulgaris
* REG IMRU, Dr. Ruth E. Gordon, Institute of Microbiology, Rutgers, The State University, New Brunswick, N.J. $ ATCC, American Type Culture Collections, Rockville, Md. § HAL, Dr. H. A. Lechevalier, Institute of Microbiology, Rutgers, The State University, New Brunswick, N.J. [j Marshfield Clinic, Marshfield, Wis.
Immunologicalcross-reactivityof pathogenicNocardia and related organisms were filled with appropriate medium and placed in flasks containing 0. I M glycine in 0.075 M sodium chloride. The dialysis bags with the medium were barely submerged in the outer phases. The flasks with the contents were then sterilized by autoclaving at 121 °C for 15 min. The system was then allowed to equilibrate at room temperature for 48 hr. The outer phase of the medium was separated and asceptically transferred into sterile flasks, and was used to grow the various actinomycetes. The thermophilic actinomycetes (T-150, T-250, T-253, T248, T-283, T-106 and T-101), with the exception of T. sacchari (T-145), were grown in the dialysate fluid from tryptic soy broth (TSB), while the latter was grown in the dialysate of nutrient broth. The remaining isolates were grown in the dialysate from TSB, supplemented with 1% glucose. The thermophilic actinomycetes M. faeni, M. caesia, M. viridinigra, S. viridis, T. vulgaris, T. candidus, T. sacchari and Pseudonocardia thermophilia were grown in stationary cultures at 55°C for 2 weeks, while the Nocardia species, Nocardiopsis species and Micropolyspora brevicatena were grown at 37°C for 2-3 weeks. Both Actinomadura madurae and A. pelletieri were grown for 4 weeks at 28°C before harvesting. At the end of the incubation period the growth was killed by adding phenol to the final concentration of 1% (v/v). The sterility of the culture was determined after 2 4 - 4 8 hr, and the broth was separated from the mycelium by filtration. The filtrate was extensively dialyzed against distilled water for 2 - 3 days at 4°C, and the retentate was passed through a 450 nm membrane filter and freeze-dried. These antigens were used to raise antisera in rabbits as well as for the antigen-antibody crossed-immunoelectrophoresis.
Immunization of rabbits White rabbits, weighing 3 - 4 kg, were injected subcutaneously with N. asteroides, N. brasiliensis and N. caviae antigens. Ten milligrams of antigens in 1 ml of sterile physiological saline were mixed with an equal amount of incomplete Freund's adjuvant. 6 - 8 injections were given at weekly intervals. The rabbits were checked at intervals for antibody production by the agar gel double diffusion method [12] and bled when strong precipitating antibodies were detected. Homologous antisera were pooled and used in the crossed-immunoelectrophoresis studies.
Agar gel double diffusion The 3 antisera were studied for precipitating antibodies using various antigen preparations by the agar gel diffusion method, as previously described [12]. The antigens were used at concentrations of 30 mg/ml for the immunodiffusion studies.
Crossed-immunoelectrophoresis The cross-reactivity of the various actinomycetes was studied by two-dimensional immunoelectrophoresis using antigens from different species of actinomycetes and antibodies of the 3 Nocardia species, as previously described [13, 14]. Approximately 5 - 7 ~1 of antigen (100-125 ~tg protein) were electrophoresed in one direction for 3 hr at 8-10 v/cm. Following electrophoresis, the gel from the plate was removed leaving a 1.5-cm wide antigen band that was replaced with gel containing 10% v/v rabbit antinocardial serum. The gel plates were subjected to electrophoresis at right angles to the first direction at 8 v/cm for 3 hr, and the
72
VISWANATHP. KURUP
plates were stained with Coomassie brilliant blue [ 15]. The number, intensity and peak height of each precipitin arc formed with the various antigen-antibody reactions were studied and compared. RESULTS The agar gel diffusion studies demonstrated that the 3 anfi-Nocardia sera reacted against homologous antigens and produced multiple precipitin arcs. Nocardia asteroides and N. caviae antigens gave strong precipitin arcs with homologous antisera, while N. brasiliensis antigens showed weaker reactions with homologous antiserum. Weak cross-reactivity was invariably detected when nocardial antigens were reacted with the 3 antisera and vice versa. Antigens from M. brevicatena showed weak cross-reactivity with the 3 noeardial antibodies. No other antigens cross-reacted with the nocardial antibodies.
Fig. 1. Crossed-immunoelectrophoresis ofN. asteroides antigens against homologous rabbit antibody. Fig. 2. Crossed-immunoelectrophoresis ofN. caviae antigens against homologous rabbit antibody. Fig. 3. Crossed-immunoelectrophoresis ofN. brasiliensis antigen against homologous rabbit antibody. Fig. 4. Crossed-immunoelectrophoresis of M. brevicatena antigens against anti-N, asteroides rabbit serum (anode at the right and top).
Immunologicalcross-reactivityof pathogenic Nocardia and related organisms
73
Antigens from 3 species of Nocardia: N. asteroides, N. caviae and N. brasiliensis, reacted with the homologous antibodies and demonstrated several precipitin arcs (Figs. 1-3) each by antigen-antibody crossed-immunoelectrophoresis technique. N. asteroides antigens revealed 16 precipitin arcs with the specific antiserum, while it gave only one precipitin arc each with anti-N, caviae and N. brasiliensis rabbit sera. N. caviae demonstrated 7 precipitin arcs against homologous antiserum, 3 - 6 arcs against anti-N, asteroides and 4 against anti-N. brasiliensis sera. N. brasiliensis antigens produced 7 precipitin arcs with homologous antiserum but only one with anti-N, caviae and 4 with anti-N, asteroides sera. None of the antigens from other species, except M. brevicatena, reacted with any of the 3 antisera. M. brevicatena antigens reacted with N. asteroides (Fig. 4), N. brasiliensis and N. caviae antisera and demonstrated 3, 3 and 1 precipitin arcs, respectively.
DISCUSSION The results indicate that N. asteroides, N. brasiliensis and N. caviae share some common antigens. Although several of the actinomycetes species included in the study are taxonomically related to Nocardia, none showed any antigenic cross-reactivity with Nocardia except the species M. brevicatena. M. brevicatena grew well at 37°C and produced colonies morphologically indistinguishable from those of the Nocardia species [ 16]. It is noteworthy that all the known isolates of M. brevicatena have been recovered from clinical materials. Interestingly, no common antigenic determinants were detected in M. brevicatena and the other species of Micropolyspora (unpublished findings). Hence, additional studies are necessary to clarify the pathogenic role and systematic position ofM. brevicatena. Species belonging to Acinornadura and Nocardiopsis were previously included in the genus Nocardia. However, based on differences in cell wall composition, these 2 genera were separated from Nocardia [17, 18]. The present study of antigenic cross-reactivity also supports this conclusion. However, additional studies are needed to determine the relationships between the Actinornadura and Nocardiopsis; the latter genera was described recently to include A. dassonvillei. Antibodies against M. faeni have been detected in the sera of patients with farmer's lung, as well as in a small percentage of apparently normal farmers. None of the nocardial antigens cross-reacted with the sera from 5 patients with farmer's lung or with 2 sera from asymptomatic farmers in whom M. faeni precipitins could be detected. This finding agrees with the antigenic cross-reactivity results that none of the thermophilic actinomycetes crossreacted with Nocardia. M. brevicatena antigens also failed to react with farmer's lung sera, but reacted with all 3 nocardial antibodies. This indicates a stronger antigenic relationship between M. brevicatena and the Nocardia species than M. brevicatena and M.faeni. Among the pathogenic actinomycetes only the antigens from the 3 Nocardia species showed crossreactivity. Acknowledgements - The technical assistance of Mary J. Falk and the editorial assistance of Mavis E. Baurle and Catherine A. Walther are gratefully acknowledged. This investigation was supported by Specialized Center of Research (SCOR) Grant HL-15389 from the National Heart, Lung and Blood Institute. Also supported by the Medical Research Service of the Veterans Administration.
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VISWANATHP. KURUP REFERENCES
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