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Evaluation of quantitative real-time PCR for rapid assessments of the exposure of sentinel fish to Myxobolus cerebralis

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Abstract

Pathogen-free rainbow trout (Oncorhynchus mykiss) aged 735 degree days were experimentally exposed to a low dose of infectious Myxobolus cerebralis (20 triactinomyxons fish−1). Three time periods were chosen for sampling that included 10 days (d), 67 d, and 5 months (mo) post exposure. Five diagnostic assays were used: (1) conventional single-round polymerase chain reaction (PCR), (2) nested PCR, (3) real-time TaqMan PCR, (4) pepsin–trypsin digest, and (5) histopathology. M. cerebralis was detected among individual rainbow trout by all of the PCR diagnostic tests employed at each of the three sampling time points. This result demonstrates that any of these three diagnostic approaches are capable of detecting the parasite from infected fish tissues under the conditions tested. Real-time PCR provided good biological evidence that parasite replication increases temporally as shown by quantification values that were significantly different (P<0.0001) at 10 d as compared to 67 d and 5 mo postexposure. Although sampling at 10 d by real-time PCR may be too early to accurately predict quantities of the parasite that will be present at 5 mo, it does forecast the proportions of fish that are likely to be infected at 67 d and 5 mo postparasite exposure. Real-time PCR could potentially be used as a quantitative diagnostic PCR tool to predict parasite load and outcome of M. cerebralis infection.

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Acknowledgements

This work was supported by the Montana Water Center, the US Fish and Wildlife Service, the Whirling Disease Foundation, and the California Department of Fish and Game. We also are grateful to K. Mukkatira and T. Waltzek for collecting parasite spores and maintaining oligochaete cultures. We also appreciate the assistance of the Lucy Whittier Molecular and Diagnostic Core Facility for the technical support.

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Correspondence to Garry O. Kelley.

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Kelley, G.O., Adkison, M.A., Zagmutt-Vergara, F.J. et al. Evaluation of quantitative real-time PCR for rapid assessments of the exposure of sentinel fish to Myxobolus cerebralis . Parasitol Res 99, 328–335 (2006). https://doi.org/10.1007/s00436-006-0166-3

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Keywords

  • Rainbow Trout
  • Polymerase Chain Reaction Assay
  • Nest Polymerase Chain Reaction
  • Polymerase Chain Reaction Test
  • Infected Fish