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Detection of multiple variable antigen types in metacyclic populations of Trypanosoma brucei
205 TRANSACTIONS OFTHEROYALSOCIETYOFTROPICALL MEDICINEANDHYGIENE,Vol. 73, No. 2, 1979
Detection
of multiple variable antigen types in metacyclic Trypanosoma brucei
populations
of
J. DAVID BARRY*, STEPHEN L. HAJDUK, KEITH VICKERMAN Department of Zoology, Glasgow University, Glasgow G12 SQQ, U.K. AND DOMINIQUE LE RAY Laboratory of’ Protozoology, Prince Leopold Institute of Tropical Medicine, Nationalestraat l&5, B-2000 Antwerp, Belgium
Summary
The identification of antigen types in tsetse salivary gland metacyclic populations of Trypanosomabrucei requires the production of monospecific antisera to the corresponding bloodstream variable antigen types. Monospecific antisera against clones from cyclically transmitted populations are difficult to prepare, however, owing to the antigenic lability of such clones. This problem has been overcome by isolating an antigenically stable clone from a syringe-infected rabbit at a time when its serum showed incipient activity towards metacyclic trypanosomes. Monospecific antisera raised against this clone reacted with up to 20% metacyclics in trypanolysis and immunofluorescence tests, confirming that a clone-derived metacyclic population of T. brucei is heterogeneous with respect to variable antigen type. Introduction
Vaccination against African trypanosomiasis is thwarted by antigenic variation, whereby the trypanosomes change the glycoprotein of their surface coat and evade the immune response of the host (GRAY & LUCKINS, 1976; VICKERMAN,1978). In recent years our understanding of this variation in the mammalian bloodstream forms of Trypanosoma brucei has improved at both the popbiation (VAN ~~EIRVENNE et al.. 1975: CAPBERN et al.. 1977: iE RAY et al., 1977) and molecular (CROSS;1975, 1978; BALTZet al., 1977; BARBET& MCGUIRE, 1978) levels. On the other hand, little is known about the surface antigens of the metacyclic forms which develop in the tsetse salivary glands and initiate the mammalian phase of the life-cycle of this trypanosome. Some work has suggested that the metacyclics carry a “basic” antigen to which the trypanosomes revert on completing cyclical development in the fly, and that the basic antigen is the first of the numerous “variable antigen types” to be detected in the blood of a mammal following cyclical transmission (GRAY, 1965, 1975; CUNNINGHAM, 1966; JENNI, 1977). Such a basic antigen would have obvious potential in vaccination. Recently, however, we have presented preliminary evidence (LE RAY et al., 1978) that the metacyclic population may be heterogeneous with respect to variable antigen type. We now present a method for the identification of metacyclic antigen types using monospecific antisera
raised against bloodstream variable antigen types and further evidence against the existence of a single metacyclic antigen type. In our previous work (LE RAY et al., 1977; 1978) trypanosomes of the Antwerp serodeme (VAN MEIRVENNEet al., 1975) were transmitted through Glossina morsitans, and two observations of significance to the present work were made: (i) Following cyclical transmission, the variable antigen types of the bloodstream trypanosome population proved much less stable than variable antigen types of trypanosomes maintained by syringe-passage in laboratory animals : this lability of antigen type makes the production of monospecificantiskia (and hence the antigenic typing of metacvclicsl difficult (LE RAY et al.. 197’i’l. (ii) Sera’cbllected ‘after a month fro& rabbits infected with the syringe-passaged line showed lytic activity against most or all of the metacyclics extruded at one time by the tsetse fly: the variable antigen type expressed by metacyclics would appear therefore not to be specific to the fly stages of the trypanosome life-cycle (LE RAY et al., 1978). In order to produce monospecific antisera to metacyclic variable antigen types the following procedure was devised. A rabbit was infected with the syringe-passaged line and by frequent collection of sera and trypanosomes for cryopreservation, an attempt was made to pin-point the time of appearance in the blood of variable antigen types expressed by metacyclics. Blood collected at, or immediately before, the time that the serum began to show lytic activity towards metacyclics should contain trypanosomes of the same variable antigen type as the metacyclics. Cloning of trypanosomes from such blood samples should give antigenically stable populations of the parasite. Monospecific antisera could then be produced against these stable variable antigen types and the activity of these antisera tested against metacyclic trypanosomes. Materials
and Methods
A rabbit (New Zealand White-Half Lop, 3.5 kg) was infected intravenously with lo6 syringe*Present Address : International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya.
206
MULTIPLE VARIABLE ANTIGEN TYPES IN METACYCLIC POPULATIONS
passaged line bloodstream trypanosomes of variable antigen type AnTat 1; for reference purposes the different variable antigen types in the repertoire of trypanosomes of the Antwerp serodeme are designated AnTat 1, AnTat 2 and so on (VAN MEIRVENNE et al., 1975). Blood samples were collected at intervals of one to three days and injected intraperitoneally into four to six-week-old female CFLP mice, from which blood was passaged to fresh mice at two to four-day intervals until enough trypanosomes (at least antilog 7*5/ml blood) were present to cryopreserve as stabilates (LUMSDEN, 1972). Serum samples collected simultaneously from the rabbit were stored at -20°C. To determine the time of appearance of metacyclic variable antigen types in the rabbit’s blood, the trypanolytic activity of the serum was tested using the method of VAN MEIRwNNE et al., (1975) against metacyclics obtained by allowing a fly carrying trypanosomes to salivate (probe) into guinea-pig serum in vitro at 37%. The methods used for maintenance of GZossina morsitans and for production of metacyclic trypanosomes were described by LE RAY et al. (1978), and cloning, indirect immunofluorescence and trypanolysis reactions, and the production of monospecific antiserum were conducted as described by VAN MEIRVENNE et al. (1975). Results Lytic activity against metacyclics was found in the rabbit’s serum collected from day nine onwards (Fig. 1). Lysis of 100% of metacyclics was not obtained with any of the samples, and two or possibly three peaks of activity occurred on days 16,23 and 29 respectively, suggesting that different metacyclic variable antigen types had arisen in the rabbit at different times.
To produce a monospecific anti-variable antigen type antiserum it is necessary first to isolate a clone expressing that antigen type. Trypanosome stabilate material from the beginning of the first peak of anti-metacyclic serum reactivity in the rabbit (nine days) was therefore selected for cloning in the hope that it would contain metacyclic variable antigen types. Each clone was tested for trypanolysis by serum taken from a second rabbit seven days after it was bitten by an infected tsetse fly: such serum had activity against most of the metacyclics in one probe (LE RAY et al., 1978). The tenth clone isolated was completely lysed by this serum, suggesting antigenic homology with the metacyclics. The variable antigen type of this clone, after comparison with known AnTats and with the other new clones, has been designated AnTat 30. To examine the level of trypanolytic activity against AnTat 30, the range of sera from the rabbit infected with the syringe line were titrated against bloodstream form AnTat 30 (Fig. 1); this activity developed by day 14, i.e. five days later than the over-all activity against metacyclics, and decreased from day 28 while over-all anti-metacyclic activity was rising again. A monospecific antiserum was raised in a third rabbit and tested against metacyclics by type specific immunofluorescence. Using salivary probes on to dry slides from flies which had originally ingested different AnTats, this antiserum labelled 11 to 20 y0 of metacyclics in all the probes examined (Fig. 2), including sequential probes from one fly (Table I). A monospecific antiserum to AnTat 30 raised in a mouse behaved similarly. Trypanolysis reactions with both antisera gave corresponding percentages of AnTat 30 in the probes.
Table I-Percentage of AnTat 30 in sequential probes from a single fly determined in immunofluorescence with rabbit anti-AnTat 30 monospecific serum Days after infecting blood meal 26 37 46
Fig. 1. Development of anti-metacyclic lytic activity in B rabbit chronically infected with a syringe passagedline of AnTat serodeme trypanosomes. The metacyclics tested were deposited by a single fly on one occasion; for each serum sample 33 trypanasomes were counted and the y. lysed calculated. Anti-AnTat 30 titres were determined as the inverses of the end-point dilutions giving at least 95/100 AnTat 30 trypanosomes lysed.
y0 AnTat 30 16 20 11
Immunofluorescence was applied to the bloodstream trypanosomes arising in mice after the infective fly bite. It was found that both the percentage and the absolute number of AnTat 30 rose in the first three to four days but became nil by day six (Table II). Using antisera against “predominant” variable antigen types of the AnTat serodeme (i.e. those which tend to arise during the GRAY, 1965; VAN first weeks of an infection; MEIRVENNE et al., 1975; CAPBERN et al., 1977) this population arising in mice was found to be a mixture of variable antigen types (Table II).
J. D. BARRY
207
et al.
Fig. 2. Indirect immunofluorescencr test on metacyclic trypanosomes in discharged saliva of a single tsetse fly using anti-AnTat AnTat 30 trypanosomes appear yellow, other metacyclics are stained orange-red by the Evans Blue counterstain. Acetone fixation. diluted 1:75, FITC-conjugate 1:200.
Table II-Percentages of AnTat 30 and other “predominant” serodeme during developing parasitaemia of mouse bitten reaction Days after fly bite 3 4 2 7 i 10
AnTat 30
3
7
47 I%
ND 0.2
ND 0 4 6 4 8
: 0 :
7: 7 0
i3
100 to 500 trypanosomes counted per sample. N.D.-not determined (less than 100 trypanosomes
Discussion Our results show that a population of metacyclic trypanosomes is heterogeneous with respect to variable antigen type, as is the first patent parasitaemia in mice following cyclical transmission. These findings rule out the existence of either a single metacyclic basic antigen or a single basic antigen type arising as the first bloodstream population-at least for the Antwerp serodeme of T. brucei. The evidence in favour of the existence of more than one metacyclic antigen type in our tsetse
30 serum. Antiserum
variable antigen types of the same by infected fly. Immunofluorescence
variable antigen types 9 14 ND i ;!ij 18 20 0
ND 0 3 9 10 0 0 0
15
18
ND 0 0 0 0
ND 0 5 14 0
1 0
: 0
present in sample).
salivary probes is several-fold. First, the metacyclic antigen type AnTat 30 identified in the present study appears to constitute only 11 to 20% of the metacyclics. Second, serum taken from the syringeinfected rabbit on day 16 at the first peak of antimetacyclic activity (Fig. 1) lysed up to 66y0 of the metacyclic trypanosomes; there must therefore have been at least one other metacyclic antigen type present in the trypanosome population during the first two weeks of infection. Third, over-all anti-metacyclic activity of the rabbit serum rose
208
MULTIPLE
VARIABLE
ANTIGEN
TYPES IN METACYCLIC
again from day 23 after infection whereas activity against AnTat 30 decreased. Fourth, lysis of 100 % of metacyclics was not achieved with any of the rabbit sera up to 28 days, although 32 to 33-day serum from similarly infected rabbits can lyse the whole metacyclic population (LE RAY et al., 1978). Fifth, we have shown previously that pooled monospecific antisera against 22 different bloodstream variable antigen types from the Antwerp serodeme have trypanolytic activity against only about 10% of metacyclics (LE RAY et al., 1978). The variable antigen type AnTat 30 was detected in every probe examined, and always within the range 11 to 20% (Table I). Whether T. brucei metacyclics always display certain variable antigen types and at constant percentage remains to be seen. The preferential expression of the AnTat 30 phenotype in metacyclic populations is of interest in relation to the problem of genetic control of antigenic variation. When a comprehensive series of metacyclic variable antigen types has been obtained it will be possible to investigate the factors controlling the phenotypic composition of the metacyclic population. Morphological transformation of trypanosomes appears to be independent of antigenic variation as demonstrated by the multiplication of AnTat 30 at the onset of the fly-induced first patent parasitaemia in the present experiment, and as shown previously (LE RAY et al., 1977) for the slender-tostumpy transformation of bloodstream forms. Our experiments support the idea that trypanosomes belonging to a particular serodeme express characteristic “predominant” variable antigen types early in the course of an infection. Thus AnTat 30 would appear to be a predominant variable antigen type of the Antwerp serodeme as it was detected in the early parasitaemia of both the cyclically infected mouse and the syringe-infected rabbit. The first variable antigen types to appear in the mouse bloodstream following AnTat 30 included those previously reported as predominant in syringe line infections with trypanosomes of this serodeme by VAN MEIRWNNE et al. (1975). By isolating clones of syringe-passaged bloodstream forms of T. brucei with variable antigen type corresponding to that of probed metacyclic trypanosomes it appears to be possible to overcome the problem posed by the antigenic lability of pleomorphic trypanosome populations in the mammalian host. Cloning from stabilates derived from syringeinfected rabbits should enable the production of monospeciiic antisera against the diverse variable antigen types present in the metacyclic population and also provide a bulk source of defined antigen. Such antigen could be used for serodiagnosis, e.g. detection of antibodies to metacyclic and predominant variable antigen types, and possibly for vaccination against trypanosomes of the same serodeme. Acknowledgement We thank Mrs. P. McKail for skilled technical assistance. This work was supported by a grant from the U.K. Ministry of Overseas Development (Scheme R3338).
POPULATIONS
References Baltz, T., Baltz, D., Pautrizel, R., Richet, C., Lamblin, G. & Degand, P. (1977). Chemical and immunological characterization of specific glycoproteins from Typanosoma equiperdum variants. FEBS Letters, 82, 93-96. Barbet, A. F. & McGuire, T. C. (1978). Crossreacting determinants in variant-specific, surface antigens of African trypanosomes. Proceedings of the National Academy of Sciences USA, 75, 1989-1993. Capbern, A., Giroud, C., Baltz, T. & Mattern, P. (1977). Trypanosoma equiperdum : etude des variations antigeniques au tours de la trypanosomose experimentale du lapin. Experimental Parasitology, 42, 6-13. Cross, G. A. M. (1975). Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology, 71, 393-417. Cross, G. A. M. (1978). Antigenic variation in trypanosomes. Proceedings of the Royal Society, Series B, 202,55-72. Cunningham, M. P. (1966). The preservation of viable metacyclic forms of Trypanosoma rhodesiense and some studies on the antigenicity of the organism. Transactions of the Royal Society of Troaical Medicine and Hvoiene. 60. 126. Gray--A. R. (1965). Antigemtvariation in a strain of Trypanosoma brucei -transmitted by Glossina morsitans and G. nalnalis. Journal of General * Microbiology, 41, 1%214. d Gray, A. R. (1975). A pattern in the development of agglutinogenic antigens of cyclically-transmitted isolates of Trypanosoma gambiense. Transactions of the Royal Society of Tropical Medicine and fiygiene, 61, 131-138. Gray, A. R. & Luckins, A. G. (1976). Antigenic variation in salivarian trypanosomes. In: Biology of the Kinetoplastida, Lumsden, W. H. R. & Evans, D. A. (Editors), 1, London: Academic Press, pp. 493-652. Jenni, L. (1977). Comparisons of antigenic types of Trypanosoma (T.) brucei strains transmitted by Glossina m. morsitans. Acta tropica, 34, 35-41. Le Ray, D., Barry, J. D., Easton, C. & Vickerman, K. (1977). First tsetse fly transmission of the serodeme of Trypanosoma brucei. “AnTat” Annales de la Socie’tt! beige de Medecine tropicale, 57, 369-381. Le Ray, D., Barry, J. D. & Vickerman, K. (1978). Antigenic heterogeneity of metacyclic forms of Trypanosoma brucei. Nature, 273, 300-302. Lumsden, W. H. R. (1972). Principles of viable preservation of parasitic protozoa. International Journal for Parasitology, 2, 327-332. Van Meirvenne. N.. Tanssens. P. G. & Maanus, E. (1975). Antigenii iariation in syringe passaged populations of Trypanosoma (Trypanozoon) brucei. I. Rationalization of the experimental approach. Annales de la Sock% beige de Medecine tropicale, 55, l-23. Vickerman, K. (1978). Antigenic variation in trypanosomes. Nature, 273, 613-617. Accepted for publication 24th July, 1978.
Detection
of multiple variable antigen types in metacyclic Trypanosoma brucei
populations
of
J. DAVID BARRY*, STEPHEN L. HAJDUK, KEITH VICKERMAN Department of Zoology, Glasgow University, Glasgow G12 SQQ, U.K. AND DOMINIQUE LE RAY Laboratory of’ Protozoology, Prince Leopold Institute of Tropical Medicine, Nationalestraat l&5, B-2000 Antwerp, Belgium
Summary
The identification of antigen types in tsetse salivary gland metacyclic populations of Trypanosomabrucei requires the production of monospecific antisera to the corresponding bloodstream variable antigen types. Monospecific antisera against clones from cyclically transmitted populations are difficult to prepare, however, owing to the antigenic lability of such clones. This problem has been overcome by isolating an antigenically stable clone from a syringe-infected rabbit at a time when its serum showed incipient activity towards metacyclic trypanosomes. Monospecific antisera raised against this clone reacted with up to 20% metacyclics in trypanolysis and immunofluorescence tests, confirming that a clone-derived metacyclic population of T. brucei is heterogeneous with respect to variable antigen type. Introduction
Vaccination against African trypanosomiasis is thwarted by antigenic variation, whereby the trypanosomes change the glycoprotein of their surface coat and evade the immune response of the host (GRAY & LUCKINS, 1976; VICKERMAN,1978). In recent years our understanding of this variation in the mammalian bloodstream forms of Trypanosoma brucei has improved at both the popbiation (VAN ~~EIRVENNE et al.. 1975: CAPBERN et al.. 1977: iE RAY et al., 1977) and molecular (CROSS;1975, 1978; BALTZet al., 1977; BARBET& MCGUIRE, 1978) levels. On the other hand, little is known about the surface antigens of the metacyclic forms which develop in the tsetse salivary glands and initiate the mammalian phase of the life-cycle of this trypanosome. Some work has suggested that the metacyclics carry a “basic” antigen to which the trypanosomes revert on completing cyclical development in the fly, and that the basic antigen is the first of the numerous “variable antigen types” to be detected in the blood of a mammal following cyclical transmission (GRAY, 1965, 1975; CUNNINGHAM, 1966; JENNI, 1977). Such a basic antigen would have obvious potential in vaccination. Recently, however, we have presented preliminary evidence (LE RAY et al., 1978) that the metacyclic population may be heterogeneous with respect to variable antigen type. We now present a method for the identification of metacyclic antigen types using monospecific antisera
raised against bloodstream variable antigen types and further evidence against the existence of a single metacyclic antigen type. In our previous work (LE RAY et al., 1977; 1978) trypanosomes of the Antwerp serodeme (VAN MEIRVENNEet al., 1975) were transmitted through Glossina morsitans, and two observations of significance to the present work were made: (i) Following cyclical transmission, the variable antigen types of the bloodstream trypanosome population proved much less stable than variable antigen types of trypanosomes maintained by syringe-passage in laboratory animals : this lability of antigen type makes the production of monospecificantiskia (and hence the antigenic typing of metacvclicsl difficult (LE RAY et al.. 197’i’l. (ii) Sera’cbllected ‘after a month fro& rabbits infected with the syringe-passaged line showed lytic activity against most or all of the metacyclics extruded at one time by the tsetse fly: the variable antigen type expressed by metacyclics would appear therefore not to be specific to the fly stages of the trypanosome life-cycle (LE RAY et al., 1978). In order to produce monospecific antisera to metacyclic variable antigen types the following procedure was devised. A rabbit was infected with the syringe-passaged line and by frequent collection of sera and trypanosomes for cryopreservation, an attempt was made to pin-point the time of appearance in the blood of variable antigen types expressed by metacyclics. Blood collected at, or immediately before, the time that the serum began to show lytic activity towards metacyclics should contain trypanosomes of the same variable antigen type as the metacyclics. Cloning of trypanosomes from such blood samples should give antigenically stable populations of the parasite. Monospecific antisera could then be produced against these stable variable antigen types and the activity of these antisera tested against metacyclic trypanosomes. Materials
and Methods
A rabbit (New Zealand White-Half Lop, 3.5 kg) was infected intravenously with lo6 syringe*Present Address : International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya.
206
MULTIPLE VARIABLE ANTIGEN TYPES IN METACYCLIC POPULATIONS
passaged line bloodstream trypanosomes of variable antigen type AnTat 1; for reference purposes the different variable antigen types in the repertoire of trypanosomes of the Antwerp serodeme are designated AnTat 1, AnTat 2 and so on (VAN MEIRVENNE et al., 1975). Blood samples were collected at intervals of one to three days and injected intraperitoneally into four to six-week-old female CFLP mice, from which blood was passaged to fresh mice at two to four-day intervals until enough trypanosomes (at least antilog 7*5/ml blood) were present to cryopreserve as stabilates (LUMSDEN, 1972). Serum samples collected simultaneously from the rabbit were stored at -20°C. To determine the time of appearance of metacyclic variable antigen types in the rabbit’s blood, the trypanolytic activity of the serum was tested using the method of VAN MEIRwNNE et al., (1975) against metacyclics obtained by allowing a fly carrying trypanosomes to salivate (probe) into guinea-pig serum in vitro at 37%. The methods used for maintenance of GZossina morsitans and for production of metacyclic trypanosomes were described by LE RAY et al. (1978), and cloning, indirect immunofluorescence and trypanolysis reactions, and the production of monospecific antiserum were conducted as described by VAN MEIRVENNE et al. (1975). Results Lytic activity against metacyclics was found in the rabbit’s serum collected from day nine onwards (Fig. 1). Lysis of 100% of metacyclics was not obtained with any of the samples, and two or possibly three peaks of activity occurred on days 16,23 and 29 respectively, suggesting that different metacyclic variable antigen types had arisen in the rabbit at different times.
To produce a monospecific anti-variable antigen type antiserum it is necessary first to isolate a clone expressing that antigen type. Trypanosome stabilate material from the beginning of the first peak of anti-metacyclic serum reactivity in the rabbit (nine days) was therefore selected for cloning in the hope that it would contain metacyclic variable antigen types. Each clone was tested for trypanolysis by serum taken from a second rabbit seven days after it was bitten by an infected tsetse fly: such serum had activity against most of the metacyclics in one probe (LE RAY et al., 1978). The tenth clone isolated was completely lysed by this serum, suggesting antigenic homology with the metacyclics. The variable antigen type of this clone, after comparison with known AnTats and with the other new clones, has been designated AnTat 30. To examine the level of trypanolytic activity against AnTat 30, the range of sera from the rabbit infected with the syringe line were titrated against bloodstream form AnTat 30 (Fig. 1); this activity developed by day 14, i.e. five days later than the over-all activity against metacyclics, and decreased from day 28 while over-all anti-metacyclic activity was rising again. A monospecific antiserum was raised in a third rabbit and tested against metacyclics by type specific immunofluorescence. Using salivary probes on to dry slides from flies which had originally ingested different AnTats, this antiserum labelled 11 to 20 y0 of metacyclics in all the probes examined (Fig. 2), including sequential probes from one fly (Table I). A monospecific antiserum to AnTat 30 raised in a mouse behaved similarly. Trypanolysis reactions with both antisera gave corresponding percentages of AnTat 30 in the probes.
Table I-Percentage of AnTat 30 in sequential probes from a single fly determined in immunofluorescence with rabbit anti-AnTat 30 monospecific serum Days after infecting blood meal 26 37 46
Fig. 1. Development of anti-metacyclic lytic activity in B rabbit chronically infected with a syringe passagedline of AnTat serodeme trypanosomes. The metacyclics tested were deposited by a single fly on one occasion; for each serum sample 33 trypanasomes were counted and the y. lysed calculated. Anti-AnTat 30 titres were determined as the inverses of the end-point dilutions giving at least 95/100 AnTat 30 trypanosomes lysed.
y0 AnTat 30 16 20 11
Immunofluorescence was applied to the bloodstream trypanosomes arising in mice after the infective fly bite. It was found that both the percentage and the absolute number of AnTat 30 rose in the first three to four days but became nil by day six (Table II). Using antisera against “predominant” variable antigen types of the AnTat serodeme (i.e. those which tend to arise during the GRAY, 1965; VAN first weeks of an infection; MEIRVENNE et al., 1975; CAPBERN et al., 1977) this population arising in mice was found to be a mixture of variable antigen types (Table II).
J. D. BARRY
207
et al.
Fig. 2. Indirect immunofluorescencr test on metacyclic trypanosomes in discharged saliva of a single tsetse fly using anti-AnTat AnTat 30 trypanosomes appear yellow, other metacyclics are stained orange-red by the Evans Blue counterstain. Acetone fixation. diluted 1:75, FITC-conjugate 1:200.
Table II-Percentages of AnTat 30 and other “predominant” serodeme during developing parasitaemia of mouse bitten reaction Days after fly bite 3 4 2 7 i 10
AnTat 30
3
7
47 I%
ND 0.2
ND 0 4 6 4 8
: 0 :
7: 7 0
i3
100 to 500 trypanosomes counted per sample. N.D.-not determined (less than 100 trypanosomes
Discussion Our results show that a population of metacyclic trypanosomes is heterogeneous with respect to variable antigen type, as is the first patent parasitaemia in mice following cyclical transmission. These findings rule out the existence of either a single metacyclic basic antigen or a single basic antigen type arising as the first bloodstream population-at least for the Antwerp serodeme of T. brucei. The evidence in favour of the existence of more than one metacyclic antigen type in our tsetse
30 serum. Antiserum
variable antigen types of the same by infected fly. Immunofluorescence
variable antigen types 9 14 ND i ;!ij 18 20 0
ND 0 3 9 10 0 0 0
15
18
ND 0 0 0 0
ND 0 5 14 0
1 0
: 0
present in sample).
salivary probes is several-fold. First, the metacyclic antigen type AnTat 30 identified in the present study appears to constitute only 11 to 20% of the metacyclics. Second, serum taken from the syringeinfected rabbit on day 16 at the first peak of antimetacyclic activity (Fig. 1) lysed up to 66y0 of the metacyclic trypanosomes; there must therefore have been at least one other metacyclic antigen type present in the trypanosome population during the first two weeks of infection. Third, over-all anti-metacyclic activity of the rabbit serum rose
208
MULTIPLE
VARIABLE
ANTIGEN
TYPES IN METACYCLIC
again from day 23 after infection whereas activity against AnTat 30 decreased. Fourth, lysis of 100 % of metacyclics was not achieved with any of the rabbit sera up to 28 days, although 32 to 33-day serum from similarly infected rabbits can lyse the whole metacyclic population (LE RAY et al., 1978). Fifth, we have shown previously that pooled monospecific antisera against 22 different bloodstream variable antigen types from the Antwerp serodeme have trypanolytic activity against only about 10% of metacyclics (LE RAY et al., 1978). The variable antigen type AnTat 30 was detected in every probe examined, and always within the range 11 to 20% (Table I). Whether T. brucei metacyclics always display certain variable antigen types and at constant percentage remains to be seen. The preferential expression of the AnTat 30 phenotype in metacyclic populations is of interest in relation to the problem of genetic control of antigenic variation. When a comprehensive series of metacyclic variable antigen types has been obtained it will be possible to investigate the factors controlling the phenotypic composition of the metacyclic population. Morphological transformation of trypanosomes appears to be independent of antigenic variation as demonstrated by the multiplication of AnTat 30 at the onset of the fly-induced first patent parasitaemia in the present experiment, and as shown previously (LE RAY et al., 1977) for the slender-tostumpy transformation of bloodstream forms. Our experiments support the idea that trypanosomes belonging to a particular serodeme express characteristic “predominant” variable antigen types early in the course of an infection. Thus AnTat 30 would appear to be a predominant variable antigen type of the Antwerp serodeme as it was detected in the early parasitaemia of both the cyclically infected mouse and the syringe-infected rabbit. The first variable antigen types to appear in the mouse bloodstream following AnTat 30 included those previously reported as predominant in syringe line infections with trypanosomes of this serodeme by VAN MEIRWNNE et al. (1975). By isolating clones of syringe-passaged bloodstream forms of T. brucei with variable antigen type corresponding to that of probed metacyclic trypanosomes it appears to be possible to overcome the problem posed by the antigenic lability of pleomorphic trypanosome populations in the mammalian host. Cloning from stabilates derived from syringeinfected rabbits should enable the production of monospeciiic antisera against the diverse variable antigen types present in the metacyclic population and also provide a bulk source of defined antigen. Such antigen could be used for serodiagnosis, e.g. detection of antibodies to metacyclic and predominant variable antigen types, and possibly for vaccination against trypanosomes of the same serodeme. Acknowledgement We thank Mrs. P. McKail for skilled technical assistance. This work was supported by a grant from the U.K. Ministry of Overseas Development (Scheme R3338).
POPULATIONS
References Baltz, T., Baltz, D., Pautrizel, R., Richet, C., Lamblin, G. & Degand, P. (1977). Chemical and immunological characterization of specific glycoproteins from Typanosoma equiperdum variants. FEBS Letters, 82, 93-96. Barbet, A. F. & McGuire, T. C. (1978). Crossreacting determinants in variant-specific, surface antigens of African trypanosomes. Proceedings of the National Academy of Sciences USA, 75, 1989-1993. Capbern, A., Giroud, C., Baltz, T. & Mattern, P. (1977). Trypanosoma equiperdum : etude des variations antigeniques au tours de la trypanosomose experimentale du lapin. Experimental Parasitology, 42, 6-13. Cross, G. A. M. (1975). Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology, 71, 393-417. Cross, G. A. M. (1978). Antigenic variation in trypanosomes. Proceedings of the Royal Society, Series B, 202,55-72. Cunningham, M. P. (1966). The preservation of viable metacyclic forms of Trypanosoma rhodesiense and some studies on the antigenicity of the organism. Transactions of the Royal Society of Troaical Medicine and Hvoiene. 60. 126. Gray--A. R. (1965). Antigemtvariation in a strain of Trypanosoma brucei -transmitted by Glossina morsitans and G. nalnalis. Journal of General * Microbiology, 41, 1%214. d Gray, A. R. (1975). A pattern in the development of agglutinogenic antigens of cyclically-transmitted isolates of Trypanosoma gambiense. Transactions of the Royal Society of Tropical Medicine and fiygiene, 61, 131-138. Gray, A. R. & Luckins, A. G. (1976). Antigenic variation in salivarian trypanosomes. In: Biology of the Kinetoplastida, Lumsden, W. H. R. & Evans, D. A. (Editors), 1, London: Academic Press, pp. 493-652. Jenni, L. (1977). Comparisons of antigenic types of Trypanosoma (T.) brucei strains transmitted by Glossina m. morsitans. Acta tropica, 34, 35-41. Le Ray, D., Barry, J. D., Easton, C. & Vickerman, K. (1977). First tsetse fly transmission of the serodeme of Trypanosoma brucei. “AnTat” Annales de la Socie’tt! beige de Medecine tropicale, 57, 369-381. Le Ray, D., Barry, J. D. & Vickerman, K. (1978). Antigenic heterogeneity of metacyclic forms of Trypanosoma brucei. Nature, 273, 300-302. Lumsden, W. H. R. (1972). Principles of viable preservation of parasitic protozoa. International Journal for Parasitology, 2, 327-332. Van Meirvenne. N.. Tanssens. P. G. & Maanus, E. (1975). Antigenii iariation in syringe passaged populations of Trypanosoma (Trypanozoon) brucei. I. Rationalization of the experimental approach. Annales de la Sock% beige de Medecine tropicale, 55, l-23. Vickerman, K. (1978). Antigenic variation in trypanosomes. Nature, 273, 613-617. Accepted for publication 24th July, 1978.