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Parasitología latinoamericana

versão On-line ISSN 0717-7712

Parasitol. latinoam. v.62 n.1-2 Santiago jun. 2007

http://dx.doi.org/10.4067/S0717-77122007000100001 

 

Parasitol Latinoam 62: 3 - 6, 2007 PLAP

ARTICULO ORIGINAL

Trypanosoma rangeli infected mouse sera reactivity with Trypanosoma cruzi synthetic peptides

 

CLAUDIO ZUNIGA*, RAMON VARGAS*, MARIA TERESA PALAU**, FELIO BELLO***, JOSÉ ANTONIO DE DIEGO**** and ULISES VERGARA*

* Unidad de Inmunología, Depto. Medicina Preventiva Animal, Facultad de Ciencias Veterinarias, Universidad de Chile.Casilla 2,Correo 15, La Granja, Santiago, Chile.
** Universidad La República, Santiago, Chile.
*** Depto.Ciencias Básicas, Universidad de La Salle. Bogotá, Colombia.
**** Laboratorio de Parasitología, Depto. Medicina Preventiva, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, España.

Correspondencia a :


ABSTRACT

In man, differential diagnosis of Trypanosoma cruzi and Trypanosoma rangeli infections represents a serious problem, not only because both parasites present similar geographical distribution, the same hosts and sometimes the same insect vector, but also because they have common antigen determinants. In this work IgM and IgG humoral responses to T. cruzi syntethic peptides in mice infected with T. cruzi and with T. rangeli were analysed. In a immunoradiometric assay (IRMA ) 6 syntethic peptides were used, denominated as clones 1, 2, SAPA, 13, 30 and 36. The results showed that sera from infected mice with T. rangeli recognized all peptides derived from T. cruzi proteins, at IgM as well as IgG level. Reactivity with peptide SAPA is discussed as previous work indicated that SAPA is not codified in the T. rangeli genome. Our results support the suggestion that crossed reactions are due to the fact that both parasites present common antigens.

Key words: Trypanosoma cruzi, Trypanosoma rangeli, cross-reaction.


 

INTRODUCTION

Two trypanosomes exist in Central and Southern America which are capable of infecting man, Trypanosoma cruzi and Trypanosoma rangeli. While T cruzi causes Chagas'disease, a complicated and devastating disease that affects around 20 million people in America12, T rangeli may be pathogenic for invertebrate host but not pathogenic for vertebrate hosts3. Even though T rangeli infection, up to now, has not been able to be associated with a specific pathology it does present a serious problem in the diagnosis of Chagas'disease, because of the appearance of crossed reactions in serological test,45. Both parasites have a similar geographical distributions, the same vertebrate host and in some regions they share the same insect vector6. Some authors have reported antigenic cross-reaction between both parasites in different immunological assays7,8.

In this work , the humoral response of mice infected with T. rangeli and T. cruzi blood trypomastigotes was analysed, thereby determining IgM and IgG reactivity to T cruzi synthetic peptides.

MATERIALS AND METHODS

Mice: Males mice of the Swiss ICO NMRI were used for each one of the infection models (Parasitology Laboratory, Universidad Autónoma de Madrid, Spain).

Parasites stocks: The T. rangeli C239 and the T. cruzi Y (Parasitology Laboratory, U.A.M., Spain ) strains were used.

Infection models: One month old mice were infected with 104 T. rangeli blood trypomastigotes and another group with 104 T. cruzi parasites. Sera from this infected animals were used in this study.

Immunoradiometric assay (IRMA) with synthetic peptides: Six synthetic peptides were used modelled according to antigen repeated units'aminoacid sequences having been previously cloned from T. cruzi10 (clones 1, 2, SAPA, 13, 30 and 36). This peptides were kindly provided by Dr. A. Engstrom from the Immunology Department of Upsala University. Sweden.

The immunoradiometric method was used following the previously described technique11. In summary, polyvinylchloride microplates (Falcon Labware, U.S.A.), were incubated for a whole night at 4° C with 50 ¡J.1 peptide diluted in a optimum concentration with pH 9.6, 0.1 M sodium bicarbonate. Later excess peptide was

eliminated in three washes with Tween 20 buffer (0.9% NaCl, 0.05% Tween 20, 0.01% Sodium Azide) and the microplates were satured with 200 yd 3% nonfat milk for 90 minutes.After washing, they were incubated for 90 minutes with 30 ¡L4,l of serial dilutions from the distinct infected mouse sera samples. After washing in Tween 20 buffer the wells were incubated with 5 x 104 c.p.m. in 30 ¡J.1 of 125I radiolabelled affinity purified goat anti-mouse IgG or goat anti-mouse IgM (Sigma, U.S.A.) diluted with 3% nonfat milk. After next being incubated for 90 minutes at room temperature, the weels were washed five times in buffer to eliminate excess radioactive material, finally the weels were cut and counted in a gamma counter (Isodata).

RESULTS AND DISCUSSION

All animals infected with T. rangeli survived however with T. cruzi infection all mice died by day 23 post-infection.

T. rangeli and T. cruzi infected mouse sera's IgM reactivity is showed in Figure 1 and Figure 2, respectively. There is reaction with all synthetic peptides, with maximum reaction level being around day 15 and staying the reactivity until day 40, after infection with T.rangeli. In both situations the greater reactivity is with peptide 1.



Figure 1. IgM reactivity in NMRI mice infected with T. rangeli C23 strain.



Figure 2. IgM reactivity in NMRI mice infected with T. cruzi Y strain.

There was also IgG response to all the peptides and again a greater reactivity with the peptide 1 is observed. IgG reactivity with T. rangeli started to climb notably on day 26 and continued until day 40 post-infection (Figure 3). Reactivity of sera from infected mice with Y strain shows a maximum level with all the peptides at the day 19 except for the peptide 1 that shows a peak in the day 15 pos-infection (Figure 4).



Figure 3. IgG reactivity in NMRI mice infected with T. rangeli strain.



Figure 4. IgG reactivity in NMRI mice infected with T. cruzi Y strain.

This results indicate that sera from infected mice by T. rangeli C23 strain recognized, at both IgM and IgG antibody level, all peptides derived from T. cruzi proteins, which were known to be localized in defined regions of the parasite12. Furthermore, no significant differences were shown with the reactivity of T. cruzi infected mouse sera. The high reactivity with peptide 1 is interesting given this antigen's flagellar localization12. Previous observations have shown that T. cruzi flagellar proteins are highly antigenic and can be effective in protection against the parasite's infectious forms1314.

Reactivity with SAPA was not in agreement with previous studies which indicates that SAPA is not codified in the T. rangeli genome15. However, this results could be explained by the fact that SAPA represents a family of distinct proteins and/or is due to genetic differences between the T. rangeli strains used. In previous works, infection with T. rangeli C23 strain

showed that it had a protector effect when faced with further infection by the T. cruzi Y strain16, therefore it would be interesting to study each one of the antigens'specific participation in the protection phenomena. Our results with synthetic peptides support the suggestion that crossed reactions are due to the fact that both parasites present common antigens.

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16.- ZÚÑIGA C, PALAU M T, PENIN P, et al. Protective effect of Trypanosoma rangeli against infection with highly virulent strain of Trypanosoma cruzi. Trop Med Int Health 1997; 5: 482-7.        [ Links ]

Correspondencia a: e-mail: clzuniga@uchile.cl

 

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