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