Detection of Clostridium proteoclasticum and closely related strains in the rumen by competitive PCR

Abstract

A competitive PCR technique was used to enumerate the proteolytic bacterium Clostridium proteoclasticum from the rumen. A PCR primer, which circumscribes this organism and several closely related strains, was designed for a variable region within their 16S rRNA genes and was used in conjunction with a universal forward primer. This primer pair was tested for specificity against 85 ruminal bacterial strains. An internal control DNA was constructed for use in competitive PCRs and was shown to amplify under the same reaction conditions and with the same amplification efficiency as the target DNA. DNA from a known number of C. proteoclasticum cells was coamplified with the internal control to construct a standard curve. Rumen samples were collected from eight dairy cows fed four diets in rotation: high nitrogen, high nitrogen supplemented with carbohydrate, low nitrogen, and low nitrogen supplemented with carbohydrate. DNA extracted from these and spiked with internal control DNA was amplified with the C. proteoclasticum primer pair. The relative intensities of the PCR products were used to quantitate the numbers of C. proteoclasticum cell equivalents from the rumen samples. The numbers ranged from 2.01 x 10(6) ml-1 to 3.12 x 10(7) ml-1. There was no significant effect on the numbers of C. proteoclasticum detected in rumen samples among cows fed the four diets. The utility of the competitive PCR approach for quantifying ruminal bacterial populations in vivo and the occurrence of C. proteoclasticum in forage-fed dairy cows are discussed.

FIG. 1

Construction of the internal control. The 830-bp PCR product was digested with AluI to give three fragments of 90, 350, and 390 bp. The fragments were ligated and PCR amplified. The 480-bp product was purified and used as the internal control.

FIG. 2

Detection limit of cPCR. DNA extracted from 10¹⁰ C. proteoclasticum cells ml⁻¹ was serially diluted and coamplified with a 2 × 10⁶ dilution of the internal control to determine the detection limit of the cPCR assay. The results are expressed as C. proteoclasticum cell equivalents based on the amount of DNA extracted per cell.

FIG. 3

Internal-control amplification efficiency. Dilutions of the internal control were coamplified with DNA from 10³ C. proteoclasticum cells, and the ratio of the intensities of internal control to the target DNA was plotted against the dilution of the internal control on a log scale.

FIG. 4

Standard curve construction. (a) DNA extracted from 10¹⁰ C. proteoclasticum cells ml⁻¹ was serially diluted and coamplified with a 10⁶ dilution of the internal control. The results are expressed as C. proteoclasticum cell equivalents based on the amount of DNA extracted per cell. (b) Ratios of the intensities of internal control to target DNA were quantitated by scanning densitometry of negative images of Polaroid photographs of ethidium bromide-stained gels.

FIG. 5

In vivo detection of C. proteoclasticum. Rumen samples were spiked with known numbers of C. proteoclasticum cells. DNA was extracted from these mixtures and assayed for the presence of C. proteoclasticum. Solid squares denote numbers of C. proteoclasticum cell equivalents detected in each sample. Open circles denote y = x. The y intercept of the line denotes no C. proteoclasticum cells added. This represents a background population of C. proteoclasticum and closely related B. fibrisolvens strains of 6.46 × 10⁶ ml⁻¹.

FIG. 6

Populations of C. proteoclasticum and closely related B. fibrisolvens strains in dairy cows under four different feeding regimens. Numbers 1 through 8 represent cows numbered 709, 710, 727, 788, 1758, 8702, 9754, and 9775, respectively. The results are the means of triplicate determinations and error bars represent standard error of the mean. The diets were fed in rotation, and rumen samples were taken during each feeding regime. CHO, carbohydrate.