Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR.

Journal of Clinical Microbiology
Esther Collantes-FernándezL M Ortega-Mora

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. ...Continue Reading

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Citations

Dec 25, 2009·Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology·Jessica P OtisHannah V Carey
Nov 26, 2003·Veterinary Parasitology·Luis Miguel Ortega-MoraGorka Aduriz
Apr 30, 2003·Veterinary Parasitology·A Criado-FornelioJ C Barba-Carretero
Oct 17, 2002·Trends in Parasitology·Andrew S Bell, Lisa C Ranford-Cartwright
Dec 15, 2012·Clinical and Vaccine Immunology : CVI·Maki NishimuraYoshifumi Nishikawa
Mar 23, 2006·Infection and Immunity·Esther Collantes-FernándezLuis M Ortega-Mora
Jul 24, 2004·The Journal of Parasitology·Esther Collantes-FernándezLuis Miguel Ortega-Mora
May 12, 2009·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Jie FuZhongming Tang
Apr 17, 2012·Veterinary Parasitology·T de Waal
Nov 10, 2015·Experimental Parasitology·Florencia Celeste MansillaAlejandra Victoria Capozzo
May 27, 2008·Veterinary Parasitology·Farida GhalmiBertrand Losson
Aug 7, 2014·Veterinary Parasitology·Caroline M MotaTiago W P Mineo
Apr 4, 2006·The Journal of Eukaryotic Microbiology·S RamamoorthyN Sriranganathan
Feb 24, 2005·Theriogenology·Ignacio FerreLuis Miguel Ortega-Mora
Aug 25, 2004·Theriogenology·Andrea Caetano-da-SilvaLuis Miguel Ortega-Mora
May 30, 2006·Veterinary Parasitology·J P Dubey, G Schares
Sep 20, 2005·Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases·Paul T Monis, Steven Giglio
Jun 5, 2014·Veterinary Parasitology·Paula Rogério Fernandes BromAndréa Caetano da Silva

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