Screening Tests for early Detection of Mastitis

via Milk Examination

Mohammad Ashraf paul

Division of Livestock Products technology

Faculty of Veterinary Sciences & Animal Husbandry, SKUAST- K,
Alusteng, Ganderbal, Kashmir
Mastitis is a parenchymal inflammation of the mammary gland, which is caused by

microorganisms, usually bacteria that invade the udder, multiply and produce toxins which are

harmful to the mammary gland. It is characterized by physical, chemical and usually

bacteriological changes in milk and pathological changes in glandular tissues. Mastitis is one of

the most important deadly diseases of milch animals, responsible for heavy economic losses due

to reduced milk yield (up to 70%), milk discard after treatment (9%) cost of veterinary services

(7%) and premature culling (14%) and also causes changes in glandular tissues, affecting quality

and quantity of milk. It is a global problem and adversely affects animal health, quantity of milk

and economics of milk production and every country including developed ones suffer huge

financial losses. This is evidenced by a recent report where in annual economic losses incurred

by dairy industry in India on account of udder infections have been estimated about Rs. 6053.21

crores. Out of this Rs. 4365.32 core (70%-80% loss) has been attributed to sub clinical version

of udder infections. Unlike the clinical mastitis, in sub clinical mastitis there are neither visual

abnormalities in milk (like blood, clots, flakes etc) nor in mammary gland (like swelling hotness,

cracks etc). Therefore, knowledge of routine physical examination of udder together with

diagnostic screening tests for early detection (i.e. during sub clinical form) of mastitis and proper

treatment of affected animals is of paramount importance in order to minimize losses

encountered due to sub clinical as well as clinical mastitis. We shall be focusing on screening

tests for early detection of mastitis via examination of milk.

EXMAINATION OF THE MILK:

Before carrying out any mastitis test, the freshly drawn milk should be examined by naked

eye for the visible abnormalities in the 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 flakes or clots in the milk or the milk may be thin or

watery and at times yellow in color.

Over a period of years many tests have been developed for the diagnosis of mastitis. For

convenience they may be divided into two groups, viz.:

(A) Indirect Tests which depend upon the development of palpable lesions in the udder or

changes in the composition of milk

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

milk.

Indirect Tests

Indirect tests are useful in determining the quality of milk, and in the absence of laboratory

facilities those which are suitable for use under field conditions may be helpful in detecting and

eliminating some of the cows that are affected with chronic mastitis. Certain indirect tests

especially the leukocyte count are needed to supplement cultural finds in the diagnosis of

mastitis
Strip Cup Test: It was Moak who first made use of strip cup test for the detection of visible

particles of milk as a means for diagnosing mastitis. This test is useful in field for physical

examination of milk. This test can be carried out by laymen. 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 flakes. The milk flakes can be seen by tilting the cups at an angle. This

test is very useful in primary screening of animals for mastitis.

Blackboard Strip Test: This test is also another type of strip cup test. This 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 flakes, 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. Lesser degree of wateriness can also be detected but

while these are probably of significance in some cases, one should be cautious in their

interpretation.

California Mastitis Test (CMT): California mastitis test is a simple, inexpensive and rapid

screening test for mastitis. The test is based on the increase in number of leukocytes and

alkalinity of the mastitis milk. These changes are due to inflammatory exudation and increased

contents of basic salts during inflammation. The original Schalm reagent (Tri-ethanolamine

sulphate and bromocresol purple) is not available in India. But recently 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 can be tested using the CMT for up to

12 hours, reliable readings can be obtained from refrigerated milk for up to 36 hours. If stored
milk is used, the milk sample must be thoroughly mixed prior to testing because somatic cells

tend to segregate wit the milk fat.

Test Procedure: A Plastic paddlle with four chambers or shallow cups are used to perform the

test. About 3 ml of milk directly striped into the labeled cups LF, LH, RF and RH from the

respective four quarters. To ensure equal quality of milk in each cup, the paddle should be titled

slightly at an angle of 450 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 the milk and reagent is shaken gently in a rotating manner of the paddle in the

horizontal plane.

Immediately after mixing or reaction must be scored within 15 seconds of mixing 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 detailed

in table below:

Table
CMT Score Description Interpretation
N (Negative) No Change Healthy quarter

T (Trace) Slime formed which disappeared with Sub Clinical mastitis

continuous movement of paddle
1 (Weak) Distinct slime, but no gel formation Sub clinical mastitis

2 (Distinct Positive) Viscous with gel formation , which adhered to Severe mastitis infection
the margin
3 (Strong Positive) The gel formation with convex projection, the Severe mastitis infection
gel did not dislodge after swirling movement of
the paddle

Sodium Lauryl Sulphate Test (SLS test): This test is similar to CMT in principle as well as

procedure. The difference is that in this test 3% sodium lauyryl sulphate is used instead of CMT
reagent. This solution (test reagent) is prepared by adding 3 g of sodium lauyryl sulphate powder

to 100 ml of distilled water. The suspension is heated to 500 C so as to make a clear solution. The

pH of the solution is adjusted to 8.0 by using HCl or NaOH as per the need.

White Side Test (WST): The test is based on the increase in number of leukocytes in mastitis

milk. The original test described by Whiteside (1939) was made by stirring 2 ml of normal

NaOH into 10 ml of milk. The development of a “viscid mass” was regarded as positive.

The test was modified by Murphy and Hanson (1941), they added one drop of normal NaOH to

five drop of milk on a glass slide and stirred the mixture for 20 seconds with a glass rod.

Negative samples were entirely free of precipitate. The amount of precipitate formed was graded

from slight to thick viscid mass and was considered to be indicative of the degree of irritation in

the udder. They regarded this procedure as superior to Bromothymol blue test for use in field as a

presumptive test for chronic bovine mastitis.

Later on, this was further modified by schalm et al (1971), they used one drop of 4% NaOH to 5

drops of milk to be tested on a clean glass slide and mixed vigorously with a glass rod for 20

seconds. The results are graded on the basis of precipitation of milk as given below:

Grading Description
Mixture remains opaque and free of particles (-) Negative
Fine dispersed particles on close inspection (+/-) Trace
A definite thickening and mixture separates (+) Distinct Positive
into a milky whey and white particles
 Mixture thickens immediately and follows (++) strong positive
glass rod

Surf Filed Mastitis Test: This test was developed by Muhammad et al (1995). This test is easy,

cheap and enough sensitive to detect all cases of sub clinical mastitis. The advantage of this test

is that the readily available house hold surf (detergent) is used as reagent. The principle of this

test is the reaction of somatic cells DNA with detergent (Surf) and leads to the formation of gel

of varying degree depending upon the number of somatic cells in the milk.

Procedure:

• 3% Surf solution-Dissolve 3 g of surf in 100 ml of clean tap water or 6 teaspoonful of

house hold detergent surf in 500 ml of clean tap water. The test solution is table for 6

months at room temperature

• A plastic paddle with four receptacles for the respective quarters of an animal can be

fabricated from locally available plastic or Bakelite. When this paddle is not at hand,

suitable container such as tea cups or glass may be substituted.

• Take equal quantity of 3% reagent and milk in the paddle or container. The mixture is

swirled for about 1 minute and then examined visually for the presence of small floccules

and gel. If floccules or gel is formed it indicates the presence of intramammary infection.

In the absence of any floccules or gel, sample is negative

Modified Aulendorfer Mastitis Probe (MAMP) Test: This test can be effectively used to detect

sub clinical mastitis in cows but it is time consuming. The test involves the use of MAMP

reagent, which contains sodium lauryl sulphate and urea. Milk samples are collected and added
to equal volume of reagent, mixed thoroughly in a test tube and left undisturbed for 24 hours at

room temperature. A positive reaction will be gelatinous product, which will be milky initially

and later becomes flaky and begins to clear from bottom depending on the intensity of reaction.

Bromothymol Blue (BTB) Test: This is a pH indicator test, after adding the milk with

Bromothymol blue, different colors are developed due to changes in pH of milk. The normal pH

of milk is 6.4 to 6.8 and isotonic with blood plasma. In mastitis, during late lactation and dry

period, the concentration of lactose and caseins in the milk is reduced whereas sodium chloride

and sodium bicarbonate pass into the alveoli from plasma to maintain isotonicity, Therefore,

during such situations milk becomes alkaline with greater amount of chlorides (Galdhar et al

2005) .In 1920 Baker and Breed used Bromothymol blue to detect changes in the pH value in

milk. Subsequently, the test has been widely used in the diagnosis of mastitis.

For this test BTB card test papers may be prepared from whatman filter paper No. 1. The

diagnostic card can be prepared by adding one drop of BTB test solution (Bromothymol blue-1.6

g in 100 ml ethanol) at 4 different spots on the paper and indicates left fore (LF), left hind (LH),

right fore (RF) and right hind (RH). One drop of suspected milk has to be put directly on the

indicator spot and observed changes in color are scored as follows : Pale green indicates normal

quarter and +, ++ and +++ (according to change in color from moderate green to dark blue

green). The disadvantage of this test is that cow in later stages of lactation may give false

positive reaction.

Bromocresol Purple Test: This test is also pH indicator test. Take 2-3 drops of 0.9%

bromocresol purple solution in a test tube and add 3 ml of freshly drawn milk. Normal milk

appears as yellow (Pale grayish purple) while mastitis milk will appear as blue or purple.
Mastrip Test: Mastrip is a cellulose based bromothymol blue strip impregnated with stabilized

ions, marketed by Dabur Ayurvet Ltd. India for the detection of mastitis. One drop of milk is

placed on the mastrip and reaction is read from the standard color matching strip. The colour

reaction is graded in table below:

Table

Grading Description
- No change in color (Yellow)
+ Light Green
++ Dark Green
+++ Bluish Green

Chloride Test: This test denotes presence of increased quantity of chloride in mastitis milk. In

normal milk, chloride level varies from 0.08 to 0.14%. There exists an inverse relationship

between the concentration of lactose and sodium chloride which depends on the stage of

lactation. Davis (1999) reported that in mastitis there is decreased amount of lactose and

increased amount of sodium chloride to maintain the normal milk pressure hence during

inflammation (mastitis)there is increase in the chloride content ( 0.14%).

Procedure:

Solution:

Solution A
 Silver nitrate -1.34 g

Distilled water -1000 ml

Solution B

Potassium Chromate -10 g

Distilled water - 1000 ml

Take 1 ml of milk and add 5 ml silver nitrate solution (solution A) followed by two drops of

potassium chromate solution (Solution B). Observe for the change of color. Development of

yellow color indicates the chloride level > 0.14 % i.e. quarter is positive for mastitis and

appearance of brownish red color indicates chloride level < 0.14 % i.e. negative to mastitis. This

test is more sensitive than the Bromothymol test

Electrical conductivity: Electrical conductivity of milk has been considered as a possible index

of mastitis. The specific conductance of milk reflects its concentration and activity of ions and is

of the order of 0.004 to 0.005 Siemens or ohms-1cm-1(4-5mScm-1) at 25oC.Higher values

represent mastitis infection in which there is an increase in the concentration of sodium and

chloride in milk. The test can be effectively carried out in a laboratory with the aid of an

electronic digital conductivity meter or in the field with the help of a portable pocket type

conductivity meter.

pH measurements: The PH of mid lactation cow milk falls between6.5 and 6.7 with 6.6 the most

usual value at 25oC.Differences in PH and buffering capacity among individual lots of

fresh milk reflect compositional variations arising from the functions of the mammary gland.

In general the pH is lower (6.0) in colostrums and higher (up to 7.5) in case of mastitis than in
normal milk of mid lactation. Colostrum and mastitis milk are known to differ radically in

proportions of the proteins and certain salts. Relating these compositional features with the early

detection of mastitis seems plausible.