week EIGHT: Taking Sterile Milk Samples & The California Mastitis Test

February 28, 2013 in Weekly Series: A Tool Box for Udder Health

week EIGHT: Taking Sterile Milk Samples & the California Mastitis Test

Image2-2Taking Sterile Milk Samples

Background
Part of mastitis control programs include microbiological analysis of milk from cows suspected of having mastitis. Culturing milk samples allows the identification of the bacteria that are causing the mastitis and the application of preventive management programs. Strict aseptic procedures must be used when collecting milk samples to avoid contamination with bacteria present on the skin of the cow, hands of the sampler and barn environment.

Equipment
• Sterile single use disposable plastic vials with tight fitting caps. Vials should be at least 15 ml capacity.

• Nitrile or latex gloves should be worn to reduce contamination of samples with bacteria present on the samplers’ hands.

• Alcohol soaked cotton, gauze or baby wipes are needed for adequate teat sanitation.

• Vials should be labeled with permanent markers to identify the cow and quarter being sampled.

• If multiple samples will be collected, racks should be used for convenient handling.

Individual Quarter Milk Sample
Udders and teats should be clean and dry prior to individual quarter sample collection.

• A strip cup can be used to examine a cow suspected with Clinical Mastitis.

• Forestripping 3 streams of milk from the teat to be sampled removes contaminated milk from the teat canal. The use of this practice will reduce the likelihood that unusable contaminated samples will be obtained.

• Teat sanitation can be accomplished through the use of Predipping with 0.5% iodine. The disinfectant must remain on the tests for 20 to 30 seconds prior to removal.

• It is important to thoroughly dry the teat with a single use cloth or paper towel.

• Special attention should be paid to the teat end to achieve adequate sanitation.

• 70% ethyl or isopropyl alcohol must be used to fully sanitize the teat end prior to obtaining the milk sample. The scrubbing of the teat end should be vigorous to fully sanitize the teat.

• Alcohol is an ideal antiseptic because it evaporates quickly and will not contaminate the milk sample. If multiple teats are sampled a separate swab must be used for each sample.

• Sanitation is not complete until the surface of the swab remains clean after it is used.

• The cap should be removed from the sample vial without touching the inside and it should be held so that the inner surface faces down. This will prevent sample contamination.

• The vial should be held at an angle so that debris does not fall into it.

• Milk from the teat to be samples can be directed at an angle into the sampling vial. A sample size of 3-5 ml is usually adequate.

• The cap should be immediately replaced after the sample is obtained.

Composite Milk Samples
Individual quarter samples are the most sensitive way to determine the type of mastitis pathogen that is present, but sometimes a “composite sample” is collected. The term composite milk sample refers to the collection of milk samples from all 4 quarters into a single sample vial. This type of sample is often used in herd screening programs for contagious mastitis pathogens such as Strep agalactia or Mycoplasma bovis. Composite samples are used to reduce the cost of sampling but generally result in some level of false negative results.

• Predipping is the first step in obtaining a composite milk sample.

• The process of teat preparation for obtaining composite milk samples is identical to that of obtaining individual quarter samples but the order of teat preparation is critical. To reduce cross teat contamination, the far teats should be sanitized before the near teats. Individual alcohol swabs should be used to sanitize each individual teat.

• After teats are prepared, an equal volume of milk should be obtained from each quarter into the same vial. The order of sampling is near teats before the far teats.

• Immediately after culturing, the milk samples should be placed on ice or in a refrigerator. The milk should be cultured within 24 hours of obtaining the sample.

• If samples cannot be cultured within 24 hours, they must be stored in a freezer as soon as possible. Isolation of staph and strep may be improved by freezing. The number of samples positive for E. coli may decline after freezing.

Summary
The correct steps for taking sterile milk cultures are:

• Use Proper Equipment
• Clean, Dry Teats and Udders
• Forestrip Teats to be Sampled
• Predip Teats
• Dry Teats
• Sanitize Teat Ends
• Take Sample
• Refrigerate or Freeze Sample

week EIGHT: Taking Sterile Milk Samples & the California Mastitis TestCalifornia Mastitis Test

Background
Identification of cows subclinically infected with mastitis is an important part of mastitis control programs. Cows with subclinical mastitis infections do not have a swollen udders or abnormal looking milk. However because an infection is present the somatic cell count in the milk will be elevated. The California Mastitis Test (CMT) is a simple, inexpensive way of detecting unseen infections. Unlike other tests that require laboratories to interpret the results, the CMT is a cowside test that gives valuable, rapid results.

Equipment
Milk collected for CMT should be collected in a hygienic manner. Samples of milk from each quarter should be collected in a clean CMT Paddle free of any milk residue. The CMT paddle has four shallow cups marked A,B,C, and D for easy identification of the individual quarter from which the milk was obtained. The CMT solution should be properly reconstituted according to package instructions.

Procedure
· About ½ teaspoon (2 cc) of milk is taken from each quarter. The is the amount that would be left in the cups when the paddle is held nearly vertical, or in an upright position.

· An equal amount of CMT reagent is added to each cup in the paddle.

· The paddle is then rotated in a circular motion to thoroughly mix the contents. The mixing should not last more than 10 seconds.

· The test must be “read” quickly because the visible reaction tends to disintegrate after about 20 seconds. The reaction is visually scored depending on the amount of gel that forms. The more gel, the higher the score.

Reading a CMT Test
· N = negative. There is no evidence of thickening in the mixture.

· T = trace. There is a slight thickening of the mixture. Trace reactions seem to disappear with a continued rotation of the paddle.

· 1= weak positive. There is a distinct thickening of the mixture, but there is no tendency to form a gel. If the paddle is rotated 20 seconds or more, the thickening may disappear.

· 2 = distinct positive. There is immediate thickening of the mixture with a slight gel formation. As the mixture is swirled, it moves toward the center of the cup, exposing the bottom of the outer edge. If the motion stops, the mixture levels out and covers the bottom of the cup.

· 3 = strong positive. A gel is formed and the surface of the mixture becomes elevated (like a fried egg). A central peak remains projected even after the paddle rotation is stopped.

Interpretation of CMT Scores
CMT scores are directly related to average somatic cell counts. The following table shows how they are related. As indicated, the somatic cell range can vary from 0 to over 5 million cells per milliliter of milk. Any reaction of trace or above indicates that the quarter has subclinical mastitis.

CMT Score Somatic Cell Range Interpretation
N (Negative) 0 – 200,000 Healthy Quarter
T (Trace) 200,000 – 400,000 Subclinical Mastitis
1 400,000 – 1,200,000 Subclinical Mastitis
2 1,200,000 – 5,000,000 Serious Mastitis Infection
3 Over 5,000,000 Serious Mastitis Infection
* Table: Jasper, D.E. 1967. Proc. Of National Mastitis Council (adapted)

Advantages of the CMT
· The CMT is fairly accurate in measuring the somatic cell concentration in milk and correlates well with other tests.
· It is sensitive.
· It is inexpensive.
· The test is simple and requires little equipment.
· The paddle is easy to clean up – simply rinse with water.

Disadvantages of the CMT
· Test scores may vary between individuals performing the test.
· Scores represent a range of somatic cells present rather than an exact count.
· Cows fresh less than 10 days or cows that are nearly dry may produce a false positive reaction. Cows should be tested closer to the middle of their lactation.
· Occasionally, acute clinical mastitis milk will not score positive if the somatic cells have been destroyed by toxins from the infecting organism.