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The Effects of Ozonated Water on Selected Mastitis-Causing Agents in vitro

Jordan Tayce, Melissa Libal1, and Jeffrey Musser1

1Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, USA

Objective– Ozonated water, at ozone concentrations that are not cytotoxic, will be bactericidal in vivo against the major pathogens which cause mastitis: Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli.

Procedures – 30ml of tryptose broth will be inoculated with each bacterium and incubated for six hours to harvest the cells in the growth phase.  The bacteria will then be centrifuged (at 10,000g for five minutes) and washed twice before being diluted to 106 and 105 cells per ml.  1ml of solution will be exposed to 1ml of ozonated water at times 10, 30, 60, 90 and 120sec.  At these times, 10ul will be removed and streaked onto a blood agar medium.  This will be incubated overnight and colonies counted the following day.  The experiment will be performed in triplicate and then repeated using a milk medium in place of saline to replicate the conditions of the cow udder.

Results – In a saline medium, Escherichia coli and Streptococcus agalactiae had complete cell death at all concentrations greater than or equal to 2.5 mg/L within ten seconds.  Staphylococcus aureus had complete cell death above 15 mg/L.  In a milk medium, ozonated water made no significant change to levels of Staphylococcus aureus or Streptococcus agalactiae at 15 mg/L, or to Escherichia coli at 25 mg/L within two minutes.

Conclusions and Clinical Relevance – Ozonated water is a very effective bactericidal agent against bacteria in a saline solution but far less effective in a milk medium.  In milk, ozone is attracted to polyunsaturated fatty acids and nucleotides that absorb ozone before it attacks the bacteria.  Further studies regarding concentrations of ozonated water in milk needed to be bactericidal or cytotoxic need to be performed.