The use of a heterogeneously controlled mouse population reveals a significant correlation of acute phase parasitemia with mortality in Chagas disease

Abstract

Chagas disease develops upon infection with the protozoan parasite Trypanosoma cruzi and undergoes an acute phase characterized by massive parasite replication and the presence of parasites in the blood. This condition is known as acute phase parasitemia. This initial stage may result in a cure, in the development of the chronic stages of the disease or in the death of the infected host. Despite intensive investigation related to the characterization of the acute and chronic phases of the disease, the cause-effect relationship of acute phase parasitemia to the outcome of the disease is still poorly understood. In this study, we artificially generated a heterogeneously controlled mouse population by intercrossing F1 mice obtained from a parental breeding of highly susceptible A/J with highly resistant C57BL/6 mouse strains. This F2 population was infected and used to assess the correlation of acute phase parasitemia with the longevity of the animals. We used nonparametric statistical analyses and found a significant association between parasitemia and mortality. If males and females were evaluated separately, we found that the former were more susceptible to death, although parasitemia was similar in males and females. In females, we found a strong negative correlation between parasitemia and longevity. In males, however, additional factors independent of parasitemia may favor mouse mortality during the development of the disease. The correlations of acute phase parasitemia with mortality reported in this study may facilitate an appropriate prognostic approach to the disease in humans. Moreover, these results illustrate the complexity of the mammalian genetic traits that regulate host resistance during Chagas disease.

Figure 1. An artificially generated F2 offspring of an F1(C57BL/6× A/J) intercross is heterogeneous for mortality and parasitemia in response to infection with the Y strain of T. cruzi.

Mice of parental resistant C57BL/6 and susceptible A/J strains and a F2 population composed of 67 individuals were infected i.p. with 1000 trypomastigotes. (A) Mortality of C57BL/6 (n = 10) and A/J (n = 10) mice was evaluated by daily inspection of cages. (B) Parasitemia of C57BL/6 and A/J mice was quantified microscopically by counting the parasites per ml of blood obtained from the tail vein at 7, 9, 11 and 13 days of infection. (C) Mortality of 67 F2 mice was evaluated by daily inspection of cages. (D) Parasitemia of 67 F2 mice was quantified microscopically by counting the parasites per ml of blood obtained from the tail vein at 7, 9, 11 and 13 days of infection. Each symbol in “D” represents an individual mouse, lines in “D” represent mean and bars in “B and D” represent standard deviation of the mean.

Figure 2. Distinct quantitative parameters can be used to assess parasitemia of infected F2 mice.

Parasitemia values (7, 9, 11 and 13 days after infection) of the 67 F2 mice infected with the Y strain of T. cruzi (same mice shown in Fig. 1D) were used to generate distinct parameters to assess parasitemia. (A) Correlation of the average number of parasites per ml of blood at days 7, 9, 11 and 13 of infection (Average) with the standard deviation of the parasitemia mean at days 7, 9, 11 and 13 of infection (Standard deviation). (B) Correlation of the Average with the value of parasitemia obtained at the day of the parasitemia peak (Peak value). (C) Correlation of the Standard deviation with the Peak value. Correlation was assessed with a Spearman test. A test of non-zero slope was applied, and the P value and r are shown. Correlation was considered significant if P<0.05.

Figure 3. Mortality of F2 mice infected with T. cruzi is associated with parasitemia.

Parasitemia of the 67 F2 mice infected with the Y strain of T. cruzi (same mice shown in Fig. 1D) was indicated for survivors (n = 20 F2 mice) and non-survivors (n = 47 F2 mice). The value of parasitemia obtained at the day of the parasitemia peak (Peak value; A), the average number of parasites per ml of blood at days 7, 9, 11 and 13 of infection (Average; B) and the standard deviation of the parasitemia mean at days 7, 9, 11 and 13 of infection (Standard deviation; C) was indicated for survivors and non-survivor F2 mice. Each symbol represents an individual mouse, lines and bars represent mean and standard deviation of the mean. Association was considered significant if P<0.05 (Mann-Whitney test).

Figure 4. The longevity of non-survivors is associated with parasitemia.

Parasitemia of the 67 F2 mice infected with the Y strain of T. cruzi (same mice shown in Fig. 1D) was categorized according to the day of death: 15–19 (n = 19 mice), 20–24 (n = 14 mice) and 25–29 (n = 14 mice) days after infection. The survivors are also indicated (n = 20). The value of parasitemia obtained at the day of the parasitemia peak (Peak value; A), the average number of parasites per ml of blood at days 7, 9, 11 and 13 of infection (Average; B), and the standard deviation of the parasitemia mean at days 7, 9, 11 and 13 of infection (Standard deviation; C) was indicated for the survivors and categorized non-survivors in the F2 mice. Each symbol represents an individual mouse, lines and bars represent mean and standard deviation of the mean. Association was considered significant if P<0.05 based on a Kruskal-Wallis test and a Dunn post-test.

Figure 5. Survival but not parasitemia of F2 mice is a trait influenced by sex.

(A) Survival curve of females (n = 32) and males (n = 35) of the 67 F2 mice infected with the Y strain of T. cruzi (same mice shown in Fig. 1D). (B–D) Parasitemia of the 67 F2 mice infected with the Y strain of T. cruzi (same mice shown in Fig. 1D) was associated with the survival status of individuals categorized by gender. The value of parasitemia obtained at the day of the parasitemia peak (Peak value; B), the average number of parasites per ml of blood at days 7, 9, 11 and 13 of infection (Average; C) and the standard deviation of the parasitemia mean at days 7, 9, 11 and 13 of infection (Standard deviation; D) was indicated by gender for survivors and non-survivor F2 mice. Each symbol represents an individual mouse, lines and bars represent mean and standard deviation of the mean. Association was considered significant if P<0.05. Mantel-Cox test (A) and Mann-Whitney test (B–D).

Figure 6. Parasitemia is inversely correlated with longevity in female but not in male mice of the F2 population.

Parasitemia of the 47 F2 mice that succumbed to infection with the Y strain of T. cruzi (same shown in Fig. 1D) was correlated with longevity according to the sex of the mice. The value of parasitemia obtained at the day of the parasitemia peak (Peak value; A, D), the average number of parasites per ml of blood at days 7, 9, 11 and 13 of infection (Average; B, E), and the standard deviation of the parasitemia mean at days 7, 9, 11 and 13 of infection (Standard deviation; C, F) was correlated with the day of death of the non-survivor F2 mice. The females (n = 19, A–C) and males (n = 28; D–F) are indicated. Correlation was assessed with a Spearman test. A test of non-zero slope was applied, and the P value and r are shown. Correlation was considered significant if P<0.05.