Multiplex Quantitative PCR Assays for the Detection and Quantification of the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces.

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, called non-O157 STEC, are important foodborne pathogens. Cattle, a major reservoir, harbor the organisms in the hindgut and shed them in the feces. Although limited data exist on fecal shedding, concentrations of non-O157 STEC in feces have not been reported. The objectives of our study were (i) to develop and validate two multiplex quantitative PCR (mqPCR) assays, targeting O-antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2); (ii) to utilize the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae), to quantify seven serogroups and three virulence genes in cattle feces; and (iii) to compare the three mqPCR assays to a 10-plex conventional PCR (cPCR) targeting seven serogroups and three virulence genes and culture methods to detect seven E. coli serogroups in cattle feces. The two mqPCR assays (1 and 2) were shown to be specific to the target genes, and the detection limits were 4 and 2 log CFU/g of pure culture-spiked fecal samples, before and after enrichment, respectively. A total of 576 fecal samples collected from a feedlot were enriched in E. coli broth and were subjected to quantification (before enrichment) and detection (after enrichment). Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥4 log CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. The three mqPCR assays detected higher proportions of postenriched fecal samples (P > 0.01) as positive for one or more serogroups compared with cPCR and culture methods. This is the first study to assess the applicability of qPCR assays to detect and quantify six non-O157 serogroups in cattle feces and to generate data on fecal concentration of the six serogroups.

Disease Severity and Microsclerotium Properties of the Sorghum Sooty Stripe Pathogen, Ramulispora sorghi.

Ramulispora sorghi causes sooty stripe of sorghum. Disease severity in irrigated and dryland plots was measured for 25 susceptible sorghum genotypes during the 2007 and 2008 growing seasons using a rating scale based upon percent leaf area infected. Disease severity ratings were approximately 1.4 points higher (P < 0.0001) on the rating scale in the irrigated plots than dryland plots for 2007 and 2008. Sooty stripe lesions were collected from each sorghum genotype in irrigated plots and assessed for mean microsclerotium production within lesions, microsclerotium size, and sporogenic germination, with significant differences apparent between genotypes for microsclerotium size (P = 0.01) and sporogenic germination (P = 0.01). There was no relationship between disease severity and microsclerotium production within leaf lesions, microsclerotium size, or sporogenic germination; however, there was a positive and significant correlation between microsclerotia production within a lesion and microsclerotium size (R2 = 0.19, P < 0.0001). Although microsclerotia from sorghum lesions varied in structural characteristics and their ability to produce spore masses, these qualities were dependent upon the sorghum genotype from which the microsclerotia were derived, because the R. sorghi population was genetically uniform as determined by internal transcribed spacer sequences and random amplified polymorphic DNA polymerase chain reaction.