ARS | Publication request: Quantification of the Sensitivity of Mycobacterium avium subsp paratuberculosis and Salmonella enterica subsp enterica to Low pH and High Organic Acids using Propidium Monoazide and Quantitative PCR

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: March 25, 2010
Publication Date: May 25, 2010
Citation: Cook, K.L., Flis, S.A., Ballard, C.S. 2010. Quantification of the Sensitivity of Mycobacterium avium subsp paratuberculosis and Salmonella enterica subsp enterica to Low pH and High Organic Acids using Propidium Monoazide and Quantitative PCR. American Society for Microbiology.

Technical Abstract: Mycobacterium avium subsp paratuberculosis (Map) and Salmonella enterica subsp enterica (S. enterica) are two pathogens that are a concern to food and animal safety due to their ability to withstand harsh conditions encountered in the natural environment and within the host during pathogenesis. Acid tolerance is one mechanism by which pathogens survive adverse conditions and acid adaptation may even increase the organisms’ virulence. The goal of this study was to evaluate the ability of these pathogens to survive the low pH and high organic acid concentrations encountered as part of the ensiling process. Viable cells were selectively quantified using the viability dye propidium monoazide (PMA) in conjunction with quantitative, real-time PCR (qPCR). Silage exudates from forage grass ensiled for 0, 25, 50, 75 and 100 days were obtained by soaking 50g silage in 200 mL water for 1 hr, removing and filtering the extracted liquid. Silage exudates were inoculated with Map (2.4±1.54 x 107 cells mL-1) or S. enterica (2.7±0.6 x 107 cells mL-1) and survival evaluated over 30 days incubation. The pH of the ensiled materials was 4.75 ± 0.09. Volatile fatty acid concentrations in ensiled materials were 5.12%, 4.13%, 0.21%, and 0.05% of lactic, acetic, propionic, and butyric acids, respectively. Within one week the concentration of S. enterica in exudates from 0, 25, and 50 days ensiling were less than 5% of initial and remained low for the remainder of the study. However, concentrations after one week incubation in exudates obtained after 75 or 100 days ensiling were 13% and 22%, respectively, and both were still 8% of initial after 30 days. Map concentrations in exudates from materials ensiled for 0, 25, or 50 days were 28%, 25% and 56% of initial levels after 30 days incubation. However, concentrations in Map exposed to exudates from day 75 and 100 silage were at or above initial levels for the duration of the study. These results suggest that Map present in manure and applied to forage grasses may survive exposure to low pH’s and high organic acids during the ensilaging process and could, therefore, be a potential route of infection if ingested by a susceptible animal.