title: Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR.
abstract: We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10(-1) tachyzoite per assay was observed with excellent linearity (R(2) = 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques (kappa = 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection.
authors.affiliation: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Collantes-Fernández
authors.forename: Angel
authors.initials: A
authors.lastname: Zaballos
authors.forename: Gema
authors.initials: G
authors.lastname: Alvarez-García
authors.forename: Luis M
authors.initials: LM
authors.lastname: Ortega-Mora
mesh_terms: D000034:Abortion, Veterinary / Q000469:parasitology*; D000818:Animals; D052160:Benzothiazoles; D001921:Brain / Q000469:parasitology; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D003048:Coccidiosis / Q000662:veterinary*; D016054:DNA, Protozoan / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D003959:Diamines; D005333:Fetus / Q000469:parasitology*; D005456:Fluorescent Dyes / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018688:Neospora / Q000302:isolation & purification* / Q000472:pathogenicity; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012339:RNA, Ribosomal, 28S / Q000235:genetics
chemical_list: D052160:Benzothiazoles; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012339:RNA, Ribosomal, 28S; C098022:SYBR Green I