title: Quantification of Ehrlichia ruminantium by real time PCR.
abstract: Ehrlichia ruminantium (ER) is the causative agent of Heartwater, one of the most common tick-borne diseases affecting ruminants in African countries and West Indies. Although ER can be used as an inactivated vaccine for wild and domestic animals, there are currently no easy and reliable methods for the quantification of this obligate intracellular bacterium. This report describes the development of a SYBR Green I based real time PCR protocol for the quantification of ER for vaccine production purposes. The method was validated for four ER strains. The external-standard-based PCR protocol developed has a large dynamic quantitative range allowing accurate ER measurement in samples containing from 10(2) to 10(8) gene copies; the method is also reproducible and precise, with intra- and inter-assay coefficients below 5%. The detection limits were validated for samples collected from bovine aortic endothelial cell culture bulks, which are commonly used to produce the ER vaccine. In contrast to the methods based upon protein content, no interference from the host cells in ER quantification was observed. Furthermore, the extended applicability of the new technique was demonstrated by monitoring ER production in cell culture thus rendering it a valuable tool to ensure consistency between vaccine lots and to evaluate optimal vaccine dosage.
authors.affiliation: IBET, Apartado 12, 2781-901 Oeiras, Portugal.
authors.forename: Cristina C
authors.initials: CC
authors.lastname: Peixoto
authors.forename: Isabel
authors.initials: I
authors.lastname: Marcelino
authors.forename: Nathalie
authors.initials: N
authors.lastname: Vachiéry
authors.forename: Albert
authors.initials: A
authors.lastname: Bensaid
authors.forename: Dominique
authors.initials: D
authors.lastname: Martinez
authors.forename: Manuel J T
authors.initials: MJ
authors.lastname: Carrondo
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
mesh_terms: D017954:Africa South of the Sahara; D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D016995:Ehrlichia ruminantium / Q000235:genetics / Q000254:growth & development*; D016873:Ehrlichiosis / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D042783:Endothelial Cells; D006357:Heartwater Disease / Q000175:diagnosis / Q000382:microbiology*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D009930:Organic Chemicals / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011804:Quinolines; D015203:Reproducibility of Results; D012418:Ruminants / Q000382:microbiology*; D014900:West Indies
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D008565:Membrane Proteins; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I