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Highly sensitive detection and quantification of the pathogen Yersinia ruckeri in fish tissues by using real-time PCR

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Abstract

Yersinia ruckeri is the causative agent of enteric redmouth diseases (ERM) and one of the major bacterial pathogens causing losses in salmonid aquaculture. Since recent ERM vaccine breakdowns have been described mostly attributed to emergence of Y. ruckeri biotype 2 strains, rapid, reproducible, and sensitive methods for detection are needed. In this study, a real-time polymerase chain reaction (PCR) primer/probe set based on recombination protein A (recA) gene was designed and optimized to improve the detection of Y. ruckeri. The primer/probe set proved to have a 100 % analytical specificity and a sensitivity of 1.8 ag μl−1, equivalent to 1.7 colony-forming units (CFU) ml−1, for purified DNA, 3.4 CFU g−1 for seeded liver, kidney, and spleen tissues, and 0.34 CFU/100 μl−1 for seeded blood, respectively. The assay was highly reproducible with low variation coefficient values for intra- and inter-run experiments (2.9 % and 9.5 %, respectively). Following optimization, the assay was used to detect changes in the bacterial load during experimental infection. Rainbow trout (Onchorhynchus mykiss) were exposed to two strains of Y. ruckeri (biotype 1 and biotype 2) by intraperitoneal inoculation. Internal organs (liver, kidney, spleen) and blood were biopsied from dead fish daily for 15 days to quantify copies of pathogen DNA per gram of tissue. The findings showed the efficacy of this real-time PCR assay to quantify Y. ruckeri cells in the fish tissues and also confirmed this assay as a non-lethal method for the detection of this pathogen in blood samples.

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Acknowledgments

This research was supported in part by Grant AGL2010-18438 for the Ministerio de Ciencia e Innovación (MICINN) Spain. A.B. acknowledges the FONACIT (Venezuela) for research fellowship.

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Authors and Affiliations

  1. Departamento de Microbiología y Parasitología, CIBUS, Universidad de Santiago de Compostela, Santiago de Compostela, 15782, Spain

    Asmine Bastardo & Jesús L. Romalde

  2. Estación de Investigaciones Hidrobiológicas de Guayana, Fundación La Salle de Ciencias Naturales, Guayana, 8051, Venezuela

    Asmine Bastardo & Carmen Ravelo

Authors
  1. Asmine Bastardo
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  2. Carmen Ravelo
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  3. Jesús L. Romalde
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Correspondence to Jesús L. Romalde.

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Bastardo, A., Ravelo, C. & Romalde, J.L. Highly sensitive detection and quantification of the pathogen Yersinia ruckeri in fish tissues by using real-time PCR. Appl Microbiol Biotechnol 96, 511–520 (2012). https://doi.org/10.1007/s00253-012-4328-1

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  • DOI: https://doi.org/10.1007/s00253-012-4328-1

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