Genomic characterization of isolated edwardsiella ictaluri from cage-cultured Pangasianodon hypophthalmus in Pahang river

Abstract

Edwardsiella ictaluri is the etiological agent of Bacillary Necrosis of Pangasius (BNP) in Pangasianodon hypophthalmus, causing substantial economic losses in freshwater aquaculture. Infected fish commonly exhibit clinical signs such as erratic swimming behaviour, patchy liver, and congested kidney. Despite its significance, the genomic characteristics, population structure, and epidemiological context of E. ictaluri in Malaysia, particularly in major production areas such as Temerloh and Pekan, remain limited. This knowledge gap hinders effective disease surveillance and control strategies in local aquaculture systems. Therefore, this study aimed to investigate the genomic characteristics, antimicrobial resistance determinants, and population-level genetic variation of E. ictaluri isolates from Temerloh and Pekan using whole-genome sequencing (WGS) and polymorphism-based approaches. Eleven E. ictaluri isolates were recovered from the tissues of clinically diseased P. hypophthalmus cultured in riverine cage systems in these areas. As all isolates originated from the same water body, one representative isolate, PLS31B, obtained from the brain of an infected fish, was selected for whole-genome sequencing (WGS) using Oxford Nanopore long-read technology. The assembled genome was 3.8 Mbp in size, with a high-contiguity N50 of 3,761,778 bp and a GC content of 57.39%. Antimicrobial resistance (AMR) analysis revealed resistance to fluoroquinolones, macrolides, and penams, highlighting potential challenges for disease management in aquaculture. Functional annotation identified 3,620 protein-coding genes and 126 RNA genes. Comparative genomic analysis indicated that PLS31B is closely related to E. ictaluri strains from China and Vietnam, suggesting potential regional connectivity or transboundary dissemination associated with the importation of fish seed. A large conserved core genome comprising of 3,246 genes was observed alongside of 51 strain-specific genes linked to those encoding outer membrane protein C precursor (OMP), ATP-binding protein, acid shock protein, and hemolysin secretion protein. Meanwhile, genetic polymorphism among all isolates was further assessed using Random Amplified Polymorphic DNA (RAPD) profiling and Sanger sequencing. Seven 10-mer primers were used for RAPD analysis, while the 23S rRNA gene region was sequenced for comparative analysis. Polymorphism analysis revealed minimal genetic differentiation among the isolates, irrespective of sampling locations or host age groups. This low genetic diversity is likely influenced by a shared aquatic environment and interconnected farming systems. In conclusion, these findings highlight the epidemiological relevance of regional dissemination pathways and guide the development of targeted control strategies, including early detection tools, preventive measures, and effective treatment approaches for BNP in P. hypophthalmus aquaculture.