Adult Litomosoides carinii, recovered from cotton rats (Sigmodon hispidus) 4–5 months post-infection (p.i.), were cultivated in vitro with emphasis on investigations into the development of intra-uterine embryonic stages. Baseline values for the embryonic status of female worms were established immediately after recovery from the hosts. In such females, on average, 16% of the intra-uterine stages were fully formed microfilariae while the remainder belonged to the early embryonic classes that were characterized. For the evaluation of culture success, apart from survival of worms in vitro, the rate of microfilariae development (mf rate) served as a major parameter. Of the five basic culture media RPMI 1640, F12, L15, NCTC 135, and IMDM, tested without supplementation, RPMI 1640 yielded by far the best results (survival = 14 days; mf rate = 41%), and was therefore chosen as the routine medium. In comparison with 5% CO2 in nitrogen, a gas phase of 5% CO2 in air was superior, although the resulting oxygen tension of 138 mmHg in the medium was not physiological. Addition of 10% newborn or foetal bovine serum to the medium in some cases distinctly influenced results. Effects of different batches of sera varied from ‘filaricidal’ to ‘very promoting’. Co-cultivation of worms and Sigmodon cells, or rhesus monkey LLCMK2 cells, only marginally improved results. Of the serum substitutes Ultroser G, BMS, and Clex, the latter had a moderately promoting effect. The protein supplements bovine serum albumin, transferrin and haemoglobin significantly improved results, and could replace certain batches of serum. Supplementation with the haemin moiety alone was less effective than with haemoglobin. The anti-oxidants glutathione plus ascorbic acid proved beneficial in combination with a serum supplement only. Results from other experiments with multiple supplementation also suggest that various supplements may act only in a synergistic manner. The longest average time that adult L. carinii survived in vitro was 3–4 weeks. The highest mf rate was 78%, which indicated that all text-abstract embryonic stages present in the uteri of a female at the start of culture completed their development to microfilariae, however, oogenesis ceased in vitro. The parameters for embryonic development employed proved to be highly sensitive for the judgement of various culture conditions.
In Vitro Cultivation of Microsporidia of Clinical Importance