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Journal of Clinical Microbiology, April 2002, p. 1194-1198, Vol. 40, No. 4
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.4.1194-1198.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Quantitative Detection of Neospora caninum in Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR

Esther Collantes-Fernández,1 Ángel Zaballos,2 Gema Álvarez-García,1 and Luis M. Ortega-Mora1*

Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid,1 Departamento de Inmunologífia y Oncología, Pharmacia-CSIC, Centro Nacional de Biotecnología, Universidad Autónoma de Madrid, Campus Cantoblanco, 28049 Madrid, Spain2

Received 1 October 2001/ Returned for modification 21 November 2001/ Accepted 7 January 2002

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 (R2 = 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.

* Corresponding author. Mailing address: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Ciudad Universitaria, s/n, 28040 Madrid, Spain. Phone: 34-91-3943713. Fax: 34-91-3943908. E-mail: luis.ortega{at}vet.ucm.es.

Journal of Clinical Microbiology, April 2002, p. 1194-1198, Vol. 40, No. 4
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.4.1194-1198.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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