Hostname: page-component-cd9895bd7-jkksz Total loading time: 0 Render date: 2024-12-26T07:44:02.989Z Has data issue: false hasContentIssue false
  • English
  • Français

In vitro cultivation of adult Litomosoides carinii: evaluation of basic culture media, gas phases and supplements

Published online by Cambridge University Press:  06 April 2009

J. Mössinger
J. Mössinger
Affiliation:
Institute of Tropical Medicine, University of Tübingen, Wilhelmstrasse 31, W-7400 Tübingen, Germany
Get access Rights & Permissions [Opens in a new window]

Extract

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.

Keywords

Litomosoides cariniiFilarioideain vitro culture conditionsembryonic developmentmicrofilarial release
Type
Research Article
Information
Parasitology , Volume 103 , Issue 1 , August 1991 , pp. 85 - 95
Copyright
Copyright © Cambridge University Press 1991

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abraham, D., Mok, M., Mika-Grieve, M. & Grieve, R. B. (1987). In vitro culture of Dirofilaria immitis third-and fourth-stage larvae under defined conditions. Journal of Parasitology 73, 377–83.CrossRefGoogle ScholarPubMed
Barrett, J. (1981). Biochemistry of Parasitic Helminths. London: Macmillan Publishers Ltd.CrossRefGoogle Scholar
Büding, E. (1949). Studies on the metabolisms of the filarial worm Litomosoides carinii. Journal of Experimental Medicine 89, 107–30.CrossRefGoogle Scholar
Comley, J. C. W. & Mendis, A. H. W. (1986). Advances in the biochemistry of filariae. Parasitology Today 2, 34–7.CrossRefGoogle ScholarPubMed
Comley, J. C. W., Szopa, T. M., Strote, G., Buttner, M., Darge, K. & Buttner, D. W. (1989). A preliminary assessment of the feasibility of evaluating promising antifilarials in vitro against adult Onchocerca volvulus. Parasitology 99, 417–25.CrossRefGoogle ScholarPubMed
Court, J. P., Martin-Short, M. & Lees, G. M. (1986). A comparison of the response of Dipetalonema viteae and Brugia pahangi adult worms to antifilarial agents in vitro. Tropical Medicine and Parasitology 37, 375–80.Google ScholarPubMed
Devaney, E. & Howells, R. E. (1983). The differentiation of microfilariae of Onchocerca lienalis in vitro. Annals of Tropical Medicine and Parasitology 77, 103–5.CrossRefGoogle ScholarPubMed
Dörner, K., Kaltwasser, J. P. & Thomas, L. (1984). Eisenstoffwechsel, Spurenelemente und toxische Metalle. In Labor und Diagnose, 2nd Edn. (ed. Thomas, L.), pp. 287–8. Marburg/Lahn: Medizinische Verlagsgesellschaft.Google Scholar
Douvres, F. W. & Urban, J. F. Jr (1983). Factors contributing to the in vitro development of Ascaris suum from second-stage larvae to mature adults. Journal of Parasitology 69, 549–58.CrossRefGoogle Scholar
Foster, L. A. & Bogitsh, B. J. (1986). Utilization of the heme moiety of hemoglobin by Schistosoma mansoni schistosomules in vitro. Journal of Parasitology 72, 669–76.CrossRefGoogle ScholarPubMed
Franke, E. D. & Weinstein, P. P. (1983). Dipetalonema viteae (Nematoda: Filarioidea): culture of third-stage larvae to young adults in vitro. Science 221, 161–3.CrossRefGoogle ScholarPubMed
Franke, E. D. & Weinstein, P. P. (1984). In vitro cultivation of Dipetalonema viteae third-stage larvae: evaluation of culture media, serum, and other supplements. Journal of Parasitology 70, 618–28.CrossRefGoogle ScholarPubMed
Franke, E. D., Riberu, W. & Wiady, I. (1990). Evaluation of medium supplements for in vitro cultivation of Wuchereria bancrofti. Journal of Parasitology 76, 262–5.CrossRefGoogle ScholarPubMed
Hawking, F. (1954). The reproductive system of Litomosoides carinii, a filarial parasite of the cotton rat. III. The number of microfilariae produced. Annals of Tropical Medicine and Parasitology 48, 382–5.CrossRefGoogle Scholar
Illgen, B. (1982). Experiments in vitro with Litomosoides carinii (Nematoda: Filarioidea) II. Influence of different sera on the release of microfilariae and experiments on the readiness for copulation of the worms. Zeitschrift für Parasitenkunde 67, 227–36.CrossRefGoogle ScholarPubMed
Lok, J. B., Mika-Grieve, M., Grieve, R. B. & Chin, T. K. (1984). In vitro development of third- and fourth stage larvae of Dirofilaria immitis: comparison of basal culture media, serum levels and possible serum substitutes. Acta Tropica 41, 145–54.Google ScholarPubMed
Mak, J. W., Lim, P. K., Sim, B. K. & Liew, L. M. (1983). Brugia malayi and B. pahangi: cultivation in vitro of infective larvae to the fourth and fifth stages. Experimental Parasitology 55, 243–8.CrossRefGoogle Scholar
Maki, J. & Weinstein, P. P. (1989). Dipetalonema viteae: survival of adult females and microfilarial release in both a chemically defined and serum-supplemented medium. Journal of Parasitology 75, 953–7.CrossRefGoogle ScholarPubMed
Miegeville, M., Bouillard, C., Marjolet, M. & Vermeil, C. (1981). Nouvelle contribution à l'étude de Dipetalonema viteae maintien en survie des adultes in vitro. Bulletin de la Société de Pathologie Exotique 74, 207–15.Google ScholarPubMed
Mössinger, J. (1990). Nematoda: Filarioidea. In In Vitro Cultivation of Parasitic Helminths (ed. Smyth, J. D.), pp. 155–86. Boca Raton: CRC Press.Google Scholar
Mössinger, J. & Wenk, P. (1986). Fecundity of Litomosoides carinii (Nematoda, Filarioidea) in vivo and in vitro. Zeitschrift für Parasitenkunde 72, 121–31.CrossRefGoogle ScholarPubMed
Mössinger, J., Wenk, P. & Schulz-Key, H. (1987). In vitro maintenance of adult Dipetalonema viteae and Litomosoides carinii (Nematoda, Filarioidea): fecundity and survival in cell-free culture systems. Zentralblatt für Bakteriologie, Mikrobiologie und Hygiene, Series A 267, 303.Google Scholar
Pollack, R. J., Lok, J. B. & Donnelly, J. J. (1988). Analysis of glutathione enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro. Journal of Parasitology 74, 353–9.CrossRefGoogle ScholarPubMed
Price, P. J. & Gregory, E. A. (1982). Relationship between in vitro growth promotion and biophysical and biochemical properties of the serum supplement. In vitro 18, 576–84.CrossRefGoogle ScholarPubMed
Ramp, T., Bachmann, R. & Köhler, P. (1985). Respiration and energy conservation in the filarial worm Litomosoides carinii. Molecular and Biochemical Parasitology 15, 11–20.CrossRefGoogle ScholarPubMed
Riberu, W. A., Atmosoedjono, S., Purnomo, S., Tirtokusumo, S., Bangs, S. J. & Baird, J. K. (1990). Cultivation of sexually mature Brugia pahangi in vitro. American Journal of Tropical Medicine and Hygiene 43, 3–5.CrossRefGoogle Scholar
Schulz-Key, H. (1990). Observations on the reproductive biology of Onchocerca volvulus. Acta Leidensia 59, 27–43.Google ScholarPubMed
Taylor, A. E. R. (1960). Maintenance of filarial worms in vitro. Experimental Parasitology 9, 113–20.CrossRefGoogle ScholarPubMed
Townson, S., Connelly, C. & Muller, R. (1986). Optimization of culture conditions for the maintenance of Onchocerca gutturosa adult worms in vitro. Journal of Helminthology 60, 323–30.CrossRefGoogle ScholarPubMed
Wang, E. J. & Saz, H. J. (1974). Comparative biochemical studies on Litomosoides carinii, Dipetalonema viteae, and Brugia pahangi adults. Journal of Parasitology 60, 316–21.CrossRefGoogle ScholarPubMed
Weinstein, P. P. (1986). Filariasis: problems and challenges. American Journal of Tropical Medicine and Hygiene 35, 221–33.CrossRefGoogle Scholar
Wenk, P., Illgen, B. & Seitz, H. M. (1978). Experiments in vitro with Litomosoides carinii (Nematoda: Filarioidea). I. Maintenance of adult females and microfilariae as well as release of microfilariae in different culture media. Zeitschrift für Parasitenkunde 55, 63–72.CrossRefGoogle ScholarPubMed