Studies on parasitemia courses and mortality in mice
infected with genetically distant Trypanosoma cruzi
clonets

ANNE WALLACE², SILVIA ORTÍZ¹, GITTITH SÁNCHEZ¹, REBECA VILLAGRA², MABEL MUGA², ALDO SOLARI

¹Program of Cellular and Molecular Biology
²Program of Immunology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile

Corresponding Author: Dr. Aldo Solari. Programa de Biología Celular y Molecular, Facultad de Medicina, Universidad de Chile, Casilla 70086, Santiago 7, Chile. E-mail: asolari@machi.med.uchile.cl

Received: January 30, 2001. In revised form: April 23, 2001. Accepted: April 27, 2001

ABSTRACT

The biological characterization of bloodstream forms of eleven Trypanosoma cruzi cloned stocks, corresponding to two genetically similar clonets (19 and 20) and one distant clonet (39), according to multilocus enzyme electrophoresis analysis, showed dissimilar parasitemia in an experimental isogenic mouse model. While clonet 39 stocks gave low parasitemias, clonets 19 or 20 stocks gave high parasitemias, independently of the inocula (10² and 10⁴ bloodstream forms) used. High parasitemia did not always associate with greater mortality. Statistical studies on mortality using a low inocula showed significantly higher mortality with clonet 39 stocks when compared to clonets 19 or 20 stocks. Finally, in order to confirm the identity of each stock studied, typing by molecular karyotype was performed before inoculating mice.

Key terms : Trypanosoma cruzi, clonets, parasitemia, mortality, bloodstream forms, multilocus enzyme electrophoresis.

INTRODUCTION

Population genetic studies have demonstrated that Trypanosoma cruzi populations exhibit a basically clonal structure (3). Therefore the natural stocks are considered clonets (35) or genotypes that appear to be identical for a given set of genetic loci. Studies on biological and behavioral variability of different T.cruzi clones have been performed. Heterogeneity among clones was found for many parameters, including doubling time, virulence and tissue tropism (11). Loss or change in virulence of T.cruzi non-cloned stocks, during long term in vitro culture or maintenance in congenic mice has been reported (24, 33, 29). For this reason, the study of cloned T. cruzi stocks in isogenic mice, infected with bloodstream forms is essential to addressing the question ofwhether phylogenetically diverse clonets have different biological behaviors.

Fortunately, previous studies with T. cruzi cloned stocks, demonstrated the constancy of pathogenicity for inbred mice after long term in vitro maintenance. Therefore these results support the approach with cloned stocks, making them meaningful and reliable (27). Studies on the biological variability among T. cruzi stocks have been reported. When separately inoculated in the same murine model, different clones isolated from an acute T. cruzi infection in humans developed clinical courses that evolved into two markedly different diseases (26).

The clinical and epidemiological consequences of T. cruzi clonal variability are still unknown. lt has been proposed that the long term clonal evolution may have had an impact on T.cruzi biological diversity. A strong statistical linkage between average data for clonets and biological parameters has been presented (18, 28).

In this work, we study in mice inoculated with bloodstream forms, the biological behavior of individual T. cruzi cloned stocks from clonets 19, 20 and 39, which display different genetic background based on multilocus enzyme electrophoresis (MLEE). These clonets represent major clones, considering they are widespread over various countries and hosts (35). The results presented in this study correspond to individual stocks of the above mentioned clonets, in order to ascertain the possible variability within each clonet.

MATERIALS AND METHODS

Trypanosoma cruzi stocks

The T. cruzi clone samples employed include stocks from three genetically different clonets:19, 20 and 39. Detailed phylogenetic information on these samples are shown in Table I, which lists the T. cruzi cloned stocks studied and their geographic and host origins. Clonets 19 and 20 appear very similar according to 15 enzyme loci and are considered genetically near to each other but distant to clonet 39 (35).

Trypanosoma cruzi infective forms

Infective trypomastigote forms were procured either by metacyclogenesis from epimastigote forms grown in Diamond media or by infection of VERO cells to generate culture derived trypomastigotes (9). Metacyclic trypomastigotes were obtained by spontaneous differentiation of epimastigote forms in axenic medium as described (30), or by incubation of epimastigote forms in TAUP medium (6). Clonets 19 and 20 differentiated in vitro to metacyclic trypomastigotes in a wide range (20-60%), while clonet 39 differentiated very poorly (3-15%). To obtain larger amounts of infective trypomastigote forms of clonet 39 stocks, and of those clonets 19 or 20 stocks with poor differentiation capacity, VERO cells were infected with the available metacyclic trypomastigotes.

Mouse infection

Trypomastigote forms obtained as described previously, were inoculated into 4 month-old, C3H/He, irradiated (4.5 Gy), male mice to obtain bloodstream trypomastigotes for further infection of immunocompetent mice. This particular mouse strain was chosen because it is associated with higher mortality after infection with T. cruzi, when compared to other H-2 haplotypes (38). In view of previously described differences in sex of host as a factor in Chagas' disease (16), ten male and ten female, normal, 4 months, C3H/He mice, were inoculated with 10⁴ bloodstream T cruzi trypomastigote forms from each cloned stock belonging to clonets 19 and 20. Due to limitations in bloodstream parasite yields resulting from the poor parasitemia of clonet 39 stocks, only 10² parasites per mouse were inoculated to 6 male and 6 female, 4-month-old C3H/He mice with the v-2148 cl 1 and v-2149 cl 1 stocks. Clonet 20, P 11 cl 3 cloned stock was used as control, following the same protocol. Parasitemia and mortality were recorded. Parasitemias were evaluated twice a week for 70 days or until death, by direct counting (4) or when negative for this method by a micro hematocrit method as described (13). Mortality data was determined after 70 or 97 days post infection when inoculations were 10⁴ or 10² parasites respectively. This difference was determined due to the longer prepatency period observed in mice inoculated with low number of parasites.

Data analysis

The genetic background and biological data (means for parasitemia maximum and mortality) were compared between the three groups of clonets, by a Student's t test.

Confirmation of Trypanosoma cruzi type

The T. cruzi cloned stocks, amplified in VERO cells, were characterized as epimastigote forms by molecular karyotype analysis before and after infection of these cells to assure that the genotypic background was maintained after parasitic differentiation and growth. The conditions used were essentially as described earlier, using the parasite gene clone 13 as probe (31). Hybridizations were done with a digoxigenin-labeled probe and development with alkaline phosphatase, NBT and BCIP as described (15).

RESULTS

Table I summarizes information on the biological behavior of the different stocks studied, including their arithmetic means and their standard deviations. Table IA includes prepatency, means of the maximum parasitemia reached by each mouse and mortality of mice inoculated with T cruzi clonets 19 and 20 stocks. Prepatency varied greatly among mice infected with stocks from the same clonet.

The expected high parasitemia and short prepatency correlation did not always occur, as can be observed between the Cutia cl 1 and 13379 cl 7 clonet 19 stocks. Mice infected with clonet 19 stocks displayed high (1089 x 10³), moderate (124 x 10³) and low (12 x 10³ ) means of maxima parasitemia, while clonet 20 stocks only induced moderate (271 x10³) and low (42 x 10³) levels.

Figures 1A and 1B depict the particular male and female parasitemia curves for each stock from clonets 19 and 20. Some differences in parasitemia courses and mortality between male and female mice can be observed. However, when a Student's t test was used, the differences were not significant (P>0.05). Nevertheless, mice infected with clonet 20 Cuica cl 1 stock exhibited the highest mortality, despite the fact that the parasitemia maximum was only one-tenth of the maximum found in this group (Esquilo cl l). Moreover, when the results of mice inoculated with Cuica cl 1 and P 209 cl 1 are compared, mice infected with the former show shorter prepatency, slightly lower parasitemia, and far greater mortality. Finally, although Cuica cl 1 and Cutia cl 2 cloned stocks generated higher mortality rates in males than in females, there were no significant differences when all clonet 19 and 20 cloned stocks were considered. These data demonstrate the high intra-clonet variability with regard to parasitemia maximun and mortality (see Table IA).

Results obtained with the 39 clonet stocks are shown in Table IB and Figure 2. Figure 2 shows parasitemia curves in mice inoculated with only 10² parasites, as mentioned in the Material and Methods section, from two clonet 39 stocks (v-2148 cl 1 and v-2149 cl 1) and one clonet 20 stock used as control (P11 cl 3). Table IB shows long prepatency periods (27 days), lower parasitemia and the greatest mortality of all the clones studied. S03 cl 5, a clonet 39 stock, was repeatedly inoculated into mice with a very low, undetermined number of bloodstream forms, always less than 10² parasites. Even though parasitemia in these mice was so low that it could only be detected by the micro hematocrit method, they died (50%) around 30 days post inoculation (not shown). These observations might represent the highly pathogenic nature of bloodstreain forms of clonet 39 stocks. It is interesting to point out the fact that mice infected with the clonet 20, P 11 cl 3 stock with the very low dose of 10² parasites presented parasitemias similar to those obtained in mice that received 10⁴ parasites. Thus, this parameter seems to depend more on the genetic background of the parasite than on the dose of inocula. Contrarily, the mortality rate appears to be higher with the lower inocula, but this fact could be explained due to a longer replication period.

Comparison of mortality means by a Student's t test, shows significant differences between clonets 19/20 and clonet 39 (P< 0.05). No significant differences are seen for these parameters between clonets 19 and 20 (P> 0.05). Finally there are no significant differences for means of maximum parasitemias between the three clonets studied.

In order to confirm the stability and discard the possibility of the parasites being mixed up during handling of the cloned stocks, all of the stocks underwent molecular karyotype analysis after parasite amplification and differentiation. Maintaining each genotypic profile provides confidence in the identity of the T. cruzi stock used to infect mice (not shown).

Figure 2. Parasitemia of two Trypanosoma cruzi clonet 39 cloned stocks (v-2148 cl 1 and v-2149 cl 1), determined in both sexes with inocula of 100 blood forms. The Trypanosoma cruzi clonet 0 cloned stock P11 cl 3 is included for comparative reasons.

DISCUSSION

Extensive population genetic studies have demonstrated that natural T. cruzi populations have a clonal structure (3). The results obtained with the employment of the isozyme electrophoresis technique, first limited to a number of enzyme systems (zymodemes) and later including a far larger number (MLEE), led to the idea that natural clones of the parasite were circulating in nature (34). Further studies on the characterization of T. cruzi zymodemes correlated with the typing performed by molecular karyotype or random-primer DNA analyses, aspects that strengthened the concept of a clonal structure for this parasite (35, 31).

The results obtained in this work demonstrate that T. cruzi clonets 19 and 20 defined by 15 gene loci, show marked differences in mortality when compared to clonet 39 stocks. The biological characterization is presented for individual stocks rather than average data for clonets (18), an advantageous procedure to asses the great variability within each clonet.

As described earlier, males were more susceptible to infection and mortality than females (16), although these differences were not statistically significant. Previous studies on the biological behavior of cloned T. cruzi stocks in a murine model, demonstrated high heterogeneity when parameters including parasitemia were considered (11). Growth kinetics of T. cruzi clones on in vitro culture (10) and their development in an invertebrate host (14, 7) have also been studied.

The observation that different T. cruzi stocks can have dissimilar biological behaviors in murine experimental models is not a new one (2, 17). Unfortunately, although these studies provided valuable information, none of them were performed with cloned stocks, the genetic classification of the parasites was limited to a low number of enzymatic loci, or the study was based on a small sample. Nevertheless, the most important contribution was that parasite types belonging to zymodeme 1 were more virulent than those classified as zymodeme 2 Bol or 2 Bra (30). Interestingly, zymodeme 2 Bol or clonet 39 is prevalent in the southern cone of South America, circulates in several geographical regions, and is frequently found in humans (34).

Two genetically close T. cruzi clonets (19 and 20) gave variable parasitemia curves among stocks, although the differences between them are not statistically significant. A similar study of biological parameters has been performed with metacyclic trypomastigotes of this same sample and again, no statistical difference was found between average data of stocks related to clonets 19 and 20 (18). Our results suggest that even though cloned stocks from these clonets share some genetic markers, they are heterogeneous with regards to important biological properties such as parasitemia course and mortality. This observation has also been detected by other investigators employing Brazilian T. cruzi clones (19).

When clonets 19 and 20 were compared to the genetically distant clonet 39, however, the former are significatively less pathogenic, despite the greater number of parasites inoculated per animal. This result differes from that of a previous report with the same sample (18). One possible explanation for this observation could be differences in the pathogenic nature of the bloodstream forms used here and the metacyclic forms employed in the earlier study. The comparison between the distant clonets is relevant, since the taxon T. cruzi appears to be composed of two major phylogenetic lineages (32, 36), originally classified as zymodemes ZI and Z2 (20, 22, 37). Natural T. cruzi stocks can be clearly divided into two major phylogenetic lineages with different ribosomal RNA gene promoters that lend credibility to the suggestion that these lineages represent distinct subspecies (12). Clonets 19 and 20 are included in the zymodeme Z 1 cluster, while clonet 39 is in the zymodeme Z 2 Bol, or second phylogenetic lineage.

We do not currently know the biological relevance of these results in human infection with different T. cruzi clonets. It is thought that in natural infection the insect transmits mixtures of T. cruzi clonets, some of them with different propagation capacities, but in certain domestic transmission habitats or geographical areas, only a limited number of clonets or pure clones are circulating, thus the question of biological relevance of T. cruzi infection in humans is approachable.

In Chile, there are endemic areas, such as the capital Santiago, where most infected persons carry the clonet 39 or zymodeme Z2 Bol (25). In the northern part of the country, this clonet and others are found in the domiciliary Triatoma infestans vector (15, 1), yet only clonet 39 seems to be transmitted to humans (5). A different situation exists in the sylvatic habitats, where the Mepraia spinolai vector carries many clonets other than 39, such as the sp 104 cloned stock studied here.

The fact that clonet 39 is prevalent in humans, along with the notion that acute cases of Chagas' disease are rarely seen in Chile compared to other geographical areas, such as Brazil, could be explained by the prevalence of clonet 39 in the extreme southern latitudes of South America. A similar situation exists in Argentina where an equivalent clonet 39 parasite type (Argentinian zymodeme 1) has frequently been found in chronic asymptomatic patients. lt is posible that this particular parasite type adapts itself to human hosts more easily than other T. cruzi clonets, since they are also transmitted by the domestic T. infestans vector (15, 1, 21, 8, 23).

Diverse biological and biochemical parameters have been considered in this study, and the genetic markers employed seem useful to shedding light on Chagas' disease pathogenicity in mice. Future epidemiological studies should endeavor to determine the importance of a particular infective T. cruzi clonet or mixture of clonets in the prognosis and treatment of Chagas' disease in humans.

ACKNOWLEDGEMENTS

This work was financiallysupported by the Economic European Community Project TS3-CT92-0155 and Fondecyt- Chile Project 193-1053.

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