ESTIMATION OF RUMINAL BACTERIOPHAGE NUMBERS BY PULSED-FIELD GEL-ELECTROPHORESIS AND LASER DENSITOMETRY

To investigate phage activity in the rumen, a method for quantifying p hage has been developed. By differential centrifugation and ultrafiltr ation, phage particles were separated and concentrated from ruminal fl uid. Linear double-stranded DNA from this fraction containing predomin ately tailed phage was isolated and separated by size, using pulsed-fi eld gel electrophoresis (PFGE). Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calcu lated and, therefore, the total numbers per milliliter of ruminal flui d to be estimated. Phage numbers were estimated to be between 3 x 10(9 ) and 1.6 x 10(10) particles ml of ruminal fluid-1. The phage populati on, as gauged by the appearance of DNA on PFGE gels, had two major com ponents. A broad region of DNA between 30 and 200 kb was always presen t on PFGE gels. It appears this region comprises DNA from a great many different phages and would include most of the temperate phages. In a ddition, discrete DNA bands ranging in size from 10 to 850 kb were fre quently observed. DNA from one such band, of 12 kb in size, was shown to consist primarily of a single DNA type, suggesting that it originat ed 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 an d growth. It appears the rumen ecosystem contains a dynamic phage popu lation that is maintained at high numbers by a significant and continu al lysis of ruminal bacteria.