Abstract
Four isolates of larvalEchinococcus multilocularis originating from Switzerland (CH/1, CH/6 and CH/22) and Alaska (A/1) were used to prepare crude homogenate or small tissue fragments (STF) in Eagle's Minimal Essential Medium with Earle's salts (EMEM/A), or 0.2 g tissue blocks (TB) which were suspended in the same medium. After addition of dimethylsulfoxide or glycerol in final concentrations of 5% and 10% (v/v), respectively, aliquots of 1.0 ml, containing either 0.1 ml crude homogenate or STF, or one block of 0.2 g, were kept in cryotubes for 30 min at +2–4°C (precooling phase), cooled subsequently to lower temperatures following a two-step or three-step schedule and finally plunged into liquid nitrogen (−196°C). After storage for one week the samples were rapidly thawed at +37°C for approximately 3 min, washed in fresh EMEM/A (37°C) and transferred into the peritoneal cavity ofMeriones for viability testing. As judged by histological examinations and metacestode weights of each 24Meriones infected with cryopreserved homogenate, STF or TB, respectively, 46%, 87% or 100% contained viable, proliferating parasites. The best proliferation rate occurred when 10% glycerol was used as cryoprotectant and after precooling a three-step freezing schedule was employed (30 min at −28°C, 30 min at −80°C, transfer to liquid nitrogen). Cooling rates were determined as 0.7, 1.0 and 1.7°C min−1 for the precooling phase, step 1 and step 2, respectively, and estimated as 65°C min−1 for step 3. These results demonstrate that metacestodes ofE. multilocularis can be successfully maintained by cryopreservation without losing their proliferative capacity in the intermediate host.
Similar content being viewed by others
References
Ashwood-Smith MJ, Farrant J (eds) (1980) Low temperature preservation in medicine and biology. Pitman Medical, Tunbridge-Wells, United Kingdom
Campbell WC, Blair LS, Egerton JR (1972) Motility and infectivity ofHaemonchus contortus larvae after freezing. Vet Rec 91:13
Cohen LM, Eveland LK (1984) Cryopreservation ofSchistosoma mansoni sporocysts. J Parasitol 70:592
Coles GC, Simpkin KG, Briscoe MG (1980) Routine cyropreservation of ruminant nematode larvae. Res Vet Sci 28:391–392
Eckert J, Pohlenz J (1978) Zur Wirkung von Mebendazol auf Metazestoden vonMesocestoides corti undEchinococcus multilocularis. Tropenmed Parasitol 27:247–262
Eckert J, Thompson RCA, Mehlhorn H (1983) Proliferation and metastases, formation of larvalEchinococcus multilocularis. 1. Animal model, macroscopical and histological findings. Z Parasitenkd 69:737–748
Ham PJ (1982) Recovery ofTaenia crassiceps cysticerci (ERS ToI derived strain) from sub-zero temperatures. J. Helminthol 56:131–133
Ham PJ, James ER (1982) Protection of cryopreservedOnchocerca microfilariae (Nematoda) from dilution shock by the use of serum. Cryobiology 19:448–457
Ham PJ, James ER (1983) Successful cryopreservation ofBrugia pahangi third-stage larvae in liquid nitrogen. Trans Roy Soc Trop Med Hyg 77:815–819
Ham PJ, James ER, Bianco AE (1979)Onchocerca spp.: cryopreservation of microfilariae and subsequent development in the insect host. Exp Parasitol 47:384–391
Ham PJ, Townson S, James ER, Bianco AE (1981) An improved technique for the cryopreservation ofOnchocerca microfilariae. Parasitology 83:139–146
James ER (1980a) Protozoa and helminths. Parasites of man and animals. In: Ashwood-Smith MJ, Farrant J (eds). Low temperature preservation in medicine and biology. Pitman Medical Tunbridge-Wells, United Kingdom, p 155–185
James ER (1980b) Cryopreservation ofSchistosoma mansoni schistosomula using 40% v/v (10 M) methanol and rapid cooling. Cryo-Letters 1:535–544
James ER (1980c) Production and cryopreservation of schistosomula. Int J Nucl Med Biol 7:125–132
James ER, Dobinson AR (1984)Schistosoma mansoni: Interactive effects of irradiation and cryopreservation on parasite maturation and immunization of mice. Exp Parasitol 57:279–286
James ER, Farrant J (1976) Studies on preservation of schistosomula ofSchistosoma mansoni andS. mattheei. Cryobiology 13:625–630
James ER, Farrant J (1977) Recovery of infectiveSchistosoma mansoni schistosomula from liquid nitrogen: A step towards storage of a live schistosomiasis vaccine. Trans Roy Soc Trop Med Hyp 71:498–500
Jensen LA, Short JA, Andersen FL (1984) The survival of protoscolices ofEchinococcus multilocularis (Cestoda) at constant temperatures. Proc Helminthol Soc Wash 51:343–345
Leef JL, Hollingdale MR, Beaudoin RL (1981) Principles of cryopreservation of protozoan parasites and erythrocytes. WHO Document Mal. 81.940
Lewis FA, Stirewalt M, Leef JL (1984a)Schistosoma mansoni: Vaccination of mice with 10-krad-irradiated; cryopreserved schistosomules Exp Parasitol 57:261–268
Lewis FA, Stirewalt M, Leef JL (1984b)Schistosoma mansoni: Radiation dose and morphologic integrity of schistosomules as factor for an effective cryopreserved live vaccine. Am J Trop Med Hyg 33:125–131
Lok JB, Mika-Grieve M, Grieve RB (1983) Cryopreservation ofDirofilaria immitis microfilariae and third-stage larvae. J Helminthol 57:319–324
Lowrie RC jr (1983) Cryopreservation of the microfilariae ofBrugia malayi, Dirofilaria corynodes, andWuchereria bancrofti. Am J Trop Med Hyg 32:138–145
Lyman JR, Marchin GL (1984) Cryopreservation ofGiardia lamblia with dimethyl sulfoxide using a Dewar flask. Cryobiology 21:170–176
Mehlhorn H, Eckert J, Thompson RCA (1983) Proliferation and metastases formation of larvalEchinococcus multilocularis. II. Ultrastructure. Z Parasitenkd 69:749–763
Mutetwa SM, James ER (1984a) Cryopreservation ofPlasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing. Cryobiology 21:329–339
Mutetwa SM, James ER (1984b) Cryopreservation ofPlasmodium chabaudi II. Cooling and warming rates. Cryobiology 21:552–558
Nguyen-Dinh P, Chemangey-Masaba S, Spencer HC, Campbell CC, Chin W, Collins WE (1981) Comparative studies on the cryopreservation of malaria parasites. WHO Document Mal 81.941
Ohnishi K, Nakao M, Inaoka T (1984) Viability and infectivity of protoscolices ofEchinococcus multilocularis stored at different temperatures Int J Parasitol 14:577–580
Parfitt JW (1971) Deep freeze preservation of nematode larvae Res Vet Sci 12:488–489
Rew RS, Campbell WC (1983) Infectivity ofHaemonchus contortus after freezing for ten years over liquid nitrogen. J Parasitol 69:251–252
Stirewalt MA, Lewis FA, Cousin CE, Leef JL (1984) Cryopreservation of schistosomules ofSchistosoma mansoni in quantity. Am J Trop Med Hyg 33:116–124
van Wyk JA, Gerber HM, van Aardt WP (1977) Cryopreservation of the infective larvae of the common nematodes of ruminants. Onderstepoort J Vet Res 44:173–194
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Eckert, J., Ramp, T. Cryopreservation ofEchinococcus multilocularis metacestodes and subsequent proliferation in rodents (Meriones). Z. Parasitenkd. 71, 777–787 (1985). https://doi.org/10.1007/BF00926803
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00926803