Exogenous nitric oxide stimulates early egress of Eimeria tenella sporozoites from primary chicken kidney cells in vitro

Abstract

Egress plays a vital role in the life cycle of apicomplexan parasites including Eimeria tenella, which has been attracting attention from various research groups. Many recent studies have focused on early egress induced by immune molecules to develop a new method of apicomplexan parasite elimination. In this study, we investigated whether nitric oxide (NO), an immune molecule produced by different types of cells in response to cytokine stimulation, could induce early egress of eimerian sporozoites in vitro. Eimeria tenella sporozoites were extracted and cultured in primary chicken kidney cells. The number of sporozoites egressed from infected cells was analyzed by flow cytometry after treatment with NO released by sodium nitroferricyanide (II) dihydrate. The results showed that exogenous NO stimulated the rapid egress of E. tenella sporozoites from primary chicken kidney cells before replication of the parasite. We also found that egress was dependent on intra-parasitic calcium ion (Ca²⁺) levels and no damage occurred to host cells after egress. The virulence of egressed sporozoites was significantly lower than that of fresh sporozoites. The results of this study contribute to a novel field examining the interactions between apicomplexan parasites and their host cells, as well as that of the clearance of intracellular pathogens by the host immune system.

Keywords: Egress; Eimeria tenella sporozoites; Nitric oxide.

Figure 1

Exogenous nitrous oxide (NO) stimulates egress of Eimeria tenella sporozoites. (A) NO concentrations released by different doses of sodium nitroferricyanide (II) dihydrate (SNP) for a variety of durations. (B) Primary chicken kidney cells (PCKs) were infected with sporozoites and incubated with different concentrations of SNP for different times. The number of free sporozoites was analyzed by flow cytometry. Data are means ± standard error of the mean (SEM) of four replicates, representing three independent experiments. **p < 0.01, comparing SNP with DMEM at the respective time point. (C) Video was captured from 20 to 25 min after SNP treatment; eight representative frames were selected and shown. Arrows indicate the egress process in two sporozoites. Bar: 20 μm.

Figure 2

Parasite development is essential for NO-induced egress. PCKs were infected with sporozoites for 12, 24, or 36 h and then incubated with 40 mM SNP for 30 min. The number of free sporozoites was analyzed by flow cytometry. Data are means ± SEM of six replicates, representing three independent experiments. *p < 0.05; **p < 0.01.

Figure 3

NO-induced egress is required for intra-parasitic Ca ²⁺. Sporozoite-infected PCKs were pre-treated with BAPTA-AM (A) or U-73122 (B) for 30 min and then incubated with 40 mM SNP for 30 min. Free parasites were analyzed by flow cytometry. Data are means ± SEM of six replicates, representing three independent experiments. **p < 0.01.

Figure 4

NO-induced egress of sporozoites is dependent on parasitic mobility. (A) Sporozoite-infected PCKs were pre-treated with 10 μM Cyto-D and then incubated with 40 mM SNP for 30 min. Free sporozoites were analyzed by flow cytometry. Data are means ± SEM of four replicates, representing three independent experiments. **p < 0.01. (B) and (C) Images captured after SNP treatment and pre-incubation with 10 μM Cyto-D. Bar: 20 μm.

Figure 5

NO-induced egress results in no host cell damage. (A) and (B) Propidium iodide (PI) staining of PCKs in flow cytometry, representing percentages of PI⁺ cells after parasite invasion, SNP treatment, or SNP induced egress, no treatment as control. Representative data from one and three independent experiments are shown. Bars indicate means ± SEM (n = 5). **p < 0.01.

Figure 6

Decreased virulence in egressed parasites. Free parasites were collected after egress and counted using blood cell counting plates. (A) Egressed parasites were added to freshly prepared PCKs and incubated for 12 h; free sporozoites were suspended in 500 μL DMEM, and 10 μL suspensions were analyzed by flow cytometry after incubation. Data are means ± SEM of five replicates, representing three independent experiments. **p < 0.01. (B) Six birds were infected with egressed parasites (2 × 10⁴) via the cloacal route. Oocyst output per bird was quantified using a MacMaster chamber at 6–9 days after infection. Bars indicate means ± SEM. **p < 0.01.