Upload
mohammad-ashraf-paul
View
112
Download
3
Embed Size (px)
Citation preview
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 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 , which adhered to the margin
Severe mastitis infection
3 (Strong Positive) The gel formation with convex projection, the gel did not dislodge after swirling movement of
the paddle
Severe mastitis infection
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 into a milky whey and white particles
(+) Distinct Positive
Mixture thickens immediately and follows glass rod
(++) strong positive
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.