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Comparison of PCR assays to detect Toxoplasma gondii oocysts in green-lipped mussels (Perna canaliculus)

  • Alicia CoupeEmail author
  • Laryssa Howe
  • Karen Shapiro
  • Wendi D. Roe
Protozoology - Original Paper
  • 13 Downloads

Abstract

Toxoplasma gondii is recognised as an important pathogen in the marine environment, with oocysts carried to coastal waters in overland runoff. Currently, there are no standardised methods to detect T. gondii directly in seawater to assess the extent of marine ecosystem contamination, but filter-feeding shellfish may serve as biosentinels. A variety of PCR-based methods have been used to confirm presence of T. gondii DNA in marine shellfish; however, systematic investigations comparing molecular methods are scarce. The primary objective of this study was to evaluate analytical sensitivity and specificity of two nested-PCR (nPCR) assays targeting dhps and B1 genes and two real-time (qPCR) assays targeting the B1 gene and a 529-bp repetitive element (rep529), for detection of T. gondii. These assays were subsequently validated for T. gondii detection in green-lipped mussel (Perna canaliculus) haemolymph using oocyst spiking experiments. All assays could reliably detect 50 oocysts spiked into mussel haemolymph. The lowest limit of detection was 5 oocysts using qPCR assays, with the rep529 primers performing best, with good correlation between oocyst concentrations and Cq values, and acceptable efficiency. Assay specificity was evaluated by testing DNA from closely related protozoans, Hammondia hammondi, Neospora caninum, and Sarcocystis spp. Both nPCR assays were specific to T. gondii. Both qPCR assays cross-reacted with Sarcocystis spp. DNA, and the rep529 primers also cross-reacted with N. caninum DNA. These studies suggest that the rep529 qPCR assay may be preferable for future mussel studies, but direct sequencing is required for definitive confirmation of T. gondii DNA detection.

Keywords

Toxoplasma gondii Polymerase chain reaction rep529 repetitive element, B1 gene, dhps gene Perna canaliculus green-lipped mussel 

Notes

Acknowledgements

We thank Heather Fritz, Jeroen Saeij, and David Arranz Solis for producing and generously providing T. gondii oocysts from the University of California, Davis. We are very grateful to Patricia Conrad, University California, Davis, for supporting collaboration with Massey University.

Funding information

Funding for this work was provided by the New Zealand Department of Conservation, the Massey University Research Foundation, the Lewis Fitch Foundation, and the McGeorge Foundation. Financial support was also received from the IVABS Doctoral Scholarship, Massey University, and the New Zealand International Doctoral Research Scholarship, Education New Zealand.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Veterinary, Animal and Biomedical Sciences, College of SciencesMassey UniversityPalmerston NorthNew Zealand
  2. 2.EpiCentre, Wool Building, University AvenueMassey University Manawatū CampusPalmerston NorthNew Zealand
  3. 3.One Health Institute and School of Veterinary MedicineUniversity of CaliforniaDavisUSA
  4. 4.Department of Pathology, Microbiology and Immunology, School of Veterinary MedicineUniversity of CaliforniaDavisUSA

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