Desjeux, P.

Tibayrenc, Michel

Horizon / Pleins textes

TR4NSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 77, NO. 1,73-75 (1983) 73 
The presence in Bolivia of two distinct zymodemes of Trypanosoma 
cruzi, circulating sympatrically in a domestic transmission cycle 
/ 
MICHEL'TIBAYRENC AND PHILIPPE DES JEUX 
ORSTOM-Imtitut Pasteur, IBBA, Embajada de Francia, Casilla 824, L a  Paz,  Bolivia 
Summary 
The enzyme profiles of 109 Bolivian stocks of Trypanosoma cruzi were determined by cellulose 
acetate electrophoresis using the four enzymes: malate dehydrogenase (oxaloacetate decarboxylating) 
(NADPf) (E.C.1.1.1.40, ME), phosphogluconate dehydrogenase (E.C.1.1.1.44, 6PGDH), phos- 
phoglucomutase (E.C.2.7.5.1, PGM) and .glucosephosphate isomerase (E.C.5.3.1.9, GPI). As 
previously, two principal zymodemes were found sympatrïcally. Both were isolated from man, one 
appeared to be more frequent at high altitude, the other appeared to be more frequent at low altitude. 
These results agree with our previous hypothesis on the genetics of T. cruzi (diploidy, lack of actual 
sexuality). 
Introduction 
Previous studies of Bolivian Trypanosoma cruzi 
stocks have confirmed the enzymic variability of t h i s  
parasite that was first demonstrated in Brazil by 
MILES et al. (1977, 1978, 1980), and allowed us to 
suggest a genetic interpretation of flagellate zymo- 
grams (TIBAYRENC et al., 1981a and b). Here, based 
on the examination of 109 Bolivian stocks, we confirm 
the presence of two principal Bolivian zymodemes, 
and reveal some interesting aspects of their geo- 
graphical distribution. The paper that follows 
(TIBAYRENC & MILES, 1983) compares the enzyme 
profiles of Bolivian and Brazilian zymodemes and 
discusses their relationships. 
Materials and Methods 
Trypanosoma cruzi stocks were collected in Bolivia from 
four geographical localities, which are briefly described 
below: 
Chiwisivi-a high river valley, at about 2,700 m and 
60 km south of the capital La Paz. The climate is temperate 
and very dry. 
The Yungas-valleys at an intermediate altitude of 
approximately 1,700 m, 100 km north-east of La Paz. The 
climate is subtropical, far more humid than at Chiwisivi, and 
there is a lush vegetation. 
Santa Cruz, more specifically the suburban village of 
Cotoca-at an altitude of 400 my 500 km south-east of La 
Paz. The climate and vegetation are also subtropical. 
Camiri-a city, also at 400 m, 300 km south of .%uta 
Cruz but with a drier climate than in Santa Cruz. 
The origins of the stocks examined are summarized in 
Table 1.. Most of the stocks were isolated directly from 
domestic or peridomestic triatomine faeces using a simple 
method, described elsewhere (Tibayrenc & Desjeux, 1982). 
The stocks were grown in GLSH liquid medium (LE b y ,  
1974) and passaged weekly. One culture tube, containing 
about 10 ml, gave sufficient material for enzyme elec- 
trophoresis-large volume cultures were not necessary for 
the examination of four enzymes, because of the sensitivity 
of cellulose acetate electrophoresis (CAE). Stocks were 
harvested routinely once only, except when specifically 
checking the stability of enzyme profiles. The packed cells 
were mixed with an equal volume of enzyme stabilizers 
(MILES et al., 19771, stood on ice for 30 min and stored at 
-70°C in a Revco deep freeze. Whole lysates were elec- 
trophoresed without centrifugation. 
The four enzymes used to examine the Bolivian T. cruzi 
stocks were as follows: malate dehydrogenase 
(oxaloacetate decarboxylating) (NADP') E.C. 1.1.1.40, 
Table I-Origins of the Typanosma cruzi stocks examined 
No. of houses Group I Group II Group III (?) 
Chiwisivi 12 64 2 O 
Yungas 9 5 4 O 
Santa Cruz 6 2 19 O 
(Cotoca) 
Camiri 4 3 1 O 
CENETROP 
H u "  
Triatoma 
6 
4 
3 
4 
1 
O 
l2 
O 
'including stock Tacmerenda (see text) 
'stock 19-79 
All stocks isolated from Triatoma infestam, except for the human stocks from CENETROP, and one stock isolated 
from Triatoma sordida at CENETROP. 
74 TWO T. C r u “  ZYMODEMES IN BOLIVIA 
PGM ME GPI SPGDH 
I 
I 
I - I  I I 
- -  _ -  - -  - - -  
I II I I I  I II I II III 
Fig. l.-E&ymC profiles of the Bolivian zymodemes f& enzymes 
PGM, ME, GPI and 6PGDH. 
ME), phosphogluconate dehydrogenase (E.C.l. 1.1.44, 
6PGDH), phosphoglucomutase (E.C.2.7.5.1, PGM), and 
glucosephosphate isomerase (E.C.5.3.1.9, GPI). The CAE 
equipment of Helena Laboratories (Beaumont, Texas) was 
employed throughout. For the enzymes ME, 6PGDH and 
GPI, we adopted the methods of KREUTZER & CHRISTEN- 
SEN (1980), but for the development of ME substituted a 
Tris-malic acid buffer, pH 7.0 (Tris 6g/l, malic acid 30g/l, 
adjusted with NaOH 4N) and, for 6PGDH and GPI used &e 
tank buffer No. 3 of SHAW & PRASAD (1970). For the 
enzyme PGM we used the method of KREUTZER & 
CHRISTENSEN (1980) without modification. 
Results and Discussion 
Table I gives the electrophoretic results for all the 
stocks examined. With the exception of one human 
stock from CENETROP (19-79), only two 
zymodemes, or combinations of pattems, were found 
(Fig. 1). Except in the case of stock 19-79, ME, 
6PGDH, PGM and GPI pattem 1 always occurred 
together as did ME, 6PGDH, PGM and GPI pattern 
2. In stock 19-79 PGM and GPI pattern 1 were 
associated with ME pattern 2 and a 6PGDH pattern 
that was not seen in the other stocks. 25 of the stocks 
were examined twice, at an interval of one month, to 
check the stability of their enzyme profiles; no 
changes in patterns were observed, except that on one 
occasion, the stock Tachiperenda appeared to show a 
mixture of both PGM patterns. 
An hypothesis on Trypanosoma CTuzi genetics was 
proposed previously, namely diploidy (TIBAYRENC et 
al., 1981a) and a lack of actual sexuality (TIBAYRENC 
et al., 1981b). The present results with a greater 
number of stocks confirm the lack of genetic exchange 
between strains 1 and 2, and the constant heterozy- 
gous state in strain 2 for enzymes 6PGDH, PGM, and 
GPI (checked for 27 stocks), which is scarcely 
compatible with sexual recombination within this 
Strain. 
The existence of two principal enzymic strains (or 
zymodemes) in Bolivia, with the exception of stock 
19-79, which requires further study, has been con- 
h e d .  It is interesting to compare the geographical 
distribution of the two Bolivian zymodemes between 
Chiwisivi and Cotoca (Table I). In Chiwisivi, from 12 
different households, 64 stocks with enzyme profile 1 
were recorded and’two with enzyme profile 2. In 
contrast, from six houses in Cotoca we recorded only 
two stocks with enzyme profile 1 but 19 with enzyme 
profile 2. In the Yungas, amongst nine stocks we 
found five with enzyme profile 1 and four with 
enzyme profile 2. In Cotoca, all the triatomine bugs 
were collected inside houses. In Chiwisivi and the 
Yungas most were collected from peridomestic 
guinea-pig houses, but, as all of the guinea-pig houses 
shared a common wall with the houses themselves, 
this distinction is not necessarily meaningful. It is not 
possible, on the basis of the present results, to decide 
if the distribution of the zymodemes constitutes really 
a cline related to geographical distance..or climate; 
further studies are required from other localities at 
similar altitudes. Both of the principal zymodemes 
were found in man (amongst stocks isolated at 
CENETROP, Santa Cruz). Furthermore, in the 
Yungas and in Cotoca, both zymodemes were re- 
corded from the same house, 
In conclusion, these observations have confìrmed 
our previous genetic interpretation (diploidy and lack 
of actual sexuality in T. “i] and the presence of at 
least two principal zymodemes in Bolivia. Both 
zymodemes occur in man and they are sympatric, 
with different geographical frequencies: the first 
zymodeme appears to be more common at high 
altitude, the second zymodeme appears to be more 
common at low altitude. Bolivia, with the different 
levels of altitude, gives excellent conditions for 
comparative ecological studies. We are beginning 
further studies to investigate relationships between 
geographical localities and zymodemes, and to study 
relationships between clinical features and 
zymodemes. 
Acknowiednements 
We thank Dr. Carlos La Fuënte, Dr. B e n j m  Ribeira 
and Dr. Christian Darras who have given nine stocks from 
CENETROP. 
References 
Kreutzer, R. D. & Christensen, H. A. (1980). Characteriza- 
tion of Leishmania spp. by isozyme electrophoresis. 
Amerkan Joumal of Tropical Medicine and Hygiene, 29, 
Le Ray, R. (1974). Les structures antig&iques de Trypano- 
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M. TIBAYRENC AND P. DESJEUX 75 
between Brazilian and Bolidan zymodemes of Ttypqno- 
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Tibayrenc, M. & Miles, M. A. (1983). A genetic comparison Accepted fm publication 9th Juay 1982. 
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The presence in Bolivia of two distinct zymodemes of Trypanosoma cruzi, circulating sympatrically in a domestic transmission cycle

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