The Survival of Protoscolices of Echinococcus Multilocularis (Cestoda) at Constant Temperatures

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ternal wall of the aorta (Chodnik, 1958, Ann. (1955, J. Am. Vet. Med. Assn. 127:499-505). Trop. Med. Parasitol. 52:145-148; Cheema and Soulsby (1965, op. cit.) recorded no evidence that Ivogli, 1978, Vet. Pathol. 15:495-505). Parasites E. poeli has pathogenic significance. However, were never found lying free in the lumen of aortas in this study many parasite nodules were some- infected with O. armillata (Al-Zubaidy, 1973, times found circumscribed along the aortic wall Trans. R. Soc. Trop. Med. Hyg. 67:436). With so that the patent lumen of the aorta was reduced E. poeli, the male resides within the nodules, to about a third of its usual size. The nodules, whereas the anterior portion of the female is fixed together with the attached parasites and obtu- in the nodule, and the rest of the body swings rating thrombi, conceivably could cause obstruc- free in the lumen of the vessel. In this survey, tion to blood flow and lead to heart failure. the female varied from 110 to 250 mm in length Thrombosis and embolism may also occur from with a maximum width of 2.5 mm, whereas the detached pieces of regressing nodules and dead males were 55-60 mm long and about 0.5 mm parasites. These factors may reduce the endur- wide. Microfilariae and embryonated eggs were ance and usefulness of severely affected animals. easily teased out of the females. Morphologic Further studies are needed to determine the de- features of the parasite, gross lesions induced, velopmental cycle of the parasite and its patho- and predilection site are similar to those reported genic effects in the Philippine water buffalo. for E. poeli. Voucher specimens of E. poeli from Bubalus Lapage (1962, Monnig's Veterinary Helmin- bubalis have been deposited in the USNM Hel- thology and Entomology, 5th ed. Williams and minthological Collection, USDA, Beltsville, Wilkins Co., Baltimore. 600 pp.) stated that in- Maryland (No. 77680). fected animals show no clinical signs. Microfi- The authors gratefully acknowledge the assis- lariae have not been associated with skin lesions tance of Drs. Primo Arambulo and Pedro Boado, comparable to those produced by E. schneideri formerly of the Veterinary Inspection Board, City in sheep as described by Jensen and Seghetti of Manila.

Proc. Helminthol. Soc. Wash. 51(2), 1984, pp. 343-345

Research Note The Survival of Protoscolices of Echinococcus multilocularis (Cestoda) at Constant Temperatures

LAURITZ A. JENSEN, ' JEFFERY A. SHORT, AND PERRON L. ANDERSEN Department of Zoology, Brigham Young University, Provo, Utah 84602

Echinococcus multilocularis, the causative 1979, J. Parasitol. 65:34), and in 1979 an au- agent of multilocular or alveolar hydatid disease, tochthonous case of alveolar hydatid disease in is primarily boreal in distribution, occurring nat- a human was identified in Minnesota (Gamble urally in foxes, coyotes, and various species of et al., 1979, J. Am. Med. Assoc. 241:904-907). rodents (Rausch, 1967, Ann. Parasitol. 42:19- Echinococcus granulosus is also considered to 63). In the contiguous United States, the tape- be a public health problem in several states in worm is endemic to several north-central and the United States (Pappaioanou et al., 1977, Am. mountain states (Leiby et al., 1970, J. Parasitol. J. Trop. Med. Hyg. 26:732-742; Schantz et al., 56:1141-1150; Kritsky et al., 1977, Am. J. Trop. 1977, Am. J. Trop. Med. Hyg. 26:121-126; Crel- Med. Hyg. 26:1046-1047; Eastman and Worley, linetal., 1982, Am. J. Epidemiol. 116:463-474). In efforts to understand the distribution of E. granulosus and the potential for increased trans- 1 Present address: Department of Microbiology, The University of Health Sciences, 2105 Independence mission, researchers have studied the impact of Blvd., Kansas City, Missouri 64124. temperature extremes upon the infective stages.

Andersen and Loveless (1978, J. Parasitol. 64: Table 1 shows the percent survival of the pro- 78-82) reported that protoscolices of E. granu- toscolices of E. multilocularis when stored with- losus in intact hydatid cysts of ovine origin could in the three treatment groups described above. survive 16 days when stored at 1°C or 10°C. Colli Protoscolices did not survive for even 1 day at and Williams (1972, J. Parasitol. 58:422-426) the temperature extremes of —10 or 50°C, re- showed that eggs of E. granulosus could survive gardless of the method of storage. These two tem- 24 hr at -30°C with no loss of infectivity to perature extremes tested in our laboratory ap- laboratory rodents, and Sweatman and Williams proximate those that occur under normal field (1963, Parasitology 53:339-390) were able to conditions in our region. In 1976, the average maintain eggs in viable condition for 7.5 mo at monthly minimum temperature at soil surface -6°C. under a short pasture grass cover was — 6.1°C Although considerably less research has been during the month of December, and the average done on the bionomics of E. multilocularis, monthly maximum temperature during August Schiller (1955, J. Parasitol. 41:578-582) found of that year at the same site was 52.1°C (Bullick that eggs of this species were still infective to red- and Andersen, 1978, Gt. Basin Nat. 38:369-378). backed voles when the tapeworms were stored The results on survival of protoscolices in our for 42 days within carcasses of arctic foxes at study suggest that these average monthly tem- —26°C. To our knowledge, no reports are yet perature extremes (with shorter periods within available on the survivability of E. multilocu- each month of lower and higher daily extremes) laris protoscolices. The purpose of our study was would definitely decrease the survival of E. mul- to determine the survival of this larval stage when tilocularis protoscolices if exposed to natural stored at constant temperatures. conditions in our region. Cotton rats that had been experimentally in- Protoscolices survived as long as 64 days when fected with an isolate of E. multilocularis (orig- stored within intact carcasses at both 0 and 5°C inally received through the courtesy of Dr. Rob- (43 and 53%, respectively); however, it is im- ert L. Rausch, School of Public Health, University portant to note that the protoscolices were not of Washington, Seattle, Washington, and then in a frozen condition when stored at 0°C. Pro- maintained via secondary perpetuation in our toscolices within carcasses and within the excised laboratory) were the source of test material used 1-g cyst samples were shown to freeze at ap- in our experiments. Rats were killed in an ether proximately -3°C, whereas those in 1 ml 0.85% jar and used as follows: the first treatment group NaCl froze at — 1°C. The ability of the organisms consisted of intact carcasses containing a 3-5- to survive as long as 64 days at 0 or 5°C illustrates mo cystic growth of E. multilocularis, the second the potential for transmission over extended time treatment group consisted of 1 -g samples of cyst- periods during seasons when such temperature ic material removed from similar source rats and ranges might exist in nature. Also, such survival transferred to 100 x 15-mm polystyrene petri capabilities as noted for the protoscolices at either dishes containing 0.85% NaCl, and the third 0 or 5°C suggest that protoscolices might be stored treatment group consisted of approximately 1,000 for several weeks in a refrigerator and still be free protoscolices stored in 1 ml of 0.85% NaCl valuable for certain experimental studies in the in stoppered, 65 x 10-mm pyrex test tubes. Suf- laboratory or as inoculum in the perpetuation of ficient numbers of each of the three treatment secondary echinococcosis in laboratory rodents. types were then placed in appropriate tempera- At 10°C only 8% of the protoscolices survived ture storage cabinets to permit an evaluation of for 32 days and none at 64 days. Survival at the survivability of protoscolices within each sample storage temperature of protoscolices within ex- after 1, 2, 4, 8, 16, 32, and 64 days storage and cised 1 -g cyst samples was 70% at 16 days and at constant temperatures of —10, 0, 5, 10, 20, 0% at 32 days. These levels agree well with those 25, 30, 40, and 50°C. Survival was determined reported for E. granulosus, wherein 22% sur- by subjecting the protoscolices to trypsin, pan- vived in excised hydatid cysts of sheep origin for creatin, and sodium taurocholate (Smyth, 1967, 16 days and 0% at 32 days (Andersen and Love- Parasitology 57:111-133) and then examining less, 1978, loc. cit.). them with a compound microscope for evagina- At constant storage temperatures near to that tion, motility, and, in questionable cases, the of normal laboratory working conditions, the presence of flame-cell activity. maximum survival time of E. multilocularis pro-

Table 1. Percent survival of protoscolices of Echinococcus multilocularis stored in carcasses of Sigmodon his- pidus, excised niultilocular hydatid cysts, or in 1 ml 0.85% NaCl at constant temperatures.

Days Temperature (°C) Medium 1 2 4 8 16 32 64 128

-10 Intact carcass 0 Cyst 0 Saline 0 0 Intact carcass 100 100 92 98 76 88 43 0 Cyst 100 100 100 35 0 Saline 91 67 0 5 Intact carcass 100 100 100 100 95 94 53 0 Cyst 100 100 93 68 70 0 Saline 100 74 83 0 10 Intact carcass 95 97 100 100 100 8 0 Cyst 100 98 100 100 70 0 Saline 90 67 10 3 1 0 20 Intact carcass 100 100 100 100 23 0 Cyst 100 100 97 0 Saline 100 100 100 0 25 Intact carcass 100 100 94 0 Cyst 100 100 100 0 Saline 100 100 95 0 30 Intact carcass 100 100 0 Cyst 100 90 0 Saline 100 93 0 40 Intact carcass 78 0 Cyst 80 0 Saline 83 0 50 Intact carcass 0 Cyst 0 Saline 0

toscolices was 16 days (23%) at 20°C, 4 days ing could facilitate increased transmission. In ad- (94%) at 25°C, and 2 days (100%) at 30°C. Bac- dition to the transmission cycle in the wild, E. terial overgrowth was apparent at these warmer multilocularis has been reported to occur infre- conditions and probably had a deleterious effect quently in domestic cats and house mice (Leiby upon the parasite. At 40°C the maximum sur- and Kritsky, 1972, J. Parasitol. 58:1213-1215), vival time was only 1 day, with survival of 78, which may maintain this cestode in urban areas. 80, and 83% within intact carcasses, in excised Also, sled dogs are known to be important in cyst material, and in 1 ml 0.85% NaCl, respec- rodent capture and subsequent transmission to tively. humans in some regions of Alaska (Rausch, 1972, In general, our results illustrate the marked Arch. Env. Hlth. 25:246-252). We suggest, there- adaptation of E. multilocularis for perpetuation fore, that individuals living in areas where E. under natural field conditions, and underscore multilocularis may occur have their dogs and cats the potential for an increased distribution of the treated at least annually with an echinococcoci- parasite into more temperate zones. Indeed, fur- dal compound and also use precautionary meas- ther spread of E. multilocularis in the United ures to prevent them from having ready access States now seems likely (Wilson and Rausch, to all rodents in that region. 1980, Am. J. Trop. Med. Hyg. 29:1340-1355), This study was supported by NIH Grant AI- and the ability of the protoscolices to survive for 10588. more than 2 mo at temperatures just above freez-