Elimination of Pathogens from Livestock Using a Combination of Yucca Saponins and Sodium Chlorate

NON-TECHNICAL SUMMARY: Foodborne bacterial disease is a major health concern in the United States. Five major bacterial pathogens cause an annual $6.9 billion in medical costs and productivity losses. New effective technologies related to the decontamination of meat are especially needed. This project will target two specific pathogenic bacteria; E. coli. 0157:H7 and Salmonella. It has been discovered recently that sodium chlorate, as a solid or aqueous solution, is effective in the killing of entero-pathogenic bacteria. The mechanism of chlorate killing these pathogens is thought to be that they contain an enzyme known as respiratory nitrate reductase that converts chlorate to chlorite, which kills the harmful bacteria. However, the use of sodium chlorate is less successful in the rumen likely due to the presence of protozoa, which are known to harbor bacteria and protect them from chemical bactericides. We propose to first isolate pure components from Yucca schidigera extract and then use them in combination with sodium chlorate to reduce or eliminate pathogenic bacteria from ruminant animals. It is anticipated that the use of Yucca schidigera extract and its purified fractions at a sufficient concentration will allow for defaunation of ruminants thereby enhancing animal production efficiency and health.

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APPROACH: I. Perform two-dimensional semi-preparative HPLC on Yucca schidigera extract and collect all fractions based on retention in normal phase and reversed-phase modes. Yucca schidigera extract fractions will be isolated and individually tested for their anti-protozoal activity in vitro: (1) The number of fractions obtained will be dictated by the number of distinct peaks that can be obtained in two chromatographic dimensions. Each collected fraction from the first dimension will be run in the second dimension to maximize the possible resolution power of the separation. This approach will maximize the resolution of components from this complex natural product mixture; (2) Measure the sapogenin content of each collected fraction; (3) Remove solvents by rotary evaporation to obtain a minimum of 10 grams of each fraction;(4) Formulate test solutions with identical mass concentrations for each Yucca schidigera extract fraction to be used in anti-protozoal testing studies. II. Measure the anti-protozoal activity of each fraction. Rumen contents will be collected from fistulated donor cows fed a general-purpose alfalfa/corn diet. Anti-protozoal activity of Yucca schidigera extract fractions will be determined on whole rumen contents or using washed, concentrated protozoa preparations. For concentrated preparations, rumen protozoa will be collected from rumen contents by sedimentation and aspiration. Yucca schidigera extract fractions will then be added for testing and the preparations will be incubated for 2 hours at 37 degrees C. Samples will then be collected, fixed and stained with molecular probes to determine protozoal populations surviving defaunation treatment. All collected Yucca schidigera extract fractions will be tested for their anti-protozoal activity at different concentrations. III. Test the combination of a Yucca schidigera extract, its extract fractions and sodium chlorate for their ability to eliminate E. coli 0157:H7 and Salmonella in-vitro. Ruminal fluid collected from a cannulated cow will be mixed 1:1 with 200 mM phosphate buffer containing cellobiose, glucose, soluble starch, and xylose and approximately 106 CFU of either a novobiocin (NO) and nalidixic acid (NA) resistant E. coli O157:H7 or Salmonella typhimurium DT104. 10 ml aliquots of the buffered ruminal fluid will then be transferred to crimp-top tubes preloaded with or without 5 mM sodium chlorate and an aliquot of each of the extract fractions. Then, these preparations will be incubated anaerobically. The bactericidal activity of the Yucca schidigera extract fraction/chlorate mixture against E. coli O157:H7 and Salmonella typhimurium DT 104 within these incubations will be determined via plating of serial 10-fold dilutions of samples collected at 0, 3, 6 and 24 h to MacConkey agar containing NO and NA and to Brilliant Green agar containing NO and chloramphenicol. These plates will be incubated overnight at 37 degrees C and suspect colonies will be confirmed via slide agglutination. This procedure will be repeated and the data will be analyzed using software called "Statistical Analysis System."
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PROGRESS: 2006/05 TO 2006/12 <BR>
We have used chemical extraction techniques and high performance liquid chromatography to isolate and characterize a number of different Yucca extracts. We have tested the extracts that contained the highest levels of saponin for their effect on bacteria and protozoa. The butanol, methanol and purified saponin preparations were effect as antiprotozoal compounds. When these Yucca extracts were combined with sodium chlorate, we demonstrated high levels of antiprotozoal activity and decreases in viable bacterial pathogen cell counts. The results of our studies indicate that several of our Yucca extract preparations when combined with sodium chlorate were very effective against rumen protozoa and bacterial pathogens such as Salmonella. A synergistic antimicrobial effect was apparent in our data.
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IMPACT: 2006/05 TO 2006/12<BR>
We have validated our, one two punch, concept of antimicrobial activity against rumen protozoa and the bacterial pathogens they harbor using Yucca saponins and sodium chlorate. Upon completion of the SBIR project we will have demonstrated that a reduction in rumen protozoa will help control bacterial pathogens like Salmonella. From both an animal production and food safety perspective our work will have economic benefits for livestock producers and health benefits for consumers.