Characterization of the effects of nitrate and tungstate, alone or combined, and Salmonella Newport inoculation on rumen microbiota, fermentation, and Salmonella survivability in vitro

Authors: Anderson, Robin; SHAW, MEGAN - St Edward'S University; Johnson, Casey; AMAT, SAMAT - North Dakota State University; Kogut, Michael; BAYNHAM, PATRICIA - St Edward'S University; Genovese, Kenneth; Crippen, Tawni; Arsenault, Ryan

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/30/2026
Publication Date: 7/23/2026
Citation: Anderson, R.C., Shaw, M.R., Johnson, C.N., Amat, S., Kogut, M.H., Baynham, P.J., Genovese, K.J., Crippen, T.L., Arsenault, R.J. 2026. Characterization of the effects of nitrate and tungstate, alone or combined, and Salmonella Newport inoculation on rumen microbiota, fermentation, and Salmonella survivability in vitro. Frontiers in Microbiology. 17. Article 1841027. https://doi.org/10.3389/fmicb.2026.1841027.
DOI: https://doi.org/10.3389/fmicb.2026.1841027

Interpretive Summary: Within the animal gastrointestinal tract, the foodborne pathogen, Salmonella, can survive by metabolizing nitrate to gain energy needed to grow. The work in this study was carried out to test if nitrate supplementation, added to increase digestion effeciency, may unintentionally promote Salmonella growth within the cow gut. The work also tested if potential anti-Salmonella treatments employing a chemical called tungstate, which is an inhibitor of nitrate metabolism, whether alone or with another beneficial nitrate-metabolizing bacterium named Paenibacillus 79R4, could decrease the survivalbility of Salmonella. Under the conditions of this work, tungstate did not inhibit, but rather increased the growth of Salmonella but this increased growth was significantly curtailed by the Paenibacillus 79R4. Results obtained from this study provide important new information on the suitability and safety of nitrate, tungstate, and Paenibacillus strain 79R4 as potential feed additives for cattle; additional work is needed to clarify the effectiveness of tungstate as an additive to reduce nitrate-metabolizing pathogens. This work will ultimately help researchers develop safe and effective feed additives to reduce the carriage of Salmonella as well as other foodborne pathogens in cattle thereby helping to ensure livestock producers can continue to produce safe and wholesome milk and meat for the Americam consumer.

Technical Abstract: To test if nitrate supplementation may enrich Salmonella within rumen microbial populations, freshly collected rumen fluid was inoculated with 104 colony forming units (CFU) of Salmonella Newport and incubated 26 h anaerobically (39oC) under simulated rumen conditions without or with 10 mM nitrate, 100 mM tungstate (a potential inhibitor of denitrification) or their combination (n = 3 tubes/treatment). Populations of coliform and lactic acid bacteria as well as S. Newport were enumerated via viable cell count and concentrations of nitrate. nitrite and ammonia were measured colorimetrically. Methane was measured by gas chromatography. Nitrate, tungstate or their combined treatment decreased (P < 0.05) methane production by the rumen microbiota by 58 to 80% compared to untreated controls (28 ± 6.0 µmol/mL culture fluid). After 26 h incubation, coliforms were increased (P < 0.05) in populations treated with nitrate, tungstate or their combination (4.08 ± 0.41, 5.32 ± 2.34 and 6.76 ± 0.08 log10 CFU/mL, respectively) compared to controls (2.83 ± 0.32 log10 CFU/mL). Salmonella were increased (P < 0.05) in populations treated with tungstate, alone or combined with nitrate (5.36 ± 1.92 and 6.27 ± 0.23 log10 CFU/mL, respectively) compared to controls or nitrate only-treated populations (2.74 ± 0.69 and 3.23 ± 0.68 log10 CFU/mL, respectively). Lactic acid bacteria responded similarly to tungstate. More nitrate was catabolized (P < 0.05) by the rumen microbiota incubations treated with nitrate alone than those treated with nitrate and tungstate combined (95 ± 0.23 versus 92 ± 0.81% of nitrate added, respectively). Nitrite accumulations were affected (P < 0.05) by treatment, respectfully achieving 0.54 ± 0.06 and 0.14 ± 0.06 µmol/mL after 6 h and 0.13 ± 0.04 and 0.18 ± 0.03 µmol/mL) after 26 h incubation in populations treated with nitrate alone or combined with nitrate. Addition of a denitrifying Paenibacillus (strain 79R4) to rumen microbiota prepared and incubated similarly enhanced methane-reducing activity of all treatments and mediated the bacterial growth-promoting effect of tungstate. Under the conditions of this experiment, tungstate but not nitrate enriched Salmonella but all treatments decreased methane production by the rumen microbiota.