Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus)

Woutrina A Smith, Ian Gardner, Edward R Atwill, Christian M. Leutenegger, Melissa A. Miller, Ronald Hedrick, Ann C. Melli, Nicole M. Barnes, Patricia A Conrad

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels (Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log10 improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected C. parvum in < 5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.

Original languageEnglish (US)
Pages (from-to)367-379
Number of pages13
JournalJournal of Microbiological Methods
Volume65
Issue number3
DOIs
StatePublished - Jun 2006

Keywords

  • Bivalve
  • Cryptosporidium
  • IMS
  • Mussel
  • Real-time PCR
  • Waterborne pathogen

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Microbiology

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