Title page for ETD etd-11152000-14490002


Type of Document Dissertation
Author Whichard, Jean Marie
Author's Email Address jwhichar@vt.edu
URN etd-11152000-14490002
Title Bacteriophage Felix O1: Genetic Characterization and Bioremedial Application
Degree PhD
Department Veterinary Medical Sciences
Advisory Committee
Advisor Name Title
Pierson, Frank William Committee Co-Chair
Sriranganathan, Nammalwar Committee Co-Chair
Keywords
  • Salmonella
  • Felix O1
  • poultry
  • bacteriophage
Date of Defense 2000-10-16
Availability unrestricted
Abstract
Bacteriophage Felix O1 was studied for applicability as a Salmonella intervention. Felix O1's potential as a Salmonella therapeutic was explored, as was its utility as a food application. Felix O1 is specific for and infects most serovars within the genus Salmonella. The entire 86.155-kb sequence of the phage's linear, double-stranded chromosome was determined. 213 open reading frames (ORFs) were found, including 23 homologues of phage genes (e<0.008). Homology searches do not indicate genes that would be expected to increase virulence of Salmonella. Thirteen T4 homologues were found, including rIIA and rIIB, rapid lysis genes of T-even phages. Site-directed mutagenesis of the rIIB region was attempted by homologous recombination with plasmids containing luxAB of Vibrio harveyi. No DrIIB luxAB+ recombinants resulted from the methods tried.

Serial in vivo passage was used to select for a longer-circulating Felix O1 mutant using the modified methods of Merril et al., (1996). No difference was found in the clearance of wild-type (WT) and Felix O1 following nine serial passages. Injection of 109pfus yielded 24-hour concentrations of 6.5 and 4.9 log10 pfus/ml plasma for WT and 9th passage, respectively. Both isolates were undetectable in plasma by 72 hours, but remained in spleens at 96 hours.

A large-plaque Felix O1 variant (LP) isolated during in vivo serial passage was compared with WT for Salmonella growth suppression. Spectrophotometric measurement of BHI cultures indicated greater suppression of S. typhi by LP than by WT, a difference not seen with S. typhimurium DT104. Both isolates suppressed 24-hour S. typhimurium DT104 growth on experimentally-contaminated chicken frankfurters at 22°C. Untreated frankfurters yielded 6.81 log10 Salmonella cfus/g, whereas WT and LP-treated samples yielded 5.01 and 4.70 log10 cfus/g, respectively. Both phages suppressed the Salmonella typhimurium DT104 growth (p<0.0001), but the isolates did not perform differently (p=0.5088). Presence of Salmonella caused a higher yield of WT phage than from the uninoculated group (p=0.0011), but did not affect LP yield (p=0.4416). With Salmonella present, the 24-hour LP concentration was lower than WT concentration. This supports the surmised LP rapid-lysis phenotype since T4 rapid-lysis mutants typically exhibit lower burst sizes than wild-type phage.

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