title: The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces.
abstract: Four quantitative PCR (qPCR) assays were evaluated for quantitative detection of Brachyspira pilosicoli, Lawsonia intracellularis, and E. coli fimbrial types F4 and F18 in pig feces. Standard curves were based on feces spiked with the respective reference strains. The detection limits from the spiking experiments were 10(2) bacteria/g feces for Bpilo-qPCR and Laws-qPCR, 10(3)CFU/g feces for F4-qPCR and F18-qPCR. The PCR efficiency for all four qPCR assays was between 0.91 and 1.01 with R(2) above 0.993. Standard curves, slopes and elevation, varied between assays and between measurements from pure DNA from reference strains and feces spiked with the respective strains. The linear ranges found for spiked fecal samples differed both from the linear ranges from pure culture of the reference strains and between the qPCR tests. The linear ranges were five log units for F4-qPCR, and Laws-qPCR, six log units for F18-qPCR and three log units for Bpilo-qPCR in spiked feces. When measured on pure DNA from the reference strains used in spiking experiments, the respective log ranges were: seven units for Bpilo-qPCR, Laws-qPCR and F18-qPCR and six log units for F4-qPCR. This shows the importance of using specific standard curves, where each pathogen is analysed in the same matrix as sample DNA. The qPCRs were compared to traditional bacteriological diagnostic methods and found to be more sensitive than cultivation for E. coli and B. pilosicoli. The qPCR assay for Lawsonia was also more sensitive than the earlier used method due to improvements in DNA extraction. In addition, as samples were not analysed for all four pathogen agents by traditional diagnostic methods, many samples were found positive for agents that were not expected on the basis of age and case history. The use of quantitative PCR tests for diagnosis of enteric diseases provides new possibilities for veterinary diagnostics. The parallel simultaneous analysis for several bacteria in multi-qPCR and the determination of the quantities of the infectious agents increases the information obtained from the samples and the chance for obtaining a relevant diagnosis.
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1790 Copenhagen V, Denmark. msta@vet.dtu.dk
authors.forename: M
authors.initials: M
authors.lastname: Ståhl
authors.forename: B
authors.initials: B
authors.lastname: Kokotovic
authors.forename: C K
authors.initials: CK
authors.lastname: Hjulsager
authors.forename: S Ø
authors.initials: SØ
authors.lastname: Breum
authors.forename: Ø
authors.initials: Ø
authors.lastname: Angen
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques; D017400:Brachyspira / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology*; D010861:Fimbriae, Bacterial / Q000235:genetics; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D020639:Lawsonia Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012680:Sensitivity and Specificity; D012703:Serotyping; D013045:Species Specificity; D013552:Swine / Q000382:microbiology*; D013553:Swine Diseases / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial