Estimation of ruminal bacteriophage numbers by pulsed-field gel electrophoresis and laser densitometry

Abstract

To investigate phage activity in the rumen, a method for quantifying phage has been developed. By differential centrifugation and ultrafiltration, phage particles were separated and concentrated from ruminal fluid. Linear double- stranded DNA from this fraction containing predominately tailed phage was isolated and separated by size, using pulsed-field gel electrophoresis (PFGE). Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calculated and, therefore, the total numbers per milliliter of ruminal fluid to be estimated. Phage numbers were estimated to be between 3 x 109 and 1.6 x 1010 particles ml of ruminal fluid-1. The phage population, as gauged by the appearance of DNA on PFGE gels, had two major components. A broad region of DNA between 30 and 200 kb was always present on PFGE gels. It appears this region comprises DNA from a great many different phages and would include most of the temperate phages. In addition, discrete DNA bands ranging in size from 10 to 850 kb were frequently observed. DNA from one such band, of 12 kb in size, was shown to consist primarily of a single DNA type, suggesting that it originated from a specific phage. It is postulated that the discrete bands are due to epidemics or blooms of phage activity from specific, probably lytic, phages. The method that has been developed will greatly enhance future investigations into the interactions between the ruminal phage population, the ruminal bacterial population, and animal nutrition and growth. It appears the rumen ecosystem contains a dynamic phage population that is maintained at high numbers by a significant and continual lysis of ruminal bacteria.