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Isoenzyme profiles and phylogenetic analysis of Giardia duodenalis isolates from Iranian patients

Abstract

The main objective of this study was to characterize the Giardia duodenalis isolates from Iranian patients in Fars Province, south of Iran by biochemical and molecular methods. Fifteen mass cultivated of G. duodenalis isolates in modified TYI-S-33 medium were analyzed using isoenzyme electrophoresis and PCR genotyping. Polyacrylamide gel electrophoresis (PAGE) of five different enzyme systems was used to characterize isolates: (i) glucose-6-phosphate dehydrogenase, (ii) glucose phosphate isomerase, (iii) malate dehydrogenase, (iv) malic enzyme, and (v) phosphoglucomutase. As well, a fragment of the SSU-rDNA (292 bp) gene was amplified by PCR using the primers RH11 and RH4. The sequencing of the PCR products and phylogenetic tree were performed. The isoenzyme electrophoretic profiles divided fifteen G. duodenalis isolates into four zymodemes. G6PD, GPI, MDH, ME, and PGM enzyme systems showed 1, 2, 2, 3, and 3 enzyme pattern, respectively. G6PD isoenzyme pattern had the most homogeneity, while isoenzyme patterns of ME and PGM had the most heterogeneity in our study. Genotyping results indicated that the zymodemes 1–4 were categorized in assemblage A based on the SSU-rDNA gene. Phylogenetic analysis showed that all four zymodemes were distributed within the cluster of assemblage A. Our results indicated that both isoenzyme and DNA analyses were useful to characterize the isolates of Giardia and distinguishing various zymodemes and assemblages. It could be suggested that the genetic diversity among isoenzymes profiles of G. duodenalis may explain the variable clinical manifestations, pathogenicity, host response, drug susceptibility, and treatment efficacy of human giardiasis.

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Acknowledgments

This is a common project between Shiraz University of Medical Sciences in Iran and University Putra Malaysia. We appreciate Vice Chancellor of Research, Shiraz University of Medical Sciences, Shiraz, Iran, for the collaboration and financial support of this research.

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Correspondence to Gholam Reza Hatam.

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Appendices

Appendix 1

Table 3 TYI-S-33 medium (Trypticase, Yeast extract, Iron-Serum)

Appendix 2

Table 4 Gel preparation (Separating and Stacking gel)

Appendix C

Table 5 Gel staining: specific reagents for each enzyme system

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Rayani, M., Unyah, N.Z., Vafafar, A. et al. Isoenzyme profiles and phylogenetic analysis of Giardia duodenalis isolates from Iranian patients. Environ Sci Pollut Res 27, 40652–40663 (2020). https://doi.org/10.1007/s11356-020-10062-1

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  • DOI: https://doi.org/10.1007/s11356-020-10062-1

Keywords

  • Giardia duodenalis
  • Isoenzyme electrophoresis
  • Phylogenetic analysis
  • SSU-rDNA gene
  • Isoenzyme profiles