title: Development of a quantitative real time PCR assay to detect and enumerate Escherichia coli O157 and O26 serogroups in bovine recto-anal swabs.
abstract: Escherichia coli O157 and O26 shedding patterns in cattle are known to vary widely. To address gaps in the understanding of the underlying factors which impact on shedding dynamics, sensitive and rapid quantitative methods which can be applied in surveillance studies on cattle are required. Current approaches for enumeration of verocytotoxigenic E. coli (VTEC) in cattle faeces are based on direct plating onto selective agars, most probable number (MPN) or real time PCR applied directly to faecal samples, all of which have limitations in terms of the labour involved or their sensitivity. The objective of this study was to develop a sensitive real time quantitative PCR assay, to quantify O157 and O26 in bovine recto-anal junction (RAJ) swabs. The approach was to target serogroup specific genes rfbE and wzx, and to couple a short enrichment, with the use of a standard calibration curve relating real time PCR cycle threshold (Ct) values against the initial concentration of the pathogen in the sample. Following initial experiments in broth culture, a 5h enrichment in modified tryptone soya broth with novobiocin (20 mg/l) (mTSBn) was found to be optimal, and a linear correlation between inocula (Log10 1 to 6 CFU ml(-1)) and the PCR Ct values for both E. coli O157 (R(2)=0.99, rsd=0.58) and E. coli O26 (R(2)=0.99, rsd=0.44) was confirmed. The developed method was then applied to bovine RAJ swab samples (n=153), which were inoculated with E. coli O157 or O26 (Log10 1 to 7 CFU swab(-1)). Calibration curves yielded correlations for E. coli O157 of R(2)=0.86, rsd=0.72 and for O26 (R(2)=0.88, rsd=0.69). In conclusion, a sensitive method for detection and enumeration of two significant VTEC serogroups in bovine RAJ samples has been developed and validated, and will support studies on the bovine shedding dynamics of these pathogens in cattle.
authors.affiliation: Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland; School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Dolapo
authors.initials: D
authors.lastname: Lawal
authors.affiliation: Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland. Electronic address: Kaye.Burgess@teagasc.ie.
authors.forename: Catherine
authors.initials: C
authors.lastname: Burgess
authors.affiliation: Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland.
authors.forename: Evonne
authors.initials: E
authors.lastname: McCabe
authors.affiliation: School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Paul
authors.initials: P
authors.lastname: Whyte
authors.affiliation: Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland.
authors.forename: Geraldine
authors.initials: G
authors.lastname: Duffy
mesh_terms: D001003:Anal Canal / Q000382:microbiology*; D000818:Animals; D058491:Bacterial Load / Q000379:methods*; D002353:Carrier State / Q000382:microbiology / Q000662:veterinary*; D002417:Cattle; D003470:Culture Media / Q000737:chemistry; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000382:microbiology / Q000662:veterinary*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012007:Rectum / Q000382:microbiology*; D012680:Sensitivity and Specificity; D065288:Serogroup; D013997:Time Factors
chemical_list: D003470:Culture Media
keywords: Cattle; Enumeration; PCR; Quantification; RAJ; Real-time