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* West Agro Inc., Kansas City, MO 64153
Mastitis Research Laboratory, Hill Farm Research Station, Louisiana State University Agricultural Center, Homer 71040
Corresponding author: C. Foret; e-mail: chris.foret{at}DeLaval.com.
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ABSTRACT |
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Key Words: iodine • Staphylococcus aureus • Streptococcus agalactiae • teat dip
Abbreviation key: TSA = trypticase soy agar, TSB = trypticase soy broth
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INTRODUCTION |
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In vitro testing was performed on both products under AOAC conditions at 4°C. The in vitro tests were performed by Udder Health Systems, Inc., Bellingham, Washington, according to AOAC (1990) specifications, except that temperature was adjusted to 4°C.
Both products demonstrated excellent kill of Staphylococcus aureus at temperatures of 20 to 25°C. However, the in vitro test results given in Table 1
show that product 1 was able to kill Staph. aureus more rapidly at low temperatures.
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The Hill Farm Research Station dairy herd of 96 cows was used in a controlled infection trial to determine the efficacy of two iodine teat dips for preventing IMI with Staph. aureus and Streptococcus agalactiae.
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MATERIALS AND METHODS |
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Sampling schedule.
The bacteriologic status of mammary quarters was determined at initiation of the trial by collecting and culturing duplicate milk samples. A third sample was collected from specific quarters and cultured when results from the first two samples differed.
Milk samples were collected and analyzed weekly during the trial. When either Staph. aureus or Strep. agalactiae was present for the first time in a previously uninfected quarter, a second sample was collected immediately and cultured. All quarters were eligible for new infections during the trial except 1) those infected with organisms of the same species as challenge organisms, and 2) those with deformed or abnormal teats. Fossomatic SCC were determined on one set of quarter milk samples collected at the initiation of the trial and monthly thereafter.
Collection of milk samples.
Before sampling, two or three streams of foremilk were discarded. Each teat apex was scrubbed for several seconds with a cotton pledget moistened with 70% alcohol. Teats on the opposite side of the udder from the technician were sanitized first, and milk samples were collected in reverse order in sterile, snap-cap plastic tubes and refrigerated at 5°C.
Laboratory culture procedures.
Samples were mixed by shaking, and a 0.01-ml aliquot was streaked on trypticase soy agar (TSA) containing 5% bovine blood. Plates were incubated at 37°C for 48 h and examined to identify microorganisms present. An IMI was confirmed when: 1) Staph. aureus or Strep. agalactiae was isolated from a clinical quarter, 2) two consecutive samples yielded 500 or more cfu/ml of the same pathogen, or 3) three consecutive samples contained 100 to 400 cfu/ml of the same pathogen.
Treatment method.
At the afternoon milking, Monday through Friday, the lower third of all four teats of each cow was experimentally exposed to a challenge suspension containing both Staph. aureus (Newbould 305) and Strep. agalactiae (McDonald 44) immediately after milking. Within 5 to 10 s thereafter, two teats (left front, right rear) were dipped full length with teat dip; the remaining two teats served as undipped controls. Teats were exposed to challenge organisms to increase the number of pathogens impinging on the teat apex, resulting in an increased rate of IMI.
Preparation of challenge suspensions.
Stock suspensions of Staph. aureus (Newbould 305) were prepared weekly. The contents of one lyophilized vial of Staph. aureus were reconstituted in 6 ml of trypticase soy broth (TSB) and incubated at 37°C for 5 to 7 h. This culture was used to inoculate 500 ml of TSB, which was incubated on a gyratory shaker for 16 h. After incubation, bacterial cells were pelleted by centrifugation, washed twice with 0.1% proteose-peptone, and resuspended to the original volume in proteose-peptone. Serial dilutions were made in proteose-peptone, and 0.1 ml was plated on TSA. Plates were incubated for 24 h at 37°C, and colonies were counted to ascertain the microbial concentration of the stock suspension. This suspension was stored at 5°C and used daily for 1 wk to prepare challenge suspensions of Staph. aureus (Hogan et al., 1990).
Streptococcus agalactiae (McDonald 44) cultures were prepared by thawing a frozen vial of Strep. agalactiae, and a 0.01-ml aliquot was streak plated onto each of five TSA plates. Plates were incubated at 37°C for 16 h and stored at 5°C to serve as stock cultures for a 1-wk period.
Daily challenge suspensions of Strep. agalactiae were prepared by inoculating 6-ml tubes of TSB with six colonies from a TSA stock plate. The 6-ml cultures were incubated for 7 h at 37°C. A 500-ml flask of TSB was inoculated with the 6-ml cultures and incubated for approximately 18 h at 37°C on a gyratory shaker. Specific aliquots of the culture were added to pasteurized milk as needed to adjust the concentration of Strep. agalactiae to approximately 5 x 107 cfu/ml.
An aliquot of the Staph. aureus stock suspension was added to the Strep. agalactiae suspension to obtain a concentration of approximately 5 x 107 cfu/ml of Staph. aureus. This bacterial suspension was taken immediately to the milking parlor to challenge teats during the afternoon milking. A plate count was conducted daily on challenge suspensions and recorded.
Statistical methods.
The Student t test was used to evaluate the statistical difference between the dipped and control quarter data. This procedure was described by Hogan et al. (1990). The statistical probability of difference in IMI between the control and dipped quarters is measured from the value of t. t = [(x1/n1) - (x2/n2)]/[(x1 + x2)/(n1n2)]0.5, where: x1 = number of new IMI in control quarters, x2 = number of new IMI in treated quarters, n1 = (number of control quarters)(days of exposure), n2 = (number of treated quarters)(days of exposure); % Reduction = 100[(x1/n1) - (x2/n2)]/(x1/n1). A teat dip is considered efficacious if the percentage of reduction is at least 40%.
Scoring of teat condition.
Characteristics of teat end and teat skin condition in dipped and control teats were scored immediately before the trial was initiated and at the conclusion of the trial to determine any effects of the teat dip products on the condition of teat ends and lateral teat skin. An ordinal scale of 1 to 5 was used. (See specifications of ordinal scale in Table 2).
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RESULTS |
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and 4. In herd 1, in which product 1 was tested, a total of 20 new Staph. aureus IMI were confirmed: 18 in control quarters and two in dipped quarters (Table 3). Twenty-six new Strep. agalactiae IMI were confirmed: 20 in control quarters and six in dipped quarters (Table 3). The teat dip reduced the infection rate 89.7% for Staph. aureus (P < 0.001) and 73.1% for Strep. agalactiae (P < 0.005).
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). Twenty-one new Strep. agalactiae IMI were confirmed: 17 in control quarters and four in dipped quarters (Table 4). This group was dipped for an extra week with S. agalactiae in an effort to increase the total number of Strep. agalactiae infections. The teat dip reduced the infection rate for Staph. aureus 86.2% (P < 0.001) and 78.4% for Strep. agalactiae (P < 0.005).
Teats in both dip groups were scored for chapping, cracks, and other forms of irritation, both before and at the end of the trial. There were no changes in teat end or teat skin characteristics during the trial. In addition, any teat irritation or abnormalities could not be attributed to use of either teat dip product. The average condition scores are listed in Table 2.
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CONCLUSIONS |
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Received for publication April 7, 2003. Accepted for publication June 7, 2003.
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REFERENCES |
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This article has been cited by other articles:
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  C. Foret, H. Aguero, and P. Janowicz Efficacy of two barrier iodine teat dips under natural exposure conditions. J Dairy Sci, June 1, 2006; 89(6): 2279 - 2285. [Abstract] [Full Text] [PDF]
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R. L. Boddie, W. E. Owens, C. J. Foret, and P. Janowicz Efficacy of a 0.1% Iodine Teat Dip Against Staphylococcus aureus and Streptococcus agalactiae During Experimental Challenge J Dairy Sci, September 1, 2004; 87(9): 3089 - 3091. [Abstract] [Full Text] [PDF]
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