In vitro culture of the strobilar stage of Echinococcus granulosus (sheep strain): A review of basic problems and results

Abstract

The basic problems of culturing Echinococcus granulosus (sheep strain) in vitro, from the protoscolex to the sexually mature adult, are reviewed. Suitable procedures for the transport, storage and sterile dissection of hydatid material are described. Protoscoleces for culture are freed from brood capsules by treatment with pepsin, washed repeatedly in saline, and evaginated in a solution of sodium taurocholate. Evaginated protoscoleces are cultured in a diphasic medium comprising approximately 80% Parker 858 (with additional glucose, K⁺ and yeast extract) plus 20% foetal calf serum over a solid base of bovine or calf serum coagulated at 76°C. Static bottle cultures or a continuous circulating system with a gas phase of 5 % CO₂+10% O₂ in N₂ are used. Foetal calf serum proved to be the most unreliable component of the culture medium, different batches varying greatly in both growth stimulating properties and in toxicity. Variations in the properties of other components (e.g. bile salts) were also noted. Detailed descriptions are given of the various stages of development of strobilated worms, especially those prior to segmentation. Identification of these stages is essential if early assessment of the growth properties is to be made. In the most successful cultures, the rate of development lagged only a few days behind that in the dog. In the majority of cultures, however, maturation required 30–50 per cent longer than in the dog. In worms developing to sexual maturity in vitro, the uterus of gravid proglottids was filled with ova; the latter failed to give rise to embryonated eggs. The receptaculum seminis of cultured worms was empty of sperm (in contrast to dog worms) indicating that impregnation had not taken place. Attempts to induce impregnation and subsequent fertilization have so far proved unsuccessful. The development of non-infective (i.e. eggless) worms, nevertheless, has an advantage from the safety point of view.