VPM 201: Veterinary Bacteriology and Mycology
Laboratory 4A: Mastitis
A. Microbial Aspects of Bovine Mastitis
Bovine mastitis is the most costly disease affecting the dairy industry. The majority of infections
can be attributed to staphylococci, streptococci and coliforms. Chronic subclinical infection with
these bacteria causes a significant loss of milk production. There is a correlation between the
degree of chronic infection in a herd, as measured by the somatic cell count, and loss in
productivity. Chronic infection is responsible for greater loss than acute and clinical mastitis.
1. Sources of bacteria in bovine mastitis
Staphylococcus aureus readily colonizes the teat orifices and teat lesions, and invades the
mammary gland; it can be found on mucosal surfaces, such as in the nose and vagina of the cow.
Streptococcus agalactiae is found in similar sites, but the prime importance of the infected udder
as a course of infection has more clearly been shown. Transmission of infection occurs at
milking. Streptococcus uberis is found in extramammary sites in the cow (skin, mucosal surfaces)
and in manure, so infection in part reflects environmental contamination and poor hygiene.
Streptococcus dysgalactiae holds a position midway between the two other streptococci.
Coliform (Escherichia coli and Klebsiella pneumoniae) mastitis generally reflects fecal
contamination of the environment. It is largely confined to cattle housed with restricted space;
wetness and high temperatures encourage growth of the bacteria. K. pneumoniae mastitis often
follows the use of sawdust bedding because the organism may be found naturally in sawdust.
Coliform mastitis is often severe and acute. Clostridium perfringens reflects fecal contamination
and causes acute, toxemic and gangrenous mastitis. Arcanobacterium pyogenes is a sporadic
cause of chronic, severe mastitis and is often associated with nosporeforming anaerobic bacteria
such as Peptoniphilus indolicus. Many other microorganisms can cause mastitis which, though
they are uncommon, can be serious in particular herds. These include Mycobacterium species,
Bacillus cereus, Pseudomonas aeruginosa, Candida species and other fungi, Norcadia species,
and Mycoplasma species such as Mycoplasma bovis.
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2. Routes of infection
Infection reaches the udder through the teat canal. Minor microscopic abrasions predispose to
infection, probably by exposing fibronectin to which S. aureus and other bacteria adhere.
Occasional infections are nosocomial because of contamination of dry cow antibiotic
preparations.
3. Natural defenses of the bovine udder
A major defense against infection is the anatomic barrier of the teat sphincter and the washout
effect of milking. The major immunoglobulin in milk is IgG but this is present in relatively small
quantities because of dilution. The predominant somatic cells in milk are the macrophages. They
have relatively poor bactericidal activity, but are important in antigen processing. Neutrophils in
milk are the major effector cell but are also poorly bactericidal, because of the inhibitory effects
of casein and fat globules which they phagocytize. The relatively poor immune defenses of the
lactating udder contrast with the efficient antibacterial nature of the non-lactating udder which is
protected by large quantities of lactoferrin and citric acid, both of which prevent bacteria from
acquiring the iron they require for growth. The udder of the cow in the postparturient period is
most susceptible to infection.
B. Assessment of Cellular Response to Intramammary Infections
The number of somatic cells in milk can be quantified directly by microscopic counting or by the
use of an electronic particle counter, or indirectly by tests in which nuclear DNA is polymerized
to a gel by a detergent.
One indirect method is the California Mastitis Test (CMT). In this method, 2mL of milk is
transferred to the cups of a special paddle and 2 mL of CMT reagent added. The mixture is
gently swirled for 10 seconds and examined for a precipitate and thickening or gelling of the
mixture. The reaction is scored as follows. The reaction may fade if shaking too hard or kept for
more than 15-20 seconds.
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Scoring System
0
– Mixture liquid, no evidence of precipitate.
Trace – Slight precipitate, especially on tipping backwards and forwards; may disappear.
1
– Distinct precipitate, no tendency to gel.
2
– Mixture thickens immediately, some suggestion of gel.
3
– Heavy gel formation.
C. Bacterial isolation and identification
1. Sampling – Samples should be taken just before milking and after discarding foremilk. Udders
and teats should be washed with clean warm water and dried with paper towels. Carefully scrub
the teat end and orifice with cotton pledget moistened with 70% alcohol. Collect milk into sterile
container without touching sample. Samples should be cultured immediately or stored at 4 ºC
until cultured, which should be within 24 hours.
2. Culturing – Warm milk to 25 ºC, shake well by inversion, and let foam disperse. For quarter
samples or composites, aliquot 0.01 mL (10.0 μL) to one quadrant (quarter) of a plate. Incubate
35 ºC for 24 and 48 hours.
3. Gram stain – A gram stain should always be done on milk from cows with severe mastitis, but
is not usually done in mild mastitis.
4. Preincubation of milk samples – This is routine practice in some laboratories but has no place
except for isolation of S. agalactiae. Milk is an excellent culture medium and one bacterial
contaminant can readily multiply.
5. Interpretation – In acute mastitis, bacteria are found in large numbers. In chronic mastitis,
bacteria may be present in low numbers but most workers require more than 5 colonies as
presumptive evidence of an intramammary source and not of contamination.
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D. Laboratory Exercises
1. Three swabs (A, B and C) are milk samples from mastitic cows. Please inoculate on BA,
MAC and Edwards agar (see Appendix B of your laboratory handbook) using a standard
quadrant streak pattern. They will be incubated overnight. Label plates carefully, otherwise
your lab tomorrow will be messed up.
2. You are provided with BA and MAC plates inoculated with two bacterial isolates (2A and 2B)
from dairy cows that developed acute mastitis. Record your information in the table below.
a. Examine the culture plates provided (growth, morphology, hemolysis, lactose fermentation…)
b. Perform gram staining
c. Results of citrate and urease tests (see lab handbook Appendix B) are provided as demo 2.
d. Based on the observations, make a tentative identification of 2A and 2B.
Growth
E. coli
Klebsiella
pneumoniae
Gram
(+/-)
BA
MAC
(Yes/No)
(Yes/No)
+
+
-
+
+
-
1
Lactose
Fermentation
2
Citrate
3
Urease
(+/-)
(+/-)
+
-
-
+
+
+
(+/-)
: Pink colonies on MAC indicate “lactose-fermenter”
: Blue means “positive”
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: Pink means “positive”
1
2
K. pneumonia produces highly mucoid colonies!!!
3. Gram stained smears (Slide sets 7-2, 7-3, 7-4, and 7-5) of milk from 4 cows with chronic
mastitis are provided. They are Nocardia (7-2), Streptococcus agalactiae (7-3), Candida
albicans (7-4), and Arcanobacterium pyogenes (7-5). Describe your microscopic observations
(morphology…).
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E. Demonstrations
1. California Mastitis Test (CMT; Appendix C) will be demonstrated at the front of the class. Is
this a direct or indirect measure of somatic cells in milk?
CMT measures somatic cells indirectly
2. Results of citrate and urease tests of 2A and 2B.
3. Merial J-VACTM vaccine for E. coli mastitis.
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Laboratory 4B: Mastitis, Bacillus and Mycobacterium
A. Bacillus
The genera Bacillus spp. are gram-positive and produce spores. Bacillus anthracis (not provided)
forms capsules, and is non-hemolytic. Other Bacillus species, such as B. cereus, are hemolytic
and mostly are found as contaminants in clinical specimens.
B. Mycobacterium
Do not take up gram-stain (some may be “beady” Gram-positive). Acid fast (Ziehl-Neelsen)
staining is required. Mycobacteria stain pink/red and the background is typically blue. For
isolation, specimens are decontaminated, and inoculated into tubed media (e.g., LowensteinJensen) and incubated for up to 8 weeks for M. tuberculosis, M. bovis and M. avium.
C. Exercises
1. Examine the BA, MAC and Edwards plates you inoculated yesterday. Conducts catalase test
and gram-staining, and complete the Table below. CAMP tests are provided as “Demo 1”. Please
make your presumptive identification of it; the table on the next page will be helpful to you.
Sample A
Sample B
Sample C
Growth on BA plate (Yes/No)
Yes
Yes
Yes
Growth on MAC plate (Yes/No)
No
No
No
Growth on Edwards plate (Yes/No)
No
Yes
Yes
+
+
+
cocci
cocci
cocci
Gram staining (+/-)
Microscopic morphology (rod, coccus?)
Hemolysis (complete, double zone…?)
Catalase (+/-)
double zone
complete
but weak
nonhemolytic
+
-
-
CAMP test (+/-)
N/A
+
-
1
N/A
-
+
Staph. aureus
Strep. agalactiae
Strep. uberis
Esculin hydrolysis (+/-)
Your presumptive I.D.
1: Edwards can differentiate between esculin-positive and negative organisms.
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Lancefield
Hemolysis
CAMP
group
Edwards
Esculin
Hippurate
agar
Staph. aureus
NA
double zone
NA
no growth
NA
NA
Strep.
B
β (weak)
+
+
-
+
arrow-head
agalactiae
Strep.
C
α
-
+
-
-
Variable
α, γ
-
+
+
+
dysgalactiae
Strep. uberis
D. Demonstrations
1. CAMP test of Samples B and C.
2. Examine the BA culture of Bacillus cereus and the gram-stained morphology of B. cereus
(Slide set #10-1). Please don’t open the culture plate!
3. Examine the photograph of B. anthracis.
4. Examine the acid-fast stained Mycobacterium in histological preparation of ileum mucosa
from a cow suspected of Johne’s disease.
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