Parasitology Research

, Volume 73, Issue 4, pp 358–365 | Cite as

Cryopreservation of Dictyocaulus viviparus third-stage larvae and Trichinella spiralis muscle larvae

  • I. Andermatt-Mettler
  • J. Eckert
  • Th. Ramp
  • B. Gottstein
Original Investigations

Abstract

In cryopreservation studies with third-stage larvae of Dictyocaulus viviparus, best results were achieved by incubating larvae in 0.05% NaOCl at 37‡ C to remove the sheath, followed by cooling at a rate of 1‡ C min per min down to about 0‡ C. After an equilibration time of 10 min at +4‡ C with or without 4% polyethylene glycol-400 as cryoprotectant, samples were frozen at the same cooling rate to an intermediate temperature of −20‡ C, maintained at this temperature for 10 min and finally plunged into liquid nitrogen for storage. Three groups of 3 calves were infected with the following batches of third-stage larvae: (a) fresh, sheated; (b) fresh, exsheathed; (c) exsheathed, cryopreserved for 13 weeks in liquid nitrogen and subsequently thawed. Although 62% of group (c) were regarded as viable in vitro, their infectivity to calves was low and only an average of 0.08% of the inoculated larvae (3000 per animal) developed into adult lungworms (=infectivity rate). Average infectivity rates of fresh, sheathed (a) and fresh, exsheathed (b) larvae were much higher (38.3% and 29.7%) and not significantly different from each other. Two of the calves inoculated with previously frozen larvae and all of the calves infected with fresh larvae excreted first-stage larvae in their faeces, but the latter groups in higher quantities. The results show that cryopreservation of exsheathed third-stage larvae of D. viviparus is possible, but for strain maintenance infection doses greater than 3000 larvae should be used for inoculation of calves. Thawed Trichinella spiralis larvae, which had been cryopreserved in tissue blocks (approx. 2 g) with addition of undiluted ethylene glycol (0.2 ml), revealed in vitro viability rates of 60% after storage at −10‡ C for 2 and 13 weeks and were still infective to rats.

Keywords

Ethylene Glycol Liquid Nitrogen Cool Rate Infectivity Rate Equilibration Time 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Ashwood-Smith MJ, Farrant J (eds) (1980) Low temperature preservation in medicine and biology. Pitman Medical, Tunbridge WellsGoogle Scholar
  2. Boch J, Supperer R (1983) VeterinÄrmedizinische Parasitologie, 3rd edn. Parey, BerlinGoogle Scholar
  3. Bunke V (1983) Dictyocaulus viviparus (Bloch 1782) beim Rind: Entwicklung, überleben und übertragung freilebender Stadien. Thesis, TierÄrztliche Hochschule HannoverGoogle Scholar
  4. Cornwell RL, Jones RM (1970) Determination of viability and infectivity of Dictyocaulus viviparus larvae after storage. Res Vet Sci 11:484–485Google Scholar
  5. Dobinson AR (1980) Preliminary studies on the cryopreservation of Trichinella spiralis. Trans R Soc Trop Med Hyg 74:111–112Google Scholar
  6. Eckert J (1986) Alternativen zu Tierversuchen in der Parasitologie. Dtsch TierÄrztl Wschr 93:380–382Google Scholar
  7. Eckert J, Inderbitzin F (1978) Arrested development of Dictyocaulus viviparus in cattle and effect of fenbendazole against inhibited stages. In: Borgsteede FHM, Armour J, Jansen J (eds) Facts and reflections III: workshop on arrested development of nematodes in sheep and cattle. Central Diergeneeskundig Instituut. Lelystad, pp 125–135Google Scholar
  8. Eckert J, Ramp Th (1985) Cryopreservation of Echinococcus multilocularis metacestodes and subsequent proliferation in rodents (Meriones). Z Parasitenkd 71:777–787Google Scholar
  9. Eisenegger H, Eckert J (1975) Zur Epizootologie und Prophylaxe der Dictyocaulose und der Trichostrongylidosen des Rindes. Schweiz Arch Tierheilkd 117:255–286Google Scholar
  10. Engvall E, Perlman P (1972) Enzyme-linked immunosorbent assay, ELISA III. J Immunol 109:129–135Google Scholar
  11. Enigk K, Düwel D (1961) Die Lebensdauer der ansteckungsfÄhigen Larven des Rinderlungenwurmes. TierÄrztl Umsch 16:415–418Google Scholar
  12. Fuller BJ, James ER (1985) Cryopreservation of isolated rat hepatocytes in pellet form. Cryo-letters 6:49–56Google Scholar
  13. Inderbitzin F (1976) Experimentell erzeugte Entwicklungshemmung von Dictyocaulus viviparus des Rindes. Thesis, ZurichGoogle Scholar
  14. Ito S (1975) Infectivity of infective larvae of Dictyocaulus viviparus stored at low temperature. J Jpn Vet Med Ass 28:406–409Google Scholar
  15. James ER (1980) Preliminary studies on the Cryopreservation of Dictyocaulus viviparus. Trans R Soc Trop Med Hyg 74:114Google Scholar
  16. James ER (1985) Cryopreservation of helminths. Parasitology Today 1:134–139Google Scholar
  17. James ER, Peacock R (1986) Studies on the Cryopreservation of Dictyocaulus viviparus (Nematoda) third-stage larvae. J Helminthol 60:65–73Google Scholar
  18. James ER, Dobinson AR, Lucas SB, Andrews BJ, Bickle QD, Taylor MG, Ham PJ (1985) Protection of sheep against Schistosoma bovis using cryopreserved radiation-attenuated schistosomula. J Helminthol 59:51–55Google Scholar
  19. James ER, Otieno M, Harrison R, Dobinson AR, Monorei J, Else JG (1986) Partial protection of baboons against Schistosoma mansoni using radiation-attenuated cryopreserved schistosomula. Trans R Soc Trop Med Hyg 80:378–384Google Scholar
  20. Jarrett WFH, McIntyre WIM, Jennings FW, Mulligan W (1957) The natural history of parasitic bronchitis with notes on prophylaxis and treatment. Vet Rec 69:1329–1336Google Scholar
  21. Leef JL, Hollingdale MR, Beaudoin RL (1981) Principles of Cryopreservation of protozoan parasites and erythrocytes. WHO Geneva (Document Mal 81.940)Google Scholar
  22. Lewis FA, Stirewalt M, Leef JL (1984) Schistosoma mansoni: radiation dose and morphologic integrity of schistosomules as factors for an effective cryopreseved live vaccine. Am J Trop Med Hyg 33:125–131Google Scholar
  23. Ramp T, Eckert J, Christen C (1986) Erfahrungen mit der Gefrierkonservierung dritter Larvenstadien von Trichostrongyliden der WiederkÄuer. Schweiz Arch Tierheilkd 128:79–86Google Scholar
  24. Rose JH (1956) The bionomics of the free-living larvae of Dictyocaulus viviparus. J Comp Pathol 66:228–240Google Scholar
  25. Sachs L (1978) Angewandte Statistik, Statistische Methoden und ihre Anwendungen, 5. Springer, BerlinGoogle Scholar
  26. Smith HJ (1984) Preconditioning of Trichinella spiralis nativa larvae in musculature to low temperatures. Vet Parasitol 17:85–90Google Scholar
  27. Taranik KT (1981) Resistance of Dictyocaulus filaria larvae to especially cold temperatures (−196‡ C). Legochnye gel'mintozy zhvachnykh zhivotnykh (Nauchnye Trudy Vaskhnil). (in Russian) Kolos 9–20Google Scholar
  28. Van Wyk JA, Gerber HM, Van Aardt WP (1977) Cryopreservation of the infective larvae of the common nematodes of ruminants. Onderstepoort J Vet Res 44:173–193Google Scholar
  29. Van Wyk JA, Gerber HM, Alves RMR (1984) Methods of infesting sheep with gastrointestinal nematodes after Cryopreservation: dosing of larvae in gelatin capsules compared to dosing of larvae in water suspension. Onderstepoort J Vet Res 51:217–221Google Scholar

Copyright information

© Springer-Verlag 1987

Authors and Affiliations

  • I. Andermatt-Mettler
    • 1
  • J. Eckert
    • 1
  • Th. Ramp
    • 1
  • B. Gottstein
    • 1
  1. 1.Institute of ParasitologyUniversity of ZürichZürichSwitzerland

Personalised recommendations