Trypanosoma cruzi

Zeitschrift for

Z Parasitenkd (1983, 69 : 283-290

Parasitenkunde Parasitology Research

9 Springer-Verlag 1983

Original Investigations The Characterization of Chilean and Bolivian Trypanosoma cruzi Stocks from Triatoma infestans by Isoelectrofocusing F. Ebert 1 and G. Schaub 2 1 Bernhard-Nocht-Institut ffir Schiffs- und Tropenkrankheiten, Abteilung ffir Protozoologie, 2000 Hamburg 4, F R G 2 [nstitut ffir Biologic I (Zoologic), Albert-Ludwigs-Universitfit, 7800 Freiburg, F R G

Abstract. Culture forms of 12 Chilean and 9 Bolivian Trypanosoma cruzi

stocks were compared isoenzymatically by the following enzymes: nonspecific esterase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and alcohol dehydrogenase. On the basis of the electrophoretic mobility of these enzymes the stocks were classified into two main groups. Ten Chilean stocks were characterized as group II; two stocks showed enzyme patterns of group I. In contrast, five Bolivian stocks were classified as belonging to group I, the other four to group II. The results show that the two groups of T. cruzi overlap in Triatoma infestans suggesting that both groups of T. cruzi are infective for man. The classification of stocks into two groups is discussed in the light of published results of Brazilian T. cruzi stocks. A strong association of groups with the transmission cycles as it seems to be in Brazil does not exist in Chile and Bolivia. Introduction

Knowledge available on the epidemiology of Chagas' disease varies from country to country in Latin America (Romana 1961; Miles 1982). Chile belongs to those countries in which epidemiological data of Chagas' disease are well known, while Bolivia is one of those countries where the real importance of the disease has not yet been demonstrated. In both Chile and Bolivia Triatoma infestans is the main vector of Trypanosoma cruzi (Brener and Andrade 1979; Schenone 1980). There is only one report of the isoenzymatic characterization of Bolivian T. cruzi stocks (Tibayrenc et al. 1981); Chilean T. cruzi stocks have not been characterized previously by electroOffprint requests to." F. Ebert

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F. Ebert and G. Schaub

p h o r e s i s . W i t h t h e n e w t e c h n i q u e o f i s o e l e c t r o f o c u s i n g it is p o s s i b l e to d e m o n s t r a t e m o r e i n t r a s p e c i f i c v a r i a n t s a m o n g T. cruzi s t o c k s ( E b e r t 1 9 8 2 a , b) t h a n f o u n d w i t h o t h e r e l e c t r o p h o r e t i c m e t h o d s . T h e p u r p o s e o f this p a p e r is t o e x a m i n e b y i s o e l e c t r i c f o c u s i n g t h e h e t e r o g e n e i t y a m o n g T. cruzi s t o c k s f r o m t h e s a m e v e c t o r s , i s o l a t e d f r o m l i m i t e d geographical endemic areas. M a t e r i a l s and M e t h o d s

Origin and Isolation of Stocks Chile. The stocks were isolated from T. inJestans by culturing rectal contents at 27~ on Brain-Heart-lnfusion-Agar (Difco), containing 15% rabbit blood and overlayed with 0.9% NaC1. The bugs were collected in Cachiyuyu, Province Atacama, North Chile (Schaub and Schottelius, in preparation). Bolivia. All of the stocks were isolated from T. infestans as described above. The bugs were collected by Miss C. Berndt on the 6 February 1982 in "El Solterito ", a suburb of Cochabamba. The cultivation of parasites, the preparation of T. cruzi extracts, the technique of isoelectrofocusing (IEF), and staining enzymes have been described previously (Ebert 1982 a). The following enzymes were used for the characterization: nonspecific esterase (NSE, E.C.3.1.1), phosphoglucomutase (PGM, E.C.2.7.5.1), glucose-6-phosphate dehydrogenase (G-6-PD, E.C. 1.1.1.49), glucosephosphate isomerase (GPI, E.C. 5.3.1.9). The enzyme alcohol dehydrogenase (NADP +) (ADH, E.C. 1.1.1.2) was focused on polyacrylamide gels at pH gradient "2-11" (Ebert 1982a), the developing components are given by Miles et al. (1980). Results

T h e e n z y m e t y p e s o f C h i l e a n a n d B o l i v i a n T. cruzi s t o c k s a r e s u m m a r i z e d in T a b l e 1. M i x e d s t o c k s c a n b e e x c l u d e d b e c a u s e all o f t h e t y p e s seen

Table 1. Enzyme types of Chilean and Bolivian T. cruzi stocks by isoelectrofocusing Original code

NSE

PGM

G-6-PD

GPI

ADH

Ch 5, Ch 12

I

I

II

I

Ib

Ch 1, Ch 2, Ch 3, Ch 4 Ch 6, Ch 7, Ch 8 Ch 9, Ch 10, Ch 11

II II

III III

III IV

IIIb III

II II

Bol 2, Bol 4, Bol 6 Bol 7, Bol 8

I

I

II

I

Ia

Bol 3, Bol 9, Bol 13 Bol 5

IX II

III III

[II III

IIIb III

II IX

group I a enzyme types possible

I

I, (II) b

I, II

I

Ia, Ib

group II a enzyme types possible

II

III, (IV) b

III, IV, V (W) b

II, III

II

" See, Ebert (1982a, b); b these types have been only found in one stock

Characterization of Chilean and Bolivian T. cruzi Stocks

285

Fig. 1. Nonspecific esterase patterns of types I and II of T. cruzi stocks after isoelectrofocusing in pH gradient " 2 - 1 1 " . 1 = Ch 5 ; 2 = Ch 8

in Table 1 have already been found on cloned stocks (see Ebert 1982a, b). Photographs of typical IEF plates are shown in Figs. 1-5. By combining the isoenzyme patterns of NSE, PGM, G-6-PD, GPI and A D H the stocks can be classified into groups I and II. Ten of the 12 Chilean stocks were characterized as belonging to group II; only two stocks (Ch 5 and Ch 12) have enzyme patterns that allow classification into group I. In contrast, five of the nine Bolivian stocks were found to be identical with stocks of group I. The stocks Bol 3, Bol 5, Bol 9 and Bol 13 belong to group II. The enzyme types could be generally compared with those already found for Brazilian and Venezuelan stocks. In the case of GPI, however, the enzyme patterns of Chilean (Ch I, Ch 2, Ch 3, Ch 4, Ch 6, Ch 7 and Ch 8) and Bolivian stocks (Bol 3, Bol 5, Bol 9 and Bol 13) were very similar to type III (Fig. 4). This type was designated temporarily as type IIIb to separate it from other stocks classified as type III. The enzyme A D H (Fig. 5) has not been used before for T. cruzi characterization by isoelectrofocusing. The T. cruzi stocks can be divided into

286


Fig. 2. Phosphoglucomutase patterns of types I and II1 of T. cruzi stocks after isoelectrofocusing in pH gradient "3-7". l = C h 12; 2=Bol 9; 3=Bol 13; 4=Bol 6

several types corresponding to groups I and II. However, the number of stocks tested by ADH was lower than in the cases of NSE, PGM, G-6-PD, and GPI. Discussion

The two main groups found among the Chilean and Bolivian T. cruzi stocks correspond with those described for Brazilian stocks (Ebert 1982b). The Brazilian stocks of group I have been found exclusively in wild animals; group II was mainly composed of stocks from human cases and domestic hosts. Similar results were obtained by starch gel electrophoresis (Miles et al. 1977). The Chilean stocks from T. infestans were found in Atacama province, near the border with Coquimbo province. In both provinces only two species of bugs occur, T. infestans and T. spinolai, T. infestans being the most

Characterization o f Chilean and Bolivian T. cruzi Stocks

287

Fig. 3. Gtucose-6-phosphate dehydrogenase patterns of types II, III and IV of T. cruzi stocks after isoelectrofocusing in pH gradient "2-11". l = C h 9 ; 2 = C h 10; 3 = C h 4 ; 4 = C h 6 ; 5=Ch 5

important (Schenone 1971, 1980). Reports of T. cruzi infections in wild animals are rare and until now have only be observed in two regions, the Atacama and Metropolian Region (Schenone t980). It appears that behaviour of vectors plays an important role in the distribution of enzyme types since this can be seen in Rhodnius prolixus in Venezuela (Ebert 1982a). T. infestans belongs to that group of bugs which are well adapted to humans and domestic animals, but it has also been found in sylvatic biotopes (Brener and Andrade 1979). Patterns of T. infestans feeding behaviour have shown that it takes small amounts of blood from rodents, opossum, and possibly bats (Minter 1976). Since T. infestans is highly significant in Chagas' disease in Chile it is not surprising that the two groups of T. cruzi are encountered in the same area and consequently found with a predominance of group II in the same vector. In T. infestans from Bolivia collected in an urban area where Chagas' disease has been described (Dias and Torrico 1943; Dias 1953) T. cruzi

288


Fig. 4. G l u c o s e p h o s p h a t e isomerase patterns o f types I, III and IIIb o f T. cruzi stocks after isoelectrofocusing in p H gradient " 5 - 9 ". 1 = Ch 5 ; 2 = C h 9 ; 3 - Ch 4; 4 = Cla 2

stocks of group I and II also have been found. However, most of the Bolivian stocks were classified as group I. Similar results were obtained by Tibayrenc et al. (1981) after investigating 73 stocks from other regions of Bolivia. The dominance of group I is difficult to interpret by the hypothesis of a relationship between enzyme patterns and transmission cycles as seems to be the case in Brazil (Miles et al. 1977; Ebert 1982b). The occurrence of groups I and II of T. cruzi in the same vector in Chile and Bolivia demonstrates that a strong association of group I with the sylvatic cycle and group II with the domestic cycle does not exist in all South American countries. The significance of both main groups of T. cruzi is not yet clear. In Brazil (Barrett et al. 1980) and in Venezuela (Miles et al. 1981 ; Ebert 1982a) it has been seen that both groups of T. cruzi can be infective for humans.

Characterization of Chilean and Bolivian T. cruzi Stocks

289

Fig. 5. Alcohol dehydrogenase patterns of types I a, I b and II of T. cruzi stocks after isoelectrofocusing in pH gradient "2-11". Ia and Ib differ only by the marked enzyme bands ( 4 ) which are sometimes absent or very weak giving optimal bands after 30 min incubation. l = C h 10; 2 = C h 7; 3=Bol 6; 4 = C h I2

An association of zymodeme 1 (identical with group I) and the absence of mega syndromes in Venezuela has been discussed by Miles et al. (1981). It seems that the two groups probably represent subspecies of T. cruzi that were originally geographically separated and adapted to different vectors and hosts (see Miles et al. 1977; Barrett et al. 1980). In Venezuela only group I has so far been found (Miles et al. 1981 ; Ebert 1982a). Studies on T. cruzi stocks isolated from humans in Ecuador (unpublished results) resulted also in classification into group I, supporting the suggestion of primary distribution of group I or zymodeme 1 (Miles et al. 1981) north of the Amazonas. The significance of sub-types of groups I and II is a matter of speculation. However, it was remarkable that most of the stocks of group II were associated with the G-6-PD-type III. This type has only been seen before

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in stocks from T. infestans in Brazil, whereas the more common types IV and V were mostly seen in stocks from humans (Ebert 1982b). It may be that the different isoenzymes are adaptions for optimal transmission in certain bugs (Godfrey 1979) or hosts. It is tempting to speculate that stocks of G-6-PD-type III probably represent those T. cruzi parasites that are most adapted to T. infestans. Acknowledgements. This investigation was supported by Deutsche Forschungsgemeinschaft. The authors are indebted to Miss C. Berndt for the collection of bugs and the technical assistance of Mrs. A. Henry-Mating is gratefully acknowledged.

References Barrett TV, Hoff RH, Mott KE, Miles MA, Godfrey DG, Teixeira R, de Souza JAA, Sherlock IA (1980) Epidemiological aspects of three Trypanosoma cruzi zymodemes in Bahia State, Brazil Trans R Soc Trop Med Hyg 74:84-89 Brener Z, Andrade Z (1979) Trypanosoma cruzi e Doenca de Chagas. Guanabara Koogan, Rio de Janeiro, p 106 Dias E (1953) Doenca de Chagas has Americas. IV. Bolivia e Paraguai. Rev Malariol Doencas Trop 5 : 11-16 Dias E, Torrico RA (1943) Estudos preliminares sobre a doenca de Chagas na Bolivia. Mem Inst Oswaldo Cruz 38:165-172 Ebert F (1982a) The use of isoelectrofocnsing on thin layer polyacrylamide and agarose gels as a method for characterization of Venezuelan Trypanosoma cruzi stocks. Tropenmed Parasitol 33 : 63-67 Ebert F (1982b) The identification of two main groups of Trypanosoma cruzi stocks from Brazil by their isoenzyme patterns of isoelectrofocusing. Tropenmed Parasitol 33 : 140 146 Godfrey DG (1979) The zymodemes of trypanosomes. Symp Br Soc Parasitol 17:31 53 Miles MA (1982) Trypanosoma cruzi: Epidemiology. In: Perspectives in Trypanomiasis Research, ed JR Baker, Res. Studies Press Miles MA, Toy6 PJ, Oswald SC, Godfrey DG (1977) The identification by isoenzyme patterns of two district strain-groups of Trypanosoma cruzi, circulating independently in a rural area of Brazil. Trans R Soc Trop Med Hyg 71:212225 Miles MA, Lanham SM, de Souza JAA, P6voa MM (1980) Further enzymic characters of Trypanosoma cruzi and their evaluation for strain identification. Trans R Soc Trop Med Hyg 74:221-237 Miles MA, P6voa MM, Prata A, Cedillos RA, de Souza AA, Macedo V (1981) Do radicalIy dissimilar Trypanosoma cruzi strains (zymodemes) cause Venezuelan and Brazilian forms of Chagas' disease? Lancet II : 1338-1340 Minter DM (1976) Effects on transmission to man of the presence of domestic animals in infested households. In: New approaches in American trypanosomiasis research, PAHO Sci Publ No 318, p 330-337 Romana C (1961) Epidemiologia y distribucion geografica de la enfermedad de Chagas. Bol Ofic Sanit Pan-Amer 51 : 390-403 Schenone H (1971) Estado de los estudios epidemiologicos sobre la enfermedad de Chagas en Chile. Bol Ofic Sanit Pan-Amer 70:250-255 Schenone H (1980) Factores biologicos y ecol6gicos en la epidemiologia de la enfermedad de Chagas en Chile. Bol Chil Parasitol 35:42-54 Tibayrenc M, Carion ML, Solignac M, Carlier Y (1981) Arguments g6n+tiques contre l'existence d'une sexualit~ actuelle chez T. cruzi. Implications Taxonomiques. CR Acad Sci (Paris) 293 : 207-209 Accepted January 19,1983