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Evaluation of a Nisin-Based Germicidal Formulation on Teat Skin of Live Cows P. M. SEARS, B. S. SMITH, W. K. STEWART, and R. N. GONZALEZ Cornell University Ithaca, NY 14850 S. D. RUBINO, S. A. GUSIK, and E. S. KULISEK Applied Microbiology, Inc. New Yo&, NY 10016

Applied Microbiology, Inc. and Public Health Research Institute New York, NY 10016


A purified preparation of the nontoxic antimicrobial peptide, nisin (AMBICIN N@), was used in the formulation of a germicidal sanitizer suitable for use on cow teats. The germicidal activity of the formulation against mastitis pathogens was measured on teat skin of live cows. The nisin-based formulation gave a mean log reduction of 3.90 against Staphylococcus aureus and 4.22 log reduction against Escherichia coli after exposure for 1 min to the germicide. This activity was comparable with that exhibited by a 1% iodophor teat dip but was significantly greater than that exhibited by the .1 and .5% iodophors and by the .5% chlorhexidine digluconate teat dips. The nisin-based formulation showed little or no potential for skin irritation after multiple application to skin, but iodophor and chlorhexidine digluconate teat dips showed significant potential for skin irritation in comparable studies. (Key words: premilking, germicide, antimicrobial peptide, nisin) INTRODUCTION

New MI are the consequence of bacteria gaining access to the mammary gland between

Received November 4, 1991. Accepted June 25, 1992. 1992 J Dairy Sci 753185-3190

milkings (2, 6) and the transfer of pathogens either between quarters within a cow or between cows at milking time (3, 6, l l ) . Prevalence of IMI in a herd is determined by the rate and the duration of the IMI (2, 5). Postmilking teat dipping is an effective method for reducing the prevalence of contagious organisms (Staphylococcus aureus and Streptococcus ugalactiae), and premilking teat dipping is effective in reducing the incidence of IMI caused by environmental pathogens. The use of premilking teat dipping has been shown to be effective in reducing new IMI (9, 12), but the risk of milk supply contamination increases when the germicide is not completely removed before milking (9, 10). The evaluation of a germicide’s potential for use as a teat sanitizer prior to milking needs to consider before field efficacy evaluation 1) the germicide’s ability to exhibit broad and rapid activity toward mastitis pathogens on the teat, 2) the formulation’s potential for skin irritation, and 3) its potential for introducing hazardous residues into milk under recommended use. The purpose of this study was to evaluate the potential of a nisin-based germicidal formulation for use as a teat sanitizer. Nisin is a nontoxic antimicrobial peptide. A preparation of nisin (Nisaplin; Aplin and Barrett, England) has been used worldwide as a preservative for cheeses and canned goods (7). Potentially, nisin is a safe alternative to more traditional chemical germicides, such as iodines and chlorhexidines.Nisin in a germicidal sanitizer that is formulated for use before milking could lower the risk of introducing poten-




(ATCC 29740), Srrep. agalactiae (Cornel1 48), Streptococcus uberis (ATCC 27958), Klebsiella pneumoniae (field strain), and Escherichia coZi (field strain). Pure suspensions were prepared for all organisms as previously reported (13) except that the final challenge suspensions were made in .l% proteosepeptone broth (Difco Laboratories). A sufficient number of frozen ampules were prepared so that a new culture was available for each trial. A frozen 1-ml ampule was thawed, added to a 6-ml tube of trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD), and incubated aerobically for 5 to 7 h at 37°C. A purity check of the 6-ml tube was made by MATERIALS AND METHODS streaking .01 ml onto trypticase soy agar containing 5% sheep blood and .l% esculin (BBL A highly purified...
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