Isolation, identification, and antimicrobial susceptibility test of the pathogen from largemouth bass (Micropterus salmoides) with white skin disease

Abstract:

[Background] In recent years, largemouth bass (Micropterus salmoides) has been prone to white skin disease during seed stage in Guangdong, Guangxi, and other places, with the symptoms obviously different from those reported clinically, which seriously endangers the production of largemouth bass seeds. [Objective] To provide scientific reference for the research and treatment of white skin disease of largemouth bass, we identified the pathogen and analyzed its growth characteristics, virulence factors, pathogenicity, and antibiotic susceptibility as well as the fish histopathological changes caused by it. [Methods] The pathogen was isolated from the focus of largemouth bass suffering from white skin disease and identified based on the morphological characteristics, physiological and biochemical indexes, and 16S rRNA and gyrB gene sequence analysis. Further, the growth curve of the strain was established, and the effects of temperature, pH, and salinity on the growth were analyzed. The activities of virulence factors were detected by plate method, and the virulence genes were screened out by PCR method. The pathogenicity of the strain and histopathological changes caused by it were confirmed by immersion challenge experiments. The susceptibility of strain to 10 antibiotics and 6 disinfectants commonly used in aquaculture was determined with microdilution method. [Results] The dominant strain ZJS18004 was isolated from the largemouth bass with white skin disease, which was identified as Aeromonas veronii. The optimum growth conditions of ZJS18004 were 30 °C, pH 8.0, and salinity 5‰. The growth curve at 30 °C showed that 0–1 h and 1–5 h were the retardation phase and logarithmic phase, respectively. Strain ZJS18004 caused hemolysis, had protease and lipase activities, and carried six virulence genes including aer, act, fla, ahyB, exu, and lip. The results of challenge experiments showed that ZJS18004 had strong pathogenicity to healthy largemouth bass at 23 °C, 28 °C, and 33 °C. The tested fish exhibited disease signs similar to those in natural infections. The pathogenicity was closely correlated with the bacterial concentration and water temperature. The infection of ZJS18004 caused obvious pathological damage such as muscle tissue lysis and necrosis of the liver, spleen, and kidney. The antimicrobial susceptibility assay revealed that ZJS18004 was susceptible to doxycycline hyclate, neomycin sulphate, thiamphenicol, and florfenicol. Six commonly used aquatic disinfectants had certain bactericidal effects on ZJS18004 in vitro, among which trichloroisocyanuric acid ranked first, followed by benzalkonium bromide. [Conclusion] A. veronii was the major pathogen of white skin disease attacking largemouth bass. The results of the present study will build a solid foundation for further analyzing the pathogenic mechanism of A. veronii and carrying out the research on accurate prevention and control.

Key words:Micropterus salmoides;Aeromonas veronii;growth characteristics;virulence gene;antibiotic susceptibility