关键词: 约氏疟原虫, 伯氏疟原虫, 肝期, 红内期, 感染

Abstract:

Objective To analyze the infection specie-specificity of Plasmodium yoelii (P.y) BY265 strain and P. berghei (P.b) ANKA-luciferase strain in rats and mice. Methods The sporozoites and merozoites of P.y and P.b were prepared. Infection was performed by injecting 1 × 10⁵ P.y and P.b sporozoites to SD rats and BALB/c mice (P.y infection, 5 SD rats/group, 5 BALB/c mice/group) and SD rats, C57BL/6J mice (P.b infection, 5 SD rats/group, 5 C57BL/6J mice/group) through tail vein, respectively. The tail vein blood samples were collected daily to prepare blood mears for microscopic examination to record the appearance time of erythrosytic stage of Plasmodium. Liver tissue samples were collected at 42 h post‑infection from the SD rats and BALB/c mice of P.y infection group to examine the relative expression level of Plasmodium 18S rRNA by real‑time quantitative PCR (qRT‑PCR), and measure the liver fluorescence signal in SD rats and C57BL/6J mice in P.b infection groups using IVIS Lumina imaging system. A dose of 1 × 10⁸, 1 × 10⁷, 1 × 10⁶ infected red blood cells （iRBCs） of P.y and P.b was used to infect SD rats (P.y infection group and P.b infection group) and susceptible mice (BALB/c and C57BL/6J) (positive control group), the tail vein blood was collected daily for smear to exam the parasitemia. The SD rats and susceptible mice were infected with P.y and P.b merozoites, respectively. The tail vein blood was collected daily for microscopic examination to calculate the parasitemia. When the parasitemia in rats and mice reached its peak, the tail vein blood smears were prepared every 8 hours for 24 hours for microscopic examination, and the proportions of rings, trophozoites, and schizonts were calculated to analyze the developmental rhythm of the rodent Plasmodium. The experimental cerebral malaria (ECM) in rats and mice were recorded. Rag2-KO SD rats, wild‑type SD rats, and susceptible mice were infected with P.y and P.b merozoites respectively, the tail vein blood smears were examined daily to calculate parasitemia. The comparison between the two groups was performed by unpaired t‑test, and the comparison between different groups of parasitemia performed by two‑way analysis of variance (two‑way ANOVA). Results All Plasmodium in the SD rats, BALB/c mice and C57BL/6J mice infected with P.y and P.b sporozoites completed the liver stage development and entered the erythrocytic stage on day 3. The qRT-PCR results showed that the relative expression levels of Plasmodium-specific 18S rRNA in SD rats and BALB/c mice infected with P.y sporozoites were (1.63 ± 0.381) and (1.00 ± 0.232) with statistically significant difference (t = 2.801, P < 0.05). The results of IVIS imaging showed that the total fluorescence intensity of SD rats infected with P.b sporozoites was (6.243 ± 1.425) × 10⁷, which was significantly higher than that of C57BL/6J mice [(1.624 ± 0.530) × 10⁷] (t = 6.077, P < 0.01). The results of erythrocyte stage iRBCs infection showed that in P.y infected groups, the peaks of parasitemia of SD rats infected with three doses were (3.500 ± 1.042)%, (2.850 ± 0.627)%, (3.400 ± 0.962)%, there was no significant difference in the trends of parasitemia among them (F = 0.145, P > 0.05), but there were significant differences in the trends of parasitemia between each group of rats and the positive control [the peak of parasitemia was (43.928 ± 9.448) %)](F = 84.040, 63.760, 58.400; all P < 0.01). In P.b infected groups, the peaks of parasitemia of SD rats were (11.468 ± 1.362)%, (7.398 ± 2.387)%, (2.984 ± 1.881)% which decreased along with infection dose decent, and the trends of parasitemia in the 1 × 10⁸ and 1 × 10⁶ dose infected rat groups was significantly different from that in the positive control (F = 13.83, 17.320; all P < 0.01), while the difference in trends of parasitemia between 1 × 10⁸ dose infected rat group and the positive control group was not significant (F = 2.234, P > 0.05). The results of merozoite infection showed that 6 days after infection, the parasitemia of SD rats and BALB/c mice in P.y infected groups were (0.902 ± 0.235)% and (17.420 ± 4.105)% respectively, which were significant different (t = 9.943, P < 0.01). The parasitemia of SD rats and C57BL/6J mice in P.b infected groups were (6.804 ± 2.978)%, (9.290 ± 1.055)% respectively, and there was no significant difference between the two groups (t = 1.759, P > 0.05). The cumulative incidences of ECM in SD rats and C57BL/6J mice in P.b infected groups were 11/15 and 13/15 respectively, with no statistically significant difference (t = 1.414, P > 0.05). The developmental rhythm analysis results showed that the developmental rhythm of SD rats was different from that of BALB/c mice, and did not exhibit a 24-hour pattern in P.y infected groups. While in P.b infected groups, the developmental rhythm of SD rats was similar to that of C57BL/6J mice, with a 24-hour rhythm. 18 days post-infection with P.y merozoites, the parasitemia in Rag2-KO SD rats, WT SD rats, and BALB/c mice with were (1.326 ± 0.908)%, 0, and (33.937 ± 3.453)% respectively, there was a significant difference of parasitemia between Rag2-KO SD rats and WT SD rats (t = 2.267, P < 0.05). Seventeen days post-infection with P.b merozoites, the parasitemia in Rag2-KO SD rats and WT SD rats were (19.685 ± 15.752)%, (0.007 ± 0.013)% (t = 2.499, P < 0.05), and parasitemia of the remaining one C57BL/6J mouse in the positive control group was 25.410%. Conclusion Sporozoites of P.y and P.b can infect rats, complete their liver stage development and enter the erythrocytic stage. The influencing factor leading to the rats resistant to P.y and P.b infection occurs at the erythrocytic stage, when the rodent Plasmodium in the rats could be cleared. P.y exhibits stronger selectivity for host species. There were no significant differences between rats and mice in P.b parasitemia at acute pahase and the occurring rate of ECM. Adaptive immunity plays an important role in complete clearance of rodent plasmodium in rats.

Key words: Plasmodium yoelii, Plasmodium berghei, Liver stage, Erythrocytic stage, Infection