Primary infection with Cystoisospora suis modulates systemic immunity and the gut microbiota during secondary infection in piglets.

Cystoisospora suis is a highly pathogenic porcine coccidian species that causes severe enteric disease in neonatal piglets. This study investigated how primary infection with C. suis modulates the gut microbiota and systemic immunity and shapes the host's response to a secondary challenge. Piglets were assigned to four groups: uninfected control (NC), early infection only (EI; 500 oocysts at 5 days), late infection only (LI; 5000 oocysts at 19 days), and early + late infection (EL; 500 oocysts at 5 days + 5000 oocysts at 19 days). Compared to the LI group, the EL group exhibited a markedly attenuated infection phenotype, demonstrating a 98.79% reduction in fecal oocyst shedding. At 8 days post-challenge, 16S rRNA sequencing and transcriptomic analyses were performed on intestinal segments and peripheral blood mononuclear cells (PBMCs). RNA sequencing revealed significant upregulation of immune-related genes in PBMCs of the LI group, including IFNG, TNF, STAT1, CCL2, CXCL10, GBP1, and GBP2, associated with inflammatory and antimicrobial responses. This response was markedly attenuated in the EL group. 16S rRNA analysis showed higher microbial alpha diversity in the cecum than in the jejunum and ileum. Post-challenge, the LI group displayed reduced ileal and cecal diversity (particularly ileum) and increased Escherichia-Shigella abundance. In contrast, the EL group maintained elevated proportions of taxa such as Lactobacillus and Clostridium_sensu_stricto_1, indicating that primary infection mitigates challenge-induced dysbiosis. Spearman correlation analysis revealed significant microbiota-immune associations. The relative abundance of genera such as Turicibacter and Clostridium_sensu_stricto_1 was negatively correlated with pro-inflammatory genes (e.g., CCL2, CCL4). In contrast, the Escherichia-Shigella was positively correlated with immune effector genes (e.g., GBP1, XCL1), reflecting an active host defense response. These findings demonstrate that primary C. suis infection modulates the host's response to secondary infection by reshaping systemic immunity and the gut microbiota, providing mechanistic insights that could inform future anticoccidial strategies in swine.