PSXII-29 - Development of an In Vitro Rumen Fermentation Model to Assess Fusobacterium necrophorum Growth Inhibition

Abstract: This experiment aimed to determine the impact of growth-inhibitory compounds on F. necrophorum subsp. necrophorum (F.n.n.), a primary etiologic agent of liver abscesses in feedlot cattle, in an in vitro rumen fermentation system. The assessment of inhibition was by quantitative PCR. We hypothesized that the addition of growth inhibitory compounds would reduce F.n.n. concentrations. A 2 x 2 x 6 factorial arrangement of treatments was used. Factors included: 1) sodium lactate (0.0 or 100 mM); 2) bacteria (0 or 1x10⁸ CFU/ml of F.n.n.); and 3) 1 mL of test compound [Control (solvent with no additive); Tylosin: 5 μg/ml; Compound A; Compound B; Compound C; and Compound D]. Prior to morning feeding, rumen contents were collected from a single ruminally-canulated steer fed a grain-based finishing diet. Rumen fluid was strained through 4 layers of cheesecloth, mixed with McDougall’s buffer (1:1 ratio; RFB), and 30 ml of the RFB was added into 100 ml-serum bottles containing the appropriate treatments. Serum bottles (n = 3 replicates per treatment) were flushed with CO₂ closed with rubber stoppers, crimped with aluminum seals, and incubated at 39ºC for 0, 6, 12, and 24 h. Following each incubation time point, a 2-ml aliquot was removed from each serum bottle under a stream of CO₂ to maintain anaerobic conditions. Immediately after sample collection, pH was determined, and DNA was extracted by boiling the sample, centrifugation, and purifying with a GeneClean Turbo DNA extraction kit. The concentration of F.n.n was determined by a quantitative PCR assay targeting the promoter region of the leukotoxin genes. Concentrations of F.n.n. from the RFB were used to adjust F.n.n. concentrations for all treatments at all time points. There was a lactate x test compound x incubation time interaction (P < 0.05) for F.n.n. concentration. In the absence of lactate, serum bottles containing tylosin or test compound treatments had lower (P < 0.05) F.n.n. concentrations by 24h post incubation. However, when sodium lactate was added, no treatments had F.n.n. concentrations lower than the control treatment at 24 h post incubation. (P > 0.05). These data suggest that compounds used in this experiment reduced the concentrations of F.n.n by 24 h post incubation in the absence of additional lactate. However, when lactate, a substrate for F.n.n, was added to the in vitro system, the reduction in F.n.n. growth was not observed.