Parasitology



PCR-based DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer


R. M. HOPKINS a1c1, C. C. CONSTANTINE a1, D. A. GROTH a2, J. D. WETHERALL a2, J. A. REYNOLDSON a1 and R. C. A. THOMPSON a1
a1 WHO Collaborating Centre for the Molecular Epidemiology of Parasite Infections and State Agricultural Biotechnology Centre, School of Veterinary and Biomedical Studies, Murdoch University, Murdoch, WA, 6150, Australia
a2 School of Biomedical Sciences, Curtin University, Bentley, Perth, Western Australia, 6102

Abstract

The potential for the non-coding intergenic rDNA spacer (IGS) to DNA fingerprint Giardia duodenalis isolates was investigated. Conserved PCR primers, specific for the flanking large and small rDNA genes, were used to amplify the IGS from 52 in vitro-cultured Giardia isolates. Four distinct IGS–PCR size groups (1·35–1–6 kb) were observed, which correlated closely with the major genetic assemblages established previously for the same isolates using isoenzyme analysis. IGS–PCR size groups A (1·42 kb), C (1·4 kb) and D (1·35 kb) corresponded to isoenzyme assemblage A, and IGS–PCR group B (1·6 kb) to isoenzyme assemblage B. Amplified products from IGS–PCR size groups A and B, which contained 50/52 isolates, were subsequently digested with 8 different restriction enzymes and their profiles compared. Analysis separated isolates within each IGS–PCR size group into 2 distinct clusters which correlated almost exactly with the same genetic groups established previously using isoenzyme electrophoresis. Within each cluster, both methods exhibited a similar capacity to distinguish between Giardia genotypes although they established different genetic relationships between individual isolates. Much of the variability associated with the IGS was attributed to isolates harbouring multiple IGS- sequence types. Restriction analysis of IGS–PCR products amplified from cloned and parent lines of a human isolate BAH 39, which contains multiple IGS variants, showed that trophozoite populations are homogeneous with respect to the types of IGS-variants they maintain. Furthermore, in vitro culture of the cloned isolate BAH39c9 over a 6-year period also failed to reveal variation in IGS–PCR digestion profiles. These results suggest that IGS–PCR RFLP profiles are inherently stable. IGS–PCR analysis was successfully applied to 11 Giardia cyst samples highlighting the potential for this approach to genotype Giardia isolates without the need for in vitro culture.

(Received September 9 1998)
(Revised December 4 1998)
(Accepted December 7 1998)


Key Words: Giardia duodenalis; protozoa; intergenic rDNA spacer; PCR; DNA fingerprinting.

Correspondence:
c1 Corresponding author: Department of Medicine, University of Western Australia, T Block, Fremantle Hospital, Alma St., Fremantle, WA, 6160, Australia. Tel: +08 9431 2523. Fax: +08 9431 2977. E-mail: Richard.Hopkins@uwa.edu.au


Metrics