Background and Aim: Concurrent infections in aquaculture species often exacerbate disease severity and complicate diagnosis and control strategies. This study investigated the pathological and molecular characteristics of co-infection with piscine intestinal coccidia and infectious spleen and kidney necrosis virus (ISKNV) associated with mass mortality in juvenile Lates calcarifer. Materials and Methods: A total of 20 moribund juvenile L. calcarifer collected during a mortality outbreak were subjected to necropsy, histopathology, and molecular analyses. Histological examination was performed using hematoxylin and eosin staining. ISKNV infection was confirmed by polymerase chain reaction targeting the major capsid protein (MCP) gene, followed by sequencing and phylogenetic analysis. Scanning electron microscopy (SEM) was used to characterize the morphology and ultrastructure of intestinal coccidial oocysts. Results: All examined fish (100%) were concurrently infected with piscine intestinal coccidia and ISKNV, indicating a strong association with the observed outbreak. Clinically, fish exhibited emaciation, discoloration, and visceral congestion. Histopathology revealed systemic ISKNV infection characterized by basophilic hypertrophic (megalocytic) cells in multiple organs, while coccidia were localized within the intestinal epithelium at epicellular, intracellular, and subepithelial positions. Severe gastrointestinal lesions, including inflammation, epithelial desquamation, and necrosis, were markedly intensified in co-infected tissues. SEM demonstrated rough-surfaced, oval oocysts consistent with piscine intestinal coccidia. Molecular analysis confirmed the presence of ISKNV, with MCP gene sequences showing 100% identity with regional and global isolates and clustering within the genotype I lineage. Phylogenetic findings further supported the close evolutionary relationship with strains from Southeast Asia and other regions. Conclusion: This study provides the first comprehensive evidence of concurrent infection with piscine intestinal coccidia and ISKNV as a significant cause of mass mortality in juvenile L. calcarifer. The synergistic pathological effects observed in the gastrointestinal tract highlight the importance of considering polymicrobial infections in disease investigations. Strengthened surveillance, improved biosecurity, and integrated disease management strategies are essential to mitigate the impact of such emerging co-infections in aquaculture systems. Keywords: aquaculture pathology, co-infection, infectious spleen and kidney necrosis virus, intestinal coccidia, juvenile fish mortality, Lates calcarifer, megalocytivirus, piscine parasitology.
Suyapoh et al. (Tue,) studied this question.