Project Number: 6612-32000-046-04
Project Type: Nonfunded Cooperative Agreement

Start Date: Sep 01, 2007
End Date: Jul 31, 2009

Objective:
The objective of this cooperative research project is to complete genomic analyses of bacterial viruses capable of lysing strains of C. perfringens that have been identified by PMSRU-ARS-USDA investigators using spot-testing and titration of strains susceptible to the isolated phages (Siragusa et al., 2004). Nucleotide sequencing and preliminary annotation has been completed by PMSRU-ARS-USDA. Cooperator will advise with completion of genomic sequence analyses in the case of identifying potential attachment (att) and packaging (pac) sites within the genome and generation of a physical genetic map of the phage genome.

Approach:
Initial screening for bacteriophages lytic for C. perfringens has been performed on filtered samples obtained from poultry (intestinal material), soil and processing drainage water (Siragusa et al., 2004). Bacterial viruses capable of lysing strains of C. perfringens were identified by spot-testing and titration of strains susceptible to the isolated phages. Lytic phage preparations have been characterized by negative staining and transmission electron microscopy. Growth of bacteriophage on the appropriate host bacteria, physical and chemical characteristics of the phages follows standard techniques (Siragusa et al., 2004). All phage tittering has and will be performed using soft agar overlays. Bacteriophage screening on various host bacteria will be performed to select phages with the highest virulence, forming transparent spots of lysis (plaques). Phage lysates were clarified by low speed centrifugation to remove cell debris, and then concentrated by differential centrifugation. Phages that hold promise for therapeutic and application purposes should be virulent on all bacteria that they infect and should preferably also be incapable of transduction. Virulence of C. perfringens phage(s) used in pilot phage therapy experiments will be examined for their ability to form clear plaques on host bacteria. Bacteria that become phage resistant in vitro may have become lysogenic. Accordingly, the DNA from such bacteria will be tested by Southern hybridization using randomly primed phage DNA as a probe.