Mastitis & Abnormal Milk: Dairy Animal Health

• Commercial raw milk must come from healthy dairy animals and be normal, not colostrum.

Abnormal Milk

• Abnormal milk comes from dairy animals treated with medicinal agents that secrete into the milk
• Milk with hazardous pesticides or herbicides is considered abnormal.
• Mastitis in dairy animals also results in abnormal milk.

Mastitis

• Mastitis is the inflammation of the udder
• The word "mastitis" is derived from "masts" (Greek for breast) and "itis" (inflammation)

Types of Mastitis

• Acute mastitis inflames udder with swelling, heat, redness, and pain
• Little to no milk is available in acute mastitis
• The milk is tinged with blood, called jurged milk, and is confused with ropy milk.
• Chronic or sub-clinical mastitis is more common in dairy cattle
• The animal appears normal in the chronic sub-clinical form and hard to detect.

Causes of Mastitis

• Mechanical injury to the udder and poor milking practices reduce animal resistance, allowing organisms in the udder to cause mastitis (streptococcus agalactia)
• Transmission of specific microorganisms from one animal to another leads to mastitis
• This transmission can occur when milking with a machine, specifically staph aureus.

Causative Organisms

• Around 55 bacteria and 27 fungi species are identified as causing mastitis.
• Main causative organisms are major and minor pathogens.

Major Pathogens

• Streptococcus agalactia is responsible for 80% of mastitis cases as present in the udder
• Frequent incomplete milking facilitates rapid increase in the number of Streptococcus agalactia in the udder, causing mastitis.
• Streptococcus agalactia is non-pathogenic for humans and easily destroyed by heat
• It leads to neonatal infection and meningitis if present in the female genital tract.
• Other major pathogens include Staphylococcus aureus, Coliforms(Klebsiella spp., Enterobacter spp., E.coli and citrobacter spp.), Streptococcus uberis, Streptococcus dysagalactia, Streptococcus bovis, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, and Pseudomonas aero genes

Minor Pathogens

• Actinmyces pyogenes and Serratia spp are major pathogens
• They cause greater compositional changes and an increase in the somatic cell count of milk
• Minor pathogens are Coagulase-negative Staphylococci (Staph. epidermidis), some yeasts and moulds (responsible for mycotic mastitis), Corynebacterium bovis, and Mycobacterium tuberculosis (responsible for tuberculus mastitis)
• Corynebacterium bovis is rarely associated with clinical mastitis but causes compositional changes or decrease in milk production.

Effects of Mastitis

• Leads to food poisoning (Staph.aureus, E.coli and high bacterial count).
• Can cause diseases like T.B. and septic sore throat in consumers.
• Results in decrease in milk production by 10% and reduce milk quality.

Effects on Milk constituents

• Decrease in lactose and casein content
• Increase in chloride, lactoglobulin and enzymes, lipase, catalase, reductase and phosphatase

Effects on Milk

• Increases the number of bacteria
• Increases the somatic cell count (monocyte and polymorph-nuclear cell) above 200,000 / ml, decreasing the shelf life.
• Increases leucocytes and fibrin (jurged milk )
• Decreases water-soluble vitamins and makes the pH of milk alkaline
• Leads to the presence of pathogenic organisms and pathological secretion
• Milk acquires an undesirable odour

Effects on Dairy Products

• Causes costly losses to processors due to its effects on milk constituents, along with an economic impact on farmers.
• High level of free fatty acids produced by the action of lipase enzyme on milk fat results in milk and dairy products with off flavor (rancid flavor).

Effects on Cheese

• It directly impacts the quality and yield of cheese.
• Increased proteolytic activity in milk causes the breaking of the original long chains of amino acids of the casein into smaller fragments
• During cheese making, milk casein doesn't coagulate properly. Small casein fragments and an increased amount of fat are lost into cheese whey, causing a decreased cheese yield and weak curd.
• An increase in pH, sodium, and chloride adversely affect rennet coagulation, which leads to long-time cheese making, lower quality cheese, and rising costs.
• Cheese has an unclean flavor, pasty texture, and higher moisture content if made from milk with a high somatic cell count
• Bacterial contamination leads to colour faults

Effects on Cream

• Lipolysis of milk fat produces free fatty acids that act as a foam depressant, which depresses the whipping properties of cream.

Effects on Fermented Dairy Products

• A high level of free fatty acids and antibacterial proteins (lactglobulin) may inhibit starter cultures used in making fermented dairy products.
• Protein breakdown leads to a weak body and undesirable separation of the yoghurt into curd and whey in the package.
• It inhibits diacetyl production, a favourable characteristic in fermented dairy products.

Effects on Heat-Treated Milk

• Due to the high amount of lactglubulin and lactalbumin, and change in salt content, milk is less stable to heat compared to normal.

Detecting Abnormal (Mastitis) Milk

• The oldest detection methods are visual observation and physical examination of the mammary glands.

Visual Observation

• It tests for a salty taste and look for fine flakes or clots, especially in fore-milk
• Milk can be grayish, yellowish, or brownish
• Clots can become larger, and milk may appear thicker or slimy.
• On standing, pus or blood may form a deposit above a clear or cloudy yellowish fluid.

Physical Examination of Mammary Glands

• Such an examination must be made immediately after milking.
• Not all cases can be detected this way, because
  • The infection can be very slight, leading to only slightly abnormal tissues,
  • Only a small part of the quarter may be involved, which can be mixed with a larger volume of normal tissue milk.

Sub-Clinical Form

• It is characterized by a lack of visible changes in the milk or the udder
• It is also characterized by a decrease in milk production, and bacteria present in the secretion with changes to the composition.
• Bacteria multiply in milk and produce toxins when they enter the mammary gland through the teat canal
• Polymorph-nuclear cells migrate from blood through capillaries lined with the alveoli and engulf the invading bacteria, resulting in an increase in the somatic cell count (SCC) of milk.
• Somatic cell count is a common measure of mammary gland health and milk quality

Testing Variety

• Varieties of tests are applied to milk in an attempt to detect irregularities which indicate that it comes from an abnormal udder.
• A single test may sometimes give a definite answer, in other cases a combination of more than one test is advisable.

Field Tests

These are a type of screening test for the detection of sub-clinical mastitis.

• Strip-cup test
• Bromo-cresol purple test papers
• Gel tests
  • White side test
  • California mastitis test (CMT or Schalm test)
• Determination of chloride content (qualitative test)

Laboratory Tests

These are used to demonstrate the presence of causative organisms

• Hotis test
• Bacteriological examination of incubated milk
• Cultivation of milk sediment

I- Field Tests

Strip-Cup Test

• Draining the fore-milk from each quarter on a shallow black dish or cup
  • Fine particles of clotted milk indicate mastitis milk.

Bromo-Cresol Purple Test Papers

• The pH value of milk is measured by pH-indicator paper.
• In mastitis, blood exudates pass into the udder, changing the pH value of milk from slightly acidic to an alkaline reaction

Types of pH paper:

• Bromo-cresol purple paper is slight purple which changes to dark purple with mastitis..
• Bromothymol blue paper is yellow which changes to greenish blue with mastitis.

Preparation of pH indicator paper:

• The papers are prepared by soaking filter paper in saturated dye solution and then dried.
  • Four strips of paper per animal are used, one for each quarter
• Procedure:
  • Fore-milk is collected onto specially prepared Bromo-cresol purple absorbent pH paper.
  • Milk gives an alkaline reaction, and a pH of more than 7.6 indicates an alkaline reaction
    • The purple color changes to dark purple.

Gel Tests: White Side Test

• It depends on the certain protein factors reaction within the nucleic acid of somatic cells combine with the sodium hydroxide.
  • This then produces the gelatinous mass to which serum solids and fat globules were adsorbed to produce a typical precipitate.
• Procedure
  • Add five drops of milk and then add two drops of NaOH (4%) mix for 20 seconds with a glass rod. Then judge.
  • A positive reaction roughly indicates the somatic cell count of milk under test. Degree of infection can be measured as the following:
• No change, homogenous mass (opaque and Milky And Free From Precipitate - SCC/mL less than 500,000.
• Fine particles appear-Traces SCC/mL 500,000-1 million
• Slight Flocculation - 1+ve 1-2 million
• Flocculation (Definite Clumps Of Coagulated Materials Or Threads May Be Found-2 +ve 2-3 million
• Gel formation (Gelatinous Mass, surrounded by a clear fluid -3 +ve SCC/mL greater than 3million

California Mastitis Test (CMT)

• This Schalm test is simple, quick, cheap and convenient.
• CMT (Schalm) reagent consists of a detergent [4% Alkyl Aryl Sulfonate + Bromo-cresol purple indicator]. The Procedure:
• Equal volumes (2 ml) of the quarter milk sample and Schalm reagent are mixed for 10 seconds with gentle rotatory movement for judgment. The white paddle or cup used to perform the process has four cups for the four quarters.
• At a concentration of 150,000-200,000 cells/mL, a precipitate forms, thickening into gel as cell concentration increases ####### CMT Test Judgement:
• No change, homogenous mass (opaque and milky and free from precipitate is measured as -ve with 0-150,000 scc/ml
• Slight precipitate appear is traces with 150,000 - 500,000 scc/ml
• Distinct precipitate with no tendency to form gel occurs with 1+ve rating and 400,000 - 1.5 millions scc/ml
• Mixture thickened immediately with some suggestion of gel formation occurs with 2+ve rating and 800,000 - 5 millions scc/ml
• Gel formation (gelatinous mass) tend to adhere to the bottom of cup with 3+ve rating and greater than 5millions scc/ml

Additional Indicators:

• Alkaline reaction is indicated by indicator deep purple color reflecting depression of secretory activity.
• Depression of secretory activity may happen because of inflammation or drying off of the gland

Precautions:

• If the milk is unrefrigerated it can only be tested accurately for around twelve hours.
• If the milk is refrigerated then it can be tested for up to 36 hours
• Somatic cells migrate with the milk fat so it is essential to ensure a representative sample.
• thorough sample mixing is a must before testing
• The CMT reaction must be scored within 10-15 seconds to avoid weak reactions.

Quantitative Determination of Chloride Test

• With mastitis cases Lactose (sugar) decreases sharply and chloride percentages increase to maintain osmotic balance.
• Add the following in a test tube. -- 1 ml of properly mixed milk sample.
  • 4 drops of 5% potassium chromate.
  • 5 ml of silver nitrate solution ( dissolving 1.3415 gm silver nitrate crystals in 1000 ml distilled water).
• The mixture is then thoroughly mixed.
• Results:Positive result is indicated by the appearance of a yellow coloration with chloride contents greater than .14%; normal milk has a brown colour.

II) Laboratory Tests:

a) Tests That Demonstrate the Presence of Causative Organisms:

A- Hotis Test:

• Used to detect Streptococci causing mastitis by adding 9. 5 ml of suspected milk sample + 0.5 ml of 0.5% sterile aqueous solution of Bromo-cresol purple.
• Indicators -- Yellow color for acid medium. -- Deep purple for alkaline medium. -- Light purple for a neutral medium.

Judgment:

-- If the color changes from purple to yellow and presence of yellow flakes, the result if positive indicating either strept. agalactiae or Streptococci with a culture containing streptococci ferment lactose to lactic acid to precipitate casein -- Yellow indicates acidity due to yellow flakes. -- No coloration change indicate a negative test. --If found positive, loop on Edwards medium that will culture violet colonies strept. agalactiac --If found positive a loop is stretched on agitated slop (37oC for 24 hours) to get a pure culture.

B- CAMP Test:

-This test is specifically designed to test streptococcus agalactiae.The test is dependent on the hemolytic power of toxins produced from micro-organisms.

• Procedure: -- The test is performed on blood 4-5% agar plates with Dextrose and Aesculin 0.1%. -- Strept strain suspected to be agalactiae must be tested perpendicular with Staph. strain. -- Incubate at 37oC for 24 hours for judgment
• -- If an arrow appears, that has a head, then is caused by toxins producing cultures.
• ------ -Agalt. = negative = the other organisms don't produce hemispheric zone.

Tests for Identification of Strept. agalactiae (a) Sodium hippurate test:

-- agalactiae has the property of hydrolyzing Sod. hippurate into benzoates --- Medium used:Add 10 gm sodium hippurate and 1.5 gm potassium dibasic phosphate to 1000 ml nutrient broth.The medium is distributed into test tubes and autoclaved --- Procedure:Inoculate the suspected culture obtained into two tubes and incubate them at 37oC for 12 hr

b). Tests of Culture: The media is as followed.

1. Blood - hemolytic v not
2. Edwards medium - streptococci
3. Mannitol salt agar - staphylococci.
4. Sabaurad agar- yeast and moulds.

a) Preparation for Culture Test:

-10 cc in a sterile milk tube 37 degrees C for duration of 24 hour.

• A from the incubated milk tested with oil and lens for -agalatactae and testing for leucocytes organisms.

C- Method of milk sediment:

1. Sterile - 10 ml mixed milk at 300 rpm for 20min.
2. Seperate sediment a stain w/ lofller to see microscope long trains and leucocytes.

Tests that measure change: Rapid Labor Test:

A. PH reaction (pH Paper) -- Milk that results to be tested is basic. PH measure/meter and a standard of normal milk The pH's below are measures for "Min," "Max" and "Average" readings:

• Normal- 6.4, 6.8, 6.6
• Mastitic- 7.6 or greater B. CI % Quantitative
• 10ml milk with N-NIO test is placed and added with nitrate +5 ml 1% is added to a solution alum sat/icon is measured and observed for 2 minutes until observation of brown colour is recognized.
• Cl solution is measured in the formulas below + as a standard: 5 mm + 11 = N (0.158), S. - 6=Amm (N (0.1 N)
• --Chloride percentage by volume (5N (0.1)) for chloride 0-0.3545 ml (1. 9ml) Solution of chloride: type for test. The chart displays the "MIN", "MAX" and "AVEARAGE" results for each test:
• Normal- .063, 1.130, .097
• Mastilic- .093, 1.170 1.130

C,Casein = caseinn x 100 (in mastisis cases, measurements decrease) Totat nitrogen = .73-71; standard is. =63/3.3

D> Cl+ (increase due to lactation/volume)

• E) Negretti

  F.) Cotalase Standard amount if observed. A Scc *MO = catalyses > increase 02 Put a sterile tube w H20 +.5 Mll04 and observe for 23 hr as measure: High B/S = High 02 The numbers measure a total ML/ Cell:

• Low=1/2 mill

• 20.30: > 0.1 Total-3/10: increase 02

• Thy bromal catalyst with H20 2: Stops/and observe for increase catalyst enzyme w M05

Amount of sediment is indicated on standard as:

• Advanced: amount of 40 to standard
• Amount 300 to 40: increase
• Nornkal standard less than .10: Amount by centrifuge for measurement of graduations:

Conrtol Of Malitia

• Proper milking; Sanitzer and a paper towel.
• Disinfection 2h of solution teat for killing of bacteria to combat surface bacteria/prevention of infection. Dry therapy.
• Clean dry area, combat bacteria .
• Milking equipment checked every 6 months.
• Detect, treat, and record the clinical trials.