Serial passage is associated with virulence attenuation in Neospora caninum and transcriptomic remodeling of invasion- and chronic-stage-associated pathways.

N. caninum is an obligate intracellular apicomplexan parasite and a major cause of abortion in cattle worldwide. However, the molecular basis underlying virulence attenuation during long-term in vitro passage remains poorly understood. Here, we performed comparative transcriptomic profiling between the virulent Nc1-09 strain and its attenuated derivative Nc1 to investigate transcriptional changes associated with virulence reduction. A total of 487 differentially expressed genes (DEGs) were identified. Functional enrichment analyses revealed prominent changes in pathways related to protein modification, host-parasite interaction, motility, and metabolism. Notably, multiple members of the surface antigen glycoprotein (SRS) family displayed marked transcriptional shifts, consistent with altered host cell interaction and immune-related functions. CRISPR/Cas9-mediated disruption of three representative DEGs (NCLIV_016320, NCLIV_024950, and NCLIV_022830) did not reproduce the attenuated phenotype, suggesting that disruption of these individual genes is insufficient to account for the full attenuated phenotype under the conditions tested. Gene set enrichment analysis further demonstrated systematic downregulation of protein modification-associated pathways in the attenuated strain. Overall, the data indicate that virulence attenuation in Neospora caninum （N. caninum） is associated with coordinated transcriptional remodeling across multiple functional pathways rather than discrete gene loss.