The resistance to human plasma of Trypanosoma brucei, T. rhodesiense and T. gambiense: III. Clones of two plasma-resistant strains

427 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPEAL

MEDICINE AND HYGIENE, VOL. 71, No. 5, 1977.

The resistance to human plasma of Trypanosoma brucei, 7. rhodesiense and T. gambiense: III. Clones of two plasma-resistant strains FRANK HAWKING* School of Biological Sciences, Brunei Uxbridge, Middlesex

Summary

Tests for resistance to human plasma were made on six clones of a stabilate of Trypanosoma rhodesiense (LUMP 10) which was calculated to contain about 3,000 resistant trypanosomes per million. Two of the clones were not resistant and four were only subresistant. Tests were also made on 12 lines (clones) of a stabilate of polymorphic trypanosomes isolated from tsetse flies. One of them, ETAT 10, had infected a laboratory worker and was found to be fully resistant to human plasma; the other lines showed only low or moderate resistance. Resistance of a strain to human plasma often depends upon a small minority of resistant trypanosomes. Strains of polymorphic trypanosomes may be classified as fully resistant, moderately resistant, subresistant, or sensitive to human plasma, if they contain respectively, all, some (e.g. one per hundred), very few (e.g. one per million) or no individuals which are resistant. Introduction

Two previous papers (HAWKING, 1977a & b) have described investigations on the resistance or susceptibility to human plasma of strains of Trypanosoma brucei, T. rhodesiense and T, gambiense. A strain is conceived as a large population of parasites differing among themselves; the behaviour of a strain in a biological test may be determined by a small minority of its component individuals. The present paper reports attempts at analysis of the composition of two human plasma resistant stabilates by examination of clones derived from them. For the purpose of these papers the name T. brucei is applied to all polymorphic strains isolated from animals or tsetse flies; T. rhodesiense to strains isolated from man in East Africa; and T. gambiense to strains isolated from man in West Africa. Materials and Methods The general methods employed were the same as those of the previous two papers (HAWKING, 1977a,b). Clones from two stabilates of trypanosomes were examined. (a) T. rhodesiense LUMP 10. This stabilate was described in HAWKING (1973). Briefly it was isolated from man in Ethiopia, 17/2/69 and preserved at -70°C. It had been passaged eight times through mice before the present work began. Six clones were isolated from it by Dr. P. J. Walker on 6112175.For convenience these are labelled BR 1 to 6 (from Brunei University). The prepatent period

*Present address: Commonwealth Institute of Helminthology, St. Albans, Her@.,England AL1 3EW.

University,

of the infections produced by single trypanosomes in young mice was four to five days. (b) ETAT clones 1 to 12 (kindly supplied by Professor W. H. R. Lumsden). These have been described in several other papers, especially those by MCNEILLAGE et al. (1969) and VAN MEIRVENNEet al. (1976), the latter giving the pedigrees of the different lines. Briefly the original stabilate TREU 164 was isolated from tsetse flies caught at Lugala, Uganda, in 1960. Clones of 12 different antigenic types (as shown by the ETAT numbers) were isolated from this by MCNEILLAGE et al. (1969). In 1974 a laboratory worker handling these stabilates became infected by one of them (ETAT 10) showing that it was infective for man (ROBERTSON & PICKENS, 1975). [Note. the ETAT numbers of these trvoanosomes refer’& the antigenic type, any one type often appearing in many different clones (with different isolation numbers). There is no evidence that different clones possessing the same antigen pattern (and the same ETAT number) are identical in their other properties including plasmaresistance. This will be considered further in the discussion.] In the present work the identity of the different antigen types studied was as follows: 5 6 1 2 3 4 Antigen type ETAT 63 1145 57 56 73 76 Stabilate LUMP 10 11 12 Antigen type ETAT 7 8 9 Stabilate LUMP 98 126 127 139 165 138 Results BR Clones

The results of testing the six BR clones with human plasma are shown in Table I. The original stabilate, LUMP 10, was resistant to human plasma with a mean prepatent period in plasma-treated mice of 2.5 days, which (according to inocula with smaller numbers of trypanosomes) corresponded to a population of about 3,000 resistant individuals uer 106. i.e. one in 300 were resistant. This may be considered to be a low degree of definite resistance. At their first testing, two of the clones (BR 2 and 6) were not resistant, and the other four (BR 1, 3, 4, 5) showed subresistance corresponding to about one resistant individual per 106. On further passage of three clones (BR 2, 5, and 6) four to eight times through mice the resistance seemed to increase somewhat; in clone 6 after six passages the mean prepatent period was 3.6 days, which suggests a proportion of perhaps 200 resistant individuals per 106.These results may be interpreted as follows: The initial absence or low degree of resistance in those clones is what would be expected when individuals for clones were selected at

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TO HUMAN

PLASMA

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Table I - Tests on BR clones and on the parent stabilate, T. rlwdesiense LUMP 10 Controls Clone

Plasma

Passage

BRl BR2

Resistant Mice infected

Mean piepatent period Days

Mice infected

Mean prepatent period* Days

313 313 313 313 313 313 313 313 313 313 313 313

1.0 1.7 1.3 1.3 0.9 1.0 1.0 1.0 1.3 1.2 0.7 1.3

414 O/4 314 414 314 214 l/4 414 314 o/4 414 414

9.5 6.3 6.5 10.3 9.9 17 56;; 6.5 3,6

?l 0 ?l 1 ?1 ?O.l ?O.l 1 ?l 0 1 200

414 313 313

0.75 2.0 3.8

515

2.5

3.5 x 103

1 2 5 8 2 2 2 5 6 1 4 6

BR3 BR4 BR5 BR6

Parent clone Inoculum : 1.65~107 1.65 x 105 1.65 x lo3

-

tryPS.

per lo6

*Mean prepatent period only of mice which became infected. Table II-

Tests on ETAT lines Controls

Plasma treated

Strain infected

Mean prepatent period Days

Mice infected

Mean prepatent period Days

313 313 313 313 313 313 313 313 313 313 313 313

0.5 0.5 0.5 0.5 0.5 0.7 0.7 0.5 0.5 0.7 0.7 0.8

414 414 414 414 414 414 414 314 414 414 414 414

4.5 3.0 3.7 3.1 4.0 ;:;

Mice

1 2 3 4 5 6 7 8 9 10 11 12

5.0 4.8 0.5 3.5 3.0

No. of resistant tryps per 106t

30 1.6~103 300 1.3 x 104 100 50 6.0 x 103 5 10 106 400 1.6~103

VAN MEIR~ENNE

et al. Prepatent periods in two mice Days 5, 4, 3, 4, 4, 3, 3, 3, 3, 1, 3, 4,

5 6 4 4 4 3 4 4 4 1* 4 4

*Highly resistant in vitro, also. fThe number of resistant trypanosomes per 106 is an approximate calculation, assuming that the inoculum contained 107 trypanosomes, as is indicated by the prepatent period of the control mice. random from a population in which only one in 300 were resistant. After such a clone has been established, however, there seems to be a tendency for resistant individuals to become more conspicuous in it, possibly by mutation and/or selective multiplication. During further experiments Clone 6 was exposed to human serum in mice (dose 0.2 ml/20 g) once and then tested again. The mean prepatent period of the plasma-treated mice (2.0 days) was now practically the same as that of the control mice (1.9 days) so that apparently almost all the individuals of the strain had become resistant (presumably through selection of resistant mutants by the plasma). On the other hand, this did not always happen because when three other clones, BR 2, 3, and 5,

were each exposed once to plasma in mice, no obvious change of resistance took place. ETAT lines The results of testing the ETAT lines are shown in Table II. One of these lines (ETAT 10, which infected a laboratory worker) was completely resistant; the mean prepatent period after exposure to plasma was only slightly less than that of the controls, so that theoretically almost 100°A of the individuals were resistant. Four of the lines (2,4, 7, and 12) were moderately resistant (more than 103 individuals per 106 being resistant) and seven of the lines (1, 3, 5, 6, 8, 9, and 11) showed only a low degree of definite resistance (five to 400 individuals per

F. HAWKING 106 being resistant). These results are broadly similar to those obtained by VAN MEIRVENNE et al. (1976) which are also indicated in the table. Their results show Line 10 as being highly resistant; with the other stabilates, the prepatent periods in their mice were mostly three to five days, which suggests that only 1,000 to five trypanosomes survived the serum and were available to produce infection. Further, in some unpublished experiments at the London School of Hygiene, Mr. L. R. Rickman found that Line 10 was highly resistant whereas the other strains showed less or no resistance. Discussion In the above experiments an examination was made of the plasma resistance of six clones (BR) derived from T. rhodesiense LUMP 10 and of 12 clones (lines) of the ETAT group. LUMP 10 was moderately resistant, but two of the clones were not resistant and four were only “subresistant”. In the ETAT group, the original stabilate was not available for testing but the clones showed great variation in their resistance, one (ETAT 10, LUMP 139) being 100 % resistant while the others showed only small proportions of resistant individuals. Assuming the properties of a clone to reproduce (for some passagesat any rate) the properties of the original trypanosome, these results are in accordance with the general concept of a strain being composed of thousands of genetically different individuals. When the strain as a whole is tested for plasma resistance the response is determined by the resistant individuals present, which may be only a small minority of the whole; but when individuals are selected by cloning from this larger population, the chances of the selected individuals being resistant are small. The resistant individuals are usually a minority and it is unlikely that they will often be included among the few specimens thus picked out. Accordingly, the populations resulting from cloning would be expected to show a lower resistance than the parent strain, and this has been the case in the above experiments. All the same it must also be remembered that clones may change after isolation, not only in their antigenic patterns but also in other properties. All the Etat lines are the result of a series of clonings (VAN ME~VENNE et al., 1976). ETAT 1, 2, 3, and 4 are original clones; ETAT 5, 6, 10, 11, and 12 are all derived by cloning from ETAT 1; and ETAT 7, 8 and 9 are derived again by cloning from 6. Yet they differ in their degrees of plasma resistance (as well as in antigens and probably in other respects). Moreover, the clones BR 2, 5, and 6 show signs of developing greater plasma resistance during six to eight passages through normal mice. General review

The experimental facts (reported in this and the two previous papers) concerning the various degrees of plasma resistance in different strains of polymorphic trypanosomes and in the different individuals which comprise each strain may now be reviewed. I. In a resistant strain, the resistant individuals are not merely the extreme end of a continuous population, but they form a special resistant subpopulation. Thus it was shown in the first of these three papers (HAVXING, 1977a) that, the prepatent period (which is an inverse indication of how many trypanosomes have resisted the plasma) was not diminished when the dose of plasma was reduced to 0.5 or 0.25 of the standard dose of 0.2 ml/20 g.

429

II. It is possible to distinguish four levels of sensitivity/ resistance: (a) Fully sensitive strains, in which no individuals resist the standard dose of plasma. These are the classical T. brucei. (b) Subresistant strains, in which a few individuals (perhaps one per million) survive to produce infection. As stated above, these few individuals are not the extreme end of a continuous variation, but they constitute a tiny subpopulation. This group contains most of the strains isolated from animals by ROBSON & RICKMAN, GEIGY, and others, and found by them to be BIIT positive. Compare also the subresistant T. brucei strains as reported in Table III of the second paper in this series (HAWKING, 1977b).

(c) Resistant strains in which most of the individuals are still sensitive but in which the resistant individuals may be 10 to 103 per million. This group contains LUMP 10 above and about half the strains isolated from man in East Africa and reported on in the preceding paper (HAWKING, 1977b). (d) Highly resistant strains in which almost all the individuals are resistant, e.g. T. gambiense, ETAT 10, and some of the T. rhodesiense strains described in the preceding paper in this series. (In the two previous papers, groups (c) and (d) have been taken together.) It is possible that the differences between these four groups are quantitative rather than qualitative, i.e. if individual trypanosomes are resistant, they may all possessapproximately the same degree of resistance, and the strains would differ because they contain none, very few, moderate numbers, or a great majority of such abnormal individuals. Nevertheless the differences between the four groups are so characteristic that they seem to indicate some fundamental difference in the genetic material. (These different levels of plasma resistance are a special feature of the T. brucei group and it is not known if they also occur in other species of trypanosomes.) III. The stability of these strains is not absolute but in some circumstances may change from one level to another. If strains (especially subresistant ones) are exposed to plasma in vitro or in vivo they tend to become more resistant--either gradually and partially, group c (cf. the various “boosted” strains in previous papers) or completely to maximally resistant group d. (Thus VAN MEIRVENNEet al. found that all the ETAT lines became highly resistant after one exposure to human serum in vitro; and in the present work BR 6 (but not BR 5) became highly resistant after one exposure in vivo). Presumably mutation occurs, followed by selection by the plasma. On the other hand if strains are passed through many laboratory animals they tend to become completely susceptible, even strains of T, gambiense; apparently the resistant individuals are gradually overgrown by quick-multiplying sensitive ones. IV. It is possible that a certain pattern of antigens (e.g. ETAT 10) might often be associated with plasma resistance, but the two are not identical because, during a relapse, the antigen changes but the resistance usually remains unchanged. Theoretical

explanation

Since these. differences of plasma-resistance are preserved through many generations of trypanosomes

430

RESISTANCE TO HUMANPLASMA

and through many passages in animals, they probably depend upon differences of the genetic material (cf. the study of arsenical-resistance by HAWKING & WALKER, 1966). Nevertheless, a complete explanation in genetic terms cannot be attempted in the present paper. Provisionally it might be suggested that there are genes r, R, and RR corresponding to the subresistant, moderately resistant and highly resistant groups respectively; but in that case the genes seem to determine the number of resistant individuals which appear in the different strains, rather than the degree of their resistance. Acknowledgements Grateful acknowledgements are due to Professor W. H. R. Lumsden and to Dr. P. J. Walker for the supply of clones, and to Professor J. D. Gillett for laboratory facilities. This work was carried out under a grant from the Overseas Development Ministry. References Hawking, F. (1973). The differentation of Trypanosoma rhodesiense from T. brucei by means of human serum. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67,517-527.

Hawking, F. (1977a). The resistance to human plasma of Trypanosoma brucei, T. rhodesiense, and T. gambiense.

OF Trypanosoma

spp.: III

1. Analysis of the composition of trypanosome strains. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70, 504-512.

Hawking, F. (1977b). The resistance to human plasma of Trypanosoma brucei, T. rhodesiense and T. gambiense. II. Survey of strains from East Africa and Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70, 513-520.

Hawking, F. & Walker, P. J. (1966). Analysis of the development of arsenical resistance in trypanosomes in vitro. Experimental Parasitology, 18, 63-86. McNeillage, G. J. C., Herbert, W. J. & Lumsden, W. H. R. (1969). Antigenic type of first relapse variants arising from a strain of Trypanosoma (Trypanozoon) brucei. Experimental Parasitology, 25, l-7. Robertson, D. H. H. & Pickens, S. (1975). Special Report. Accidental laboratory infection with Trypanosoma brucei rhodesiense. A case report. Communicable Diseases Scotland Weekly Reuort. 918175.iii-iv. Van Meirvenne, N., Magnus, E: & Janssens;P. G. (1976). The effect of normal human serum on trypanosomes of distinct antigenic type (ETAT 1 to 12) isolation from a strain of Trypanosoma brucei rhodesiense. Annales de la Societe beige de Medicine

Accepted for publication

tropicale, 56, 55-63.

2nd June 1977