The cultivation of Trypanosomes in Arthropod tissue culture
The literature relating to the development of the technique of arthropod tissue culture, its application to biological research, and the cultivation of trypanosomes is reviewed.Cultivation of tissues and organs from Glossina morsitans morsitans was investigated using several culture media. Of the media tested, modified Trager's medium was found to be the best for the growth and maintenance of tissues. Successful cultures of developing adult tissues from ticks, Rhipicephalus appendiculatus, R.bursa and Amblyomma hebraeum and from partially engorged adult Ixodes ricinus were obtained in VP4 medium.The greatest success in the cultivation of Trypanosoma brucei and T.congolense in association with Glossina tissues was achieved in the presence of complete alimentary tract of pupae older than 21 days in 15 -20a1 hanging -drops of modified Trager's medium incubated at 28 °C. Growth of the trypanosomes also occurred in cultures in which the flagellates were separated from the insect tissues by a semi -permeable membrane. No multiplication of the parasites was obseived in (a) culture medium alone, (b) medium containing extracts of alimentary tract and (c) medium in which alimentary canal had been cultured for 3 or 4 days.In all the cultures in which multiplication of the trypanosomes occurred, it was accompanied by morphological transformation. Bloodstream trypomastigotes in the inoculum changed into long slender forms similar to those seen in the midgut of the tsetse fly. Infective stages were not observed.Complete digestive tract from G.morsitans submorsitans, G.austeni and G.palpalis was as suitable as that from G.m.morsitans for the cultivation of the trypanosomes and the same applied to the alimentary tract of the non- haematophagous fly Sarcophaga, a dipteran closely related to Glossina. On the other hand, cultures of comparable tissues from mosquitoes, Aedes aegypti and ticks, R.appendiculatus failed to support the growth of trypanosomes.Fly transmissible as well as laboratory adapted strains of T.brucei and T.congolense could be cultivated in tsetse organ cultures. Negative results were obtained, however, with laboratory adapted strains of T.gambiense and T.vivax and with the monomorphic T.evansi which is not cyclically transmitted in nature by tsetse flies.Stercorarian species, Trypanosoma musculi, T.lewisi, T.mzilophagium and T.theileri were also grown in the presence of tsetse alimentary tract. All these species transformed into and multiplied in forms resembling those formed in the hindgut and rectum of their arthropod vectors. Unlike the cultures of the salivarian trypanosomes, those of T.musculi and T.lewisi remained infective to laboratory rodents for many days.Amino acid analyses of haemolymph of pupae, pre- emerged and adult G.morsitans revealed that of the 21 amino acids, proline was present in highest concentrations in all the stages examined. There were also considerable differences in the concentrations of the individual amino acids in these three stages. The results of the amino acid analyses formed bases for the design of culture media which were capable of supporting growth of T.brucei and T.congolense.