DIVERSITY OF TRYPANOSOMA CRUZI STOCKS AND CLONES DERIVED FROM CHAGAS DISEASE PATIENTS : I-BEHAVIORAL CHARACTERIZATION IN VITRO

the behavior of naturally occurring Trypanosoma cruzi populations. Several authors1 3 4 5 8 9 11 14 15 have suggested that the variation in T. cruzi populations may be related to variation in the epidemiological and clinical aspects of Chagas disease in man. The enormous behavioral heterogeneity of this protozoan has been shown by both in vitro and in vivo studies1 3 5 11 and by schizodeme3 4 20 23 and RFLP profiles14 21 27. Variation of the parasites described in the above reports suggests that further studies should be carried out with clonal populations derived from Chagas disease patients with well-defined pathology. Examination of the T. cruzi isolates from heart and esophageal patients and their resulting clonal lines (each derived from a single cell yielding a genetically homogeneous population) will shed light on the role played in this scenario by a particular parasite isolate or a subpopulation thereof. This paper reports on the behavioral characteristics of T. cruzi isolates and their clonal lines derived from two patients with the cardiac and the digestive forms of this disease.


MATERIAL AND METHODS
Trypanosoma cruzi stocks and clones.In this study we isolated T. cruzi by xenodiagnosis from two patients with chronic Chagas disease.The parasite stock hSLU239 was obtained from a patient with the cardiac disease, whereas stock mSLU142 derived from a patient with the digestive form (megaesophagus) of the disease.The metacyclic trypomastigotes passed in the feces of first instar nymphs of Dipetalogaster maximus used for xenodiagnosis were used to inoculate Balb/c mice.One week after inoculation the blood trypomastigotes were obtained by cardiac puncture.The red cells were lysed in 0.78% ammonium chloride and the parasite forms were used to infect primary cultures of fetal mouse skeletal muscle cells 26 .The cell cloning experiments consisted of infecting murine skeletal muscle cell culture in 24 well plates with a single trypomastigote 9 26 .The parasite suspension in culture medium was diluted to contain a single cell per 5µl.This cell dilution aliquot was placed on sterile cover slip in Petri dish and examined with an inverted microscope.Each 5µl aliquot containing a single parasite was transferred to a well of the plate.One week thereafter infected host cells in each well was observed with an inverted microscope.In a parallel control In this study, we isolated Trypanosoma cruzi from chronic Chagas heart disease and from megaesophagus patients.The parasite stock hSLU239 (heart disease) yielded clones h1 and h2, whereas stock mSLU142 (megaesophagus) yielded clones m1, m2, m3 and m4.The parasite growth kinetics, doubling time and differentiation in axenic liquid medium showed broad behavioral diversity.It was shown that a particular pattern of behavior for a parental stock could not necessarily be assigned for subsequent clones.This study indicates that i) each Chagas disease patient is infected with several T. cruzi populations; ii) clonal lines derived from patient samples may have different biological characteristics from the original isolate; and that iii) additional behavioral and/or molecular markers are required for further characterization of Trypanosoma cruzi stocks and clones derived from Chagas disease patients in order to identify correlations with pathology.experiment, no growth was observed in the wells receiving an aliquot of medium in which the parasite could not be seen microscopically.
Gr owth kinetics, doubling time and differentiation.The T. cruzi stocks and clones were maintained in liver infusion tryptose (LIT) medium 6 .The behavioral characterization of the parasite consisted of growth kinetics, doubling time and percent of differentiation.For the growth kinetics studies, stocks and clones of the parasite harvested from tissue culture were transferred in triplicate to LIT medium.After three passages with 5 dayintervals, the exponential growth phase parasites were used to begin the study.Each initial inoculum of 1 x 10 6 cells was seeded in fresh LIT medium and the parasite growth was d e t e r m i n e d e v e r y o t h e r d a y u n t i l l a t e stationary phase.Aliquots of the parasite culture forms were counted in triplicate using a Neubauer cell counting chamber and an inverted microscope at 100X magnification.Smears of the parasite forms were fixed in methanol, stained by Giemsa's method and mounted on coverslip.The glass slides were examined under the microscope at 1000X magnification.The percent of epimastigotes and trypomastigotes were estimated from 300 flagellates counted in each smear.

RESULTS
In this study, the method used for parasite cloning ascertained that each subsequent clonal line derived from a single trypomastigote.The parasite stock hSLU239, isolated from a heart disease patient, yielded clones h1 and h2.In addition, the parasite stock mSLU142, isolated from a megaesophagus patient, yielded clones m1, m2, m3, and m4.
Figure 1 shows the means and standard deviation of the parasite growth for a period of 22 days.Figure 1A shows the growth kinetics of the T. cruzi stock hSLU239 and of its clones h1 and h2.The parental stock and clone h1 showed similar dividing profile with quick proliferation, and reached high cell number (≅ 2-3 x 10 7 /ml) after 8 days.In contrast, clone h2 derived from same parental stock showed slow proliferation and substantial fewer cells (≅ 2 x 10 6 /ml) after 8 days of growth.
Figure 1B shows percent of parasite d i f f e r e n t i a t i o n f r o m e p i m a s t i g o t e s t o trypomastigotes in the axenic medium.These observations were carried out through 22 days of parasite growth.The T. cruzi stock hSLU239 showed maximum percent differentiation of circa of 3% at days 12 and 14, whereas clone h1 reached 15% at day 18, and clone h2 reached 10% at day 20.
Figure 2A shows the growth kinetics of the T. cruzi stock mSLU142 and of its four clones.The parental stock and clone m2 showed moderate growth: stock mSLU142 reached approximately 1 x 10 7 cell culture forms/ml after 10 days in axenic culture, whereas clone 189 m2 reached ≅ 3 x 10 6 cells/ml at day 12.In marked contrast, clones m3 and m4 multiplied very quickly and reached approximatelly 7 x 10 7 cells/ml after 12 days of growth.Figure 2B shows that m SLU142 had moderate differentiation reaching a maximum of 5 to 6 per cent between days 14 to 20. Clones m2 and m4 showed the lowest differentiation (1%) fr om days 12 to 16, whereas clone m 3 epimastigotes differentiated to circa 15% trypomastigotes, at day 16 in LIT medium.
The T. cruzi population doubling time (DT) was indicated by the number of hours required for two-fold increase of the parasite population.
Table 1 shows the DT observed for each parasite stock and clone.Interestingly, the DT of stock hSLU239 (65.4 ± 0.9h) was similar to that of stock mSLU142 (65.6 ± 4.9h).The DT of both T. cruzi stocks, however, differed significantly from their clones.Clone h1 DT reached 37.5 ± 0.9, which was much lower than that of clone h2 that required 87.2 ± 4.3h.The parasite clones derived from the parental mSLU142 also showed marked divergence.Clone m2 showed DT of 56.4 ± 3.6h, whereas clones m3 and m4 showed 38.4 ± 1h.In marked contrast, clone m1 showed DT of 124.8 ± 3.6h.An attempt to correlate the T. cruzi growth kinetics with the parasite population doubling time is shown in Figure 3.It was observed that clones h2 and m1 that had very long DT showed slow growth.Furthermore, clones m3 and m4 that had very short DT presented very  high growth yields reaching high cell countings after 10 days in culture (Figure 2).However, clone h1 that had very short DT presented moderate growth (Figure 1).In addition, stock hSLU239 that had moderate DT showed high growth kinetics similar to those shown by clones m3 and m4.
In brief, DT in LIT medium allowed classification of the populations of T. cruzi stocks and clones into three groups: I-long DT (from 87.4 to 124h) composed of clones h2 and m1; II-moderate DT (from 56.4 to 65.6), consisted of stocks hSLU239, mSLU142 and clone m2; III-short DT (from 37.5 to 38.2h), which comprised clones h1, m3 and m4.

DISCUSSION
A stable behavioral marker for each single parasite population used to infect a singeneic host has been sought in order to determine whether the outcome of lesions in Chagas disease may be related to Trypanosoma cruzi superinfections.In this paper we describe several parasite populations (h1, h2, m1-m4) in two stocks derived from patients with chronic Chagas disease, which showed different kinetics of growth, doubling time and differentiation in axenic culture.This study suggests that a particular pattern of in vitro behavior of a parental T. cruzi stock can not necessarily be extrapolated to its clones.For example, the growth kinetics of clone h2 were much slower than those observed for the parental hSLU239.Also, clones m3 and m4 demonstrated more rapid growth kinetics than the parental mSLU142.However, clone m1 showed significantly slower growth than the parental mSLU142.Moreover, the parasite population doubling time varied from one clone to another regardless of stock origin.In this regard, clones h2 and m1 (group I) showed the longest doubling time, in contrast with clones h1, m3 and m4 (group III) that showed from one-half to one-third of the doubling time.A correlation between the kinetics of growth and the population doubling time was not seen in all cases.This observation is hard to explain because the factors associated with the regulation of T. cruzi population growth in axenic culture are basically unknown.Furthermore, variability between stock and derivative clonal lines is also found for the parasite differentiation in culture.Clones h1 and h2 differentiated more extensively than their parental stock hSLU239, whereas clones m2 and m4 showed minimal differentiation.
It has been postulated that a particular feature of a T. cruzi population might be corr elated to the pathology of Chagas disease 1 3 8 10 11 .The behavioral characteristics of the parasite stocks and derived clones reported here, however, are in keeping with the idea that each Chagas disease patient is infected with several T. cruzi populations having broad biological diversity 8 22 24 .Some behavioral aspects of a particular parasite population may change from time to time 7 12 25 .In addition, it has been shown that selection of some T. cruzi populations may take place in the intestine of the insect-vector 13 .Sampling (xeno) with D. maximus may not have isolated all the biological types.In this respect, the parasite populations described in this paper will be used in the future to determine if the T. cruzi stocks and clones maintained in vitro and in vivo can change their phenotypic profiles.
The results of this study highlight the dificulty of understanding the role that parasite variation plays in the highly variable pathogenesis of disease.In order to circumvent this difficulty, an attempt has been made to determine stable isoenzyme and DNA marker of these clones, such as polymorphisms of DNA sequences encoding the glycolytic enzymes piruvate kynase 2 , fructose bi-phosphate aldolase 19 , glucose phosphate isomerase 18 and glyceraldehyde-3-phosphate dehydrogenase 16 for each parasite population 17 .In conclusion, we believe that additional behavioral and molecular markers are required for further characterization of the various T. cruzi clones present in the parasite stocks derived from Chagas disease patients.
Lauria-Pires L, Santana JM, Tavares FS, Teixeira ARL.Diversity of Trypanosoma cruzi stocks and clones derived from Chagas' disease patients: I-Behavioral characterization in vitro.Revista da Sociedade Brasileira de Medicina Tropical 30:187-192, mai-jun, 1997.Este estudo indica que i) cada paciente chagásico é infectado com várias subpopulações de T. cruzi; ii) linhagens clonais derivadas de cada estoque do parasito podem ter características biológicas diferentes do isolado original de paciente chagásico; e que iii) marcadores comportamentais e/ou moleculares adicionais são necessários para melhor caracterização de estoques de T. cruzi e seus clones derivados de pacientes com doença de Chagas, a fim de identificar as possíveis correlações com a patologia.P a l a v r a s -c h a v e s : Tr y p a n o s o m a c r u z i .Caracterização do comportamento.Diversidade genética.
187 DIVERSITY OF TRYPANOSOMA CRUZI STOCKS AND CLONES DERIVED FROM CHAGAS DISEASE PATIENTS: I-BEHAVIORAL CHARACTERIZATION IN VITRO L. Lauria-Pires, J.M. Santana, F.S. Tavares and A.R.L. Teixeira

Figure 1 -
Figure 1 -A) Profiles of Trypanosoma cruzi growth in LIT medium; B) Patterns of the parasite differentiation from epimastigotes to trypomastigotes in the axenic culture: Stock hSLU239 ( ) and its clones h1 ( ) and h2 ( ).Each finding represents the means ± standard deviation in triplicate countings of at least 300 parasites.

Figure 2 -
Figure 2 -A) Profiles of Trypanosoma cruzi growth in LIT medium; B) Patterns of the parasite differentiation from epimastigotes to trypomastigotes in the axenic culture: Stock mSLU142 ( ) and its clones m1 ( ), m2 ( ), m3 () and m4 ( ).Each finding represents the means standard deviation in triplicate countings of 300 parasites.

Figure 3 -
Figure 3 -Patterns of Trypanosoma cruzi growth kinetics and population doubling time in axenic culture.Doubling time is shown on the axis; vertical bars represent maximum yield of cells.Notice that stock mSLU142 that had moderate doubling time presented low growth, whereas clones m3 and m4 that had short doubling time showed very high growth yield.

Table 1 -
Doubling time of Trypanosoma cruzi stocks and clones in LIT medium at 28 o C.