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Variability of in vitro culture characteristics, including metacyclic trypomastigote production, in different stocks of Trypanosoma congolense
Acta Tropica, 50(1992)135-140
135
© 1991 Elsevier Science Publishers B.V. All rights reserved. 0001-706X/91/$03.50 ACTROP 00175
Variability of in vitro culture characteristics, including metacyclic trypomastigote production, in different stocks of Trypanosoma congolense I.A. Frame 1, C.A. R o s s 2 a n d A . G . L u c k i n s 2 1London School of Hygiene and Tropical Medicine, Keppel Street, London, U.K., and 2Centrefor Tropical Veterinary Medicine, University o['Edinburgh Easter Bush, Roslin, Midlothian, U.K. (Received 4 April 1991; accepted 24 June 1991)
Six cloned stocks of Trypanosoma congolense, isolated from the same area of Eastern Zambia, were maintained in vitro as insect form cultures producing infective metacyclic trypanosomes. Although the same general culture conditions were applied, different handling regimes were required for optimum growth of each stock. During primary isolation, many differences were found in the culture characteristics of the stocks. The time taken for cytoadherence to occur varied from 14 to 62 days, while the interval between attachment and the appearance of infective metacyclic trypanosomes ranged from 9 to 94 days. There was a 10-fold difference in the numbers of metacyclic forms produced by different stocks. Time in culture appeared to have little effect on the production of metacyclic forms, and it is probable that in vitro characteristics of T. congolense depend on the genetic constitution of individual stocks or clones. Key words: Trypanosomacongolense culture in vitro; Metacyclic trypanosomes
Introduction It is now possible to culture in vitro all the morphological forms of Trypanosoma eongolense that have been described in the tsetse fly vector (Gray et al., 1981, 1984). Cultures consisting predominantly of the epimastigote stage produce infective metacyclic trypanosomes which can be used in studies on antigenic variation (Crowe et al., 1983; Luckins et al., 1986; Prain and Ross, 1988), biochemistry (Ross et al., 1987; Ross, 1987) and epidemiology (Frame et al., 1990). Selection of stocks for different types of investigation therefore depends to a certain extent on their ability to produce metacyclic trypanosomes. This paper describes the in vitro characteristics of six stocks of T. congolensewhich were isolated from the same area of Eastern Zambia. The cultures were established and maintained using similar methods to those described by Gray et al. (1981, 1984). There were, however, considerable differences in handling requirements and growth characteristics of different stocks in vitro and we have found that this variability extended to metacyclic production. Correspondence address." Dr. C.A. Ross, CTVM, Easter Bush, Roslin, Midlothian EH25 9RG, U.K. [Fax 031-445-5099]
136 Materials and Methods
Trypanosomes The stocks used in this paper were obtained as uncloned isolates from the London School of Hygiene and Tropical Medicine. The original isolation numbers, T R E U numbers of the uncloned parent stocks and their cloned derivatives have been described elsewhere (Frame et al., 1990). Trypanosome stocks were each cyclically transmitted by G. morsitans obtained from puparia supplied by the Tsetse Research Laboratory, Langford, Bristol. Emerged tsetse were infected and maintained as described by Gray et al. (1981).
Establishment and maintenance Of in vitro cultures The techniques used for initiation and maintenance of cultures were essentially as described by Gray et al. (1981, 1984) although primary cultures were initiated in Falcon (Primaria) T-25 tissue culture flasks. Cultures were maintained in 4 ml medium which was changed at 48 h intervals. Once established, cultures were incubated with 4 ml medium for two successive 48 h intervals followed by a 72 h incubation with 8 ml medium.
Metacyclic to,panosorne production The total number of metacyclic forms and their proportion relative to other insect forms in culture supernatants of five stocks were examined over a 14 day period involving six harvests. Two T-25 flasks were initiated for each of the five stocks examined. Trypanosomes were passaged from mature cultures 40 days before the first harvest. The number of trypanosomes produced in each culture was determined after DE-52 anion exchange chromatography, using the method described by Ross (1987). Briefly, two 0.5 ml samples of freshly harvested supernatant from each flask were passed through 2 ml (packed volume) DE-52 cellulose columns. Metacyclic forms were eluted with 2.5 ml phosphate buffered saline pH 8.0 containing 1% (w/v) glucose (PSG). The number per ml in the total eluate (3 ml) was obtained by haemocytometer count.
Results and Discussion
All six cloned Zambian stocks of T. congolense were successfully adapted to insect form culture in vitro and the times taken for those adaptations are shown in Table 1. The attachment of epimastigotes is an important feature in the establishment of insect form culture. However, although the presence of attached epimastigotes is essential for metacyclic form production (Hendry and Vickerman, 1988), their presence does not indicate that metacyclic production is imminent. Attached epimastigotes were first observed between 14 days (TREU 1881) and 62 days (TREU 2037) post-initiation, but the interval between attachment and the appearance of infective metacyclic forms ranged from 9 days (TREU 1881) to 94 days (TREU 1896). The variation in time taken for the different trypanosome stocks to establish attached
137 TABLE 1 Origin, designation, time of epimastigote attachment and time of infectivity to mice of the cloned Zambian T. congolense cultures initiated after dissection of infected tsetse flies (Glossina morsitans)
Stock
TREU TREU TREU TREU TREU TREU
1881 1885 1894 1896 2034 2037
Source
Attachment of epimastigotes (days)
Infectivity to mice (days)
Proventriculus Midgut Proventriculus Proventriculus Proventriculus Proventriculus
14 27 33 16 44 62
23 44 104 I l0 107 107
epimastigotes, and subsequently mature, could be due partly to the way in which the cultures were manipulated and might not necessarily be a reflection of each stock's ability to adapt to culture in vitro. It is interesting to note, however, that TREU 1881 which produced the largest numbers of metacyclic forms also achieved established epimastigote forms in the shortest period (14 days) and produced infective metacyclics by day 23 after initiation, whilst TREU 2037 produced the smallest numbers of metacyclics and took the longest time to demonstrate epimastigote attachment and metacyclic production. While epimastigotes of stock TREU 1896 established rapidly (Table 1), subsequent metacyclic production was very slow. In addition, cryopreservation and resuscitation of TREU 1896 insect forms was extremely difficult and it was often impossible to obtain metacyclic producing cultures. Therefore, no data are presented for TREU 1896 in Figs. I and 2. Fig. 1 shows the absolute numbers of metacyclics per T-25 culture flask produced for each stock at each of the six harvests. There was a 10-fold difference in the numbers of metacyclics produced by TREU 1881 compared to those produced by cultures of TREU 2037, TREU 1894 and TREU 1885, and each stock showed little day to day variation in the numbers of metacyclics counted over the period of observation. The majority of trypanosomes in established culture supernatants were epimastigore forms, while the proportions of metacyclic forms present could vary widely. These showed a similar pattern to the absolute numbers of metacyclic forms produced, and are to some extent stock specific (Fig. 2). TREU 2037 produced fewer metacyclic forms relative to other insect forms whilst TREU 1881 produced the largest proportion. TREU 1894, TREU 1885 and TREU 1881 showed a rise in the proportion of metacyclic forms to other insect forms at the 2nd and 5th harvests. These harvests corresponded to the second medium change after the 72 h culture period. Although all six stocks were maintained in the same medium and under the same conditions, cultures of each stock required different handling requirements to optimize metacyclic production. Cultures of TREU 1881 were washed vigorously and the epimastigote carpet was scraped extensively during medium changes. This treatment was necessary to prevent cultures requiring more frequent changes of medium. In contrast, cultures of TREU 2037 were treated with gentler washes and scraped
138
14
13
~]lsth .... t
[]4thh ..... t
rh N r
10
[ ] 2nd h..... t
•
~]'~i/
[ ] 3rd t...... t
[ ] 6th h..... t
5th h..... t
g9 8 6 s
Nit
~j~:~ll]
/
1
TREU2037 T R E U 2 0 3 4
TREU1894
TREU1885
TREU1881
TRYPANOSOME STOCKS Fig. 1. The number of metacyclic trypanosomes per T-25 culture flask produced in vitro from T. congolense, T R E U 2037, T R E U 2034, T R E U 1894, T R E U 1885 and T R E U 1881 over six consecutive harvests. Vertical lines represent 2 x S.E.
11-
[ ] 1st harvest
[ ] 4th harvest
9-
I~ 2rid harvest
•
Z < 8et >-
[ ] 3rd harvc.st
[ ] 6th harvest
t¢l 10O
7-
I-
5tll harvest
64-
~ •
3-
1- ~ TREU2037 TREU2034
TREU1894 TREU1885 TREU 1881
TRYPANOSOME STOCKS Fig. 2. The relative proportions of metacyclic trypanosomes compared to other insect forms produced in vitro from T. congolense T R E U 2037, T R E U 2034, T R E U 1894, T R E U 1885 and T R E U 188I over six consecutive harvests. Vertical lines represent 2 x S.E.
only occasionally. While these different handling requirements would certainly influence the total number of insect forms harvested, they do not explain the differences in proportions of metacyclic forms compared to other insect forms observed in the different stocks. Gray et al. (1984) also noted differences in the numbers of metacyclic forms produced in vitro by different stocks of T. congolense; one from East Africa
139 a n d one f r o m West Africa. T h e y a t t r i b u t e d this to differences in a d a p t a t i o n to in vitro culture. T h e p o s s i b l e p h y s i o l o g i c a l differences between stocks which might c o n t r i b u t e to their a d a p t a b i l i t y in vitro are n o t k n o w n . In the case o f these six Z a m b i a n isolates, a l t h o u g h all were collected from a small, well-defined area, they are antigenically u n r e l a t e d a n d represent six different s e r o d e m e s ( F r a m e et al., 1990). F o u r o f the stocks, T R E U 1881, 1894, 1896 a n d 2034 have been c h a r a c t e r i s e d e n z y m a t i c a l l y a n d s h o w n to b e l o n g to four different z y m o d e m e s ( G a s h u m b a et al., 1988). In a d d i t i o n , these a u t h o r s c o n s t r u c t e d a d e n d o g r a m a c c o r d i n g to dissimilarities between enzyme profiles, which s h o w e d that T R E U 1894 exhibited greater differences from 1881, 1896 a n d 2034 t h a n these stocks did from each other. A s s o c i a t i o n between clinical disease a n d v e c t o r / z y m o d e m e type has been suggested for T. cruzi (Miles et al., 1981; Miles, 1983). A l t h o u g h no similar trends have yet been established for T. congolense, it is possible t h a t such a s s o c i a t i o n s d o exist. It is also w o r t h speculating t h a t the wide range o f m o r p h o l o g i c a l a n d biological characteristics shown by T. congolense in the insect v e c t o r a n d the v e r t e b r a t e host ( H o a r e , 1972; Stephen, 1986) might also be d i s p l a y e d when the parasites are c u l t u r e d in vitro.
Acknowledgements We wish to t h a n k Dr. D . G . G o d f r e y w h o supplied the stocks o f Trypanosoma congolense. T h e w o r k was f u n d e d by the Overseas D e v e l o p m e n t A d m i n i s t r a t i o n , London,
References Crowe, J.S., Barry, J.D., Ross, C.A., Luckins, A.G. and Vickerman, K. (1983) All metacyclic variable antigen types of Trypanosoma congolense identified using monoclonal antibodies. Nature 306, 389 391. Frame, I.A., Ross, C.A. and Luckins, A.G. (1990) Characterisation of Trypanosoma congolense serodemes in stocks isolated from Chipata district, Zambia. Parasitology 101,235-241. Gashumba, J.K., Baker, R.D. and Godfrey, D.G. (1988) Trypanosoma congolense: the distribution of enzyme variants in East and West Africa. Parasitology 96, 475 486. Gray, M.A., Cunningham, I., Gardiner, P.R., Taylor, A.M. and Luckins, A.G. (1981) Cultivation of infective forms of Trypanosoma congolense at 28°C from trypanosomes in the proboscis of Glossina morsitans. Parasitology 82, 81-95. Gray, M.A., Ross, C.A.,Taylor, A.M. and Luckins, A.G. (1984) In vitro cultivation of Trypanosoma congolense: the production of infective metacyclic trypanosomes in cultures initiated from cloned stocks. Acta Trop. 41,343-353. Hendry, K.A.K. and Vickerman, K. (1988) The requirement for epimastigote attachment during division and metacyclogenesis in Trypanosoma congolense. Parasitol. Res. 74, 403 408. Hoare, C.A. (1972) The trypanosomes of mammals. A zoological monograph. Blackwell, Oxford. Luckins, A.G., Frame, I.A., Gray, M.A., Crowe, J.S. and Ross, C.A. (1986) Analysis of trypanosome variable antigen type in cultures of metacyclics and mammalian forms of Trypanosoma congolense. Parasitology 93, 99-109. Miles, M.A. (1983) The epidemiology of South American trypanosomiasis - - biochemical and immunological approaches and their relevance to control. Trans. R. Soc. Trop. Med. Hyg. 77, 5-23. Miles, M.A., Povoa, M.M., de Souza, A.A., Lainson, R., Shaw, J.J. and Keneridge, D.S. (1981) Chagas' disease in the Amazon Basin: II. The distribution of Trypanosoma cruzi zymodemes 1 and 3 in Para State, North Brazil. Trans. R. Soc. Trop. Med. Hyg. 75, 667-674.
140 Prain, C.J. and Ross, C.A. (1988) Trvpanosoma congolense: interactions between trypanosomes expressing different metacyclic variable antigen types in vitro and in vivo. Parasitology 97, 277-286. Ross, C.A. (1987) Trypanosoma congolense: differentiation to metacyclic trypanosomes in culture depends on the concentration of glutamine or proline. Acta Trop. 44, 293 301. Ross, C.A., Cardoso de Almeida, M.L. and Turner, M.J. (1987) Variant surface glycoproteins of Trypanosoma congolense bloodstream and metacyclic forms are anchored by a glycolipid tail. Mol. Biochem. Parasitol. 22, 153 158. Stephen, L.E. (1986) Trypanosomiasis. A Veterinary Perspective. Pergamon, Oxford.
135
© 1991 Elsevier Science Publishers B.V. All rights reserved. 0001-706X/91/$03.50 ACTROP 00175
Variability of in vitro culture characteristics, including metacyclic trypomastigote production, in different stocks of Trypanosoma congolense I.A. Frame 1, C.A. R o s s 2 a n d A . G . L u c k i n s 2 1London School of Hygiene and Tropical Medicine, Keppel Street, London, U.K., and 2Centrefor Tropical Veterinary Medicine, University o['Edinburgh Easter Bush, Roslin, Midlothian, U.K. (Received 4 April 1991; accepted 24 June 1991)
Six cloned stocks of Trypanosoma congolense, isolated from the same area of Eastern Zambia, were maintained in vitro as insect form cultures producing infective metacyclic trypanosomes. Although the same general culture conditions were applied, different handling regimes were required for optimum growth of each stock. During primary isolation, many differences were found in the culture characteristics of the stocks. The time taken for cytoadherence to occur varied from 14 to 62 days, while the interval between attachment and the appearance of infective metacyclic trypanosomes ranged from 9 to 94 days. There was a 10-fold difference in the numbers of metacyclic forms produced by different stocks. Time in culture appeared to have little effect on the production of metacyclic forms, and it is probable that in vitro characteristics of T. congolense depend on the genetic constitution of individual stocks or clones. Key words: Trypanosomacongolense culture in vitro; Metacyclic trypanosomes
Introduction It is now possible to culture in vitro all the morphological forms of Trypanosoma eongolense that have been described in the tsetse fly vector (Gray et al., 1981, 1984). Cultures consisting predominantly of the epimastigote stage produce infective metacyclic trypanosomes which can be used in studies on antigenic variation (Crowe et al., 1983; Luckins et al., 1986; Prain and Ross, 1988), biochemistry (Ross et al., 1987; Ross, 1987) and epidemiology (Frame et al., 1990). Selection of stocks for different types of investigation therefore depends to a certain extent on their ability to produce metacyclic trypanosomes. This paper describes the in vitro characteristics of six stocks of T. congolensewhich were isolated from the same area of Eastern Zambia. The cultures were established and maintained using similar methods to those described by Gray et al. (1981, 1984). There were, however, considerable differences in handling requirements and growth characteristics of different stocks in vitro and we have found that this variability extended to metacyclic production. Correspondence address." Dr. C.A. Ross, CTVM, Easter Bush, Roslin, Midlothian EH25 9RG, U.K. [Fax 031-445-5099]
136 Materials and Methods
Trypanosomes The stocks used in this paper were obtained as uncloned isolates from the London School of Hygiene and Tropical Medicine. The original isolation numbers, T R E U numbers of the uncloned parent stocks and their cloned derivatives have been described elsewhere (Frame et al., 1990). Trypanosome stocks were each cyclically transmitted by G. morsitans obtained from puparia supplied by the Tsetse Research Laboratory, Langford, Bristol. Emerged tsetse were infected and maintained as described by Gray et al. (1981).
Establishment and maintenance Of in vitro cultures The techniques used for initiation and maintenance of cultures were essentially as described by Gray et al. (1981, 1984) although primary cultures were initiated in Falcon (Primaria) T-25 tissue culture flasks. Cultures were maintained in 4 ml medium which was changed at 48 h intervals. Once established, cultures were incubated with 4 ml medium for two successive 48 h intervals followed by a 72 h incubation with 8 ml medium.
Metacyclic to,panosorne production The total number of metacyclic forms and their proportion relative to other insect forms in culture supernatants of five stocks were examined over a 14 day period involving six harvests. Two T-25 flasks were initiated for each of the five stocks examined. Trypanosomes were passaged from mature cultures 40 days before the first harvest. The number of trypanosomes produced in each culture was determined after DE-52 anion exchange chromatography, using the method described by Ross (1987). Briefly, two 0.5 ml samples of freshly harvested supernatant from each flask were passed through 2 ml (packed volume) DE-52 cellulose columns. Metacyclic forms were eluted with 2.5 ml phosphate buffered saline pH 8.0 containing 1% (w/v) glucose (PSG). The number per ml in the total eluate (3 ml) was obtained by haemocytometer count.
Results and Discussion
All six cloned Zambian stocks of T. congolense were successfully adapted to insect form culture in vitro and the times taken for those adaptations are shown in Table 1. The attachment of epimastigotes is an important feature in the establishment of insect form culture. However, although the presence of attached epimastigotes is essential for metacyclic form production (Hendry and Vickerman, 1988), their presence does not indicate that metacyclic production is imminent. Attached epimastigotes were first observed between 14 days (TREU 1881) and 62 days (TREU 2037) post-initiation, but the interval between attachment and the appearance of infective metacyclic forms ranged from 9 days (TREU 1881) to 94 days (TREU 1896). The variation in time taken for the different trypanosome stocks to establish attached
137 TABLE 1 Origin, designation, time of epimastigote attachment and time of infectivity to mice of the cloned Zambian T. congolense cultures initiated after dissection of infected tsetse flies (Glossina morsitans)
Stock
TREU TREU TREU TREU TREU TREU
1881 1885 1894 1896 2034 2037
Source
Attachment of epimastigotes (days)
Infectivity to mice (days)
Proventriculus Midgut Proventriculus Proventriculus Proventriculus Proventriculus
14 27 33 16 44 62
23 44 104 I l0 107 107
epimastigotes, and subsequently mature, could be due partly to the way in which the cultures were manipulated and might not necessarily be a reflection of each stock's ability to adapt to culture in vitro. It is interesting to note, however, that TREU 1881 which produced the largest numbers of metacyclic forms also achieved established epimastigote forms in the shortest period (14 days) and produced infective metacyclics by day 23 after initiation, whilst TREU 2037 produced the smallest numbers of metacyclics and took the longest time to demonstrate epimastigote attachment and metacyclic production. While epimastigotes of stock TREU 1896 established rapidly (Table 1), subsequent metacyclic production was very slow. In addition, cryopreservation and resuscitation of TREU 1896 insect forms was extremely difficult and it was often impossible to obtain metacyclic producing cultures. Therefore, no data are presented for TREU 1896 in Figs. I and 2. Fig. 1 shows the absolute numbers of metacyclics per T-25 culture flask produced for each stock at each of the six harvests. There was a 10-fold difference in the numbers of metacyclics produced by TREU 1881 compared to those produced by cultures of TREU 2037, TREU 1894 and TREU 1885, and each stock showed little day to day variation in the numbers of metacyclics counted over the period of observation. The majority of trypanosomes in established culture supernatants were epimastigore forms, while the proportions of metacyclic forms present could vary widely. These showed a similar pattern to the absolute numbers of metacyclic forms produced, and are to some extent stock specific (Fig. 2). TREU 2037 produced fewer metacyclic forms relative to other insect forms whilst TREU 1881 produced the largest proportion. TREU 1894, TREU 1885 and TREU 1881 showed a rise in the proportion of metacyclic forms to other insect forms at the 2nd and 5th harvests. These harvests corresponded to the second medium change after the 72 h culture period. Although all six stocks were maintained in the same medium and under the same conditions, cultures of each stock required different handling requirements to optimize metacyclic production. Cultures of TREU 1881 were washed vigorously and the epimastigote carpet was scraped extensively during medium changes. This treatment was necessary to prevent cultures requiring more frequent changes of medium. In contrast, cultures of TREU 2037 were treated with gentler washes and scraped
138
14
13
~]lsth .... t
[]4thh ..... t
rh N r
10
[ ] 2nd h..... t
•
~]'~i/
[ ] 3rd t...... t
[ ] 6th h..... t
5th h..... t
g9 8 6 s
Nit
~j~:~ll]
/
1
TREU2037 T R E U 2 0 3 4
TREU1894
TREU1885
TREU1881
TRYPANOSOME STOCKS Fig. 1. The number of metacyclic trypanosomes per T-25 culture flask produced in vitro from T. congolense, T R E U 2037, T R E U 2034, T R E U 1894, T R E U 1885 and T R E U 1881 over six consecutive harvests. Vertical lines represent 2 x S.E.
11-
[ ] 1st harvest
[ ] 4th harvest
9-
I~ 2rid harvest
•
Z < 8et >-
[ ] 3rd harvc.st
[ ] 6th harvest
t¢l 10O
7-
I-
5tll harvest
64-
~ •
3-
1- ~ TREU2037 TREU2034
TREU1894 TREU1885 TREU 1881
TRYPANOSOME STOCKS Fig. 2. The relative proportions of metacyclic trypanosomes compared to other insect forms produced in vitro from T. congolense T R E U 2037, T R E U 2034, T R E U 1894, T R E U 1885 and T R E U 188I over six consecutive harvests. Vertical lines represent 2 x S.E.
only occasionally. While these different handling requirements would certainly influence the total number of insect forms harvested, they do not explain the differences in proportions of metacyclic forms compared to other insect forms observed in the different stocks. Gray et al. (1984) also noted differences in the numbers of metacyclic forms produced in vitro by different stocks of T. congolense; one from East Africa
139 a n d one f r o m West Africa. T h e y a t t r i b u t e d this to differences in a d a p t a t i o n to in vitro culture. T h e p o s s i b l e p h y s i o l o g i c a l differences between stocks which might c o n t r i b u t e to their a d a p t a b i l i t y in vitro are n o t k n o w n . In the case o f these six Z a m b i a n isolates, a l t h o u g h all were collected from a small, well-defined area, they are antigenically u n r e l a t e d a n d represent six different s e r o d e m e s ( F r a m e et al., 1990). F o u r o f the stocks, T R E U 1881, 1894, 1896 a n d 2034 have been c h a r a c t e r i s e d e n z y m a t i c a l l y a n d s h o w n to b e l o n g to four different z y m o d e m e s ( G a s h u m b a et al., 1988). In a d d i t i o n , these a u t h o r s c o n s t r u c t e d a d e n d o g r a m a c c o r d i n g to dissimilarities between enzyme profiles, which s h o w e d that T R E U 1894 exhibited greater differences from 1881, 1896 a n d 2034 t h a n these stocks did from each other. A s s o c i a t i o n between clinical disease a n d v e c t o r / z y m o d e m e type has been suggested for T. cruzi (Miles et al., 1981; Miles, 1983). A l t h o u g h no similar trends have yet been established for T. congolense, it is possible t h a t such a s s o c i a t i o n s d o exist. It is also w o r t h speculating t h a t the wide range o f m o r p h o l o g i c a l a n d biological characteristics shown by T. congolense in the insect v e c t o r a n d the v e r t e b r a t e host ( H o a r e , 1972; Stephen, 1986) might also be d i s p l a y e d when the parasites are c u l t u r e d in vitro.
Acknowledgements We wish to t h a n k Dr. D . G . G o d f r e y w h o supplied the stocks o f Trypanosoma congolense. T h e w o r k was f u n d e d by the Overseas D e v e l o p m e n t A d m i n i s t r a t i o n , London,
References Crowe, J.S., Barry, J.D., Ross, C.A., Luckins, A.G. and Vickerman, K. (1983) All metacyclic variable antigen types of Trypanosoma congolense identified using monoclonal antibodies. Nature 306, 389 391. Frame, I.A., Ross, C.A. and Luckins, A.G. (1990) Characterisation of Trypanosoma congolense serodemes in stocks isolated from Chipata district, Zambia. Parasitology 101,235-241. Gashumba, J.K., Baker, R.D. and Godfrey, D.G. (1988) Trypanosoma congolense: the distribution of enzyme variants in East and West Africa. Parasitology 96, 475 486. Gray, M.A., Cunningham, I., Gardiner, P.R., Taylor, A.M. and Luckins, A.G. (1981) Cultivation of infective forms of Trypanosoma congolense at 28°C from trypanosomes in the proboscis of Glossina morsitans. Parasitology 82, 81-95. Gray, M.A., Ross, C.A.,Taylor, A.M. and Luckins, A.G. (1984) In vitro cultivation of Trypanosoma congolense: the production of infective metacyclic trypanosomes in cultures initiated from cloned stocks. Acta Trop. 41,343-353. Hendry, K.A.K. and Vickerman, K. (1988) The requirement for epimastigote attachment during division and metacyclogenesis in Trypanosoma congolense. Parasitol. Res. 74, 403 408. Hoare, C.A. (1972) The trypanosomes of mammals. A zoological monograph. Blackwell, Oxford. Luckins, A.G., Frame, I.A., Gray, M.A., Crowe, J.S. and Ross, C.A. (1986) Analysis of trypanosome variable antigen type in cultures of metacyclics and mammalian forms of Trypanosoma congolense. Parasitology 93, 99-109. Miles, M.A. (1983) The epidemiology of South American trypanosomiasis - - biochemical and immunological approaches and their relevance to control. Trans. R. Soc. Trop. Med. Hyg. 77, 5-23. Miles, M.A., Povoa, M.M., de Souza, A.A., Lainson, R., Shaw, J.J. and Keneridge, D.S. (1981) Chagas' disease in the Amazon Basin: II. The distribution of Trypanosoma cruzi zymodemes 1 and 3 in Para State, North Brazil. Trans. R. Soc. Trop. Med. Hyg. 75, 667-674.
140 Prain, C.J. and Ross, C.A. (1988) Trvpanosoma congolense: interactions between trypanosomes expressing different metacyclic variable antigen types in vitro and in vivo. Parasitology 97, 277-286. Ross, C.A. (1987) Trypanosoma congolense: differentiation to metacyclic trypanosomes in culture depends on the concentration of glutamine or proline. Acta Trop. 44, 293 301. Ross, C.A., Cardoso de Almeida, M.L. and Turner, M.J. (1987) Variant surface glycoproteins of Trypanosoma congolense bloodstream and metacyclic forms are anchored by a glycolipid tail. Mol. Biochem. Parasitol. 22, 153 158. Stephen, L.E. (1986) Trypanosomiasis. A Veterinary Perspective. Pergamon, Oxford.