Survival in animals co-infected with T. gondii and T. musculi/T. lewisi is associated with splenomegaly
As shown in supplementary table 1, rats are high resistant to T. gondii infection, as all showed zero mortality. However, infection of T. lewisi may kill rats of SD, BN, LEW and WST strains, except F344. In generally, additional T. gondii infection may not change the mortality caused by T. lewisi, only Tl,Tg group of BN rats showed significant high mortality. Considering the parasitemia, T. lewisi could survive in the blood for more than 40 days in four strains of rats (BN, F344, SD, WST) (Supplementary figure 1), the LEW strain rats were so resistant to T. lewisi that no parasitemia was observed. In WST groups, all rats developed similar levels of parasitemia. In F344 and SD groups, lower parasitemia peaks of T. lewisi were found in those that were co-infected with T. gondii than in singly infection groups (Tl vs Tl,Tg/Tg,Tl: F344, p = 0.0079/0.0005; SD, p < 0.0001/0.0001), while BN missed many data due to animal deaths. Spleen indexes [spleen weight (mg)/body weight (g)] in rats on 60 dpi (Table 1) showes T. lewisi infection resulted significant splenomegaly, which T. gondii resulted splenomegaly F344 rats but reverse effect in other rats. In case of co-infection, all rats had higher spleen indexes than the T. gondii groups.
As shown in Table 2 and supplementary figure 2, T. musculi is nonlethal to mice with 100% survival. However, mice are highly susceptible to T. gondii infection (even with the avirulent Pru strain) and showed various levels of mortality, as C57BL/6, the most susceptible mice, survived only 15% (2/13) and BALB/c, the most resistant mice, all alive. In co-infected groups, there was generally a decreased survivability compared to both singly infected controls, but only significant for the “Tm,Tg” group of BALB/C mice (p = 0.025).
During the infection of T. musculi (30 days) only the parasitemia found in “Tm,Tg” group of BALB/C mice was significantly higher than the “Tm” group at their peaks between days 9 or 12 (p = 0.0418), while other groups were not (Supplementary figure 2). Importantly, most C57BL/6 mice died before showing high parasitemia, indicating the death may due to immune-storm of co-infection.
Spleen indexes in surviving mice on 60 dpi (Table 3) showes either T. gondii or T. musculi infection resulted significant splenomegaly, whereas co-infection generally enhanced the splenomegaly, compared with the T. gondii groups. Data for C57BL/6 mice were limited due to few was survived.
Toxoplasma gondii cyst burden in the brains of co-infected animals
We investigated the variance in susceptibility to T. gondii in mice and rats when co-infected with T. musculi (mice) and T. lewisi (rats). A surprising result was that mice of Swiss Webster and NIH improved their resistance, to a greater or lesser degree, to the T. gondii PRU strain when co-infected with T. musculi (Figure 1). Heavy cyst burdens were detected in the brains of mice infected with T. gondii only (Mean±SEM of group “Tg”: BALB/c, 355±71.81, n=13; NIH, 3629±626.9, n=10; Swiss Webster, 23198±3472, n=3), while lower numbers of brain cysts were detected in the brains of T. musculi pre-infected groups (Mean±SEM of group “Tm, Tg”: BALB/c, 206±96.36 , n=6; NIH, 1353±508.4, n=8; Swiss Webster, 124.3±15.67, n=3) with the difference being significant in the NIH (p = 0.015) and Swiss Webster strains (p < 0.001) (Figure 1). However, when T. musculi was coinfected with T. gondii at the same time (Tm+Tg) or subsequently (Tg, Tm) there was little influence on cyst burden, in the mice strains except in the case of Swiss Webster where a significant drop in cyst number occurred when T. gondii infected mice were post-infected with T. musculi. Cyst burden in C57BL/6 mice could not be determined due to high lethality (more than 85%) after infection with T. gondii.
Interestingly, in contrast to the mice, we found (Figure 2) that rat strains F344 and BN pre-infected with T. lewisi showed a greater susceptibility to T. gondii than the T. gondii singly infected rats (Mean±SEM of group “Tl, Tg”: F344, 2103±234.6, n=5; BN, 936.0±259.5, n=5; SD, 90.0±60.0, n=5; vs group “Tg”: F344, 216.0±47.6, n=5; BN, 0±0, n=5; SD, 0±0, n=5). However, there was no significant difference between the other coinfected groups (Tg, Tl i.e. post-infection with T. lewisi) and the T. gondii infected groups (Tg). In the case of the remaining rat strains, WST and LEW, these are very resistant to T. gondii infection and no Toxoplasma cysts were found at all (by microscopic examination and T. gondii-PCR) even when coinfected with T. lewisi (data not shown).
Variation in NO/urea production and iNOS/arginase expression in the peritoneal macrophages of mice or rats is related to the proliferation status of T. gondii
It has been previously demonstrated that there is competition for the substrate arginine between the enzymes iNOS and arginase . Therefore, we analyzed the level of NO/urea production and iNOS/arginase expression in peritoneal macrophages isolated from Sprague- Dawley rats (SD) and Swiss Webster mice which were pre-infected with T. lewisi and T. musculi, respectively. Increased amounts of NO production was found in primary cultured peritoneal macrophages taken from mice pre-infected with T. musculi (25.82±1.88 µM from T. musculi pre-infected mice after culture for 24 hrs compared with 4.44±2.80 µM from control mice at 24 hrs, p = 0.0004) (Figure 3A). In addition, a higher level of iNOS mRNA expression (reference gene, GADPH) was observed in peritoneal macrophages from mice pre-infected with T. musculi whereas iNOS mRNA expression could not be detected in control mouse peritoneal macrophages (Figure 3C). Western blot analysis (reference protein, β-actin) demonstrated a slightly higher expression of iNOS protein in T. musculi pre-infected mouse than normal mouse peritoneal macrophages. Measurement of arginase mRNA expression and urea production, in primary cultured peritoneal macrophages from mice, showed higher levels in the control group (0.34±0.04 mM from control mice after 24 hrs culture and 0.23±0.05 mM from T. musculi pre-infected mice at 24 hrs, p = 0.043) (Figure 3B). However, there is no distinct difference in arginase expression between control and T. musculi pre-infected mice by western blot analysis (Figure 3D). This suggest that the drop in urea production in the pretreated mice was probably due to competition, by increased iNOS expression. for the arginase/iNOS substrate arginine.
Unlike the effect of T. musculi on mouse, NO production was significantly lower in T. lewisi pre-infected rats (15.26±2.03 µM) than control rats (35.34±3.34 µM, p < 0.0001), and after only 12 hrs of culture this difference already existed (p < 0.0001) (Figure 4A). In addition, it can be seen that the expression of iNOS mRNA (reference gene, GADPH) and protein (reference protein, β-actin) were clearly decreased in SD rats after T. lewisi infection (Figure 4C and 4D). However, the urea production and mRNA/protein expression of arginase were significantly higher in T. lewisi pre-infected rats (Figure 4B, 4C and 4D).
It is known that NO is an important effector molecule involved in the resistance of rat/mouse peritoneal macrophages to T. gondii infection. In addition, it has been confirmed that a lower level of NO production was detected in the peritoneal macrophages of T. lewisi infected SD rats. Therefore, we expected that there would be a proliferation of T. gondii in macrophages from rats pre-infected with T. lewisi. Indeed, the proliferation of T. gondii tachyzoites (RH strain) dramatically increased in peritoneal macrophages from T. lewisi pre-infected SD rats (Figure 5) and this was associated with the observed lowered NO production. The reproductive rate of T. gondii tachyzoites is clearly inhibited in the peritoneal macrophages from the T. lewisi uninfected control SD rats.
In contrast in mice, it seems that the increase in NO production after infection with T. musculi confers no obvious resistance to proliferation of T. gondii tachyzoites in the peritoneal macrophages of mice (data not shown). However, in vivo, the infection of mice with T. musculi was observed to extend the survival time of hosts infected with T. gondii tachyzoites (RH strain). This was especially notable when the host was infected T. musculi before T. gondii (Supplementary figure 3).