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Studies on tapeworm physiology

XI. In vitro cultivation of Echinococcus granulosus from the protoscolex to the strobilate stage

Published online by Cambridge University Press:  06 April 2009

J. D. Smyth
J. D. Smyth
Affiliation:
Department of Zoology, Australian National University, Canberra, Australia
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Extract

Some factors controlling differentiation of the protoscoleces of Echinococcus into strobilate or cystic stages, in vitro, have been identified. Development in a strobilate direction requires (a) evagination of the scolex, (b) contact of the evaginated scolex with a suitable nutritive substrate. These conditions are satisfied by treating fresh protoscoleces with pepsin and by evaginating them with trypsin + pancreatin + dog bile and subsequently culturing in diphasic media.

The most satisfactory diphasic system so far developed consisted of a base of bovine serum (coagulated by heating) with a liquid phase of Parker 199 medium +20% hydatid fluid and a gas phase of 8% O2+5% CO2 in N2. In this diphasic system, protoscoleces have been cultured to the 3-proglottid strobilate stage with the anlagen of the male and female genitalia formed: the first proglottid appeared after 30 days culture in vitro compared with 16 days in vivo; three proglottids were formed after 60 days. Development in vitro stopped just short of mature egg-producing worms.

That an intimate contact of a ‘placental’ nature takes place between the parasite and the solid proteinaceous base is shown by the fact that strobilization never took place in cultures lacking bases or in those in which the serum base was separated from the protoscoleces by a cellulose membrane. Cytological and ultra-structure studies of an evaginated protoscolex revealed that fully developed microtriches were present only in the rostellar region and not in the post-sucker region. It is concluded that it is this anterior region which makes initial contact with the nutritive substrate—a contact which appears to provide the ‘strobilization stimulus’. It is not known if the ‘strobilization stimulus’ is stimulatory (i.e. nervous) or nutritive or both. Contact with a non-nutritive base (i.e. agar) does not induce strobilization.

In any one sample of protoscoleces, after a high initial evagination, only about 30–40% finally remained evaginated and continued to show sucker activity. The degree of evagination appears to be characteristic for any sample of protoscoleces and may be related to the source of the hydatid cysts. It may prove to be a genetically determined character.

Evaginated protoscoleces became inactive under certain conditions and the degree of activity seems to be related to stimulatory and nutritive factors. The activity of evaginated protoscoleces was used as a criterion for assessing the effectiveness of a culture medium or condition. Evaginated protoscoleces which lost their activity developed small posterior bladders as did the majority of unevaginated protoscoleces. In both cases, posterior bladders enlarged and secreted a PAS-positive layer which formed the laminated membrane of a miniature hydatid cyst.

The well-known appearance of vesicular forms in cultures appears to be related to unfavourable culture conditions in vitro, such as a low or high pH or a low or high O2 tension.

It is pointed out that since the morphogenesis of Echinococcus protoscoleces can now be controlled to a considerable extent in vitro and since the contents of any one cyst have the same genotype, this organism provides exceptionally interesting material for studies on morphogenesis.

Acknowledgement is made to the Australian Wool Board, the National Institutes of Health, U.S. Public Health Services Grant No. AI-04707–03 and the World Health Organizati on for generous financial assistance in support of this work.

The skilful technical assistance of Mr A. B. Howkins and Mrs Mary Barton is also acknowledged.

Type
Research Article
Information
Parasitology , Volume 57 , Issue 1 , February 1967 , pp. 111 - 133
Copyright
Copyright © Cambridge University Press 1967

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