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In vitro cultivation of Anisakis simplex: pepsin increases survival and moulting from fourth larval to adult stage

This paper describes the in vitro cultivation of the 3rd-larval stage (L3) of Anisakis simplex to adulthood in a much simpler and easier to prepare medium than those described to date. The adult males obtained are between 3.8 and 6.5 cm long and the females between 4.5 and 8.0 cm. Some individually cultivated females laid eggs which had an average size of 44.4×50.5 μm. The culture conditions were as follows: medium RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum and 1% commercial pepsin, at pH 4.0 and a temperature of 37 °C, and in air atmosphere with 5% CO2. The pepsin was found to be the key to the success of the culture. The average survival of the worms in the culture increased from 50 to 88 days, due to the fact that the survival of the adults practically doubled (increasing by 1.9 times). Furthermore, the number of worms that completed the 4th moulting (M4) increased by 4.2 times, from 22.9 to 95.6%. This culture medium may facilitate, due to its simplicity, the study of anisakids, or at least of A. simplex, constituting another step towards achieving a complete in vitro life-cycle for these parasites.