A real-time PCR assay for the detection and quantification of Fusobacterium necrophorum and Fusobacterium varium in ruminal contents of cattle*

Objectives Our objectives were to develop and vali- date a quantitative PCR assay for the detection and quantification of the 2 subspecies of Fusobacterium nec- rophorum (ssp. necrophorum and ssp. funduliforme) and a recently recognized species, Fusobacterium varium, in ruminal contents of cattle collected at slaughter. Materials and Methods A quantitative PCR assay with primers and probes designed to target hgdA, which encodes for 2-hydroxyglutaryl dehydratase, for F. necroph- orum (hgdA-n) and F. varium (hgdA-v), and the leukotox- in promotor region, lktA-n and lktA-f, for the 2 subspecies of F. necrophorum were developed and validated. A total of 345 ruminal fluid samples were collected in an abattoir immediately after slaughter, which included 181 (52.5%) samples from cattle with apparently healthy, nonabscessed livers and 164 (47.5%) from cattle with abscessed livers. Basal media with lactate or lysine as the major energy source and each with or without josamycin, vancomycin, and norfloxacin were used to enrich ruminal samples that were below the lower limit of quantification. Results and Discussion The ssp. necrophorum was quantified in 22% and 29% of samples from cattle with nonabscessed and abscessed livers, respectively. The ssp. funduliforme and F. varium were prevalent in almost all ruminal samples (98% to 100%) and were not different between cattle with or without abscessed livers. The mean concentrations of the 2 subspecies and F. varium were 103 to 104 cfu/mL and were not different between cattle with abscessed or nonabscessed livers. Implications and Applications A major finding of the study was that only a small proportion of the ruminal contents harbored the ssp. necrophorum, which suggested that it is not a normal member of the ruminal microbial community. In contrast, the ssp. funduliforme and F. vari- um were prevalent in all ruminal contents tested. Because F. varium is a pathogen, the question whether the species contributes to the development of liver abscesses needs to be investigated.