title: Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus).
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 log(10) 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.
authors.affiliation: Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, 95616, USA.
authors.forename: Woutrina A
authors.initials: WA
authors.lastname: Miller
authors.forename: Ian A
authors.initials: IA
authors.lastname: Gardner
authors.forename: Edward R
authors.initials: ER
authors.lastname: Atwill
authors.forename: Christian M
authors.initials: CM
authors.lastname: Leutenegger
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Miller
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Hedrick
authors.forename: Ann C
authors.initials: AC
authors.lastname: Melli
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Barnes
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Conrad
mesh_terms: D000818:Animals; D003458:Cryptosporidium / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000032:analysis; D019085:Fluorescent Antibody Technique, Direct; D018189:Immunomagnetic Separation; D049878:Mytilus / Q000469:parasitology*; D033661:Oocysts / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
chemical_list: D016054:DNA, Protozoan; D019914:Taq Polymerase