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Vaccination with Trypanosoma rangeli induces resistance of guinea pigs to virulent Trypanosoma cruzi
Veterinary Immunology and Immunopathology 157 (2014) 119–123
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Vaccination with Trypanosoma rangeli induces resistance of guinea pigs to virulent Trypanosoma cruzi B. Basso a,∗ , E. Moretti a , R. Fretes b a Cátedra de Pediatría y Neonatología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba & Coordinación Nacional de Control de Vectores, Córdoba, Argentina b Cátedra de Histología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Argentina
a r t i c l e
i n f o
Article history: Received 10 July 2013 Received in revised form 1 October 2013 Accepted 21 October 2013 Keywords: Vaccination Chagas’ disease Guinea pigs Trypanosoma rangeli Trypanosoma cruzi
a b s t r a c t Chagas’ disease, endemic in Latin America, is spread in natural environments through animal reservoirs, including marsupials, mice and guinea pigs. Farms breeding guinea pigs for food are located in some Latin-American countries with consequent risk of digestive infection. The aim of this work was to study the effect of vaccination with Trypanosoma rangeli in guinea pigs challenged with Trypanosoma cruzi. Animals were vaccinated with fixated epimastigotes of T. rangeli, emulsified with saponin. Controls received only PBS. Before being challenged with T. cruzi, parasitemia, survival rates and histological studies were performed. The vaccinated guinea pigs revealed significantly lower parasitemia than controls (p < 0.0001–0.01) and a discrete lymphomonocytic infiltrate in cardiac and skeletal muscles was present. In the chronic phase, the histological view was normal. In contrast, control group revealed amastigote nests and typical histopathological alterations compatible with chagasic myocarditis, endocarditis and pericarditis. These results, together with previous works in our laboratory, show that T. rangeli induces immunoprotection in three species of animals: mice, guinea pigs and dogs. The development of vaccines for use in animals, like domestic dogs and guinea pigs in captivity, opens up new opportunities for preventive tools, and could reduce the risk of infection with T. cruzi in the community. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chagas’ disease is endemic in Latin America and affects nearly 18 million people. Symptomatic cardiac disease develops in 30% of infected people (Coura, 2007). The disease is caused by the protozoan parasite Trypanosoma cruzi. Guinea pigs are among the important natural reservoirs of Chagas’ disease (Pizarro et al., 2007), in the rural environment of Andean communities, in South America. Local farms that breed guinea pigs for food, present a risk of infection via the digestive tract if the consumed animals are infected with T. cruzi and not properly cooked. Moreover,
∗ Corresponding author at: Güemes 383, B◦ General Paz CP: 5000, Córdoba, Argentina. Tel.: +54 351 4520648. E-mail address: [email protected] (B. Basso). 0165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2013.10.011
local trade of these animals is common practice and they are also exported to different countries. In several Latin American places, another nonpathogenic trypanosome than can infect humans, Trypanosoma rangeli occurs (Tejera, 1920; D’Alessandro, 1974; ˜ Anez et al., 1985; Cuba Cuba, 1998). Both parasites share endemic areas, vectors, and present a strong antigenic relationship (Basso et al., 1987; Stevens et al., 1999). As for other parasitic diseases, no effective vaccine for humans is yet available, in spite of the numerous experimental approaches performed with different antigenic materials, ranging from subcellular fractions to recombinant antigens, or even plasmid DNA encoding antigens of T. cruzi (Taibi et al., 1995; Costa et al., 1998; Wrightsman and Manning, 2000; Planelles et al., 2001; Garg and Tarleton, 2002; Limon-Flores et al., 2010; Gupta and Garg, 2013). In our laboratory, we have developed a mouse model for
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vaccination with T. rangeli. Mice were protected both by fixed and live epimastigotes of a Colombian strain of T. rangeli maintained in our laboratory in axenic cultures (Basso et al., 1991; Introini et al., 1998) and two strains of different origin (Basso et al., 2008). In comparison with the non-vaccinated infected mice, the immunisation schedule induces a strong reduction in parasitemia and mortality levels, as well as absence of histological lesions. Moreover, this vaccine also induces protection of dogs (Basso et al., 2007). The aim of this study was to analyse the evolution of T. cruzi infection in guinea pigs previously vaccinated with fixated epimastigotes of T. rangeli, in order to determine if the protection against T. cruzi infection is a common feature among different animal species. 2. Materials and methods 2.1. Animals Groups of guinea pigs (1 month old) were maintained under standard conditions in an animal room. Experiments were started after two weeks.
microscope to count the parasites in a Neubauer chamber. Survival rates were checked daily. 2.5. Histological studies Guinea pigs from both experimental groups were killed with CO2 . The cardiac and quadriceps muscle were immediately removed from each guinea pig, fixated in 10% buffered formalin (pH 7.0), and embedded in paraffin. 5 m-thick sections of each organ were stained with haematoxylin–eosin. At least 25 areas from each section were checked for parasites and histopathology under a 40× objective lens in a blind study. 2.6. Ethical standards All experiments reported herein were conducted in compliance with the Animal Welfare Act and in accordance with the principles set forth in the “Guide for the Care Use of Laboratory Animals”, Institute of Laboratory Animal Resources National Research Council, National Academy Press, 1996. 2.7. Statistical analysis
2.2. Parasites The Tulahuen strain of T. cruzi was maintained by weekly sub inoculations in Balb/c albino mice. Bloodstream trypomastigotes used for challenging inoculations were obtained by cardiac puncture on day 14 postinfection. A Colombian strain of T. rangeli was used. Epimastigote forms were cultivated in monophasic medium as previously published (Basso et al., 1980). Parasites were harvested in the exponential phase of growth. The obtained epimastigotes were washed three times in PBS at 10,000 g for 20 min at 4 ◦ C, fixated with glutaraldehyde (0.1%) and washed again. Just before immunisation, 1 × 109 parasites/ml were emulsified with saponin, as adjuvant. 2.3. Vaccination and infection schedule The 45-day-old guinea pigs, n = 4 in each experiment were vaccinated as previously described (Basso et al., 1991). Briefly, vaccinated animals (V) were inoculated intradermically with a volume of 0.2 ml containing 1 × 108 fixated epimastigotes (0.1 ml epimastigotes plus 0.1 ml of saponin) on days 0, 7 and 21. The same number of control animals (C) received 0.2 ml sterile PBS. All guinea pigs were infected 7 days after the last immunisation, by intraperitoneal inoculation of 50,000 T. cruzi blood trypomastigotes. 2.4. Parasitemia The parasite counts were performed using the technique described by Hoff (1974). Briefly, heparinised blood from guinea pigs was diluted using 0.85% NH4 Cl solution in an Eppendorf tube, and shaken for 5 min. Then, it was centrifuged at 800 × g, at 4 ◦ C for 10 min. The supernatant was slowly discarded, and the pellet was examined under the
The experimental data were subjected to the Student’s t-test. All results at a level p < 0.05 were considered statistically significant. 3. Results and discussion Fig. 1 shows the results of a parasitemia curve of a representative experiment, performed with T. rangelivaccinated and non-vaccinated guinea pigs. Both groups were infected with T. cruzi as described in Section 2. As can be seen, until the 15th day post-infection, no differences in the parasitemias developed by the vaccinated and control groups were observed. Nevertheless, from the 20th post-infection day, the vaccinated group presented significantly lower parasitemias than the control group: C: 30.7 ± 1.2 × 104 /ml; V: 15 ± 2.4; (p < 0.01). At 30th p.i. the values were: C: 62.7 ± 10.5; V: 6.5 ± 1.2 × 104 /ml. This trend was observed until the end of the study on the 60th post-infection day. Moreover, from the 40th day, the parasitemia in vaccinated guinea pigs was almost negligible C: 59.5 ± 10.5; V: 0.8 ± 0.3 × 104 /ml and 60th day: C: 21.2 ± 1.8; V: 0.3 ± 0.4 (p < 0.0001). The histological studies of the heart of the Control group (Fig. 2A and B) revealed the typical histopathological alterations compatible with acute chagasic myocarditis, endocarditis and pericarditis with nests of amastigotes, the multiplicative form of T. cruzi, and numerous mononuclear cell infiltrates. In fact, the histological studies of the heart of this group revealed the presence of amastigote nests in the ventricular wall in 4/6 animals. The mean of amastigotes/nest was 16.0 ± 12.7 and the mean number of nest/field was 1.2 ± 0.9. In contrast, the T. rangeli-vaccinated guinea pigs infected with T. cruzi (Fig. 2C) showed low histopathological alterations, i.e. only focal and scarce infiltration of mononuclear cells in epicardic area and absence of amastigote nests in tissue (0/6) (p = 0.015). The difference
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Fig. 1. Parasitemia levels in guinea pigs vaccinated with T. rangeli. Control Group (C), unvaccinated and infected with T. cruzi; Vaccinated Group (V), vaccinated and infected animals. Each time point represents the mean ± S.E.M. of four guinea pigs of a representative experiment. Asterisks indicate significant differences between V and C groups (Student’s t-test p < 0.01 to <0.0001).
between Control vs. Vaccinated group was significant when the infiltrates were <10 cells/infiltrate (p = 0.037) and in the total number of infiltrates (p = 0.02). In skeletal muscle the results were similar: amastigote/nests in control
guinea pigs 8.4 ± 7.7 and the mean number of nest/field was 0.8 ± 0.6. In contrast, the vaccined-infected animals revealed absence of amastigotes in tissue (0/6), p = 0.036. Furthermore, histological studies performed in the control
Fig. 2. Histological sections of the heart of Control (A and B) and Vaccinated (C) animals, hematoxylin–eosin, 400×. Slices of the Control group show nests of amastigotes (A, thick arrow) and mononuclear cell infiltration (B, thin arrows). Section from vaccinated-infected animals shows focal mononuclear cell infiltrates (C, thin arrow). No amastigote nests were observed.
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group sacrificed in the chronic phase, on 120th postinfection day, showed minor histological lesions, whereas in the vaccinated group no histopathological lesions were detected in the chronic phase (data not shown). We have previously demonstrated that the immunisation schedule used in this work elicited B and T cell specific responses to T. cruzi, as well as the secretion of a particular pattern of cytokines and a strong reduction in parasitemia level and mortality rate in infected mice (Basso et al., 1991, 2004; Introini et al., 1998). Indeed, more than 95% of mice survived a lethal T. cruzi infection and displayed strongly reduced parasite burden during the acute phase, associated with high levels of antibodies and strong cellular responses, elevated serum levels of IL12 and IFN␥, low levels of proinflammatory cytokines (IL6 and TNF␣) and normal levels of IL10, as well as absence of histopathological lesions. The immunisation produced an adequate balance of Th1 and Th2 responses, both necessary to induce protection. We also demonstrated that this immunisation schedule is effective in dogs (Basso et al., 2007). In the present work we observed a different course of T. cruzi infection in control and vaccinated groups. The latter group displayed significantly lower parasitemia than controls, as well as scarcer inflammatory cell infiltration than non-vaccinated infected guinea pigs. In most of the vaccinated animals, histological evaluation revealed normal tissue structures. Moreover, no nests of amastigotes were observed in cardiac and skeletal muscles of these animals. This finding highlights a significant difference in comparison to the control group. Regarding survival, no animal from any group died during the study (120th post-infection day), probably because this species is a natural reservoir of the parasite. The results of the present work demonstrate, for the first time, protection in guinea pigs immunised with nonpathogenic T. rangeli and challenged with a high number of virulent T. cruzi trypomastigote forms. These results are consistent with those reported by other authors, in mouse models (Zuniga et al., 1997; Araujo et al., 1999; Palau et al., 2003). In conclusion, the results of the present work show that the antigens involved in the humoral immunity induced by T. rangeli are able to protect guinea pigs, which are natural reservoirs of T. cruzi. It is important to highlight this significant protection because guinea pigs could play an important role in the transmission to humans when they consume food from infected animals in endemic area. This could be avoided through vaccination of guinea pigs with T. rangeli in breedings farms. Lee et al. (2010) showed in a computational model that vaccination against Chagas’ disease is very cost-effective, in many cases providing both cost savings and health benefits, even at low infection risk and vaccine efficacy. Moreover, from the point of view of pre-clinical research, this is the third species in which protection is induced, besides mice and dogs, through immunisation with epimastigotes of T. rangeli, with the vaccination model herein presented. Taking together, these findings and those obtained in dogs could open up new expectatives for veterinary vaccination and lead to new opportunities for the
epidemiological control of Chagas’ disease, through vaccination of domestic reservoirs, which jointly with vector control could reduce the risk of infection in rural communities.
Acknowledgements Special thanks to Ms. Patricia Gil, laboratory technician, and to Mrs. María Fernanda Garstein for her contribution to the translation and revision of this paper. Financial support: This work was performed with the financial support of Secretaría de Ciencia y Tecnología, Universidad Nacional de Córdoba (SECYT-UNC) and Servicio Nacional de Chagas (Argentina).
References ˜ Anez, N., Velandia, J., Rodríguez, A.M., 1985. Trypanosoma rangeli Tejera, 1920. VIII. Response to reinfections in two mammals. Mem. Inst. Oswaldo Cruz 80, 149–153. Araujo, Z., El Bouhdidi, A., Heremans, H., Van Marck, E., Castes, M., Carlier, Y., 1999. Vaccination of mice with a combination of BCG and killed Leishmania promastigotes reduces acute Trypanosoma cruzi infection by promoting an IFN-gamma response. Vaccine 17, 957–964. Basso, B., Moretti, E., Dominguez, M., 1980. Cultivo de Trypanosoma cruzi en medio monofásico con aireación para obtención de antígenos. Medicina (Bs. As.) 40, 428–432. Basso, B., Moretti, E., Vottero-Cima, E., 1987. Antigenic relationships between Trypanosoma cruzi and Trypanosoma rangeli. Rev. Iber. Parasitol. 47, 15–21. Basso, B., Moretti, E., Vottero-Cima, E., 1991. Immune response and Trypanosoma cruzi infection in Trypanosoma rangeli-immunized mice. Am. J. Trop. Med. Hyg. 44, 413–419. Basso, B., Cervetta, L., Moretti, E., Carlier, Y., Truyens, C., 2004. Acute Trypanosoma cruzi infection: IL-12, IL-18, TNF, sTNFR and NO in T. rangeli-vaccinated mice. Vaccine 22, 1868–1872. Basso, B., Castro, I., Introini, V., Gil, P., Truyens, C., Moretti, E., 2007. Vaccination with, Trypanosoma rangeli reduces the infectiousness of dogs experimentally infected with Trypanosoma cruzi. Vaccine 25, 3855–3858. Basso, B., Moretti, E., Fretes, R., 2008. Vaccination with fixed epimastigotes of different strains of Trypanosoma rangeli protects mice against Trypanosoma cruzi infection. Mem. Inst. Oswaldo Cruz 103, 370–374. Costa, F., Franchin, G., Pereira-Chioccola, V.L., Ribeirao, M., Schenkman, S., Rodrigues, M.M., 1998. Immunization with a plasmid DNA containing the gene of transialidase reduces Trypanosoma cruzi infection in mice. Vaccine 16, 768–774. Coura, J.R., 2007. Chagas’ disease: what is known and what is needed? A background article. Mem. Inst. Oswaldo Cruz 102, 113–122. Cuba Cuba, A., 1998. Review of the biologic and diagnostic aspects of Trypanosoma (Herpetosoma) rangeli. Rev. Soc. Bras. Med. Trop. 31, 207L 220. D’Alessandro, A., 1974. Trypanosoma rangeli. Acta Med. del Valle 5, 139–142. Garg, N., Tarleton, R.L., 2002. Genetic immunization elicits antigen-specific protective immune responses and decreases disease severity in Trypanosoma cruzi infection. Infect. Immun. 70, 5547–5555. Gupta, S., Garg, N.J., 2013. TcVac3 Induced Control of Trypanosoma cruzi Infection and Chronic Myocarditis in Mice. PLoS ONE 8 (3), e59434, http://dx.doi.org/10.1371/journal. Hoff, R., 1974. A methods of counting and concentrating living Trypanosoma cruzi in blood lysed with ammonium chloride. J. Parasitol. 60, 527–528. Introini, M.V., Basso, B., Moretti, E., 1998. Experimental Chagas disease: I. Study of different immunization conditions in the infection course. Bol. Chil. Parasitol. 53, 45–51. Lee, B.Y., Bacon, K.M., Connor, D.L., Willig, A.M., Bailey, R.R., 2010. The potential economic value of a Trypanosoma cruzi (Chagas disease) vaccine in Latin America. PLoS Negl. Trop. Dis. 4 (12), e916, http://dx.doi.org/10.1371/journal. Limon-Flores, A.Y., Cervera-Cetina, R., Tzec-Arjona, J.L., Ek-Macias, L., Sánchez-Burgos, G., Ramirez-Sierra, M.J., Cruz-Chan, J.V., VanWynsberghe, N.R., Dumonteil, E., 2010. Effect of a combination DNA vaccine
B. Basso et al. / Veterinary Immunology and Immunopathology 157 (2014) 119–123 for the prevention and therapy of Trypanosoma cruzi infection in mice: role of CD4+ and CD8+ T cells. Vaccine 46, 7414–7419. Palau, M.T., Mejia, A.J., Vergara, U., Zuniga, C.A., 2003. Action of Trypanosoma rangeli in infections with virulent Trypanosoma cruzi populations. Mem. Inst. Oswaldo Cruz 98, 543–548. Pizarro, J.C., Lucero, D., Steven, L., 2007. A method for the identification of guinea pig blood meal in the Chagas’ disease vector, Triatoma infestans. Kinetoplastid Biol. Dis. 6, 1, http://dx.doi.org/10.1186/1475-9292-6-1. Planelles, L., Thomas, M.C., Alonso, C., Lopez, M.C., 2001. DNA immunization with Trypanosoma cruzi HSP70 fused to the KMP11 protein elicits a cytotoxic and humoral immune response against the antigen and leads to protection. Infect. Immun. 69, 6558–6563.
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Stevens, J.R., Teixeira, M.M., Bingle, L.E., Gibson, W.C., 1999. The taxonomic position and evolutionary relationships of Trypanosoma rangeli. Int. J. Parasitol. 29, 749–757. Taibi, A., Espinoza, A.G., Ouaissi, A., 1995. Trypanosoma cruzi: analysis of cellular and humoral response against a protective recombinant antigen during experimental Chagas’ disease. Immunol. Lett. 48, 193–200. Wrightsman, R.A., Manning, J.E., 2000. Paraflagellar rod proteins administered with alum and IL-12 or recombinant adenovirus expressing IL-12 generates antigen-specific responses and protective immunity in mice against Trypanosoma cruzi. Vaccine 18, 1419–1427. Zuniga, C.A., Palau, T., Penin, P., Gramallo, C., de Diego, J., 1997. Protective effect of Trypanosoma rangeli against infections with a highly virulent strain of Trypanosoma cruzi. Trop. Med. Int. Health 2, 482–487.
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Vaccination with Trypanosoma rangeli induces resistance of guinea pigs to virulent Trypanosoma cruzi B. Basso a,∗ , E. Moretti a , R. Fretes b a Cátedra de Pediatría y Neonatología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba & Coordinación Nacional de Control de Vectores, Córdoba, Argentina b Cátedra de Histología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Argentina
a r t i c l e
i n f o
Article history: Received 10 July 2013 Received in revised form 1 October 2013 Accepted 21 October 2013 Keywords: Vaccination Chagas’ disease Guinea pigs Trypanosoma rangeli Trypanosoma cruzi
a b s t r a c t Chagas’ disease, endemic in Latin America, is spread in natural environments through animal reservoirs, including marsupials, mice and guinea pigs. Farms breeding guinea pigs for food are located in some Latin-American countries with consequent risk of digestive infection. The aim of this work was to study the effect of vaccination with Trypanosoma rangeli in guinea pigs challenged with Trypanosoma cruzi. Animals were vaccinated with fixated epimastigotes of T. rangeli, emulsified with saponin. Controls received only PBS. Before being challenged with T. cruzi, parasitemia, survival rates and histological studies were performed. The vaccinated guinea pigs revealed significantly lower parasitemia than controls (p < 0.0001–0.01) and a discrete lymphomonocytic infiltrate in cardiac and skeletal muscles was present. In the chronic phase, the histological view was normal. In contrast, control group revealed amastigote nests and typical histopathological alterations compatible with chagasic myocarditis, endocarditis and pericarditis. These results, together with previous works in our laboratory, show that T. rangeli induces immunoprotection in three species of animals: mice, guinea pigs and dogs. The development of vaccines for use in animals, like domestic dogs and guinea pigs in captivity, opens up new opportunities for preventive tools, and could reduce the risk of infection with T. cruzi in the community. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chagas’ disease is endemic in Latin America and affects nearly 18 million people. Symptomatic cardiac disease develops in 30% of infected people (Coura, 2007). The disease is caused by the protozoan parasite Trypanosoma cruzi. Guinea pigs are among the important natural reservoirs of Chagas’ disease (Pizarro et al., 2007), in the rural environment of Andean communities, in South America. Local farms that breed guinea pigs for food, present a risk of infection via the digestive tract if the consumed animals are infected with T. cruzi and not properly cooked. Moreover,
∗ Corresponding author at: Güemes 383, B◦ General Paz CP: 5000, Córdoba, Argentina. Tel.: +54 351 4520648. E-mail address: [email protected] (B. Basso). 0165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2013.10.011
local trade of these animals is common practice and they are also exported to different countries. In several Latin American places, another nonpathogenic trypanosome than can infect humans, Trypanosoma rangeli occurs (Tejera, 1920; D’Alessandro, 1974; ˜ Anez et al., 1985; Cuba Cuba, 1998). Both parasites share endemic areas, vectors, and present a strong antigenic relationship (Basso et al., 1987; Stevens et al., 1999). As for other parasitic diseases, no effective vaccine for humans is yet available, in spite of the numerous experimental approaches performed with different antigenic materials, ranging from subcellular fractions to recombinant antigens, or even plasmid DNA encoding antigens of T. cruzi (Taibi et al., 1995; Costa et al., 1998; Wrightsman and Manning, 2000; Planelles et al., 2001; Garg and Tarleton, 2002; Limon-Flores et al., 2010; Gupta and Garg, 2013). In our laboratory, we have developed a mouse model for
120
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vaccination with T. rangeli. Mice were protected both by fixed and live epimastigotes of a Colombian strain of T. rangeli maintained in our laboratory in axenic cultures (Basso et al., 1991; Introini et al., 1998) and two strains of different origin (Basso et al., 2008). In comparison with the non-vaccinated infected mice, the immunisation schedule induces a strong reduction in parasitemia and mortality levels, as well as absence of histological lesions. Moreover, this vaccine also induces protection of dogs (Basso et al., 2007). The aim of this study was to analyse the evolution of T. cruzi infection in guinea pigs previously vaccinated with fixated epimastigotes of T. rangeli, in order to determine if the protection against T. cruzi infection is a common feature among different animal species. 2. Materials and methods 2.1. Animals Groups of guinea pigs (1 month old) were maintained under standard conditions in an animal room. Experiments were started after two weeks.
microscope to count the parasites in a Neubauer chamber. Survival rates were checked daily. 2.5. Histological studies Guinea pigs from both experimental groups were killed with CO2 . The cardiac and quadriceps muscle were immediately removed from each guinea pig, fixated in 10% buffered formalin (pH 7.0), and embedded in paraffin. 5 m-thick sections of each organ were stained with haematoxylin–eosin. At least 25 areas from each section were checked for parasites and histopathology under a 40× objective lens in a blind study. 2.6. Ethical standards All experiments reported herein were conducted in compliance with the Animal Welfare Act and in accordance with the principles set forth in the “Guide for the Care Use of Laboratory Animals”, Institute of Laboratory Animal Resources National Research Council, National Academy Press, 1996. 2.7. Statistical analysis
2.2. Parasites The Tulahuen strain of T. cruzi was maintained by weekly sub inoculations in Balb/c albino mice. Bloodstream trypomastigotes used for challenging inoculations were obtained by cardiac puncture on day 14 postinfection. A Colombian strain of T. rangeli was used. Epimastigote forms were cultivated in monophasic medium as previously published (Basso et al., 1980). Parasites were harvested in the exponential phase of growth. The obtained epimastigotes were washed three times in PBS at 10,000 g for 20 min at 4 ◦ C, fixated with glutaraldehyde (0.1%) and washed again. Just before immunisation, 1 × 109 parasites/ml were emulsified with saponin, as adjuvant. 2.3. Vaccination and infection schedule The 45-day-old guinea pigs, n = 4 in each experiment were vaccinated as previously described (Basso et al., 1991). Briefly, vaccinated animals (V) were inoculated intradermically with a volume of 0.2 ml containing 1 × 108 fixated epimastigotes (0.1 ml epimastigotes plus 0.1 ml of saponin) on days 0, 7 and 21. The same number of control animals (C) received 0.2 ml sterile PBS. All guinea pigs were infected 7 days after the last immunisation, by intraperitoneal inoculation of 50,000 T. cruzi blood trypomastigotes. 2.4. Parasitemia The parasite counts were performed using the technique described by Hoff (1974). Briefly, heparinised blood from guinea pigs was diluted using 0.85% NH4 Cl solution in an Eppendorf tube, and shaken for 5 min. Then, it was centrifuged at 800 × g, at 4 ◦ C for 10 min. The supernatant was slowly discarded, and the pellet was examined under the
The experimental data were subjected to the Student’s t-test. All results at a level p < 0.05 were considered statistically significant. 3. Results and discussion Fig. 1 shows the results of a parasitemia curve of a representative experiment, performed with T. rangelivaccinated and non-vaccinated guinea pigs. Both groups were infected with T. cruzi as described in Section 2. As can be seen, until the 15th day post-infection, no differences in the parasitemias developed by the vaccinated and control groups were observed. Nevertheless, from the 20th post-infection day, the vaccinated group presented significantly lower parasitemias than the control group: C: 30.7 ± 1.2 × 104 /ml; V: 15 ± 2.4; (p < 0.01). At 30th p.i. the values were: C: 62.7 ± 10.5; V: 6.5 ± 1.2 × 104 /ml. This trend was observed until the end of the study on the 60th post-infection day. Moreover, from the 40th day, the parasitemia in vaccinated guinea pigs was almost negligible C: 59.5 ± 10.5; V: 0.8 ± 0.3 × 104 /ml and 60th day: C: 21.2 ± 1.8; V: 0.3 ± 0.4 (p < 0.0001). The histological studies of the heart of the Control group (Fig. 2A and B) revealed the typical histopathological alterations compatible with acute chagasic myocarditis, endocarditis and pericarditis with nests of amastigotes, the multiplicative form of T. cruzi, and numerous mononuclear cell infiltrates. In fact, the histological studies of the heart of this group revealed the presence of amastigote nests in the ventricular wall in 4/6 animals. The mean of amastigotes/nest was 16.0 ± 12.7 and the mean number of nest/field was 1.2 ± 0.9. In contrast, the T. rangeli-vaccinated guinea pigs infected with T. cruzi (Fig. 2C) showed low histopathological alterations, i.e. only focal and scarce infiltration of mononuclear cells in epicardic area and absence of amastigote nests in tissue (0/6) (p = 0.015). The difference
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Fig. 1. Parasitemia levels in guinea pigs vaccinated with T. rangeli. Control Group (C), unvaccinated and infected with T. cruzi; Vaccinated Group (V), vaccinated and infected animals. Each time point represents the mean ± S.E.M. of four guinea pigs of a representative experiment. Asterisks indicate significant differences between V and C groups (Student’s t-test p < 0.01 to <0.0001).
between Control vs. Vaccinated group was significant when the infiltrates were <10 cells/infiltrate (p = 0.037) and in the total number of infiltrates (p = 0.02). In skeletal muscle the results were similar: amastigote/nests in control
guinea pigs 8.4 ± 7.7 and the mean number of nest/field was 0.8 ± 0.6. In contrast, the vaccined-infected animals revealed absence of amastigotes in tissue (0/6), p = 0.036. Furthermore, histological studies performed in the control
Fig. 2. Histological sections of the heart of Control (A and B) and Vaccinated (C) animals, hematoxylin–eosin, 400×. Slices of the Control group show nests of amastigotes (A, thick arrow) and mononuclear cell infiltration (B, thin arrows). Section from vaccinated-infected animals shows focal mononuclear cell infiltrates (C, thin arrow). No amastigote nests were observed.
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group sacrificed in the chronic phase, on 120th postinfection day, showed minor histological lesions, whereas in the vaccinated group no histopathological lesions were detected in the chronic phase (data not shown). We have previously demonstrated that the immunisation schedule used in this work elicited B and T cell specific responses to T. cruzi, as well as the secretion of a particular pattern of cytokines and a strong reduction in parasitemia level and mortality rate in infected mice (Basso et al., 1991, 2004; Introini et al., 1998). Indeed, more than 95% of mice survived a lethal T. cruzi infection and displayed strongly reduced parasite burden during the acute phase, associated with high levels of antibodies and strong cellular responses, elevated serum levels of IL12 and IFN␥, low levels of proinflammatory cytokines (IL6 and TNF␣) and normal levels of IL10, as well as absence of histopathological lesions. The immunisation produced an adequate balance of Th1 and Th2 responses, both necessary to induce protection. We also demonstrated that this immunisation schedule is effective in dogs (Basso et al., 2007). In the present work we observed a different course of T. cruzi infection in control and vaccinated groups. The latter group displayed significantly lower parasitemia than controls, as well as scarcer inflammatory cell infiltration than non-vaccinated infected guinea pigs. In most of the vaccinated animals, histological evaluation revealed normal tissue structures. Moreover, no nests of amastigotes were observed in cardiac and skeletal muscles of these animals. This finding highlights a significant difference in comparison to the control group. Regarding survival, no animal from any group died during the study (120th post-infection day), probably because this species is a natural reservoir of the parasite. The results of the present work demonstrate, for the first time, protection in guinea pigs immunised with nonpathogenic T. rangeli and challenged with a high number of virulent T. cruzi trypomastigote forms. These results are consistent with those reported by other authors, in mouse models (Zuniga et al., 1997; Araujo et al., 1999; Palau et al., 2003). In conclusion, the results of the present work show that the antigens involved in the humoral immunity induced by T. rangeli are able to protect guinea pigs, which are natural reservoirs of T. cruzi. It is important to highlight this significant protection because guinea pigs could play an important role in the transmission to humans when they consume food from infected animals in endemic area. This could be avoided through vaccination of guinea pigs with T. rangeli in breedings farms. Lee et al. (2010) showed in a computational model that vaccination against Chagas’ disease is very cost-effective, in many cases providing both cost savings and health benefits, even at low infection risk and vaccine efficacy. Moreover, from the point of view of pre-clinical research, this is the third species in which protection is induced, besides mice and dogs, through immunisation with epimastigotes of T. rangeli, with the vaccination model herein presented. Taking together, these findings and those obtained in dogs could open up new expectatives for veterinary vaccination and lead to new opportunities for the
epidemiological control of Chagas’ disease, through vaccination of domestic reservoirs, which jointly with vector control could reduce the risk of infection in rural communities.
Acknowledgements Special thanks to Ms. Patricia Gil, laboratory technician, and to Mrs. María Fernanda Garstein for her contribution to the translation and revision of this paper. Financial support: This work was performed with the financial support of Secretaría de Ciencia y Tecnología, Universidad Nacional de Córdoba (SECYT-UNC) and Servicio Nacional de Chagas (Argentina).
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