Pathogen isolation, identification and pathomorphological observation of ulcerative syndrome of largemouth bass in winter and spring

Objective In recent years, outbreak of an epidemic disease with significant ulcers on the pond-farmed largemouth bass (Micropterus salmoides) happen in winter and spring. This study aims to identify the underlying etiology.

Method To clarify its etiology, disease fish samples from Dongpo and Hongya District, Meishan were collected and analyzed using pathogenesis, pathology and molecular biology methods.

Result A large number of long filamentous and less-branched mycelia with 10−30 μm diameter were observed in the ulcerated tissues. Pathologically, we observed necrosis or lysed muscle fibers, inflammatory cell infiltration in the muscle tissue, and distribution of numerous fungal granulomas consisting of epithelioid cells, fibroblasts, and central located mycelia in the lesion tissues. By using periodic acid-silver metheramine (PASM) staining, the mycelia in the fungal granulomas center displayed black colour. We confirmed the infection of Aphanomyces invadans in the lesion muscle tissues by PCR using species-specific primers that target the ITS gene of A. invadans. The A. invadans (BW1 and BW3) pure cultures were isolated by glucose and peotone (GP) agar and then incubated in sterilized pond water. Prosporangium formation was observed in the culture after 12 h incubation period, which was consistent with the characteristics of Aphanomyces. Based on ITS gene sequencing of BW1 and BW3, their ITS genes were found to have 99.64% and 99.46% sequence similarities with A. invadans, and the two strains clustered with A. invadans in the phylogenetic tree.

Conclusion The ulcer disease of largemouth bass in winter and spring has been caused by A. invadans.