In vitro viability test for the eggs of Echinococcus granulosus: a rapid method

Parasitology Research volume 110, pages925–930(2012)Cite this article

Abstract

In this study an attempt was made to develop an efficient, rapid, simple, and reproducible method for the in vitro viability test of Echinococcus granulosus eggs. The eggs were obtained from an experimentally infected dog and kept at 4°C until use. To prepare the dead or damaged eggs, the eggs were heated in hot water (69–72°C for 10 min), preserved in 70% ethyl alcohol (16 days) or exposed to direct sunlight (18 h). Sodium hypochlorite (0.5–0.7%) was used for the hatching process, and the hatched oncospheres were stained with 0.1% eosin for the viability test. With 0.5% sodium hypochlorite, the hatching rates for viable eggs and eggs killed or damaged by heat (69°C), 70% ethyl alcohol, and direct sunlight were 96%, 97.5%, 91.5%, and 94.6% respectively and there was no significant difference between the hatching rate for viable and dead or damaged eggs (p > 0.05). After staining with 0.1% eosin, the rates of the viable oncospheres hatched from viable eggs and the eggs killed or damaged by heat (69°C), 70% ethyl alcohol, and direct sunlight were 97.5% 3.6%, 7%, and 10.5%, respectively. The difference between the rates of viable oncospheres hatched from viable and dead or damaged eggs was extremely significant (P < 0.0001). With 0.7% sodium hypochlorite, the hatching rates for viable and dead eggs (killed by 72°C for 10 min) were 99.1% and 99.9%, respectively. In this condition, the rate of viable oncospheres was an average of 98.5% for viable eggs and 0.0% for dead ones. The results of this study showed that hatching of eggs by 0.7% sodium hypochlorite and staining of hatched oncospheres by 0.1% eosin are practical methods for the differentiation of viable and nonviable (dead) eggs of Echinococcus granulosus.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

US$ 39.95

Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2

References

  1. AVMA (American Veterinary Medical Association) (1993) Report of the AVMA panel on euthanasia. J Am Vet Med Assoc 202:229–249

    Google Scholar 

  2. Chapalamadugu KC, Busboom JR, Nelson ML, Hancock DD, Tang J, Jasmer DP (2008) Taenia taeniaeformis: effectiveness of staining oncospheres is related to both temperature of treatment and molecular weight of dyes utilized. Vet Parasitol 151:203–211

    PubMed  Article  CAS  Google Scholar 

  3. Ciarmela ML, Sanchez Thevenet P, Alvarez HM, Minvielle MC, Basualdo JA (2005) Effect of Paecilomyces lilacinus on the viability of oncospheres of Taenia hydatigena. Vet Parasitol 131:61–64

    PubMed  Article  CAS  Google Scholar 

  4. Ciftci IH, Esme H, Sahin DA, Solak O, Sezer M, Dilek ON (2007) Effect of octenidine dihydrochloride on viability of protoscoleces in hepatic and pulmonary hydatid diseases. J Natl Med Assoc 99:674–677

    PubMed  Google Scholar 

  5. Colli CW, Williams JF (1972) Influence of temperature on the infectivity of eggs of Echinococcus granulosus in laboratory rodents. J Parasitol 58:422–426

    PubMed  Article  CAS  Google Scholar 

  6. Coman BJ, Rickard MD (1977) A comparison of in vitro and in vivo estimates of the viability of Taenia pisiformis eggs aged under controlled conditions, and their ability to immunise against a challenge infection. Int J Parasitol 7:15–20

    PubMed  Article  CAS  Google Scholar 

  7. Diker AI, Tinar R, Senlik B (2007) Viability of Echinococcus granulosus protoscolices at different conditions. Vet Parasitol 150:84–87

    PubMed  Article  Google Scholar 

  8. Esme H, Ciftçi IH, Solak O, Dilek ON (2007) Investigation of the germicidal effect of usnic acid, betadine, savlosol, and desderman on the protoscolexes of lung hydatid cysts. Türkiye Parazitol Derg 31:101–104

    PubMed  Google Scholar 

  9. Gallie GJ, Sewell MM (1970) A technique for hatching Taenia saginata eggs. Vet Rec 86:749

    PubMed  Article  CAS  Google Scholar 

  10. Gavidia CM, Gonzalez AE, Barron EA, Ninaquispe B, Llamosas M, Verastegui MR, Robinson C, Gilman RH (2010) Evaluation of oxfendazole, praziquantel and albendazole against cystic echinococcosis: a randomized clinical trial in naturally infected sheep. PLoS Negl Trop Dis 4(2):1–8, 23

    Article  Google Scholar 

  11. Gemmell MA (1978) The effect of weather on tapeworm eggs and its epidemiological complications. Weather and Parasitic Animal Disease; World Meteorological Organization technical Note 159: 83–94

  12. Gold D (1997) Assessment of the viability of Schistosoma mansoni schistosomula by comparative uptake of various vital dyes. Parasitol Res 83:163–169

    PubMed  Article  CAS  Google Scholar 

  13. Heath DD, Smyth JD (1970) In vitro cultivation of Echinococcus granulosus, Taenia hydatigena, T. ovis, T. pisiformis and T. serialis from oncosphere to cystic larva. Parasitology 61:329–343

    PubMed  Article  CAS  Google Scholar 

  14. Ito A, Yamada T, Ishiguro T (1996) Vaccination of rats with frozen eggs, ethanol-fixed eggs and frozen oncospheres with or without embryophoric blocks of Taenia taeniaeformis. Kisechugaku Zasshi 45:330–332

    Google Scholar 

  15. Kopper JJ, Mansfield LS (2010) Development of improved methods for delivery of Trichuris muris to the laboratory mouse. Parasitol Res 107:1103–1113

    PubMed  Article  Google Scholar 

  16. Kváč M, Květoňová D, Salát J, Ditrich O (2007) Viability staining and animal infectivity of Cryptosporidium andersoni oocysts after long-term storage. Parasitol Res 100:213–217

    PubMed  Article  Google Scholar 

  17. Laws GF (1968) The hatching of taeniid eggs. Exp Parasitol 23:1–10

    PubMed  Article  CAS  Google Scholar 

  18. Lightowlers MW, Mitchell GF, Bowtell DDL, Anders RF, Rickard MD (1984) Immunisation against Taenia taeniaeformis in mice: studies on the characterization on antigen from oncospheres. Int J Parasitol 14:321–333

    PubMed  Article  CAS  Google Scholar 

  19. Mackie A, Parnell IW (1967) Some observations on Taeniid ovicides: the effects of some organic compounds and pesticides on activity and hatching. J Helminthol 41:167–210

    Article  Google Scholar 

  20. Moazeni M, Alipour-Chaharmahali MR (2011) Echinococcus granulosus: in vitro effectiveness of warm water on protoscolices. Exp Parasitol 127:14–17

    PubMed  Article  Google Scholar 

  21. Moazeni M, Larki S (2010) In vitro effectiveness of acidic and alkline solutions on scolices of hydatid cyst. Parasitol Res 106:853–856

    PubMed  Article  Google Scholar 

  22. Moazeni M, Nazer A (2010) In vitro effectiveness of garlic (Allium sativum) extract on scolices of hydatid cyst. W J Surg 34:2677–2681

    Article  Google Scholar 

  23. Morseth DJ (1965) Ultrastructure of developing taeniid embryophores and associated structures. Exp Parasitol 16:207–216

    PubMed  Article  CAS  Google Scholar 

  24. Morseth DJ (1966) Chemical composition of embryophoric blocks of Taenia hydatigena, Taenia ovis and Taenia pisiformis eggs. Exp Parasitol 18:347–354

    PubMed  Article  CAS  Google Scholar 

  25. Negita T, Ito A (1994) In vitro hatching of oncospheres of Taenia taeniaeformis using eggs isolated from fresh, frozen, formalin-fixed and ethanol-fixed segments. J Helminthol 68:271–272

    PubMed  Article  CAS  Google Scholar 

  26. Nieland ML (1968) Electron microscope observation on the egg of Taenia taeniaeformis. J Parasitol 54:957–969

    PubMed  Article  CAS  Google Scholar 

  27. Sakamoto T (1981) Electron microscopical observation on the egg of Echinococcus multilocularis. Mem Fac Agr Kagoshima Univ 17:165–174

    Google Scholar 

  28. Sánchez Thevenet P, Jensen O, Drut R, Cerrone GE, Grenovero MS, Alvarez HM, Targovnik HM, Basualdo JA (2005) Viability and infectiousness of eggs of Echinococcus granulosus aged under natural conditions of inferior arid climate. Vet Parasitol 133(1):71–77

    Article  Google Scholar 

  29. Silverman PH (1954) Studies on the biology of some tape worms of the genus Taenia. 1. Factors affecting hatching and activation of taeniid ova, and some criteria of their viability. Ann Trop Med Parasitol 48:207–215

    PubMed  CAS  Google Scholar 

  30. Stevenson P (1983) Observations on the hatching and activation of fresh Taenia saginata eggs. Ann Trop Med Parasitol 77:399–404

    PubMed  CAS  Google Scholar 

  31. Swiderski Z (1982) Echinococcus granulosus: embryonic envelop formation. Proceeding of 10th International congress of Electron Microscopy 3: 513

  32. Takemoto Y, Negita T, Ohnishi K, Suzuki M, Ito A (1995) A simple method for collecting eggs of taeniid cestodes from fresh, frozen or ethanol-fixed segments. Int J Parasitol 25:537–538

    PubMed  Article  CAS  Google Scholar 

  33. Thompson RCA, Lymbery AJ (1995) Echinococcus and hydatid disease. CAB International, Wallingford

    Google Scholar 

  34. Veit P, Bilger B, Schad V, Schäfer J, Frank W, Lucius R (1995) Influence of environmental factors on the infectivity of Echinococcus multilocularis eggs. Parasitology 110:79–86

    PubMed  Article  Google Scholar 

  35. Wachira TM, Macpherson CN, Gathuma JM (1991) Release and survival of Echinococcus eggs in different environments in Turkana, and their possible impact on the incidence of hydatidosis in man and livestock. J Helminthol 65:55–61

    PubMed  Article  CAS  Google Scholar 

  36. Wang IC, Ma YX, Kuo CH, Fan PC (1997) A comparative study on egg hatching methods and oncosphere viability determination for Taenia solium eggs. Int J Parasitol 27:1311–1313

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are thankful to Shiraz University for providing financial assistance (Grant number 87-GR-VT-24). We kindly thank Mr. A. Mootabi Alavi for his kind assistance in performing this study.

Author information

Affiliations

  1. Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, 71345-1731, Iran

    Mohammad Moazeni & Ehsan Rakhshandehroo

Authors
  1. Mohammad Moazeni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ehsan Rakhshandehroo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mohammad Moazeni.

Electronic supplementary material

Below is the link to the electronic supplementary material.

WMV 3232 kb

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Moazeni, M., Rakhshandehroo, E. In vitro viability test for the eggs of Echinococcus granulosus: a rapid method. Parasitol Res 110, 925–930 (2012). https://doi.org/10.1007/s00436-011-2578-y

Download citation

Keywords

  • Methylene Blue
  • Ethyl Alcohol
  • Sodium Hypochlorite
  • Direct Sunlight
  • Viability Test