PROGRESS IN PROTOZOOLOGY
PROGRESS IN PROTOZOOLOGY PROGRESS IN PROTOZOOLOGY
2.32 I. CUNNINGHAM tion with Dr. S. K. M o 1 o o, was able to etablish cultures of bloodstream forms of a West African stock of T. vivax in feeder layers of goat embryo fibroblast-like cells grown in Eagle's MEM with Earle's salts supplemented with inactivated goat serum. The cultures were maintained for several weeks and were infective to mice. The cultures yielded up to 3 to 8 X 10 5 organisms/ml and the trypanosomes possessed a surface coat. These methods for the cultivation of infective trypanosomes offer possibilities of studying processes such as antigenic variation, pleomorphism among bloodstream populations or cell differentiation in the absence of the mammalian host. They might also be useful in development of vaccines and drug testing. It would, however, be desirable if the infective forms could be grown in larger quantities in cell- or tissue-free systems. Entamoeba and Trichomonadidae Dr. Diamond reviewed the types of media and techniques currently employed in the initiation and establishment of axenic cultures of these protozoa. There are many similarities in the development of axenic culture systems for enteric amoebae and trichomonads. Further refinements and modifications of the available media might still improve the yields of parasites, but satisfactory levels of growth have been obtained for most investigations. Cloning procedures were discussed in their role for providing uniform populations of organisms and as a means of more critically assessing their viability. Relatively few Entamoeba or Trichomonadidae have been cultivated axenically. The availability of these culture systems would be a useful source of material for studies on biochemistry, physiology and immunology of these organisms. Our knowledge of the metabolism and nutritional requirements of these protozoa awaits the formulation of completely defined media. Furthermore, such media would facilitate investigations of the physical and chemical nature of their biomembranes, a subject of intense interest at present. With few exceptions, the interval between the isolation of these protozoa and their adaptation to axenic cultivation is exceedingly long. Therefore, it is possible that selection and/or mutation could have occurred during this period. There is a need to reduce this period in order to conserve the characteristics of the freshly isolated population. With the exception of Entamoeba invadens, a reptilian parasite, the cystic stage of Entamoeba does not form spontaneously, and furthermore, encystation cannot be induced in axenic cultures. Thus, this important life cycle stage cannot be studied in other Entamoeba under axenic conditions. Knowledge of the biology of cysts is required http://rcin.org.pl
229 for the development of methods to control the transmission of these amoebae, especially the human pathogen, E. histolytica. It is important to develop methods to induce encystation under the unique conditions in axenic cultures. Leishmania Dr. B. M. Honigberg presented the paper on the cultivation of Leishmania submitted by Dr. Larry Hendricks who was unable to attend the Congress. The cultivation methods of the two developmental stages of this parasite were dealt with separately. (1) Promastigotes Methods to cultivate promastigote forms morphologically similar to those found in the sandfly have existed for many years. This extracellular, flagellated organism grows optimally at 30°C. The early workers used monophasic or biphasic blood-containing media which have been superceded by the semi-defined or defined liquid media more suitable for assessing the condition and growth of the parasites and for immunological or biochemical investigations. Commercially available insect media of Grace; Mitsuhashi and Maramorosch, and Schneider (Hendricks et al. 1978, Chi Ids et al. 1978) and the mammalian cell media, e.g., Eagle's MEM and TC 199 (B e r e n s et al. 1976) can generate satisfactory yields of promastigotes of a wide variety of Leishmania sspp. These media available commercially, and prepackaged lyophilized with 30% foetal bovine serum in serum vials, have been useful for the diagnosis of both cutaneous and visceral leishmaniasis from both the old and new world. The insect media can support the large scale production of promastigotes for metabolic studies. Several culture systems used for this purpose include the chemostat (S c h a e f e r et al. 1970) and the fermenter (E n d e r s et al. 1977). A typical bulk culture system consisted of promastigotes grown in 9-liter quantities of Schneider's Drosophila medium supplemented with 30% foetal bovine serum. Yields of up to 5 X 10 7 organisms/ml produced 16 ml of packed cells of several strains of L. braziliensis and L. mexicana. The tsetse fly-trypanosome medium SM (C u nn i n g h a m 1977) mixed 1 : 1 with RPMI 1640 and enriched with 30% foetal bovine serum has been used routinely for large scale production of promastigotes (5—10 ml of packed cells) of strains of L. donovani from East Africa, L. chagasi from Central America and cutaneous leishmania from Central and South America and the Middle East. http://rcin.org.pl
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- Page 9 and 10: 183 In his abstract Mignot (1981) s
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- Page 13 and 14: 187 algae by phycologists (Bourelly
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- Page 35 and 36: 209 vine et al. 1980), there is no
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- Page 39 and 40: 213 evidence for the assumption of
- Page 41 and 42: REFERENCES 215 Bardele C. F. 1977:
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- Page 51 and 52: 225 brate cycle in vitro. Further c
- Page 53: 227 could support the development o
- Page 57 and 58: IN VITRO CULTIVATION OF PARASITIC P
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- Page 63 and 64: 237 Rai Choudhuri A. N.. Chowdhuri
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2.32<br />
I. CUNN<strong>IN</strong>GHAM<br />
tion with Dr. S. K. M o 1 o o, was able to etablish cultures of bloodstream<br />
forms of a West African stock of T. vivax in feeder layers of goat embryo<br />
fibroblast-like cells grown in Eagle's MEM with Earle's salts supplemented<br />
with inactivated goat serum. The cultures were maintained<br />
for several weeks and were infective to mice. The cultures yielded up to<br />
3 to 8 X 10 5 organisms/ml and the trypanosomes possessed a surface coat.<br />
These methods for the cultivation of infective trypanosomes offer<br />
possibilities of studying processes such as antigenic variation, pleomorphism<br />
among bloodstream populations or cell differentiation in the absence<br />
of the mammalian host. They might also be useful in development<br />
of vaccines and drug testing. It would, however, be desirable if the<br />
infective forms could be grown in larger quantities in cell- or tissue-free<br />
systems.<br />
Entamoeba and Trichomonadidae<br />
Dr. Diamond reviewed the types of media and techniques currently<br />
employed in the initiation and establishment of axenic cultures of<br />
these protozoa. There are many similarities in the development of axenic<br />
culture systems for enteric amoebae and trichomonads. Further<br />
refinements and modifications of the available media might still improve<br />
the yields of parasites, but satisfactory levels of growth have been<br />
obtained for most investigations. Cloning procedures were discussed<br />
in their role for providing uniform populations of organisms and as<br />
a means of more critically assessing their viability. Relatively few Entamoeba<br />
or Trichomonadidae have been cultivated axenically. The<br />
availability of these culture systems would be a useful source of material<br />
for studies on biochemistry, physiology and immunology of these<br />
organisms. Our knowledge of the metabolism and nutritional requirements<br />
of these protozoa awaits the formulation of completely defined<br />
media. Furthermore, such media would facilitate investigations of the<br />
physical and chemical nature of their biomembranes, a subject of intense<br />
interest at present. With few exceptions, the interval between the<br />
isolation of these protozoa and their adaptation to axenic cultivation is<br />
exceedingly long. Therefore, it is possible that selection and/or mutation<br />
could have occurred during this period. There is a need to reduce this<br />
period in order to conserve the characteristics of the freshly isolated<br />
population. With the exception of Entamoeba invadens, a reptilian parasite,<br />
the cystic stage of Entamoeba does not form spontaneously, and<br />
furthermore, encystation cannot be induced in axenic cultures. Thus,<br />
this important life cycle stage cannot be studied in other Entamoeba<br />
under axenic conditions. Knowledge of the biology of cysts is required<br />
http://rcin.org.pl