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Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the design of primers for rapid differentiation from Entamoeba histolytica

Published online by Cambridge University Press:  06 April 2009

S. Novati ,
M. Sironi ,
S. Granata ,
A. Bruno ,
S. Gatti ,
M. Scaglia  and
C. Bandi
S. Novati
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy
M. Sironi
Affiliation:
Istituto di Patologia Generate Veterinaria, Università di Milano, Via Celoria 10, 20133 Milano, Italy
S. Granata
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy Dipartimento di Genetica e Microbiologia, Università di Pavia, Via Abbiategrasso 207, 27100 Pavia, Italy
A. Bruno
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy
S. Gatti
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy
M. Scaglia
Affiliation:
Laboratorio di Parassitologia Clinica, Università di Pavia-I.R.C.C.S. Policlinico San Matteo, Viale Taramelli 5, 27100 Pavia, Italy
C. Bandi*
Affiliation:
Istituto di Patologia Generate Veterinaria, Università di Milano, Via Celoria 10, 20133 Milano, Italy Dipartimento di Genetica e Microbiologia, Università di Pavia, Via Abbiategrasso 207, 27100 Pavia, Italy
*
* Corresponding author: Dr C. Bandi, Istituo di Patologia Generale Veterinaria, Università Milano, Via Celoria 10, 20133 Milano, Italy.
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Summary

Since 1993, strains of Entamoeba histolytica sensn lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. histolytica, this sequence shows 1·7 % nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from both E. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

Keywords

Entamoeba disparEntamoeba histolyticaribosomal RNAspecies identificationRFLPdirect sequencing

Information

Type
Research Article
Information
Parasitology , Volume 112 , Issue 4 , April 1996 , pp. 363 - 369
Copyright
Copyright © Cambridge University Press 1996

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References

Acuna-Soto, R., Samuelson, J., De Girolami, P., Zarate, L., Millan-Velasco, F., Schoolnick, G. & Wirth, D. (1993). Application of the polymerase chain reaction to the epidemiology of pathogenic and nonpathogenic Entamoeba histolytica. American Journal of Tropical Medicine and Hygiene 48, 58–70.CrossRefGoogle Scholar
Andrews, B. J., Mentzoni, L. & Bjorvatn, B. (1990). Zymodeme conversion of isolates of Entamoeba histolytica. Transactions of the Royal Society of Tropical Medicine and Hygiene 84, 63–5.Google Scholar
Bandi, C., Damiani, G., Magrassi, L., Grigolo, A., Fani, R. & Sacchi, L. (1994). Flavobacteria as intracellular symbionts in cockroaches. Proceedings of the Royal Society of London, B 257, 43–8.Google ScholarPubMed
Bhattacharya, S., Bhattacharya, A. & Diamond, L. S. (1988). Comparison of repeated DNA from strains of Entamoeba histolytica and other Entamoeba. Molecular and Biochemical Parasitology 27, 257–62.Google Scholar
Bhattacharya, S., Bhattacharya, A., Diamond, L. S. & Soldo, A. T. (1989). Circular DNA of Entamoeba histolytica encodes ribosomal RNA. Journal of Protozoology 36, 455–8.Google Scholar
Blanc, D. & Sargeaunt, P. G. (1991). Entamoeba histolytica zymodemes: exhibition of γ and δ bands only of glucose phosphate isomerase and phosphoglucomutase may be influenced by starch content in the medium. Experimental Parasitology 72, 87–90.CrossRefGoogle ScholarPubMed
Brumpt, E. (1925). Etude sommarie de l' ‘Entamoeba dispar‘ n. sp. Amibe à kystes quadrinuclees, parasite de l'homme. Bulletin de l' Académie de Médecine (Paris) 94, 943–52.Google Scholar
Clark, C. G., Cunnick, C. C. & Diamond, L. S. (1992). Entamoeba histolytica: is conversion of ‘nonpathogenic’ amebae to the ‘pathogenic’ form a real phenomenon? Experimental Parasitology 74, 307–14.Google Scholar
Clark, C. G. & Diamond, L. S. (1991). Ribosomal RNA genes of ‘pathogenic’ and ‘nonpathogenic’ Entamoeba histolytica are distinct. Molecular and Biochemical Parasitology 49, 297–302.CrossRefGoogle ScholarPubMed
Clark, C. G. & Diamond, L. S. (1993). Entamoeba histolytica: an explanation for the reported conversion of nonpathogenic amoebae to the pathogenic form. Experimental Parasitology 77, 456–60.CrossRefGoogle Scholar
Cruz-Reyes, J. A., Spice, W. M., Rehman, T., Gisborne, E. & Ackers, J. P. (1992). Ribosomal DNA sequences in the differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica. Parasitology 104, 239–46.CrossRefGoogle ScholarPubMed
Diamond, L. s. & Clark, G. (1993). A redescription of Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911) separating it from Entamoeba dispar Brumpt, 1925. Journal of Eurkaryotic Microbiology 40, 340–4.CrossRefGoogle ScholarPubMed
Dobson, S. J., Colwell, R. R., McMeekin, T. A. & Franzmann, p. D. (1993). Direct sequencing of the polymerase chain reaction-amplified 16S rRNA gene of Flavobacterium gondwanense sp. nov. and Flavobacterium salegens sp. nov., two new species from hypersaline antartic lake. International Journal of Systematic Bacteriology 43, 77–83.CrossRefGoogle Scholar
Gatti, S., Cevini, C., Marchi, L., Novati, S. & Scaglia, M. (1995). Entamoeba histolytica autochthonous isolates from mentally retarded Italian patients. Parasitology Research 81, 148–51.Google Scholar
Hillis, D. M., Larson, A., Davis, S. K. & Zimmer, E. A. (1990). Nucleic acids III: sequencing. In Molecular Systematics (ed. Hillis, D. M. & Moritz, C.), pp. 318–70. Sunderland, Massachusetts: Sinauer Associates.Google Scholar
Huber, M., Koller, B., Gitler, C., Mirelman, D., Revel, M., Rozenblatt, S. & Garfinkel, L. (1989). Entamoeba histolytica ribosomal RNA genes are carried on palindromic circular DNA molecules. Molecular and Biochemical Parasitology 32, 285–96.Google Scholar
Jackson, T. F. H. G., Gathiram, V., Suparsad, S. & Anderson, C. A. (1992). Stability of the zymodemes of Entamoeba histolytica in cultures. Archives of Medical Research 23, 71.Google Scholar
Katzwinkel-Wladarsch, S., Löscher, T. & Rinder, H. (1994). Direct amplification and differentiation of pathogenic and nonpathogenic Entamoeba histolytica DNA from stool specimens. American Journal of Tropical Medicine and Hygiene 51, 115–18.CrossRefGoogle ScholarPubMed
Leipe, D. D., Gunderson, J. H., Nerad, T. A. & Sogin, M. L. (1993). Small subunit ribosomal RNA + of Hexamita inflata and the quest for the first branch in the eukariotic tree. Molecular and Biochemical Parasitology 59, 41–8.Google Scholar
Maidak, B. L., Larsen, N., McCaughey, M. J., Overbeek, R., Olsen, G. J., Fogel, K., Blandy, J. & Woese, C. R. (1994). The Ribosomal Database project. Nucleic Acids Research 22 (Suppl.), 3485–7.CrossRefGoogle ScholarPubMed
Martinez-Palomo, A., Martinez-Robles, A. & De La Torre, M. (1973). Selective agglutination of pathogenic strains of Entamoeba histolytica induced con A. Nature, New Biology 245, 186–7.Google Scholar
Mirelman, D., Bracha, R., Chayen, A., Aust-Kettis, A. & Diamond, L. s. (1986 a). Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence. Experimental Parasitology 62, 142–8.Google Scholar
Mirelman, D., Bracha, R., Wexler, A. & Chayen, A. (1986 b). Changes in isoenzyme patterns of a cloned culture of non-pathogenic Entamoeba histolytica during axenization. Infection and Immunity 54, 827–32.CrossRefGoogle Scholar
Mukherjee, R. M., Bhol, K. C., Mehra, S., Maitra, T. K. & Jalan, K. N. (1993). Zymodeme alteration of Entamoeba histolytica isolates under varying conditions. Transactions of the Royal Society of Tropical Medicine and Hygiene 87, 490–1.Google Scholar
Ochman, H. & Wilson, A. c. (1987). Evolution in bacteria: evidence for a universal substitution rate in cellular genomes. Journal of Molecular Evolution 26, 74–86.CrossRefGoogle ScholarPubMed
Que, x. & Reed, s. L. (1991). Nucleotide sequence of a small subunit ribosomal RNA (16S-like rRNA) gene from Entamoeba histolytica: differentiation of pathogenic from nonpathogenic isolates. Nucleic Acids Research 19, 5438.CrossRefGoogle ScholarPubMed
Ramachandran, S., Bhattacharya, A. & Bhattacharya, s. (1993). Nucleotide sequence analysis of the rRNA transcription unit of a pathogenic Entamoeba histolytica strain HM-1: IMSS. Nucleic Acids Research 21, 2011.Google Scholar
Robinson, G. L. (1968). The laboratory diagnosis of human parasitic amoebae. Transactions of the Royal Society of Tropical Medicine and Hygiene 62, 285–94.Google Scholar
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. 2nd Edn.Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press.Google Scholar
Sargeaunt, P. G. (1988). Zymodemes of Entamoeba histolytica. In Amoebiasis. Human Infection by Entamoeba histolytica (ed. Ravdin, J. I.), pp. 370–421. New York: Churchill Livingstone Publications.Google Scholar
Sargeaunt, p. G. (1992). Entamoeba histolytica is a complex of two species. Australian Microbiologist 13, 359–61.Google Scholar
Sargeaunt, P. G. (1994). Entamoeba histolytica and E. dispar. Transactions of the Royal Society of Tropical Medicine and Hygiene 88, 254.CrossRefGoogle ScholarPubMed
Sargeaunt, P. G. & Williams, J. E. (1978). Electrophoretic isoenzyme patterns of Entamoeba histolytica and Entamoeba coli. Transactions of the Royal Society of ropical Medicine and Hygiene 72, 164–6.CrossRefGoogle ScholarPubMed
Sargeaunt, P. G., Williams, J. E. & Grene, J. D. (1978). The differentiation of invasive and non-invasive Entamoeba histolytica by isoenzyme electrophoresis. Transactions of the Royal Society of Tropical Medicine and Hygiene 72, 519–21.Google Scholar
Sarceaunt, P. G., Jackson, T. F. H. G., Wiffen, S. R. & Bhojnani, R. (1988). Biological evidence of genetic exchange in Entamoeba histolytica. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 862–7.Google Scholar
Scaglia, M., Gatti, S., Bruno, A., Cevini, C., Marchi, L. & Sargeaunt, P. G. (1991). Autochthonous amoebiasis in institutionalized mentally-retarded patients: preliminary evaluation of isoenzyme patterns in three isolates. Annals of Tropical Medicine and Parasitology 85, 509–13.Google Scholar
Schlötterer, C., Hauser, M.-T., Von Haeseler, A. & Tautz, D. (1994). Comparative evolutionary analysis of rDNA ITS region in Drosophila. Molecular Biology and Evolution 11, 513–22.Google ScholarPubMed
Sehgal, D., Mittal, V., Ramachandran, S., Dhar, S. K., Bhattacharya, A. & Bhattacharya, S. (1994). Nucleotide sequence organisation and analysis of the nuclear ribosomal DNA circle of the protozoan parasite Entamoeba histolytica. Molecular and Biochemical Parasitology 67, 205–14.CrossRefGoogle ScholarPubMed
Sidow, A. & Thomas, W. K. (1994). A molecular evolutionary framework for eukaryotic model organisms. Current Biology 4, 596–603.Google Scholar
Sogin, M. L. (1989). Evolution of eukaryotic microorganisms and their small subunit ribosomal RNAs. American Zoologist 29, 487–99.CrossRefGoogle Scholar
Tannich, E. & Burchard, G. D. (1991). Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro. Journal of Clinical Microbiology 29, 250–5.CrossRefGoogle ScholarPubMed
Vargas, M. A. & Orozco, E. (1993). Entamoeba histolytica: changes in the zymodeme of cloned nonpathogenic trophozoites cultured under different conditions. Parasitology Research 79, 353–6.CrossRefGoogle ScholarPubMed
Walsh, J. A. (1986). Problems in recognition and diagnosis of amoebiasis: estimation of the global magnitude of morbidity and mortality. Reviews of Infectious Diseases 8, 228–38.Google Scholar