MASTITIS DIAGNOSIS

Presented by: Adil Rasool ParaY B.V.Sc.&A.H. 4th year, SKUAST-K VB-2011-1049

SIGNIFICANCE OF BOVINE MASTITIS ? Loss in milk production.

Poor milk quality.

Mastitis reduces milk by 21 % and Butter fat by 25 %.

Reduction in price.

Discarding of milk after the antibiotic treatment.

Additional treatment costs.

Increased labour costs.

Premature culling.

Reduced productive-life of cattle.

The most costly disease affecting dairy cattle throughout the world.

Increased costs for surveillance.

Lower value for culled cattle meat.

Inferior quality milk is unfit for the human consumption and dangerous for health.

Tuberculosis, Brucellosis, Staphylococcal toxemia, Streptococcal sore throat, Scarlet fever and Gastroenteritis etc can be transmitted via this milk .

Therefore it is necessary to screen out the affected animal of the herd for timely treatment or culling.

Mastitis development in an infected udder

Definition Inflammation of mammary gland accompanied by physical and

pathological changes of udder and physical, chemical and bacteriological changes of milk with or without systemic reaction.

1- Case history 2- Clinical examination a) Animal b) Udder c) Teat 3- Milk examination

Diagnosis

I) Case History

ASK ABOUT:

1. Current amount of milk.

2. Milking technique a) Hand milking. b) Milking machine.

3. Milking hygiene.

4. Type of food.

5. Number and course of previous lactation.

6. Previous mastitis and treatment.

II) Clinical Examination

Examination of animal: Determine animal temperature, pulsation, respiration, superfacial

mucous membranes and superfacial lymph nodes. 1. Presence or absence of systemic reaction to avoid septicemia and bacteremia. 2. Detect specific signs for specific diseases.

Examination of udder:

1. Inspection: observe udder from rear, front and sides.

Normally udder appear symmetric, one mass, without any abnormalities and hind quarters larger than fore quarter. Abnormally: a) Swelling in one or more quarters or atrophy. b) Presence of vesicle, ulcers, scar and wound as in cases of FMD, Pox .

2. Palpation:

Normally udder appear as fine grains or spongy when free from milk, no cardinal signs of inflammation, Pliable skin under hand and Supramammary lymph node not felt.

Abnormally Coarse grains and spongy when contain milk due to clotted milk

in case of chronic inflammation, cardinal signs of inflammation, not pliable udder with adherence to underlying tissue and enlarged and inflamed supramammary lymph node.

Examination of teat 1. Presence of fibrous cord at the base of teat or at teat opening. 2. Presence of fibrous cord within teat canal. 3. Dilated and obstructed teat opening.

III) Milk examination

1. Physical examination

A. Color: Normally is White in buffalo and whitish yellow in cow Physiological Discoloration Yellow: ration contain high carotene, Colostrum,

Some breeds as Jersey cow. Blood stained: high producing animal. Pathological Discoloration Red: Dicumarol toxicity, Leptospirosis,

Staphylococcosis Green: Corynebacterium Yellow: Most of bacterial Mastitis.

B. Odor Normally milky odor

Abnormally ① Fetid (Putrefied) odor: Gangrenous Mastitis, Dry cow Mastitis .② Fecal odor: Coliform Mastitis. ③ Acetone odor: Ketosis .④Rancid odor: Milking in bad ventilated place or leaving milk for

2 days in environment after milking .⑤Antiseptic odor: excessive dipping of teat.

C. Consistency: Normally Milky consistency

Abnormally: ① Watery milk (decrease consistency) >Streptococcal mastitis

. ② Increase consistency > corynebacterial mastitis.

All physical changes can be detected by Strip Cup Test

Strip Cup Test: Field test used for detection of physical changes that occur in mastitic milk by pouring few amount of each quarter separately on shiny fine meshed plate over a cup then observe results

Result: ① Presence of physical changes (abnormal color, consistency) +ve Mastitis

② Absence of physical changes not indicative of absence of mastitis.

2. Chemical examination

A. pH: Normally 6.4 – 6.8 (average 6.6) during mastitis become alkaline due to high sodium,

chloride and bicarbonate and low casein, lactose and fat

Buffalo’s milk - from 6.6 to 6.9. Detection by: pH paper, pH meter, Bromothymol

blue test and Bromocresol purple tests Advantages: user friendly, cost effective and rapid. Disadvantage: not as sensitive as other tests.

Bromocresol Purple Test: based on alteration of pH of milk. 2-3 drops of 0.9%Bromocresol Purple Solution is added

to 3ml of milk. Normal milk following addition of solution will appear as

yellow while Mastitis milk will appear as blue or purple.

Bromothymol blue test: BTB test solution (1.6 gm bromothymol blue in 100 ml

ethanol) BTB card test paper may be prepared in the laboratory from

Whatman filter paper No.1. One drop of the suspected milk has to be put directly on the

spot and the change of the color is to be noted, the change of color may be scored as “(pale green) i.e, normal

quarter and “+” , “++” , “+++” (according to the change of color from moderate green to dark green).

cow in later lactation may give false positive reaction.

B. Chloride: Normally 0.08 – 0.14 %.

Detection by: Chloride Test Test is as follows: Solution A: Silver nitrate = 1.3415 gm, Distilled water = 1000ml. Solution B: Potassium Chromate = 10 gm, Distilled water = 100ml.

Procedure:1. Take 1 ml of milk in a test tube.2. Add 5 ml of solution A.3. Then add 2 drops of solution B to the mixture.4. The mixture is to be mixed well by inverting the tube.

AgNO3 + Milk Chloride = AgCl-PPT AgNO3 + K2CrO4 = Ag2CrO4 (Brownish Red Silver Chromate)

Interpretation: A yellow color denotes more than 0.14 % chloride in the milk and a

brownish red color indicates less than above amount.

Electrical Conductivity (EC) Test Elevation in levels of ions such as sodium, potassium,

calcium, magnesium and chloride during inflammation. Can be detected by Hand held mastitis detector, as well as

by EC-meter. Milk samples with EC ≥ 300 are considered to be from

healthy and uninfected quarters, whereas, those with EC ≤ 250 are considered to be from SCM suspected quarters.

Advantage: can be used ‘on-site’. Disadvantage: non-mastitis-related variations in EC. Quarters infected with S. aureus and Streptococcus

agalactiae - lower conductivity values than quarters infected with environmental streptococci.

C. Hotis test: used to differentiate between Strept. agalactia and Staph. aureus

0.5ml of 0.5% aqueous Bromocresol Purple solution is mixed to 9.5ml of milk in sterile test tube. This is mixed thoroughly and incubated at 37^C for 24 hours.

Streptococcus agalactiae, if present in the milk will produce canary yellow colonies along the side of test tube.

D. Monomast test (Immunodiffusion assay) (Radial immunodiffusion) used to measure serum albumin level which increase in mastitic milk due to injury of udder.

NaOH test

The test is based on the increase in number of leukocytes in mastitis milk. The original test was described by Whiteside (1939) and was modified by Schalm et al., (1971).

For this purpose, 4% NaOH solution was prepared taking 4g NaOH mixed with 96ml distilled water.

Three ml of milk sample was mixed with 3ml of NaOH solution.

The gel formation indicated a positive result.

Somatic cell count (SCC): WBCs + Sloughed epithelial cells Factors affecting SCC: ① Age: old ˃ Young (Heifer) ② Seasonal incidence: Summer ˃ Winter Afternoon ˃ Morning ③ Frequency of lactation: ↑ frequency ↑ SCC ④ Period of post-calving and Late stage of pregnancy: ↑ SCC

(Physiologically) ⑤ Hygienic Measures: ↓Hygienic Measures ↑ SCC

Permissible limit: less than 200,000 cells/ml milk

Methods of SC Counting A. Direct Method: Direct Microscopic Counting – Direct Somatic Cell

Counter B. Indirect Method: Using Chemical Reaction:

1. Take a clean slide and divide it in to two Squares of 1cm2 areas with the help of diamond pencil.

2. Put 10μl (0.01ml) of milk on each area and allow for air dry.

3. Then put the slide in Xylene for 2-3 minutes for defatting.

4. After defatting allow the slide for air drying again and then fixation of smear by 95% ethanol for 5 minutes.

After fixation again allow slide for air drying and then stain by 10 % Giemsa solution for 30 minutes.

5. Now wash with tap water and observe under oil immersion (100X) lens of microscope and counting of leukocytes is done.0-200,000 number of cells per ml of milk is considered normal and more than this is considered positive. More than 500, 0000 cells per ml of milk is taken as +++ mastitis.

Procedure of determination SCC / ml of milk

LINEAR SCORE: A SCC scoring system that divides the SCC of composite milk into 10 categories from 0-9, known as linear score.

The linear score is a base 2 logarithm of the SCC (in cells/mL), whereby linear score = log2 (SCC/l00 000) + 3.

Likewise, to calculate SCC (in cells/mL) from the linear score (LS), the following formula is used: SCC = 100 000 X 2^(LS - 3).

The proportion of neutrophils in the SCC is very low « 11 %) in healthy quarters but is markedly increased in quarters with intramammary infection (to > 90%) .

Accordingly, the percentage of neutrophils in the SCC may provide a useful indication of intramammary infection.

Fossomatic SCC This counter operates on the principle of optical fluorescence. Ethidium

bromide penetrates and intercalates with nuclear DNA, and the fluoresecent signal generated is used to estimate the SCC in milk.

Advantages: rapid and automated. Disadvantages: the device is expensive (US$7000) and complex to

use.

Delaval cell counter This counter operates on the principle of optical fluorescence, whereby

propidium iodide is used to stain nuclear DNA to estimate the SCC in milk.

Advantages: rapid and the device is easily transportable. Disadvantage: relatively expensive.

CALIFORNIA MASTITIS TEST (CMT)

(CMT) is a rapid, accurate, cow-side test to help determine somatic cell counts (SCC) in a specific cow.

A small sample of milk (aprox. ½ teaspoon) from each quarter is collected into a plastic paddle that has 4 shallow cups marked A, B, C and D.

An equal amount of CMT reagent (sodium lauryl sulphate+bromocresol purple) is added to the milk.

The paddle is rotated to mix the contents. In approximately 10 seconds, read the score

while continuing to rotate the paddle. Because the reaction disappears within 20

seconds, the test must be read quickly.

SURF FIELD MASTITIS TEST

Procedure of Surf Field Mastitis Test and its interpretation:a) Procedure1. Prepare a 3% solution of the household detergent viz. Surf Excel. • Dissolve 5-6 teaspoonfuls of the Surf Excel powder in ½ liter of

ordinary water. Pour this solution into a plastic bottle, apply a lid and place the bottle in a dark place. This reagent is good for about 3 months.

2. Collect 10-15ml of milk from each teat in separate container like tea cups, or the Surf Field Mastitis Test paddle.3. Mix the milk from an individual teat and the Surf solution (3%) in

approximately equal proportions (i.e. add 10-15 ml 3% Surf solution).

4. Rotate the mixture of milk and the Surf solution for about 15-20 seconds.5. Examine the mixture for thickening or any other change.

Unique attributes of Surf Field Mastitis Test

a. Compatibility with the technical capabilities of farmers who happen to be mostly illiterate in the developing countries.

b. Desirable sensitivity (72.81 and 66.22 in cows and buffaloes respectively) of detection compared to California Mastitis Test (75.73 and 70.27 cows and buffaloes) and the gold standard of mastitis diagnosis i.e. Microbiological examination of aseptically collected milk samples.

c. Availability of the required reagent, i.e. Surf Excel Powder in almost every village.

d. User friendly nature of the test.

. SAIC has recognized the Surf Field Mastitis Test as a success story in the book entitled “SUCCESS STORIES ON TRANSFER OF FARM TECHNOLOGY IN SAARC COUNTRIES” published in 1998 by SAARC Agricultural Information Centre (SAIC) ,Dhaka.(www.saic-dhaka.org). -- video on SFMT

EnzymesMany of the indigenous enzymes increase in milk during inflammation.

The enzymes dealing with the synthesis of milk decrease and the enzymes related to inflammation increase.

The enzymes originating from phagocytes increase exponentially, and they include N-acetyl--D-glucosaminidase (NAGase), beta-glucuronidase and catalase.

The activity of the enzymes originating from the blood also increase, for example plasminogen which then is locally activated to plasmin, a proteolytic enzyme that degrades fibrin and casein.

NAGase

NAGase is an intracellular, lysosomal enzyme.

Milk NAGase activity correlates very closely with SCC.

In mastitis caused by major pathogens, milk NAGase level is significantly higher than in mastitis due to minor pathogens .

It gave an average of 17% false positive and 2% false negative diagnoses.

A fluorometric NAGase assay based on microtitration tray technology has been developed.

The method has a high capacity and is based on a within test quarter comparison, where other quarters are compared with the lowest value of the particular cow.

Milk arginase activity may be used as an additional laboratory method for the diagnosis of subclinical mastitis.

it has been reported to synthesize the amino acid proline required for casein synthesis.

It has been found that arginase activity increases in inflamatory processes (BACHETTI et al., 2004).

It is proposed that arginase passes into the milk in the event of mammary gland inflammation, with a subsequent rise in milk arginase activity. This may be of importance in the diagnosis of subclinical mastitis.

Milk arginase activity is measured (U/mg protein ) by spectrophotometer, using a modification of the thiosemicarbazide-diacetylmonoxime urea (TDMU) method.

Milk arginase

Other enzymes

Different lipases, esterases, phosphatases, lactate dehydrogenase, etc have potential in mastitis detection.

The concentration of the proteolytic enzyme plasmin has also been shown to increase in mastitis. Plasminogen leaks from the blood into milk and is converted to plasmin.

Milk plasmin activity is positively correlated with SCC. An increase of SCC from 100 000/mL to 1 300 000/mL was associated with a 2.3 fold increase in plasmin activity.

A fluorometric method for the determination of plasmin has been developed.

Acute phase proteinsIn cattle, most sensitive acute phase proteins are haptoglobin and serum amyloid A (SAA), - acute inflammation, and 1-acid glycoprotein - chronic conditions.

Can increase over 100-fold.

1-acid glycoprotein has a low relative rise, reacts slowly and indicates more chronic inflammation.

The first acute phase proteins measured from milk and used as indicators of inflammation are bovine serum albumin and 1-trypsin inhibitor .

Using a threshold value of 0.02 mg/mL for milk haptoglobin and 0.55 g/mL for milk SAA, both tests had a high specificity (100%) with no false positive results, and a reasonable sensitivity (86 and 93) for the diagnosis of mastitis.

One advantage is that these proteins are not present at all in healthy cow.

The C-reactive protein is not regarded as an acute phase protein in cattle, but has Indicators of inflammation to detect mastitis been tested as an indicator for mastitis.

correlation between the concentration of the C-reactive protein in milk and SCC was low (r = 0.32).

Lactose Mastitis results in decreased biosynthesis of lactose. The lactose concentration of milk could be used as

an indicator of mastitis, since it clearly decreases during inflammation.

The percent decrease is, however, relatively small, which compromises the use of lactose to detect mastitis.

Berning and Shook compared SCC, NAGase and lactose, and found out that log NAGase and SCC were most responsive to changes in infection status, but lactose did not perform so well.

Recently lactose was proposed to be one of the most useful markers of mastitis for the future use with a proposed threshold value of 4.7%.

3. Bacteriological examination Aim:

①Isolation and Identification of microorganism

②Culture and Sensitivity test to detect drug of choice Sampling

Sample Preparation: incubation at 37oc overnight 18 hrs then centrifugation at 3000 rpm/ 20 min

3 layers formed (Cream for TB – Whey for Mycoplasma – Deposit for others)

Laboratory Procedures:

A. Direct smear: staining by giemsa, gram, ziehl neelsen, newman's stains according to suspected microorganism.

B. Culturing on specific media: sabouraud dextrose agar (Mycotic), baired parker media (Staph.), edward's media (Strept.), Macconkey (enterobacteriaceae).

C. Culture and Sensitivity test (C & S): colony brain heart infusion broth 37oc 18 hrs Mueller hinton agar Dispense antibiotic discs on agar measure inhibition zone for each Antibiotic and according to these inhibition zones select drug of choice.

S. agalactia A. pyogens

K.pnaeumoniaeS. aureus

P. aerugenosa

Yeast on SDA and blood agar

Candida colonies on SDA

RECENT LABORATORY DEVELOPMENTS

Immunoassays ELISAs have only been developed for some of the most

prevalent pathogens, such as S. aureus, Escherichia coli and Listeria monocytogenes.

For example, S. aureus antibody test kit (SAATK) (Veterinary Medical Research and Development [VMRD], USA) was assessed as a primary screen for cows suspected of having an S. aureus infection.

However, microbial culture of the milk of ELISA-positive cows was required for confirmation.

An ELISA to determine the level of antibodies produced against L. monocytogenes was also developed.

Nucleic acid testing The genome sequences of many of the major mastitis causing pathogens are

now available and can be utilized to develop nucleic acid-based testing methods, such as PCR.

Such tests are generally more expensive than, for example, immunoassays.

However, they are highly sensitive and specific, can be performed rapidly.

Multiplex PCR and ‘real-time’ PCR assays that can simultaneously detect different mastitis- causing organisms in milk samples have been described.

The most recently developed assay is capable of detecting 11 of the major

mastitis-associated pathogens, including E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis.

Nucleic acid sequence based amplification (NASBA), which is used for quantification of RNA, has an advantage over PCR methods in that it is capable of discriminating between dead and living organisms, and real-time NASBA for the detection of Bacillus cereus in milk has been reported.

The application of real-time PCR or NASBA could revolutionize veterinary diagnostics by reducing sample analysis times significantly and allowing the simultaneous analysis of a large number of samples for multiple organisms.

Hematology and serum biochemistry

Clinical mastitis episodes associated with Gram-negative bacteria frequently cause a profound leukopenia, neutropenia, lymphopenia and monocytopenia as a result of the endotoxemia. as well an increased packed cell volume.

In contrast, the leukogram in cattle with clinical mastitis associated Bovine mastitis with Gram-positive bacteria is normal or mildly increased.

Biopsy of mammary tissue A biopsy of mammary tissue can be used for histological and

biochemical evaluation in research studies. The use of a rotating stainless steel cannula with a retractable blade at the cutting edge has been described for obtaining biopsy material from COWS .

Despite some postoperative bleeding, milk yield and composition in the biopsied gland were affected only transiently.

Ultrasonography of the mammary gland. Two-dimensional ultrasonographic images of the gland

cistern, parenchymal tissue and teat are easily obtained using a 5, 7.5,‘ or 8.5 MHz linear array transducer.

Imaging should be performed in two planes, sagittal to the teat (and therefore perpendicular to the ground), and transverse to the teat (and therefore horizontal to the ground).

The injection of sterile 0.9% NaCl through teat cannula into the gland provides a practical contrast agent.

The superficial supennamary lymph nodes can be ultrasounded using a 7.5 MHz linear transducer, with the lymph node being well demarcated from the surrounding tissues.

Mean lymph node length was 7.4 cm (range 3.5-15 .0 cm) and mean depth was 2.5 cm (range, 1.2-5 .7 cm) . Mastitis produces an increased heterogeneous echogenicity to the milk in the gland cistern, compared to an uninfected quarter.

RECENT DEVELOPMENTS IN ‘COW-SIDE’ TESTS

A thermal camera can detect temperature changes of 1 to 1.5^C. A strong correlation (R2 = 0.92) between skin surface

temperature and SCCs has been observed.

Electronic tongue Developed by Mottram and coworkers. a chemical-array-based sensor. able to detect chloride, potassium and sodium ions

released during mastitis in addition to inorganic and organic cations and anions.

successfully discriminates between normal and mastitic milk samples with a specificity and sensitivity of 96% and 93%, respectively.

Electronic nose Developed by Eriksson and coworkers a gas-sensor array system consisted of several gas sensors that interact with volatile

substances, including sulphides, ketones, amines and acids.

Biochips (lab-on-a-chip) Moon and colleagues Disposable microchips, used with a portable reader system to

measure milk SCC. milk sample is mixed with a lysis solution to burst the somatic

cells, and a fluorescent dye is added to stain the DNA. The sample is then applied to the microchip, which uses a

capillary flow to allow even distribution of the sample, and the fluorescence is measured with the portable reader system

A lactate screenprinted sensor that contains lactate oxidase printed onto the sensor surface has been developed .

Lactate oxidase reduces lactate and produces electrons, which generates a current that is measured using a potentiostat and that can be correlated to the concentration of lactate present.

Pemberton and coworkers developed an electrobiochemical sensor using a screen-printed carbon electrode (SPCE) that could detect NAGase via its ability to convert the substrate 1-naphthyl N-acetyl-b-Dglucosaminidine to 1-naphthol, which was subsequently detected by the electrode .

The limit of detection of this NAGase assay is 10 mU/mL.

Akerstedt and colleagues developed a competitive biosensor assay using surface plasmon resonance to monitor the interaction between Hp and Hb to discriminate between sub-clinical mastitic and non-mastitic milk .

The diagnosis of clinical mastitis is not difficult if a careful clinical examination of the udder is carried out as part of the complete examination of a cow with systemic clinical findings.

The diagnosis of mastitis depends largely upon the detection of clinical abnormalities of the udder and gross abnormalities of the milk or the use of an indirect test like the CMT to detect subclinical mastitis.

Other mammary abnormalities that must be differentiated from clinical mastitis include periparturient edema, rupture of the suspensory ligament, cancerous growths, hematoma, etc. These are not accompanied by abnormalities of the milk unless there is hemorrhage into the udder.

Differentiation of the different causes of mastitis is difficult on the basis of clinical findings alone but must be attempted, especially in peracute cases where specific treatment must be given before results of laboratory examinations are available.

DIFFERENTIAL DIAGNOSIS

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