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LSU Historical Dissertations and Theses
Graduate School
1987
Physiological Aspects of Bovine Mammary
Involution: a Biochemical and Morphological
Investigation.
Lorraine Marie Sordillo
Louisiana State University and Agricultural & Mechanical College
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Sordillo, Lorraine Marie, "Physiological Aspects of Bovine Mammary Involution: a Biochemical and Morphological Investigation."
(1987). LSU Historical Dissertations and Theses. 4423.
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Order N u m b e r 8728219
P hysiological aspects of bovine m am m ary involution: A
biochem ical and m orphological investigation
Sordillo, Lorraine Marie, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1987
U MI
300 N. ZeebRd.
Ann Arbor, MI 48106
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PHYSIOLOGICAL ASPECTS OF BOVINE MAMMARY INVOLUTION:
A BIOCHEMICAL AND MORPHOLOGICAL INVESTIGATION
A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Dairy Science
by
Lorraine M. Sordillo
B.S., University of Massachusetts, 1981
M.S., University of Massachusetts, 1984
August 1987
ACKNOWLEDGMENT
The author would like to express sincere
Stephen
the
C.
Nickerson
course
of
dissertation.
for his enduring
these
His
support
investigations
contributions were
appreciation
and
to Dr.
and guidance during
preparation
invaluable
of
and his
this
patience
most appreciated.
Gratitude
excellent
is extended
facilities
to
to Dr.
carry
W.
out
Nelson Philpot
this
research
for providing
in
the
Mastitis
Heseach Laboratory, Hill Farm Research, Homer, LA.
The author is grateful to members of the dissertation advisory
committee,
Haldiman,
Drs.
Robert
Ronald
J.
W.
Adkinson,
Siebeling,
John
and
E.
Leonard
Chandler,
C.
Jerrold T.
Kappel
for
their
contributions and evaluation of this research.
The author is thankful for the friendship of her colleagues at
the Mastitis
Research
Laboratory.
Special
thanks
are extended to
Nancy Boddie and Frances McKenzie for their expert assistance.
The author would like to express appreciation to her family for
their support and being there when
they were most needed.
thanks
is
parents,
and
deepest
Howard
encouragement
appreciation
and
and
Jacquelyn
support
kept
expressed
Neill,
her
to
whose
"reaching
the
never
for
the
Special
author's
ending
stars"
throughout her academic endeavors.
Finally,
appreciation
the
to
author
her
wishes
loving
to
express
husband,
Jeff,
sincere
for
his
gratitude
and
support
and
patience throughout the long months of study and research.
ii
TABLE OF CONTENTS
A C K N O W L E D G M E N T .................................
LIST OF TABLES ..............................
LIST OF FIGURES ............
LIST OF APPENDIX FIGURES .........................................
A B S T R A C T .......
x
xiv
xvii
Chapter
II.
v
vii
LIST OF APPENDIX TABLES ............... ........
I.
ii
Page
LITERATURE REVIEW ........................................
1
Introduction ...........................................
Functional transitions of the mammary gland .......
Mastitis and the dry period ........................
Physiology of Mammary Gland Involution ..............
Cessation of milk synthesis and secretion .........
Merits of a dry period .........
Functional morphology during i n v o l u t i o n .......
Functional morphology during lactogenesis..........
Association of corpora amylacea with
nonlactating tissue ..............................
Secretion composition of the nonlactating gland ...
Susceptibility to Mastitis During the Dry Period ....
..............
Economics of mastitis c o n t r o l
Factors affecting bovine mastitis ..................
Defense Systems of the Mammary Gland .................
Anatomical defense mechanisms ......................
Cellular aspects of mammary immunity ..............
Humoral immune system ...............................
Chemical defense mechanisms ........................
Enhancement of mammary defenses ....................
References ..............................
1
1
2
3
3
A
6
7
8
10
13
13
13
17
17
19
22
23
24
28
SECRETION COMPOSITION DURING BOVINE MAMMARY
INVOLUTION AND THE INTERRELATIONSHIP WITH MASTITIS ---
39
Abstract ................................................
Introduction .........................................
Materials and Methods ................
Experimental design .................................
Microbiological procedures ...............
Milk somatic cells ..................................
Total protein and butterfat
......
Compositional analyses ..............................
40
41
43
43
44
44
45
45
iii
Chapter
Page
Results ..................................................
Discussion ..............................................
References ........................................... .. •
Tables and Figures .............................
III.
IV.
V.
46
50
55
58
MORPHOLOGICAL CHANGES IN THE BOVINE MAMMARY GLAND DURING
INVOLUTION AND LACTOGENESIS..............................
74
Abstract .........................................
Introduction ..........................................
Materials and Methods ................................
Experimental design ................................
Tissue preparation .................................
Morphometric analysis
.............................
Ultrastructural examination .......................
Microbiological procedures ........................
Results ...............................................
Discussion ............................................
References ........................................
Tables and Figures ...................................
75
76
77
77
78
78
79
80
81
87
95
98
QUANTIFICATION AND IMMUNOGLOBULIN CLASSIFICATION OF
PLASMA CELLS IN NONLACTATING BOVINE MAMMARY TISSUE ---
112
Abstract ............................
Introduction .......................................
Materials and Methods .................................
Experimental design .................................
Microbiological procedures .........................
Tissue preparation ..................................
Staining procedure for histochemical analysis ....
Morphometric analysis ...............................
Results .................................................
Discussion
........................................
References ...........
Tables and Figures ........................
113
114
116
116
117
117
118
119
120
122
126
129
SUMMARY AND CONCLUSIONS .................................
136
References .............................................
142
A P P E N D I X ..................................................
144
VITA ......................................................
199
APPROVAL SHEET
203
LIST OF TABLES
Table Number
1
2
3
4
5
6
7
8
9
10
11
Page
Frequency of bacterial isolates in bovine
mammary secretion from drying off through
early lactation ........................................
58
Effect of bacterial infection on numbers of total
somatic cell and differential cell counts in
mammary secretion of nonlactating cows ..............
59
Frequency of bacterial isolates from bovine
mammary foremilk samples from drying off through
lactogenesis ................
98
Histological analysis* of bovine mammary tissue
from drying off through lactogenesis .................
99
Cytological analysis* of bovine mammary epithelium
from drying off through lactogenesis ............
100
Effect o| infection status on histological
analysis of nonlactating bovine
mammary tissue ........................................
101
Effect o| infection status on cytological
analysis of nonlactating bovine
mammary tissue ........................................
102
Ultrastructural analysis* of alveolar cells in
bovine mammary tissue from drying off
through lactogenesis ..................................
103
Effect of infection status on ultrastructural
analysis of alveolar cells in nonlactating bovine
mammary t i s s u e .......................................
104
Cytological comparison of infiltrating cells
in uninfected mammary tissue from drying off
through lactogenesis ..................................
105
Frequency of bacterial isolates from bovine
mammary foremilk samples from drying off through
lactogenesis ...........................................
129
v
LIST OF TABLES (continued)
Table Number
12
Enumeration of specific plasma cgll glasses
(mean number of cells per 6 X 10 pm tissue area)
in the bovine mammary gland from drying off
through lactogenesis in uninfected and infected
quarters ............................................
13
Effect of infection status on distribution
(mean number of cells per 6 X 10 pm tissue area)
of specific plasma cell classes in the
bovine mammary gland from drying off through
lactogenesis
.......................................
LIST OF FIGURES
Figure Number
1
2
3
4
5
6
7
Page
Changes in total somatic cell counts in bovine
mammary secretion from drying off through
the first 14 days of lactation .....................
61
Changes in concentrations of lactoferrin and
citrate and the citrate to lactoferrin molar
ratio in bovine mammary secretion in uninfected
quarters from drying off through the first 14
days of lactation.............................
63
Effect of infection status on percent fat
and total protein in bovine mammary secretion
from drying off through the first 14 days
of lactation ........................................
65
Effect of infection status on pH of bovine mammary
secretion from drying off through the first 14
days of lactation ................ .........
67
Changes in concentrations of bovine serum albumin
in mammary secretion from drying off through
the first 14 days of lactation .....................
69
Effect of infection status on concentrations of
immunoglobulin G in bovine mammary secretion
from drying off through the first 7 days of
l a c t a t i o n ............... . ...........................
71
Changes in concentration of a-lactalbumin in
bovine serum from drying off through the
first 14 days of l a c t a t i o n ........................
73
8
Mammary tissue typically obtained at drying off
and calving exhibiting minimal stromal area (S)
with larger proportions of epithelium (E) and
distended lumina (L) occupying the tissue area.
Fully active epithelial cells were characterized
by the basally located nuclei (N), large cytoplasmic
to nuclear ratio, and presence of numerous secretory
vesicles (SV) in the apical cytoplasm X 935 .....
107
9
Mammary tissue obtained at 14 days after drying
off appeared nonactive with a large proportion
of stromal area (S) with minimal luminal area (L).
The shrunken alveoli (A) were characterized by a
layer of closely packed cells, and the limited
luminal areas stained deeply basophilic X 935 ....
vii
107
LIST OF FIGURES (continued)
Figure Number
10
11
12
13
1A
15
16
Page
Portion of an alveolus typically obtained at
drying off and calving characterized by
polarized cells with a basal nuclei (N) and
supranuclear Golgi (G). Abundant rough
endoplasmic reticulum (R), mitochondria (M),
and apically situated fat (F) and secretory
vesicles (SV) occupied the cytoplasmic area
with microvilli protruding from the apical
surface X 6,075 .....................................
107
Nonactive epithelial cells 1A days after
drying off characterized by a small
cytoplasmic to nuclear ratio and irregularly
shaped nuclei (N).
The cytoplasm consisted
only of Golgi dictyosomal membranes (G) and
scattered mitochondria, but no rough endoplasmic
reticulum cisternae.
The apical surface
(arrows) lacked extensive microvilli and the
alveolar lumen (L) contained an accumulation of
electron-dense material.
S, stroma X 6,075 .....
107
Portion of an alveolus obtained 7 days prior to
to calving demonstrating fluid accumulation.
F,
fat; G, Golgi aparatus; L, lumen; N, nucleus; S,
stroma; and SV, secretory vesicle X
7,560 .......
109
Corpora amylacea (CA) were most frequently
observed filling the alveolar lumen
X 687 .......
Ill
Polymorphonuclear leukocytes (P) exhibited
phagocytic vacuole containing mammary secretion
components (arrows), and macrophages (M) with
internalized cellular debris (arrowheads)
X 5,625 ..............................................
Ill
Positive staining of plasma cells (arrows)
located in the subepithelial stroma (S) and
within the alveolar epithelial (E) lining of
mammary tissue involuted for 7 days X 1,A60 ......
133
Clusters of
within the
parenchyma
epithelium
133
positive staing plasma cells
stromal area (S) of mammary
obtained at parturition.
E,
X 1,A60 ..................................
viii
LIST OF FIGURES (continued)
Figure Number
17
Page
Clusters of plasma cells exhibiting
abundant parallel lamellae of rough
endoplasmic reticulum containing granulated,
electron-lucent material (arrows) X 6,320 .......... 135
ix
LIST OF APPENDIX TABLES
Table Number
la
2a
3a
4a
5a
6a
7a
8a
Page
Sources of variation, degrees of freedom, and mean
squares for numbers of total somatic cells and
differential cell counts in bovine mammary
secretion from uninfected quarters and those
infected with minor and major pathogens ...........
145
Source of variation, degrees of freedom, and mean
squares for numbers of total somatic cells and
differential cell counts in bovine mammary
secretion from uninfected and infected quarters ....
146
Sources of variation, degrees
of freedom, and mean
squares for concentrations of
lactoferrin and
citrate in bovine mammary secretion from
uninfected quarters and those infected with minor
and major pathogens ..................................
147
Sources of variation, degrees
of freedom, and mean
squares for concentrations of
lactoferrin and
citrate in bovine mammary secretion from uninfected
and infected quarters ..............
148
Sources of variation, degrees
of freedom, and mean
squares for percent fat and total protein in bovine
mammary secretion from uninfected quarters and
those infected with minor and major pathogens .....
149
Sources of variation, degrees of freedom, and mean
squares for percent fat and total protein in bovine
mammary secretion from uninfected and infected
quarters ..............................................
150
Sources of variation, degrees of freedom, and mean
squares for concentration of immunoglobulin G in
bovine mammary secretion from uninfected quarters
and those infected with minor and major
pathogens .....................................
151
Sources of variation, degrees of freedom, and mean
squares for concentration of immunoglobulin G in
bovine mammary secretion from uninfected and
infected quarters ....................................
152
x
LIST OF APPENDIX TABLES (continued)
Table Number
9a
Sources of variation, degrees of freedom, and mean
squares for pH and concentrations of bovine serum
albumin and a-lactalbumin in bovine mammary
secretion from uninfected quarters and those
infected with minor and major pathogens ..........
10a
Sources of variation, degrees of freedom,
and mean squares for pH and concentrations
of bovine serum albumin and a-lactalbumin
from uninfected and infected quarters ............
11a
Sources of variation, degrees of freedom,
and mean squares for concentrations of
a-lactalbumin in bovine blood sera
.........
12a
Sources of variation, degrees of freedom,
and mean squares for numbers of infiltrating
leukocytes within the epithelial lining of
bovine mammary tissue from uninfected quarters and
those infected with minor and major pathogens ....
13a
Sources of variation, degrees of freedom,
and mean squares for numbers of infiltrating
leukocytes within the epithelial lining of bovine
mammary tissue from uninfected and infected
quarters ................................... ........
14a
Sources of variation, degrees of freedom, and
mean squares for numbers of infiltrating
leukocytes within alveolar lumina of bovine
mammary tissue from uninfected quarters and
those infected with minor and major pathogens ....
15a
Sources of variation, degrees of freedom, and
mean squares for numbers of infiltrating
leukocytes within alveolar lumina of bovine
mammary tissue from uninfected and infected
quarters ........................... ................
16a
Sources of variation, degrees of freedom, and
mean squares for numbers of infiltrating
leukocytes within the subepithelial stroma
of bovine mammary tissue from uninfected quarters
and those infected with minor and major
pathogens .......... ................................
LIST OF APPENDIX TABLES (continued)
Table Number
17a
18a
19a
20a
21a
22a
23a
Page
Sources of variation, degrees of freedom, and
mean squares for numbers of infiltrating
leukocytes within the subepithelial stroma of
bovine mammary tissue from uninfected and
infected quarters
................................
161
Sources of variation, degrees of freedom,
and mean squares for percent tissue area
composed of epithelium, lumen, and stroma in
bovine mammary glands from uninfected quarters
and those infected with minor and major
pathogens .............................................
162
Sources of variation, degrees of freedom,
and mean squares for percent tissue area
composed of epithelium, lumen, and stroma
in uninfected and infected bovine mammary
glands ................................................
163
Sources of variation, degrees of freedom,
and mean squares for percent tissue area
composed of nonactive, moderately active,
and fully active secretory epithelium in bovine
mammary glands from uninfected quarters and
those infected with minor and major pathogens .....
164
Sources of variation, degrees of freedom,
and mean squares for percent tissue area
composed of nonactive, moderately active, and
fully active secretory epithelium in bovine
mammary glands from uninfected and infected
quarters ..............................................
165
Sources of variation, degrees of freedom,
and mean squares for numbers of corpora amylacea
found within the lumen, epithelium, and stroma
of bovine mammary tissue from uninfected
quarters and those infected with minor and
major pathogens .......................................
166
Sources of variation, degrees of freedom,
and mean squares for numbers of corpora amylacea
found within the lumen, epithelium, and stroma
of bovine mammary tissue from uninfected and
infected quarters
.............................
167
xii
LIST OF APPENDIX TABLES (continued)
Table Number
24a
25a
26a
27a
Page
Sources of variation, degrees of freedom,
and mean squares for percent alveolar cell area
composed of nucleus, cytoplasm, rough endoplasmic
reticulum, Golgi, mitochondria, fat, secretory
vesicles, and stasis vacuoles in bovine mammary
tissue from uninfected quarters and those infected
with minor and major pathogens .....................
168
Sources of variation, degrees of freedom,
and mean squares for percent alveolar cell area
composed of nucleus, cytoplasm, rough endoplasmic
reticulum, Golgi, mitochondria, fat, secretory
vesicles, and stasis vacuoles in bovine mammary
tissue from uninfected and infected quarters ......
169
Sources of variation, degrees of freedom,
and mean squares for percent tissue area composed
of immunoglobulin G^, G 2 * A, and M plasma cells
in bovine mammary tissue from uninfected quarters
and those infected with minor and major
pathogens .............................................
170
Sources of variation, degrees of freedom,
and mean squares for percent tissue area composed
of immunoglobulin G., G^, A, and M plasma cells
in bovine mammary tissue from uninfected and
infected quarters .............
171
xiii
LIST OF APPENDIX FIGURES
Figure Number
la
2a
3a
4a
5a
6a
7a
8a
9a
10a
Page
Changes in percent monocytes in bovine
mammary secretion by week of involution
and infection status .................................
172
Changes in percent lymphocytes in bovine
mammary secretion by week of involution
and infection status .................................
173
Changes in percent polymorphonuclear leukocytes
in bovine mammary secretion by week of involution
and infection status ...............................
174
Changes in concentrations of lactoferrin
in bovine mammary secretion by week of
involution and infection status...................
175
Changes in concentrations of citrate in
bovine mammary secretion by week of involution
and infection status................................
176
Changes in percent fat in bovine mammary
secretion by week of involution and
infection status....................................
177
Changes in percent protein in bovine mammary
secretion by week of involution and infection
status.......................................
178
Changes in concentration of bovine serum
albumin in bovine mammary secretion by week
of involution and infection status.................
179
Changes in concentration of a-lactalbumin in
bovine mammary secretion by week of involution
and infection status................................
180
Changes in percent tissue area composed of
epithelium from bovine mammary glands by
week of involution and infection status...........
181
xiv
LIST OF APPENDIX FIGURES (continued)
Figure Number
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
Page
Changes in percent tissue area composed of
lumen from bovine mammary glands by week of
involution and infection status ...................
182
Changes in percent tissue area composed of
stroma from bovine mammary glands by week of
involution and infection status ...................
183
Changes in percent alveolar area
composed of nonactive secretory cells
from bovine mammary glands by week
of involution and infection status .......
184
Changes in percent alveolar area
composed of moderately active secretory
cells from bovine mammary glands by week
of involution and infection status ...............
185
Changes in percent alveolar area composed
of fully active secretory cells from bovine
mammary glands by week of involution and
infection status
..............................
186
Changes in percent epithelial cell area
composed of nucleus from bovine mammary
tissue by week of involution and infection
status .........................
187
Changes in percent epithelial cell area
composed of unoccupied cytoplasm from
bovine mammary tissue by week of involution
and infection status .........
188
Changes in percent epithelial cell area
composed of rough endoplasmic reticulum from
bovine mammary tissue by week of involution
and infection status ...............................
189
Changes in percent epithelial cell area
composed of Golgi apparatus from bovine
mammary tissue by week of involution and
infection status ...................................
190
Changes in percent epithelial cell area
composed of mitochondria from bovine
mammary tissue by week of involution and
infection status ...............
191
xv
LIST OF APPENDIX FIGURES (continued)
Figure Number
21a
22a
23a
24a
25a
26a
27a
Page
Changes in percent epithelial cell area
composed of fat from bovine mammary tissue
by week of involution and infection status ......
192
Changes in percent epithelial cell area
composed of secretory vesicles from bovine
mammary tissue by week of involution and
infection status .................................
193
Changes in percent epithelial cell area
composed of milk stasis vacuoles from bovine
mammary tissue by week of involution and
infection status ........................
194
Changes in numbers of immunoglobulin G^producing plasma cells in bovine mammary
tissue by week of involution and infection
status ...........................................
195
Changes in numbers of immunoglobulin G ^ producing plasma cells in bovine mammary
tissue by week of involution and infection
status ..............................................
196
Changes in numbers of immunoglobulin Aproducing plasma cells in bovine mammary
tissue by week of involution and infection
status ..............................................
197
Changes in numbers of immunoglobulin Mproducing plasma cells in bovine mammary
tissue by week of involution and infection
status ..............................................
198
xvi
ABSTRACT
Quarter
milk
secretion
samples
and
blood
serum
for
compositional analysis were collected weekly from 29 cows beginning
at
drying
off
and
continuing
until
2
wk
biopsies were
taken from 5 additional animals
beginning
drying
at
off
through
postpartum.
Quarter
at weekly
intervals
parturition.
Histological
and
cytological parameters
of tissues were correlated with biochemical
characteristics
secretions.
of
Increased
tight
junction
permeability and decreased synthetic ability of secretory epithelium
became
evident
by
changes
in
mammary
secretion
composition
tissue morphology during the first 2 wk of involution.
lactoferrin,
immunoglobulin
Somatic cell
counts,
serum
protein,
and serum concentrations of a-lactalbumin increased while
fat,
albumin,
and
G,
pH,
total
citrate, and the citrate to lactoferrin molar ratio decreased.
Morphometric analysis of tissue demonstrated increases in stroma and
• -
i,
nonactive secretory epithelium with decreases in epithelium,
lumen,
and
wk
fully
active
involution.
secretory
epithelium
during
function and structure was
percent
2
of
These biochemical and structural
changes reversed beginning 2 wk prepartum,
had
first
Decreases in organelles associated with milk synthesis
and secretion were observed also.
quarters
the
typical
significantly
polymorphonuclear
of
higher
and by parturition,
lactating
somatic
leukocytes,
but
glands.
cell
lower
counts,
cell
Infected
pH,
concentrations
and
of
lactoferrin and percent lymphocytes compared to uninfected quarters.
Tissue
from
infected
quarters
also
xvii
had
less
synthetic
and
secretory
cells,
ability with higher percentages
but
lower
quarters.
percentages
Plasma
cell
of
lumen
populations
increased gradually from drying off,
wk
prepartum,
gestation.
isotypes
and
followed
were more
than
uninfected
by
material
bacterial
pathogens
nonlactating
mammary
of
G2
were
A
and
infected
cisternae
antibody
apparently
tissue.
uninfected
mammary
during
Ultrastructural
reticulum
indicative
bovine
to
elicited
These
the
M.
an
data
last
most
the
natural
wk
with
minor
provide
M
revealed
flocculent
Exposure
immune
of
pathogens
examination
with
2
numerous
Immunoglobulin
engorged
synthesis.
the
to
minor
response
in
information
concerning the quantitation and distribution of components
in the mammary
tissue
reached peak concentrations
and
in tissue
quarters.
endoplasmic
in
immunoglobulins
numerous
stroma and nonactive
compared
significantly
Immunoglobulins
cells
rough
dropped
of
involved
immmune system which may be manipulated to enhance
defense
mechanisms
of
gland.
xviii
the
involuted
bovine
mammary
C H A P T E R
I
LITERATURE REVIEW
Introduction
Functional transitions of the mammary gland
Functional activity of the mammary gland varies from a dormant
phase in nonlactating animals to a vigorous level during lactation.
During
successive
reproductive
cycles,
transformation
of
mammary
cells from an involuted to a secretory state is highly dependent on
hormonal
(20),
influences.
peak milk
results,
nutritional
(41,104),-
and
neurohormonal
Likewise, cessation of copious milk secretion following
yield
is under
in part,
determined
by
similar
control.
Involution
and
mammary
mammary
number
gland
peak
reductions
cells
resulting
involution
following
(63).
in
in the
rat
by a reduction in total secretory cell numbers as
deoxyribonucleic
acid
Previous studies have reported considerable decreases
size
(60,123)
from
(31,33).
lactation
synthetic
Relatively
in
and
lysosomal
Gradual
the
cow
reduction
may
secretory
little
digestion
be
in
is
(84).
in both cell
during
milk
attributed
capabilities
information
(DNA)
rat
yield
also
to
of
remaining
available
regarding
the demise of existing mammary secretory cells with each functional
transition.
Autoradiography of rat mammary glands suggests as much
1
2
as
a
75%
carry-over
of
secretory cells
from
one
lactation
to
the next (93).
The extent to which alveoli persist through involution into the
subsequent
Adequate
lactation
in
proliferation
ruminant
and
mammary
differentiation
glands
of
is
mammary
unknown.
secretory
epithelium during the nonlactating period was shown to be essential
for
optimal
lactation
of
synthetic
both
cows
and
and
secretory
goats
function
in
(73,115,116).
the
ensuing
Mechanisms
which
regulate cellular differentiation and the onset of lactation need to
be further defined,
and a greater understanding of
these processes
may provide new approaches for increasing milk production in dairy
cattle.
Mastitis and the dry period
Although the dynamics
understood
clearly,
of
lactogenesis
susceptibility of
the
and
involution
are not
bovine mammary gland to
bacterial infection is greatest during these functional transitions.
Neave
and
infection
times
coworkers
(67)
found
the
incidence
of
new
intramammary
(IMI) during the first 3 wk of involution to be 7 to 10
greater
than during
lactation.
Moreover,
the new infection
rate during the dry period was thought to account for the level of
mastitis in subsequent lactations (106).
Studies demonstrated that
unmilked quarters were more susceptible to IMI than milked quarters
as a result of cessation of the flushing action associated with the
milking process and fluid accumulation
(69).
Cows producing large
quantities of milk at drying off were found to be more susceptible
to new IMI during the early dry period
(79).
The high rate of IMI
3
during periods of mammary transition has been associated also with
lower
levels
(81).
The
logical
of
protective
early dry period
points
Unfortunately,
during
natural
of attack
and the
the
for
histochemical
involution,
not understood.
and
factors
control
occurring
interaction of
secretion
period
of
within
IMI with
are
mastitis.
the
udder
involution are
Further details concerning changes in both mammary
morphology and biochemistry with respect to
are
mammary
periparturient
the
changes
in
warranted and
may
lead
to
innovative
immunological function
approaches
for
mastitis
control.
Physiology of Mammary Gland Involution
Cessation of milk synthesis and secretion
Bovine
mammary
gland
involution
can
be
characterized
as
progressing through three distinct phases: a) gradual involution; b)
initiated
involution;
and
c)
senile
involution
(45).
Gradual
involution occurs during the course of a normal lactation following
peak
milk
yield.
yield which
total
This
results
is
presumably
secretory cell numbers
activity of
remaining
manifested
from
(65)
by
a
cells as observed
naturally-occurring
decrease
gradual
and/or
in
reduction
depressed
in mice
involution has a more drastic effect on the
than does
a
milk
in
synthetic
(121).
Initiated
"drying off" process
involution following peak
lactation.
Abrupt cessation of milking in the goat was found to reduce mammary
secretion rate up to 80% by day 3 of the dry period (25).
Mammary
4
distension, with a concomitant increase in intramammary pressure,
is
thought to be responsible for arresting milk secretion in the goat
(91).
In
also
to
Gradual
number
addition, infiltration of phagocytic cells has been shown
expedite
the
reduction
is
in
referred
involution
milk
to
yield
as
process
with
senile
in
the
advancing
rat
(31,100).
or
lactation
age
involution.
Changes
in
milk
production are thought to be due to mammary gland deterioration and
reduction in secretory tissue brought about
by increased incidence
of mastitis in older animals (45).
Merits of a dry period
A
customary
procedure
implemented
by
dairy
farmers
is
to
initiate involution during the seventh to eighth month of pregnancy.
Previous studies
is
related
indicated that duration of the nonlactating period
critically
lactation (17,107,117).
to
secretory
activity
during
the
ensuing
Dairy cows which averaged 10 to 40 days dry
produced less milk in the following lactation than cows having a dry
period
of
40
to
60
days
(17).
Moreover,
cows which were milked
continually throughout pregnancy produced 33%
less milk during the
subsequent lactation compared to their twins with a 2 mo dry period
(117).
Benefits
improvements
lactation.
in
derived
the
from
cow's
a
dry
nutritional
period
status
involve
for
the
more
than
forthcoming
Favorable effects of involution on subsequent milk yield
result from regeneration and/or reactivation of secretory epithelium
before the next lactation begins.
Biosynthetic
population
of
such
activity
cells
of
play
secretory
decisive
cells
roles
in
and
the
determining
total
milk
5
yield.
During lactation, total mammary DNA declines following peak
lactation and continues to decrease as lactation progresses in mice
(42).
Because mitotic activity is absent in secretory cells during
established lactation
in mice
(42),
loss
in cell numbers
decreased DNA content cannot be replenished
majority
of
cellular
hamsters
occurs
(2,4,105).
in
proliferation
nonlactating
Previous
studies
have
during lactation.
in ruminants,
mammary
the
The
guinea pigs,
glands
found
based on
during
greatest
and
pregnancy
increases
in
mammary DNA content of goats (3), guinea pigs (4), and heifers (118)
to
occur
in
the
last
trimester
of
pregnancy
and
continuing
occasionally to day 5 of lactation.
The
plant
examine
alkaloid,
factors
development.
which
Mode
colchicine,
regulate
of
milk
colchicine
has
been used extensively to
production
action
and mammary
is
disruption
intracellular microtubular integrity necessary for mitosis
exocytotic
mechanisms
(74,94).
gland
of
(53) and
Prepartum intramammary infusion of
colchicine in heifers altered secretion composition and lowered milk
production
in
cytological
supported
subsequent
evidence
the
suppressed
(73,115).
the
in
concept
irreversibly
In
both
a
recent
lactation
bovine
that
a
period
of
and
colchicine
active
of
a
findings
reduction
support
in
the
total
treatment
epithelia
last
interfered apparently with mitosis
mammogenesis.
secretory
contention
mammary
and
glands
infusion during the
Lower
observed with colchicine-treated udder halves,
from
mammary
colchicine
differentiation
study,
Histological
caprine
prepartum
trimester of pregnancy in goats
during
(1).
that
cell
an
milk
yields
were
resulting presumably
numbers
adequate
(116).
dry
These
period
is
6
essential
for
cellular
proliferation,
epithelial
development,
and
optimal milk production.
Functional morphology during involution
Helminen
and
Ericsson
(30,31,32,33)
studied
the
histological
and ultrastructural changes of rat mammary glands during involution.
They found that once milk cessation occurred, the rat mammary gland
became distended with milk, and alterations in milk producing cells
became visible within approximately 24 h.
alveoli
and
ducts
degeneration
of
increased
secretory
Accumulation of milk
intramammary
cells
alveolar and lobular structures.
with
pressure,
subsequent
and
in
caused
disruption
of
Milk stasis became evident with an
accumulation of fat droplets and secretory vesicles, and a reduction
in
size
of
involution
the
rough
progressed,
reduced substantially
pyknotic
endoplasmic
and
the
secretory
reticulum
and
became
concomitant increase in cytosomes.
numerous
fat
droplets
autophagocytosis
by
synthetic
in size and number
cytoplasm
organelles
(31,99).
vacuolated
(31,32,99).
As
became
Nuclei appeared
extensively
with
a
Macrophage-like cells containing
appeared.
lysosomes
(RER)
By
within
48
the
h,
an
epithelium
increase
was
in
found,
accompanied by leukocytic infiltration and notable reduction in cell
volume (33).
After 72 h into involution, macrophages were observed
often between epithelial cells and
debris
(31).
to
Degenerative
within
48
72 h
intact
(31,32).
after
However,
cells
ingesting fragments
were
weaning,
the
rat
shed
leaving
mammary
into
only
of cellular
alveolar
lumens
basement membranes
gland
did
not
regress
7
entirely
as
many
alveoli
persisted.
Many
myoepithelial
cells
remained while secretory cells
were eliminated, and appeared
an important role in bridging
gaps where necrotic epithelial cells
had sloughed,
to play
thereby preventing total loss of organized structure
(31).
In
the
fully
involuted
caprine mammary
gland,
secretory tissue decreased proportionately to
total
(115).
to
rat
was
observed
in
the
fully
of
increased amounts of
intralobular and interlobular connective tissue
what
area
involuted
In contrast
mammary
gland,
sloughing of epithelial cells into alveolar lumina was not apparent
in the
goat.
Instead,
electron-dense
alveoli
proteinaceous
exhibited small
material,
and
lumina filled with
alveolar
epithelial
cells were in an undifferentiated state.
Functional morphology during lactogenesis
Histological
growth
and
increased
gestation
cytological
rapidly
(125).
Cowie
evidence
in
cows
between
(19)
observed
showed
lobulo-alveolar
days 110
limited
and
140
changes
in
of
the
structure of primigravid goat mammary glands during the first half
of
pregnancy.
occurred
between
periparturient
intense
However,
period
mammary
parenchyma
60
a
period
and
in
growth
120
both
and
(39,42,115).
of
days
mice
rapid
Prepartum
advanced
of
alveolar
gestation
and goats was
mammary
The
associated with
differentiation
goat
(19).
growth
of
secretory
tissue
exhibited
characteristics indicative of copious milk synthesis and secretion,
and as parturition approached,
synchronous
increases
in
total area of stroma decreased with
luminal
and
epithelial
areas
(115).
8
Cytological examination indicated gradual differentiation of mammary
epithelium during the last trimester of pregnancy with an increased
cytoplasmic to nuclear ratio, a higher degree of cellular polarity,
and more apically located secretory vesicles.
Association of corpora amylacea with nonlactating tissue
Corpora
observed
amylacea
frequently
are
in
spherical,
bovine
lamellated
mammary
inclusion
tissues.
Notice
bodies
of
their
appearance in bovine mammary glands dates back to the early 1900's
(82).
Biochemical
analysis
found
bovine
corpora
amylacea
to
be
composed of dicalclum and monocalcium phosphates (28), alkaline and
acid phosphatases,
proteins,
and
lipids
studies of bovine corpora amylacea
(52).
Early morphological
indicated complex heterogeneous
structures composed of a number of distinct concentric layers (59).
Ultrastructurally,
morphological
displayed
forms.
often
casein-like
corpora
amylacea
Dense
several
material
bodies
appeared
to
of the total amyloid population.
fibrils.
than
dense
Fibrils
approximately
10 nm
forms
were
were
be
and
arranged
in
deeply
lamellated striations.
components of these amyloid bodies.
basophilic
appeared
2
basophilic
Centrally
deposited
basic
among
and
located,
fibrillar
These structures comprised 70%
Fibrillar bodies
appeared
in
to
(30%) were
contain
parallel
less
only amyloid
arrays
measuring
in diameter and displayed often a filamentous
network (10,76).
Occurrence of bovine corpora amylacea throughout the lactation
cycle was reviewed extensively by Nickerson et al. (76).
Prevalence
9
of corpora amylacea Increased gradually from early to late lactation
and
ultimately
progressed,
peaked
numbers
during
of
early
corpora
involution.
gradually
As
involution
decreased
toward
lactogenesis.
Although
the
origin
and
demise
of
corpora
amylacea
are
not
understood clearly, previous research suggests they are derived from
aggregation
of
casein
micelles
in
alveolar
milk.
It
has been
postulated that mechanisms of aggregation in the initial stages of
amylaceum formation involve co-precipitation of casein with calcium
phosphate (10).
(62,76) who
synthetic
This concept is consistent with theories of others
suggested
and
corpora
secretory
amylacea
processes.
developed
Recent
from deposits
studies
on
of
growth
patterns of bovine corpora amylacea suggest the development of these
structures
although
is
not
restricted
nucleation
appears
to
a particular
to
occur
stage
within
of
lactation,
alveolar
lumens.
Gradual increases in size and number of corpora from parturition to
late
lactation
indicate
that
development
of
the
structures
accelerates as lactation progresses (114),
Amyloid
concentrations
found within
during lactation have been implicated in
of
involution
(76).
with
by
filling
luminal
the bovine
mammary
gland
milk stasis and the onset
spaces and clogging
small
ducts
Accumulation of corpora during late lactation may interfere
mechanisms
reduced
milk
of
yield
milk
up
to
synthesis
30%
(14).
and
It
secretion,
has
been
resulting
theorized
in
that
corpora diminish throughout the dry period by the phagocytic action
of
macrophages
and
multinucleated
giant
cells
(MGC)
(75).
Phagocytosis appears to be instrumental in reducing concentration of
10
amyloid
prior
to
the
subsequent
lactation
and
preventing
accumulation throughout the productive lifetime of the animal.
Secretion composition of the nonlactating gland
Several
biochemical
changes
in
following cessation of lactation.
milk
constituents
(casein,
secretion
composition
Synthesis and secretion of major
lactose,
and fat)
decrease considerably
by the fourth day of involution in the cow (29,131).
mammary
gland,
a
ribonucleic acid
during
earlier
(mRNA)
stages
concentrations
bicarbonate,
increases
(25,91).
substantial
of
and
with
decrease
in
both
In the rabbit
casein
and its transcription can also
of
initiated
cessation
serum
of
messenger
be detected
involution
(120).
Conversely,
(Ig),
sodium,
chloride,
immunoglobulins
bovine
occur
albumin
lactation
(BSA)
in
increase,
cows
(29)
and
and
pH
goats
These compositional changes are correlated closely to the
breakdown of secretory epithelium and reduced metabolic activity of
remaining cells during involution.
In cows and goats,
concentrations
fluid
volume
involution
in milk
reduction
(77,91,112)
gradual
increases
in serum protein and
are synchronous with
between
the
resulting
in
third
the
the period
and
ion
of mammary
seventh
concentration
day
of
the
of
2
components.
Previous research suggested also that these changes may
result
a
from
loss
of
alveolar
cell
integrity
plasma constituents and ions into alveolar lumina
and Peaker (A9.51) examined changes
allowing
(25,AO).
entry
of
Linzell
in colostrum composition in the
goat at about the time of parturition.
They found that lactose and
11
potassium
concentrations
in
the
prelactating
gland
decreased
when
tight junctions between adjacent secretory cells became "leaky".
the lactating
gland,
tight
junctions
became
impermeable,
In
enforcing
polarized transport of serum-derived components via a transcellular
pathway,
and
resulted
in
increased
lactose
and
potassium
concentrations following parturition.
A
continuous
derived
from
increase
de
novo
in
concentration
synthesis
during
the
involution suggests
that the
change
cell activity as opposed
in
alveolar
of
milk
constituents
initial
involutionary process
to
stages
also
of
involves
complete
a
cellular
dissolution.
The iron-binding protein, lactoferrin (Lf), is a major
whey protein
in secretion of fully involuted bovine mammary glands
(108).
Lactoferrin
epithelium
and,
neutrophilic
increase
by
is
to
a
leukocytes
the
thought
fourth
lesser
(PMN)
day
formed
from
secreted
Citrate
after
as the
by
CoA
vesicles
concentrations
the
onset of
and
extent,
involution
and
(81).
concentrations
remain
within
Golgi
decrease
secretory
polymorphonuclear
process continues.
from
by
elevated
as
levels of citrate decrease
oxaloacetate
not
by
Lactoferrin
Conversely,
derived
do
synthesized
involution
involutionary
acetyl
be
(54,55).
of
involution progresses (129).
gradually
to
until
This
Milk citrate
mitochondria,
components
and
(24,133).
approximately
indicates
is
7 days
that metabolic
activity in secretory epithelial cells continues past the third, and
up to the seventh day after drying off.
molar
rat io decreases
gradually
However,
from drying
decline as the gland involutes (81).
off
the citrate to Lf
and
continues
to
12
Lactogenesis is initiated hormonally near the end of pregnancy,
and is characterized by biochemical and morphological changes in the
mammary gland which have been defined loosely in 2 stages (26).
The
first
cytological
and
accompanied
by
comprises
enzymatic
the
prepartum
differentiation
appearance
of
period
of
precolostral
during
alveolar
fluid.
which
cells
Stage
is
2
begins
just
before
parturition and is noted by the onset of copious colostral secretion
(26,29).
As
parturition
approaches,
major
changes
in
the
composition of colostrum were demonstrated in cows (29), goats (51),
and rats
(13).
biochemical
However,
changes
mammary
cells.
readily
allow
chloride
junctions
Ln
with
prelactating
of
from blood
become
in goats
correlated
passage
ions
only
(50,115)
structural
glands,
sucrose,
to milk
and rats
differentiation
"leaky"
lactose,
(13) were
Ig,
and vice versa.
tight
and
of
junctions
sodium
and
At parturition,
less permeable and block paracellular movement of
serum proteins and
ions into milk
(50).
Instead,
all transport
is
via the transcellular route resulting in decreased levels of sodium,
chloride, and Ig, with increased levels of potassium, a-lactalbumin,
and lactose (49,51).
concentration
parturition
of
a-lactalbumin
(35).
a-lactalbumin,
reflect
Recently, results have demonstrated changes in
The
and
cellular
in
dry
mammary
transient
redevelopment
gland
increases
of
cow
mammary
contention
that
drying off and during
integrity
of
the
changes
in
lactogenesis
tissue
blood-milk
barrier
and
locally
prepartum
as
well
as
may
the
These findings support
secretion
result
through
synthesizes
observed
onset of milk synthesis and secretion (35).
the
secretions
composition
from changes
metabolic
following
in both the
activity
of
13
existing alveolar cells.
Susceptibility to Mastitis Purine the Dry Period
Economics of mastitis control
Mastitis is a general term which refers to an inflammation of
the
mammary
gland.
microorganisms
estimated
have
Most mastitis
within
the
results
gland.
In
from presence of
the
bovine,
it
living
has
been
that approximately one half of the dairy cows world-wide
some
form
of
mastitis
(57).
Apart
from
the
debilitating
effects of the disease on the animal, production losses have proven
to
be
exorbitant.
Dollar
services
and drugs,
animals,
and
(21).
mastitic
the
greatest
by
subclinical
production
caused
shown
subclinically-infected
that
occur
increased culling
discarding
However,
losses
or
loss
from
costs
of
veterinary
rate of chronically-infected
antibiotic-contaminated
occurs
mastitis.
quarters
from
In
reduced
fact,
produce
up
it
milk
milk
has
been
to 45%
less
milk than uninfected quarters (92).
Factors affecting bovine mastitis
Current control programs consist of correct use of functionally
adequate
milking
systems,
disinfection
of
teats
immediately
after
milking, prompt treatment of clinical cases, antibiotic treatment of
all
quarters
cows.
in
at
drying
off,
and
culling
of
chronically-infected
Although these procedures have proven to be highly effective
lactating animals,
most offer
little protection against new IMI
14
during
the
dry
perlad.
Further
progress
mastitis
In
control
procedures are clearly needed not only to reduce new infection rate,
but also to eliminate existing infections.
Intramammary infections occur when microorganisms gain entrance
to the gland via the streak canal and colonize the duct system and
alveoli.
Several
penetrability
vectors
ofthese
equipment,
and milking
the
is
gland
invading
often
pathogenic
metabolism
cause
resulting
in
composition
an
and
decreases
in
been
organisms,
hygiene
identified
i.e,
(36).
favorable
to
The
irritation
to
increases
trace
the
solids,
of
delicate
calcium,
milking
environment
of
multiplication
of
secretory
Changes
leukocyte,
of
facilitate
bacterial
Ig,
concentrations,
concentrations
solids-not-fat, total
and
reaction.
in
mineral
internal
survival
Byproducts
inflammatory
which
environment,
bacteria.
include
chloride),
have
growth
parenchyma,
in
secretion
ion (sodium
with
lactose,
and
and
concomitant
total
protein,
and
potassium
phosphorus,
(71).
Research
mammary
has
gland
to
indicated
that
bacterial
susceptibility
pathogens
is
of
related
the
bovine
critically
to
functional transitions that occur during involution and lactogenesis
(22,67).
first
While
establishment
of
IMI
is
greatest
during
the
3 days following drying off (110), the lowest incidence of new
infection occurs
in the fully
involuted gland approximately 3 to 5
wk following drying off (67).
susceptible
the
new
dry
pathogens
to Gram-positive
period
(103,122)
Moreover, the mammary gland is highly
pathogens
and
highly
during
the
resistant
in the fully involuted gland (6,23).
early
to
stages
of
Gram-negative
15
Hensons
for
susceptibility
However,
dry
more
studies
IMi
rate
the
dry
have
susceptible
intervals
period
high
throughout
several
markedly
regular
the
to
(69,122).
exceeded
at
drying off,
period
shown
new
are
that
and changes
poorly
unmilked
infection
than
in
understood.
quarters
those
are
milked
at
Because new infection rates during the
those
of
lactation,
it
was
suggested
that
pathogens in the streak canal were not flushed out in the absence of
regular
milking.
At
which may also have
This,
however,
during
does
drying
an effect
not
colostrogenesis.
related
to
the
size
off,
teat
sanitation
on susceptibility
explain
the
high
Susceptibility
and
shape
of
the
to
is
discontinued
to mastitis
rate
of
new
infection
streak
infection
may
canal.
(36),
It
also
has
be
been
suggested that heightened susceptibility in the early dry period and
during
colostrogenesis
is
due
to
the
relative
bacterial pathogens penetrate the streak canal.
in
intramammary
shortening
pressure
and
dilation
penetration of bacterial
streak
canals
penetrated
inoculation
(18).
as
following
of
pathogens
immediately
the
or
teat
the
after
cistern,
eliminated
cessation
streak
(79).
drying
but
entirely
were
from
ease
with
Temporary increases
of milking
canal,
Bacteria
off
restricted
dry
may
thus
to
28
and
the
days
of
bacterial
inhibitors,
may
into
often
site
of
or more
It was postulated that changes within the streak canal,
development
cause
allowing
inoculated
multiplied
cows
which
such
make penetration more
difficult in later stages of involution.
Progressive
during
after
changes
involution
bacterial
may
in the
also
penetration
composition
influence
of
of mammary
establishment
the streak canal.
of
secretions
infection
Involuted bovine
16
mammary gland*
appeared to be more susceptible to Escherichia coll
and Klebsiella pneumonia (6,23) just prior to parturition as opposed
to
the
early
fluctuating
Lactoferrin
or
mid dry
citrate
to
sequesters
period.
Lf
This
molar
iron
ratios
during
related
involution
to
(5).
from the environment which is required
by these bacteria for normal growth.
an
phenomenon was
Enteric bacteria also possess
iron-sequestering system involving citrate (111).
The degree of
growth inhibition when both citrate and Lf are present is related to
their
molar
decreased
to
in late
be an
vitro.
ratio.
Therefore,
Conversely,
just
the
citrate
lactation and through
increase
encountered
as
to
Lf
molar
ratio
involution,
there appeared
in growth inhibition of coliform
test strains in
a
decrease in the citrate to Lf molar ratio,
prior
to
parturition,
resulted
in
a
as
subsequent
increase in coliform growth.
Dry
cow
therapy
is
currently
recommended
treatment of IMI during involution.
nature
of
mastitis
infection,
dry
and
cow
the
therapy
for prevention
and
However, because of the complex
diversity
products
in
are
organisms
not
always
that
cause
effective
in
preventing new infections or eliminating those already in existence.
Consequently, mastitis remains widespread in most dairy herds.
Manipulation
natural
protective
secretion
control
gland
of
may
a
systems
provide
procedures.
is
mammary
complex
an
The
physiology
associated
alternative
natural
system
which
in
an
attempt
with
to
mammary
less
enhance
tissue
effective
defense mechanism
includes
to
mastitis
of
the mammary
nonspecific
resistance,
antibody production, and cell-mediated immunity (CMI) (72,128).
efficiency of
this
defense
system
and
is a major
The
factor which governs
17
establishment
of
mastitis.
immunological
potential
into an effective,
control program.
treatment
and
concern.
animal
A
of
better
the
economical,
understanding
mammary
gland
of
could
evolve
and practical nonantibiotic mastitis
Widespread use of antibiotics and germicides
prevention
the
of
bovine
mastitis
has
raised
for
public
Improper use of mastitis treatment may cause residues
products
Alternative
and
present
methods
of
serious
mastitis
health
control
problems
which
in
to consumers.
rely
less
on
antibiotics would be advantageous from a public health standpoint.
Defense Systems of the Mammary Gland
Anatomical defense mechanisms
Nonspecific
anatomic
The
structures,
streak
canal
infection (64).
end
that
protective
tissues
phagocytic
provides
the
of
cells,
the
mammary
gland
and antibacterial
primary
line
of
include
proteins.
defense
against
Mastitis-causing organisms first must traverse teat
to establish
susceptibility
factors which
factors
to
increase
pathogens within
infection
new
within
infection
survivability
the streak canal.
is
the
gland.
influenced
It
greatly
or penetrability of
This structure
follows
by
bacterial
is surrounded by
smooth muscle fibers which function in maintaining tight closure of
the canal.
The ability of certain bacterial pathogens to penetrate
the mammary gland
surrounding
is related to the tonus of the sphincter muscles
the streak canal
(64).
Cows with patent streak canals
are more susceptible to mastitis (69).
18
Ultrastructural
revealed
the
material
fills
barrier
against
observations
mesh-like
the
of
character
lumen
of
pathogenic
the
of
the
the
canal
bacteria.
bovine
streak
canal
keratin
lining.
This
and
provides
It was
an
effective
demonstrated
in cows
inoculated experimentally with Staphylococcus aureus that the nature
of keratin may inhibit progressive movement of cocci from the streak
canal
to the gland cistern
(12).
The keratin lining streak canals
of susceptible quarters was found to be much thinner and less dense
compared to resistant quarters
(56).
Moreover,
removal of keratin
from the streak canal of bovine mammary glands was found to increase
susceptibility to Streptococcus agalactiae infection (64).
New
to
bacterial
populations to which teats of the dry udder are exposed.
Neave and
Oliver
IMI
rates
also
appear
to
be
related
(68) demonstrated a positive correlation between the numbers
of S. aureus applied to teats of dry cows and occurrence of new IMI.
They
also
milking,
have
found
that
in
the
absence
of
repeated
exposure
during
S. aureus numbers on teat skin diminished greatly.
shown
that
cessation
of
milking
favored
Gram-positive cocci into the teat cistern (98).
involuted
glands
appeared
susceptible
resistant to Streptococcus uberis.
to
Others
penetration
of
Lactating and early
S.
aureus,
but
more
However, isolations of S. uberis
from teat skin and orifices increased greatly after 21 days into the
dry period.
Reasons
for the high rate of new IMI and changes in bacterial
flora of teat ends during the dry period remain unclear.
suggest,
that
Gram-positive
bacteria
adhere
ductal epithelium of bovine mammary glands
more
(27).
readily
Findings
to
the
S. aureus and S.
19
agalactiae
colonize
Streptococcus
organisms
mastitis
better
faecalis, E.
shown
most
to
often
on
ductal
coli, or
adhere
during
Corynebacterium
better
the
epithelium
early
are
do
bovis.
Those
which
cause
those
dry period.
than
Since unmilked
quarters are more susceptible to new infection than those which are
milked regularly,
the
streak
canal
it follows that pathogens adhere more readily to
when
the
flushing
action
of
the milking
process
ceases.
Cellular aspects of mammary immunity
Once bacteria breach the streak canal,
population
of
leukocytes
somatic cells.
somatic
within
the mammary
cells
include
PMN,
from uninfected glands
cells/ml
gland
referred
to
as
Electron microscopic studies have shown that mammary
macrophages,
percentage of epithelial cells (48).
milk
they are attacked by a
(72).
In
and
a
small
The concentration of cells
is generally
infected glands,
lymphocytes,
1.0 x
10^
to
3.0
x
in
10^
bacterial products and factors
i
released
from
migration
of
affected
leukocytes
tissues
evoke
inflammation
from blood to milk with
resulting
in
levels as high as
mi 11 ions/ml.
Phagocytosis
of
invading
pathogens
line of defense against mastitis (89).
and macrophages
to 90 7„ of the
is
considered
the
second
Polymorphonuclear leukocytes
are the principal phagocytic cells and comprise 80
cells
in uninfected
bovine milk
(48).
It has
been
demonstrated that more than 5.0 x 10^ leukocytes/ml of foremilk are
required
to
process,
PMN
protect
against
accumulate
in
IMI
(102).
mammary
During
tissue
and
the
milk
inflammatory
through
the
20
process
of
chemotaxis.
Breakdown
products
of
epithelium,
leukocytes, and bacteria serve as chemotactic agents to increase the
influx
of
PMN.
Leukocyte
levels
increase
only
after
microbial
populations have increased, causing tissue irritation and damage.
substantial
time delay
the mammary
gland
milk
and
leukocytes
invasion,
become
occurs
appearance
of
essential
for
are
the time
between
initiation
PMN
of
in milk
defense
irritation
(103).
A
in
Although
against
microbial
Lapse allows a sufficient period for bacteria to
established.
Previous
studies
have
also
demonstrated
deficiencies in the ability of milk PMN to phagocytose mammary gland
pathogens (66,101).
Lower phagocytic and bactericidal properties of
milk PMN,
compared
reduction
in milk PMN glucose (66);
complement
(101);
in
to blood
milk
(132);
PMN,
c)
have
been
attributed
to:
a)
38%
b) deficiencies in opsonins and
binding
of
casein
to
PMN
surfaces
d) loss of PMN pseudopods due to fat ingestion (130); and e)
depletion of hydrolytic enzymes within PMN following fat and casein
ingestion (95).
Macrophages may also play an important role in the phagocytosis
and
intracellular
killing
of
invading
microorganisms.
These
phagocytes are believed to be the first leukocyte type that bacteria
encounter
in
streak canal.
glands,
uninfected
quarters
Although PMN are most numerous
colostrum,
macrophages
and
previously
and
are the
nonlactating
secretion
predominant
glands
(38,45).
during
cell
In
type
the
upon
involution
uninfected
(47),
lactating
involuted mammary gland,
macrophages actively ingest fat globules and appear often as
foamy cells (48).
the
in milk from infected
early
of
breaching
large
Bovine mammary macrophages bear Fc receptors for
21
IgG^
and
pathogens
IgG^
as
which
well
promote
(34).
Ingestion
Although
and
the
killing
major
role
of
of
bacterial
these
cells
appears to be removal of foreign material and cell debris, they also
may play an important role in antigen processing,
the
magnitude
of
lymphocyte
response
involution, macrophages may make
bacterial
antigens to,
have been
shown to enhance the
in the
initial
and in regulating
bovine
(83).
contact with,
lymphoid cells (126).
During
and present
Moreover, macrophages
transformation of blood lymphocytes
in response to phytohemagglutinin in vitro (83).
Migratory lymphocyte populations
of mammary gland immunity.
Data from CMI studies demonstrated that
73% T-lymphocytes
and
peripheral
lymphocyte
blood
B-lymphocytes
found
27%
in
constitute the cellular basis
B-lymphocytes
comprise
population.
normal
bovine
the
total
Percentages
milk
during
approximately 50% and 20%, respectively (16).
bovine
of
T-
lactation
were
B-lymphocytes respond
to antigenic stimulation by multiplication and differentiation
2
morphologically
cells
Ig,
Some
and
and
memory
leading
to
cells
functionally
cells.
proliferation
acquire
antibody-secreting
plasma
discrete
Antigens
RER
of
cells.
bind
populations:
to
sensitized
and
specific
other
into
plasma
surface
B-lymphocyte
eventually
The
and
clones.
develope
population
of
into
cells
derived from stimulated antigen-sensitive B-lymphocytes possesses Ig
receptors
long-lived
of
the same
memory
cells
specificity
which
have
as
their
the
parent.
ability
These
to initiate
are
a
heightened response to a second dose of antigen (124).
T-lymphocytes react to specific antigen in a similar fashion by
differentiation
into
2 cell
populations.
Like
the
B-lymphocytes,
22
memory
cells
maintain
sensitivity over an extended period of time
and will respond to subsequent antigen exposure.
of
synthesizing antibody as B-lymphocytes,
T-lymphocytes
suppressors
takes
and
on
an
effector
helpers.
However,
instead
the other population of
function
Activated
and
acts
as
T-lymphocytes
both
release
lymphokines that enhance recruitment, activation, and immobilization
of
macrophages
cells
and
release
PMN
in
substances
infected
which
tissue areas
stimulate
(119).
B-lymphocyte
T-helper
response
to
antigenic stimulation.
Humoral immune system
Immunoglobulin concentrations of mammary secretion may play an
important
role
in
local
immunity
to
antibody in milk varies with Ig class.
infection.
The
th
in
lactation
mammary
cycle
secretion
and
is
varies
dependent
on
during
lowest
involution
Evidence
cells
during
in
suggests
small
and
lactation
(1 mg/ml)
colostrogenesis
antibodies
vesicles
pass
(50
into
originating
at
Immunoglobulin
considerably
the
tmeability of milk secreting tissue (72).
IgM are
of
Both IgG^ and IgG^ are serum
derived while IgA and IgM are of local origin (70).
cone -ntration
origin
degree
throughout
of
vascular
Levels of IgG, IgA, and
and
to
increase
150
gradually
mg/ml)
(8,A3).
secretion
through
mammary
the
border.
During
basal
inflammation, however, Ig levels are elevated due to increased tight
junction
permeability
and
passage
of
serum
components
into
milk
(128).
Mammary
gland
opsonizing bacterial
antibodies
antigens
IgG^,
and
IgG 2 »
and
IgM
function
facilitating phagocytosis
by
by PMN
23
and macrophages.
AntigBn-antibody immune complexes, either alone or
with complement, can bind to Fc and C3b receptors on phagocytic cell
surfaces
(34).
Activation
destroy
pathogenic
of
complement
organisms.
pathways
Bactericidal
can
lyse
and
consequences
of
antibody-complement complexes are a function of bacterial cell wall
thickness
and
are
ineffective
against
Gram-positive
Although IgA does not function as an opsonin,
in
toxin
neutralization,
bacterial
bacteria.
it has been implicated
agglutination,
and
preventing
mammary
secretion
bacterial adherence to cell membranes (78).
Chemical defense mechanisms
Nonspecific
include
Lf,
peroxide
bacteriostatic
lysozyme,
(LP)
and
system.
fully
the
proteins
lactoperoxidase/thiocyanate/hydrogen
Lactoferrin
secretions
of
Lactoferrin
concentrations
of
involuted
become
is
a
bovine
major
whey
mammary
elevated
by
day
protein
glands
4 of
involution
bacteria
(81).
because
Lactoferrin
of
its
is
iron-chelating
unavailable for bacterial growth.
iron
requirements
and
bacteriostatic
are
ability
off
for
(109).
involution
and continue to increase linearly as involution progresses.
to Lf molar ratio decreases gradually from drying
Citrate
to day
a
which
in
7 of
variety
makes
of
iron
Gram-negative bacteria have high
consequently
more
influenced
by
Lf
concentrations than Gram-positive organisms (97).
Lysozyme hydrolyzes the 1-4, B-linkage between muramic acid and
N-acetylglucosamine
of
bacterial
cell
wall
peptidoglycan
(127).
24
donuBtitratioHs of lyaoaytne are extremely low in both bovine milk and
PMN; production is thought to occur via local synthesis or diffusion
from blood.
Although lysozyme levels are too low to be effective,
it has been shown to
lyse bacteria
in the presence of complement,
and after lysis, to stimulate opsonic activity of IgM, and increase
the bactericidal activity of IgM plus complement (96).
The
LP
system
bactericidal
(96).
for
in
lacteal
Gram-positive
Lactoperoxidase
whereas
bovine
thiocyanate
is
some
synthesized
(SCN )
glycoside hydrolysis.
and
is
secretion
Hydrogen
from
peroxide
shown
Gram-negative
locally
derived
was
in
(^C^)
as
be
bacteria
mammary
serum
to
a
tissue,
result
of
is not present
in
milk, but is produced metabolically by streptococci (96,97).
The LP
system inhibits bacterial growth when lactoperoxidase combines with
^2^2
to oxidize SCN .
then
modifies
which
are
the
The resulting intermediary oxidation product
sulfhydryl
necessary
for
groups
glucose
organisms are catalase positive,
of
bacterial
transport.
Since
exogenous
the LP system can protect against IMI
cell
membranes,
Gram-negative
is required
before
with E. coli.
Enhancement of mammary defenses
Although
considerable
information
is
available
regarding
natural defense systems of the mammary gland, a practical method of
enhancing resistance
techniques
being
for
stimulating
developed.
killing,
mammary
attempts
gland
to infection has not been elucidated.
local
Considering
have
been
the
made
immune
mechanisms
are
currently
in
bacterial
importance
of
to
leukocytosis
induce
PMN
Several
to establish a protective PMN barrier.
in
Infusion
the
of
25
small
amounts
prevented
of
E.
coli
subsequent
endotoxin
establishment
induced
of
a
PMN
response
experimental
S.
which
agalactiae
infection (11). Other studies have established subclinical infection
with
C.
bovis
stimulate
PMN
or
Staphylococcus
influx
upon
epidermidis
subsequent
which
exposure
to
appeared
more
to
pathogenic
bacteria (7) and provide resistance to infection by S. aureus (90).
Research efforts have been directed also toward eliciting local
PMN
migration
within
the
polyethylene device (IMD).
gland
cistern
using
an
intramammary
The IKD is a sterile plastic loop which
is inserted through the streak canal into the gland cistern where it
remains
for
increases
levels,
in
lactations
leukocyte
e.g.,
quality of
loss
several
(86).
numbers
in
> 900,000 cells/ml
IMD-fitted
quarters
in milk production
foremilk
(87).
(37).
significant
is offset
Further
implantation
decreases
studies
are
provokes
samples
Reports
However,
in milk
yield
adverse
before
the
to
chronic
protective
indicate that milk
adversely,
by preventing
examining
necessary
IMD
is not affected
have resulted from infection (86,88).
reports
The
losses
and
any
that would
a more recent study
for
side
devices
IMD-fitted glands
effects
can
be
of
IMD
used
for
mastitis control.
Attempts have been made also to increase nonspecific components
of
immunity
prevalent
during
(81).
resistant
protective
and
The
fully
involution
when
involuted
from nonlactating cows
factors
bacteriostatic
including
proteins
such
new
mammary
to IMI due to distinct changes
Mammary secretion
Ig,
early
infections
gland
highly
in secretion composition.
contains
elevated natural
phagocytes,
as
is
are
Lf
(45,112),
lymphocytes,
and
lower
26
concentrations of casein, lactose, and citrate which can be utilized
for
bacterial
resistance
to
growth
and
colonization.
mastitis.
Intramammary
These
changes
injection
of
enhance
colchicine,
plant lectins (concanavalin A and phytohemagglutinin), and endotoxin
during
the
bacterial
occurs
early
dry
growth
normally
period
by
over
in
cows
have
been
accelerating
involution,
several
These
wk.
shown
a
to
inhibit
process
treatments
which
resulted
in
increased levels of natural protective factors in mammary secretion
during
the
(9,81).
period
when
the
gland
is most
susceptible
to new
IMI
In a more recent study, pathogenesis of S. uberis infection
in the mouse was modified effectively by intramammary injections of
pokeweed
mitogen
(PWM)
Immunostimulation
of
prior
to
mammary
experimental
glands
with
challenge
PWM
at
(113).
drying
off
accelerated mammary involution, enhanced antimicrobial defenses, and
facilitated a marked cellular response which reduced the severity of
experimental S. uberis infection (113).
Considerable
suitable
vaccine
effort has
against
been directed toward development of a
bovine
mastitis.
Unfortunately,
several
problems of vaccination during lactation are associated with mammary
immunology:
defense
milk
compared
fat
phagocytic
most
a)
and
requiring
successful
to
contains
casein
d)
relatively
secretion
efficiency;
bacteria;
mastitis;
milk
have
c)
from
the
is an
heterogeneity
of
components
fully
inhibitory
milk
few
involuted
effects
excellent
on
of
immune
gland;
mammary
PMN
growth medium
microorganisms
which
b)
for
cause
and e) the extensive surface area of secretory epithelium
immunological
attempts
at
surveillance
vaccination
(15).
have
Although
been
reported
several
under
27
experimental conditions (15,44,46,58), a better understanding of the
immune response of mammary tissue to bacterial
infection
is needed
to overcome the limitations of vaccines against bovine mastitis.
Numerous
gland
studies
have
demonstrated
the
bovine
mammary
is most susceptible to invasion by mastitis pathogens during
early involution (36,69,79,80,81,106).
the
that
disease
Progress
in
have
concentrated
developing
nonlactating
dairy
an
primarily
effective
cows
However,
has
on
mastitis
been
efforts to control
lactating
control
limited
understanding of the mammary immune system.
by
animals.
program
an
for
inadequate
The mammary gland has a
natural ability to prevent invasion by pathogenic bacteria; however,
physiological
synthesis
transition
and
capability.
secretion
or
has
been
from,
of
of
new
IMI
with
mastitis
a
shown
An understanding of the
interrelationship
development
to,
state
to
of
inhibit
active
this
milk
defense
involutionary process and
involution
control
is
procedures
necessary
which
will
the
for
be
effective in nonlactating glands.
The
objectives
of
this
study
were:
a)
to
describe
the
biochemical changes in mammary secretion composition from involution
through lactogenesis and compare these with morphological changes in
mammary parenchyma;
mammary
secretion
lactogenesis;
plasma
cells
and
b) to compare infection status with changes
and
c)
to
associated
tissue
morphology
during
identify and quantitate
with
compare with infection status.
nonlactating
involution
the
mammary
Ig classes
tissue
in
and
of
and
28
References
1.
Akers, R. M . , and S. C. Nickerson.
1983.
Effects of prepartum
blockade of microtubule formation on
milk production and
biochemical
differentiation
of
the mammary epithelium
in
Holstein heifers.
Int. J. Biochem. 15:771.
2.
Anderson, R. R.
41:118.
3.
Anderson, R. R. , J. R. Harness, A. F. Snead, and M. S. Salah.
1981.
Mammary growth pattern in goats during pregnancy and
lactation.
J. Dairy Sci.
64:427.
4.
Anderson, R. R . , M. S. Salah, J. R. Harness, and A. F. Snead.
1982.
Mammary growth patterns in guinea pigs during puberty,
pregnancy and lactation.
Biol. Reprod.
17:599.
5.
Bishop, J. C., F. S. Schanbacher, C. C. Ferguson, and K. L.
Smith. 1976.
tn vitro growth inhibition of mastitis-causing
coliform bacteria by bovine apo-lactoferrin and reversal of
inhibition
by
citrate
and
high
concentrations
of
apo-lactoferrin.
Inf. Immun. 14:911.
6.
Bramley, A. J.
1976.
Variations in the susceptibility of
lactating and non-lactating bovine udders to infection when
infused with Escherichia coli.
J. Dairy Res.
43:205.
7.
Bramley, A. J. 1978.
The effect of subclinical Staphylococcus
epidermidis
infection
with
Streptococcus
agalatiae
and
Escherichia coli. Br. Vet. J.
134:146.
8.
Brandon, M. R., D. L. Watson, and A. K. Lascelles.
1971.
The
mechanisms
of
transfer
of
immunoglobulins
into mammary
secretion of cows.
Aust. J. Exp. Biol. Med. Sci. 49:613.
9.
Breau, W. C., and S. P. Oliver.
1986.
Growth inhibition of
environmental mastitis pathogens during physiologic transitions
of the bovine mammary gland.
Am. J. Vet. Res.
47:218.
10.
Brooker, B. E.
1978.
The origin, structure, and occurrence of
corpora amylacea .in the bovine mammary gland and milk.
Cell
Tissue Res.
191:525.
11.
Brownlie, J.
1979.
The effect of an intramammary infusion of
endotoxin on the establistiment of experimental mastitis by
Streptococcus agalatiae in the cow.
J. Hyg. 83:103.
1975.
Mammary growth in
sheep.
J. Anlm. Sci.
29
12.
Chandler, R. L . , A. W. D. Lepper, and J. Wilcox.
1969.
Ultrastructural observations of the bovine teat duct.
J. Comp.
Pathol. 79:315.
13.
Chatterton, R. T. Jr., J. A. Harris, and R. M. Wynn.
1975.
Lactogenesis
in the rat:an ultrastructural
study
of the
initiation of the
secretory process. J. Reprod. Fert.
43:479.
14.
Chumakov, V. P., and M. A. Fel'dshtein.
1984.
Lacteal calculi
in the bovine udder.
Veterinariya (Moscow) 5:49.
15.
Colditz,
I.
G.,
and
D.
L.
Watson.
1985.
The
immunophysiological basis for vaccinating ruminants against
mastitis.
Aust. Vet. J.
62:145.
16.
Concha,
C. ,
0.
Holmberg,
and
B.
Morein.
1981.
Characterization of bovine mammary lymphocytes at different
periods of lactation. Page 806 iji The ruminant immune system.
J. E. Butler, ed.
Plenum Press, New York, N. Y,
17.
Coppock, C. E . , R. W. Everett, R. P. Natzke, and H. R. Ainslie.
1974.
Effect of dry period length on Holstein milk production
and selected disorders at parturition.
J, Dairy Sci. 57:712.
18.
Cousins, C. L . , T. M. Higgs, E. R. Jackson, F. K. Neave, and F.
H.
Dodd.
1980.
Susceptibility
of the
bovine
udder
to
bacterial infection in the dry period.
J. Dairy Res.
47:11.
19.
Cowie, A. T.
1971.
The hormonal control of milk secretion.
Page 123 iji Lactation.
I. E. Falconer, ed.
Butterworths,
London.
20.
Cowie, A. T., I. A. Forsyth, and I. C. Hart.
control of lactation. Springer-Verlag, Berlin.
21.
Dobbins, C. N.
Assoc. 170:1129.
22.
Dodd,
F.
Strategy
170:1124.
23.
Eberhart, R. J.
A s s o c . 170:1160.
24.
Faulkner A., and M. Peaker,
1982.
Reviews of the progress of
Dairy Science: secretion of citrate into milk.
J. Dairy Res.
49:159.
25.
Fleet, I. R . , and M. Peaker.
1978.
Mammary infection and its
control at the cessation of lactation in the goat.
J. Physiol.
279:491.
1977.
Mastitis
H., D. R. Westgarth,
of mastitis
control.
1977.
losses.
and
J.
J.
1980.
Am.
Hormonal
Vet.
T. F. Griffin.
Am.
Vet.
Med.
Coliform mastitis.
J.
Am.
Vet.
Med.
1977.
Assoc.
Med.
30
26.
Fleet, 1. H., J. H. Goode, M. H. Hamon, M. S. Laurie, J. L.
Linzell, and M. Peaker.
1975.
Secretory activity of the goat
mammary glands during pregnancy and the onset of lactation.
J.
Physiol.
251:763.
27.
Frost, A. J., D. D. Wanasinghe,
and J. B. Woolcock.
1977.
Some factors affecting selective adherence of microorganisms in
the bovine mammary gland.
Inf. Immunol. 15:245.
28.
Hadwen, S., and R. Gwatkin.
1939.
The detection of abnormal
cow's milk by microscopic methods.
Can. J. Res.
17:225.
29.
Hartmann, P. E.
1973.
Changes in the composition and yield of
mammary secretion of cow during the initiation of lactation.
J. Endocrinol.
59:231.
30.
Helminen,
H. J., and J. L.
E. Ericsson.
1968.
Studies
on
mammary gland involution. I. On
the ultrastructure of the
lactating mammary gland. J. Ultrastruct. Res. 25:193.
31.
Helminen,
H. J., and J. L.
E. Ericsson.
1968.
Studies
on
mammary gland involution. II.
Ultrastructural evidence for
auto- and heterophagocytosis.
J. Ultrastruct. Res.
25:214.
32.
Helminen,
H. J., and J. L.
E. Ericsson.
1968.
Studies
on
mammary gland involution.
III.
Alterations outside auto- and
heterophagocytic pathways
for cytoplasmic degradation.
J.
Ultrastruct. Res. 25:228.
33.
Helminen,
H. J., and J. L.
E. Ericsson.
1971.
Effects
of
enforced milk stasis on mammary gland epithelium, with special
reference to changes in lysosomes and lysosomal enzymes.
Exp.
Cell Res.
68:411.
34.
Howard, L. J., G. Taylor, and J. Brownlie.
1980.
Surface
receptors
for
immunoglobulin
on
bovine
polymorphonuclear
neutrophils and macrophages.
Res. Vet, Sci.
29:128.
35.
Hurley, W. L. , and J. J. Rejman.
1986.
p-lactoglobulin and
a-lactalbumin in mammary secretions during the dry period:
Parallelism of concentration changes.
J. Dairy Sci. 69:1642.
36.
Jain, N. C.
1979.
Common mammary pathogens
Infection and mastitis.
J. Dairy Sci. 62:128.
37.
Jaster, E. H. , A. R. Smith, and T. A. McPherson.
1982.
The
effect of an intramammary device on milk production, somatic
cells, conductivity, and bovine serum albumin in dairy cows.
J. Dairy Sci. 65 (Suppl. 1):175.
(Abstract),
38.
Jensen, D. L. , and R. J. Eberhart.
bovine milk.
Am. J. Vet. Res. 36:619.
1975.
and
factors
Macrophages
in
in
31
39.
Jones,
K. A.
1978.
Changes
in the activity of lactose
synthesis in the goat udder during pregnancy.
J. Dairy Res.
46:35.
40.
Kitchen, B. J.
1981.
Review of the progress of Dairy Science:
Bovine
mastitis:
Milk
compositional
changes
and
related
diagnostic tests.
J. Dairy Res.
48:167.
41.
Knight R.
39:193.
42.
1980.
Feeding
Knight,
C.
H. ,
and
proliferation
in mice
relation to milk yield.
for milk quality.
ADAS Quat.
M.
Peaker.
1982.
during
pregnancy
and
Quart. J. Exp. Physiol.
Rev.
Mammary
cell
lactation
in
67:165.
43.
Larson,
B.
L . , H.
L.
Leary,
and
J.
E.
Devery.
1980.
Immunoglobulin production and transport by the mammary gland.
J. Dairy Sci.
63:665.
44.
Lascelles, A. K.
1979,
The immune system of the ruminant
mammary gland and its role in the control of mastitis.
J.
Dairy Sci.
62:154.
45.
Lascelles, A. K., and C. S. Lee.
1978.
Involution of the
mammary
gland.
Page
115
jji Lactation,
a
comprehensive
treatise.
B. L. Larson, ed. Academic Press, New York, N. Y.
46.
Lascelles, A. K . f and G. H. McDowell.
1974.
Localized humoral
immunity with particular reference to ruminants.
Transplant.
Rev. 19:170.
47.
Lee, C. S., and P. M. Outteridge.
colostrum, milk,
and involution
reference to uttrastruct.ural and
J. Dairy Res.
48:225.
48.
Lee,
C.
S.,
F.
B.
P.
Wooding,
and
P.
Kemp.
1980.
Identification, properties,* and differential counts of cell
populations using electron microscopy of dry cow secretions,
colostrum, and milk from normal cows.
J. Dairy Res. 47:39.
49.
Linzell.
J.
L.,
and
M.
Peaker.
1971.
Intracellular
concentrations
of
sodium,
potassium and
chloride
in the
lactating mammary gland and their relation to the secretory
mechanism.
J. Physiol. 216:683.
50.
Linzell , J. L. , and M. Peaker.
1971.
mammary ducts. J. Physiol.
216:701.
51.
Linzell, ,J. L. , and M. Peaker.
1974.
Changes in colostrum
composition and in permeability of mammary epithelium at about
the time of parturition in goats. J. Physiol.
243:129.
1981.
Leucocytes of sheep
secretion, with particular
lymphocyte sub-populations.
The
permeability
of
32
52.
Lomakina, 0. M.
3:48.
1982.
Lacteal calculi.
Veterinariya (Moscow)
53.
Margolis, R. L . , and L. Wilson.
1978.
Mitotic mechanism based
on intrinsic microtubule behavior.
Nature
272:450.
54.
Masson,
P.
L . , and J.
F.
Heremans.
1966.
Studies
on
lactoferrin, the iron-binding protein of secretion.
Protides
Biol. Fluids Proc. Colloq. 14:115.
55.
Masson,
P.
L. , J.
F.
Heremans,
Lactoferrin,
an
iron
binding
leukocytes.
J. Exp. Med.
130:643.
56.
McDonald, J. S.
1970.
Microscopic observations of teat canals
from
susceptible
and
resistant
bovine mammary
glands:
A
preliminary report, jm Proc. 6th Int. Conf. Cattle Diseases.
97:103.
57.
McDonald, J. S.
Sci.
62:117.
58.
McDowell,
G.
H. ,
and
A.
K.
Lascelles.
1971.
Local
immunization of ewes with staphylococcal cell and cell-toxoid
vaccines.
Res. Vet. Sci. 12:258.
59.
McFadyean, J.
1930.
The corpora anylacea of the mammary gland
of the cow.
J. Comp. Pathol.
43:291.
60.
Mena F., P. Pacheco, and C. E. Grosvenor.
1980.
Effect of
electrical stimulation of mammary nerve upon pituitary and
plasma
prolactin
concentrations
in
anaesthetized
rats.
Endocrinol. 106:458.
61.
Miller, R. H. , A. J. Guidry, M. J. Paape, A. M. Dulin, and L.
A.
Fulton.
1986.
Relationship
between
immunoglobulin
concentrations
in
milk
and
phagocytosis
by
bovine
polymorphonuclear leukocytes.
Vet. Immunol. Immunopathol. (In
Press).
62.
Morrill, C. C.
1938.
A histopathological study of the bovine
udder.
Cornell Vet. 28:196*
63.
Munford, R. E.
1964.
A review of anatomical and biochemical
changes in the
mammary gland with particular reference to
quantitative methods of assessing mammary development.
Dairy
Sci. Abstr.
26:293.
64.
Murphy, J. M. , and
0. M. Stuart.
1953.
The effect of
introducing small numbers of Streptococcus agalactiae (Cornell
Strain 48) directly into the bovine teat cavity.
Cornell Vet.
43:290.
1979.
Symposium:
and
E.
protein
Shonne.
1969.
in
neutrophilic
Bovine mastitis.
J.
Dairy
33
65.
Nagai, J . , and N. K. Sarkar.
1978.
Relationship between milk
yield and mammary gland development in mice.
J. Dairy Sci.
61:733.
66.
Naidu, T. C. , and F. H. S.
leukocytes from bovine blood
37:47.
67.
Neave, F. K. , F. H. Dodd, and E. Henriques.
1950.
infections in the "dry period".
J. Dairy Res.
17:37.
68.
Neave, F.
K., and J. Oliver.
1962.
The relationship between
the number of mastitis pathogens placed on the teats of dry
cows, their survival, and the amount of intramammary infection
caused.
J. Dairy Res.
29:79.
69.
Neave, F. K. , J. Oliver, F. H. Dodd, and T. M.
Rate of infection of milked and unmilked udders.
35:127.
70.
Newby, T.
J, , and J. Bourne.
1977.
The nature
immune system of the
bovine mammary gland,
118:461.
71.
Newstead,
D.
F.
1973.
composition and properties.
72.
Nickerson, S. C.
1985.
Immune mechanisms of the bovine udder:
An overview.
J. Am. Vet. Med. Assoc.
187:41.
73.
Nickerson, S. C., and R. M. Akers.
1983.
Effects of prepartum
blockade
of
microtubule
formation
on
ultrastructural
differentiation of mammary epithelium in Holstein heifers.
Int. J. Biochem.
15:777.
74.
Nickerson, S. C. , J. J. Smith, and T. W. Keenan.
1980.
Role
of microtubules in milk secretion - Action of colchicine on
microtublules and exocytosis of secretory vesicles in rat
mammary epithelial cells.
Cell Tiss. Res.
207:361.
75.
Nickerson,
S.
C.,
and L. M.
Sordillo.
1985.
Role of
macrophages and multinucleated giant cells in the resorption of
corpora amylacea in the involuting bovine mammary gland.
Cell
Tiss. Res.
240:397.
76.
Nickerson, S. C. , L. M. Sordillo, N. T. Boddie, and A. M.
Saxton.
1984.
Prevalence and ultrastructural characteristics
of bovine mammary corpora amylacea during the lactation cycle.
J. Dairy Sci.
68:709.
77.
Nonnecke, B. J., and K. L. Smith.
1984.
Biochemical and
antibacterial properties of bovine mammary secretion during
mammary involution and at parturition.
J. Dairy Sci. 67:2863.
Newbould.
and milk.
1973.
Glycogen in
Can. J. Comp. Med.
Udder
Higgs.
1968.
J. Dairy Res.
of the local
J.
Immunol.
Effects
of mastitis
on
milk
N. Z. J. Dairy Sci. Tech. 8:52.
34
78.
Norcross, N. L.
1977.
Immune response of the mammary gland
and role of immunization in mastitis control.
J. Am. Vet. Med.
Assoc. 170:1228.
79.
Oliver,
J., F. H.
Dodd,
and F. K. Neave.
1956.
Udder
infections in the dry period.
IV.
The relationship between
the new infection rate in the early dry period and the daily
milk yield at drying-off when lactation was ended by either
intermittent or abrupt cessation of milking.
J. Dairy Res.
23:204.
80.
Oliver, S. P., and B. A. Mitchell.
1983.
Susceptibility
bovine mammary glands to infections during the dry period.
Dairy Sci. 66:1162.
81.
Oliver, S.
P., and K. L. Smith.
1982.
Milk yield and
secretion
composition
following
intramammary
infusion
of
colchicine.
J. Dairy Sci. 65:204.
82.
Ottolenghi,
D.
functionirenden
Entwicklongsmech.
83.
Outteridge, P. M . , and C. S. Lee.
1981.
Cellular immunity in
the
mammary
gland
with
particular
reference
to
T,
B
lymphocytes, and macrophages.
Page 513 in The ruminant immune
system.
J. E. Butler, ed. Plenum Press, New York, N. Y.
84.
Paape, M. J., A. J. Krai, and R. H. Miller.
1972.
Collagen of
rat mammary glands
during post-lactational involution.
J.
Dairy Sci. 55:1185.
85.
Paape, M. J. , and A. J. Guidry.
1977.
Effect of fat and
casein on intracellular killing of Staphylococcus aureus by
milk leukocytes.
Proc. Soc. Exp. Biol. Med. 155:588.
86.
Paape, M. J. , W. D. Schultze, A. J. Guidry, W. M. Kortum, and
B. T. Weinland.
1981.
Effect of an intramammary polyethylene
device on the concentration of leukocytes and immunoglobulins
in milk and on the leukocyte response to Escherichia coli
endotoxin and challenge exposure with Staphylococcus aureus.
Am. J. Vet. Res. 42:774.
87.
Paape, M.
.1., W.
D.
Schultze, and W. M.
Kortum.
1979.
Leukocyte
response
in
mammary quarters
fitted
with
an
intramammary device.
Page 320 Jjt. Proc. 17th Annu. Mtg, Am.
Soc. Anim. Sci.
(Abstract).
88.
Paape, M.
J., W. D.
Schultze, and R. R.
Peters.
1984.
Intramammary devices in bovine mastitis control.
J. Am. Vet.
Med. Assoc.
184:1362.
1901.
Beitrag
zur
milchdrusen.
Arch.
58:581.
histologie
Mikrosk.
of
J.
der
Anat.
35
89. Paape,
M.
Leukocytes
pathogens.
J.,
W.
P. Wergin,
and A.
J.
Guidry.
1979.
- second line of defense against invading mastitis
J. Dairy Sci. 62:135.
90. Pankey, J. W., S. C. Nickerson, R. L. Boddie, and J. S. Hogan.
1985.
Effects
of
Corynebacterium
bovis
infection
on
susceptibility to major mastitis pathogens.
J. Dairy Sci.
68:2684.
91. Peaker, M.
1980.
The effect of raised intramammary pressure
on mammary function in the goat in relation to the cessation of
lactation.
J. Physiol.
Lond.
301:415.
92. Philpot, W. N.
1967.
Influence of
milk production and milk composition.
subclinical mastitis on
J. Dairy Sci. 50:978.
93. Pitkow, H. S., R. P. Reece, and G. G. Waszilyzcsak.
integrity
of
mammary
alveolar
cells
in
two
lactations.
Proc. Soc. Exp. Biol. Med.
139:845.
1972.
The
consecutive
94. Heaven, E. P., and G. M. Reaven.
1977.
Distribution and
content of microtubules in relation to the transport of lipid.
J. Cell Biol.
75:559.
95. Reinitz, D. M. , and M. J. Paape.
1979.
Deficiency in the
lowering
of pH in
phagosomes
of
milk
polymorphonuclear
leukocytes (PMN) following phagocytosis.
J. Dairy Sci. 62:122.
96. Reiter, B.
1978.
Review of
Antimicrobial systems in milk.
the progress of Dairy
J. Dairy Res. 45:131.
Science:
97. Reiter, B. , and A. J. Bramley.
1975.
Defense mechanisms of
the udder and their relevence to mastitis control. Page 210 in
Proc. Int. Dairy Fed. Seminar on Mastitis Control.
Brussels,
Belgium.
98. Reiter, B . , M. E. Sharpe, and T. M. Higgs.
1970.
Experimental
infection of the non-lactating bovine udder with Staphylococcus
aureus and Streptococcus uberis. Res. Vet. Sci.
11:18.
99.
Richards, R. C. , and G. K. Benson.
1971.
Ultrastructural
changes accompanying involution of the mammary gland in the
albino rat.
.1. Endocrinol. 51:127.
100.
Richards, R. C., and G. K. Benson. 1971.
Involvement of the
macrophage system in the involution of the mammary gland in the
albino rat.
J. Endocrinol. 51:149.
101. Russell, M. W . , and B. Reiter.
1975.
Phagocytic deficiency of
bovine
milk
leukocytes:
An
effect
of
casein.
J.
Reticuloendothel. Soc. 18:1.
36
102.
Schalm,
O.
W . , J.
Lasmanis,
and E. J.
Carroll.
1966.
Significance of
leukocytic
infiltration
into the milk
in
experimental Streptococcus agalactiae mastitis in cattle.
Am.
J. Vet. Res. 27:1537.
103.
Schalm,
0.
M . , J.
Lasmanis,
and E. J.
Carroll.
Experimental Pseudomonas aeruginosa mastitis in cattle.
Vet. Res.
28:697.
1967.
Am. J.
104. Sejrsen K., R. M. Akers, E. M. Fitzgerald, H. A. Tucker, and J.
T. Huber.
1980.
Effect of plane of nutrition of mammary
development pre- and
postpuberty. J.Dairy Sci.
63(Suppl.
1):125.
(Abstract).
105. Sinha, K. N . , R. R. Anderson, and C. W. Turner.
1970.
Growth
of the mammary gland of the golden hamster, Mesocricetus
auratus. Biol. Reprod.
2:185.
106. Smith, A., F. H. Dodd, and F. K. Neave,
1968.
The effect of
intramammary infection during the dry period on the milk
production of
the affected quarter at the start of the
succeeding lactation.
J. Dairy Res.
35:287.
107. Smith, A., J. V. Wheelock, and F. H. Dodd.
1966.
milking throughout pregnancy on milk yield in the
lactation.
J. Dairy Sci.
49:895.
108. Smith,
K.
L.,
H.
R.
Conrad,
and R.
Lactoferrin and
IgGimmunoglobulins
from
mammary glands.
J. Dairy Sci.
54:1427.
Effect of
successive
M.
Porter.
involuted
1971.
bovine
109. Smith,
K. L . , and S.
P. Oliver.
1981.
Lactoferrin:
A
component of nonspecific defense of the involuting bovine
mammary gland.
Page 535 jji The ruminant immune system.
J. E.
Butler, ed. Plenum Press, New York, N. Y.
110. Smith, K. L. , S. P. Oliver, and P. S. Schoenberger.
1979.
Effect of endotoxin infusion on bovine mammary gland involution
and resistance to infection during the early dry period.
J.
Dairy Sci.
62 (Suppl. 1)s124. (Abstr.)
111. Smith, K. L . , and F. L. Schanbacher.
1977.
Lactoferrin as a
factor of resistance to infection of the bovine mammary gland.
J. Am. Vet. Med. Assoc.
170:1224.
112. Smith, K. L. , and D. A. Todhunter.
1982.
The physiology of
mammary glands during the dry period and the relationship to
infection.
Page 87 i_n Proc. 21st Annu. Mtg. Natl. Mastitis
Counc., Inc., Arlington, VA.
37
113. Sordillo, L. M. , and S. C. Nickerson.
1986.
Morphological
changes caused by experimental Streptococcus uberis mastitis in
mice
following
intramammary infusion of pokeweed mitogen.
Proc. Soc. Exp. Biol. Med.
182:522.
114. Sordillo, L. M. , and S. C. Nickerson.
1986.
and histochemical characterization of bovine
amylacea.
J. Histochem. Cytochem.
34:593.
Growth patterns
mammary corpora
115.
Sordillo, L. M. , S.
P. Oliver, R.
T. Duby, and R.
Rufner.
1984.
Effects of colchicine on milk yield, composition, and
cellular differentiation during caprine lactogenesis.
Int. J.
Biochem.
16:1135.
116.
Sordillo, L. M., S.
P. Oliver, and S. C. Nickerson.
1984.
Caprine mammary differentiation and initiation of lactation
following prepartum colchicine infusion.
Int. J. Biochem.
16:1265.
117. Swanson,
E.
W.
1965.
Comparing continuous
sixty-day dry periods in successive lactations.
48:1205.
milking with
J. Dairy Sci.
118. Swanson, E. W. , and J. I. Poffenbarger, 1979.
Mammary gland
development of dairy heifers during their first gestation.
62:702.
119.
Targowski,
S.
P.,
and D. T. Berman.
1975.
Leukocytic response
of bovine mammary gland to injection of killed cells and cell
walls of Staphylococcus aureus. Am. J. Vet. Res. 36:1561.
120. Teyssot, B . , and L. M. Houdebine.
1981.
Role of progesterone
and glucocorticoids in the transcription of the 3-casein and
28-S ribosomal genes in the rabbit mammary gland.
Eur. J.
Biochem.
114:597.
121. Thatcher, W. W . , and H. A. Tucker.
1968.
Intensive nursing
and lactational performance during extended lactation.
Proc.
Soc. Exp. Biol. Med.
128:46.
122. Thomas, C. L., F. K. Neave, F. H. Dodd, and T. M. Higgs.
1972.
The susceptibility of milked and unmilked udder quarters to
intramammary infection.
J. Dairy Sci.
39:113.
123. Tindal, .1. S.
1978.
Neuroendocrine control of lactation. Page
67 Jjn
Lactation, a comprehensive treatise. B. L. Larson, ed.
Plenum Press, New York, N. Y.
124. Tizard,
I.
immunology.
R.
1977.
An
Introduction
Saunders, Philadelphia, PA.
to
veterinanry
38
125. Turner, C. W., H. Yammamoto, and H. L. Ruppert.
1956.
experimental induction of growth of the cow's udder and
initiation of milk secretion.
J. Dairy Sci.
39:1717.
126. Unanue,
E.
phagocytes.
127.
R.
1976.
Secretory
Am. J. Pathol. 83:396.
function
of
The
the
mononuclear
Vakil,
J.
R . , R. C.
Chandler, and R.
M.
Perry.
1969.
Susceptibility of several microorganisms to milk lysozymes.
J.
Dairy Sci. 52:1192.
128. Watson, D. L. , M. R. Brandon, and A. K. Lascelles.
1972.
Concentrations of
immunoglobulins
in mammary secretion of
ruminants
during
involution
with
particular
reference
to
selective transfer of IgG.
Aust. J. Exp. Biol. Med. Sci.
50:535.
129.
Welty, F. K., K. L. Smith, and F. L.
Schanbacher.
Lactoferrin
concentration during involution of the
mammary gland.
J. Dairy Sci.
59:224.
1976.
bovine
130. Wergin,
W.
P.,
and
M.
J.
Paape.
1977.
Structure
of
polymorphonuclear leukocytes (PMN) isolated from bovine blood
and milk.
J. Cell Biol. 75:102.
131. Wheelock, J. V., A. Smith, F. H. Dodd, and R. L. J. Lyster.
1967.
Changes in the quantity and composition of mammary gland
secretion
in the
dry period
between
lactations
I.
The
beginning of the dry period.
J. Dairy Res.
34:199.
132.
Wisniowski,
J.,
K.
Ronanivkowa, and H.
Grojewski.
1965.
Phagocytosis phenomenon in the mammary glands of cows.
I. The
opsonizing factor and the phagocytic activity of leukocytes in
milk and blood.
Bull. Vet. Inst. Pulway. 9:140.
133. Zulak I. M., and T. W. Keenan.
1983.
a Golgi apparatus rich fraction from
gland.
Int. J. Biochem.
15:747.
Citrate accumulation by
lactating bovine mammary
C H A P T E R
Running head:
II
Bovine Mammary Secretion Composition
Key
words:
Involution, Lactogenesis,
Mastitis, Mammary, Bovine
SECRETION COMPOSITION DURING
RELATIONSHIP WITH MASTITIS
BOVINE
Secretion
MAMMARY
Composition,
INVOLUTION
AND
THE
L. M. SORDILLO, S. C. NICKERSON, R. M, AKERS1 , and S. P. OLIVER.2
Louisiana
University
71040
Agricultural
Experiment
Agricultural Center, Hill
Station,
Louisiana
Farm Research Station,
State
Homer
d e p a r t m e n t of Dairy Science, VPI & S U . , Blacksburg 24061.
2
Department
37901.
of
Animal
Correspondence sent to:
Science,
University
of
Tennessee,
Knoxville
Lorraine M. Sordillo-Gandy
Department of Animal Science
University of Tennessee
P.O. Box 1071
Knoxville, Tennessee
37901-1071
Approved
for
publication
by
the
Director
of
the
Louisiana
Agricultural Experiment Station as manuscript number 87-80-1144.
39
Abstract
Quarter
mammary
compositional
drying
off
revealed
secretion
analyses
through
that
collected
early
30%
Staphylococcus
were
samples
for
from
lactation.
contained
bacteriological
29
dairy
aur e u s , Corynebacterium
cows
Bacteriological
coagulase-negative
bovis, or
and
from
analysis
staphylococci,
streptococci.
As
involution progressed, somatic cell counts, percent protein, pH, and
concentrations of
increased
citrate
while
to
uninfected
higher
percent
quarters,
cell
lactation.
detected
fat,
lactoferrin
somatic
early
serum albumin,
molar
those
ratio
which
counts
in
quarters.
percentages
polymorphonuclear
Percentages
of
fat
of
infected
during
numbers
percentages
of
citrate,
had
of
infected
of
fat
first.
the
quarters
and
2 wk
higher
of
concentrations
compared
sampling
to
involute by
period.
contained
pH
protein
lactation.
of
Infected
lactoferrin
uninfected
However,
to
quarters.
significantly
quarters
the
Results
14 to 21 days post drying off.
and
were
not
quarters
but
to
had
lower
infected
concentrations
mammary
lower
uninfected
during
to
compared
and
significantly
compared
Compared
lymphocytes
serum albumin and citrate were similar between all
most
the
colostrogenesis
uninfected
of
and
macrophages
leukocytes
and
immunoglobulin G
decreased.
However,
higher
quarters.
were
especially
significantly
of
and
concentrations
Differences
between
lactoferrin,
quarters
also
during
secretion
from
percentages
quarters
during
contained
nonlactating
suggest
that
Intramammary
altered normal secretion composition during lactogenesis.
of
of
the
lower
period
udders
infection
Introduction
The
bovine
mammary
gland
is most
susceptible
to
invasion
by
bacterial pathogens during functional transitions from lactation to
involution and
from
involution to
lactogenesis
(14,18).
Incidence
of new intramammary infection (IMI) during the first 3 wk of the dry
period may be 7 times greater than during lactation (14).
The fully
involuted mammary gland appears to be highly resistant to new IMI,
but as parturition approaches, susceptibility to IMI increases.
Secretions
of
concentrations
fully
of
involuted
leukocytes,
udders
contain
immunoglobulins
high
(Ig)>
and
bacteriostatic whey proteins that have been implicated as resistance
factors to IMI (10,21).
secretion
that
are
(9).
macrophages
function
Immunoglobulin
by
bacteria
Immunoglobulin
A
colonization
of
multiplication,
(15,22).
that
sequestering
polymorphonuclear
secretion
IgG^,
for
in milk
IgG 2 » and
may
play
epithelial
neutralizing
bacterial
iron (2,24).
invading
is of humoral
phagocytosis
Lactoferrin (Lf)
inhibits
and
phagocytosing
in mammary
In the bovine,
prepare
Predominant leukocyte types present in dry
a
IgM function
by
role
surfaces,
toxins,
and
leukocytes
(PMN)
pathogens
(7).
or
local origin
as opsonins
macrophages
and
and
PMN.
in preventing
bacterial
inhibiting
bacterial
agglutinating
bacteria
is a major whey protein of dry secretion
growth
in
presence
of
bicarbonate
by
The iron-binding activity of Lf in vitro
is diminished by citrate (2);
consequently,
ratio plays a role in udder defense.
the citrate to Lf molar
42
Suscep tibil ity or resistance to mastitis is
related to changes
in antibacterial components of secretions during the lactation cycle
(3,17).
The
rate
of
involution apparently
change
in
these
is not sufficient
components
during
to prevent new
IMI.
early
Other
biochemical components that undergo changes during this time include
protein,
which
fat,
can
to
bovine
be measured
permeability.
thought
pH,
lactations
(23),
attack
mastitis
for
the
albumin
to monitor
Because
account
serum
new
for
IMI
the
and
a-lactalbumin
secretory activity
and glandular
occurring
level
nonlactating
control.
(BSA),
The
of
during
infection
period
purpose
is
of
a
involution
in
subsequent
logical
this
are
point
study
was
of
to
examine effects of IMI on lacteal secretions during the nonlactating
and
early
lactating
periods
to
determine
if presence
affected the processes of involution and lactogenesis.
of
bacteria
43
Materials and Methods
Experimental design
Twenty-nine
were
used.
infusion
and
At
Jersey
cows
drying off,
products
off;
Mammary
containing
7,
14,
prepartum;
Duplicate
status,
quarter
21,
at
to
quantitate
BSA.
Total
total
protein and
somatic
determined also.
venipuncture,
general
samples
after
and
at
Research
treated with
procaine
(Quartermaster,
days
parturition;
Farm
Station
intramammary
penicillin G
Upjohn Co. , Kalamazoo,
were
collected
drying
off;
7
14
and
14
days
at
and
drying
7
days
postpartum.
foremilk samples (10 ml) were used to determine infection
and
Data were
28
Hill
1,000,000 units
secretion
and
the
animals were
1 g dihydrostreptomycin
Ml).
from
and
fat,
concentrations
differential
pH,
and
of
cell
Ig,
Lf,
counts,
citrate,
percentages
and
of
the citrate to Lf molar ratio were
Blood samples were collected at the above times by
to quantitate concentration of a-lactalbumin in sera.
analyzed
linear
by
model
least
square
procedure
to
analysis
of variance using
determine
effects
of
wk
the
during
involution and infection status on biochemical composition of bovine
mammary secretion.
The statistical analysis included effect of cow,
wk during involution,
infection status, and interaction of wk during
involution with infection status.
Y...
ijk
Where:
The following model was used:
= p + x. + (3. + a p . . + e...
l
ijk
u = effect common to all cows,
Xj = fixed effect of ith wk during involution,
P. - fixed effect of
J
ith infection status,
J
AA
t (S..
= fixed effect due to interaction between ith wk
during involution and jth infection status,
e.,, = random error associated with each observation,
ijk
The
effect
square
of
means
cow
was
absorbed.
Preplanned
comparisons
from the overall model were made
of
least
by pairwise T-test.
Means were contrasted between infection status group within a time
period
and
between
time period within
infection status group.
No
for
in
other comparisons were made.
MicrobioloRical procedures
Samples
were
processed,
examined
(A), and identified to species
identified
using
the
API
level.
microbial
Staphylococcal
Staph-Ident
System
growth
as
isolates were
(Analytab
Products,
Plainview, NY) and streptococcal isolates were identified by the API
20S System (Analytab Products).
minor
(coagulase-negative
Microorganisms were classified as
staphylococci
and
Corynebacterium
bovis)
and major (streptococci and Staphylococcus aureus) pathogens.
Milk somatic cells
Somatic
Cell
cell
Counter
differential
counts
(Foss
(SCC)
Electric
were
determined
Ltd.,
using a Fossomatic
Hillerod,
Denmark),
cell counts were determined on dried milk film stained
with GS-Wright's stain (General Scientific, Richmond, VA).
smear,
200
and
cells were differentiated microscopically at
expressed as percent of total cells counted.
as macrophages,
lymphocytes, and PMN.
For each
1000 X and
Cells were classified
45
Total protein and butterfat
A
Fossomatic
Milk
Analyzer
(Foss
Electric
determine total protein and butterfat.
Ltd.)
was
used
to
Assays were performed on 10
ml-milk samples preserved with potassium dichromate ( ^ C ^ O ^ )
at the
Dairy Herd Improvement Association Laboratory, Baton Rouge, LA.
Compositional analyses
Skim
milk
and
whey
Immunoglobulin,
Lf,
and
fractions
BSA
electroimmunodLffusion
(EID)
Rabbit
and
anti-IgG
Pel-Freez
(Fab^
Biologicals,
were
on
the
Rogers,
were
quantitated
cellulose
IgGj
prepared
as
in
acetate
standard
were
in
(19).
wheys
plates
by
(17).
purchased
from
AR, and diluted with a 1:4 dilution
of high resoiution buffer, pH 8.8 (Gelman Instrument Co., Ann Arbor,
MI)
in
distilled
standards
with
were
water.
prepared
Antiserum
from purified
.0125 M sodium phosphate buffer,
BSA standards were
Quantification
of
purchased
citrate
to
skim
Lf
bovine Lf
pH 7.4.
from Miles
in
bovine
milk
and
(24),
and
Lf
diluted
Rabbit anti-BSA and
Laboratory,
was
bovine
by
a
Kankakee,
modification
IL.
of
(25).
Blood samples were centrifuged at 1,240 X g for 25 min and sera
frozen
and
stored
at
-20°C
until
needed.
Concentration
of
a-lactalbumin in whey samples was determined by radioimmunoassay (1)
using a Beckman L-4000 gamma counter.
46
Results
Frequencies
and
early
quarters
of
bacterial
lactating
increased
periods
markedly
isolates
are
in
from
D-0
throughout
Table
to
1.
D+7
the nonlactating
Percent
and
uninfected
D+14.
At
D+21,
percent uninfected quarters began to decrease gradually through C+14
to
levels
markedly
observed
from
D-0
staphylococci
D-0.
to
(CNS)
Staphylococcus
warneri.
at
Isolations
early
isolates
the
bovis
decreased
Coagulase-negative
Staphylococcus
simulans, Staphylococcus
throughout
C.
lactation.
included
Coagulase-negative
of
epidermidis,
hyicus, and
Staphylococcus
staphylococci were the most
sampling
period.
frequent
Percent
quarters
containing CNS and S. aureus decreased from D-0 to early involution
(D+7
and D+14),
early
lactation
streptococci
was
but
increased
to
gradually
percentages
most
frequent
beginning
observed
from D-0
at
to
at
D-0.
D+28
but
D+21
through
Isolation
of
remained
low
from C-14 through C+14.
Somatic
cell
from D-0 to D+7,
(Figure
time,
1).
they
quarters
counts
uninfected
quarters
increased
then decreased steadily to minimum levels at C+14
Although
had
from
infected
significantly
(Table 2).
Somatic
quarters
higher
SCC
were
more
variable
compared
to
cell counts in quarters
over
uninfected
infected with
major pathogens were elevated significantly over uninfected quarters
from C-7 to C+7 (Figure 1).
Percentages
increased
from
of
D-0
macrophages
(33.14
concentrations at D+28
+
(38.64 +
and
.96
lymphocytes
and
26.88
1.55 and 37.38 +
for
+
all
1.03)
1.51).
quarters
to
peak
Beginning
47
at C-14, percentages of these cell types decreased,
reaching lowest
levels at C+14 (30.48 + 1.62 and 27.10 + 1.74) (Appendix Figures
la
and
on
2a).
There
macrophage
However,
no
significant
concentration
uninfected
percentages of
PMN
was
compared
(Table
quarters
lymphocytes,
to
2,
infected
effect
of
Appendix
had
Tables
The
status
la
significantly
and significantly
quarters.
infection
and
higher
2a).
total
lower percentages
proportion
PMN
all
involution progressed,
D+28
1.97).
Polymorphonuclear leukocyte numbers increased
at C-14
(27.61
significantly
+
2.09),
continuing
lowest
in
quarters decreased as
(22.56 +
with
of
of
levels at
through
the
pre-
and postpartum periods, and peaking at C+14 (41.46 + 2.05, Appendix
Figure 3a).
Lactoferrin
concentrations
significantly
from
nonlactating
period
C-0,
Lf
through
levels
the
uninfected
minor
D-0
dropped
quarters
D+7,
(Figure
early
pathogens
to
in
uninfected
and
2,
remained
Appendix
significantly
lactating
(10.87
(10.09 +
+
and
sampling
.38
mg/ml)
1.17 mg/ml),
quarters
high
through
the
4a)).
At
Figure
continued
periods.
and
those
quarters
increased
to
decrease
Compared
infected
to
with
infected with major
pathogens had significantly lower total concentrations of Lf (7.78 +
1.04 mg/ml) (Appendix Tables 3a and 4a).
Citrate values and the citrate to Lf molar ratio in uninfected
quarters decreased markedly from D-0 to D+7 and remained low during
the
nonlactating
citrate
markedly
periods.
period
concentrations
and
remained
There
was
no
(Figure
2,
Appendix
Figure
5a).
At
C-0,
and the citrate to Lf molar ratio increased
high
during
significant
the
early
effect
of
lactating
infection
sampling
status
on
48
citrate concentrations.
Changes
In percent fat and protein were observed with respect
to sampling period (Figure 3, Appendix Figures 6a and 7a).
fat
from both uninfected
and
Percent
infected quarters decreased gradually
as involution progressed from D-0 through C-7.
At C-0,
the percent
fat
increased markedly and continued to increase through C+14 where
it
reached
content
stable
of
mammary
gradually as
following
Infected
values
in
uninfected
secretions
from
involution
continued
parturition,
reaching
quarters
had
lower
quarters.
uninfected
up
through
lowest
quarters
C-7,
of
then
fat
However,
protein
increased
percentages
percentages
throughout most of the sampling period.
The
decreased
at
and
C+14.
protein
infected quarters
had significantly higher percentages of protein at C-0 compared with
uninfected quarters (Appendix Tables 5a and 6a).
The pH of mammary secretions from both uninfected and
infected
quarters increased markedly from D-0 to D+7, and remained high until
D+28
(Figure
C-0.
Slight
infected
infected
4).
At C-14,
increases
quarters
with
in
at
major
pH decreased
pH
were
C+7 and
pathogens
reaching
observed
C+14.
had
lowest
for
levels
uninfected
Secretion from
significantly
higher
at
and
quarters
pH
values
from C-0 to C+14 compared to uninfected quarters (Appendix Tables 9a
and 10a).
Concentration
from
D-0
contjmiod,
period.
levels
to
D+7
USA
At
of
BSA
in
(Figure
5,
contentremained
C-0,
observed
at:
BSA
D-0
all
quarters
Appendix
high
concentrations
and
continued
increased
Figure
through
decreased
to
significantly
8a). As
the C-7
involution
sampling
significantly
decrease as
to
lactation
49
continued.
between
No
differences
uninfected
and
in
BSA
infected
concentration
quarters
were
(Appendix
detected
Tables
9a
and
10a).
Concentrations of IgG in mammary secretions of both uninfected
and
infected
increase
6).
quarters
reaching
increased
peak
from D-0
concentrations
to D+7
at
and
C-14
and
continued
C-7
to
(Figure
Compared to uninfected quarters (17.86 + 1.23), secretion from
infected
quarters
(16.15
+
1.75)
had
throughout most of the sampling period.
IgG were higher
at C-14
in quarters
less
IgG
However,
concentration
concentrations of
infected with major pathogens
and higher at C-0 in quarters infected with minor pathogens compared
to uninfected quarters (Appendix Table 8a).
Concentration
of
a-lactalbumin
in
sera
of
cows
over
nonlactating period increased significantly from D-0 to D+7
7).
At
D+14,
significantly
a-lactalbumin
concentrations.
concentrations
the
and
a-lactalbumin
levels
levels
low
increased
A marked
continued
remained
decrease
to
content
of
serum
through
C-14.
significantly
was
decline
information on a-lactalbumin content
(Figure
decreased
At
C-0,
reaching
observed
following
through
C+14.
in mammary
the
C-0
and
Additional
secretion
in the Appendix on Tables 9a and 10a and on Figure 9a.
peak
is
found
50
Discussion
At D+7, numbers of infected quarters were much lower compared with
D-0.
Decreased
nonlactating
period
administration
with
C.
D+7
of
bovis
lactation.
and
numbers
in
remained
this
study
at
low
quarters
was
D-0.
during
most
Percent
throughout
the
the
likely
due
quarters
dry
early
to
infected
period
and
into
Although infections with CNS and S. aureus were lower at
remained
D-0
infected
antibiotics
lower
C-0 and during early
at
of
(32%).
These
throughout
lactation
C-14,
frequency of
isolation at
(43%) was higher than that observed
findings
suggest
that
the
bovine
udder
is
highly susceptible to IMI by CNS and S. aureus during the peripartum
period.
Susceptibility to mastitis is associated with the physiological
transitions of the mammary gland either to or from a state of active
milk synthesis and secretion (13,19).
characterized
the
dry
period
composition
Major
antibacterial
(17,21),
with
respect
changes
in
Although several studies have
components of mammary
our
to
understanding
susceptibility
biochemical
secretion during
of
to
the
changes
mastitis
composition
and
is
in
vague.
antibacterial
properties of mammary secretion were found during the early and late
stages
days
of
of
involution.
Involution
Increases were observed within
in numbers
of SCC;
percentages
the
first
7
of macrophages,
lymphocytes, and protein; pH; and concentrations of Lf, BSA, and IgG
reaching
peak
or
approached
(from
decreased
and
stable
values
C-7
to
C-0)
remained
low
by
D+14
to
concentrations
D+21.
of
during the early
As
these
parturition
parameters
lactating period.
51
Conversely,
and
percentages of
the citrate
PMN
to Lf molar
and
fat,
ratio
concentration
declined
of
citrate,
within the first
wk
of involution while progressively increasing from C-7 to C+14,
Levels
of SCC,
percent protein,
pH,
and concentrations of BSA
and IgG in bovine mammary secretion during involution reflect degree
of
cellular
(8).
integrity
In this study,
reported
and
permeability
of
the
blood-milk
barrier
transient changes in SCC were similar to those
in previous studies.
It was hypothesized
that the influx
of cells during the early dry period was a function of the cessation
of
milk
removal
populations
migration
from
of
and
D+7
fluid
to D+14
leukocytes
concentration
effect
decrease
may
to a dilution
gland
during
mammary
the
in
macrophages
and
Macrophages
and
from
secretions
From the
colostrum
and
milk
and/or
became
fully involuted
during
system and/or
of
Differential
early
the
14
cell
increased
resorption.
removal
first
an
following
approximately
lymphocytes
from
fluid
population
the
Elevated
circulatory
process.
during
(7,10).
resulted
from
effect
revealed
PMN
have
the
leukocyte
milking
secretion
may
resulting
subsequent
be due
resorption
cell
days
major
The
parturition
cells
equal
a
from
the
counts
of
numbers
of
of
involution.
cell
types
in
gland and PMN were prevalent in
lactation.
These data
are
consistent with the Findings of others (7,11,12).
Changes in concentrations of Lf, citrate, and the citrate to Lf
molar
mammary
ratio
are
gland
indicative
(19,24).
of
the
Increased
functional
Lf
transition
concentration
nonlactating mammary gland is a marker for involution and
of
in
the
the
decreased
synthetic and secretory ability of mammary epithelial cells (19,24).
52
Results
of
suggesting
this
the
study demonstrated
udder
reaches
that
a fully
Lf
levels
involuted
peaked on D+14,
state between D+14
and D+28 when Lf reaches maximum concentrations.
Changes
mammary
in
citrate
secretory
concentration
activity,
which
were
decreased
monitored
to
significantly
follow
by
D+7.
The subsequent increase in citrate concentration, and the citrate to
Lf molar ratio during
the last 7 days of gestation were attributed
to the onset of milk synthesis and secretion.
Changes
determined
activity
in
and
serum
used
during
concentrations
involution
as
the
of
cells.
Consequently,
route
of
increases
structural
in
of
a-lactalbumin
of
remaining
mammary
concentrations
to
in serum
the
rose
concentrations
differention
of
prior
mammary
to
cells,
a
serum
reduced
last
wk
of
dramatically
parturition
the
which
secretory
indicates
During
as
follow
levels at parturition as reported in (6).
serum
and
Decreased
D+14
cells.
D+7,
D+28
mammary
blood.
after
By
through
appears
were
synthetic
significantly
between
to
cell
(1,6).
decreased
junctions
milk
a-lactalbumin
period
a-lactalbumin
a-lactalbumin
peak
tight
from
capability
gestation,
reaching
in
of
increased
concentrations
alteration
synthetic
indicator
a-lactalbumin
suggests
concentrations
an
nonlactating
progressed,
paracellular
concentrations
onset
Transient
indicate
of
milk
synthesis and secretion, and fluid accumulation resulting in leakage
of milk to blood.
Compositional
been reviewed (8).
yield,
reductions
changes
in mastitic
milk
during
lactation
have
Mastitis generally results in a decrease in milk
in
concentrations
of
lactose
and
fat,
and
53
increases
in SCC,
been attributed
and
pH,
to
disruption
BSA,
Ig,
and Lf
(8),
Such alterations
have
inflammatory damage of mammary secretory tissue
of
the
blood-milk
barrier
(8).
During
the
early
lactating period,
significantly lower percentages of fat and higher
pH
from
in
secretion
quarters
from
reflect
this
infected quarters,
infected
loss
quarters
in cellular
compared
to
integrity.
In
SCC were also higher during
early lactation compared to uninfected quarters.
uninfected
secretions
involution and
Elevated leukocyte
numbers may be indicative of an inflammatory response of the mammary
gland to
bacterial toxins.
numerous
cell
previous
study
secretion
(7).
in
type
during
also
from
found
infected
quarters
the dry and early
PMN
were the most
lactating
PMN to
be
the predominant
quarters
at
all
Neutrophilia during
uninfected
In infected quarters,
stages
of
periods.
cell
the
A
type
dry
in
period
the early dry and periparturient periods
appears
ineffective
in
rates of new IMI during this critical time.
reducing
the
high
Paape and Guidry (20)
have shown that phagocytosis and intracellular bacteriolysis by PMN
are
inhibited by the indiscriminant
mammary secretion.
phagocytosis
of
ingestion of fat and casein
Results of this study support the
bacteria
by
mammary
PMN
and
concept
macrophages
in
that
is
less
vary
with
effective during periods of functional transition.
Immunoglobulin
respect
to
stage
serum-derived
(15).
In
the
and
concentration
of
lactation
selectively
lactating
in
and
presence
transferred
gland,
secretions
of
into
concentrations
dramatically during acute mastitis (8).
gland ruptures
lacteal
IMI.
Most
mammary
of
IgG
IgG
is
secretions
increased
Inflammation of the mammary
the blood-milk permeability
barrier allowing
IgG
to
54
passively
enter
nonlactating
lacteal
secretions
from
quarters
had
uninfected
serum.
In
this
slightly
study,
higher
IgG
concentrations when compared to infected quarters during most of the
sampling
and
period.
This
Ig-producing
may
plasma
be
explained
cell
function
by compromised
as
reported
lymphocyte
previously
in
quarters infected with chronic staphylococcal mastitis (16).
Elevated concentrations of IgG during colostrogenesis
is likely due
to selective transport via receptors on mammary secretory cells (5).
However,
significantly
quarters
observed
at
higher
C-14
concentrations
and C-0 compared
of
IgG
in
infected
to uninfected quarters
may be due to rupture of the blood-milk barrier.
Fully involuted mammary glands exhibit
IMI
(3,19,24).
Secretions
from
involuted
increased resistance to
mammary
glands
contain
elevated natural protective factors including phagocytes and Lf, but
lower
concentrations
utilized
by
Previous
studies
from
invading
(3,17).
A
bacteria
demonstrated
nonlactating
vitro
of casein,
lactose,
for
the
and citrate which
colonization
inhibitory
and
action
mammary
glands
on mastitis-causing
decline
in
citrate
the
to
Lf
can
growth
of
(21).
secretion
organisms
molar
be
ratio,
in
and
elevation of Lf and IgG concentrations have been shown to contribute
to
the
antimicrobial
Lactoferrin
sequester
interferes
iron
from
action
with
mammary
against
the
environmental
ability
secretions.
of
In
these
this
organisms.
bacteria
study,
to
infected
quarters had lower Lf concentrations compared to unifected quarters.
Lower
levels
natural
of this
defense
antibacterial
potential
of
the
component
gland,
may
allowing
have
reduced
colonization
minor and major pathogens during the peripartum period.
the
by
55
References
1.
Akers, R. M . , T. B. McFadden, W. E. Beal, A. J. Guidry, and H.
M. Farrell.
1986.
a-lactalbumin
Dairy Res.
2.
Bishop,
in
serum,
1976.
coliform
S.
bacteria
by
by
C.,
growth
bovine
citrate
apo-lactoferrin.
W.
and tissue culture media.
Schanbacher,
In vitro
inhibition
Breau,
milk
J.
In Press.
J. C. , F.
Smith.
3.
Radioimmunoassay for measurment of bovine
C. Ferguson,
and
K.
C.
inhibition of mastitis-causing
apo-lactoferrin
and
Inf. Immun.
and S.
C.
high
and
reversal
of
concentrations
of
14:911.
P. Oliver.
1986.
Growth
inhibition of
environmental mastitis pathogens during physiologic transitions
of the bovine mammary gland.
4.
Brown,
R. W. , D. A.
W.
Schultze.
the
D.
diagnosis
Counc.,
5.
Howard,
Barnum,
1981.
of
Am. J. Vet. Res.
D. E. Jasper,
L. J.,
G.
for
bovine
mastitis.
Taylor,
and
immunoglobulin
W. L. , and
a-lactalbumin
2nd Ed.,
in
J.
J.
mammary
J.Brownlie.
on
bovine
Jensen,
Natl.
in
Mastitis
Rejman.
D.
differential
I,.,
cell
and
R.
counts
bovine mammary gland.
in
Surface
29:128.
1986. (3-lactoglobulin and
secretions
J.
1980.
polymorphonuclear
Res. Vet. Sci.
Parallelism of concentration changes.
7.
and
Microbiological procedures for use
neutrophils and macrophages.
Hurley,
S. McDonald,
Inc., Arlington, VA.
receptors
6.
J.
47:218.
during
dry
J. Dairy Sci.
Eberhart,
secretions
Am. J. Vet. Res.
the
1981.
of
the
42:743.
period:
69:1642.
Total
and
nonlactating
56
8.
Kitchen, B. J.
Bovine
1981,
mastitis:
Milk
diagnostic tests.
9.
Larson,
B.
Review of the progress of Dairy Science:
compositional
J. Dairy Res.
L. ,
H.
L.
and
and
related
48:167.
Leary,
Immunoglobulin production
changes
and
J.
transport
E.
Devery.
1980.
by the mammary gland.
J. Dairy Sci. 63:665.
10.
Lascelles, A.
mammary
gland.
treatise.
11.
Lee,
K.,and
C.
C.
S.
Page
115
S.,
G.
H.
of
macrophages
Lee,
C.
S.,
F.
Identification,
populations
B.
Lactation,
and A.
in
the
K.
removal
P.
Wooding,
electron
and
McDonald,
Sci.
14.
Neave,
J. S.
1979.
fat
The
from
P.
of
dry
Kemp.
counts
cow
the
1980.
of
cell
secretions,
J. Dairy Res.
47:39.
Symposium: Bovine mastitis.
F.
K. , F.
Nickerson,
S. C.
H.
Dodd,
and
"dry period".
E.
J.
J.
Nonnecke,
B. J.,
sf.apy lucocca I
Henriques.
Dairy Res.
1950.
Dairy
and
J.
mastitis
A.
on
udder:
187:41.
Harp.1985.
mitogenic
lymphocytes. J. Dairy Sci, 68:3323.
Udder
17:37.
1985.Immune mechanisms of the bovine
An overview. J. Am. Vet. Med. Assoc.
16.
1969.
62:117.
infections in the
15.
York, NY.
10:34.
differential
microscopy
the
comprehensive
of
and
colostrum, and milk from normal cows.
13.
a
of
Lascelles.
Res. Vet. Sci.
properties,
using
Involution
Academic Press, New
McDowell,
involuting mammary gland.
1978.
in
B. L. Larson, ed.
importance
12.
Lee.
Effect
of
responses
chronic
of
bovine
57
17.
Nonnecke,
R.
J.,
antibacterial
and
K.
L.
Smith.
1984,
properties
of
bovine
mammary
mammary involution and at parturition.
18.
Oliver,
S.
P.,
and B.
A.
Biochemical
secretion
J. Dairy Sci.
Mitchell.
1983.
and
during
67:2863,
Susceptibility
of
bovine mammary glands to infections during the dry period.
19.
Dairy Sci.
66:1162.
Oliver,
P.,
S.
secretion
Paape,
casein
M.
J. , and
on
22.
whey
23.
Smith,
killing
proteins
and
infection
of
the
Smith,
K.
component
L.,
of
1977.
of
during
affected
Effect
1975.
enzymes:
of
of
fat
and
aureus
by
155:588.
Formation and role
Relation
1985.
to
mammary
P.
Immune defences at
J. Dairy Res.
the
The effect of
period
on
the
start
at
the
milk
of
the
Lactoferrin:
A
35:287.
Oliver.
defense
52:599.
1968.
dry
quarter
J. Dairy Res.
S.
and
infusion
Staphylococcus
and F. K. Neave.
nonspecific
mammary gland.
Butler, ed.
and
yield
58:1048.
in ruminants.
succeeding lactation.
25.
Guidry.
Proc. Soc. Exp. Biol. Med.
A., F. H. Dodd,
production
Milk
65:204.
Sheldrake, R. F., and A. J. Husband.
intramammary
24.
J.
J. Dairy Sci.
mucosal surfaces
1982.
intramammary
F. L . , and K. L. Smith.
unusual
function.
A.
Smith.
following
intracellular
Schanbacher,
of
L.
J. Dairy Sci.
milk leukocytes.
21.
K.
composition
colchicine.
20.
and
J.
1981.
of
the
involuting
bovine
Page 535 jni The ruminant immune system.
J. E.
Plenum Press, New York, NY.
White, J. C. D . , and D. T. Davies.
citric acid in milk and milk sera.
1963.
The determination of
J. Dairy Res. 30:171.
TABLE 1.
Frequency of bacterial
1ac tat ion .
isolates
in bovine mammary secretion
from drying off through early
Samp 1ing Per iod ^
Bac ter i a ^
0
CB
0+0
n
n
c.
CNS
SA
STP
n
r>
n
0.
■o
Tota 13
^Days relative
65
(58.0)
12
(10.7)
19
( 17.0)
13
(11.6)
0+7
0+14
D+21
0 + 28
C-lit
100
76
(88.4)
57
(71.0)
57
(79.2)
(69.8)
2
(3.2)
11
(17.5)
(88.5)
0
(0)
2
2
(2.3)
(2.5)
1
0.4)
1
if
(0.9)
(^ - 7)
12
(15-0)
7
(9.7)
8
2
6
(7.D
(2.3)
3
(2.7)
4
2
(3-5)
(2.3)
1 12
113
to drying off
86
(D) or calving
ifk
C+14
Tota 1
55
(60.4)
55
(57.9)
620
(70.0)
(1.1)
2
(2.2)
4
(4.2)
18
(22.8)
21
(22.1)
19
(20.9)
22
(23.2)
(15-1)
11
(11.6)
15
(16.5)
13
(13-7)
89
(10.1)
1
(1.1)
17
(1.9)
C-7
51
(64.6)
0
(0)
6
10
(7-5)
5
(6.9)
(9-5)
(12.7)
3
(3.8)
2
(2.8)
0
(0)
0
(0)
63
79
80
72
C-0
60
(63.2)
1
2
(2.1)
95
C+7
0
(0)
91
95
26
(2.9)
134
886
(C).
^Pathogens isolated at time of sampling; 0, uninfected; CB, Corynbacterium b o v i s ; CN5, c o a g u 1ase-negative
staphylococci; SA, Staphylococcus aureus; and S T P , streptococci.
^Total
number of quarter samples.
59
TABLE 2. Effect of bacterial infection on numbers of total somatic
cell and differential cell counts in mammary secretion of
nonlactating cows.
Infection Status^
Parameter
0
INF
MINOR
MAJOR
2. 28b +
.12
2.99a +
.22
MAC
(%)
34.6Ia +
.51
33.51a +
.88
32.36a + 1.51
34.61a + 1.26
LYM
U)
34.26a +
.55
30.91b +
.95
31.99b + 1.62
29.15b + 1.35
PMN
(%)
29.92b +
.64
34.33a + 1.11
34.41a + 1.91
34.87a + 1.59
see
2.50b +
.38
3.83a +
.34
(106 )
Pathogens isolated at time of sampling; 0, uninfected; INF,
bacteriologically positive; MINOR, Corynebacterium bovis and
coagulase-negative staphylococci; MAJOR, Staphylococcus aureus and
streptococci.
b
’ Means between infection status with different superscripts differ
(P<.05).
q
60
Figure
1-
Changes
in total
somatic
cell
counts
In bovine mammary
secretion from drying off through the first 14 days of lactation.
Means
within
(P<.05) .
sampling
period
with
different
letters
(a,b)
differ
D-0
D+7
D+14
D+21
D+28
C-14
C-7
C-0
C+7
C+14
SAMPLING PERIOD
Uninfected
50
Minor
IIIMajor
o>
62
Figure 2.
Changes
in concentration of lactoferrin and
the citrate to lactoferrin molar ratio
citrate and
in bovine mammary secretion
in uninfected quarters from drying off through the first 14 days of
lactation.
Means
with different
letters
(a,b,c,d)
differ
(P<.05).
*The citrate to lactoferrin molar ration was calculated from citrate
and lactoferrin least square means at each sampling period.
■O L actoferrin
ItrateiL actoferrin
ab
■3.0 o
CITRATE
& LACTOFERRIN (m g /m l)
15-
2.0 2
-1.5
2.0-
1.0
to
ab
1.0
D-0
D+
D+U
0+21
D+28
C-7
C-0
C+7
C+14
SAMPLING PERIOD
0)
U
64
Figure
3.
protein
first
14
Effect
in
bovine
days
of
of
infection
mammary
status
secretion
lactation.
Means
different letters (a,b) differ (P<.05).
on
from
percent
drying
within
fat
off
sampling
and
total
through
period
the
with
D-0
D+7
D+14
D+21 D+28 C-14
C-7
SAMPLING PERIOD
^ ■ i % Fat Uninfected
111111111% Fat Infected
C-0
C+7
C+14
31111% Protein Uninfected
WS A % Protein Infected
o>
ui
66
Figure
4.
secretion
Means
Effect
infection
from drying off
within
(PC.05).
of
sampling
status
through the
period
with
on
first
different
pH
of
bovine
14 days
letters
of
mammary
lactation.
(a,b)
differ
D+7
D+14 D+21 D+28 C-14 C-7
SAMPLING
■ Uninfected
PERIOD
Minor
'SSS/S* Major
68
Figaro
mammary
5.
Chongoo
secretion
lactation.
in
from
concentrations
drying
off
of
bovine
through
the
serum
first
albumin
in
14
of
days
Means with different letters (a,b,c,d) differ (PC.05),
BOVINE SERUM ALBUMIN (mg/ml)
OI
[Ha
o
a
QHo
■ii"
O■
#m
□
t
□ — Iff
SAMPLING
o
4
□ — Hu
m
to
CB
I
o
PERIOD
JD—
O
-IT
0
1
m
O
O
+
■
o4
- «
£
69
CHa
Ota
10
10>
70
Figure
6.
Effect
of
infection
status
on
concentrations
of
immunoglobulin G in bovine mammary secretion from drying off through
the first 7 days of lactation.
differ (PC.05).
Means with different
letters
(a,b)
IMMUNOGLOBULIN
^
a
M
O
U
O
ft
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ft
O
a
SAMPLING
08
PERIOD
S
J5.
o
HIHIIHIIIIIIIIIIHIIIIIIH
u
{D
P
G (mg/ml)
72
Figure 7.
Changes in concentration of a-lactalbumin in bovine serum
from drying off through the first
14 days of lactation.
different letters (a,b,c) differ (PC.05).
Means with
a
I
1400 -
1200ab
•t-LACTALBUMIN
(nahil)
1000-
★
%
800
\
\
800-
%
400-
c
***»»!
I
200-
DO
D-7
D*14
D*21
D*28
C-14
SAMPLING PERIOD
C*7
CO
C+7
C*14
03
C H A P T E R III
Running head:
Morphology of Bovine Mammary Involution
Key words: Bovine, Mammary Ultrastructure, Involution, Lactogenesis.
MORPHOLOGICAL CHANGES IN THE BOVINE MAMMARY GLAND DURING INVOLUTION
AND LACTOGENESIS.
L. M. SORDILLO and S. C. NICKERSON
Louisiana
Agricultural
Experiment
Station,
Louisiana
University Agricultural Center, Hill Farm Research Station,
71040.
Correspondence sent to:
State
Homer,
L. M, Sordillo-Gandy
Department of Animal Science
University of Tennessee
P.O. Box 1071
Knoxville, Tennessee 37901-1071
Approved
for
publication
by
the
Director
of
the
Louisiana
Agricultural Experiment Station as manuscript number 87-80-1143.
74
75
Abstract
Morphological
changes
involution were examined.
cows
beginning
electron
gradual
at
reduction
off
through
examination
in synthetic
of
and
demonstrated
epithelium
fully
with
active
involution.
increases
concomitant
secretory
Electron
mammary
and
decreases
epithelium
during
indicated
activity
and
in
of
nonactive
epithelium,
during
analysis
this
and
a
alveolar
Light microscopic morphologic
in stroma
microscopic
secretion
mammary
Light
tissue
the
of
demonstrated a decreased number of organelles
synthesis
bovine
parturition.
secretory
epithelium as involution progressed.
analysis
during
Quarter biopsies were taken weekly from 5
drying
microscopic
occurring
time.
secretory
lumen,
first
alveolar
2
and
wk
of
epithelium
associated with milk
These changes
reversed
gradually beginning 2 wk prepartum, and by the time of calving, cell
structure exhibited morphology typical of lactating glands.
Tissue
from infected quarters had less synthetic and secretory ability as
indicated
by
nonactive
significantly
cells,
but
lower
higher
percentages
percentages
of
of
lumen
stroma
and
and
moderately
active cells compared to uninfected quarters. Infected quarters also
had more
compared
leukocytes
to
macrophages
removed
uninfected
and
milk
infiltrating the epithelium,
quarters.
polymorphonuclear
components
Large
numbers
of
rough
endoplasmic
plasma
and
reticulum,
Microscopic
leukocytes
cellular
cells,
these
during
distended
local
and stroma
examination
suggested
debris
exhibiting
suggested
during the periparturient period.
lumen,
of
cells
involution.
cisternae
antibody
of
production
76
Introduction
Morphological
involution
and
changes
lactogenesis
laboratory
animals
information
is
cessation
of
available
the
have
in
describing
these
the
mammary
bovine
importance
of
a
of
40
to
60
days
have
processes
(2).
gland
previously
relatively
in
little
following
the
Research
has
gland.
nonlactating
been
mammary
reported
However,
duration on milk yield of dairy cows
period
during
been
(3,4,7,11).
milking
demonstrated
occurring
period
of
adequate
Dairy cows having a dry
shown
to
produce
more milk
in
subsequent lactations than those with a less than 40 day dry period.
It has been hypothesized that mammary cells lost following peak milk
production during
cells during
is
also
effect
has not
a
of
lactation
are
the nonlactating
time
of
bacterial
increased
replaced
with new,
period (7).
more
efficient
The nonlactating period
susceptibility
to
mastitis,
and
the
infection on mammary tissue during this period
been determined.
An understanding of
the extent
the bovine mammary gland regresses during involution,
to which
the nature of
secretory cell growth and redevelopment in the prepartum period, and
the
relationship
process may
subsequent
The
purpose
occurring
of
lead
intramammary
infection with
to new approaches
the
to maximize milk
involutionary
production
in
lactations and possibly shorten the nonlactating period.
of
in
this
the
study
bovine
was
to
mammary
examine
gland
the
structural
during
changes
involution
and
lactogenesis, and to determine the effect of infection on changes in
tissue morphology.
77
Materials and Methods
Experimental design
Five Jersey cows from the Hill Farm Research Station were used.
Foremilk
samples
collection
gland
and
quarter.
were
collected
used to
evaluate
Mammary
tissue
aseptically
prior
infection status
samples
were
of
to
tissue
each
mammary
obtained
by
needle
biopsy technique from each quarter at drying off; 7 and 14 days post
drying
off;
and
14 and
7 days
prepartum.
At parturition,
animals
3
were slaughtered and
each
quarter.
approximately 1 cm
All tissue
examination.
Data
using
the general
effects
of
during
wk
wk
involution
x
linear
prepared
least
for
squares
analysis
model procedure
and
microscopic
to
infection
determine
status
infection status,
infection
status.
The
and
following model was
Y. .. = p + x. + p. + xp, . + e ,
ijk
l
'j
ij
ijk
p = effect common to all cows,
x^ =
fixed effect of ith wk during involution,
Pj =
fixed effect of jth infection status,
xp., =
ij
on
interaction of
used:
Where:
of
The statistical analysis included effect
wk during involution,
during
by
involution
morphological parameters.
of cow,
samples were
were analyzed
variance
of tissue was obtained from
fixed effect due to interaction between
ith wk during involution and jth infection
status,
e . .. = random error associated with each
ijk
observation
78
The
effect
of
cow
was
absorbed.
Preplanned
comparisons
of
least
squares means from the overall model were made by pairwise T-test.
Means were contrasted between
period and
between
infection status group within a time
time period within
infection
status
group.
No
other comparisons were made.
Tissue preparation
Cows were anesthetized locally prior to biopsy with 2 ml of 2%
Lid-o-cain
(Butler Co.,
Columbus,
OH).
A small
puncture
was made
through the skin with a scalpel blade above the gland cistern, and
approximately 3 cm x 2 mm of tissue was removed using a disposable
biopsy
needle
(Travenol,
application
of
obtained
slaughter
at
Dallas,
antibiotics.
fixed
in
2.5%
37°C)
for
and
topical
tissue
samples
glutaraldehyde
in
2
was
embedded
in
obtained
on
toluidine
dehydrated
epoxy
resins.
a
Porter
blue
for
in
a
Thick
Blum
graded
light
series
of
ethanol,
(0.5
to
1.0
sections
MT-5000
Tissue
M
in 0.1 M cacodylate-buffered osmium tetroxide (pH 7.0 at
h,
h.
0.1
post-fixed
1.5
at
samples
by
buffer
for
7.0
followed
cacodylate
5°C)
(pH
were
Biopsy
TX),
microtome
microscopy.
and
then
and
pm)
were
stained
with
Ultrathin
sections
approximately 60 nm thick were stained with 5.0% uranyl acetate
in
50% methanol for 20 min followed by 0.4% lead citrate for 10 min and
examined using a Philips EM 300 electron microscope at 60 kV.
Morphometric analysis
Quantitative
percentage
mammary
morphologic
tissue
area
analysis
composed
was
of
used
to
determine
interalveolar
stroma,
79
epithelium,
and
replications
of
magnification
alveolar
100
of
microscope.
A
lumen.
contact
X
630
points
using
reference
For
grid
were
a
in
each
counted
Zeiss
the
further as
(1)
nonactive,
sample,
per
Standard
microscope
fixed points used in the counting process.
characterized
tissue
slide
10
at
a
18
research
ocular
provided
Alveolar epithelium was
(2)
moderately active,
or
(3) fully active.
Tissue specimens of mammary parenchyma were also examined for
the
presence
of
polymorphonuclear
Prevalence
of
quantitated
corpora
leukocytes
these
with
epithelial
amylacea,
lining,
cell
(PMN),
macrophages,
mast
populations
respect
to
lumen,
and
tissue
or
cells,
and
in
plasma
corpora
location
stroma)
lymphocytes,
amylacea
(i.e.
10
cells.
were
within
randomly
the
selected
microscopic fields per tissue sample at X 630.
Ultrastructural examination
Based on light microscopic observations,
quarters
were
selected
to
Morphometric
analysis
through
approximate
apical,
the
basal,
and
of
evaluate
secretory
midportion
tissue areas from all
secretory
epithelial
of
the
Solari
method
activity.
cells
sectioned
nucleus
and
showing
lateral cell membranes was performed using
20 cm micrographs at a magnification of X 9,500.
the
cell
(19)
was
used
to
determine
12 x
A modification of
percentages
of
cell
area occupied by nucleus, membrane-free cytoplasm, rough endoplasmic
reticulum
secretory
(RER),
vesicles,
Golgi
and
apparatus,
milk
stasis
nuclear ratio was determined also.
mitochondria,
vacuoles.
The
fat
droplets,
cytoplasmic
to
80
Microbiological procedures
Prior
collected
to
tissue
collection,
duplicate
for microbiological analysis.
foremilk
samples
were
Presumptive identification
of bacterial isolates was determined by methods decribed by Brown et
al.
(1).
Intramammary
infection
was
defined
as
same microorganism from duplicate foremilk samples.
isolation
of
the
81
Results
Frequencies
are
were
in Table
3.
classified
classified
of
as
bacterial
Because
as
isolates
of
the
uninfected
infected
low number
or
with
during
of
infected.
minor
(
the
sampling
isolates,
quarters
Quarters were
Corynebacterium
period
further
bovis
or
coagulase negative staphylococci), or major ( Staphylococcus aureus,
Streptococcus uberis, or Nocardia) pathogens.
Percentages of
from
uninfected
tissue
and
area
infected
involution progressed reaching
area
peaked
percentages
decreased,
(Table
of
composed of
A,
quarters
lowest
Appendix
epithelium
and
decreased
and
lumen
gradually
as
levels at D+1A while stromal
Figures
lumen
continuing through C-0,
epithelium
10a-12a).
began
to
increase
At
C-1A,
as
stroma
reaching values observed at D-0.
Compare Figures 8 and 9.
Percent
of epithelial
area
composed of
nonactive
cells
from
uninfected and infected quarters increased significantly from D-0 to
D+7
peaking
percent
at D+1A
nonactive
(Table
5,
epithelium
Appendix Figure
began
to
13a).
decrease,
significantly lower percentage at C-0 compared to D+1A.
percent
epithelial
Figure
epithelium
began
percentage
at
increase,
Percent
reaching
of
a
epithelial
fully
significantly
area
composed
active
Conversely,
(Appendix
percent
fully
a
significantly from D-0 to D+7 reaching a minimum at D+1A
C-1A,
of
reaching
decreased
At
composed
C-1A,
cells
lAa).
area
At
active
higher
of moderately
active
to
C-0.
cells
82
fluctuated
very
little
from
D-0
to
C-14,
but
was
of
stroma
significantly
higher at C-7 compared to D-0.
Percent
tissue
areas
epithelia
were
regardless
of pathogen,
and
Likewise,
7).
percentages of
of sampling
quarters
composed
significantly
higher
compared
infected
to
from
had
quarters,
quarters
(Tables
significantly
lumen and moderately active epithelium
time.
nonactive
infected
uninfected
quarters
and
6
lower
irrespective
No significant differences were observed between
infected with minor or major pathogens
in histological or
cytological analyses.
Ultrastructural
analysis
demonstrated
that at D-0,
cells exhibited abundant parallel RER cisternae,
dictyosomal
cytoplasm,
casein
components,
several
micelles,
nuclear
ratio.
numerous
apically
apical
mitochondria
located
fat
secretory
droplets,
Microvilli
were
and
present
a
on
epithelial
supranuclear Golgi
scattered
about
vesicles
containing
large
the
the
cytoplasmic
apical
to
surfaces,
tight junctions between cells were intact, and nuclei were oval and
basally located (Figure 10).
At
D+7,
considerable
changes
compared to D-0 (Table 8).
RER
Large
cisternae,
fat
irregular
droplets
in
cytoplasmic
with
a
Golgi
shape,
to
occupied
and
structure were
components,
most
there
of
was
ratio.
significant
(Appendix Figures 16a-23a).
fine
observed
Cytoplasm exhibited significantly fewer
dictyosomal
nuclear
concomitant
in
a
the
and
cytoplasm,
significant
Stasis
decrease
mitochondria.
nuclei
decrease
vacuoles
in
secretory
in
were
the
increased
vesicles
83
At
D+14
continued
Golgi,
to
and
C-14,
increase
percent
cell
area
significantly,
while
composed
of
percentages
and mitochondria decreased (Table 8, Figure 11).
nuclei
of
RER,
Epithelial
cells exhibited increases in numbers of stasis vacuoles, as well as
a high
percentage
of cytoplasmic area composed
of fat.
Lysosomes
were observed within the cytoplasm.
At C-7, percentages of cell area occupied by nuclei, unoccupied
cytoplasm,
RER,
fat, and stasis vacuoles decreased, while percentages of
Golgi,
significantly
mitochondria,
in
and
comparison
secretory
with
cells
vesicles
obtained
cytoplasmic to nuclear ratio also increased.
at
increased
C-14.
The
Epithelial cells began
to exhibit secretory activity with appearance of small fat droplets,
and vesicles
containing casein micelles
that accumulated under the
apical plasma membrane.
The nuclei of some epithelial cells took on
a smooth ovoid shape and
microvilli became evident along the apical
surface.
The alveolar
lumen
granules.
The outline of the alveolar structure appeared
the
secretory material
was
accumulated
filled
in
with
lumina
fat
and
and
protein
rounded as
epithelial
cells
became cuboidal and flattened (Figure 12).
At C-0, ultrastructural examination showed that,
with
C-7,
percentages
while percent
polarized
region,
of
Golgi
Fat and nuclei
and exhibited
supranuclear
secretory
decreased.
abundant
Golgi
and
and
apparatus
vesicles
Epithelial
parallel
with
in comparison
RER
increased
cells appeared
in
secretory
the
basal
vesicles
containing casein micelles, and numerous mitochondria throughout the
cytoplasm.
apical
Secretory vesicles and fat droplets accumulated
cytoplasm.
Microvilli
on
the apical surface were
in the
shown
to
84
protrude
into
the
alveolar
lumen,
nuclei
were
oval
and
basally
located, and there was a larger cytoplasmic to nuclear ratio (Figure
1 0) .
Comparison
revealed
fat,
no
of
tissue
differences
from
uninfected
in percent
Golgi
or stasis vacuoles (Table 9).
significantly
cytoplasm,
vesicles
25a).
higher
and
apparatus,
However,
percentages
of
infected
mitochondria,
infected
nuclei
quarters
and
quarters had
organelle-free
but significantly lower percentages of RER and secretory
compared
For
the
to
uninfected
most
part,
ultrastructural
analysis
with
major
minor
and
tissues
were
no
significant
observed
pathogens.
(Appendix
between
However,
Tables
24a
and
differences
in
quarters
quarters
infected
infected
with
major pathogens had significantly lower percentages of RER compared
to uninfected quarters or those infected with minor pathogens.
Corpora amylacea were observed most frequently during the first
14 days
of
the dry period.
The majority of
corpora
were
seen
in
alveolar lumina where they often completely filled the luminal space
and
caused
flattening
microscopic
abutting
the
of
observations
apical
the
epithelium
demonstrated
cytoplasm
Corpora amylacea were observed
of
the
(Figure
dense
degenerating
to a
granular
Electron
material
epithelial
lesser extent
epithelial lining and in the interalveolar stroma.
13).
cells.
in the alveolar
Infection status
had no effect on the prevalence of corpora amylacea (Appendix Tables
22a and 23a).
Lymphocytes were the most prevalent cell type in the epithelial
lining,
followed
by
macrophages
and
PMN.
Macrophages
increased
significantly from D-0 to D+7 and continued to increase through C-0.
85
Lymphocyte populations
fluctuated throughout the sampling period as
numbers increased significantly from D-0 to D+14, but then decreased
significantly at C-14 and C-0 compared to concentrations at C-7.
significant changes
in numbers of PMN were observed throughout
sampling period.
lining
quarters
and
no
differences
macrophages
(Appendix
uninfected
the
Comparison of cell numbers within the epithelial
demonstrated
lymphocytes
No
Tables
quarters,
between
12a
those
and
which
in
numbers
uninfected and
13a).
were
infected
However,
infected
had
of
compared
to
significantly
higher numbers of PMN (1.22 + ,78 vs 6.05 + .77).
Macrophages
alveolar
fat,
and
lumina.
casein,
and
PMN
Many
were
the
exhibited
cellular
debris
most
prevalent
cytoplasmic
(Figure
cell
vacuoles
14).
increased
gradually
throughout
the
in
containing
Lymphocytes
observed less frequently throughout the sampling period.
macrophages
types
were
Numbers of
sampling
period
reaching significantly higher concentrations at C-0 compared to D-0.
Lymphocyte
populations
were
highest
at
D+14
significantly lower at C-7 in comparison.
PMN
numbers
Comparison
of
were
total
observed
cell
found
C-0,
but
were
No significant changes in
throughout
numbers
and
the sampling
within
the
period.
alveolar
lumen
demonstrated no differences between uninfected and infected quarters
in numbers of lymphocytes.
Conversely, when compared to uninfected
quarters, those which were infected had significantly higher numbers
of macrophages (5.76 + 1.26 vs
12.33 + 1.25) and
13.86 + 1.63) (Appendix Tables
14a and 15a).
PMN (3.47 + 1.65 vs
86
Subepithelial
leukocyte
type
infiltration.
followed
(Table
stromal
10).
gradually
by
from
parturition,
cells,
D-0
that
were
Macrophages
lymphocytes,
Plasma
concentrations
areas
were
plasma
C-7
most
the
cells,
macrophages,
through
were
the
and
where
significantly
most
mast
prevalent
cells,
they
higher
than
concentrations
at
C-0
from
D-0
compared
reaching
to
all
other
period.
differences
Comparison
in levels
of
total
of macrophages,
cell
and
PMN
increased
at
peak
D-0.
At
remained high
Mast cell numbers
significantly
sampling
significant differences in PMN numbers were observed
sampling
of
cell
reached
numbers of plasma cells and macrophages
gradually
site
lymphocytes
while lymphocyte numbers decreased significantly.
decreased
common
times.
throughout
numbers
lymphocytes,
lower
No
the
indicated
mast
cells,
no
or
plasma cells within the stroma of uninfected and infected quarters.
However,
numbers
quarters
(10.10
of
+
PMN
1.26)
were
significantly
compared
to
higher
uninfected
in
quarters
infected
(1.53
1.27) throughout the sampling period (Appendix Tables 16a and 17a).
+
87
Discussion
Although considerable information is available in rats and mice
regarding
alterations
morphological
lactation
extent
of
in
changes
the
mammary
accompanying
bovine
have
not
to which milk producing
milking,
with
in
and
species
animals
cannot
involution
and
morphological
changes
lactogenesis
data
the
bovine
rate
of
and
following cessation
to
bovine.
onset
The
varies
available
effectively
involution,
the
elucidated.
during
in
in
and
regress
extrapolated
lactogenesis
involution
been
Consequently,
be
during
tissues
redevelopment
(7).
structure
the
This
mammary
in
greatly
laboratory
processes
study
tissue
of
examined
during
the
nonlactating period.
Morphological changes during involution indicated marked shifts
in the secretory activity of the gland during
nonlactating
period
followed
approached.
Following
the
by
more
first
wk
the first wk of
gradual
of
changes
involution,
as
the
calving
secretory
activity of mammary epithelium decreased as evidenced by a reduction
in
alveolar
area.
luminal
area
with
a
concomitant
increase
in
stromal
Luminal spaces shrank when secretion no longer displaced the
alveolar
area.
resorbed,
Once
stromal
milk
areas
sythesis
also
and
expanded proportionately
the reduced alveolar luminal area.
epithelium
ceased
revealed
an
mammary
fluid
was
to compensate for
Cytological analysis of mammary
increase
in
prevalence
of
nonactive
cells with a concurrent decrease in fully active cells through D+14.
Lee and Lascelles
gland.
(8)
reported
similar
changes
in the ewe mammary
They found as regression of the parenchyma progressed, there
88
was
a corresponding
increase
in stroma
within
16 days
of weaning
( 8).
Ultrastructure
closely
The
cells
cell
of
described
cytoplasm
parallel
bovine
alveolar
previously
contained
cisternae,
well
cells
from
D-0
lactating
abundant
developed
at
RER
Golgi
resembled
glands
with
(5,12).
polarized
apparatus,
and
and
numerous
mitochondria, all of which are indicative of synthetic activity.
On
D+7,
regressive
ultrastructural
activity.
level
changes
which
became
indicated
evident
reduction
at
in
the
secretory
The pronounced accumulation of large fat droplets in the
cytoplasm and reduced luminal area were changes observed also in rat
mammary
glands
3
days
following
weaning
(16).
Early
changes
in
bovine alveoli also included alterations in numbers and organization
of
cytoplasmic
were
broken
reduced
organelles.
up
into
considerably
decreased.
observed
Such
after
Rough
irregular
in
size,
reductions
1 wk
in
endoplasmic
strands,
and
reticulum
Golgi
numbers
synthetic
of
and
dry closely resembled
cisternae
dictyosomes
were
mitochondria
were
secretory
changes
organelles
reported
in rat
(3,16) and mouse (19) mammary glands on the second to third day of
induced involution.
Stasis vacuoles were first observed in epithelial cells at D+7,
but
increased
in
number
to
D+14
and
intramammary pressure associated with
may
trigger
stasis
and
the
formation
failure
of
the
C-14.
initial
of stasis vacuoles
fusion
and
The
build
stages of
due to
release
up
of
involution
intracellular
mechanism
(19,23).
89
Studies
of
mammary
gland
Involution
in
the
mouse
suggest
these
intracellular protein granules and other secretory products may be
removed
(19).
during
involution
through
digestion
in
lysosomal
vacuoles
Many epithelial cells in this study exhibited vacuolation of
the
cytoplasm
with
electron-dense
granules.
Previous
studies
suggested these electron dense granules were lysosomes (3).
The
mammary
postweaning.
At
gland
the
of
ewes
light
involutes
microscopic
completely
level,
by
32
degenerative
days
cells
disappeared, and the alveolar remnants were lined with a few layers
of closely packed epithelial cells (8).
involution of mammary glands
the
same
extent
as
noted
The present study suggested
from pregnant cows did not regress to
in
other
species.
Morphological
data
indicated bovine mammary glands regressed markedly between D+14 and
C-14
although
Histological
still
not
to
examination
discernible,
albumin
Although
areas
in
and
a
total
previously
mammary
filled
from
tissue
with
nonlactating
protein
lactoferrin
portion
of
as
noted
found
a
in
sheep.
luminal
spaces
deeply
basophilic,
In a companion study (22), changes in mammary
composition
increases
degree
but
proteinaceous fluid.
secretion
the
of
and
during
the
cows
revealed
concentrations
this
same
proteinaceous
of
stage
fluid
significant
bovine
of
serum
involution.
occupying
luminal
is of serum origin (bovine serum albumin), elevated levels of
locally produced whey proteins (lactoferrin) suggests some synthetic
capabilities
in
the
maximum regression.
bovine
mammary
gland
during
this
period
of
90
Ultrastructural
C-14
found
the
examination
cytoplasmic
of
mammary
to nuclear
tissue
ratio
was
from
D+14
smallest
and
at
this
point and the minimal amount of cytoplasm was occupied by numerous
stasis
vacuoles
and
fat
droplets.
dedifferentiating and appeared
the
general
lobulo-alveolar
Although epithelial cells were
less
capable of synthetic activity,
framework
remained
intact.
Bovine
alveoli apparently did not involute to the same degree as observed
in other
species.
In
the
rat,
no
alveoli were observed
15 days
after weaning, and only small ducts passing through dense connective
tissue
remained
(16).
alveolar
lumens,
Previous
studies
Epithelial
cells were
leaving only
suggested
that
the
found
to slough
basement membrane
myoepithelial
cells
into
intact.
survived
the
destructive processes and functioned as a framework to prevent total
loss of organized structure in the fully involuted rat mammary gland
(15).
Corpora amylacea were most numerous during the first 14 days of
the
dry
corpora
period.
Previous studies
amylacea
during
involution (13).
bodies
and
the
have
later
noted the
stages
of
Morphological relationships
mammary
parenchyma
suggest
prevalence
lactation
and
of
early
between these amyloid
they
may
suppress
milk
secretion by engorging luminal spaces and obstructing small ducts.
Changes
glands
in
showed
histological
a
marked
structure
rise
in
of
rat and
mouse
mammary
alveolar diameter
and
corresponding fall in alveoli per unit area at parturition (11).
the
present
microscopic
study,
level
similar
in
changes
prepartum
were
bovine
observed
mammary
at
the
tissue.
a
In
light
During
91
the
last
2
wk
of
gestation,
increased
activity was apparent by higher
by
accumulated,
luminal spaces became engorged.
lumina
resulted
stromal area.
became
more
As
cell
a
of
enlarged
As
secretory
mammary
fluid
Expansion of alveolar
compression
approached,
while nonactive
cytoplasm
stroma.
concomitant
parturition
numerous
epithelial
percentages
in
and
percentages of epithelium and lumen
accompanied
area
lower
synthetic
fully
of
surrounding
active
cells decreased.
significantly
cells
At
and
also
C-7,
the
contained
numerous fat droplets at the basal region and accumulated secretory
vesicles
at
indicative
similar
the
the
of
apical
milk
synthesis
lobulo-alveolar
third
or
region,
second
showing
and
secretion
development
day
the
prepartum
did
(5),
not
typical
(12).
morphology
In
become
indicating
the
mouse,
complete
marked
until
species
variation.
Infection status influenced normal changes in mammary structure
during
and
involution
major
indicated
and
pathogens
by
lactogenesis.
had
less
significantly
secretory vesicles,
Quarters
synthetic
lower
and
infected
secretory
percentages
of
with
minor
activity
lumen,
RER,
as
and
but significantly higher percentages
of stroma
and nonactive epithelium compared to uninfected quarters.
Quarters
infected
with
major
pathogens
had
also
significantly
higher
percentages of membrane-free cytoplasm and lower percentages of RER
which further substantiate impaired synthetic ability.
The presence
of bacteria within the gland during the nonlactating period appeared
to
have
a
deleterious
effect
on
development
of
secretory
cells.
These findings support the contention that infections during the dry
period
may
interfere
with
normal
mammary
secretory
cell
92
differentiation and decrease milk yields in subsequent lactations.
Jensen and Eberhart
numerous
cell
prevalent
type
(6)
during
initially,
found that macrophages were
most
decreased
of
as
during
Previous
studies
involution
have
period.
period
decreased
demonstrated
of
mammary
revealed
first
tissue
wk
of
the
infiltrating
an
the
near
presence
leukocytes
increase
involution.
alveolar
and
parturition.
of
leukocytes
lumen, and underlying connective tissue
Numbers
elevated at parturition also.
within
were
progressed,
in nonlactating rat and mouse mammary glands (3,17).
quantification
PMN
The proportion of lymphocytes
and then
infiltrating the epithelium,
dry
the dry
increased as parturition approached.
increased
the
the most
lumen
in
in all
of
In this study,
nonlactating
cell
types
macrophages
bovine
during
and
PMN
the
were
Large numbers of foamy cells observed
were
similar
morphologically
to
the
fat-laden macrophages described by others in involuted mouse mammary
glands
(8,17).
observed
The
presence
of fat,casein,
and
cellular
in the cytoplasm of both PMN and macrophages
findings of others
resorption
of
that
milk
these cells
components
play an
supports
important role
and facilitating
the
debris
the
in the
removal
of
degenerated epithelial cells from the involuting mammary gland (9).
Mast
cells were most numerous
the first 2 wk of the dry period,
as parturition
was
observed
connective
release
resulting
approached.
in
involuted
tissue
heparin
in
of
and
but were observed less frequently
A similar pattern in mast cell numbers
ewe
other
mammary
organ
histamine
increased
in the connective tissue during
vascular
glands
systems
under
(8).
have
Mast
been
pathological
permeability
(18).
cells
in
reported
to
conditions,
Increased
93
concentrations of these cells during involution has been attributed
to the increasing prominence of stroma as a consequence of alveolar
degeneration
(8).
However,
mast
cell numbers could have decreased
at C-0 from degranulation as a result of the inflammation associated
with parturition.
Macrophages have been shown to be the predominant cell type in
secretions from noninfected
involuted glands
(10).
However, during
mammary infection, PMN may accumulate to over 50 x 10^
(14).
In
greatly
this
the
study,
mean
infection
number
parenchymal tissue.
of
status
leukocytes
of
quarters
infiltrating
influenced
the
mammary
Compared to uninfected quarters, those infected
with major pathogens had consistently higher numbers
throughout
cells per ml
the nonlactating period.
of
leukocytes
Numbers of macrophages and PMN
within the epithelial lining, lumen, and stroma of infected quarters
were
especially high
Presence
during
of
bacteria
inflammation
resulting
blood
to
milk,
involution.
elevated
revealed
material.
and
the
within
in
first
the
amplified
exceeding
and
gland
last wk
may
migration
levels
of
involution.
have
of
normally
evoked
leukocytes
associated
an
from
with
Plasma cell numbers of infected tissue were especially
at
C-0.
grossly
In
a
Ultrastructural
distended
recent
RER
study
examination
cisternae
(21),
of
engorged
these
cells
with
flocculent
immunocytochemical
techniques
were used to demonstrate immunoglobulin secretory activity by these
plasma
cells.
Exposure
to
bacterial
pathogens
may
have
enhanced
plasma cell proliferation and local antibody production in response
to antigenic stimulation.
94
Results
of
this
study
suggest
the
bovine
mammary
gland
involutes gradually over a 2 wk period.
Bovine epithelial cells do
not regress to the same extent observed
in rat mammary glands
appear
to maintain some synthetic and secretory activity during the
nonlactating period.
normal
and
mammary
The presence of intramammary infection altered
structure
during
involution
and
may decrease milk yields in subsequent lactations.
lactogenesis
which
95
References
1. Brown, R. W . , D. A. Barnum, D. E. Jasper, J. S. McDonald, and W.
D.
Schultze.
1981.
Microbiological
procedures
for use
in the
diagnosis of bovine mastitis.
2. Coppock,
1974.
C. E . , R. W.
Effect
of
Everett,
dry period
R.
P. Natzke,
length on Holstein milk production
and selected disorders at parturition.
3.
Helminen,
H.
J.,
and J.
L.E.
mammary gland involution. II.
and heterophagocytosis.
4.
Helminen,
enforced
H.
J.,
milk
reference
stasis
to changes
Cell Res.
Ericsson.
in
L.E.
Ericsson.
gland
lysosomes
and
Page 3
counts
iji Lactation,
in
A.
mammary gland.
secretions
K. ,
Effects of
epithelium,
lysosomal
with special
enzymes.
Exp.
a comprehensive
treaties.
B.
L.
and
of
1981.
the
Total and differential
nonlactating
bovine
mammary
42:743.
C.
S.
Lee.
1978.
Involution
of
the
Page 115 in Lactation, a comprehensive treatise.
B. L. Larson, ed.
Academic Press, New York, N. Y.
8. Lee, C. S., and A. K. Lascelles.
in
1971.
Academic Press, New York, N. Y.
Am. J. Vet. Res.
7. Lascelles,
25:214.
Cytology and fine structure of the mammary
6. Jensen, D. L . , and R. J. Eberhart.
gland.
Studies on
68:411.
Larson, ed.
cell
1968.
Ultrastructural evidence for auto-
on mammary
5. Hollmann, K. H. 1978.
gland.
J. Dairy Sci. 57:712.
J. Ultrastruct. Res.
and J.
and H. R. Ainslie.
involuting mammary
allergic response.
glands
of
1969.
ewes
The histological changes
in relation
Aust. J. Exp. Biol. Med. Sci.
to
the
47:613.
local
96
9.
Lee, C. S.,
G. H. McDowell,
Importance
of
macrophages
in
involuting mammary gland.
10.
Lee,
C.
S.,
F.
Identification,
populations
and A. K.
B.
removal
Res. Vet. Sci.
P.
Wooding,
properties,
using
the
and
electron
Munford, R. E.
changes
in
1964.
the
microscopy
quantitative methods
Sci. Abstr.
12.
The
from
the
of
dry
Kemp.
1980.
counts
cow
of
cell
secretions,
J. Dairy Res.
47:39.
anatomical and biochemical
with
particular
of assessing mammary
reference
development.
to
Dairy
26:293.
Nickerson, S. C., and R. M. Akers.
blockade
of
microtubule
differentiation
of
Int. J. Biochem.
13.
gland
fat
P.
differential
A review of
mammary
of
1969.
10:34.
and
colostrum, and milk from normal cows.
11.
Lascelles.
Nickerson,
S.
1983.
formation
mammary
epithelium
Effects of prepartum
on
in
ultrastructural
Holstein
heifers.
15:777.
C.,
and
L.
M.
Sordillo.
1985.
Role
of
macrophages and multinucleated giant cells in the resorption of
corpora amylacea in the involuting bovine mammary gland.
Tiss. Res.
14.
Paape,
240:397.
M.
J.,
Leukocytes
Randor,
W.
Richards,
changes
Wergin,
and
A.
J.
line of defense against
Guidry.
1979.
invading mastitis
J. Dairy Sci. 62:135.
C.
R.
P.
glands of the rat.
16.
P.
- second
pathogens.
15.
Cell
R.
C.,
1972.
J. Anat.
and
accompanying
albino rat.
Myoepithelium in involuting mammary
G.
112:355.
K. Benson.
involution
J. Endocrinol.
of
51:127.
the
1971.
Ultrastructural
mammary
gland
in
the
97
17.
Richards,
R. C., and
G. K. Benson.
1971.
Involvement of the
macrophage system in the involution of the mammary gland in the
albino rat.
18.
J. Endocrinol.
Riley, J. F.
cells,
1959.
histamine,
The mast cells.
19.
Sekhri,
of
mouse
Solari,
I.
and
heprin
in
tissue
glands.
I,
conditions.
Cytomorpholgy
J. Natl. Cancer Inst.
1973.
Etude
iji
1967.
of
Studies
the
normal
39:459.
quantitative d'organes ou de tissus.
Ann. Biol. Anim. Bioch.
13:247.
Sordillo, L.
M.,
Quantification
in
Mast
E. and S. Livingstone, LTD.
Methodes d'estimation des volumes.
cells
mast cells:
in pathological
J. F. Riley, ed.
mammary
A.
Biophys.
21.
Histamine
K. K., D. R. Pitelka, and K. B. DeOme.
mammary gland.
20.
51:149.
S. C. Nickerson,
and
and
immunoglobulin
nonlactating
bovine
S. P.
Oliver.
classification
mammary
tissue.
J.
of
1987.
plasma
Dairy
Sci.
(submitted).
22.
Sordillo, L. M . , S. C. Nickerson,
and
S.
P.
Oliver.
1987.
Secretion composition during bovine mammary involution and the
interrrelationship
with
mastitis.
Int.
J.
Biochem.
(submitted).
23.
Wellings S. R . , and K. B. DeOme
(1963)
Electron microscopy of
milk secretion in the mammary gland of the C3H/Crg mouse.
III.
Cytomorphology
Inst
30:241-267
of
the
involuting
gland.
J
Natl
Cancer
98
TABLE 3. Frequency of bacterial isolates from bovine mammary
foremilk samples from drying off through lactogenesis.
Sampling Period^
D-0
D+7
D+14
C-14
N
55
11
50
10
45
9
%
N
0
0
15
3
7.
N
5
1
%
N
C-7
C-0
55
11
55
11
50
10
20
4
10
2
5
1
5
1
0
0
5
1
10
2
5
1
0
0
35
7
30
6
25
5
20
4
25
5
30
6
N
0
0
0
0
0
0
0
0
5
1
10
2
%
N
5
1
5
1
5
1
5
1
5
1
5
1
Organism
None
coagulase (-)
staphylococci
C. bovis
S. aureus
S. uberis
Nocardia
7.
7.
^Days relative to drying off (D) or calving (C).
%, Percentage of total quarters within each sampling period.
N, Number of quarters within each sampling period.
99
TABLE 4. Histological analysis*
drying off through lactogenesis.
of
Tissue
bovine
mammary
tissue
from
C-7
C-0
SamplinR: Period^
D-0
classification
D+7
D+14
C-14
Epithelium
X
SE
45.36a
1.93
41.14ab
1.32
38.59b
1.18
41.89a
1.36
43.73a 45.82a
1.93
1.51
Lumen
X
SE
16.87ab
2.92
13.59b
1.99
13.38b
1.79
15,48ab
2.06
20.49a 20.88a
2.92
2.28
Stroma
X
SE
37.66cd
2.68
45.61ab
1.83
48.21a
1.64
42.70bcd 35.98d 33.53d
1.89
2.09
2.67
Data are expressed as mean percent of tissue area.
2
Days relative to drying off (D) or calving (C).
& b c d
’ ’ 1 Means
between
differ (P<.05).
sampling periods with different
superscripts
100
TABLE 5.
Cytological analysis* of bovine mammary
drying off through lactogenesis.
Sampling Period
Epithelial
D-0
classification
Nonactive
Moderately
active
Fully active
D+7
D+14
48.51a
3.69
C-14
epithelium from
2
C-7
X
SE
23.49C
6.03
36.90b
4.12
X
SE
35.89b
6.65
48.51ab 46.45ab 50.7lab 57.74a
5.21
4.69
4.55
4.07
X
SE
40.46a
6.90
14.54cd
4.72
5.02d
4.22
29.55bc 25.62°
4.25
4.72
19.73°
4.87
C-0
18.79C
6.01
45.15ab
6.64
16.50cd 35.90a
5.41
6.89
Data are expressed as mean percent of tissue area.
2
Days relative to drying off (D) or calving (C).
3 b c
' ' Means between sampling periods with different superscripts
differ (PC.05).
101
TABLE 6. Effect of infection status on histological analysis^ of
nonlactating bovine mammary tissue.
Infection Status
Tissue
0
Classification
INF
2
MINOR
MAJOR
Epithelium
X
SE
41.95a
.73
41.74a
.73
45.03a
1.73
41.29a
.87
Lumen
X
SE
21.25a
1.10
16.42b
1.10
12.56b
2.62
16.53b
1.32
Stroma
X
SE
36.60b
1.01
42.09a
.99
42.69a
2.39
42.55a
1.21
Data are expressed as mean percent of tissue area.
2
Infection status at time of sampling: 0, uninfected; INF, infected;
MINOR, Corynebacterium bovis and coagulase negative staphylococci;
MAJOR, Staphylococcus aur e us, Streptococcus uberis, and Nocardia.
3 b
’ Means between infection status with different superscripts differ
(PC.05).
102
TABLE 7, Effect of infection status on cytological analysis*
nonlactating bovine mammary epithelium.
Infection i Status
Epithelial
Classification
Nonactive
Moderately
active
Fully active
0
INF
MINOR
of
2
MAJOR
X
SE
21.99b
2.29
32.26a
2.27
35.093
5.40
34,35a
2.72
X
SE
52.66a
2.52
h
44. 37
2.50
4 8.14ab
5.96
V|
41.43
3.00
X
SE
25.39a
2.62
23.33a
2.57
16.423
6.18
24.28a
3.11
Data are expressed as mean percent of tissue area.
2
Infection status at time of sampling: 0, uninfected; INF, infected;
MINOR, Corynebacterium bovis and coagulase negative staphylococci;
MAJOR, Staphylococcus aureus, Streptococcus uberis, and Nocardia.
3 b
* Means between infection status with different superscripts differ
(PC.05).
TABLE 8.
Ultrastructural
1actogenesi s .
analysis^ of alveolar cells
in bovine mammary tissue from drying off through
Samp 1inq Per iod2
Parameter 3
D-0
D+14
D+7
C - 14
C-7
C-0
NUC
25-95 c ±2.29
32. 19b± K 57
4 3 . 8 5 a± 1 .40
4 1.27a± 1-52
2 8.20c +1.79
22.27c ±2.29
CYT
l8.45b± 2 . 15
2 0 . I4b ± l .46
2 2 .49b± T . 3 1
2 7.75a ±1.42
18.17 b± 1.68
17.9!b± 2 . 15
RER
19.I5a ± 1 .84
12.03b± 1 .26
4.68c + 1 .24
5.46 c ± 1 .21
l8.00a± 1 .44
17 *84a ± 1.83
GOL
11.44b± 1 .23
8.34c ± .84
2.74d ± .75
3.45d± .81
11.11b+ .96
I8.68b± 1 .22
M !T
13.42a ± 1 .21
5-99c ± .83
3.63d + .75
3 .84d + .80
9-91b ± .95
8.68b± 1 .22
FAT
6.45bc±2.00
I7.00a± 1 .37
l6.69a ± 1 .23
i
14.69a b ± 1.32
12.08b + 1 .57
8.23b ± 1 .99
SEC
4.50a± 1 .23
2.05b± .77
,02c + .69
• 18C ± .75
2.73a b ± -88
6 , 0 4 a ± l .12
STA
.11a ±1.11
•93a ± .76
2 .44a± .68
1.07a ± .73
.21a± .87
.50a ± 1 .11
2.85a ± .71
2 . 11b ± .32
1.28c ± .49
1.42c ± .63
2.55a b ± .71
3.49a ± .82
C Y T :NUC
^Data are expressed as mean percentage contribution ± S.E.
^Days relative to drying off (D) or calving
to a total cell area.
(C).
3parameters measured:
NUC, nucleus; CYT, unoccupied cytoplasm; RER, rough endoplasmic reticulum; GOL,
Golgi apparatus; MIT, mitochondria; FAT, fat droplets; SEC, secretory vesicles; STA, stasis vacuoles;
and CYT:NUC, cytoplasmic to nuclear ratio.
a,b,c,dMeanS between sampling periods with different
superscripts differ
(P<.05).
104
TABLE 9. Effect of Infection status on ultrastructural analysis* of
alveolar cells in bovine mammary tissue.
Infection Status^
0
Parameter
INF
MINOR
MAJOR
NUC
X
SE
31.12b
.85
34.30a
.86
30.69ab
2.06
34.98a
1.03
CYT
X
SE
19.46b
.79
22.09a
.79
20.56ab
1.92
22.34a
.97
RER
X
SE
13.66a
.68
11.39b
.69
14.49a
1.64
10.49b
.83
GOL
X
SE
9.58a
.48
8.68a
.48
10.03a
1.09
8.38a
.55
MIT
X
SE
8.28a
.45
7.23a
.45
7.16a
1.09
7.30a
.55
FAT
X
SE
12.13a
.72
12.10a
.73
13.72a
1.79
11.79a
.90
SEC
X
SE
3.79a
.41
2.27b
.41
1.41b
1.01
2.55b
.51
STA
X
SE
1.093
.40
.86a
.41
.38a
.99
1.19a
.50
Data are expressed as mean percentage contribution to a total cell
area.
Infection status at time of sampling: 0, uninfective; INF,infected;
MINOR, Corynebacterium bovis and coagulase negative staphylococci;
and MAJOR, Staphylococcus aureus, Streptococcus uberis, and
Nocardia.
3 b
’ Means between infection status with different superscripts differ
(PC.05).
105
TABLE 10.
Cytological comparison of infiltrating cells in
uninfected mammary tissue from drying off through lactogenesis.
Sampling Period
Cell
Types
Epithelium
MAC
D-0
D+7
2
D+l A
C-14
C-7
C-0
X
SE
10.93C
2.57
24.25b
2.71
25.88b
3.01
21.94b
3.20
33.68a
2.57
27.61ab
2.66
LYM
X
SE
42.41b
7.16
58.47ab 66.93a
7 .54
8.39
48.92b
8.91
73.513
7.16
44.86b
7.40
PMN
X
SE
.15b
.71
X
SE
,62b
2.77
Lumen
MAC
1.46a
1.81
2.20a
2.01
.99a
2.13
.97a
1.71
1.85a
1.77
6.56ab
2.92
7.88ab
3.25
4.42ab
3.46
5.12ab
2.78
9.96a
2.87
LYM
X
SE
PMN
X
SE
.78b
3.60
5.19a
3.08
6.22a
4.22
2.98a
4.28
X
SE
32.96b
6.76
42.46b
7.12
65.78a
7.93
LYM
X
SE
23.19b
3.22
32.99a
3.39
PMN
X
SE
1.50a
2.80
MAST
X
SE
PLM
X
SE
Stroma
MAC
.14b
.28
.98a
.29
1.34a
3.61
4.28a
3.37
51.00ab
8.41
71.35a
6.77
61.55a
7.00
32.26a
3.78
34.72a
4.01
42.97a
3.22
27.79b
3.33
.47a
2.95
.94a
3.29
2.74a
3.48
1.80a
2.80
1.72a
2.89
10.29a
1.96
8.59a
2.06
9.08a
2.29
7.88a
2.43
5.213
1.96
2.52b
2.02
16.10b
5.31
20.17b
5.59
23.37b
6.60
41.47a
5.31
.37ab
.28
.46ab
.29
1.07a
.32
29.92ab
6.22
.36ab
.34
27.35ab
5.49
Data are expressed^as m^an number of cells enumerated per unit
tissue area (6 x 10
pm
).
2
Days relative to drying off (D) and calving (C).
Cell types are: MAC, macrophages; LYM, lymphocytes; PMN,
polymorphonuclear leukocyte; MAST, mast cells; and PLM, plasma
cells.
3 b
' Means between sampling period with different superscripts differ
(PC.05).
106
Fig. 8.
Mammary tissue typically obtained at drying off and calving
exhibiting
minimal
stromal
area
epithelium (E) and distended
Fully
active
epithelial
(S)
with
larger
proportions
of
lumina (L) occupying the tissue area.
cells
were
characterized
by
the
basally
located nuclei (N), large cytoplasmic to nuclear ratio, and presence
of numerous secretory vesicles (SV) in the apical cytoplasm
Fig.
9.
Mammary
tissue
obtained
at
14
days
after
X 935.
drying
off
appeared nonactive with a large proportion of stromal area (S) with
minimal
luminal
characterized
area
by a
(L).
The
shrunken
layer' of closely
alveoli
packed cells,
(A)
and the
were
limited
luminal areas stained deeply basophilic X 935.
Fig.
typically obtained at drying
off
and calving characterized by polarized cells with a basal nuclei
(N)
and
10.
Portion of
supranuclear
an alveolus
Golgi
(G).
Abundant
rough
endoplasmic
(R), mitochondria (M), and apically situated fat (F)
vesicles
(SV)
occupied
the
cytoplasmic
protruding from the apical surface.
Fig.
11.
Nonactive
characterized
irregularly
by
shaped
a
epithelial
small
nuclei
Golgi dictyosomal membranes
and secretory
with
microvilli
L, lumen X 6,075.
cells
14 days
cytoplasmic
(N).
area
reticulum
The
to
after
nuclear
cytoplasm
drying
off
ratio
and
consisted
(G) and scattered mitochondria,
rough endoplasmic reticulum cisternae.
only
of
but no
The apical surface (arrows)
lacked extensive microvilli and the alveolar lumen (L) contained an
accumulation of electron-dense material X 6,075.
107
108
Fig.
12.
Portion of an alveolus obtained at 7 days prior to calving
demonstrating
fluid accumulation.
F,
fat;
G,
Golgi
apparatus;
lumen; N, nucleus; S, stroma; and SV, secretory vesicle X 7,560.
L,
109
.‘V
f4
.
110
Fig.
13.
Corpora
amylacea
filling the alveolar lumen.
Fig.
14.
vacuoles
Polymorphonuclear
containing
macrophages
5,625.
(M)
with
mammary
(CA)
were
most
frequently
E, epithelium and S, stroma
leukocytes
secretion
internalized
(P)
exhibited
components
cellular
debris
observed
X 650.
phagocytic
(arrows),
and
(arrowheads)
X
111
C H A P T E R
Running head:
IV
Plasma Cell Isotypes in Mammary Tissue
Key words:
Involution,
Lactogenesis,
Plasma
Cells,
Mammary Biopsy, Immunohistochemistry, Immunoglobulin.
Mastitis,
QUANTIFICATION AND IMMUNOGLOBULIN CLASSIFICATION OF PLASMA CELLS IN
NONLACTATING BOVINE MAMMARY TISSUE.
L. M. SORDILLO and
S. C. NICKERSON
Louisiana
Agricultural
Experiment
Station,
Louisiana
University Agricultural Center, Hill Farm Research Station,
71040
Correspondence sent to:
State
Homer,
L. M. Sordillo-Gandy
Department of Animal Science
University of Tennessee
P.O. Box 1071
Knoxville, Tennessee 37901-1071
Approved
for
publication
by
the
Director
of
the
Louisiana
Agricultural Experiment Station as manuscript number 87-80-1148.
112
113
Abatract
Plasma cell populations in bovine mammary tissue were examined
during involution using electron microscopic and immunohistochemical
techniques.
at
weekly
Plasma
This
Biopsies were taken from each quarter of 5 Jersey cows
intervals
cells
were
association
epithelium
numbers
and
beginning
observed
may
milk.
cells
of
gradually
gestation.
were
nonlactating
the
to
period
from
cells
were
parturition.
epithelial
antibody
off,
cells.
through
plasma
cell
reached
peak
and dropped significantly during the
numerous
G^-
and
isotypes
followed
more
of
drying
by
numerous
in
pathogens than uninfected quarters.
G 2_producing
observed
plasma
during
immunoglobulin
immunoglobulin A-producing plasma cells.
plasma
through
alveolar
transport
Immunoglobulin
most
off
Immunoglobulin-producing
concentrations 2 wk prepartum,
last wk
drying
proximal
facilitate
into
increased
at
M-
the
and
Immunoglobulin M-producing
tissue
infected
with
minor
Ultrastructural examination of
these cells revealed rough endoplasmic reticulum cisternae engorged
with
flocculent
Exposure
to
material,
minor
indicative
pathogens
may
of
have
antibody
enhanced
synthesis.
plasma
cell
proliferation and local antibody production in response to antigenic
stimulation.
nonlactating
Results
period
in
of
plasma
bovine
cell
mammary
distribution
tissue
indicate
over
the
times
when
local immunostimulation may be most effective in enhancing
to intramammary infection.
immunity
114
Introduction
Immunoglobulins are an important soluble component of
immunity.
Immunoglobulin
bacteria for phagocytosis
does not
function
neutralization,
adhesion
to
secretion
are
cells
in
by leukocytes
as an opsonin,
bacterial
cell
the
but has been
from
(15).
blood,
subepithelial
and
(10,16).
agglutination,
membranes
derived
G 2»
(Ig) isotypes G ^ ,
and
opsonize
implicated in toxin
preventing
produced
connective
M
Immunoglobulin A
Immunoglobulins
or
mammary
in
locally
tissue.
bacterial
by
Most
mammary
plasma
IgG
is
serum-derived, whereas IgA and IgM are primarily of local origin (3,
7).
In
ruminants,
secretions
for
IgG^
all
is
stages
the
of
predominant
lactation
isotype
and
is
in
mammary
transported
selectively from blood to milk in the absence of udder inflammation
(3).
Production
of
IgA
and IgM
by
plasma
cells
creates
a
concentration gradient adjacent to the basement membrane of alveoli
and these Igs are transported in
epithelia and released into
pinocytotic vesicles across mammary
milk (5,16).
Systemic immunization of ruminants has been shown to reduce the
severity
of
mastitis,
but
does
not
prevent
occurrence
of
new
intramammary infection (IMI) (14,17). Failure has been attributed to
the
blood-milk
reaching milk
in
lacteal
enhancing
barrier which may prevent most
(7).
demonstrated
the
Ig
from
Local immunization to elevate Ig concentrations
secretions
udder
circulating
has proven
defense
role
of
to
be a more
mechanisms
plasma
cells
(10,17).
in
effective means
Previous
antibody
of
studies
production
in
115
ruminant mammary glands
function
in
udder
(12,18).
defense
This
against
locally
synthesized Ig may
infection
(9),
but
information is needed concerning the prevalence and Ig
plasma
cells
in mammary
tissues.
Immunocytochemical
basic
isotypes of
methods
were
used previously to demonstrate Ig-producing cells in normal mammary
glands
of
cows
(19),
data
are
However,
no
isotype
concentrations
nonlactating
this
time.
period
Because
and
during
available
in
the
udder
describing
bovine
inflammation
changes
mammary
Ig may
plasma
gland
and in response to bacterial
locally-synthesized
in
(11,18).
cell
during
the
infection during
provide
a
form of
specialized immunological protection of bovine mammary tissue,
this
study examined changes in plasma cell populations during involution
and through parturition
to develop a basis
for
future
local immunostimulation and enhancing Ig production.
attempts
at
116
Materials and Methods
Experimental design
Five Jersey cows from the Hill Farm Research Station were used.
Approximately 5 ml of foremilk were collected aseptically prior to
tissue
collection
and
used
to
evaluate
infection status.
Mammary
tissue samples were obtained by needle biopsy from each quarter at
drying off,
to
7 and 14 days after drying off, and 14 and 7 days prior
parturition.
At
parturition,
animals
were
slaughtered
and
of tissue was obtained from each quarter.
All
3
approximately 1 cm
tissue
samples
ultrastructural
analysis
determine
of
were
processed
examination.
variance
effects
using
for
Data were
the
of wk during
general
included
effect
of
analyzed
linear
involution and
each of the 4 Ig-producing plasma cell
analysis
immunohistochemical
cow,
wk
least
model
squares
procedure
to
infection status
on
isotypes.
during
by
and
The
statistical
involution,
infection
status, and interaction of wk during involution by infection status.
Dependent
variables
were
IgG^-, IgG^-,
IgA-,
plasma cells enumerated per unit tissue area.
and
IgM-producing
The following model
was used:
Y... = p + T. + 3. + x3. . + e.,.
ijk
i
j
ij
ijk
where:
p = effect common to all cows,
l
= fixed effect of ith wk,
3j = fixed
x 3 . . = fixed
ij
effect of jth infection status,
effect due to interaction between ith wk
and jth infection status,
117
e , ,, = random error associated with each observation,
ijk
The
effect
of
square means
cow was
absorbed.
Preplanned
from the overall model were made
comparisons
of
least
by pairwise T-test.
Means were contrasted between status group within a time period and
between
time
period
within
a status
group.
No
other
comparisons
and
presumptive
were made.
Microbiological procedures
Quarter
secretion
samples
were
cultured,
identification of bacterial isolates was as described by Brown (2).
Intramammary
infection
was
defined
as
isolation
of
the
same
microorganism from duplicated foremilk samples.
Tissue preparation
Mammary
tissues
examination were
anesthetized
Columbus,
obtained
locally
OH).
for
with
immunohistochemical
and
2
prepared
ml
of
and
ultrastructural
for microscopy.
2%
Lid-o-cane
Cows were
(Butler,
Co.,
A small puncture was made through the skin with a
scalpel blade above the gland cistern and approximately 3 cm x 2 mm
of
tissue was
Dallas, TX).
site.
For
removed
using a disposable
biopsy needle
(Travenol,
Antibiotics were administered topically at the biopsy
light
microscopy,
biopsy
samples
and
tissue
samples
obtained at slaughter were fixed for 48 h in 10% formalin buffered
118
to pH 7.4 with .025 M phosphate.
sectioning
by
infiltration
Scientific
Products,
McGaw
and
Mammary tissues were prepared for
embedding
Park,
IL).
in
Paraplast
For
electron
(American
microscopy,
biopsy samples and tissue sample obtained at slaughter were fixed in
.1
M
cacodylate
buffered
2.5%
glutaraldehyde,
followed
by
.1
M
cacodylate buffered osmium tetroxide, dehydrated in a graded series
of ethanol, washed in propylene oxide,
and embedded in epoxy resin.
Tissue blocks were sectioned on an MT-5000 ultramicrotome, and 60 run
thick sections stained with 5.0%
uranyl acetate in 50% methanol for
20 min followed by .4% lead citrate for 10 min.
Staining procedure for histochemical analysis
Sections
(2pm
thick)
were
(PAP)
staining
peroxidase-antiperoxidase
Naperville, IL).
with
a
3%
to
primary
hydrogen
suppress
antibody
using
kit (Miles
a
Scientific,
Following deparaffinization, sections were treated
peroxidase activity,
serum
stained
peroxide
solution
and incubated at
nonspecific
(rabbit
to destroy
endogenous
room temperature with normal
protein
binding.
antibovine-IgG^,
IgG 2 »
A
drop
IgA,
of
or
20%
IgM),
provided by Dr. A. Guidry (USDA, Beltsville, MD), was placed on the
section to react with the antigen in a humidity chamber for 30 min.
Free
the
antibody was washed off with
link antibody
(porcine antirabbit
ensure one free binding site.
Tris buffer,
.05 M Tris buffer (pH 7.6), and
incubated for
added
in excess
Sections were washed again
20 min
reagent was removed by washing
IgG) was
in PAP complex,
in Tris buffer.
to
in .05 M
and unbound PAP
Sections were then
incubated for 40 min in a substrate solution (.3% hydrogen peroxide
119
In water and amino-ethyl-carbasole) forming a reddish precipitate at
antigen
sites.
hematoxylin,
Sections
were
rinsed,
counterstained
in
Mayer's
and coverslipped with glycerol-gelatin for microscopic
examination.
Morphometric analysis
Quantitative
Ig-producing
Standard
counted
plasma
18
in
research
10
2
pm /section).
of
plasma
morphometric
cells.
analysis
Sections
microscope
randomly
at
selected
was
were
X
1000
used
observed
and
microscopic
to
enumerate
with
plasma
fields
a
Zeiss
cells
/
(6.0
were
x
10
A
Tissue specimens were examined also for the location
cells
with
respect
ultrastructural characteristics.
to
mammary
epithelium
and
120
Results
Frequencies
of
are in Table 11.
were
bacterial
as
uninfected,
infected
with
the
infected
minor
staphylococci
or
pathogens
Staphylococcus
(
during
sampling
period
Because of the low numbers of isolates, quarters
classified
positive),
isolates
Corynebacterium
(bacteriologically
pathogens
bovis),
aureus,
(coagulase
or
infected
Streptococcus
negative
with
major
uberis.
or
Nocardia).
Plasma
cell numbers/unit area for IgG^,
IgA, and IgM isotypes
across uninfected and infected quarters increased significantly from
D-0
to
D+7,
while
IgG 2 ~producing
significantly
at
D+14
(Table
Immunoglobulin
Gj
cells
remained
numbers
of
IgG 2 _ , IgA-,
significantly
and
Immunoglobulin
G^-
predominant
and
Appendix
elevated
cells
increased
Figures
24a-27a).
through
IgM-producing
plasma
relatively
lower
remained
and
12,
plasma
IgG 2 _producing
plasma
C-0.
At
cells
C-7,
decreased
through
cells
C-0.
were
the
isotypes observed throughout the sampling period, while
IgA- and IgM-producing plasma cells were less abundant.
No
significant
IgA-producing
infected
plasma
quarters
IgM-producing
differences
cells
were
(Appendix
plasma
cells
in
detected
Tables
were
numbers
26a
and
of
IgG^-,
between
27a).
significantly
IgG 2 _ ,
uninfected
Mean
higher
number
in
or
and
of
quarters
infected with minor pathogens compared to all other quarters (Table
13).
Compared
to
uninfected
quarters,
those
infected
with
minor
pathogens had higher numbers of all plasma cell isotypes while those
infected with major pathogens had lower numbers of these cells.
121
Examination
of
immunohistochemically
stained
tissues
demonstrated that the majority of plasma cells were located in the
subepithelial stroma proximal to,
but not directly associated with
the alveolar epithelium (Figures 15 and 16).
observed
lodged
between
lining
alveoli
(Figure
these
cells
revealed
the
15).
basal
Electron
typical
antibody-producing plasma cells.
condensed
chromatin
and
portions
Some plasma cells were
of
epithelial
microscopic
ultrastructure
cells
examination
exhibited
of
by
The nuclei exhibited peripherally
prominent
nucleoli.
The
cytoplasm
was
filled with abundant, parallel arrays of rough endoplasmic reticulum
(RER) which contained an electron-lucent substance (Figure 17).
122
Discussion
This
study demonstrated that the bovine mammary gland has the
cellular machinery available for Ig synthesis during the dry period.
Plasma
cells
were
observed
most
frequently
proximal
to
basal
surfaces of epithelial cells lining alveolar lumina of nonlactating
tissue.
Ultrastructural
typical
morphology
of
examination
of
these
antibody-producing
cells
cells
exhibited
observed
by
the
others
(12) with abundant and dilated RER cisternae containing granular or
flocculent material previously identified as immunoglobulin.
Others
have noted the proximity of parenchymal plasma cells with epithelia
(8,12)
and
suggested
direct
access
for
antibody
transport
through
alveolar cells and into milk with minimal diffusion into surrounding
connective tissue.
increased
Mean number of plasma cells for all Ig classes
significantly
concentrations at C-14,
from
D-0
to
D+7
and
Increasing numbers
D+14,
reaching
peak
of Ig-producing plasma
cells with advancing pregnancy was shown also in the mammary glands
of
rats
(8).
Elevated
concentrations
of
Ig
in
bovine
mammary
secretions were reported during the periparturient period (4).
More
recently,
total IgG in mammary secretion from nonlactating cows was
shown
also
to
Sordillo
et
Ig-producing
reach
al.,
peak
1986,
cells
at
concentrations
unpublished
C-14
reported
2
data).
in
wk
prepartum
Increased
this^ study
(L.
M.
numbers
of
supports
the
contention that the bovine mammary gland produces Ig locally as well
as
the
accumulates
last
2
Ig
wk
proliferation or
of
from serum.
gestation
Enhancing
by
either
Ig concentrations
stimulating
during
plasma
cell
increasing productivity of existing cells may help
123
to
protect
the
Alternatively,
gland
when
susceptibility
to
IMI
is
increased.
because the bovine mammary gland is most susceptible
to IMI during the first 3 wk of involution,
local immunostimulation
of mammary glands at drying off may result
in elevated plasma cell
populations when numbers are normally low.
Yurchak
et
al.
(19)
localize
Ig-producing
tissue.
He
plasma
cells
numbers.
predominant
Numbers of
plasma
found
that
while
the
In
this
used a fluorescent antibody technique to
mammary
other
study,
isotypes
cells
in
lactating
tissue
isotypes
IgG^
and
throughout
the
had
bovine
mainly
were
present
IgG 2
cells
entire
mammary
IgG-producing
only
were
in small
also
nonlactating
the
period.
Ig-producing plasma cells in nonlactating bovine mammary
tissue during colostrogenesis apparently reflect the concentrations
of
Ig
in
secretions
periparturient
Ig/ml
of
period,
of which
IgG
cows
during
colostrum
is
about
this
normally
85%,
time.
contains
IgM about
7%,
During
up
and
to
the
150
mg
IgA about
5%
(3,A). Although most IgG is derived from blood, high concentrations
of IgG cells
in the underlying connective tissue may contribute to
total concentrations in mammary secretion.
Because most IgA and IgM
are
of
produced
secretion
locally
may
be
(3,5),
explained
lower
by
levels
fewer
IgA
these
and
IgM
Ig
in mammary
cells
in
the
underlying connective tissue observed in this study.
Prevalence of some cells may have resulted from local antigenic
stimulation via the teat canal and/or following blast transformation
induced in peripheral lymphoid tissue.
No differences in numbers of
IgG^-,
cells
IgG 2 ",
uninfected
and
or
IgA-producing
infected
plasma
quarters.
These
were
findings
detected between
are
consistent
124
with
those
of
Nickerson
and
Heald
(11)
who
found
no
significant
difference
in numbers of IgG- and IgA-staining plasma cells between
S.
infected
aureus
and
control
quarters.
However,
in
quarters
infected with minor pathogens, numbers of IgM-producing plasma cells
were higher at most sampling times compared to all other quarters.
Minor
pathogens
gland
and
may
are
isolated
produce
frequently
elevated
from
somatic
the
cell
bovine
counts.
mammary
However,
infections caused by minor pathogens go unnoticed often as they only
produce a mild or subclinical form of mastitis.
have shown a marked
infected
study
with
suggests
pathogens
of
plasma
coagulase
cell
response
negative
colonization
of
Boddie et al.
in tissues
staphylococci.
the
streak
by
in previous lactations may have resulted
mammary
lymphocytes
Ig-producing
plasma
and
subsequent
cells.
from quarters
Data
canal
from
these
this
minor
in sensitization
proliferation
Conversely,
(1)
lower
into
numbers
of
Ig-producing plasma cells in quarters infected with major pathogens
suggest
cell
depressed
function.
lymphocyte
Similar
proliferation
findings
have
or
been
compromised
plasma
reported previously by
Nonnecke and Harp (13).
They found depressed lymphocyte response to
mitogenic
in
stimulation
quarters
with
chronic
staphylococcal
mastitis.
Studies
have
shown
that
local
immunization
of
bovine mammary
glands with staphylococcal vaccines provided a substantial degree of
protection
against
Intramammary
induced
infusion
local
lactation
experimental
(9).
of
antigens
production
Most
of
Ig
staphylococcal
challenge
(17).
during
period
sheep
which
the
dry
persisted
locally-produced
Ig
into
following
in
the
ensuing
antigenic
125
stimulation in ruminants was IgA and to a less extent,
IgM (6,7,9).
This study demonstrated higher IgM-producing plasma cell numbers in
quarters
periods
Although
infected
of
with
antigenic
relatively
minor
stimulation
fewer
cell
from
numbers
demonstrated in nonlactating
Ig-producing plasma
pathogens
of
possibly
due
teat
canal
IgA
and IgM
to
longer
colonization.
cells were
bovine mammary glands, numbers of
isotypes
peaked along with
IgG^
these
and
IgG 2
cells approximately 2 wk prior to parturition.
Importance of locally synthesized Ig in mammary defense against
infection
has
Ig-producing
been
plasma
demonstrated
cells
at
D-0
susceptible to IMI at this time.
of
Ig-producing
plasma
cells
(7).
may
Low
render
the
udder
of
more
Alternatively, high concentrations
in
the
bovine
mammary
prepartum should provide a source of immunity
when incidence of new IMI
concentrations
is high.
tissue
2
wk
during lactogenesis
Local antigenic stimulation of
nonlactating bovine mammary glands at D-0 and C-14 may stimulate and
amplify,
respectively,
the
local
antibody
response to
mastitis
pathogens and help protect against new IMI during the nonlactating
and early lactating periods.
126
References
1.
Boddie, R. L . , S. C. Nickerson,
1986.
W.
E. Owens,
and J. L. Watts.
Udder microflora in nonlactating heifers.
Agri-practice
(submitted).
2.
Brown,
R. W . , D. A.
W. D. Schultze.
the
diagnasis
Barn urn, D.
1981.
of
E. Jasper, J. S. McDonald,
Microbiological
bovine
mastitis.
procedures
2nd
Ed.,
and
for use
in
Natl.
Mastitis
the
mammary
Counc., Inc. Arlington VA.
3.
Butler,
J.
E.
secretions.
1974.
Page
Immunoglobulins
217 jui Lactation.
B.
of
L.
Larson
and V.
R.
Smith, ed. Academic Press, New York, NY.
4.
Guidry, A. J., J. E. Butler, R. E. Pearson, and B. T. Weinland.
1980.
IgA,
IgGp
IgM,
throughout lactation.
5.
Larson,
B.
L.,
H.
and BSA in serum and mammary secretion
Vet. Immunol. Immunopathol. 1:329.
L.
Leary,
Immunoglobulin production and
and
J.
transport
E.
by
Devery.
1980.
the mammary gland.
J. Dairy Sci. 63:655.
6.
Lascelles,
mammary
A.
gland
Dairy Sci.
7.
K.
1979.
and
its
The
role
in
the
system
of
control
of
Lascelles, A. K., and G. H. McDowell.
Rev.
the
ruminant
mastitis.
J.
62:154.
immunity with particular
8.
immune
reference to
1974.
Localized humoral
ruminants.
Transplant.
19:170.
Lee, C. G . , P. W.
Ladds,
and D.
L. Watson.
1978.
Immunocyte
populations in the mammary gland of the rat at different stages
of pregnancy and lactation.
Res. Vet. Sci.
24:322.
127
9.
McDowell,
G.
H.,
and
A.
K.
Lascelles.
immunization of ewes with staphylococcal
vaccines.
10.
Res. Vet. Sci.
Nickerson, S. C.
An overview.
11.
Nickerson,
reaction
C.
Nickerson,
and
C.
W.
experimental
S.
C.,
Distribution,
187:41.
Heald.
1982.
Staphylococcus
J. Dairy Sci.
J.
W.
location,
the uninfected,
cell and cell-toxoid
Immune mechanisms of the bovine udder:
bovine mammary tissue.
12.
Local
12:258.
J. Am. Vet. Med. Assoc.
S.
to
1985.
1971.
Pankey,
Cells
aureus
in
local
infection
in
65:105.
and
N.
T,
and ultrastructure
Boddie.
1984.
of plasma cells in
lactating bovine mammary gland.
J. Dairy Res.
51:209.
13.
Nonnecke,
B.
J.,
staphylococcal
lymphocytes.
14.
and
J.
A.
mastitis
on
J. Dairy Sci.
Evaluation
vaccines
of
against
15.
mitogenic
and
Staphylococcus
Sheldrake, R. F., and A. J. Husband.
Watson,
D.
L.
1980.
33:403.
chronic
of
bovine
a
and S. C. Nickerson.
commercial
aureus
bacterin
using
as
mastitis
1985.
Immune defences at
J. Dairy Res. 52:599.
Immunological
gland and its secretion.
Sci.
of
J. Dairy Sci. 68:726.
mucosal surfaces in ruminants.
16.
Effect
responses
J. L. Watts,
protein A
experimental challenge.
1985.
68:3323.
Pankey, J. W . , N. T. Boddie,
1985.
Harp.
functions
Comparative review.
of the mammary
Aust.
J.
Biol.
128
17.
Watson,
D.
L . , and A.
systemic
immunization
antibody
production
K.
Lascelles.
1975.
The
Influence of
during mammary involution on subsequent
in
the
mammary
gland.
Res.
Vet.
Sci.
18:182.
18.
Willoughby,
R.
A.
1966.
immunohistochemical
formation.
19.
Yurchak,
study
of
Am. J. Vet. Res.
A.
M.,
J.
E.
Flourescent localization
the cow.
Bovine
J. Dairy Sci.
the
cellular
mastitis:
sites
of
an
antibody
27:522.
Butler,
of
staphylococci
and
T.
immunoglobulins
54:1324.
B.
Tomasi.
1971.
in the tissues of
129
TABLE 11. Frequency of bacterial isolates from bovine mammary
foremilk samples from drying off through lactogenesis.
Sampling Period*
Organism
D-0
D+7
D+14
C - 14
C-7
C-0
None
%
N
55
11
50
10
45
9
55
11
55
11
50
10
coagulase (-)
staphylococci
%
N
0
0
15
3
20
4
10
2
5
1
5
1
C. bovis
%
N
5
1
0
0
5
1
10
2
5
1
0
0
N
35
7
30
6
25
5
20
4
25
5
30
6
S. uberis
%
N
0
0
0
0
0
0
0
0
5
1
10
2
Nocardia
%
N
5
1
5
1
5
1
5
1
5
1
5
1
S. aureus
7.
*Days relative to drying off (D) or calving (C).
%, Percentage of total quarters within each sampling period.
N, Number of quarters within each sampling period.
130
TABLE 12.
Enumeration^ o£ specific plasma cell classes
(mean
number of cells/6 x 10 pm tissue area) in the bovine mammary gland
from drying off through lactogenesis
in infected and
uninfected quarters.
Sampling Period^
Immunoglobulin
Class
D+7
D-0
D+14
igc1
X
S.E.
7.39C
6.31
26.llb
4.34
IgG2
X
S.E.
19.31b
4.70
24.54b
3.24
IgA
X
S.E.
2.05°
3.31
IgM
X
S.E.
12.26d
3.46
C-14
C-7
C-0
35.72ab
4.45
28.68b
4.94
23.51b
6.30
2.88
32.16a
3.32
28.71b
3.68
28.llb
4.69
21.46b
2.28
23.46b
2.03
31.04a
2.34
21.99b
2.59
20.06b
3.30
20.89c
2.51
29.18b
2.23
37.43a
2.57
28.92b
2.85
26.25bc
3.64
41.23a
3.87
32.89a
*Days relative to drying off (D) and calving (C).
’ * ’ means between
differ (P<.05).
sample
period with different superscripts
131
TABLE 13.
Effect of ^injection status on distribution (mean
number of cells/6 x 10 pm tissue area) of specific plasma cell
classes
in the bovine mammary gland from drying off through
lactogenesis.
Infection Status*
Immunoglobul in
Class
0
INF
MINOR
MAJOR
IgGj
X
S.E.
28.713
2.42
26.84a
2.36
28.47a
5.67
24.14a
2.85
Ib g 7
Z
X
S.E.
28.14a
1.80
26.65a
1.75
29.69a
4.22
24.75a
2.12
X
21.63a
S.E.
1.27
19.623
1.26
20.38a
2.97
18.01a
1.50
23.37b
1.48
34.62a
3.27
19.39b
1.65
IgA
IgM
X
23.47b
S.E.
1.40
Pathogens isolated at time of sampling: 0, uninfected; INF,
infected: MINOR, Corynebacterium bovis and coagulase negative
staphylococci; MAJOR, Staphylococcus aureus and streptococci.
b
’ Means within immunoglobulin class with different superscripts
differ (PC.05).
132
FIGURE
15-16.
Nonlactating
immunohistochemically
for
bovine
presence
of
mammary
tiaaue
immunoglobulin
atained
Gj-producing
plasma cells.
(15)
Positive
subepithelial
staining
stroma
of
(S)
plasma
and
cells
within
the
(arrows)
alveolar
lining of mammary tissue involuted for 7 days.
located
in
the
epithelial
(E)
L, lumen X 1,460.
(16) Clusters of positive staining plasma cells (arrows) within the
stromal (S) area of mammary parenchyma obtained at parturition.
epithelium
X 650.
E,
/* '
/V '
* $►*
•♦•.’ ir?
*
•
•>
*
i• '
*
■
'fl.
’■ • ■ v
N
.
t -
«
V>
10 um
«T
»,
«*
''
' Vi r?; '
"<r>
.v
y
10 )jm
134
FIGURE
17.
lamellae
Clusters
of
rough
of
endoplasmic
electron-lucent material
6,320.
plasma
cells
exhibiting abundant
reticulum
(arrowheads).
containing
N, nucleus
parallel
granulated,
and S,
stroma X
135
vyy*-vr.
C H A P T E R
V
SUMMARY AND CONCLUSIONS
Biochemical
glands
from
and
D-0
morphological
through
lactogenesis
events which occur during
milk
synthesis
and
throughout
analyzed
to
counts,
Lf,
transition
secretion.
collected
composition
the
percentages
citrate,
clarified
Quarter
for
IgG,
and
changes
the
the
and
status.
in
of fat and protein,
and
bovine
foremilk
period
infection
analyzed
of
mammary
physiological
to or from a state of active
sampling
determine
was
examination
were
bacteriologically
Mammary
SCC,
pH,
samples
secretion
differential
cell
concentrations of BSA,
citrate
to
Lf
molar
ratio.
Concentrations of a-lactalbumin in blood serum was determined also.
Quarter
cows
biopsies
beginning
were
at
taken at weekly
D-0
through
C-0.
intervals
from 5 additional
Histological
and
cytological
parameters evaluated were the characterization of parenchymal tissue
area;
classification
epithelium;
and
frequency and
ultrastructural
location of
changes
infiltrating
of
mammary
leukocytes;
and
immunocytochemical quantification and classification of Ig-producing
plasma cells.
Prior
biochemical
structural
involution
to
this
changes
changes
and
study,
in
in
no data were available which correlated
secretion
mammary
and
tissue
lactogenesis.
serum
during
Changes
136
in
composition
bovine
the
with
mammary
biochemical
137
composition of mammary secretion during the first 2 wk of involution
included
decreased
citrate,
and
decreased
concentrations
the
citrate
synthetic
to
Lf
ability
involution progressed.
of normal
of
molar
milk
components
ratio),
mammary
which
epithelial
(fat,
indicate
cells
as
The concomitant increase in parameters which
indicate the loss of tight junction permeability (SCC, BSA, Lf, IgG,
pH,
and
the
total protein)
influx
barrier.
of
serum
As C-0
composition
were
reflect the loss of cellular
derived
components
through
integrity and
the
blood-milk
approached, reverse trends in secretion and serum
observed.
At
about
the
last
wk
of
gestation,
decreases in SCC, total protein, BSA, Lf, and IgG with a concomitant
increase in fat,
levels
of
citrate,
the citrate to Lf molar ratio, and serum
a-lactalbumin
reflected
the
cellular
redevelopment
of
mammary tissue and the onset of milk synthesis and secretion.
Changes in mammary structure as involution progressed coincided
with
altered
concentration
Histological
tissue
during
secretion
composition
associated
and
with
the
ultrastructural
the
first
wk
and
of
serum
a-lactalbumin
cessation
changes
in
involution
of
lactation.
mammary
parenchymal
indicated
a
gradual
reduction in synthetic and secretory activity of alveolar epithelium
also.
in
Changes at the
stroma
and nonactive
secretory epithelium,
regression
light microscopic
of
level included an
epithelium with a concomitant decrease
lumen, and fully active cells.
epithelial
increase
cells
also was
evident
by
in
A progressive
the
decreased
numbers of cytoplasmic organelles and increased prevalence of stasis
vacuoles at the electron microscopic level.
138
Approximately
Beginning
2
prepartum,
wk
cell
structure
gradually assumed the morphology typically found in lactating bovine
mammary glands with
secretory cells,
increased
secretory
epithelial
cells
changes
in
lumen,
and
fully active
but accompanied by decreased amounts of stroma and
nonactive
cytoplasmic
epithelium,
cells.
A
was
apparent
also
organelles
secretion
and
progressive
by
secretory
composition
and
the
differentiation
increased
vesicles.
of
numbers
These
tissue morphology
of
transient
throughout
the non-lactating period provide an accurate indicator of secretory
cell
involution
lactogenesis.
functional
and
subsequent
Results
transition
of
this
from
occurs during the first and
development
study
lactation
last
support
to
during
the
bovine
contention
involution
that
and vice versa
2 wk of the nonlactating period,
respectively.
The
non-lactating
period
has
been
shown
to
be
critically
related to the incidence of new intramammary infection (2,4,6,7,12).
Most new infections occur during the first
2 wk of
involution and
from 1 wk prior to through 1 wk after parturition (2,6,20).
affecting susceptibility and resistance to mastitis
period have not been studied extensively.
study
has
pathogens
allowed
on
the
some
speculation
process
of
as
mammary
Factors
during the dry
Observations made in this
to
the
affect
involution
of
as
mastitis
well
as
identifying specific factors which may be responsible for decreased
resistance to infection during this critical time.
The presence of mastitis-causing organisms
early
in nonlactating and
lactating glands was associated with alterations in secretion
composition and tissue morphology when compared to normal uninfected
139
glands.
Total
infiltrating
from
SCC
In
mammary
mastitic
secretion
parenchymal
quarters
and
mean
tissues
compared
to
number
were
of
leukocytes
consistantly
uninfected
quarters.
higher
Elevated
leukocyte numbers, especially PMN, are indicative of an inflammatory
reaction,
which may have resulted from release of bacterial toxins
(9,14,16,17).
Differential
morphometric
analysis
higher
numbers
glands.
An
digest
of
important
bacteria
ability
of
compromised
PMN
and
PMN
to
in
the
cell
counts
of
mammary
pseudopodia
mammary tissue
revealed significantly
in
infected glands
compared to
function
thereby
of mammary PMN
reduce
phagocytize
is
bacterial
mastitis
nonlactating
frequently
and
exhibited
and cellular debris.
and
tissues
of
and
of
gland
causing
due
become
to
degranulated.
cytoplasmic
vacuoles
uninfected
to
numbers
engulf
and
(14).
The
organisms
the
ingestion of accumulated fat and casein (13,22);
lose
secretion
may
indiscriminate
consequently,
In
be
this study,
containing
fat,
they
PMN
casein,
Although PMN were prevalent in both secretions
infected
quarters,
their
role
in
fluid
resorption
during involution may have rendered them less efficient in defending
the gland against bacterial infection.
Immunoglobulins
present
in
mammary
secretions
defend
the
mammary gland through opsonization and formation of antibody-antigen
complexes
in conjunction with complement
(3,9).
Neutralization of
toxins and promotion of inflammation are other important biological
functions
of
mammary
Ig
(1,9,11).
Immunoglobulins
in
lacteal
secretions are either of humoral origin or are produced locally by
plasma cells in the sub~mucosa (8).
specific
plasma
cell
isotypes
This study described change's in
in
mammary
tissue
during
the
140
nonlactating
period
Concentrations
of
and
IgG
in
in
response
secretion
to
were
bacterial
often
quarters compared to uninfected quarters.
lower
Numbers
infection.
in
infected
of Ig-producing
plasma cells in mammary tissue were also lower in quarters infected
with
major
pathogens
compared
to
uninfected
quarters.
These
findings suggest that mastitis caused by major pathogens may depress
lymphocyte
reported
proliferation
previously
opsonizing
Ig
in
or
by
compromise
others
secretions
(10).
from
plasma
cell
Lower
quarters
function
as
concentrations
of
infected
with
major
pathogens may also be responsible for inferior performance of PMN in
mastitic secretion and tissue during the nonlactating period.
Recently, the ability of IgG 2 » IgM, and IgA to enhance in vitro
phagocytosis
by
demonstrated
that
study,
quarters
mammary
PMN
IgM
the
was
infected
with
were
evaluated
(5).
Results
most
important
opsonin.
minor
pathogens
had
In
this
significantly
higher numbers of IgM-producing plasma cells at most sampling times
compared
streak
to
canal
resulted
in
all
other
quarters.
by
minor
pathogens
sensitization
proliferation
antibodies
control
can
into
are
in
lactations
lymphocytes
plasma
opsonins,
by exploiting
colonization
previous
mammary
Ig-producing
important
be made
of
Prolonged
perhaps
cells.
may
the
have
subsequent
Because
progress
the mammary
synthesize IgM via local stimulation.
and
of
gland's
in
IgM
mastitis
ability
Mean number of plasma
to
cells
in mammary tissue for all Ig classes reached peak concentrations at
C-14.
Stimulating
plasma
cell
productivity
at
this
time
could
elevate opsonizing levels of Ig in secretion and enhance protection
of the gland during times of increased susceptibility to IMI.
141
Lactoferrin
wheys
of
la
bovine
restricted
by
a
nonspecific
mammary
the
antimicrobial
secretion
ability
of
(19).
bacteria
to
protaln
Bacterial
compete
protein-bound iron in mammary secretion (15).
found
growth
with
In
is
Lf
for
Concentrations of Lf
increase up to 100-fold in secretions of fully involuted glands and
at times,
concentrations may equal or exceed that of IgG (19).
has been shown also that Lf,
provided
a
powerful
It
in conjunction with specific antibody,
antimicrobial
environment
(18,21).
In
this
study, Lf concentrations in mammary secretion from infected quarters
were significantly lower compared to uninfected quarters.
quarters
often
had
uninfected quarters.
lower
concentrations
of
IgG
Infected
compared
to
Lower levels of these antibacterial components
in secretion may have reduced the natural defense potential of the
gland, allowing colonization of mastitis-causing organisms.
These data provide information regarding the quantification and
distribution
during
gland
the nonlactating
is
defense
levels
of essential
protected
by
mechanisms,
of
these
components
period.
a
the
manipulate
the
defense
pathogenic
bacteria
may
system
presence
of
of
mammary
to
primary
bacterial
Additional
potential
lead
the
immune
system
Although the nonlactating mammary
complex
components.
of
the
gland
an
effective
program focused on the nonlactating dairy herd.
secondary
infection
research
of
and
in
which
altered
could
response
mastitis
to
control
142
References
1.
Colditz,
I.
G.,
and
D.
L.
Watson. 1985.
The
immunophysiological basis for vaccinating ruminants against
mastitis.
Aust. Vet. J.
62:145.
2.
Dodd, F.
Strategy
170:1124.
3.
Howard, L. J., G. Taylor, and J. Brownlie.
1980.
Surface
receptors
for
immunoglobulin
on
bovine
polymorphonuclear
neutrophils and macrophages.
Res. Vet. Sci.
29:128.
4.
Jain, N. C.
1979.
Common mammary pathogens
infection and mastitis.
J. Dairy Sci. 62:128.
5.
Miller, R. H . , A, J. Guidry, M. J. Paape, A. M. Dulin, and L.
A.
Fulton.
1986.
Relationship
between
immunoglobulin
concentrations
in
milk
and
phagocytosis
by
bovine
polymorphonuclear leukocytes.
Vet. Immunol. Immunopathol. (In
Press).
6.
Neave, F.
K., F.
H. Dodd, and E. Henriques. 1950.
infections in the "dry period".
J. Dairy Res.
17:37.
7.
Neave, F.
K., J. Oliver, F. H. Dodd, and T. M. Higgs.
1968.
Rate of infection of milked and unmilked udders. J. Dairy Res.
35:127.
8.
Newby, T. J., and J. Bourne.
1977.
The nature of the local
immune system of the bovine mammary gland.
J.
Immunol.
118:461.
9.
Nickerson, S. C.
1985.
Immune mechanisms of the bovine udder:
An overview.
J. Am. Vet. Med. Assoc.
187:41.
H., D. R. Westgarth,
of
mastitis
control.
and T. F. Griffin.
J.
Am.
Vet.
Med.
and
1977.
Assoc.
factors
in
Udder
10.
Nonnecke,
B. J.,and K. L. Smith.
1984.
Biochemical and
antibacterial properties of bovine mammary secretion during
mammary involution and at parturition.
J. Dairy Sci. 67:2863.
11.
Norcross, N. L.
1977.
Immune response of the mammary gland
and role of immunization in mastitis control.
J. Am. Vet. Med.
Assoc. 170:1228.
12.
Oliver, S. P., and B, A. Mitchell.
1983.
Susceptibility of
bovine mammary glands to infections during the dry period.
J.
Dairy Sci.
66:1162.
143
13.
Paape, M. J., and A. J. Guidry.
1977.
Effect of fat and
casein on intracellular killing of Staphylococcus aureus by
milk leukocytes.
Proc. Soc. Exp. Biol. Med. 155:588.
14.
Paape,
M.
Leukocytes
pathogens.
15.
Reiter, B. , and A. J. Bramley.
1975.
Defense mechanisms of
the udder and their relevence to mastitis control. Page 210 in
Proc. Int. Dairy Fed. Seminar on Mastitis Control.
Brussels,
Belgium.
16.
Schalm,
0.
W.,
J. Lasmanis,
and
E.
J. Carroll.
1966.
Significance of leukocytic
infiltration
into the milk in
experimental Streptococcus agalactiae mastitis in cattle.
Am.
J. Vet. Res. 27:1537.
17.
Schalm,
0.
M . , J. Lasmanis,
and
E.
J. Carroll.
Experimental Pseudomonas aeruginosa mastitis in cattle.
Vet. Res.
28:697.
1967.
Am. J.
18.
Smith,
K.
L . , H.
R.
Conrad,
and R.
Lactoferrin and IgG immunoglobulins from
mammary glands.
J. Dairy Sci.
54:1427.
1971.
bovine
19.
Smith, K. L., and S. P. Oliver.
1981.
Lactoferrin:
A
component of nonspecific defense of the involuting bovine
mammary gland.
Page 535 in The ruminant immune system.
J. E.
Butler, ed. Plenum Press, New York, N. Y.
20.
Smith, K. L., S. P. Oliver, and P. S. Schoenberger.
1979.
Effect of endotoxin infusion on bovine mammary gland involution
and resistance to infection during the early dry period.
J.
Dairy Sci.
62 (Suppl. 1):124. (Abstr.)
21.
Smith, K. L . , and F. L. Schanbacher.
1977.
Lactoferrin as a
factor of resistance to infection of the bovine mammary gland.
J. Am. Vet. Med. Assoc.
170:1224.
22.
Wergin,
W.
P.,
and M.
J.
Paape.
1977.
Structure
of
polymorphonuclear leukocytes (PMN) isolated from bovine blood
and milk.
J. Cell Biol. 75:102.
J . , W.
P. Wergin,
and A.
J.
Guidry.
1979.
- second line of defense against invading mastitis
J. Dairy Sci. 62:135.
M.
Porter.
involuted
APPENDIX
144
145
TABLE la.
Sources
of variation,
degrees of freedom, and mean
squares for numbers of total somatic cells and differential cell
counts in bovine mammary secretion from uninfected quarters and
those infected with minor and major pathogens.
Source of
Variation2
Variable1 Mean Square
LYM
MAC
df3
SCC
Week
9
67.37***
157.03
711.60***
1393.00***
Status
2
74.03***
104.42
774.70***
972.91***
18
35.17***
73.59
120.46
218.13
116.27(681)
160.46(681)
Week*Status
Error (df)
8.04(870)100.96(681)
PMN
V a r i a b l e s were: SCC, total somatic cell counts; Mac, macrophages;
LYM, lymphocytes; and PMN, polymorphonuclear
leukocytes.
V a r i a t i o n s were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
***P<.002.
146
TABLE 2a.
Sources of variation,
degrees of freedom,
and mean
squares for numbers of total somatic cells and differential cell
counts
in bovine mammary secretion from uninfected and infected
quarters.
Source of
Variation2
dfJ SCC
Variable1 Mean Squar e
PMN
LYM
MAC
Week
9
72.72***
238.96**
1074.93***
2013.01***
Status
1
63.26**
120.92
1114.64**
1925.69***
Week*Status
9
39.59***
Error (df)
8.36(823)
45.60
101.11(691)
130.11
216.85
116.43(691) 160.99(692)
^Variables were:
SCC, total somatic cell counts; MAC, macrophages;
LYM, lymphocytes; and PMN, polymorphonuclear leukocytes.
2Variations were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
* *P<.0l.
***P<.001.
147
TABLE 3a.
Sources
of
variation,
degrees of freedom, and mean
squares for concentrations of lactoferrin and citrate in bovine
mammary secretion from uninfected quarters and those infected with
minor and major pathogens.
Source of
Variation2
df a
Variable1 Mean Square
LF
C1T
Week
9
2750.3***
Status
2
281.59*
0.01
18
100.31
0.26
Week*Status
Error (df)
72.37(791)
8.31***
0.11(801)
V a r i a b l e s were: I.F, lactoferrin and CIT, citrate.
V a r i a t i o n s were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
^Degrees of freedom.
*P<.05.
***P<.001.
148
TABLF, 4a.
Sources of variation, degrees of freedom, and mean
squares for concentrations of lactoferrin and citrate in bovine
mammary secretion from uninfected and infected quarters.
Source of
Variation-*
df 3
Week
9
4175.83***
Status
1
303.19*
0.24
Week*Status
9
74.55
0.16
73.02(801)
0.11(811)
Error (df)
Variable1 Mean Square
LF
CIT
11.91***
‘Variables were: Lf, lactoferrin and CIT, citrate.
^Variations were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
^Degrees of freedom.
* P < .05.
* * * P < .001
149
TABLF. 5a.
Sources of variation, degrees of freedom, and mean
squares
for percent fat and total protein in bovine mammary
secretion from uninfected quarters and those infected with minor and
major pathogens.
Source of
Variation1
Week
Status
Week*Status
Error (df)
df2
9
2
18
Variable Mean Square
Fat
71.50***
Protein
243.04***
3.69
49.02***
9.63*
24.58***
5.94(485)
5.13(483)
V a r i a t i o n s were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
2I)egrees of freedom.
* P < .05.
* * * P < .001.
150
TABLE 6a.
Sources of variation, degrees of
squares
for percent fat and total protein
secretion from uninfected and infected quarters.
Variable Mean Square
Protein
Fat
Source of
Variation1
df2
Week
9
Status
1
2.00
Week*Status
9
11.88*
Error (df)
freedom, and mean
in bovine mammary
85.92***
5.99(493)
311.32***
18.39
38.85***
5.40(491)
1Variations were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
^Degrees of freedom.
*P<.05.
***P<.001.
151
TABLE 7a.
Sources of variation, degrees of freedom, and mean
squares for concentration of immunoglobulin G in bovine mammary
secretion from uninfected quarters and those infected with minor and
major pathogens.
Source of
Variation1
df*
Week
8
Status
2
Week*Status
Error
16
407
Mean Square
5368.42***
59.07
1191.35***
241.04
V a r i a t i o n s were: Week, w e e k of the sampling period; Status, quarter
infection status; and week * status, interaction between w e e k of
sampling period and quarter infections status.
^Degrees of freedom.
***P<.001
152
TABLE 8a.
Sources of variation, degrees of freedom, and mean
squares for concentration of immunoglobulin G in bovine mammary
secretion from uninfected and infected quarters.
Source of
Variation1
df2
Week
8
Status
1
165.36
Week*Status
8
919.67***
Error
416
Mean Square
6684.88***
264.06
1Variations were: Week, we e k of the s a mpling period; Status, quarter
infection status; and week * status, interaction between w e e k of
s ampling period and quarter infections status.
2Degrees of freedom.
***P<.001.
153
TABLE 9a.
Sources of variation, degrees of freedom, and mean
squares for pH and concentrations of bovine serum albumin and
a-lactalbumin in bovine mammary secretion from uninfected quarters
and those infected with minor and major pathogens.
Source of
Variation2
df3
pH
Week
9
2.14***
Status
?.
0.95***
Week*Status
18
Error (df)
V a r i a b l e s were:
Variable1 Mean Square
BSA
316.57***
15.75
LAC
764.33***
1003.04***
0.10**
7.23
223.11*
0.05(810)
9.30(801)
140.64(759)
BSA, bovine serum albumin and LAC, a“ lactalbumin.
^Variations were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
*P<.05.
* * P < .01.
***P<.001.
154
TABLE 10a.
Sources of variation, degrees of freedom, and mean
squares for pH and concentrations of bovine serum albumin and
a-lactalbumin in bovine mammary secretion from uninfected and
infected quarters.
Source of
Variation^
dfJ
Week
9
4.22***
Status
1
1.50***
Week*Status
9
0.11*
6.69
189.07
0.05(820)
9.30(811)
142.31(769)
Error (df)
V a r i a b l e s were:
pH
Variable1 Mean Square
BSA
471.01***
32.36
LAC
1085.69***
2262.31***
BSA, bovine serum albumin and LAC, a-lactalbumin.
2Variations were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
■^Degrees of freedom.
* P < .05.
* * * P < .001.
155
Sources of variation, degrees of freedom, and
mean
TABLE 11a.
squares for concentrations of a-lactalbumin in bovine blood sera.
Source of
Variation1
Animal
Week
Error
df2
27
9
149
Mean Square
382066.93
2562106.10***
518536.98
V a r i a t i o n s were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
^Degrees of freedom.
***F<.001.
156
TABLE 12a.
Sources of variation, degrees of freedom, and mean
squares for numbers of infiltrating leukocytes within the epithelial
lining of bovine mammary tissue from uninfected quarters and those
infected with minor and major pathogens.
Source of
Variation2
PMN
Variable1 Mean Square
MAC
df3
LYM
Week
5
121.44
1150.53
Status
2
46.47
977.14
366.14***
Week*Status
10
101.16
457.95
65.80**
Error
94
69.34
524.47
28.12
63.43*
V a r i a b l e s were:
MAC, macrophages; LYM, lymphocytes; and PMN,
polymorphonuclear leukocytes.
V a r i a t i o n s were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
* P < .05.
**P<.01.
***P<.001.
157
TABLE 13a.
Sources of variation, degrees of freedom, and mean
squares for numbers of Infiltrating leukocytes within the epithelial
lining of bovine mammary tissue from uninfected and infected
quarters.
Source of
Variation2
df3
We ek
5
Status
1
26.08
276.34
Week*Status
5
122.62
340.05
76.30*
100
69.33
536.11
30.76
Error
MAC
Variable1 Mean Square
LYM
PMN
494.09***
1936.42**
133.04***
536.91***
1Variables were: MAC, macrophages; LYM, lymphocytes; and PMN,
polymorphonuclear leukocytes.
2Variations were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
*P<.05.
**P<.01.
***p<.om.
158
TABLE 14a.
Sources of variation, degrees of freedom, and mean
squares for numbers of infiltrating leukocytes within alveolar
lumina of bovine mammary tissue from uninfected quarters and those
infected with minor and major pathogens.
Source of
Variation2
df3
Week
5
168.56*
2.03*
Status
2
790.95***
0.89
1573.77***
167.16**
1.03
375.65***
0.79
122.43
Week*Status
10
Error
94
MAC
67.85
Variable1 Mean Square
LYM
PMN
385.84**
MAC, macrophages; LYM, lymphocytes; and PMN,
V a r i a b l e s were:
polymorphonuclear leukocytes.
2Variations were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
*P<.05.
**P<.01.
***P<.001.
159
TABLE 15a.
Sources of variation, degrees of freedom, and mean
squares for numbers of Infiltrating leukocytes within alveolar
lumina of bovine mammary tissue from uninfected and infected
quarters.
Source of
Variation2
df
MAC
Variable1 Mean Square
LYM
PMN
Week
5
583.40***
0.30***
Status
1
995.36***
0.64
2486.67***
Week*Status
5
124.35
0.71
546.42***
100
80.76
0.83
136.20
Error
962.59***
^Variables were: MAC, macrophages; LYM, lymphocytes; and PMN,
polymorphonuclear leukocytes.
2Variations were: Week, week of the sampling period; Status, quarter
Infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
* * * P < .001.
160
TABLE 16a.
Sources of variation, degrees of freedom, and mean
squares
for
numbers
of
infiltrating
leukocytes
within
the
subepithelial stroma of bovine mammary tissue from uninfected
quarters and those infected with minor and major pathogens.
Source of
Variation2
df^
Week
5
Status
2
44.68
284.43
Week*Status
10
16.48
506.55
696.82
198.39*
Error
94
41.06
284.82
438.98
103.07
MAST
141.71**
Variable1 Mean Square
PLMS
MAC
LYM
1422.21***
839.99
1864.48**
PMN
450.08*** 139.32
21.51
889.39***
238.37**
78.01
V a r i a b l e s were: MAST, mast cells; PLMS, plasma cells; MAC,
macrophages; LYM, lymphocytes; and PMN, polymorphonuclear leukocytes.
2Variations were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
* P < .05.
**P<.01.
* * * P < .001.
161
TABLE 17a.
Sources of variation, degrees of freedom, and mean
squares
for
numbers
of
infiltrating
leukocytes
within
the
subepithelial stroma of bovine mammary tissue from uninfected and
infected quarters.
Variable1 Mean Square
LYM
PLMS
MAC
Source of
Variation2
dfJ
Week
5 231.89*** 1654.16*** 1757.46** 765.04***
Status
1
69.26
440.26
416.29
12.00
1690.03***
Week*Status
5
7.25
530.31
561.62
164.25
1690.03***
100
39.98
294.90
479.14
108.72
364.53***
Error
MAST
PMN
354.88***
V a r i a b l e s were: MAST, mast cells; PLMS, plasma cells; MAC,
macrophages; LMN, lymphocytes; and PMN, polymorphonuclear leukocytes.
V a r i a t i o n s were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
**P<.01.
***P<.001.
162
TABLE 18a.
Sources of variation, degrees of freedom, and mean
squares for percent tissue area composed of epithelium, lumen, and
stroma in bovine mammary glands from uninfected quarters and those
infected with minor and major pathogens.
Variable Mean Square
Epithelium
Lumen
Source of
Variation1
df?-
Week
5
8904.60**
12556.84
Status
2
5047.43
36772.58**
Week*Status
10
4592.32
5846.47
2984.80
Error
94
2674.46
6134.37
5139.48
Stroma
3942.19***
37304.16***
^Variations were: Week, w e e k of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
ZDegrees of freedom.
**P<.01.
***P<.001.
163
TABLE 19a.
Sources of variation, degrees of freedom, and mean
squares for percent tissue area composed of epithelium, lumen, and
stroma in uninfected and infected bovine mammary glands.
Source of
Variation1
df 2
Variable Mean Square
Epithelium
Lumen
Week
5
4057,46
Status
1
169.56
Week*StatuB
5
Error
100
Stroma
45268.69***
75620.65***
51189.2.5**
68088.89***
5731.9
3029.16
2347.81
2716.27.
6220.97
5014.37
V a r i a t i o n s were: Week, week of the sampling period; Status, quarter
infection status; and week * Btatus, interaction between week of
sampling period and quarter infections status.
20egrees of freedom.
**P<.01.
***P<.001.
164
TABLE 20a.
Sources of variations, degrees of freedom, and mean
squares for percent tissue area composed of nonactive, moderately
a cti v e , and fully active secretory epithelium in bovine mammary
glands from uninfected quarters and those infected with minor and
major pathogens.
Source of
Variation2
df3
NON
Variable Mean Squares1
FULLY
MOD
Week
5
15.21***
Status
2
16.32**
4.91
18.73***
12.05*
2.90
Week*Status
10
2.85
1.74
2.95
Error
94
2.61
3.18
3.42
1Variables
were:
NON,
nonactive
secretory
epithelium;
NOD,
moderately active secretory epithelium; and FULLY, fully active
secretory epithelium.
V a r i a t i o n s were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
*3
Degrees of freedom.
*P<.05.
* * P < .01.
***P<.001.
165
TABLE 21a.
Sources of variation, degrees of freedom, and mean
squares for percent tissue area composed of nonactive, moderately
active, and fully active secretory epithelium in bovine mammary
glands from uninfected and infected quarters.
Source of
Variation2
df3
Variable Mean Square1
MOD
FULLY
NON
Week
5
35.24***
6.27
Status
1
23.08***
15.45*
Week*Status
5
1.72
1.65
3.04
100
2.66
3.15
3.39
CM
CO
*
Error
48.74***
Variables
were:
NON,
nonactive
secretory
epithelium;
MOD,
moderately active secretory epithelium; and FULLY, fully active
secretory epithelium.
2Variations were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
3Degrees of freedom.
* P < .05.
***p< .001 .
166
TABLE 22a.
squares for
epithelium,
quarters and
Sources of variation, degrees of freedom, and mean
numbers of corpora amylacea found within the lumen,
and stroma of bovine mammary tissue from uninfected
those infected with minor and major pathogens.
Source of
Variation1
df2
Week
5
Status
2
Week*Status
Error
Variable Mean Square
Epithelium
Lumen
Stroma
10.05**
.15
.09
7.00
.39
.05
10
2.64
.10
.26
94
2.56
.15
.56
1Variations were: Week, w e e k of the sampling period; Status, quarter
infection status; and w e e k * status, interaction between w e e k of
sampling period and quarter infections status.
2Degrees of freedom.
**F<.0I.
167
TABLE 23a.
Sources of variation, degrees of freedom, and mean
squares for numbers of corpora amylacea found within the lumen,
epithelium, and stroma of bovine mammary tissue from uninfected and
infected quarters.
Source of
Variation1
df2
Variable Mean Square
Epithelium
Lumen
Stroma
Week
5
12.33***
.09
.33
Status
1
10.49*
.10
.01
Week*Status
5
2.37
.13
.49
100
2.56
.15
.53
Error
V a r i a t i o n s were: Week, week of the sampling period; Status, quarter
infection status; and week * status, interaction between week of
sampling period and quarter infections status.
^Degrees of freedom.
*P<-05.
***P<.001.
TABLE 24a.
Sources of variation, degrees of freedom, and mean squares for percent alveolar cell area
composed of nucleus, cytoplasm, rough endoplasmic reticulum, Golgi, mitochondria, fat, secretory
vesicles, and stasis vacuoles in bovine mammary tissue from uninfected quarters and those infected with
minor and major pathogens.
Source of
Variation2
df 3
NUC
CYT
Week
5
890.63***
Status
2
161.08*
75.68
RER
Variable Mean Square 1
GOL
MIT
FAT
SEC
STA
191.86*** 520.40*** 373.98*** 154.16*** 191.20*** 58.07*** 10.69
117.37**
17.90
10.44
13.52
27.22
2.42
Week*Status
10
35.62
30.33
29.04
26.35*
9.91
29.72
7.52
6.99
Error
98
37.69
33.19
24.22
10.78
10.67
28.79
9.13
8.90
Variables were: NUC, nucleus; CYT, cytoplasm; RER, rough endoplasmic reticulum;
mitochondria; FAT, fat droplets; SEC, secretory vesicles; and STA, stasis vacuoles.
Variations were: Week, week of the sampling period; Status, quarter infection
status, interaction between week of sampling period and quarter infections status.
GOL,
status;
Golgi;
MIT,
and week *
^Degrees of freedom.
*P<.05.
**P<.01.
***P<.001.
168
TABLE 25a.
Sources of variation, degrees of freedom, and mean squares for percent alveolar cell area
composed of nucleus, cytoplasm, rough endoplasmic reticulum, Golgi, mitochondria, fat, secretory
vesicles, and stasis vacuoles in bovine mammary tissue from uninfected and infected quarters.
Source of
Variation^
df3
NUC
CYT
RER
Variable Mean Square1
GOL
MIT
FAT
STA
SEC
1320.44*** 210.54*** 734.66*** 571.02*** 262.95*** 475.49*** 141.34*** 25.26*
Week
5
Status
1
235.01*
160.68*
119.65*
19.25
25.70
.02
Week*Status
5
41.73
33.59
39.10
12.42
10.14
53.77
9.86
13.40
104
38.48
32.73
24.84
12.29
10.53
27.64
8.90
8.54
Error
54.26*
^Variables were: NUC, nucleus; CYT, cytoplasm; RER, rough endoplasmic reticulum;
mitochondria; FAT, fat droplets; SEC, secretory vesicles; and STA, stasis vacuoles.
2Variations were: Week, week of the sampling period; Status, quarter infection
status, interaction between week of sampling period and quarter infections status.
GOL,
1.23
Golgi;
status;
MIT,
and week *
3Degrees of freedom.
*P<.05.
***P<.001.
'
169
170
TABLE 26a.
Sources of variation, degrees of freedom, and mean
squares for percent tissue area composed of immunoglobulin G j , G2 ,
A, and M plasma cells in bovine mammary tiewuo from uninfacted
quarters and those infected with minor and major pathogens.
Source of
Variation1
df2
Week
5
Status
2
Week*Status
Error
IgGi
Variable Mean Square
IgA
. I£G2
Ir M
282.50
825.21***
775.71***
216.07
152.27
125.69
846.59***
10
354.78
132.94
125.59
123.63
93
285.96
158.69
78.69
95.35
1413.41***
V a r i a t i o n s were: Week, we e k of the sampling period; Status, quarter
infection status; and w e e k * status, interaction between w e e k of
s ampling period and quarter infections status.
2 Degrees of freedom.
***P<.00I.
171
TABLE 27a.
Sources of variation, degrees of freedom, and mean
squares for percent tissue area composed of immunoglobulin G j , G2 ,
A, and M plasma cells in bovine mammary tissue from uninfected and
infected quarters.
Variable Mean Square
IgA
IgG-,
Source of
Variation1
df 2
Week
5
Status
1
61.29
49.49
82.41
2.00
Week*Status
5
450.07
151.96
124.13
121.92
99
286.14
156.49
82.09
112.34
Error
IgG,
2512.55***
IgM
797.20*** 1728.51*** 1770.03***
1Variations were: Week, w e e k of the sampling period; Status, quarter
infection status; and w e e k * status, interaction between w e e k of
sampling period and quarter infections statUB.
2 Degrees of freedom.
***P<.00I.
FIGURE la
status.
Changes in percent monocytes in bovine mammary secretion by week of involution and infection
50r
45
40
35
I
30
25*— 1
D-0
0+7
0+14
UNINFECTED
□
MAJOR
D+21
D+28
C-14
TIME
C-7
MINOR
C-0
C+7
C+14
FIGURE
2a.
Changes
in
percent
lymphocytes
in
bovine
mammary
secretion
by week
of
infection status.
50
I
40
cn
Ui
r—
V
a
X
a
x
>_i
«
30
I
In
CJ
20
10
0*— 1
D-0
■ ■
l~
0+7
P+14
UNINFECTED
1 MAJOR
D+21
D+28
C-14
TIME
C-7
MINOR
C-0
C+7
C+14
involution
FIGURE 3a.
Changes in percent
involution and infection status.
polymorphonuclear
leukocytes
in bovine
mammary
secretion by week of
60
50 -
40
CD
X
a.
o
CT
X
LjJ
2
S«
30
I
20
10
Ql-J
D-0
D+7
D+14
I B
UNINFECTED
□
MAJOR
□+21
D+28
C-14
TIME
C-7
MINOR
C-0
C+7
C+14
FIGURE 4a.
Changes in concentrations of lactoferrin in bovine mammary secretion by week of involution
and infection statue.
40
LACTOFERRIN
(MG/ML)
30
20
10
0-0
0+7
0+14
0+21
D+28
C-14
C-7
TIM
E
UNINFECTED
I
I MAJOR
MINOR
C-0
C+7
■ffl
C+14
FIGURE 5a.
Changes in concentrations
of citrate
in bovine mammary secretion by we e k of Involution and
infection status.
C3
z
UJ
<
IX
D-0
D+7
D+14
0+21
D+2S
C-14
C-7
TIME
■ ■
UNINFECTED
□
MAJOR
MINOR
C-0
C+7
C+14
FIGURE
6a.
Changes
in
percent
fat
in
bovine
mamm a r y
secretion
by we e k
of
involution
status.
<
b.
M
0-0
0+7
0+14
■ ■
UNINFECTED
□
HAJON
D+Zl
0+28
C-14
C-7
MINOR
C-0
C+7
C+14
and
infection
FIGURE 7a.
status.
Changes
in percent protein in bovine mammary secretion by we e k of involution and infection
15
12
PROTEIN
I
86
*
01— 1
□-0
D+7
D+14
UNINFECTED
EZD
D+21
D+28
C-14
TIM
E
C-7
C-0
C+7
C+14
M
INO
R
MAJOR
'vj
00
Changes in concentration
FIGURE 8a.
involution and infection status.
of
bovine
serum
albumin
in bovine
C-14
TIM
E
C-7
mammary
secretion
by week
of
D
CD
Z
13
II
UJ
m
ui
z
»-l
>
o
03
linl
D-0
0+7
D+14
UNINFECTED
□
D+21
0+28
C-0
C+7
C+14
M
INO
R
MAJOR
179
FIGURE 9a. Changes in concentration of a-lactalbumin in bovine mammary secretion by week of involution
and infection status.
LACTALBUMIN
(MG/ML)
25,
0-0
D+7
0+14
H i
UNINFECTED
□
MAJOR
0+21
D+20
C-14
TIME
C-7
MINOR
C-0
C+7
C+14
FIGURE 10a.
Changes in percent tissue area composed of epithelium from bovine mammary glands by we e k of
involution and infection status.
X EPITHELIAL AREA
60
TIM
E
UNINFECTED
C D
MAJOR
MINOR
FIGURE 11a.
Changes in percent
involution and infection status.
tissue area composed
of
lumen
from bovine
mammary
glands
by week
of
40
X LU
M
IN
A
L AREA
30
C-7
□+14
C-0
TIM
E
UNINFECTED
□
M
INO
R
MAJOR
182
tissue
area
composed
of
stroma
from bovine mammary
gland
by week
of
X STR
O
M
A
L AREA
FIGURE 12a.
Changes in percent
involution and infection status.
D+14
TIM
E
IHI
UNINFECTED
□
MAJOR
M
INO
R
183
% NQNACTIVE
EPITHELIUM
FIGURE 13a.
Changes in percent alveolar area composed of nonactive secretory cells from bovine mammary
glands by week of involution and infection status.
1
D+i4
■ ■
UNINFECTED
L. _ 1 MAJOR
C -i4
MINOR
X MODERATELY
ACTIVE
EPITHELIUM
FIGURE 14a.
Changes in percent alveolar area composed of moderately active secretory cells from bovine
mammary glands by w e e k of involution and infection status.
0+7
D-0
UNINFECTED
CD
MAJOR
0+14
C-14
TIME
C-7
MINOR
C-0
fully
X FULLY
ACTIVE
EPITHELIUM
FIGURE 15a.
Changes in percent alveolar area composed of
mammary glands by week of involution and infection status.
D+14
C-14
TIME
IHI
UNINFECTED
□
MAJOR
MI N O R
active
secretory
cells
from
bovine
FIGURE 16a.
Changes in percent epithelial cell area composed of nucleus
week of Involution and infection status.
PERCENT
NUCLEUS
90
0+14
UNINFECTED
1
J
MAJOR
from bovine mammary tissue by
X UNOCCUPIED
CYTOPLASMIC
AREA
FIGURE
17a.
Changes in percent epithelial cell area composed
mammary tissue by week of involution and infection status.
C-14
UNINFECTED
□
MAJOR
MINOR
of
unoccupied
cytoplasm
from
bovine
X ROUGH
ENDOPLASMIC
RETICULAR
FIGURE 18a.
Changes in percent epithelial cell area composed of rough endoplasmic reticulum from bovine
mammary tissue by week of involution and infection status.
D-0
D+7
UNINFECTED
C Z I MAJOR
0+14
C-14
TIME
C-7
MINOR
C-0
X G0L6I APPARATUS
AREA
FIGURE 19a.
Changes in percent epithelial cell area
tissue by week of involution and infection status.
UNINFECTED
1
1 HAJOR
composed
of Golgi
apparatus
from bovine
mammary
% MITOCHONDRIA
FIGURE 20a.
Changes in percent epithelial cell area c omposed of m i t o c h o n d r i a from b o vine m a m m a r y tissue
by w e e k of involution and infection status.
D+14
UNINF-ECTED
□
MAJOR
MINOR
X FAT AREA
FIGURE 2 la.
Changes in percent epithelial cell area composed of fat from bovine mammary tissue by week
of involution and infection status.
©' ™
0-0
■■I
1—
—
™
0+7
UNINFECTED
MAJOR
™
0+14
™
™
C-14
TIME
C-7
MINOR
™
C-0
X SEC
R
ETO
R
Y VESICLE AREA
FIGURE 22a.
Changes in percent epithelial cell area composed of secretory vesicles
tissue by week of involution and infection status.
I
D-0
D+7
UNINFECTED
MAJOR
0+14
C-7
MINOR
C-0
from bovine mammary
of
milk
stasis
X STASIS
VACUOLE
AREA
FIGURE 23a.
Changes In percent epithelial cell ar e a composed
mammary tissue by we e k of involution and infection status.
1-
V
a.
D-0
i
0+7
■ ■
UNINFECTED
□
MAJOR
m
D+14
I
C-14
TIME
V//A
I-ih ^
C-7
MINOR
C-0
vacuoles
from
bovine
IgGl
PLASMA
CELLS
PER
TISSUE
FIGURE 24a.
Changes in numbers of immunoglobulin Gi-producing plasma cells In bovine mammary tissue by
week of involution and infection status.
D+14
UNINFECTED
□
MAJOR
C-14
TIME
MINOR
IgG
2 PL
A
SM
A CELLS/TISSUE AREA
FIGURE 25a.
Changes in numbers of immunoglobulin G 2~producing plasma cells in bovine mammary tissue by
week of involution and infection status.
D+14
I B
UNINFECTED
C D
MAJOR
C -1 4
TIME
MINOR
IgA PL
A
SM
A CELLS/TISSUE AREA
FIGURE 26a.
Changes in numbers of immunoglobulin A-producing plasma cells in bovine mammary tissue by
veek of involution and infection status.
C-14
■ ■
UNINFECTED
CD
MAJOR
MINQR
IgM PL
A
SM
A CELLS/TISSUE AREA
FIGURE 27a.
Changes in numbers of immunoglobulin M-producing plasma cells in bovine mammary tissue by
week of involution and infection status.
D+14
TIME
■ ■
UNINFECTED
□
MAJOR
MINOR
VITA
NAME:
Lorraine Marie Sordillo
BIRTH:
November 5, 1959,
Winchester, Massachusetts
EDUCATION:
1977
Graduated from Malden High School
Malden, Massachusettes
1981
B.S., University of Massachusetts, Amherst
Major:
Animal Science;
Minor:
Zoology
1984
M.S., University of Massachusetts, Amherst
Major:
Animal Science/Lactation Physiology
Thesis: "Caprine Mammary Gland Development and
Initiation of Lactation Following
Intramammary Colchicine Infusion."
EMPLOYMENT:
1984-1986
Graduate Research Assistant
Mastitis Research Laboratory
Hill Farm Research Station
Louisiana State University
1981-1983
Graduate Teaching Assistant
Department of Veterinary and Animal Sciences
University of Massachusetts
1980-1981
Research Laboratory Assistant
Department of Veterinary and Animal Sciences
University of Massachusetts
MEMBERSHIPS:
(A)
(B)
(C)
(D)
(E)
Alpha Zeta Fraternity
American Dairy Science Association
Louisiana Society for Electron Microscopy
Mastitis Research Workers Conference, 1983-present
Society for Experimental Biology and Medicine
HONORS AND AWARDS:
Arceneaux Memorial Award
Electron
Microscopy.
Microscopy, 1985.
for Outstanding Student Research in
Louisiana
Society
for
Electron
American Dairy Science Association Annual Award for Outstanding
Research Paper Presentation, 1985.
199
200
PUBLICATIONS :
Articles in Refereed Journals;
1.
Sordillo, L. M., S. P. Oliver, R. T. Duby, and R. Rufner.
1984.
Effects of colchicine on milk yield, composition,
and cellular differentiation during lactogenesis in the
caprine mammary gland.
Int. J. Biochem.
16:1135.
2.
Sordillo, L. M . , S. P. Oliver, and S. C. Nickerson.
1984.
Caprine mammary differentiation and the initiation of
lactation following prepartum colchicine infusion.
Int. J.
Biochem.
16:1265.
3.
Nickerson, S. C., and L. M. Sordillo.
1985.
Role of
macrophages
and
multinucleated
giant
cells
in
the
resorption of corpora amylacea in the involuting bovine
mammary gland.
Cell Tiss. Res.
240:397.
4.
Nickerson, S. C . , L. M. Sordillo, N. T. Boddie, and A. M.
Saxton.
1985.
Prevalence
and
ultrastructural
characteristics of bovine mammary corpora amylacea during
lactation and involution.
J. Dairy Sci.
68:709.
5.
Sordillo,
patterns
mammary
34:593.
L. M . , and S. C. Nickerson.
1986.
Growth
and histochemical
characterization of bovine
corpora
amylacea.
J.
Histochem.
Cytochem.
6.
Sordillo,
L. M., and S. C. Nickerson.
1986.
Morphologi­
cal changes caused by experimental Streptococcus uberis
mastitis
in mice
following
intramammary
infusion
of
pokeweed mitogen.
Proc. Soc. Exp. Biol. Med.
182:522.
7.
Nickerson,
S.
C.,
and L. M. Sordillo.
1986.
Origin,
fate, and properties of multinucleated giant cells and
their association with milk synthesizing tissues of the
bovine mammary gland.
Immunobiology. (Submitted).
8.
Sordillo, L. M . , S. C. Nickerson, S. P. Oliver, and R. M.
Akers.
1987.
Secretion composition during bovine mammary
involution and the relationship with mastitis.
Int. J.
Biochem.
(Submitted).
9.
Sordillo, L. M . , S. C. Nickerson, and S. P. Oliver.
1987.
Quantification and immunoglobulin classification of plasma
cells in nonlactating bovine mammary tissue.
J. Dairy
Sci.
(Submitted).
201
P U B L I C A T I O N S ; (continued)
Articles in Refereed Journals;
10.
(continued)
Sordillo, L. M . , S. C. Nickerson, and S. P. Oliver.
1987.
Morphological changes in the bovine mammary gland during
involution
and
lactogenesis.
Am.
J.
Vet.
Res.
(Submitted).
Research Papers Presented at Annual Meetings;
1.
Poos, M. I.,
and L. M. Sordillo.
1982.
type of housing and supplementation on
dairy calves
from birth to weaning.
65(Supplement 1):121.
(Abstract).
The effects of
performance of
J. Dairy Sci.
2.
Sordillo,
L. M . , S. P. Oliver, and S. P. Baker.
1982.
Influence
of
iodinated
casein
feeding
during
late
lactation on bovine mammary involution.
J. Dairy Sci.
65(Supplement 1):73.
(Abstract).
3.
Pekala, R. R., R. T. Duby, S. P. Oliver, G. Gnatek, A.
Pike, L.
M.Sordillo,
and A. Schmidt.
1982.
Plasma
progesterone levels during the estrous cycle, pregnancy,
and early lactation in goats.
Presented at the 10th New
England Endocrinology Conference, October 16, 1982, Mount
Holyoke College, South Hadley, Massachusetts.
4.
Sordillo, L. M., and S. P. Oliver.
1983.
Suppression of
milk
secretion
and
changes
in composition
following
intramammary infusion of colchicine near parturition.
J.
Dairy Sci.
66 (Supplement 1):104.
(Abstract).
5.
Nickerson, S. C., L. M. Sordillo, and J. W. Pankey.
1984.
Differential leukocytic response of internal bovine teat
end tissue to presence of intramammary infection.
J.
Dairy Sci.
67(Supplement l):85.
(Abstract),
6.
Sordillo, L. M . , and S. C. Nickerson.
1984.
Distribution
and ultrastructural changes of bovine mammary plasma cells
following
intramammary
infection
with
Staphylococcus
aureus. J. Dairy Sci.
67(Supplement l):170.
(Abstract).
7.
Sordillo, L. M . , S. P. Oliver, and R, T. Duby.
1984.
Caprine mammary development following prepartum colchicine
infusion.
J.
Dairy
Sci.
67(Supplement
1):94.
(Abstract).
8.
Sordillo, L. M . , S, C. Nickerson, N. T. Boddie, and J. W.
Pankey.
1984.
Morphologic and histochemical aspects of
bovine
mammary
corpora
amylacea.
J.
Dairy
Sci.
67(Supplement 1):174.
(Abstract).
202
9.
Sordillo, L. M . , and S. C. Nickerson.
1985.
Origin,
growth, and resorption of bovine mammary corpora amylacea
during
the
lactation
cycle.
Louisiana
Society
for
Electron Microscopy Newsletter.
11:9.
(Abstract).
10.
Sordillo, L. M., and S. C. Nickerson.
1985.
Resorption
of bovine mammary corpora amylacea during involution by
macrophages and multinucleate giant cells.
J. Dairy Sci.
68(Supplement 1):103.
(Abstract).
11.
Sordillo, L. M , , and S. C. Nickerson.
1986.
Pathogenesis
of
Streptococcus
uberis
mastitis
following
immunostimulation with pokeweed mitogen.
J. Dairy Sci.
69(Supplement l):199.
(Abstract).
12.
Sordillo, L. M . , S. C. Nickerson, and S. P. Oliver.
1986.
Biochemical and morphological changes
in
the bovine
mammary
gland
following
involution.
J.
Dairy
Sci.
6 9 (Supplement 1):162.
(Abstract).
13.
Sordillo, L. M.
1986.
Local antibody production by
bovine mammary plasma cells during involution and in
response to intramammary infection.
Mastitis Research
Workers Conference, Hershey, PA.
14.
Sordillo, L. M . , S, C. Nickerson, and S. P. Oliver.
1987.
Morphological
evaluation
of
bovine
mammary
tissue
throughout involution and colostrogenesis. J. Dairy Sci.
(Submitted).
15.
Sordillo, L. M., S. C. Nickerson, and
S. P. Oliver. 1987.
Influence of bacterial isolation on composition of bovine
mammary
secretion
throughout
involution
and
early
lactation.
J. Dairy Sci. (Submitted).
16.
Sordillo, L. M., S. C. Nickerson, and
S. P. Oliver. 1987.
Immunoglobulin
classification
of plasma
cells
in
nonlactating
bovine
mammary
tissue.
J.
Dairy
Sci.
(Submitted).
Other Publications:
1.
Sordillo, L. M.
1984.
Caprine mammary development and
initiation of lactation following intramammary colchicine
infusion.
Thesis.
University of Massachusetts, Amherst,
MA.
2.
Nickerson, S. C. , and L. M. Sordillo.
1985.
Corpora
amylacea in the bovine mammary gland:
prevalence and
ultrastructural
relationships
with
milk
synthesizing
tissue.
Louisiana
Society
for
Electron
Microscopy
Newsletter.
11:12.
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate:
Lorraine M, Sordillo
Major Field;
Dairy Science
Title of Dissertation:
Physiological Aspects of Bovine Mammary Involution:
A Biochemical and Morphological Investigation
Approved:
J'
/y
Major Professor and.Chairman
Dean of the Graduate School
EXAMINING COMMITTEE:
S, C. Nickerson (Chairman)
R, W. Adkinson
mu.
E. Chandler
r. T. Haldiman
L
• tf 6
L. C.
fl. Kappel
TCflnnpl I
d J L
W. N. Philpot
Date of Examination:
R. J. Siebeling
September 11, 1986