A total infectome framework for resolving complex disease etiology in aquaculture.

Traditional pathogen isolation can establish disease causality but often fails in complex infections involving unknown or multiple microbial backgrounds, whereas metatranscriptomics comprehensively captures the total infectome without directly resolving the primary etiological agent. Here, using overwintering syndrome (OWS) in grass carp, a widespread but unresolved aquaculture disease in China, as a disease model, we developed an integrated strategy that links comparative total infectome analysis with targeted isolation, experimental infection, and post-challenge infectome validation. Multi-organ metatranscriptomic profiling of healthy and diseased grass carp identified 107 microorganisms spanning viruses, bacteria, and eukaryotic microbes. Comparative infectomics prioritized Flavobacterium psychrophilum through its consistent disease enrichment, lesion-site abundance, multi-organ dissemination, and recurrence across affected hosts. Infection with F. psychrophilum alone reproduced the clinical signs, tissue pathology, mortality patterns, and infectome signatures of natural OWS, establishing it as the primary etiological agent. Temperature-shift experiments further showed that disease progression is promoted by warming from low temperature conditions, explaining the seasonal emergence of OWS. This work resolves a major grass carp disease and provides a generalizable framework for converting unbiased pathogen discovery into causal inference in complex infectious disease systems.