Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes

Sacchi, Claudio Tavares; Lemos, Ana Paula Silva de; Casagrande, Silvana Tadeu; Mori, Alice Massumi; Almeida, Carmecy Lopes de

doi:10.1590/S0036-46651995000400002

Abstracts

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

Corynebacterium diphtheriae; Ribotyping; Epidemiology

No presente estudo, nós reportamos os resultados de uma análise, baseada na ribotipagem de cepas de C. diphtheriae intermedius isoladas de uma criança de 9 anos com difteria e seus 5 contatos. Análise quantitativa por RFLP de rRNA foi usada para determinar a relação destas 7 cepas de C. diphtheriae fornecendo dados de interesse epidemiológico. Nós também testamos estas cepas para toxicidade in vitro usando método de difusão de Elek e in vivo usando método de cultura celular com células VERO. Os resultados de hibridização revelaram que as 5 cepas de C. diphtheriae isoladas dos contatos e uma isolada do caso (cepa isolada do nariz) tiveram padrões idênticos com as 4 enzimas de restrição usadas, ribotipo B. A distância genética deste ribotipo e o ribotipo A (cepa isolada da garganta do caso), que nós inicialmente assumimos ser responsável pela doença do paciente, foi de 0,450 mostrando pouca relação genética entre estes dois ribotipos. Nós não encontramos diferenças relativas à produção de toxina usando método de cultura celular entre as cepas. Em conclusão, o uso de RLFP de rRNA gene foi satisfatório em detectar pequenas diferenças em cepas de C. diphtheriae toxigênicas epidemiologicamente relacionadas e em fornecer informação sobre a relação genética entre elas.

MICROBIOLOGY

Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes

Relação genética de cepas de Corynebacterium diphtheriae isoladas de caso e seus contatos por RLFP de rRNA gene

Claudio Tavares Sacchi^I; Ana Paula Silva de Lemos^I; Silvana Tadeu Casagrande^I; Alice Massumi Mori^I; Carmecy Lopes de Almeida^II

^IBacteriology Divison, Adolfo Lutz Institute, São Paulo, Brazil

^IICenter for Epidemiological Surveillance of Osasco, São Paulo, Brazil

^{Correspondence to} Correspondence to: Claudio T. Sacchi Bacteriology Divison, Adolfo Lutz Institute Av. Dr. Arnaldo 351 01246-902 São Paulo, Brazil Phone: 55.11.8510111. Fax: 55.11.8533505 Electronic mail address: SACCHI@USP.BR

SUMMARY

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

Keywords:Corynebacterium diphtheriae; Ribotyping; Epidemiology.

RESUMO

No presente estudo, nós reportamos os resultados de uma análise, baseada na ribotipagem de cepas de C. diphtheriae intermedius isoladas de uma criança de 9 anos com difteria e seus 5 contatos. Análise quantitativa por RFLP de rRNA foi usada para determinar a relação destas 7 cepas de C. diphtheriae fornecendo dados de interesse epidemiológico. Nós também testamos estas cepas para toxicidade in vitro usando método de difusão de Elek e in vivo usando método de cultura celular com células VERO. Os resultados de hibridização revelaram que as 5 cepas de C. diphtheriae isoladas dos contatos e uma isolada do caso (cepa isolada do nariz) tiveram padrões idênticos com as 4 enzimas de restrição usadas, ribotipo B. A distância genética deste ribotipo e o ribotipo A (cepa isolada da garganta do caso), que nós inicialmente assumimos ser responsável pela doença do paciente, foi de 0,450 mostrando pouca relação genética entre estes dois ribotipos. Nós não encontramos diferenças relativas à produção de toxina usando método de cultura celular entre as cepas. Em conclusão, o uso de RLFP de rRNA gene foi satisfatório em detectar pequenas diferenças em cepas de C. diphtheriae toxigênicas epidemiologicamente relacionadas e em fornecer informação sobre a relação genética entre elas.

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Recebido pa publicação em 30/01/1995

Aceito para publicação em 28/06 1995

1. ALTWEGG, M. & MAYER, L.W. - Bacterial molecular epidemiology based on a non-radioactive probe complementary to ribosomal RNA. Res. Microbiol., 140: 325-333, 1989.
2. ALTWEGG, M.; HICKNAN-BRENNER, F.W. & FARMER III, J.J. - Ribosomal RNA gene restriction pattern provide increased sensitivity for typing Salmonella typhi strain J. infect. Dis., 160: 145-149, 1989.
3. BJÖRKHOLM, B.; BÖTTIGER, M.; CHRISTENSON, B. & HAGBERG, L. - Antitoxic antibody levels and the outcome of illness during an outbreak of diphtheria among alcoholics. Scand. J. infect. Dis., 18: 235-239, 1986.
4. BOEHRINGER MANNHEIM BIOCHEMICALS. - The Genius system user's guide for filter hybridization. Indianapolis, Boehringer Mannheim Biochemicals, 1992.
5. BRENNER, D.J.; MCWHORTER, A.C.; KNUTSON, J.K.L. & STEIGERWALT, A.G. - Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J. clin. Microbiol., 15: 1133-1140, 1982.
6. BROOKS, R. - Guidelines for the laboratory diagnosis of diphtheria. Geneva, WHO, 1981. (LAB/81.7).
7. BURROWS, W. & MOULDER, J.W. - Corynebacterium (The diphtheria bacillus). In: BURROWS, W. & MOULDER, J.W. Textbook of microbiology. 19. ed. Philadelphia, W.B. Saunders Company, 1968. p.642-656.
8. COYLE, M.B.; GROMON, M.B.; RUSEL, J.Q. et al. - The molecular epidemiology of three biotypes of Corynebacterium diphtheriae in the Seattle outbreak of 1972-1982. J. infect. Dis., 159: 670-679, 1989.
9. EFSTRATION, A.; TILEY,S.M.; SANGRADOR, A. et al. - Invasive disease caused by multiple clones of Corynebacterium diphtheriae. (Letter). Clin. infect. Dis., 17: 136, 1993.
10. GRIMONT, F. & GRIMONT, P.A.D. - Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur/Microbiol. (Paris), 137B: 165-175, 1986.
11. GROMAN, N.; CIANCIOTTO, N.; BJORN, M. & RABIN, N. - Detection and expression of DNA homologous to the tax gene in nontoxinogenic isolates of Corynebacterium diphtheriae. Infect. Immun., 42: 48-56, 1983.
12. IRINO, K.; GRIMONT, F.; CASIN, I.; GRIMONT, P.A.D. & THE BRAZILIAN PURPURIC FEVER STUDY GROUP - rRNA gene restriction patterns of Haemophilus influtenzae biogroup aegyptius strains associated with Brazilian purpuric fever. J. clin. Microbiol., 26: 1535-1538, 1988.
13. JORDENS, J.Z. & PENNINGTON, T.H. - Characterization of N. meningitidis isolates by ribosomal RNA gene restriction patterns and restriction endonuclease digestion of chromosomal DNA. Epidem. Infect., 107: 253-262, 1991.
14. KIEHL BAUCH, J.A.; PLIKAYTIS, B.D.; SWAMINATHAN, B.; CAMERON, D.N. & WACHSMUTH, I.K. - Restriction fragment length polymorphisms in the ribosomal genes for species identification and subtyping of aerotolerant Campylobacter species. J. clin. Microbiol., 29: 1670-1676, 1991.
15. LEONARD, R.B.; NOWOWIEJSKI, D.J.; WARREN, J.J.; FINN, D.J. & COYLE, M.B. - Molecular evidence of person-to-person transmission of a pigmented strain of Corynebacterium striatum in intensive care units. J. clin. Microbiol., 32: 164-169, 1994.
16. MICHEL, J.L.; RAPPUOLI, R.; MURPHY, JR. & PAPPENHEIMER, A.M.JR. - Restriction endonuclease map of the nontoxigenic corynephage yc and is relationship to the toxigenic corynephage Bc. J. Virol., 42: 510-518, 1982.
17. MIYAMURA, K.; NISHIO, S.; ITO, A.; MURATA, R. & KONO, R. - Micro cell culture for determination of diphtheria toxin and antitoxin titles using VERO cells. I. Studies of factors affecting the toxin and antitoxin titration. J. biol. Stand., 2: 189-201, 1974.
18. MIYAMURA, K.; TAJIRI, E.; ITO, A.; MURATA, R. & KONO, R. - Micro cell culture for determination of diphtheria toxin and antitoxin titres using VERO cells. II. Comparation with the rabbit skin method and practical application for sero-epidemiological studies. J. biol. Stand., 2: 203-209, 1974.
19. MURPHY, W.J.; MALEY, V.H. & DICK, L. - Continued high incidence of diphtheria in a well-immunized community. Publ. Hlth . Rep. (Wash.), 71: 481-486, 1956.
20. OLSEN, G.J. - rRNA - based phylogeny: phylogeny analysis using ribosomal RNA. In: BERGAN, T. & NORRIS, J.R. Methods in enzymology. London, Academic Press, 1988. v. 2, p. 793-812.
21. PAPPENHEIMER, A.M. & MURPHY, J.R. - Studies on the molecular epidemiology of diphtheria. Lancet, 2: 923-926, 1983.
22. RAPPUOLI, R.; MICHEL, J.L. & MURPHY, J.R. - Restriction endonuclease map of corinebacteriophage w_ctox+, isolated from the Park-Williams no. 8 strain of Corynebacterium diphtheriae. J. Virol., 45: 524-530, 1983.
23. RAPPUOLI, R.; MICHEL, J.L. & MURPHY, J.R. - Integration of corynebacteriophages ßtox+, wtox+, and ytox+, into two attachment sites on the Corynebacterium diphtheriae chromosome. J. Bact., 153: 1202-1210, 1983.
24. RAPPUOLI, R.; PERUGINI, M. & FALSEN, E. - Molecular epidemiology of the 1984-1986 outbreak of diphtheria in Sweden. New Engl. J. Med., 318: 12-14, 1988.
25. SACCHI, C.T.; TONDELLA, M.L.C.; BRANDILEONE, M.C.C.; MELLES, C.E.A. & PAULA, M.D.N. - Corynebacterium diphtheriae isolada de sangue. Rev. Inst. Adolfo Lutz., 45: 73-79, 1985.
26. SACCHI, C.T.; RAMOS, S.R.; MELLES, C.E.A. et al. - Estudo bacteriológico de cepas de Corynebacterium diphtheriae isoladas no estado de Săo Paulo, Brasil, no período de 1980 a 1986. Rev. Inst. Adolfo Lutz., 47: 31-37, 1987.
27. SARAGEA, A.; MAXIMESCU, P. & MEITERT, E. - Corynebacterium diphtheriae: microbiological methods used in clinical and epidemiological investigations. In.: BERGAN, T. & NORRIS, J.R. Methods in microbiology. London, Academic Press, 1979. v. 13, p 161-176.
28. SCHILLER, J.; GROMAN, N. & COYLE, M.B. - Plasmids in Corynebacterium diphtheriae and difteroids mediating erytromycin resistence. Antimicrob. Agents Chemother., 18: 814-821, 1980.
29. SOTO, A.; PITCHER, D.G. & SORIANO, F. - A numerical analysis of ribosomal RNA gene patterns for typing clinical isolates of Corynebacterium group D2. Epidem. Infect., 107: 263-272, 1991.
30. SOTTNEK, F.O. & MILLER, J.M. - Isolation and identification of Corynebacterium diphtheriae. Atlanta, Centers for Disease Control, 1980. (revised 1982).
31. TONDELLA, M.L.C.; SACCHI, C.T. & NEVES, B.C. - Ribotyping as an additional molecular marker for studying Neisseria meningitidis serogroup B epidemic strains. J. clin. Microbiol., 32: 2745-2748, 1994.
32. TOSHACH, S.; VALENTINE, A. & SIGURDSON, S. - Bacteriophage typing of Corynebacterium diphtheriae. J. infect. Dis., 136: 655-660, 1977.
33. WEHRLE, P.F. - Diphtheria. In: EVANS, A.S. & BRACHMAN, P.S. Bacterial injections of humans. Epidemiology and control. 2. ed. New York, Plenum Medical Book Company, 1991. p. 227-237.
34. WOODS, T.C.; HELSEL, L.O.; SWAMINATHAN, B. et al. - Characterization of N. meningitidis serogroup C by multilocus enzyme electrophoresis and ribosomal DNA restriction profiles (Ribotyping). J. clin. Microbiol., 30: 132-137, 1992.
35. YOGEV, G.; HALACHMI, D.; KENNY, G.E. & RAZIN, S. - Distinction of species and strains of mycoplasmas (Mollicutes) by genomic DNA fingerprints with an rRNA probe. J. clin. Microbiol., 26: 1198-1201, 1988.

Correspondence to:

Claudio T. Sacchi

Bacteriology Divison, Adolfo Lutz Institute

Av. Dr. Arnaldo 351

01246-902 São Paulo, Brazil

Phone: 55.11.8510111. Fax: 55.11.8533505

Electronic mail address:

SACCHI@USP.BR

Publication Dates

Publication in this collection
21 Sept 2006
Date of issue
Aug 1995

History

Received
30 Jan 1995
Accepted
28 June 1995

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. ALTWEGG, M. & MAYER, L.W. - Bacterial molecular epidemiology based on a non-radioactive probe complementary to ribosomal RNA. Res. Microbiol., 140: 325-333, 1989.

[2] 2. ALTWEGG, M.; HICKNAN-BRENNER, F.W. & FARMER III, J.J. - Ribosomal RNA gene restriction pattern provide increased sensitivity for typing Salmonella typhi strain J. infect. Dis., 160: 145-149, 1989.

[3] 3. BJÖRKHOLM, B.; BÖTTIGER, M.; CHRISTENSON, B. & HAGBERG, L. - Antitoxic antibody levels and the outcome of illness during an outbreak of diphtheria among alcoholics. Scand. J. infect. Dis., 18: 235-239, 1986.

[4] 4. BOEHRINGER MANNHEIM BIOCHEMICALS. - The Genius system user's guide for filter hybridization. Indianapolis, Boehringer Mannheim Biochemicals, 1992.

[5] 5. BRENNER, D.J.; MCWHORTER, A.C.; KNUTSON, J.K.L. & STEIGERWALT, A.G. - Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J. clin. Microbiol., 15: 1133-1140, 1982.

[6] 6. BROOKS, R. - Guidelines for the laboratory diagnosis of diphtheria. Geneva, WHO, 1981. (LAB/81.7).

[7] 7. BURROWS, W. & MOULDER, J.W. - Corynebacterium (The diphtheria bacillus). In: BURROWS, W. & MOULDER, J.W. Textbook of microbiology. 19. ed. Philadelphia, W.B. Saunders Company, 1968. p.642-656.

[8] 8. COYLE, M.B.; GROMON, M.B.; RUSEL, J.Q. et al. - The molecular epidemiology of three biotypes of Corynebacterium diphtheriae in the Seattle outbreak of 1972-1982. J. infect. Dis., 159: 670-679, 1989.

[9] 9. EFSTRATION, A.; TILEY,S.M.; SANGRADOR, A. et al. - Invasive disease caused by multiple clones of Corynebacterium diphtheriae. (Letter). Clin. infect. Dis., 17: 136, 1993.

[10] 10. GRIMONT, F. & GRIMONT, P.A.D. - Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur/Microbiol. (Paris), 137B: 165-175, 1986.

[11] 11. GROMAN, N.; CIANCIOTTO, N.; BJORN, M. & RABIN, N. - Detection and expression of DNA homologous to the tax gene in nontoxinogenic isolates of Corynebacterium diphtheriae. Infect. Immun., 42: 48-56, 1983.

[12] 12. IRINO, K.; GRIMONT, F.; CASIN, I.; GRIMONT, P.A.D. & THE BRAZILIAN PURPURIC FEVER STUDY GROUP - rRNA gene restriction patterns of Haemophilus influtenzae biogroup aegyptius strains associated with Brazilian purpuric fever. J. clin. Microbiol., 26: 1535-1538, 1988.

[13] 13. JORDENS, J.Z. & PENNINGTON, T.H. - Characterization of N. meningitidis isolates by ribosomal RNA gene restriction patterns and restriction endonuclease digestion of chromosomal DNA. Epidem. Infect., 107: 253-262, 1991.

[14] 14. KIEHL BAUCH, J.A.; PLIKAYTIS, B.D.; SWAMINATHAN, B.; CAMERON, D.N. & WACHSMUTH, I.K. - Restriction fragment length polymorphisms in the ribosomal genes for species identification and subtyping of aerotolerant Campylobacter species. J. clin. Microbiol., 29: 1670-1676, 1991.

[15] 15. LEONARD, R.B.; NOWOWIEJSKI, D.J.; WARREN, J.J.; FINN, D.J. & COYLE, M.B. - Molecular evidence of person-to-person transmission of a pigmented strain of Corynebacterium striatum in intensive care units. J. clin. Microbiol., 32: 164-169, 1994.

[16] 16. MICHEL, J.L.; RAPPUOLI, R.; MURPHY, JR. & PAPPENHEIMER, A.M.JR. - Restriction endonuclease map of the nontoxigenic corynephage yc and is relationship to the toxigenic corynephage Bc. J. Virol., 42: 510-518, 1982.

[17] 17. MIYAMURA, K.; NISHIO, S.; ITO, A.; MURATA, R. & KONO, R. - Micro cell culture for determination of diphtheria toxin and antitoxin titles using VERO cells. I. Studies of factors affecting the toxin and antitoxin titration. J. biol. Stand., 2: 189-201, 1974.

[18] 18. MIYAMURA, K.; TAJIRI, E.; ITO, A.; MURATA, R. & KONO, R. - Micro cell culture for determination of diphtheria toxin and antitoxin titres using VERO cells. II. Comparation with the rabbit skin method and practical application for sero-epidemiological studies. J. biol. Stand., 2: 203-209, 1974.

[19] 19. MURPHY, W.J.; MALEY, V.H. & DICK, L. - Continued high incidence of diphtheria in a well-immunized community. Publ. Hlth . Rep. (Wash.), 71: 481-486, 1956.

[20] 20. OLSEN, G.J. - rRNA - based phylogeny: phylogeny analysis using ribosomal RNA. In: BERGAN, T. & NORRIS, J.R. Methods in enzymology. London, Academic Press, 1988. v. 2, p. 793-812.

[21] 21. PAPPENHEIMER, A.M. & MURPHY, J.R. - Studies on the molecular epidemiology of diphtheria. Lancet, 2: 923-926, 1983.

[22] 22. RAPPUOLI, R.; MICHEL, J.L. & MURPHY, J.R. - Restriction endonuclease map of corinebacteriophage w_ctox+, isolated from the Park-Williams no. 8 strain of Corynebacterium diphtheriae. J. Virol., 45: 524-530, 1983.

[23] 23. RAPPUOLI, R.; MICHEL, J.L. & MURPHY, J.R. - Integration of corynebacteriophages ßtox+, wtox+, and ytox+, into two attachment sites on the Corynebacterium diphtheriae chromosome. J. Bact., 153: 1202-1210, 1983.

[24] 24. RAPPUOLI, R.; PERUGINI, M. & FALSEN, E. - Molecular epidemiology of the 1984-1986 outbreak of diphtheria in Sweden. New Engl. J. Med., 318: 12-14, 1988.

[25] 25. SACCHI, C.T.; TONDELLA, M.L.C.; BRANDILEONE, M.C.C.; MELLES, C.E.A. & PAULA, M.D.N. - Corynebacterium diphtheriae isolada de sangue. Rev. Inst. Adolfo Lutz., 45: 73-79, 1985.

[26] 26. SACCHI, C.T.; RAMOS, S.R.; MELLES, C.E.A. et al. - Estudo bacteriológico de cepas de Corynebacterium diphtheriae isoladas no estado de Săo Paulo, Brasil, no período de 1980 a 1986. Rev. Inst. Adolfo Lutz., 47: 31-37, 1987.

[27] 27. SARAGEA, A.; MAXIMESCU, P. & MEITERT, E. - Corynebacterium diphtheriae: microbiological methods used in clinical and epidemiological investigations. In.: BERGAN, T. & NORRIS, J.R. Methods in microbiology. London, Academic Press, 1979. v. 13, p 161-176.

[28] 28. SCHILLER, J.; GROMAN, N. & COYLE, M.B. - Plasmids in Corynebacterium diphtheriae and difteroids mediating erytromycin resistence. Antimicrob. Agents Chemother., 18: 814-821, 1980.

[29] 29. SOTO, A.; PITCHER, D.G. & SORIANO, F. - A numerical analysis of ribosomal RNA gene patterns for typing clinical isolates of Corynebacterium group D2. Epidem. Infect., 107: 263-272, 1991.

[30] 30. SOTTNEK, F.O. & MILLER, J.M. - Isolation and identification of Corynebacterium diphtheriae. Atlanta, Centers for Disease Control, 1980. (revised 1982).

[31] 31. TONDELLA, M.L.C.; SACCHI, C.T. & NEVES, B.C. - Ribotyping as an additional molecular marker for studying Neisseria meningitidis serogroup B epidemic strains. J. clin. Microbiol., 32: 2745-2748, 1994.

[32] 32. TOSHACH, S.; VALENTINE, A. & SIGURDSON, S. - Bacteriophage typing of Corynebacterium diphtheriae. J. infect. Dis., 136: 655-660, 1977.

[33] 33. WEHRLE, P.F. - Diphtheria. In: EVANS, A.S. & BRACHMAN, P.S. Bacterial injections of humans. Epidemiology and control. 2. ed. New York, Plenum Medical Book Company, 1991. p. 227-237.

[34] 34. WOODS, T.C.; HELSEL, L.O.; SWAMINATHAN, B. et al. - Characterization of N. meningitidis serogroup C by multilocus enzyme electrophoresis and ribosomal DNA restriction profiles (Ribotyping). J. clin. Microbiol., 30: 132-137, 1992.

[35] 35. YOGEV, G.; HALACHMI, D.; KENNY, G.E. & RAZIN, S. - Distinction of species and strains of mycoplasmas (Mollicutes) by genomic DNA fingerprints with an rRNA probe. J. clin. Microbiol., 26: 1198-1201, 1988.

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