Co-infection with Trypanosoma cruzi protects mice against early death by neurological or pulmonary disorders induced by Plasmodium berghei ANKA
© Egima et al; licensee BioMed Central Ltd. 2007
Received: 12 March 2007
Accepted: 09 July 2007
Published: 09 July 2007
The objective of this study was to investigate whether the infection of C57BL/6 mice by P. berghei ANKA, which causes severe malaria, was modulated by co-infection with Trypanosoma cruzi.
Groups of C57BL/6 mice were infected either with P. berghei ANKA, T. cruzi strain G, or with both parasites. The presence of parasites was checked by microscopic examination of blood samples. Symptoms of neurological or respiratory disorders, as well as mortality, were registered. Breakdown of the blood brain barrier was determined by injecting the dye Evans blue. Histological sections of the lung were prepared and stained with hematoxilin-eosin.
All mice infected only with P. berghei ANKA died within 7–11 days post-infection, either with symptoms of cerebral malaria or with respiratory abnormalities. The animals co-infected with T. cruzi strain G survived longer, without any of the referred to symptoms. Protection against the early death by severe malaria was effective when mice were given T. cruzi 15 days before P. berghei inoculation. Breakdown of the blood brain barrier and extensive pulmonary oedema, caused by malaria parasites, were much less pronounced in co-infected mice. The degree of protection to severe malaria and early death, conferred by co-infection with T. cruzi, was comparable to that conferred by treatment with anti-CD8 antibodies.
Co-infection with T. cruzi protects C57BL/6 against the early death by malaria infection, by partially preventing either the breakdown of the blood brain, and cerebral malaria as a consequence, or the pulmonary oedema.
Multiple infections by parasitic agents that cause diverse clinical manifestations occur frequently and increase or decrease of overall pathogenic impact can be influenced by synergistic or competitive interactions between parasite species [1, 2]. Malaria is prevalent throughout tropical regions where concomitant infections occur frequently. Double infections with Plasmodium spp. and Ascaris lumbricoides, or triple infections with the two parasites plus Trichuris trichiura, without synergism or antagonism among parasites, have been reported in Zaire . Concomitant parasitism by agents of malaria and lymphatic filariasis, with no indication of interaction between the two infections, has been detected in India and in Guyana [4, 5]. On the other hand, it has been found that Senegalese children lightly infected with Schistosoma haematobium had lower Plasmodium falciparum densities, suggestive of negative interactions between both parasites . It has been pointed out that, if worms have in fact deleterious effect on malaria, treatment of helminthic infection would offer an affordable and effective means to roll back malaria . Experiments in mice have shown that malaria-filaria co-infection causes more severe anaemia and loss of body mass than Plasmodium chabaudi malarial infection alone , and that concomitant P. chabaudi and Schistosoma mansoni infections increase malaria parasitaemia and suppress spleen cell proliferative and Th2 responses to S. mansoni soluble egg antigen . Using Plasmodium berghei ANKA, which causes lethal cerebral malaria in C57BL/6J mice, Legesse et al.  found that superinfection with S. mansoni enhanced malaria parasite development, increasing parasitaemia and mortality.
As regards the mixed infection by malaria parasite and another protozoan, the data are sparse. The possibility, for instance, of co-infection with Trypanosoma cruzi, the agent of Chagas' disease, has not been examined. In Brazil, as there has been an increase in cases of Chagas' disease in the Amazon, where malaria prevails, recommendations have been made to include the microscopic analysis of blood smears for direct search of T. cruzi in patients with fever in the routine survey for malaria parasites . Thirty years ago, Krettli  reported that in double infection with P. berghei NK65 and T. cruzi about 40% of mice chronically infected with T. cruzi relapsed to the acute phase when inoculated with P. berghei, while some decrease in P. berghei parasitaemia was observed. Since then, the question has not been further addressed. This study aimed at investigating if, and to what extent, the agents of malaria and Chagas' disease exerted their effects to each other's course of infection. To that end, P. berghei ANKA, which produces cerebral malaria in C57BL/6 mice, and a T. cruzi strain from the Amazon, which produces subpatent infection, were used.
Parasites and infection of mice
P. berghei ANKA was conserved as stabilates of 107 parasitized erythrocytes in liquid nitrogen. When needed, the parasites were defrosted and injected intraperitoneally into C57BL/6 mice and seven days later these served as the inoculum to infect mice. C57BL/6 mice bred in the animal facility (CEDEME), at Universidade Federal de São Paulo, were used throughout. All procedures and experiments conformed to the regulations of the institutional Ethical Committee for animal experimentation. In all assays, mice were infected with P. berghei by inoculating intraperitoneally 106 parasitized erythrocytes. Parasitaemia was monitored by reading Giemsa-stained blood smears. The T. cruzi G strain, isolated from an opossum in the Brazilian Amazon  was maintained cyclically in Swiss mice and in liver infusion tryptose medium. Infective metacyclic trypomastigotes from cultures at the stationary growth phase were purified by passage through DEAE-cellulose column, as described earlier . For standard co-infection experiments, C57BL/6 mice were inoculated intraperitoneally with 106 T. cruzi metacyclic forms, and 15 days later they received 106 erythrocytes parasitized with P. berghei ANKA. The T. cruzi parasitaemia was checked by counting the parasites in 5 μl fresh blood collected from the mouse tail, under phase-contrast microscope.
Injection of Evans blue into mice and removal of the brain
The dye Evans blue was prepared as a 1% solution in PBS and each mouse received 0,2 ml through intraorbital route. When P. berghei-infected mice showed signs of neurological disorder, such as deviation of the head, convulsion and paralysis, they were given the dye and one hour later the brain was removed. Each time a P. berghei-infected mouse received the dye, a co-infected mouse and a mouse infected only with T. cruzi, were injected with the dye and their brain collected one hour later. The brains were stored at 4°C in a petri dish with PBS, and at the end of the experiment their images were recorded with a digital camera.
Preparation of histological sections of mouse lung
Experiments in which P. berghei-infected mice presented respiratory disorders, right after death their lungs were collected and fixed in 10% neutral buffered formalin for 24 h. Afterwards, the organs were gradually dehydrated in ethanol solution at different concentrations, followed by immersion in xylol, and then embedded in paraffin. Serial sections, 5 μm thick, were prepared and stained with hematoxylin-eosin. Equivalent numbers of symptomless mice, co-infected with T. cruzi, were also killed and their organs processed as above.
Treatment of mice with anti-CD8 antibodies
Mice infected with P. berghei ANKA only, or co-infected with T. cruzi, were treated with anti-CD8 antibodies (0.3 ml/mouse of ascitic fluid from clone TIB 21O by intraperitoneal injection), on days 0 and 5 of malaria infection.
Results and Discussion
Co-infection with T. cruzi protects mice from early death by P. berghei ANKA
Parasitaemia of P. berghei reached ~16% at the peak in mice with single infection, and around 20–30% in doubly infected animals (Figure 1B). On the other hand, T. cruzi blood forms, which are not detectable in a single infection unless the animals are immunosuppressed by irradiation or by treatment with cyclophosphamide, could be visualized in most animals co-infected with P. berghei (Figure 1C). This finding is compatible with the immunosuppressive effect induced by malaria infection [15–17]. Although rendered positive by co-infection, T. cruzi parasitaemia levels were very low as compared to those of virulent strains .
Symptoms of cerebral malaria are absent and breakdown of brain blood barrier is less pronounced in mice co-infected with T. cruzi
One of the symptoms of C57BL/6 mice with P. berghei ANKA is the neurological disorder leading to paralysis, and ultimately to coma and death, characteristic of cerebral malaria. In the experiment described in Figure 1, the majority of mice infected with P. berghei presented signs of neurological damage before dying. In repeat experiments, such abnormality was not seen in mice co-infected with T. cruzi.
Co-infection with T. cruzi protects mice from extensive pulmonary edema induced by malaria parasites
Protection to severe malaria and early death conferred by co-infection with T. cruzi is similar to that observed in P. berghei ANKA-infected mice treated with anti-CD8 antibodies
The mechanism by which co-infection with T. cruzi confers protection against severe malaria is unknown. An interesting possibility is that in mixed infection the sequestration of CD8+ T cells in organs or tissues that are targets of T. cruzi, such as the heart and skeletal muscles, results in reduced availability of these cell types for localization in the brain. That possibility exists. Predominance of CD8+ T lymphocytes in inflammatory cardiac and skeletal muscles has been observed in mice with acute T. cruzi infection . In that study, CD8+ T cells (47.0–58.9%) significantly outnumbered CD4+ cells (9.3–18.6%). As regards the pulmonary oedema in severe malaria infection, it still remains to be determined what induces it. If CD8+ T cells are also involved, the preferential recruitment of these cells to organs that are targets of T. cruzi infection, which do not include lungs, might prevent the accumulation of CD8+ T cells in sufficient numbers to produce more severe pulmonary alterations.
This study shows for the first time the modulatory effect of co-infection with T. cruzi on malaria infection that otherwise leads to an early death by cerebral malaria or extensive pulmonary oedema. In most regions where malaria is endemic, mixed infections are frequent. Therefore, the varied outcomes of malaria infection may result, at least in part, from positive or negative regulatory effects of other pathogens harbored by the patients.
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasil. Cells producing anti-CD8+antibodies were provided by Dr. Mauricio Martins Rodrigues, Universidade Federal de São Paulo.
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