333 - Development of a 4-plex Quantitative PCR Assay for the Detection and Quantification of Species and Subspecies of Fusobacterium Necrophorum and Fusobacterium Varium in Bovine Rumen Fluid

Abstract: The Fusobacterium genus contains Gram-negative, rod-shaped anaerobic bacteria. Fusobacterium necrophorum and its two subspecies, necrophorum and funduliforme, are normal ruminal inhabitants of cattle. Subsp. necrophorum is the primary causative agent of liver abscesses in feedlot cattle. Fusobacterium varium is also a normal rumen inhabitant, but its role in ruminal fermentation and involvement in liver abscesses is unknown. Currently, no PCR assays exist to quantify F. varium and both subspecies of F. necrophorum. Therefore, our objective was to develop, validate, and determine the applicability of a quantitative PCR assay for quantification of F. varium and the two subspecies of F. necrophorum. Assay targets were the species-specific hgdA gene, encoding for (R)-2-hydroxyglutarylCoA dehydratase subunit alpha, and the subspecies-specific promoter sequence of the leukotoxin gene, lktA. Assay specificity was validated with different species and strains of Fusobacterium, and other bacterial species. Assay sensitivity was determined using pure cultures. Applicability of the assay for quantification of F. necrophorum and F. varium was determined using ruminal fluid from slaughtered cattle. Species and subspecies-level specificity was confirmed. Pure culture quantification limits (CFU/ml) were 2.1x10³ for the F. necrophorum species, 5.6x10² for subsp. necrophorum, 1.5x10³ for subsp. funduliforme, and 1.8x10³ for F. varium. Analysis of ruminal fluid collected from cattle with healthy livers (n=83) and abscessed livers (n=68) indicated that 97% of samples contained F. funduliforme, with mean concentrations of 7.8 x10³, and 8.1x10³, respectively. Additionally, subsp. necrophorum and F. varium were more prevalent in ruminal fluid from cattle with abscessed livers (41% and 59%) compared with samples from cattle with healthy livers (31% and 41%). In conclusion, subsp. funduliforme appears to be the normal inhabitant of the rumen, while subsp. necrophorum and F. varium are more prevalent in the rumen of cattle with abscessed livers than those with healthy livers.