title: Development of an immunomagnetic bead separation-coupled quantitative PCR method for rapid and sensitive detection of Cryptosporidium parvum oocysts in calf feces.
abstract: Cattle feces are the environmental vehicle for the zoonotic Cryptosporidium oocysts, but there are drawbacks associated with reliability of the existing methods for the detection of oocysts in the feces. Quantification of the immunomagnetic bead separation (IMS) coupled with real-time TaqMan PCR (qPCR) was accomplished by comparing the fluorescence signals obtained from the calf fecal samples of Cryptosporidium parvum oocysts with those obtained from standard dilutions of C. parvum oocysts. TaqMan qPCR assays were developed for the detection of C. parvum based on 18S rDNA gene. This IMS-qPCR assay allowed a reliable quantification of C. parvum oocysts over seven orders of magnitude with a baseline sensitivity of 8.7 oocysts. The newly developed IMS-qPCR technique proved specific as confirmed by negative reactivity against a wide panel of non-parvum Cryptosporidium oocysts. As a field application, experimentally infected calves (15 infected and 9 non-infected) were screened for oocysts shedding on 16, 18, and 21 days postinfection. Acid-fast staining microscopy of infected calves revealed oocysts in the feces of 11, 7, and 4 calves, respectively, compared to 15, 15, and 12 in case of screening by IMS-qPCR. Taken together, the proposed IMS-qPCR method significantly improved the diagnostic capacity for C. parvum infection in calves, making the technique a useful, sensitive, reliable, and time-saving.
authors.affiliation: National Research Center for Wildlife Diseases, Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Gao
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.forename: Said
authors.initials: S
authors.lastname: Amer
authors.forename: Jing
authors.initials: J
authors.lastname: Luo
authors.forename: Chengmin
authors.initials: C
authors.lastname: Wang
authors.forename: Shaoqiang
authors.initials: S
authors.lastname: Wu
authors.forename: Baohua
authors.initials: B
authors.lastname: Zhao
authors.forename: Hongxuan
authors.initials: H
authors.lastname: He
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000469:parasitology; D016785:Cryptosporidium parvum / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology*; D018189:Immunomagnetic Separation / Q000379:methods / Q000662:veterinary*; D033661:Oocysts; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors