the most common disease in dairy farm. there are many physical, chemical and microbiological changes in udder. for lab diagnosis there are many tests.

1. Dr. Dina A. B. Awad
Lecturer food hygiene and control , faculty of veterinary medicine
Benha university, Egypt

2. Mastitis
-the most costly disease of dairy animals.
- ranked as number one disease of dairy animals.
- mastitis shortens lactation period of each animal
- reduces milk yield per lactation.
- mastitis impairs the quality of milk and milk products

3. Mastitis means
Inflammation in udder pathological
changes on udder and physical
and chemical changes in milk
Clinical
mastitis
Sub clinical
mastitis
Chronic
mastitis

4. Clinical Mastitis

5. Chronic mastitis

6. Sub clinical mastitis
• Characterized by
no change in
milk and udder
but the
microorganism
present and
multiplicats in
milk

7. Many microorganisms
Like

8. california Mastitis Test (CMT)
This is a subjective test which allows us to estimate the quantity of somatic cells in a cow in each
quarter. It should be done every two weeks if there is no monthly individual control on the farm,
and in every animal at the beginning or end of lactation. Sometimes is also called Rapid Mastitis
Test (RMT).
The person who does the CMT must consider the following standards:
• Stripping before sampling is very important because the first jets can give false positives.
• CMT has to be done at the beginning of the milking because it is easier to identify due to the
quantity of pathogens.
The table below shows the relationship between the CMT result and its equivalence according
to the SCC.
Biosensors for On-Farm Diagnosis of Mastitis

9. Major Mastitis Pathogen Isolation
Streptococus agalactiae
Gram positive small colonies
catalase neg
CAMP positive -hemolytic
Streptococus species
Gram positive small colonies
catalase neg esculin positive
CAMP negative non-hemolytic
Staphylococcus aureus
large colonies (cream-tan color)
Gram positive
Staphylococcus species
large colonies (white to yellow)
Gram positive
Staph aureus
Staph sp
-
Coagulase Test
CAMP Test
growth
BA
TKT
BA
TKT
-hemolytic colonies
Strep ag
Strep sp
No
growth
No
growth
No
growth
+
Sears, 2003

10. E. coli
Blood agar:
large colonies (grey, moist)
Gram negative rods
pink zone around colonies
Kkebsiella sp
Blood agar:
large colonies (grey, mucoid )
Gram negative rods
Coliforms
pink center “fish eye”
BA
BA
Sears, 2003

11. A B
Coliforms Streptococci Staphylococci
C D
E F
Staph sp
 hemolysis
-
Coagulase Test
+
beta-hemolytic colonies esculin positive CAMP Test
Strep ag
Strep sp
C D
MC: pink zone around colonies MC: pink center “fish eye”
Staphylococci
Streptococci
Coliforms
E. coli Klebsiella sp
Strep ag Strep sp
Staph aureus Staph sp
A B
A
E F
BA: large greyish colonies
MC
BA

12. Other Mastitis Pathogens (less common)
Pseudomonas spp.
Gram-negative rods
MacConkey agar:translucent, shiny colonies
A. pyogenes
“Summer mastitis”result of wounds, injuries and forms
incurable abscess in the udder.
Pasteurella spp.
Colonies are grey, very mucoid and confluent
Gram-negative rods, bipolar staining
Do not grow on MacConkey agar

13. Yeast
White and dry-smooth or wrinkled
Serratia spp.
Gram-negative rod
MacConkey agar: translucent colonies, often with red
pigment (bottom)

14. Big Two
• Streptococcus agalactia (Strep. ag.)
• Staphylococcus aureus (Staph. aureus)

15. Changes occurs in milk due to mastitis
Physical appearance - color, flakes, clots, etc
pH - normal 6.5 increase to 7 or more but in s.agalactia decrease in P H .
Cellularity - increase in SCC
- increase leucocytic count (poly morphonuclear leucocyte)
Chloride - increase Cl (Koestler No.)
Catalase - tissue damage - O2 release
-Increase in sediment and dirt .
- Decrease in potassium .
-Increase in lactalbumin and lactglobulin.
Decrease in lactose %, casein %and casein No.

16. 1. Wash udder with water and soap then apply cotton
with alcohol.
2. Collect an aseptic milk sample.
3. Discard first 2 streams of milk .
4. Collect composite or quarter milk sample
5. Refrigrate (or ice) sample at collection
Collection of samples for mastitis
detection

17. 3. Discard first 2
streams of milk .
4. Collect composite or
quarter milk sample
5. Refrigrate (or ice)
sample at collection

18. mastitis
Field test (inside farm) Lab. Test
Strip
cup
test
Bromo
cresol
purple
test
Gel test
White
Side
test
Schalm
test
or C.M.T
Bact.ex. Rapid test
Hotis test Milk sed.
CAMP ,Na.hippurate
test, edward media
Kielers test

19. Field test
(inside the farm)
Udder diagram illustrating how mastitis
develops in an infected udder.
Environmental and contagious
microorganisms invade the udder though
the teat cistern. They then multiply within
the alveolus where they are attacked by
neutrophils (white blood cells) while
damaging the milk-producing epithelial
cells of the bovine udder. The image is
taken from Viguier et al, 2009.

20. A- strip cup test
drain few drops in shallow pan --------
then wait few min.

21. B- Bromocresol purple test
Preperation
promocresol purpl
filter paper
-- result
-Still yellow-------------- -ve
-- become purple -------------
----mastitic milk

22. White side test & schalm test (CMT)

23. Principle :reaction SCC &NaoH
Procedure: on black glass plate 5drops milk sample
&2 drops NaoH 4%----------------- MIX
Result:
Rough count scc
degree
Result
Less than 500,000 cfu
/ ml
-ve
Homogenous fluid
1-2 millions
+ ve
Slight flocculation
2-3 million
+ +
Flocculation and flakes
More than 3 million
+ + +
Gel formation (clear fluid)

24. California Mastitis Test
Principle :(CMT) is an indirect test used to determine the somatic cell
count in milk. Reaction between reagent and DNA of bacterial cell and
somatic cell
Procedure:
Clean teat and remove a single steam of milk from each quarterCollect milk (about 2
ml) in appropriate well: A, right fore; B, right rear; C, left fore D, left rear
Add equal amount (2 ml) and CMT solution Rotate mixture gently for 20 seconds
Result: Determine agglutination score:
Score Milk gelling Somatic Cell Count (cell/ml) MilkQuality
Neg none <10,000-200,000 Good
Trace slight 150,000-500,000 Good
1 Mild 400,000-1,500,000 In-between
2 moderate 800,000-5,000,000 Bad
3(gel formation strong over 5,000,000 Bad

26. Laboratory test

27. Used for detection of st.agalactia and st.dys agalactia.
9.5 ml milk sample +0.5 ml bromocresol purple
Mixing yellow color canary flakes on wall t.t
Confirmed by CAMP TEST ,Na hippurate test ,cultivate
on Edward media
Hotis test

28. CAMP test
• Principle:
– Streptococcus agalactia produce extracellular protein (CAMP factor)
which make hydrolysis RBCS
– CAMP act synergistically with staph.aureus to cause lysis of RBCs
– CAMP is an abbreviation for the names of the 4 individuals who
originally described the test.
• Procedure:
– Single streak of Streptococcus to be tested and a Staph. aureus are
made perpendicular to each other
– 3-5 mm distance was left between two streaks
– After incubation, a positive result appear as an arrow - head shaped
zone of complete hemolysis.

29. 2. CAMP test
 The hemolytic activity of Staphylococcal Beta lysin on
RBCs is increased by an extracellular factor produced by
S. agalactia called the CAMP factor.
 This test is don by making a single streak of
Streptococcus (to be identified) on sheep blood agar
perpendicular to a strain of S. aureus known to produce
betalysin.
 The two streak lines must not touch one another. The
plate is incubated for 24hours .
 The positive result is expressed by a zone of increased
lysis assuming the shape of an arrow-head at the
junction of the two streak lines.
29

30. Strep uberis
Staph aureus -  hemolysin
CAMP Test for
Streptococcus agalactiae
Strep dyagalactiae
enterococci
Strep agalactiae

Strep sp
Strep ag
MacConkey Agar

31. CAMP test

32. 32

33. Na hippurate test
• Principle
• Streptococcus agalactia secrets enzyme
hippuricase which can hydrolyze hippuric acid.
• The products of the hydrolysis of sodium
hippurate are sodium benzoate

34. Nahippurate media +loopful of positive
hotis test ---- incubate at 37 c -5 day ------
then add 0.5 ml fecl3 and 2ml H2so4

35. -Violet color from ferric benzote
-White color test benzoic acid

37. 10ml of milk sample ----incubate --------
staining or cultuvation on specific media

38. 10 ml milk sample take sediment detect
density of cells –field (Kielers test)
Loopful from sediment mix with sterile saline --
on elliptical area of slide
Dry and stain with tolludine dye then examined
under oil immersion lense

40. Thank you for attention
Presented by :
Dr. Dina A. B. Awad
Lecturer of food hygiene department
Faculty of veterinary medicine
Benha university , Egypt
Dr.Dina A.B. Awad 40