National Mastitis Council

Recommended Protocol for Determining Efficacy of a Premilking Teat Dip
Based on Reduction of Naturally Occurring New Intramammary Infections

Experimental Design
Predip teats of half of the quarters in the product being tested. Experimental design can be either a split-herd or split-udder design. Predip all teats of half the cows in the experimental product in a split-herd design. Cows in the remainder of the herd serve as controls. In this situation, take care to ensure that cows are balanced by: 1) parity; 2) stage of lactation; and 3) bacteriological status of quarters. Ensure that the two groups are milked by the same personnel in the same facility. When a half-udder design is used, either predip two diagonal teats or teats on either the right or left side of each udder with the experimental product. Determine number of new intramammary infections (IMI) that occur in quarters with teats predipped in the experimental product and in control non-predipped quarters.

Milking Procedures
Milking routine for cows or quarters in predip and control groups should be consistent except for predipping the treatment group. Teats on control quarters should be forestripped, washed, and dried with single service paper towels in preparation for milking. Teats on treatment quarters should be forestripped, washed and experimental predip product applied with a minimum contact time of 15-30 seconds as recommended on the label, and thoroughly dried with a single service paper towel. Detail descriptions of teat preparation techniques including the manner that teats are washed and concentration of sanitizer in wash solution are imperative. Apply a product of known efficacy (based upon NMC recommended protocols for evaluating efficacy of postmilking teat dips) as a postmilking teat dip to all teats after milking machine removal. The same postmilking teat dip product must be used on both treatment and control teats.

Selecting Experimental Herds, Cows, and Quarters
Trials should be conducted in at least two herds. Conduct trials in herds where wholehearted cooperation of managers to comply with experimental procedures can be attained. Monitor milking equipment and milking management practices carefully and regularly to minimize machine-mediated infections. This is especially necessary in commercial herds where constant supervision by the investigator will not be practical. All quarters are eligible for new IMI except quarters with teats that are abnormal due to previous injury. Exclude quarters with teats that are injured during the trial for the remainder of that lactation; such quarters may re-enter the trial after a dry period if the injury has healed.

Sampling Schedule and Procedures
Collect duplicate or two consecutive single quarter milk samples aseptically to determine existing infections in the herd at the beginning of the trial. Obtain a third sample when results of the first two samples do not agree. Culture single quarter milk samples monthly or duplicate sample bimonthly during the trial. Culture duplicate samples from cows calving and herd additions prior to inclusion in the trial. When any quarter develops clinical mastitis, collect and culture duplicate milk samples from all four quarter before any treatment is administered. Collect and culture duplicate milk samples from individual cows at drying off or upon leaving the herd. Collect all samples consistently either immediately before of after a regular milking using standard procedures (1).

Criteria for Diagnosing Infections
Examine all milk samples bacteriologically and identify organisms isolated according to standard procedures (1). In determining that a quarter is free of infection when it enters the trial, no pathogens may be recovered from either of two of the initial samples. Diagnose an IMI when the same bacterial species is isolated from: 1) both of the duplicate samples taken bimonthly or from clinical quarters or; 2) two consecutive monthly samples taken during the trial. The status of a quarter should be recorded as a bacteriologically-negative clinical case when bacteriological results of duplicate samples from a clinical quarter do not match.

An individual quarter is eligible for only one infection per bacterial species during a lactation (i.e., only one Escherichia coli infection per quarter per lactation). Quarters infected in one lactation may be included in the trial in the subsequent lactation if it is determined that the infection was eliminated during the dry period.

Trial Size and Duration
Duration of each trial should be at least 12 months to include each season of the year. The length of a trial required to demonstrate efficacy of a predip will depend on the number of quarters available initially and the rate of new IMI in control and treated groups. Guidelines for estimating the number of quarters required, the probable duration of a trial and the point at which a trial may be terminated after 12 months are those detailed previously in the postmilking teat dip experimental challenge protocol (2). These guideline are applied to total IMI and separately to each species of bacteria against which efficacy is tested.

Data Presentation
The report of a trial should include: 1) duration of the trial; 2) number of quarters in the trial at the onset and on the date of each monthly or bimonthly sampling; 3) number of new IMI, categorized by bacterial species or type that occurred in control and predipped quarters; 4) the percentage differences in new IMI between predipped and control quarter for each bacterial species; 5) the number of new clinical cases, categorized by bacteriological status, that occurred in control and predipped quarters; and 6) the percentage difference in new clinical cases between predipped and control quarters.

1. Harmon, R.J., R..J. Eberhart, D.E. Jasper, B.E. Langlois, and R.A. Wilson. 1990. Microbiological Procedures for the Diagnosis of Bovine Udder Infection. 3rd edition. National Mastitis Council, Inc., Arlington, VA.

2. Hogan, J.S., D.M. Galton, R.J. Harmon, S.C. Nickerson, S.P. Oliver, and J.W. Pankey. 1990. Protocols for evaluating efficacy of postmilking teat dips. J. Dairy Sci 73:2580.

Protocol developed by the National Mastitis Council Research Committee; Published in the National Mastitis Council 1991 Annual Meeting Proceedings, pg. 157.

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