Variation analysis of pathogenic Vibrio spp. and Pseudomonas spp. in Changhai mollusc farming waters using real-time PCR assay during 2011–2014

ABSTRACT

Bacterial disease has caused high mortality of breeding molluscs from 2009 to 2011 in the Changhai area (Dalian, China). Vibrio spp. and Pseudomonas spp. have been detected as major pathogenic agents for aquatic animals in this area. In the present study, four virulence genes including vsm, toxR, aprX and carA were targeted to develop a real-time PCR assay for the quantitative detection of Vibrio splendidus, V. parahaemolyticus, Pseudomonas fluorescens and P. putida, respectively. The sensitivity and specificity of the assays were verified by experimental samples, and the variation tendencies of pathogenic V. splendidus, V. parahaemolyticus, P. fluorescens and P. putida strains from June to September were also detected in mollusc farming waters during 2011–2014. The concentration of V. splendidus increased from June to July, reduced in August, and then increased again in September. The highest count of V. parahaemolyticus appeared in July, and then dramatically decreased from August to September. Conversely, the counts of P. fluorescens and P. putida remained at lower levels from June to August, and then dramatically peaked in September. All four pathogenic bacteria displayed similar fluctuation tendencies of count variation in each year, and their concentrations were found to have a correlation with the average annual temperature. The variation tendency of pathogenic bacteria with temperature suggested that temperature was one of the most important factors to regulate the bacterial growth in a farming area, which could further provide information for the early warning of disease outbreak by using a convenient real-time PCR assay.

RESPONSIBLE EDITOR:

• Hongyue Dang

KEYWORDS:

• Vibrio splendidus
• Vibrio parahaemolyticus
• Pseudomonas fluorescens
• Pseudomonas putida
• pathogenic bacteria
• variation tendency
• real-time PCR

Acknowledgements

The authors are grateful to all the laboratory members for their technical advice and helpful discussion.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the earmarked fund for Modern Agro-industry Technology Research System, CARS-48, and the fund from the National & Local Joint Engineering Laboratory of Ecological Mariculture.