Module 6. Mastitic milk

Lesson 26

DETECTION OF MASTITIC MILK

26.1 Introduction

Tests are based either on the detection of compositional change in milk such as increase in pH,  increased chloride,  increased catalase content or on the detection of causative micro-organism or somatic cells by  direct microscopic count, Hotis test and blood agar plating etc. Sampling is done aseptically.   Discard first milk into strip cup.  Take sample directly into sterile sample container. For herd test, mixed quarter samples and for detail test, individual quarter samples are taken for testing of mastitis.

26.2 Tests Based on Compositional Changes

26.2.1 Examination of milk

Before carrying out any test for detection of mastitis, the freshly drawn milk should be examined for the visible abnormalities in milk. In dry period milk turns too watery. Appearance of udder secretion in advanced cases of chronic mastitis is usually abnormal in appearance at irregular intervals. In case of acute mastitis, the secretion becomes grossly altered. The visible abnormalities may be the presence of flecks or clots in milk or milk may be thin or watery and sometimes yellow in color.

For convenience these mastitis milk tests are divided into two groups:

(A) Indirect Tests that depend upon the development of palpable lesions in udder or changes in the composition of milk 

(B)  Direct or cultural tests to determine the presence and identity of mastitis micro-organisms in milk.

26.2.1.1 Indirect tests

These are useful in determining the quality of milk. In the absence of lab facilities these are suitable under field conditions may be helpful in detecting and isolating the animals that are affected with chronic mastitis. Certain indirect tests, especially the leukocyte count are needed to supplement cultural finding in the diagnosis of mastitis

26.2.1.2 Appearance of milk

Gross changes in milk may be observed at the time of milking such as the presence of flecks, or clots in milk. This is the most common means of detection of clinical mastitis. Stripping the first few squirts of milk from each quarter into a strip cup at the beginning of milking is a preferred method of detecting flecks or clots in the milk.

26.2.1.3 Strip cup test

This test is useful under field conditions for physical examination of milk. This is a simple test used for finding out the presence of fibrin, mucous and clots of milk in foremilk that is an indication of mastitis. Most cases of acute mastitis and 10% of chronic infections are detected by this test. In this test, enamel plate divided in four strip cups is used. The bottom of the plate is black colored, so that it gives a good contrast to easily observe the milk flecks. The milk flecks can be seen by tilting the cups at an angle. This test is quite useful in primary screening of animals for mastitis detection.

26.2.1.4 Blackboard strip test

This test is another type of strip cup test that usually consists of some flat material with a smooth black surface, cut to fit at an angle lengthwise in a shallow basin, such as a small rectangular baking pan. As the streams of milk from each teat are milked on the sloping black surface, any pinpoint flecks, clots, or thick milk are revealed, just as with the strip cup test. Marked wateriness, much of which is not detectable by any other stable test, can easily be shown by the blackboard test.

26.2.1.5 pH

The pH of normal milk varies between 6.6 - 6.8. The milk from infected udders is usually alkaline in reaction (pH 7.0 to 7.4) and this can be detected by observing the color change shown by a suitable pH indicator, bromothymol blue, added to milk. Mastitic milk has >6.8 pH.5 ml milk is mixed with 1ml of 0.04% bromothymol blue and color change is observed.  Blue green to green color is indicative of mastitis, whereas normal milk shows yellow color. Milk in advanced lactation also has an alkaline reaction and therefore gives a positive test and thus a negative test cannot be taken as evidence of absence of infection. The test can detects about 70% of infected cases and hence, may be supported by other tests.

26.2.1.6 Chloride test

The chloride content increases in milk of animals suffering from mastitis.   Animals in early or in late lactation may give false positive reaction to chloride test. Normal milk has a chloride content of 0.08 to 0.14%. Abnormal milk has more than 0.14%chloride content. For test measure 5 ml of 0.134 % silver nitrate solution and transfer into a test tube. Add two drops of 10 % potassium chromate indicator and this gives red color.  Add exactly 1 ml of milk to the contents of tube and mix.  Observe the color of mixture. If the sample contains an abnormally high percentage of chloride, the red color will change to yellow to indicate a positive test.  A brownish-red color indicates a negative test.

26.2.1.7 Catalase test

Living cells including leucocytes contain the enzyme catalase. The number of leucocytes in the milk increases during infection of udder and hence catalase test is used to measure the increase in catalase depending on the ability of it to break down hydrogen peroxide.  The presence of catalase is examined by evolution of oxygen on adding hydrogen peroxide. To 15 ml of milk in a test tube, add 5ml of 1% H2O2 solution.  Invert tube and incubate at 37C for 3h. Oxygen collects at upper portion. In normal milk the amount of oxygen liberated will be about 2 ml.  More than 2.5 ml of gas collected in the top of the tube is presumed to be due to an abnormal infected udder.  Milk from chronic cases may produce as much as 10 ml of gas.  Milk of animals in early or late lactation may give false positive tests to catalase.

26.2.1.8 Somatic cell count

An increase in the number of somatic cells in milk indicates that mastitic milk is present. This can be estimated by any of the following procedures:

Detection of Somatic Cells: Several methods for detection of mastitis are available for detecting somatic cells in milk, including the California Mastitis Test (CMT), the Wisconsin Mastitis Test (WMT; on-farm test), Microscopic Somatic Cell Count, and Electronic Somatic Cell Counting.

CMT is a simple, inexpensive and rapid screening test for mastitis. The test is based on the increase in number of leucocytes and alkalinity of mastitic milk. These changes are due to inflammatory exudation and increased contents of basic salts during inflammation. B.V Biologicals, India launched a CMT reagent along with plastic paddle by the name of CMT kit. The accuracy of this method is found to be 88.66%. Fresh, unrefrigerated milk is tested using the CMT for up to 12 h; reliable readings can be obtained from refrigerated milk for up to 36 h.

26.2.1.8.1 Procedure

A plastic paddle with four chambers or shallow cups is used to perform the test. About 3 ml of milk directly stripped into the labeled cups from the respective four quarters. To ensure equal quality of milk in each cup, the paddle should be titled slightly to allow overflow of excess of the milk samples, if any in any cup. Then approximately equal quantity of the test reagent (CMT reagent) is added to each cup. The mixture of milk and reagent is shaken gently in a rotating manner of paddle in the horizontal plane.

The CMT and WMT detect formation of a gel, when DNA in somatic cells reacts with a detergent. The reaction occurs on a paddle (CMT) and is graded (i.e. negative, trace, 1, 2, 3), or in a tube (WMT) and is measured in millimeters. CMT or WMT results can be used as rough estimates of the number of somatic cells in milk.

The different steps of CMT protocols are:

Step 1: Clean teats, strip a few squirts onto the ground, then collect several milliliters from each quarter into the respective wells.

Step 2: Tilt the plate in order to better estimate the volume of milk. Add a volume of CMT solution to each well that is approximately equal to the volume of milk in that well.

Step 3: Mix the CMT solution and milk by swirling the paddle.

Step 4: Positive reactions will be indicated by a gelatinous mass that collects near the center of the well as it is being swirled. Note the purple color of the gelatinous mass in this well.   

 Immediately after mixing reaction must be scored within 15 s of because weak reaction will disappear after that time. Any reaction of trace (T) or higher indicates that the quarter has sub-clinical mastitis. The reaction is graded by intensity of gel formation as below:

Table 26.1

CMT Score

Description

Interpretation

N (Negative)

No change

Healthy quarter

T(Trace)

Slime formed that disappeared with continuous movement of paddle

Sub-clinical mastitis

1(Weak)

Distinct slime, but no gel formation

Sub-clinical mastitis

2(Distinct positive)

Viscous with gel formation that adhered to margin

Severe mastitis infection

3(Strong positive)

Gel formation with convex projection, the gel did not dislodge after swirling movement of the paddle

Severe mastitis infection

 

26.2.1.9  Sodium lauryl sulphate test

It is presumptive determination of somatic cells and the severity of mastitis is based on the increase in viscosity of milk on adding sodium lauryl sulphate solution (4% in a 15% teepol solution, adjust pH to 12.0). Two ml of milk is mixed with 2 ml of test solution by shaking gently for 20 s in a tube and observed for coagulation of leucocytes.

Table 26.2 Sodium lauryl sulphate test and inference thereof

Observation(s)

Leucocyte count/ml

Condition

No viscous layer

<100,000

No mastitis

Slight viscous layer

100,000-500,000

Sub-acute

Central viscous cone disappears after stopping rotation

500,000-5,000,000

Acute

Central cone persists

>5,000,000

Acute

 

a)      Direct leucocyte count: The presence of ≥5,00,000 somatic cells per ml of milk is an indicative of mastitis. It is performed in a similar way as DMC by staining with Newmans stain.

26.2.2 Tests based on the detection of causative microorganisms

26.2.2.1 Microscopic examination

This is helpful in detecting the admixture of mastitic milk with herd milk. Presence of long chains of streptococci is indicative of mastitis due to Streptococcus agalactiae, whereas occurrence of cells in grape like bunches in milk suggests Staphylococcal mastitis.

26.2.2.2 Hotis test

It gives the most accurate information about mastitis infection.  It is based on the fact that Streptococcus agalactiae, when growing in milk, produces a characteristic colony like mass of cells adhering to the sides of test tube.  By the introduction of an acid indicator (bromocresol purple), the identification of these colonies or 'buttons' is facilitated by their characteristic yellow color. The appearance of yellow colonies of micro-organisms along the sides of tube or in bottom indicates infection with Streptococcus agalactiae. For this, 9.5ml of milk is mixed with 0.5ml of 0.5% aqueous bromocresol purple and incubated at 37C for 24-48 hrs a test tube.

Table 26.3 Interpretation of hotis test

Yellow colonies           

Presence of streptococcus

Flocculent on side of test tube

 Presence of especially Streptococcus agalactiae.           

Rusty brown color colonies          

 Presence of Staphylococcus aureus.

 

26.2.2.3 Blood agar test

Pre incubated (37˚C over night) mastitic milk sample are streaked on blood agar and incubated at 37C for 24hand examined for colony formation and haemolysis.

Table 26.4 Detection of specific causative organism using blood agar test

Observation

Micro-organism

Small colonies, α or β-haemolysis will occur or no haemolysis will occur in some cases

Streptococcus agalactiae

 α-haemolysis(small zone around colonies and green discoloration)

Streptococcus dysagalactiae

No reaction

Streptococcus uberis

Large colonies than streptococci, β-haemolysis(a wide zone of clearance around colonies)

Staphylococcus aureus

 

26.2.3.4 CAMP (Christie, atkin, munch and peterson) test

The CAMP test is quite specific for the detection of mastitis caused by Streptococcus agalactiae. A standard culture of Staphylococcus aureus is streaked vertically down across the center of a blood agar plate. The suspected streptococci plates are cross streaked horizontally at an angle taking care that this streaks do not come into contact with Staphylococcus aureus streak. After incubation at 37C overnight and observe for zones. Clear zone between the streaks of Staphylococcus aureus and milk sample indicates that is positive for mastitis of Streptococcus agalactiae.

26.3 Electrical Conductivity

Electrical conductivity of milk increases during mastitis due to increases in Na+ and Cl- and decreases in K+ and lactose. Changes in conductivity can be detected by hand-held or in-milk line instrumentation. The latter is the basis for the computerized milking systems that track electrical conductivity measurements on milk of cows at each milking. This data can be analyzed by computer programs to that have milk electrical conductivity that is altered from normal.