Advertisement

Development and Evaluation of Modified Cryopreservation for Long-Term Storage of Blastocystis Subtypes 1–3 and 6

  • 11 Accesses

Abstract

Background

Blastocystis is a protozoan parasite living in the intestine of humans and a wide range of animals. Although Blastocystis grows in several cultivation media, axenification and serial cultivations for long time are the main challenges of the researchers. Therefore, the long-term storage of subtypes/strains of Blastocystis using cryopreservation provides a suitable source of this parasite for the physiological, biochemical, and biological studies.

Methods

In the current study, seven xenic isolates including two separated isolates from ST1-3 and one isolate from ST6 were cryopreserved using a standard method with minor modifications. After 3 months, all isolates were recovered and cultivated in DMEM medium.

Results

The findings of the method showed all seven isolates were successfully recovered in DMEM medium. In addition, all isolates remained viable after several sub-cultures.

Conclusions

It seems that cryopreservation is a simple method that can provide a suitable condition for the long-term storage of Blastocystis.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Fig. 1
Fig. 2

Data availability

The data associated with this manuscript are included in the article.

References

  1. 1.

    Clark CG, Stensvold CR (2016) Blastocystis: isolation, xenic cultivation, and cryopreservation. Curr Protoc Microbiol:18(43):20A.1.1–20A.1.8. https://doi.org/10.1002/cpmc.18

  2. 2.

    Stensvold CR, Clark CG (2016) Current status of Blastocystis: a personal view. Parasitol Int 65(6 Pt B):763–771. https://doi.org/10.1016/j.parint.2016.05.015

  3. 3.

    Burgess SL, Gilchrist CA, Lynn TC, Petri WA (2017) Parasitic protozoa and interactions with the host intestinal microbiota. Infect Immun 85(8):e00101–17. https://doi.org/10.1128/IAI.00101-17

  4. 4.

    Javanmard E, Mohammad Rahimi H, Niyyati M, Asadzadeh Aghdaei H, Sharifdini M, Mirjalali H, Zali MR, Karanis P (2019) Molecular analysis of Blastocystis sp. and its subtypes from treated wastewater routinely used for irrigation of vegetable farmlands in Iran. J Water Health 17(5):837–844. https://doi.org/10.2166/wh.2019.045

  5. 5.

    Suresh K, Smith H (2004) Comparison of methods for detecting Blastocystis hominis. Eur J Clin Microbiol Infect Dis 23(6):509–511

  6. 6.

    Alinaghizade A, Mirjalali H, Mohebali M, Stensvold CR, Rezaeian M (2017) Inter- and intra-subtype variation of Blastocystis subtypes isolated from diarrheic and non-diarrheic patients in Iran. Infect Genet Evol 50:77–82. https://doi.org/10.1016/j.meegid.2017.02.016

  7. 7.

    Jalallou N, Iravani S, Rezaeian M, Alinaghizade A, Mirjalali H (2017) Subtypes distribution and frequency of Blastocystis sp isolated from diarrheic and non-diarrheic patients. Iran J Parasitol 12(1):63–68

  8. 8.

    Parija SC, Jeremiah S (2013) Blastocystis: taxonomy, biology and virulence. Trop Parasitol 3(1):17

  9. 9.

    Kumarasamy V, Anbazhagan D, Subramaniyan V, Vellasamy S (2018) Blastocystis sp., parasite associated with gastrointestinal disorders: an overview of its pathogenesis, immune modulation and therapeutic strategies. Curr Pharm Des 24(27):3172–3175. https://doi.org/10.2174/1381612824666180807101536

  10. 10.

    Nourrisson C, Wawrzyniak I, Cian A, Livrelli V, Viscogliosi E, Delbac F, Poirier P (2016) On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal caco-2 cell monolayers. Parasitol 143(13):1713–1722. https://doi.org/10.1017/S0031182016001396

  11. 11.

    Balakrishnan DD, Kumar SG (2014) Higher caspase-like activity in symptomatic isolates of Blastocystis spp. Parasit Vectors 7:219. https://doi.org/10.1186/1756-3305-7-219

  12. 12.

    Evans DB, Godfrey D, Lanham S, Lanotte Gv, Modabber F, Schnur L, et al (1989). In: David E (ed) Handbook on isolation, characterization and cryopreservation of Leishmania Geneva

  13. 13.

    Zheng H, Chen Y, Lu F, Liu M, Yang X, Fu X, Zhao Y, Huang B, Huang S, Kasper LH (2012) Cryopreservation of Toxoplasma gondii in infected murine tissues. Parasitol Res 111(6):2449–2453

  14. 14.

    Palmer D, Buening G, Carson C (1982) Cryopreservation of Babesia bovis for in vitro cultivation. Parasitol 84(3):567–572

  15. 15.

    Kilvington S, White D (1991) A simple method for the cryopreservation of free-living amoebae belonging to the genera Naegleria and Acanthamoeba. Eur J Protist 27(2):115–118

  16. 16.

    Miyake Y, Karanis P, Uga S (2004) Cryopreservation of protozoan parasites. Cryobiol 48(1):1–7

  17. 17.

    Rezaei Riabi T, Mirjalali H, Haghighi A, Rostami Nejad M, Pourhoseingholi MA, Poirier P, Delbac F, Wawrzyniak I, Zali MR (2018) Genetic diversity analysis of Blastocystis subtypes from both symptomatic and asymptomatic subjects using a barcoding region from the 18S rRNA gene. Infect Genet Evol 61:119–126. https://doi.org/10.1016/j.meegid.2018.03.026

  18. 18.

    Riabi TR, Haghighi A, Mirjalali H, Gol SMA, Karamati SA, Ghasemian M, Monfared AB, Aghamohammadi E, Zojaji H (2017) Study of prevalence, distribution and clinical significance of Blastocystis isolated from two medical centers in Iran. Gastroenterol Hepatol Bed Bench 10(Suppl1):S102–S107

  19. 19.

    Padilla-Vaca F, Ankri S, Bracha R, Koole LA, Mirelman D (1999) Down regulation of Entamoeba histolytica virulence by monoxenic cultivation with Escherichia coli O55 is related to a decrease in expression of the light (35-kilodalton) subunit of the Gal/GalNAc lectin. Infect Immun 67(5):2096–2102

  20. 20.

    Eckert J (1988) Cryopreservation of parasites. Experientia 44(10):873–877

  21. 21.

    Ragavan ND, Govind SK, Chye TT, Mahadeva S (2014) Phenotypic variation in Blastocystis sp. ST3. Parasite Vectors 7(1):404

  22. 22.

    Karamati SA, Mirjalali H, Niyyati M, Rezaei Riabi T, Yadegar A, Asadzadeh Aghdaei H, Haghighi A, Seyyed Tabaei SJ, Zali MR (2019) Comprehensive study of phenotypic and growth rate features of Blastocystis subtypes 1-3 and 6 in symptomatic and asymptomatic subjects. Iran J Parasitol 14(2):204–213

  23. 23.

    Costa AO, Gomes MA, Rocha OA, Silva EF (2006) Pathogenicity of Entamoeba dispar under xenic and monoxenic cultivation compared to a virulent E. histolytica. Rev Instit Med Trop de Sao Paulo 48(5):245–250

  24. 24.

    Moreira D, Santarém N, Loureiro I, Tavares J, Silva AM, Amorim AM, Ouaissi A, Cordeiro-da-Silva A, Silvestre R (2012) Impact of continuous axenic cultivation in Leishmania infantum virulence. PLoS Negl Trop Dis 6(1):e1469

Download references

Acknowledgements

This article has been extracted from the thesis written by Mr. Seyed Ahmad Karamati in School of Medicine, Shahid Beheshti University of Medical Science (Registration No: M341). The project was also financially supported by Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences (grant No. 887). The authors would like to thank all the staffs of the Foodborne and Waterborne Diseases Research Center for their collaboration.

Funding

This study was financially supported by the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran (grant no. 887).

Author information

Conceived and designed the experiments: HM SAK MN. Performed the experiments: HM SAK. Analyzed the data: HM SAK. Contributed reagents/materials/analysis tools/positive samples: MN AH HAA MRZ. Wrote the paper: HM SAK SJST. All authors read and approved the final version of the manuscript.

Correspondence to Maryam Niyyati or Hamed Mirjalali.

Ethics declarations

Conflict of interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

This study did not use any human participants, human data or human tissue and, therefore, did not require ethical approval.

Informed consent

This study did not use any individual person’s data in any form and, therefore, did not require consent for publication.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Karamati, S.A., Asadzadeh Aghdaei, H., Niyyati, M. et al. Development and Evaluation of Modified Cryopreservation for Long-Term Storage of Blastocystis Subtypes 1–3 and 6. Acta Parasit. (2020) doi:10.2478/s11686-019-00157-2

Download citation

Keywords

  • Blastocystis
  • Subtypes
  • Cryopreservation
  • Long-term storage